Patent Application
20210189346
The present invention relates to the field of cell culture, preferably hepatocyte culture, and in particular 3D cell culture, preferably 3D hepatocyte culture.
Hepatocytes are the major parenchymal cells of the liver and represent up to 70-85% of the liver mass. Hepatocytes are highly differentiated cells that carry out most of the hepatic functions, which pertain notably to metabolism, detoxification and systemic homeostasis. In the liver, hepatocytes normally are long-lived quiescent cells. However, upon injury or loss of functional mass, hepatocytes are able to proliferate, thus allowing liver regeneration.
The establishment of in vitro cultures of hepatocytes has long been pursued with the aim of developing in vitro models able to faithfully recapitulate key liver functions. Such models can be used in research to understand and study normal liver functions. They can also be used to understand and study liver pathologies, for example viral hepatitis, and assess the ability of candidate drugs to restore liver functions. Moreover, given the central role of the liver in metabolism and detoxification, in vitro cultures of hepatocytes are relevant models to predict the metabolism and toxicity of new drugs. In particular, cultures of primary human hepatocytes (PHH) are considered the gold standard model for assessing in vitro drug metabolism, drug-drug interaction and hepatotoxicity. Finally, in vitro cultures of primary human hepatocytes (PHH) also represent a promising approach in the treatment of liver diseases or disorders, for example through the development of extracorporeal devices, hepatocyte transplantation or transplantation of three-dimensional (3D) hepatocyte structures as an alternative to organ transplantation.
Thus, numerous hepatocyte culture systems have been designed, starting with the conventional two-dimensional (2D) monolayer cultures of primary hepatocytes, in particular of primary human hepatocytes. However, in such cultures, the primary hepatocytes de-differentiate and rapidly lose hepatocyte-specific functions. Moreover, conventional 2D monolayer cultures do not allow the long-term survival of primary hepatocytes. More complex culture systems have been developed, notably to allow longer survival and better maintenance of hepatocyte-specific functions. For example, primary hepatocytes have been cultured in a sandwich configuration, between two layers of gelled extracellular matrix proteins. Hepatocytes have also been co-cultured, for instance with non-parenchymal liver cells (Baffet et al., 1991), hepatocellular carcinoma (HCC) cells (Jang et al., 2016) or 3T3 fibroblasts (Berger et al., 2016). More recently, three-dimensional (3D) cultures have been showed to be particularly suited for obtaining hepatocytes with a stable phenotype, able to retain morphology, viability and hepatocyte-specific functions. In particular, primary human hepatocyte free sphere-shaped aggregates can be obtained, for example when hepatocytes are cultured in ultra-low attachment plates (Bell et al., 2016) or when hepatocytes are encapsulated or embedded in a matrix or a scaffold, such as a polysaccharide scaffold, for example alginate (WO2013/087843). Critically, none of the above-mentioned culture systems enable proliferation of the cultured primary human hepatocytes.
Proliferation can be observed using alternative cell systems, such as hepatocyte-like cell lines, either originating from tumors or obtained by oncogenic immortalization (i.e., by cell transformation), or stem-cell derived hepatocyte-like cells. For example, HuH-7 and HepG2 are hepatocyte-like cell lines derived from human hepatocellular carcinoma with little differentiated functions. Fa2N-4 and Hepa RG are human immortalized cell lines, the latter retaining many characteristics of primary human hepatocytes. However, even with these alternative cell systems, proliferation and differentiation are usually mutually exclusive. Moreover, cancer cell lines and immortalized cell lines do not constitute the optimal in vitro model to recapitulate physiological human liver biology.
To date, proliferation of cultured primary hepatocytes has only been observed with rodent hepatocytes. Spontaneous proliferation has been observed with mouse hepatocytes (Frémin et al., 2007) while stimulation with a growth factor has been able to induce proliferation of rat hepatocytes. For example, US 2005/0244959 describes a method to induce active in vitro proliferation of rat hepatocytes through the addition of at least a cytokine and a growth factor, preferably TNFα and EGF, to the rat hepatocyte culture. However, as with cancer cell lines and immortalized cell lines, rodent hepatocyte cultures do not constitute the optimal in vitro model to recapitulate physiological human liver biology.
Thus, studying and taking advantage of the proliferative capacity of hepatocytes for pharmaceutical or therapeutic applications through the use of an in vitro model remain a major challenge. In particular, there is still a need for a 3D culturing method enabling the in vitro proliferation of human primary hepatocytes.
The present invention thus relates to a method of culturing animal cells allowing to obtain 3D animal cell structures comprising proliferative animal cells, said proliferative animal cells retaining their phenotype, e.g., their differentiated state, and their functions. The method of the invention comprises a first step of culturing the animal cells in a non-adherent culture vessel, preferably a low or ultra-low attachment culture vessel, a second step of embedding the animal cells in a collagen matrix or in a gelatin matrix, in particular a GelMa matrix, and a third step of culturing the animal cells embedded in the collagen matrix or in the gelatin matrix, in particular in the GelMa matrix. The method of the invention is particularly suited for the culture of primary human hepatocytes and allows to obtain proliferative PHH spheroids, i.e., acinus-like structures with a hollow lumen, comprising proliferative primary human hepatocytes. The present invention also relates to a spheroid comprising proliferative primary human hepatocytes and uses thereof, for example for engineering an artificial liver model or an artificial liver organ, or for assessing in vitro the toxicity and/or the effects of a drug or a compound.
The present invention relates to a method of culturing animal cells to obtain 3D animal cell structures comprising proliferative animal cells, said method comprising:
According to one embodiment, the present invention relates to a method of culturing animal cells to obtain 3D animal cell structures comprising proliferative animal cells, said method comprising:
thereby obtaining 3D animal cell structures comprising proliferative animal cells.
In one embodiment, at step b) of the method as described hereinabove, the animal cells are transferred to a culture medium comprising collagen, preferably fibrillar collagen, at a concentration ranging from about 0.25 mg/mL to about 3 mg/mL. In one embodiment, said fibrillar collagen is selected from the group comprising or consisting of type I collagen, type II collagen, type III collagen, type V collagen, type VI collagen, type XI collagen, type XXIV collagen, type XXVII collagen and any mixtures thereof.
According to one embodiment, the present invention relates to a method of culturing animal cells to obtain 3D animal cell structures comprising proliferative animal cells, said method comprising:
thereby obtaining 3D animal cell structures comprising proliferative animal cells.
In one embodiment, at step b) of the method as described hereinabove, the animal cells are transferred to a culture medium comprising methacrylated gelatin (GelMa) at a concentration ranging from about 1% (w/v) to about 20% (w/v).
In one embodiment, at step a) of the method as described hereinabove, the animal cells are cultured in a non-adherent culture vessel, preferably a low or ultra-low attachment culture vessel, for a period ranging from about 1 h to about 96 h.
In one embodiment, at step c) of the method as described hereinabove, the animal cells embedded in the collagen or gelatin matrix are cultured for at least about 2 days.
In one embodiment, the method as described hereinabove further comprises:
In one embodiment, the method as described hereinabove further comprises:
In one embodiment, the animal cells as described hereinabove are primary animal cells. In one embodiment, the animal cells as described hereinabove are primary hepatocytes, preferably primary human hepatocytes, and the 3D animal cell structures comprising proliferative animal cells as described hereinabove are spheroids, preferably said spheroids have an acinus-like structure with a hollow lumen, comprising proliferative primary hepatocytes, preferably proliferative primary human hepatocytes.
The present invention also relates to a spheroid comprising proliferative primary hepatocytes embedded in a collagen or gelatin matrix, preferably wherein said spheroid has an acinus-like structure with a hollow lumen. In one embodiment, said spheroid is embedded in a methacrylated gelatin (GelMa) matrix. In one embodiment, said proliferative primary hepatocytes are proliferative primary human hepatocytes.
The present invention also relates to the use of a spheroid comprising proliferative primary human hepatocytes for engineering an artificial liver model or an artificial liver organ, preferably wherein said spheroid has an acinus-like structure with a hollow lumen.
The present invention also relates to the use of a spheroid comprising proliferative primary human hepatocytes for assessing in vitro the liver toxicity, in particular liver genotoxicity, and/or the effects of a drug or a compound, preferably wherein said spheroid has an acinus-like structure with a hollow lumen. In one embodiment, said use is for assessing in vitro the liver genotoxicity of a drug or a compound, preferably wherein said spheroid has an acinus-like structure with a hollow lumen.
The present invention also relates to an in vitro method of assessing the toxicity and/or the effects of a drug or a compound, the method comprising:
In the present invention, the following terms have the following meanings:
The present invention relates to a method of culturing animal cells comprising:
thereby obtaining 3D animal cell structures comprising proliferative animal cells.
According to one embodiment, the present invention relates to a method of culturing animal cells comprising:
thereby obtaining 3D animal cell structures comprising proliferative animal cells.
According to one embodiment, the present invention relates to a method of culturing animal cells comprising:
thereby obtaining 3D animal cell structures comprising proliferative animal cells.
In one embodiment, the present invention relates to a method of culturing animal cells comprising:
thereby obtaining 3D animal cell structures comprising proliferative animal cells.
According to one embodiment, animal cells are proliferative if at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%, preferably at least about 20%, more preferably at least about 40%, of the total number of animal cells are positive for a proliferation marker such as, for example, positive for BrdU incorporation (BrdU+), positive for EdU incorporation (EdU+), positive for Ki67 expression (Ki67+), or positive for cyclin D1 expression (cyclin D1*).
The method of the invention is suitable for the culture of virtually any type of animal cells requiring cell-cell interactions to survive and differentiate.
In one embodiment, the animal cells cultured according to the method of the invention are primary animal cells.
In one embodiment, the animal cells as described hereinabove are mammal cells. In another embodiment, the animal cells as described hereinabove are primate cells. In another embodiment, the animal cells as described hereinabove are human cells.
In one embodiment, the animal cells as described hereinabove are somatic or differentiated animal cells. In another embodiment, the animal cells as described hereinabove are stem cells, in particular somatic stem cells.
In one embodiment, the animal cells as described hereinabove are not human embryonic stem cells.
Examples of animal cells that can be cultured according to the method of the invention include, without being limited to, parenchymal liver cells such as hepatocytes; non-parenchymal liver cells such as Kupffer cells, cholangiocytes, fibroblasts, sinusoidal endothelial cells and stellate cells; lung cells; heart cells; kidney cells; colon cells; skin cells; testis cells; eye cells; brain cells; and any mixtures thereof.
In one embodiment, the animal cells as described hereinabove are selected from the group comprising or consisting of liver cells such as parenchymal liver cells (e.g., hepatocytes) and non-parenchymal liver cells (e.g., Kupffer cells, cholangiocytes, fibroblasts, sinusoidal endothelial cells and stellate cells); lung cells such as pneumocytes; heart cells such as cardiomyocytes, nodal cells and myocardial endocrine cells; colon cells such as enterocytes, caliciform cells (also referred to as Goblet cells), Paneth cells, and enteroendocrine cells; skin cells such as keratinocytes, melanocytes, corneocytes; kidney cells such as kidney epithelial cells; testis cells such as Leydig cells and Sertoli cells; eye cells such as photoreceptor cells, ocular cells and keratocytes; brain cells such as neuronal cells and glial cells; and any mixtures thereof.
In one embodiment, the animal cells as described hereinabove are selected from the group comprising or consisting of hepatocytes, Kupffer cells, cholangiocytes, fibroblasts, sinusoidal endothelial cells, stellate cells, pneumocytes, cardiomyocytes, nodal cells, myocardial endocrine cells, enterocytes, caliciform cells (also referred to as Goblet cells), Paneth cells, enteroendocrine cells, keratinocytes, melanocytes, corneocytes, kidney epithelial cells, Leydig cells, Sertoli cells, photoreceptor cells, keratocytes, neuronal cells, glial cells, and any mixtures thereof.
In one embodiment, the animal cells to be cultured with the method of the invention are selected from the group comprising or consisting of hepatocytes, Kupffer cells, cholangiocytes, fibroblasts, sinusoidal endothelial cells, stellate cells and any mixtures thereof; preferably said animal cells are primary animal cells, more preferably human primary cells.
In one embodiment, the animal cells to be cultured with the method of the invention are hepatocytes, preferably primary hepatocytes, more preferably primary human hepatocytes (PHH).
In one embodiment, the animal cells to be cultured with the method of the invention are isolated animal cells. In one embodiment, the animal cells to be cultured with the method of the invention are obtained from a sample of tissue previously taken from a subject, such as, for example, from a biopsy previously taken. In one embodiment, the animal cells to be cultured with the method of the invention are kept frozen after having been obtained from a sample of liver tissue previously taken from a subject.
In one embodiment, the primary human hepatocytes to be cultured according to the method of the invention are obtained from a sample of liver tissue previously taken from a subject. In one embodiment, the primary human hepatocytes to be cultured according to the method of the invention are kept frozen after having been obtained from a sample of liver tissue previously taken from a subject.
According to the method of the invention, the animal cells are first cultured in a non-adherent culture vessel. Without wishing to be bound by any theory, the Applicant suggests that first culturing the animal cells in a non-adherent culture vessel enables the animal cells to form aggregates and thus enhances cell-cell interactions. Cell-cell interactions are essential for the subsequent development, structuration and growth of 3D animal cell structures comprising proliferative animal cells, such as, for example, spheroids.
The Applicant indeed showed that directly adding animal cells to a culture medium comprising collagen and culturing the animal cells thus embedded in a collagen matrix without first culturing the animal cells in a non-adherent culture vessel did not allow to obtain 3D animal cell structures comprising proliferative animal cells. In particular, the Applicant showed that primary human hepatocytes must first be cultured in a non-adherent culture vessel before being transferred to a culture medium comprising collagen or gelatin, in particular methacrylated gelatin (GelMa), and cultured in a collagen matrix or in a gelatin matrix, in particular a GelMa matrix, in order to obtain spheroids comprising proliferative primary human hepatocytes.
As used herein, the term “non-adherent culture vessel” refers to a culture vessel that is not conductive to the attachment of cells, in particular animal cells, to said vessel. In other words, cells, in particular animal cells, being cultured in a non-adherent culture vessel do not attach, or very little, and do not spread to the inner surface of said culture vessel (i.e., to the walls and/or bottom of said culture vessel).
Non-adherent culture vessels include, without being limited to, culture vessels made of glass, untreated culture vessels, low attachment culture vessels, and ultra-low attachment culture vessels.
As used herein, the term “untreated culture vessel” refers to a culture vessel that did not undergo the chemical treatment or the coating commonly applied to culture vessels in order to enhance cell attachment to the culture vessels. In the field of cell culture, “treated culture vessel” commonly refers to a culture vessel, such as a plate or a dish, usually made of polystyrene, that underwent a chemical treatment or a coating in order to enhance the attachment of cells, in particular animal cells, to the inner surface of the culture vessel. Indeed, polystyrene is a very hydrophobic polymer to which cells, in particular animal cells, have difficulty attaching.
In one embodiment, the non-adherent culture vessel as described hereinabove is selected from the group comprising or consisting of culture vessels made of glass, untreated culture vessels, low attachment culture vessels and ultra-low attachment culture vessels; preferably said non-adherent culture vessel is a low or ultra-low attachment culture vessel.
Examples of low and ultra-low attachment culture vessels include, without being limited to, low and ultra-low attachment dishes, low and ultra-low attachment flasks and low and ultra-low attachment plates, such as, for example, low and ultra-low attachment multi-well plates or low and ultra-low attachment microplates.
Low attachment culture vessels and ultra-low attachment culture vessels are characterized by the presence of a coating, usually a hydrophilic gel, that is covalently bound to the inner surface of the culture vessel. Said coating inhibits specific and nonspecific immobilization and thus prevents cell attachment to the inner surface of the culture vessel, thereby maintaining cells into a suspended state.
Low and ultra-low attachment culture vessels, in particular low attachment plates (LAP) and ultra-low attachment (ULA) plates, are readily available from suppliers and include, for example, Corning® Costar®, InSphero®, S-Bio® or Perkin Elmer® low and ultra-low attachment plates.
Alternatively, methods to prepare low attachment culture vessels, in particular low attachment plates, are well known to those skilled in the art. Such methods include, without being limited to, coating untreated culture vessels with 1% agarose or coating untreated culture vessels with poly-2-hydroxyethyl methacrylate (also referred to as poly-HEMA).
In one embodiment, the animal cells to be cultured with the method of the invention were stored frozen. Thus, in one embodiment, the animal cells to be cultured with the method of the invention are first thawed.
Methods to thaw previously frozen animal cells are well-known in the field of animal cell culture.
In one embodiment, the animal cells to be cultured with the method of the invention are first cultured in a low attachment plate (LAP) or in an ultra-low attachment plate (ULA) such as, for example, Corning® Costar®, InSphero®, S-Bio® or Perkin Elmer® low or ultra-low attachment plates.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above for at least about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, or 15 h.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above for at most about 96 h, 84 h, 72 h, 60 h, 48 h, 36 h, 24 h, 23 h, 22 h, 21 h, or 20 h.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above for a duration ranging from about 1 h to about 96 h, preferably from about 5 h to about 48 h, more preferably from about 10 h to about 20 h.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above at a concentration of at least about 102, 5×102, or 103 cells per cm2.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above at a concentration of at most about 106, 5×105 or 105 cells per cm2.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above at a concentration ranging from about 102 to about 106 cells per cm2, preferably from about 103 to about 105 cells per cm2, more preferably from about 1×104 to about 9×104 cells per cm2.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above, at a concentration ranging from about 105 to about 5×105 cells per cm2, preferably from about 2×105 to about 2.5×105 cells per cm2. In one embodiment, the animal cells are cultured in a low or ultra-low attachment plate at a concentration ranging from about 106 to about 5×106 cells per well, preferably from about 2×106 to about 2.5×106 cells per well, said well preferably having a surface of about 10 cm2.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above at a concentration of about 102, 103, 104, 105, or 106 cells per cm2.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above at a concentration of about 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, or 9×104 cells per cm2.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above at a concentration of at least about 5×103, 104, or 5×104 cells per ml of culture medium.
In one embodiment, the animal cells are cultured in non-adherent culture vessel as described above at a concentration of at most about 2×107, 107, or 5×106 cells per ml of culture medium.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above at a concentration ranging from about 5×103 to about 2×107 cells per ml of culture medium, preferably from about 104 to about 107 cells per ml of culture medium, more preferably from about 105 to about 106 cells per ml of culture medium.
In one embodiment, the animal cells are cultured in the non-adherent culture vessel as described above at a concentration of about 104, 105, 106, or 107 cells per ml of culture medium.
Selecting conditions suitable for the culture of animal cells is well known to those skilled in the art. Thus, the culture conditions to be used for culturing animal cells in the non-adherent culture vessel as described above according to the method of the invention, such as, for example, culture medium, temperature, humidity and levels of CO2, will be apparent to those having skill in the art and will depend on the animal cells to be cultured.
Culture media that may be used according to the method of the invention include natural media and synthetic media, such as, for example, serum-containing media, serum-free media, xeno-free media notably for human cell culture, protein-free media, chemically defined media.
Examples of culture media include, without being limited to, William's E medium, Basal Medium Eagle (BME), Eagle's Minimum Essential Medium (EMEM), Minimum Essential
Medium (MEM), Dulbecco's Modified Eagles Medium (DMEM), Ham's F-10, Ham's F-12 medium, Kaighn' s modified Ham's F-12 medium, DMEM/F-12 medium, and McCoy's 5A medium.
Culture media according to the present invention also include media suitable for the culture of a particular type of animal cells, such as, for example, culture media suitable for the culture of hepatocytes (e.g., William's E Medium).
According to the present invention, the culture medium may be supplemented with additional substances such as salts, carbon sources, amino acids, serum and serum components, vitamins, minerals, reducing agents, buffering agents, lipids, nucleosides, antibiotics, cytokines, and growth factors.
According to one embodiment, the animal cells to be cultured with the method of the invention are primary animal cells, preferably primary human cells, and they are cultured in the non-adherent culture vessel as described above in a medium suitable for the culture of primary animal cells, preferably primary human cells.
Media suitable for the culture of primary animal cells are commercially available. The medium suitable for the culture of primary animal cells, preferably primary human cells will be apparent to those skilled in the art and will depend on the primary animal cells to be cultured (e.g., hepatocytes, pneumocytes, cardiomyocytes, keratinocytes, melanocytes, corneocytes, or neuronal cells).
According to one embodiment, the animal cells to be cultured with the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, and they are cultured in the non-adherent culture vessel as described above in a medium suitable for the culture of hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes.
Examples of media suitable for the culture of hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, include, without being limited to, Dulbecco's Modified Eagles Medium (DMEM), William's E Medium, Hepatocyte Culture Medium, Basal HepaRG Medium, HBM Basal Medium, and Hepatocyte Basal Medium.
According to the present invention, said media suitable for the culture of hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, may be supplemented, such as, for example, with L-glutamine, albumin, penicillin, streptomycin, insulin, transferrin, sodium pyruvate, sodium selenite, hydrocortisone or dexamethasone, HGF (hepatocyte growth factor), EGF (epidermal growth factor) and/or FCS (fetal calf serum) also referred to as FBS (fetal bovine serum).
In one embodiment, said media suitable for the culture of hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, may be supplemented, such as, for example, with gentamicin, L-glutamine, albumin, penicillin, streptomycin, insulin, transferrin, sodium pyruvate, sodium selenite, hydrocortisone or dexamethasone, HGF (hepatocyte growth factor), EGF (epidermal growth factor) and/or FCS (fetal calf serum) also referred to as FBS (fetal bovine serum).
In one embodiment, the animal cells to be cultured according to the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes and they are cultured in a low or ultra-low attachment culture vessel, preferably a low or ultra-low attachment plate, in William's E Medium supplemented with penicillin, streptomycin, insulin, glutamine (L-glutamine) and albumin, and optionally supplemented with transferrin, sodium selenite, hydrocortisone, HGF (hepatocyte growth factor), EGF (epidermal growth factor) and/or FCS (fetal calf serum), also referred to as WE HH (William's E medium human hepatocytes).
In one embodiment, the animal cells to be cultured according to the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes and they are cultured in a low or ultra-low attachment culture vessel, preferably a low or ultra-low attachment plate, in WE HH medium comprising penicillin, streptomycin, insulin, glutamine (L-glutamine), albumin and optionally FCS (fetal calf serum).
In one embodiment, the WE HH medium of the invention comprises penicillin at a concentration ranging from about 50 to about 200 U/mL, preferably about 100 U/mL; streptomycin at a concentration ranging from about 50 to about 200 μg/mL, preferably 100 μg/mL; insulin at a concentration ranging from about 5 to about 30 μg/mL, preferably about 5 μg/mL or about 15 μg/mL; glutamine (L-glutamine) at a concentration ranging from about 1 to about 10 mM, preferably about 2 mM; albumin at a concentration ranging from about 0.01 to about 1% (w/v), preferably about 0.1% (w/v); and FCS (fetal calf serum) at a concentration ranging from about 0 to about 20% (v/v), or from 1% to about 20% (v/v), and preferably about 10% (v/v).
In one embodiment, the animal cells to be cultured according to the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes and they are cultured in a low or ultra-low attachment culture vessel, preferably a low or ultra-low attachment plate, in a culture medium, preferably in William's E Medium, which does not comprise or is not supplemented with FCS (fetal calf serum).
In one embodiment, the animal cells to be cultured according to the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes and they are cultured in a low or ultra-low attachment culture vessel, preferably a low or ultra-low attachment plate, in WE HH medium supplemented with penicillin, streptomycin, insulin, glutamine (L-glutamine), albumin and hydrocortisone and optionally supplemented with transferrin, sodium selenite, HGF (hepatocyte growth factor), EGF (epidermal growth factor) and/or FCS (fetal calf serum).
In one embodiment, the WE HH medium of the invention is supplemented with penicillin, streptomycin, insulin, glutamine (L-glutamine) and albumin, with or without FCS (fetal calf serum).
In one embodiment, the WE HH medium of the invention is supplemented with penicillin, streptomycin, insulin, glutamine (L-glutamine), albumin, hydrocortisone, transferrin, sodium selenite, HGF (hepatocyte growth factor) and EGF (epidermal growth factor), with or without FCS (fetal calf serum).
In one embodiment, the WE HH medium of the invention comprises insulin at a concentration ranging from about 5 to about 30 μg/mL, preferably ranging from about 5 to about 15 μg/mL, more preferably about 5 μg/mL or about 15 μg/mL; transferrin at a concentration ranging from about 0 to about 10 μg/mL, or from about 0.1 to about 10 μg/mL, and preferably about 5.5 μg/mL; sodium selenite at a concentration ranging from about 0 to about 10 μg/mL, or from about 0.1 to about 10 μg/mL, and preferably about 5 μg/mL; hydrocortisone at a concentration ranging from about 0.1 to about 50 μM, preferably about 1 μM; rhHGF (recombinant human hepatocyte growth factor) at a concentration ranging from about 0 to about 10 ng/mL, or from about 0.1 to about 10 ng/mL, and preferably about 2.5 ng/mL; rhEGF (recombinant human epidermal growth factor) at a concentration ranging from about 0 to about 1 ng/μL, or from about 0.01 to about 1 ng/μL, and preferably about 0.05 ng/μL; FCS (fetal calf serum) at a concentration ranging from about 0 to about 20% (v/v), or from about 1% to about 20% (v/v), and preferably about 10% (v/v); glutamine (L-glutamine) at a concentration ranging from about 1 to about 10 mM, preferably about 2 mM; albumin at a concentration ranging from about 0.01 to about 1% (w/v), preferably about 0.1% (w/v); penicillin at a concentration ranging from about 50 to about 200 U/mL, preferably about 100 U/mL; streptomycin at a concentration ranging from about 50 to about 200 μg/mL, preferably 100 μg/mL.
In one embodiment, the WE HH medium as described hereinabove further comprises gentamicin.
Thus, in one embodiment, the animal cells to be cultured according to the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes and they are cultured in a low or ultra-low attachment culture vessel, preferably a low or ultra-low attachment plate, in WE HH medium supplemented with gentamicin, penicillin, streptomycin, insulin, glutamine (L-glutamine), albumin and hydrocortisone, with or without FCS (fetal calf serum).
In one embodiment, the WE HH medium as described hereinabove comprises gentamicin at a concentration ranging from about 1 to about 100 μg/mL, preferably about 50 μg/mL.
Thus, in one embodiment, the animal cells to be cultured according to the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes and they are cultured in a low or ultra-low attachment culture vessel, preferably a low or ultra-low attachment plate, in WE HH medium supplemented with gentamicin at a concentration ranging from about 1 to about 100 μg/mL, preferably about 50 μg/mL; penicillin at a concentration ranging from about 50 to about 200 U/mL, preferably about 100 U/mL; streptomycin at a concentration ranging from about 50 to about 200 μg/mL, preferably 100 μg/mL; insulin at a concentration ranging from about 5 to about 30 μg/mL, preferably ranging from about 5 to about 15 μg/mL, more preferably about 5 μg/mL; glutamine (L-glutamine) at a concentration ranging from about 1 to about 10 mM, preferably about 2 mM; albumin at a concentration ranging from about 0.01 to about 1% (w/v), preferably about 0.1% (w/v); with or without FCS (fetal calf serum) at a concentration ranging from about 1% to about 20% (v/v), and preferably about 10% (v/v).
In one embodiment, the animal cells to be cultured according to the method of the invention are human cells, preferably primary human cells such as primary human hepatocytes, and they are cultured in the non-adherent culture vessel, preferably a low or ultra-low attachment culture vessel, at about 37° C. and about 5% CO2.
According to the method of the invention, culturing the animal cells in the non-adherent culture vessel as described above allows to obtain aggregates of said animal cells.
In one embodiment, after culturing the animal cells in the non-adherent culture vessel as described above at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% ,75% or 80%, preferably at least about 50%, of the animal cells are aggregated.
Methods to observe the formation of cell aggregates are well-known to one skilled in the art and include, for example, fluorescence microscopy such as two-photon excited fluorescence (TPEF) microscopy.
According to the method of the invention, after being first cultured in a non-adherent culture vessel as described above, the animal cells are transferred to a culture medium comprising collagen or truncated collagen, i.e., gelatin, and are thus embedded in a collagen or gelatin matrix. Without wishing to be bound by any theory, the Applicant suggests that embedding the animal cells in a collagen or gelatin matrix provides the animal cells with a microenvironment enabling the proliferation of the animal cells and the formation of 3D animal cell structures comprising proliferative animal cells. In particular, the Applicant suggests that embedding primary human hepatocytes (PHH) in a collagen matrix or a gelatin matrix, in particular a methacrylated gelatin (GelMa) matrix, provides the PHH with a microenvironment enabling the proliferation of the PHH and the formation of PHH spheroids according to the invention, i.e., spheres delimited by a single layer of well-organized PHH forming acinus-like structures with a hollow lumen.
The Applicant indeed showed that continuously culturing the animal cells in a non-adherent culture vessel as described above without ever embedding the animal cells in a collagen or gelatin matrix did not allow to obtain 3D animal cell structures comprising proliferative animal cells. In particular, the Applicant showed that primary human hepatocytes first cultured in a non-adherent culture vessel such as a low attachment plate must be transferred to a culture medium comprising collagen or gelatin, in particular methacrylated gelatin (GelMa), and embedded in a collagen matrix or in a gelatin matrix, in particular a GelMa matrix, in order to obtain spheroids comprising proliferative primary human hepatocytes, wherein said spheroids are acinus-like structures with a hollow lumen, delimited by a single layer of well-organized PHH forming.
According to the present invention, collagen is added to a culture medium as described hereinabove in order to obtain a culture medium comprising collagen, and thus to transfer the animal cells first cultured in a non-adherent culture vessel in said culture medium comprising collagen.
As used herein, the term “collagen” encompasses the different sub-families of collagens and truncated collagen also referred to as gelatin.
Thus, in one embodiment, the animal cells are transferred to a culture medium comprising truncated collagen, i.e., gelatin.
In one embodiment, the animal cells are transferred to a culture medium comprising fibrillar collagen.
In one embodiment, the animal cells are transferred to a culture medium comprising gelatin derived from fibrillar collagen.
Examples of fibrillar collagen include, without being limited to, type I collagen, type II collagen, type III collagen, type V collagen, type VI collagen, type XI collagen, type XXIV collagen and type XXVII collagen.
In one embodiment, the animal cells are transferred to a culture medium comprising fibrillar collagen selected from the group comprising or consisting of type I collagen, type II collagen, type III collagen, type V collagen, type VI collagen, type XI collagen, type XXIV collagen, type XXVII collagen and any mixtures thereof.
In one embodiment, the animal cells are transferred to a culture medium comprising gelatin derived from fibrillar collagen selected from the group comprising or consisting of type I collagen, type II collagen, type III collagen, type V collagen, type VI collagen, type XI collagen, type XXIV collagen, type XXVII collagen and any mixtures thereof.
In one embodiment, the animal cells are transferred to a culture medium comprising fibrillar collagen selected from the group comprising or consisting of type I collagen, type III collagen, and any mixtures thereof.
In one embodiment, the animal cells are transferred to a culture medium comprising gelatin derived from fibrillar collagen selected from the group comprising or consisting of type I collagen, type III collagen, and any mixtures thereof.
In one embodiment, the animal cells are transferred to a culture medium comprising type I collagen.
In one embodiment, the animal cells are transferred to a culture medium comprising gelatin derived from type I collagen. In one embodiment, the animal cells are transferred to a culture medium comprising type A gelatin, in particular type A gelatin derived from type I collagen.
Examples of type I collagen that may be used according to the method of the invention include, without being limited to, rat type I collagen, bovine type I collagen, porcine type I collagen, human type I collagen from human placenta, recombinant human type I collagen, type I collagen from rabbit skin, ovine type I collagen and type I-related fibrillar collagen from jelly fish and sea materials.
In one embodiment, the animal cells are transferred to a culture medium comprising collagen as described hereinabove at a concentration of at least about 0.25 mg/mL, 0.30 mg/mL, 0.35 mg/mL, 0.40 mg/mL, 0.45 mg/mL, 0.5mg/mL, 0.55 mg/mL, 0.6 mg/mL, 0.65 mg/mL, 0.7 mg/mL, or 0.75 mg/mL. In another embodiment, the animal cells are transferred to a culture medium comprising collagen as described hereinabove at a concentration of at least about 0.70 mg/mL, 0.71 mg/mL, 0.72 mg/mL, 0.73 mg/mL, 0.74 mg/mL or 0.75 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising collagen as described hereinabove at a concentration of at most about 4 mg/mL, 3.75 mg/mL, 3.5 mg/mL, 3.25 mg/mL, 3 mg/mL, 2.75 mg/mL, 2.5mg/mL, 2.25 mg/mL or 2 mg/mL. In another embodiment, the animal cells are transferred to a culture medium comprising collagen as described hereinabove at a concentration of at most about 4 mg/mL, 3.9 mg/mL, 3.8 mg/mL, 3.7 mg/mL, 3.6 mg/mL, 3.5 mg/mL, 3.4 mg/mL, 3.3 mg/mL, 3.2 mg/mL, 3.1 mg/mL or 3 mg/mL. In another embodiment, the animal cells are transferred to a culture medium comprising collagen as described hereinabove at a concentration of at most about 3 mg/mL, 2.9 mg/mL, 2.8 mg/mL, 2.7 mg/mL, 2.6 mg/mL, 2.5 mg/mL, 2.4 mg/mL, 2.3 mg/mL, 2.2 mg/mL, 2.1 mg/mL or 2 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising collagen as described hereinabove at a concentration ranging from about 0.25 mg/mL to about 3 mg/mL, preferably from about 0.5 mg/mL to about 2.5 mg/mL, more preferably from about 0.75 mg/mL to about 1.5 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising collagen at a concentration of about 0.75 mg/mL, 1 mg/mL, 1.25 mg/mL or 1.5 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising gelatin as described hereinabove at a concentration of at least about 15% (w/v), 20% (v/w), 25% (w/v), 30% (w/v), 35% (w/v), 40% (w/v), 45% (w/v) or 50% (w/v). Thus, in one embodiment, the animal cells are transferred to a culture medium comprising gelatin as described hereinabove at a concentration of at least about 150 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL, 350 mg/mL, 400 mg/mL, 450 mg/mL or 500 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising gelatin as described hereinabove at a concentration of at most about 60% (w/v), 55% (w/v), 50% (w/v), 45% (w/v) or 40% (w/v). Thus, in one embodiment, the animal cells are transferred to a culture medium comprising gelatin as described hereinabove at a concentration of at most about 600 mg/mL, 550 mg/mL, 500 mg/mL, 450 mg/mL or 400 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising gelatin as described hereinabove at a concentration ranging from about 15% (w/v) to about 60% (w/v), preferably from about 20 (w/v) to about 50% (w/v). Thus, in one embodiment, the animal cells are transferred to a culture medium comprising gelatin as described hereinabove at a concentration ranging from about 150 mg/mL to about 600 mg/mL, preferably from about 200 mg/mL to about 500 mg/mL.
In a particular embodiment, the animal cells are transferred to a culture medium comprising methacrylated gelatin (GelMa).
In one embodiment, GelMa is obtained by the methacrylation of gelatin as described hereinabove with methacrylic anhydride. In one embodiment, GelMa is obtained by the methacrylation of gelatin derived from type I collagen with methacrylic anhydride. In one embodiment, GelMa is obtained by the methacrylation of type A gelatin. In one embodiment, GelMa is obtained by the methacrylation of type A gelatin derived from type I collagen with methacrylic anhydride.
In one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove at a concentration of at least about 1% (w/v), 2% (w/v), 2.5% (w/v), 3% (w/v), 4% (w/v) or 5% (w/v). Thus, in one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove at a concentration of at least about 10 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 40 mg/mL or 50 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove at a concentration of at most about 20% (w/v), 15% (w/v), 10% (w/v), 9% (w/v), 8% (w/v), 7% (w/v), 7.5% (w/v), 6% (w/v) or 5% (w/v). Thus, in one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove at a concentration of at most about 200 mg/mL, 150 mg/mL, 100 mg/mL, 90 mg/mL, 80 mg/mL, 70 mg/mL, 75 mg/mL, 60 mg/mL or 50 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove at a concentration ranging from about 1% (w/v) to about 20% (w/v), preferably from about 2.5% (w/v) to about 10% (w/v), more preferably from about 4% (w/v) to about 6% (w/v). Thus, in one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove at a concentration ranging from about 10 mg/mL to about 200 mg/mL, preferably from about 25 mg/mL to about 100 mg/mL, more preferably from about 40 mg/mL to about 60 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising GelMa at a concentration of about 5% (w/v), i.e., at a concentration of about 50 mg/mL.
In one embodiment, the concentration of collagen, truncated collagen (i.e., gelatin) or methacrylated gelatin comprised in the culture medium as described hereinabove is referred to as the final concentration of collagen, truncated collagen (i.e., gelatin) or methacrylated gelatin in the culture medium.
In one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove and further comprising a photoinitiator to induce polymerization upon light exposure.
Examples of photoinitiators include, without being limited to, 2,2′-azobis[2-methyl-n-(2-hydroxyethyl)propionamide] (also known as VA-086), 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propanon (also known as Irgacure 2959), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (also known as LAP or BioKey), 2′,4′,5′,7′-tetrabromofluorescein disodium salt (also known as eosin Y or EY).
In one embodiment, the photoinitiator is selected from the group comprising or consisting of 2,2′-azobis[2-methyl-n-(2-hydroxyethyl)propionamide] (also known as VA-086), 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propanon (also known as Irgacure 2959), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (also known as LAP or BioKey), and 2′,4′,5′,7′-tetrabromofluorescein disodium salt (also known as eosin Y or EY), preferably the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (also known as LAP or BioKey).
In one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove and further comprising a photoinitiator as described hereinabove at a concentration of at least about 0.01% (w/v), 0.05% (w/v), 0.075% (w/v), 0.1% (w/v), 0.25% (w/v), or 0.5% (w/v). Thus, in one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove and further comprising a photoinitiator as described hereinabove at a concentration of at least about 0.1 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 2.5 mg/mL or 5 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove and further comprising a photoinitiator as described hereinabove at a concentration of at most about 2% (w/v), 1.5% (w/v), 1% (w/v), 0.75% (w/v), 0.5% (w/v), 0.25% (w/v), 0.1% (w/v) or 0,05% (w/v). Thus, in one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove and further comprising a photoinitiator as described hereinabove at a concentration of at most about 20 mg/mL, 15 mg/mL, 10 mg/mL, 7.5 mg/mL, 5 mg/mL, 2.5 mg/mL, 1 mg/mL or 0.5 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove and further comprising a photoinitiator as described hereinabove at a concentration ranging from about 0.01% (w/v) to about 2% (w/v). Thus, in one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove and further comprising a photoinitiator as described hereinabove at a concentration ranging from about 0.1 mg/mL to about 20 mg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising GelMa as described hereinabove and further comprising a photoinitiator as described hereinabove at a concentration of about 1% (w/v), i.e., at a concentration of about 1 mg/mL.
In one embodiment, the culture medium in which collagen or gelatin is added to obtain a culture medium comprising collagen or gelatin and the culture medium previously used to culture the animal cells in a non-adherent culture vessel as described hereinabove are different media. In another embodiment, the culture medium in which collagen or gelatin is added to obtain a culture medium comprising collagen or gelatin and the culture medium previously used to culture the animal cells in a non-adherent culture vessel as described hereinabove are the same media.
As mentioned hereinabove, the culture medium to be used according to the method of the invention will be apparent to those skilled in the art and will depend on the animal cells to be cultured. Culture media that may be used according to the method of the invention are described hereinabove.
According to one embodiment, the animal cells to be cultured with the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, and they are transferred to a medium suitable for the culture of hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, said culture medium further comprising collagen or gelatin as described hereinabove.
Examples of media suitable for the culture of hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes are listed hereinabove.
In one embodiment, the animal cells to be cultured according to the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes and they are transferred in WE HH as defined hereinabove, said WE HH further comprising collagen or gelatin as described hereinabove. In one embodiment, the animal cells to be cultured according to the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes and they are transferred in WE HH as defined hereinabove, said WE HH further comprising methacrylated gelatin as described hereinabove. In one embodiment, the animal cells to be cultured according to the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes and they are transferred in WE HH as defined hereinabove, said WE HH further comprising methacrylated gelatin and a photoinitiator, preferably lithium phenyl-2,4,6-trimethylbenzoylphosphinate, as described hereinabove.
In one embodiment, the WE HH medium is supplemented with penicillin, streptomycin, insulin, glutamine (L-glutamine), albumin, transferrin, sodium selenite, hydrocortisone, HGF (hepatocyte growth factor) and EGF (epidermal growth factor), and optionally gentamicin and/or FCS (fetal calf serum).
In one embodiment, the WE HH medium comprises penicillin at a concentration ranging from about 50 to about 200 U/mL, preferably about 100 U/mL; streptomycin at a concentration ranging from about 50 to about 200 μg/mL, preferably 100 μg/mL; insulin at a concentration ranging from about 5 to about 30 μg/mL, preferably ranging from about 5 to about 15 μg/mL, preferably about 15 μg/mL; glutamine (L-glutamine) at a concentration ranging from about 1 to about 10 mM, preferably about 2 mM; albumin at a concentration ranging from about 0.01 to about 1% (w/v), preferably about 0.1% (w/v); transferrin at a concentration ranging from about 0 to about 10 μg/mL, or from about 0.1 to about 10 μg/mL, and preferably about 5.5 ug/mL; sodium selenite at a concentration ranging from about 0 to about 10 μg/mL, or from about 0.1 to about 10 μg/mL, and preferably about 5 μg/mL; hydrocortisone at a concentration ranging from about 0.1 to about 50 μM, preferably about 1 μM; rhHGF (recombinant human hepatocyte growth factor) at a concentration ranging from about 0 to about 10 ng/mL, or from about 0.1 to about 10 ng/mL, and preferably about 2.5 ng/mL; rhEGF (recombinant human epidermal growth factor) at a concentration ranging from about 0 to about 1 ng/μL, or from about 0.01 to about 1 ng/μL, and preferably about 0.05 ng/μL; and FCS (fetal calf serum) at a concentration ranging from about 0 to about 20% (v/v), or from about 1% to about 20% (v/v), and preferably about 10% (v/v); and optionally gentamicin at a concentration ranging from about 1 to about 100 μg/mL, preferably about 50 μg/mL.
In one embodiment, the animal cells are transferred to a culture medium comprising collagen or gelatin as described hereinabove at a concentration of at least about 103, 5.103, or 104 cells/mL of culture medium comprising collagen or gelatin.
In one embodiment, the animal cells are transferred to a culture medium comprising collagen or gelatin as described hereinabove at a concentration of at most about 107, 5×106 or 106 cells/mL of culture medium comprising collagen or gelatin.
In one embodiment, the animal cells are transferred to a culture medium comprising collagen or gelatin as described hereinabove at a concentration ranging from about 103 cells/mL to about 107 cells/mL, preferably from about 104 cells/mL to about 106 cells/mL, more preferably from about 1×105 to about 9×105 cells/mL of culture medium comprising collagen or gelatin.
In another embodiment, the animal cells are transferred to a culture medium comprising collagen or gelatin as described hereinabove at a concentration ranging from about 3×105 cells/mL to about 4.5×105 cells/mL, preferably from about 3.25×105 cells/mL to about 4×105 cells/mL, more preferably from about 3.5×105 cells/mL to about 3.75×105 cells/mL of culture medium comprising collagen or gelatin.
In one embodiment, the animal cells are transferred to a culture medium comprising collagen or gelatin as described hereinabove at a concentration of about 3×105, 3.25×105, 3.5×105, 3.65×105, 3.75×105, 4×105, 4.25×105 or 4.5×105 cells/mL of culture medium comprising collagen or gelatin. In another embodiment, the animal cells are transferred to a culture medium comprising collagen as described hereinabove at a concentration of about 3.65×105 cells/mL of culture medium comprising collagen or gelatin.
In one embodiment, after transfer of the animal cells to a culture medium comprising collagen or gelatin as described hereinabove, the pH of the resulting mix is adjusted at a value ranging from about 7 to about 8, preferably at a value of about 7.4. Methods to adjust the pH of a culture medium are well-known to the person skilled in the art. For example, the pH of a culture medium may be increased as required with the addition of an appropriate volume of a NaOH solution. Alternatively, the pH of a culture medium may be lowered as required with the addition of an appropriate volume of a HCl solution.
Selecting conditions suitable for the culture of animal cells is well known to those skilled in the art. Thus, the culture conditions to be used according to the method of the invention, such as, for example, culture vessel, temperature, humidity and levels of CO2, will be apparent to those having skill in the art and will depend on the animal cells to be cultured.
In one embodiment, the mix resulting from the transfer of animal cells to a culture medium comprising collagen or gelatin as described hereinabove is poured into a culture vessel such as a plate or a multi-well plate, for example, a 24-well plate, a 48-well plate or a 96-well plate. It will be evident to those skilled in the art that the volume poured in the culture vessel will depend on the size of the culture vessel, for example on the size of the plate. In the case of a multi-well plate, the volume poured per well will depend on the size of the well and thus on the size of the multi-well plate.
Thus, in one embodiment, about 100 μL of the mix resulting from the transfer of animal cells to a culture medium comprising collagen or gelatin as described hereinabove are poured per well of a 96-well plate, about 300 μL are poured per well of a 48-well plate, and about 400 μL are poured per well of a 24-well plate.
According to one embodiment of the method of the invention, the mix resulting from the transfer of animal cells to a culture medium comprising collagen or gelatin as described hereinabove is incubated in a culture vessel, preferably a multi-well plate, for at least about 10 min, 20 min, 30 min, 45 min, 1 h, 2 h, 3 h, or 4 h, preferably for at least about 2 h.
In one embodiment, the animal cells to be cultured according to the method of the invention are human cells, preferably primary human hepatocytes, and the mix resulting from the transfer of said human cells, preferably primary human hepatocytes, to a culture medium comprising collagen is incubated as described hereinabove at about 37° C. and about 5% CO2, humidity 80-100%, preferably 85-95%.
According to one embodiment of the method of the invention, after an incubation at 37° C. of at least about 10 min, 20 min, 30 min, 45 min, 1 h, 2 h, 3 h, or 4 h, preferably of at least about 2 h, the collagen comprised in the culture medium as described hereinabove is polymerized and the animal cells are thus embedded in a collagen matrix.
According to another embodiment of the method of the invention, the mix resulting from the transfer of animal cells to a culture medium comprising methacrylated gelatin (GelMa) as described hereinabove is illuminated with light having a wavelength ranging from about 250 nm to about 530 nm, preferably from about 365 to about 405 nm. In one embodiment, the mix resulting from the transfer of animal cells to a culture medium comprising methacrylated gelatin (GelMa) as described hereinabove is illuminated with light having a wavelength of about 365 nm or 405 nm.
In one embodiment, the mix resulting from the transfer of animal cells to a culture medium comprising methacrylated gelatin (GelMa) as described hereinabove is illuminated as described hereinabove for a time ranging from about 10 seconds to about 10 minutes, preferably from about 30 seconds to about 5 minutes.
It is well-known in the field that the time of illumination will depend on the wavelength and on the intensity of the light. Accordingly, the intensity of the light will depend on the wavelength of the light and on the time of illumination.
Examples of light intensity include intensities ranging from about 1 mW/cm2 to about 150 mW/cm2.
According to the method of the invention, after illumination, preferably with a light having a wavelength of about 365 nm or 405 nm, for a time of about 60 seconds, the methacrylated gelatin (GelMa) comprised in the culture medium as described hereinabove is polymerized and the animal cells are thus embedded in a GelMa matrix.
The method of the invention comprises a third step of culturing the animal cells embedded in the collagen or gelatin matrix as described hereinabove and thus allows to obtain 3D animal cell structures, preferably spheroids, comprising proliferative animal cells.
According to the method of the invention, culture medium is added to the culture vessel comprising the collagen or gelatin matrix as described hereinabove.
In one embodiment culture medium is added to the culture vessel comprising the collagen or gelatin matrix as described hereinabove in a ratio ranging from about 1.5:1 to about 1:1.5 with respect to the previously added volume of the mix resulting from the transfer of animal cells to a culture medium comprising collagen or gelatin as described hereinabove. In one embodiment culture medium is added to the culture vessel comprising the collagen or gelatin matrix as described hereinabove in a ratio of about 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, or 1:1.5, preferably in a ratio of about 1:1, with respect to the previously added volume of the mix resulting from the transfer of animal cells to a culture medium comprising collagen or gelatin as described hereinabove.
In one embodiment, the culture medium added as described hereinabove and the culture medium used to obtain the collagen or gelatin matrix of the invention are different media. In another embodiment, the culture medium added as described hereinabove and the culture medium used to obtain the collagen or gelatin matrix of the invention are the same media. Preferably, the same culture medium is used in the second step and in the third step of the method of the invention.
As mentioned hereinabove, the culture medium to be used according to the method of the invention will be apparent to those skilled in the art and will depend on the animal cells to be cultured. Culture media that may be used according to the method of the invention are described hereinabove.
According to one embodiment, the animal cells embedded in the collagen or gelatin matrix according to the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, and they are cultured in a medium suitable for the culture of hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes.
Examples of media suitable for the culture of hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes are listed hereinabove.
In one embodiment, hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, are embedded in the collagen matrix or in the gelatin matrix, in particular the GelMa matrix, according to the invention and are cultured in WE HH as defined hereinabove.
In one embodiment, the WE HH medium is supplemented with penicillin, streptomycin, insulin, glutamine (L-glutamine), albumin, transferrin, sodium selenite, hydrocortisone, HGF (hepatocyte growth factor) and EGF (epidermal growth factor), and optionally gentamicin and/or FCS (fetal calf serum).
In one embodiment, the WE HH medium comprises penicillin at a concentration ranging from about 50 to about 200 U/mL, preferably about 100 U/mL; streptomycin at a concentration ranging from about 50 to about 200 μg/mL, preferably 100 μg/mL; insulin at a concentration ranging from about 5 to about 30 μg/mL, preferably ranging from about 5 to about 15 μg/mL, preferably about 15 μg/mL; glutamine (L-glutamine) at a concentration ranging from about 1 to about 10 mM, preferably about 2 mM; albumin at a concentration ranging from about 0.01 to about 1% (w/v), preferably about 0.1% (w/v); transferrin at a concentration ranging from about 0 to about 10 μg/mL, or from about 0.1 to about 10 μg/mL, and preferably about 5.5 ug/mL; sodium selenite at a concentration ranging from about 0 to about 10 μg/mL, or from about 0.1 to about 10 μg/mL, and preferably about 5 μg/mL; hydrocortisone at a concentration ranging from about 0.1 to about 50 μM, preferably about 1 μM; rhHGF (recombinant human hepatocyte growth factor) at a concentration ranging from about 0 to about 10 ng/mL, or from about 0.1 to about 10 ng/mL, and preferably about 2.5 ng/mL; rhEGF (recombinant human epidermal growth factor) at a concentration ranging from about 0 to about 1 ng/μL, or from about 0.01 to about 1 ng/μL, and preferably about 0.05 ng/μL; and FCS (fetal calf serum) at a concentration ranging from about 0 to about 20% (v/v), or from about 1% to about 20% (v/v), and preferably about 10% (v/v); and optionally gentamicin at a concentration ranging from about 1 to about 100 μg/mL, preferably about 50 μg/mL.
Selecting conditions suitable for the culture of animal cells is well known to those skilled in the art. Thus, the culture conditions to be used for culturing animal cells embedded in the collagen or gelatin matrix according to the method of the invention, such as, for example, medium renewal, temperature, humidity and levels of CO2 will be apparent to those having skill in the art and will depend on the animal cells to be cultured.
In one embodiment, the culture medium added to the culture vessel comprising the collagen or gelatin matrix as described hereinabove is changed every 1, 2, 3, 4 or more days, preferably every 2 days.
In one embodiment, the culture medium added to the culture vessel comprising the collagen or gelatin matrix as described hereinabove is changed every 24 h, 36 h, 48 h, 60 h, 72 h or more, preferably every 48 h.
The frequency at which to change the culture medium will be apparent to those skilled in the art and will depend on the purpose of the culture.
In one embodiment, the animal cells embedded in the collagen or gelatin matrix according to the method of the invention are human cells, preferably primary human hepatocytes, and they are cultured at about 37° C. and about 5% CO2, humidity 80-100%, preferably 85-95%.
In one embodiment, the animal cells embedded in the collagen or gelatin matrix according to the method of the invention are cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or 35 days.
In one embodiment, the animal cells embedded in the collagen or gelatin matrix according to the method of the invention are cultured for at least about 1, 2, 3, 4, 5, or 6 weeks.
According to one embodiment, the animal cells embedded in the collagen matrix or in the gelatin matrix, in particular the GelMa matrix, according to the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, and they are cultured for at least about 2 days in order to obtain 3D animal cell structures, preferably spheroids, comprising proliferative hepatocytes.
According to one embodiment, primary hepatocytes, preferably primary human hepatocytes, are proliferative if at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%, preferably at least about 20%, more preferably at least about 30%, even more preferably at least about 40%, of the total number of primary hepatocytes, preferably primary human hepatocytes, are positive for a proliferation marker such as, for example, positive for BrdU incorporation (BrdU+), positive for EdU incorporation (EdU+), positive for Ki67 expression (Ki67±), or positive for cyclin D1 expression (cyclin D1+).
In one embodiment, the total number of primary hepatocytes, preferably primary human hepatocytes, is assessed through the detection of a hepatocyte marker, such as, for example, albumin (alb) expression.
In one embodiment, primary hepatocytes, preferably primary human hepatocytes, are proliferative if at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%, preferably at least about 20%, more preferably at least about 30%, even more preferably at least about 40%, of the total number of Alb+ primary hepatocytes are positive for a proliferation marker such as, for example, positive for BrdU incorporation (BrdU+), positive for EdU incorporation (EdU+), positive for Ki67 expression (Ki67+), or positive for cyclin D1 expression (cyclin D1+).
In one embodiment, the total number of primary hepatocytes, in particular of Alb+ primary hepatocytes, correspond to the total number of primary hepatocytes, in particular of Alb+ primary hepatocytes observed or considered when assessing a proliferation marker, for example through the measurement of mRNA expressions or protein expressions and/or levels.
In one embodiment, the method of the invention further comprises a step to induce one or more additional wave(s) of proliferation of the animal cells in culture in the collagen or gelatin matrix as described hereinabove.
In one embodiment, the method of the invention further comprises a step of transiently inhibiting the MAPK MEK1/2-ERK1/2 pathway in the animal cells in culture in the collagen or gelatin matrix as described hereinabove.
The Applicant indeed showed that transiently inhibiting the MAPK MEK1/2-ERK1/2 pathway in animal cells in culture in the collagen matrix as described hereinabove induced an additional wave of proliferation of said animal cells. In particular, the Applicant showed that primary human hepatocytes underwent an additional wave of proliferation induced by the addition of an inhibitor of the MAPK MEK1/2-ERK1/2 pathway to the culture.
As used in the present invention, inhibitors of the MAPK MEK1/2-ERK1/2 pathway encompass MEK1/2 inhibitors, MEK1 inhibitors, MEK2 inhibitors, ERK1/2 inhibitors, ERK1 inhibitors, and ERK2 inhibitors.
Thus, in one embodiment, the method of the invention further comprises a fourth step (i. e. , step d)) of adding a MEK1/2 inhibitor, a MEK1 inhibitor, a MEK2 inhibitor, an ERK1/2 inhibitor, an ERK1 inhibitor, or an ERK2 inhibitor to the culture of animal cells embedded in the collagen matrix or in the gelatin matrix, in particular the GelMa matrix, as described hereinabove.
Examples of MEK inhibitors include, without being limited to, U0126, PD98059, PD184352, CL-1040, FR180204, and BUD-523.
In one embodiment, the inhibitor of the MAPK MEK1/2-ERK1/2 pathway as described above is added to the culture of animal cells embedded in the collagen or gelatin matrix at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days after the start of the culture of the animal cells embedded in a collagen or gelatin matrix according to the invention.
In one embodiment, the inhibitor of the MAPK MEK1/2-ERK1/2 pathway as described above is added to the culture of animal cells embedded in the collagen or gelatin matrix as described hereinabove for about 24 h, 36 h, 48 h, 60 h, or 72 h, preferably for about 48 h.
Thus, in one embodiment, the culture medium comprising the inhibitor of the MAPK MEK1/2-ERK1/2 pathway as described above is replaced with culture medium without MAPK MEK1/2-ERK1/2 pathway inhibitor after about 24 h, 36 h, 48 h, 60 h, or 72 h, preferably after about 48 h.
In one embodiment, the addition of an inhibitor of the MAPK MEK1/2-ERK1/2 pathway as described above is repeated to induce additional wave(s) of proliferation when desired.
In one embodiment, the 3D animal cell structures, preferably spheroids, comprising proliferative animal cells obtained according to the method of the invention are embedded in the collagen matrix or in the gelatin matrix, in particular the GelMa matrix.
In one embodiment, the method of the invention further comprises a step to isolate the 3D animal cell structures, preferably spheroids, comprising proliferative animal cells from the collagen matrix or from the gelatin matrix, in particular the GelMa matrix.
In one embodiment, the 3D animal cell structures, preferably spheroids, comprising proliferative animal cells are isolated from the collagen or gelatin matrix after enzymatic digestion of the collagen. Thus, in one embodiment, the 3D animal cell structures, preferably spheroids, comprising proliferative animal cells, are isolated from the collagen or gelatin matrix after incubation with a collagenase or an enzyme mixture with collagenolytic activity.
In one embodiment, the method of the invention further comprises a final step (e.g., step e)) of incubating the 3D animal cell structures, preferably spheroids, comprising proliferative animal cells embedded in the collagen matrix or in the gelatin matrix, in particular the GelMa matrix, with a collagenase or an enzyme mixture with collagenolytic activity.
Examples of enzyme mixtures with collagenolytic activity include, without being limited to,
Liberase® and Accutase®.
The present invention thus relates to a method of culturing animal cells, preferably primary animal cells, comprising:
The present invention also relates to a method of culturing animal cells, preferably primary animal cells, comprising:
In one embodiment, the present invention relates to a method of culturing animal cells, preferably primary animal cells, comprising:
The present invention also relates to a method of culturing animal cells, preferably primary animal cells, comprising:
In one embodiment, the present invention relates to a method of culturing animal cells, preferably primary animal cells, comprising:
In one embodiment, the animal cells to be cultured with the method of the invention are primary human hepatocytes (PHH) and the 3D animal cell structures comprising proliferative animal cells obtained at step c) are spheroids comprising proliferative PHH.
According to one embodiment, the animal cells to be cultured with the method of the invention are primary animal cells, in particular primary human cells, and the method of the invention allows to obtain 3D animal cell structures comprising proliferative primary animal cells, in particular primary human cells.
In one embodiment, the animal cells to be cultured with the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, and the method of the invention allows to obtain spheroids comprising proliferative hepatocytes, in particular proliferative primary hepatocytes, preferably proliferative primary human hepatocytes.
In one embodiment, the animal cells to be cultured with the method of the invention are hepatocytes, in particular primary hepatocytes, preferably primary human hepatocytes, and the method of the invention allows to obtain spheroids comprising proliferative hepatocytes, in particular proliferative primary hepatocytes, preferably proliferative primary human hepatocytes, wherein said spheroids have an acinus-like structure with a hollow lumen.
Thus, the invention also relates to a method for inducing in vitro proliferation of primary hepatocytes, preferably primary human hepatocytes, comprising:
Another object of the invention is a spheroid comprising proliferative primary hepatocytes, preferably proliferative primary human hepatocytes (PHH).
According to one embodiment of the invention, said spheroid comprises primary hepatocytes which retain their differentiated state and their hepatic functions throughout the time of their culture in the collagen matrix or in the gelatin matrix, in particular a GelMa matrix, as described hereinabove.
According to one embodiment of the invention, the spheroid comprising primary hepatocytes, preferably primary human hepatocytes, of the invention has an acinus-like structure with a hollow lumen.
In one embodiment, the spheroid according to the present invention thus appears as a sphere delimited by a single layer of well-organized primary hepatocytes, preferably primary human hepatocytes, forming an acinus-like structure with a hollow lumen.
Methods to assess the differentiated state of primary hepatocytes include, without being limited to, detection and intracellular localization of epithelial markers such as E-cadherin; and detection and intracellular localization of mesenchymal markers such as N-cadherin, vimentin, cytokeratin 8, and cytokeratin 18.
As used herein, “hepatic functions” encompass the polarization, protein expression, protein secretion, and enzymatic activities specific to hepatocytes.
Thus, hepatic functions may be assessed for example though the determination of the expression of fetoprotein, albumin, and/or aldolase B; the determination of the secretion of albumin; the determination of the expression, intracellular localization and/or activity of xenobiotic metabolism enzymes such as phase I, phase II, and/or phase III enzymes; the determination of the expression, intracellular localization and/or activity of drug transporters such as MRP2 and/or MRP3.
In one embodiment, the spheroid of the invention comprises proliferative primary hepatocytes, preferably proliferative PHH, which can be further characterized as being at least one of the following:
In one embodiment, the spheroid of the invention comprises polarized proliferative primary hepatocytes, preferably polarized proliferative PHH. In one embodiment, the spheroid of the invention comprises proliferative primary hepatocytes, preferably proliferative PHH, expressing fetoprotein, albumin, and/or aldolase B. In one embodiment, the spheroid of the invention comprises proliferative primary hepatocytes, preferably proliferative PHH, secreting albumin. In one embodiment, the spheroid of the invention comprises proliferative primary hepatocytes, preferably proliferative PHH, expressing phase I, phase II, and/or phase III metabolism enzymes. In one embodiment, the spheroid of the invention comprises proliferative primary hepatocytes, preferably proliferative PHH, expressing drug transporters such as MRP2 and/or MRP3.
In one embodiment, the spheroid of the invention is embedded in a collagen matrix or in a gelatin matrix, in particular a GelMa matrix. In another embodiment, the spheroid of the invention is not embedded in a collagen matrix or in a gelatin matrix, in particular a GelMa matrix. Thus, in one embodiment, the spheroid of the invention is an isolated spheroid comprising proliferative primary hepatocytes. In one embodiment, the spheroid of the invention is in suspension in a medium suitable for the culture of primary hepatocytes.
In one embodiment, the spheroid of the invention is obtained or susceptible to be obtained according to the method as described hereinabove. Thus, in one embodiment, the spheroid of the invention is obtained or susceptible to be obtained according to the method comprising:
In one embodiment, the spheroid of the invention as described hereinabove comprises proliferative primary human hepatocytes.
In one embodiment, the spheroid of the invention has a diameter ranging from about 50 μm to about 150 μm.
In one embodiment, the spheroid of the invention has a diameter ranging from about 20 μm to about 130 μm, preferably from about 30 μm to about 100 μm, more preferably from about 40 μm to about 90 μm, even more preferably from about 45 μm to about 80 μm.
In one embodiment, the spheroid of the invention has a volume ranging from about 4 pL to about 2000 pL, preferably about from about 100 pL to about 600 pL.
In one embodiment, the spheroid of the invention comprises from about 2 to about 150 proliferative primary hepatocytes, preferably from about 5 to about 100 proliferative primary hepatocytes, more preferably from about 5 to about 50 proliferative primary hepatocytes.
The invention also relates to the use of a spheroid comprising proliferative primary hepatocytes as described hereinabove for engineering an artificial liver model or an artificial liver organ.
The invention also relates to the use of a spheroid comprising proliferative primary hepatocytes as described hereinabove for assessing in vitro the toxicity and/or the effects of a drug or a compound.
Examples of parameters that may be examined to assess the toxicity and/or the effects of a drug or a compound include, without being limited to, cell morphology and motility, matrix reorganization and collagen synthesis, cell death, cell proliferation, cell metabolism, cell polarity, and cell differentiation.
Example of methods to assess the morphology and motility of hepatocytes include, without being limited to, contrast light microscopy, confocal microscopy, auto-fluorescence imaging of hepatocytes or of hepatocyte spheroids by biphoton microscopy imaging; time-lapse motility imaging, and invadosomes/pMLC, MLCK/RhoKinase immunolocalizations.
Examples of methods to assess matrix reorganization and collagen synthesis include, without being limited to, imaging of hepatocyte spheroids with Second Harmonic Generation (SHG) microscopy.
Examples of methods to assess cell death include, without being limited to, imaging of nuclear fragmentation (TUNEL assay), methods to assess apoptosis of hepatocytes such as caspase imaging assays and caspase activity assays, and methods to assess necrosis of hepatocytes (staining with YOYO).
Examples of methods to assess proliferation of hepatocytes include, without being limited to, biphoton cells numeration from 3D z-stack images, determination of BrdU incorporation, determination of EdU incorporation, determination of a mitotic index, and immuno-histochemical staining or measurement of the expression of Ki67, β-tubulin, cyclin D1, cyclin D2, cyclin D3, cyclin E, cyclin A2, cyclin B, Cdk1, Cdk2 and/or Aurora A.
Examples of methods to assess the xenobiotic metabolism or the hepatic functions of hepatocytes include, without being limited to, assays to determine the enzymatic activities, protein and mRNA expression, regulation of phase I and phase II xenobiotic metabolism enzyme such as CYPs, GSTs, UGTs, NATs and associated nuclear factors CAR, PXR, AhR; MRP2 activity (related to cholestatic status) through fluorescein diacetate processing; and assays to determining lipid content such as oil red staining, or bodipy 493/503 labelling.
In one embodiment, the spheroid comprising proliferative primary hepatocytes as described hereinabove is used for assessing the liver genotoxicity of a drug or a compound.
Examples of parameters that may be examined to assess the liver genotoxicity of a drug or a compound include, without being limited to, hepatocyte proliferation, DNA replication, chromosomes aberrations (such as, for example, single and double-strand breaks, loss, formation of micronuclei), presence of DNA damages, and presence of mutations.
Thus, for example, the liver genotoxicity of a drug or a compound may be assessed through detection of micronuclei formation, comet assay, detection of phosphorylation of the histone H2Ax, and exome sequencing (for example to establish hotspot signatures linked to xenobiotic exposure) performed on spheroids comprising proliferative PHH according to the present invention.
The invention also relates to an in vitro method of assessing the toxicity and/or the effects of a drug or a compound, the method comprising:
In one preferred embodiment, said in vitro method comprises:
As mentioned hereinabove, the parameters that may be examined to assess the toxicity and/or the effects of a drug or a compound on the 3D animal cell structures comprising proliferative animal cells, in particular on the spheroids comprising proliferative PHH, include, without being limited to, cell morphology and motility, matrix reorganization and collagen synthesis, cell death, cell proliferation, cell metabolism, cell polarity, and cell differentiation.
In one embodiment, the method of the invention is for assessing the liver genotoxicity of a drug or a compound.
As mentioned hereinabove, the parameters that may be examined to assess the liver genotoxicity of a drug or a compound on the spheroids comprising proliferative PHH, include, without being limited to, hepatocyte proliferation, DNA replication, presence of chromosomes aberrations (such as, for example, single-strand breaks or double-strand breaks, loss, micronuclei formation), presence of DNA damages, and presence of mutations.
Thus, the liver genotoxicity of a drug or a compound may be assessed, for example, through detection of micronuclei, comet assay, detection of the presence of phosphorylation of the histone H2Ax, and exome sequencing (for example to establish xenobiotic signatures) performed on spheroids comprising proliferative PHH according to the present invention.
In one embodiment, the 3D animal cell structures comprising proliferative animal cells, preferably spheroids comprising proliferative PHH, are embedded in the collagen matrix or in the gelatin matrix, in particular the GelMa matrix, as described hereinabove.
In one embodiment, the 3D animal cell structures comprising proliferative animal cells, preferably spheroids comprising proliferative PHH, are contacted with a drug or a compound after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days of culture in the collagen matrix or in the gelatin matrix, in particular the GelMa matrix. In another embodiment, the 3D animal cell structures comprising proliferative animal cells, preferably spheroids comprising proliferative PHH, are contacted with a drug or a compound after at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days of culture in the collagen matrix or in the gelatin matrix, in particular the GelMa matrix.
The method of the invention for culturing animal cells allows to obtain 3D animal cell structures which comprises proliferative animal cells, said proliferative animal cells retaining their phenotype, e.g., their differentiated state, and their functions.
As illustrated in the Examples below, the Applicant showed that the method of the invention is particularly suited for the culture of primary human hepatocytes and allows to obtain spheroids comprising proliferative primary human hepatocytes. Of particular interest, the method of the invention allows to obtain spheroids comprising proliferative primary human hepatocytes even in the absence of growth factors such as HGF (hepatocyte growth factor) and/or EGF (epidermal growth factor). The method of the invention also allows to obtain spheroids comprising proliferative primary human hepatocytes even in the absence of FCS (fetal calf serum).
In particular, the Applicant surprisingly found that primary human hepatocytes retain their differentiated state and their hepatic functions and are able to undergo a first wave of proliferation comprising at least two cell cycles during the first week of culture in the collagen matrix or in a gelatin matrix, in particular a GelMa matrix, according to the method of the invention. The Applicant also found that primary human hepatocytes are able to undergo a second wave of proliferation during the second week of culture in the collagen matrix according to the method of the invention. Further additional wave(s) of proliferation can occur when the MEK/ERK is transiently inhibited with an inhibitor such as the MEK inhibitor U0126.
Notably, the Applicant showed that proliferation of the primary human hepatocytes only occurs when the primary human hepatocytes are first cultured in a non-adherent culture vessel such as a low attachment culture vessel, before being embedded and cultured in a collagen matrix or in a gelatin matrix, in particular a GelMa matrix, according to the method of the invention as described hereinabove. Primary human hepatocytes continuously cultured in a low attachment culture vessel, without ever being embedded in a collagen matrix or in a gelatin matrix, in particular a GelMa matrix, for a similar time and in the same culture medium, were not able to proliferate. Similarly, primary human hepatocytes directly embedded and cultured in a collagen matrix, for a similar time and in the same culture medium, were not able to proliferate.
The method of the invention thus allows to obtain non-transformed proliferative human hepatocytes essential for the further development of in vitro models with both therapeutic and pharmaceutical applications. For example, the capacity to expand primary human hepatocytes will most likely have an impact on the development of hepatocyte, spheroid or organoid transplant aiming at restoring liver function in a subject. The capacity to expand primary human hepatocytes will also improve in vitro PHH models used to predict the liver toxicity, and in particular the liver genotoxicity, of candidate drugs and environmental contaminants.
The present invention is further illustrated by the following examples.
Materials and Methods
Material
Cells
Human liver samples were obtained from patients undergoing liver resection for primary hepatocellular carcinoma or liver metastases through the Centre de Ressources Biologiques (CRB)-Sante of Rennes (CHRU Pontchaillou, Rennes, France). The research protocol was conducted under French legal guidelines and the local institutional ethics committee. The clinical characteristics of the human liver samples are detailed in Table 1 below.
Human hepatocytes were isolated by a two-step collagenase perfusion procedure, and parenchymal cells were maintained in a modified William's E medium, called WE HH (human hepatocytes) comprising penicillin (100 U), streptomycin (100 μg/mL), insulin (15 μg/mL), glutamine (2 mM), albumin (0.1% (w/v)), transferrin (5.5 μg/mL), sodium selenite (5 μg/mL), hydrocortisone (1 μM), HGF (hepatocyte growth factor) (2.5 ng/mL), EGF (epidermal growth factor) (0.05 ng/μL) with or without FCS (fetal calf serum) (10% (v/v)).
Reagents
The cell proliferation reagent WST-1, Liberase (10 μg/mL) and colcemid (1 μM) were obtained from Roche. Cisplatin was obtained from Mylan. Type I collagen, ethoxyresorufin, methoxyresorufin, Hoechst, 5(6)-Carboxy-2′,7′-dichlorofluorescein diacetate (CDFDA, 10 μM), 1× ITS (insulin, transferrin, selenium) and anti β-actin antibody (A-5441, 1/1000) were obtained from Sigma-Aldrich. BrdU (RPN201V, 1:1000) and the anti-BrdU antibody (RPN202, 1:100) were obtained from GE Healthcare (Chicago, USA). The rhEGF was obtained from Promega (Fitchburg, USA) and the rhHGF from R&D Systems. The anti-HSC-70 antibody (sc-7298, 1:5000) was obtained from Santa-Cruz Biotechnology. The anti-Ki67 (MA5-14520, 1:400) antibody was obtained from Invitrogen. Antibodies against phospho-p44/42 MAPK (Thr202/Tyr204) (9106, 1:1000), E-cadherin (3195, 1:100), pro-apoptosis Bcl-2 family (1:500), and pro-survival Bcl-2 family (1:500) were obtained from Cell Signaling. The anti-N-cadherin antibody (610921, 1:100) was obtained from BD Biosciences. Antibodies against MRP2 (ab3373, 1:100) and Cyclin D1 (ab16663, 1:100) were obtained from Abcam. The anti-vimentin antibody (M0725, 1:1000) was from obtained Dako. The anti phospho-histone H3 (Ser10) antibody (06-570, 1:100) was obtained from Merck and the anti-albumin antibody (A80-229A, 1:100) was obtained from Bethyl Laboratories, Inc. The MEK inhibitor U0126 (50 μM) was obtained from Promega.
Methods
Primary Human Hepatocyte 2D Culture
As a control, aggregates of primary human hepatocytes (PHH) formed in low attachment plates as described below were seeded in standard multi-well plates and cultured in the same medium, i.e., the WE HH medium described hereinabove.
Primary Human Hepatocyte 3D Culture
As shown on
Culture in a low attachment plate: isolated human hepatocytes were incubated at 37° C., 5% CO2, humidity 85-95% during one night (about 15 to 20 h) in 6-well low attachment plates (Corning®, Costar®) at a concentration of 2×106 to 2.5×106 cells per well, in WE or WE HH. Inclusion in a collagen matrix followed by culture of hepatocytes embedded in the collagen matrix: type I collagen at 3 mg/mL or 6 mg/mL from Sigma-Aldrich was diluted into WE HH culture medium to obtain a collagen solution at the desired concentration. The human hepatocytes were added at a concentration of 3.65×105 cells/mL to the WE HH comprising collagen and the pH was adjusted at 7,4 with NaOH 0.1 N. The resulting mix of human hepatocytes and DMEM HH comprising collagen was poured into 96-well plates (100 μl) or 48-well plates (300 μl) and incubated at 37° C., 5% CO2, humidity 85-95%. After at least 2 h, the gels were polymerized and a volume of WE HH medium equal to the volume of gel was added. The hepatocytes embedded in the gel matrix were cultured in the modified WE HH as described above for at least 2 days at 37° C., 5% CO2, humidity 85-95%. The inclusion of the hepatocytes in a collagen matrix thus allowed for a 3D culture of the hepatocytes.
Hereafter, in Example 1, the term “3D culture” refers to a culture of PHH established as described hereinabove, with a first incubation (or culture) of the PHH in a low attachment plate followed by the inclusion of the PHH in a collagen matrix, according to the method of the invention.
However, any mention of a time of culture, e.g., day 5 of culture, refers to the time of culture in a collagen matrix (or in a standard multi-well plate for the 2D control condition). In other words, any time of culture mentioned hereafter does not include the period of incubation (or culture) in a low attachment plate.
Collagen Gels Inclusion in Paraffin
After fixation in formol (also known as formaldehyde) 4%, collagens gels were dehydrated by successive incubations in alcohol and xylene baths at increasing concentrations before being impregnated with paraffin using EXCELSIOR ES tools (Thermo Fisher Scientific, Waltham, USA). After impregnation, gels were included in paraffin blocs and 4 μm cuts were made.
Hepatocyte DNA Synthesis
Incorporation of the thymidine analog BrdU was used as an index of cell proliferation. After 24 h of incorporation, cells in collagen gels were fixed in ethanol-glycine and included in paraffin as indicated above. BrdU positive cells were detected using an anti-BrdU antibody (RPN202, 1:100).
Immunohistochemistry
Immuno-histochemical staining was performed on the Discovery Automated IHC stainer using the Discovery Rhodamine kit (Ventana Medical Systems, Tucson, USA). Following deparaffination with Ventana EZ Prep solution at 75° C. for 8 min, antigen retrieval was performed using Tris-based buffer solution CC1 (Ventana Medical Systems, Tucson, USA) at 95° C. to 100° C. for 36 min. Endogen peroxidase was blocked with 3% H2O2 (Ventana Medical Systems, Tucson, USA) for 8 min at 37° C. After rinsing with reaction buffer (Roche, Basel, Switzerland), slides were incubated at 37° C. for 60 min with an appropriate dilution of the desired primary antibodies. After rinsing, signal enhancement was performed using the Ventana Rhodamine kit and incubation with the secondary antibody anti-Rabbit HRP (Roche, Basel, Switzerland) was carried out for 16 min After removal from the instrument, slides were manually rinsed, stained with albumin (1:100), with the secondary antibody for albumin detection (Donkey anti-goat 655, 1:250) and with Hoechst (1:1500), and coverslipped.
RT-qPCR Analysis
Cells were extracted from the collagen gels by the action of the Liberase enzyme blend 10 μg/mL (Roche, Basel, Switzerland) for 15 min at 37° C. Then, total RNA was extracted from cell pellets using NucleoSpin RNA (Macherey-Nagel, Hoerdt, France) and the concentration of total RNA was measured with a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, USA). Total RNA was used for cDNA synthesis using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Saint Aubin, France). Real-time PCR for all genes was performed using SYBR green technology with the Power SYBR Green PCR master mix (Applied Biosystems, Saint Aubin, France) and the CFX384 Real-Time System (Biorad) according to the manufacturer's recommendations. Primer sequences used are described in Tables 2 and 3 below.
The amplification curves were analyzed with the Biorad CFX Manager software using the comparative regression method. GAPDH was used for the normalization of expression data. The relative amount of measured mRNA in samples was determined using the 2-ΔΔCT method where ΔΔCT=(CTtarget-CTGAPDH)sample−(CTtarget-CTGAPDH)calibrator. Final results were expressed as the n-fold differences in target gene expression in tested samples when compared with the mean expression value of calibrator. Values are given for one experiment representative of at least three independent experiments.
Immunoblotting Analysis
Cells were extracted from the collagen gels by the action of the Liberase enzyme blend 10 μg/mL (Roche, Basel, Switzerland) for 15 min at 37° C. Protein samples were extracted from cell pellets and dosed. Protein samples were then separated using SDS-PAGE and transferred onto nitrocellulose membranes in a transfer buffer (25 mM Tris, 200 mM glycine, Ethanol 20%). The blots were blocked with 5% low fat milk in Tris-buffer saline (TBS) (65 mM Tris pH 7.4, 150 mM NaCl) at room temperature for 1 h. The blots were incubated overnight with the desired primary antibodies at 4° C. The blots were washed with TBS and incubated for 1 h with a mouse-IgG HRP or a rabbit-IgG HRP secondary antibody (1:1000) in 5% low fat milk in TBS at room temperature. The blots were then washed with TBS. Immunocomplexes were visualized with a Fujifilm LAS-3000 imager (Fujifilm, Tokyo, Japan) after a chemiluminescent reaction using the Immobilon Western Chemiluminescent HRP substrate (Millipore, Merck, Darmstadt, Germany). Densitometric analyses of the bands were carried out with MultiGauge software (Fujifilm, Tokyo, Japan).
MRP2 Transporter Activity
A fluorescence-based efflux assay was used to investigate MRP2 transporter activity in 3D primary human hepatocyte (PHH) cultures. The membrane permeable and non-fluorescent substrate 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDFDA) enters into hepatocytes where its hydrolysis by intracellular non-specific esterases results in a fluorescent product. This product, which is a substrate of the membrane transporter MRP-2, is effluxed from hepatocytes into the bile canaliculi.
The 3D cultures were incubated 10 min with CDFDA (10 μM). The dye-solution was then replaced by serum-free medium for 2 h. The cultures were washed twice with PBS solution before the fluorescence was assessed using microscopy.
CYP Activities Measurement
Ethoxyresorufin O-deethylation (EROD) and methoxyresorufin O-deethylation (MROD) associated with CYP 1A1/2 and 1A2 activities, respectively, were measured in cultured hepatocytes as described by Burke and Mayer (Burke and Mayer, 1983). Briefly, cells were washed with phosphate-buffer saline at 37° C. before being incubated with salicylamide (1.5 mM) to block phase II-conjugation enzymes. 7-Ethoxyresorufine or 7-Methoxyresorufine was added 1 min later and the oxidation of the substrates was measured by fluorescence detection every 2 min during 20 min at 37° C. The reaction rate was linear with time. After the assessment of P450 1A1/2 and 1A2 activities, the colorimetric WST-1 based assay was performed to normalize said activities with the quantity of viable cells. Values (pmol/min/OD) are correspond to the mean values±standard deviation of triplicate measurements.
Mitotic Index
To measure the mitotic index of PHH cultivated in 3D cultures established according to the method of the invention, a microtubule-depolymerizing drug, colcemid, was used to arrest cells in metaphase. Cells were treated during 24 h with colcemid before being fixed in formol 4%. The quantification of the mitotic index was performed by immunostaining of the phosphorylated histone H3.
Imaging
Observation and imaging of the cultured hepatocytes were carried out using two-photon excited fluorescence (TPEF) microscopy (also referred to as non-linear, multiphoton, or two-photon laser scanning microscopy). TPEF microscopy is an alternative to confocal and deconvolution microscopy that is particularly suited for deep and high-resolution three-dimensional imaging. TPEF enables observation of endogenous auto-fluorescent unstained or stained samples. In tandem with Second Harmonic Generation (SHG) which allows observation of hyper polarizable fibrillar proteins such as type 1 or type 3 collagen, these methods provide images of spatially resolved 3-dimensionnal structures of cells and collagen matrix. The SHG imaging system consisted of a confocal SP5 scanning head (Leica Microsystems, Mannheim, Germany) mounted on a DMI 6000 inverted microscope (Leica Microsystems) and equipped with a MAITAI femtosecond laser (Spectra Physics, Santa Clara, Calif.). A high NA water immersion objective (LUMFL 60 W×1.1 NA; Olympus, Tokyo, Japan) was used. The SHG signal was collected in the forward direction using a water immersion condenser (S1, NA=0.9; Leica Microsystems). A 405-20 bandpass filter was placed before the photomultiplier tube and oil immersion objective 10×/0.4 HC PL APO oil or 20×/0.7 HC PL APO oil.
Statistical Analysis
Results are expressed as mean values±standard deviation. Data were analyzed with two-tailed Student's t-test. Differences were considered significant when p<0.05 (*), p<0.01 (**), or p<0.001 (***). All experiments were performed at least three times.
Results
3D culture in collagen matrix after pre-culture in LAP allows long term survival of adult primary human hepatocytes
As described hereinabove, primary human hepatocytes were first cultured in a low attachment plate for 15 h before being embedded in collagen gels in the presence of an appropriate growth factors/cytokines cocktail. As shown on
All these observations thus demonstrate that adult primary human hepatocytes can form acini-like structures, i.e., spheroids, when embedded in a collagen gel. This process appears to be dynamic and spheroids seem able to remodel the collagen matrix over culture time.
Then, long-term survival of the primary human hepatocytes (PHH) was evaluated by analyzing a panel of pro-apoptotic and pro-survival factors. As shown on
Taken all together, these data demonstrate that PHH in 3D culture do not undergo apoptosis.
3D culture in collagen matrix after pre-culture in LAP allows stable differentiation of adult primary human hepatocytes
As shown on
Taken all together, these data indicate that the protein expression in PHH in 3D culture established as described hereinabove is significantly different from that in PHH in control 2D culture and closely resembles that of in situ hepatocytes.
Adult primary human hepatocytes in 3D culture in collagen matrix after a pre-culture in LAP display hepatic functions
To ascertain the impact of 3D culture on human hepatocyte differentiation, analyses by RT-qPCR were carried out to investigate the mRNA expression levels of alpha-fetoprotein, albumin, aldolase B, and phase xenobiotic metabolism enzymes (also referred to as detoxifying enzymes). As shown on
RT-qPCR analysis also showed a strong induction of the expression of the transcription factors involved in the regulation of detoxifying pathways (CAR, PXR and AhR) in the PHH in 3D culture as compared to that in PHH in control 2D cultures (
As shown on
Formation of PHH spheroids in 3D culture in collagen matrix after a pre-culture in LAP
The size and morphology of the hepato-spheroids were assessed over two weeks of culture (
As indicated above, the PHH forming the spheroids were polarized, as could be observed notably with the exclusive localization of the drug-transporter MRP2 at the apico-lateral region and the apical/bile canalicular domain Moreover, as indicated above, the PHH forming the spheroids retained their differentiated state and their hepatic functions.
Expression of hypoxia and/or apoptosis markers was not detected in the spheroids obtained with the method of the invention. Moreover, the size of the spheroids did not decrease over time during the 3D culture. On the contrary, as shown on
Proliferation of adult primary human hepatocytes in 3D culture in collagen matrix after a pre-culture in LAP
As mentioned above, the size of the spheroids gradually increased over time. Notably, the number of hepatocyte spheroids with a diameter inferior to 60 μm decreased whereas the number of spheroids with a diameter superior to 60 μm progressively increased (
Next, the number of Ki67 and BrdU positive cells was quantified over the first 8 days of culture. Both Ki67 and BrdU are makers of cell proliferation, and Ki67 and BrdU positive cells are cells undergoing S phase. Hepatocytes were isolated from 8 different patients (see Table 1) and Ki67 staining or BrdU labeling were analyzed both in 3D and 2D cultures. Albumin, a marker of mature hepatocyte function, was detected as a positive control to quantify only Alb+ hepatocytes. As shown on
BrdU±/Alb+hepatocytes observed between days 2 and 7 demonstrated the presence of proliferative primary human hepatocytes in the 3D cultures established as described hereinabove. No Ki67+ hepatocytes could be detected in the control 2D cultures, stimulated by the same growth factors/cytokines cocktail. RT-qPCR analysis of the expressions of the cell cycle markers Cdk2, cyclin D1, PCNA, P21 and P27 showed that said cell cycle markers were highest expressed in 3D cultures at day 4 as compared to 3D day 2 and to 2D days 2 and 4 (
The results clearly demonstrate for the first time that 3D cultures in collagen gels established as described hereinabove provide a microenvironment enabling adult primary human hepatocytes to proliferate, in particular in the presence of a growth factor cocktail. By contrast, PHH in control 2D cultures did not proliferate, even in the presence of the same growth factor cocktail.
Analyses of Ki67 (
Transient MEK/ERK pathway inhibition induces a new wave of proliferation
As described hereinabove, analysis of all the 3D cultures established according to the method of the invention from hepatocytes isolated from different donors (HL-a to HL-j, see Table 1) demonstrated the proliferation capability of adult human hepatocytes between day 2 and 15, with small variations in the proliferation response depending on the donor. No further proliferation could be detected after two weeks of culture. It was previously shown that transient activation of the MEK/ERK pathway stimulates DNA replication in rodent hepatocytes, and that inhibition of the MEK/ERK pathway blocks rodent hepatocyte proliferation. It was also shown that sustained activation of the MEK/ERK pathway can have a negative role on rat hepatocyte proliferation (Fremin et al., 2009) and that overactivation of the cascade can also inhibit cell replication in the human hepatocarcinoma cell line HuH-7 (Guegan et al., 2015). Proliferation of the primary human hepatocytes was assessed after the MAPK MEK1/2-ERK1/2 pathway was transiently inhibited for 48 h with the MEK inhibitor U0126 in 3D cultures established as described hereinabove at day 12 or 15. Control of ERK1/2 phosphorylation confirmed a strong inhibition of ERK1/2 activity at the end of the U0126 treatment in all culture conditions (
Materials and Methods
Material
Cells
Primary human hepatocytes (PHH) were obtained as described hereinabove.
Methods
Primary Human Hepatocyte Culture
Primary human hepatocytes 3D cultures were set up as described hereinabove (see Example 1). Briefly, PHH were first cultured in a low attachment plate. Then, the aggregates of PHH thus obtained were embedded in a collagen matrix and further cultured in the collagen matrix.
Culture Medium
The PHH were cultured in WE HH medium, defined hereinabove (see Example 1).
Immunohistochemistry
Immuno-histochemical staining was performed as described hereinabove to assess the proliferation of the PHH.
Results
The importance of the growth factor stimulation for the proliferation of PHH in the 3D culture according to the invention was further studied. PHH in collagen matrix were cultured as described hereinabove in WE HH medium depleted of EGF, HGF, and/or ITS (insulin, transferrin and sodium selenite). PHH in collagen matrix were also cultured as described hereinabove in WE HH medium depleted of FCS. As shown in Table 5 below, the proliferation of PHH cultured in the different culture media was assessed by quantification of the number of Cyclin D1+/Alb+ cells with respect to the percentage of positive cells in the culture control (WE HH medium) and related to the number of cells detected.
When grown in WE HH medium comprising hydrocortisone at a concentration of 50 μM (“Hydro N”), i.e., a concentration 50-fold higher than that in the control condition, PPH displayed a percentage of proliferation similar to that observed when they were grown in the control condition. Single depletion of each of the growth factor present in the cocktail (“-EGF”, “-HGF” or “-ITS”) only resulted in a partial decrease in PHH proliferation (at most a 30% decrease). Moreover, neither the concomitant depletion of EGF, HGF and ITS (“-EGF/-HGF/-ITS”) nor the single depletion of fetal calf serum (“-FCS”) abolished the proliferation of PHH. These results thus demonstrate that the PHH can proliferate in 3D cultures according to the invention even in absence of EGF, in the absence of HGF, and/or in the absence of ITS. Strikingly, the observed results also demonstrate that the PHH can proliferate in 3D cultures according to the invention even in absence of fetal calf serum (FCS).
The effect of collagen stiffness on the proliferation of PHH was also assessed. As shown in Table 6 below, the proliferation of PHH cultured in the indicated collagen matrices was assessed by quantification of the number of Cyclin D1+/Alb+ cells with respect to the percentage of positive cells in the collagen matrix control (1.5 mg/mL) and related to the number of cells detected. Increasing type I collagen concentration from 1.5 mg/mL to 3 mg/mL or 4 mg/mL induced a decrease in PHH proliferation of about 50% and 80%, respectively. By contrast, decreasing type I collagen concentration from 1.5 mg/mL to 0.75 mg/mL did not induce a decrease in PHH proliferation. These results thus demonstrate that increasing the collagen concentration of the 3D matrix, and thus the matrix stiffness, inhibits the proliferation of hepatocytes in a concentration-dependent manner
Strikingly, PHH embedded directly in 3D collagen gels and cultured in WE HH medium without a prior incubation in a low attachment plate displayed a very low rate of proliferation (16.9%, see Table 6). Similarly, PHH cultured in a low attachment plate in WE HH medium without ever being embedded in a 3D collagen matrix displayed a very low rate of proliferation (11.7%, see Table 6).
Taken all together, these results demonstrate that the 3D microenvironment and controlled collagen stiffness are major factors for inducing human adult primary hepatocyte proliferation. Moreover, these results show that both a first incubation (or culture) of the PHH in a non-adherent culture vessel, e.g., a low attachment plate, and the subsequent inclusion of the PHH in a collagen matrix are required to induce the proliferation of adult PHH.
Materials and Methods
Material
Cells
Primary human hepatocytes (PHH) were obtained as described hereinabove (see Example 1).
Methods
Primary Human Hepatocyte Culture
Primary human hepatocytes 2D and 3D cultures in a collagen matrix were set up as described hereinabove (see Example 1). PHH were cultured in WE HH medium as described hereinabove.
Comet Assay
PHH either in control 2D culture (2D) or in 3D culture established according to the method of the invention in a collagen matrix (3D) were incubated with the indicated genotoxic drugs at the indicated concentrations (see Table 7) from the start of the culture. After 9 days, PHH were extracted from the collagen matrix by the action of purified collagenase. The cell pellets were resuspended in 0.5% low-melting point agarose and laid on conventional microscope slides covered with regular agarose. The electrophoresis migration was processed and after staining with propidium iodide, at least 100 images were acquired using a fluorescence microscope. The images were analyzed using the Comet Assay IV software. The extent of DNA damage in individual cells was evaluated by the percentage of tail DNA.
γH2Ax Immunostaining after Treatment of PHH
PHH in 3D culture in a collagen matrix established according to the method of the invention (3D) were incubated with the indicated genotoxic drugs at the indicated concentrations (see Table 7) from the start of the culture. After 9 days, immuno-histochemical staining was performed as described hereinabove to detect the presence of phosphorylated H2AX (i.e., γH2Ax), a marker of DNA double-strand break, in the PHH.
Results
To assess the sensitivity of the spheroids comprising proliferative PHH as a model for testing drug-induced genotoxicity, the effects of two classes of compounds on said spheroids were studied: alkylating agents, i.e., cisplatin (phosphorylated histone γH2Ax assay, see
To confirm these results, the number of yH2Ax positive cells in 3D cultures established according to the method of the invention in a collagen matrix was quantified by immunostaining. Very high levels of DNA damages were thus observed with the 3 tested drugs: cisplatin induced DNA damages in about 90% of the PHH, 4-ABP in about 50% of the PHH, and AFB1 in about 35% of the PHH (
These results thus demonstrate that, with their highly differentiated and proliferating status, the spheroid comprising proliferative PHH constitute a relevant model for testing the genotoxicity of environmental and chemical compounds.
Materials and Methods
Material
Cells
Primary human hepatocytes (PHH) were obtained as described hereinabove (see Example 1).
Reagents
Methacrylated gelatin was obtained from the ART Bio-encres (Accelerateur de Recherches Technologiques de l'Inserm, Bordeaux). Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was obtained from TCI.
Methods
Primary Human Hepatocyte Culture in Gelatin Methacrylate (GelMa)
In a manner similar to that shown on
Culture in a low attachment plate: as indicated above (see Example 1), isolated human hepatocytes were incubated at 37° C., 5% CO2, humidity 100% during two days (about 48 h) in 6-well low attachment plates (Corning®, Costar®) at a concentration of about 3×106 cells well, in a modified William's E medium comprising gentamicin (50 μg/mL), penicillin (100 U/mL), streptomycin (100 μg/mL), insulin (5 μg/mL), L-glutamine (2 mM), albumin (0.1% (w/v)) and with or without FCS (fetal calf serum) (10% (v/v)).
Preparation of medium comprising 5% GelMa: small crushed fragments of freeze-dried GelMa (up to 2 mm in length) were dissolved at 37° C. for a minimum of 8 h in a modified William's E medium comprising gentamicin (50 μg/mL), penicillin (100 U/mL), streptomycin (100 mg/mL), insulin (15 μg/mL), L-glutamine (2 mM), albumin (0.1% (w/v)), transferrin (5.5 μg/mL), sodium selenite (5 μg/mL), hydrocortisone (1 μM), HGF (hepatocyte growth factor) (2.5 ng/mL), EGF (epidermal growth factor) (0.05 ng/μL) and with or without FCS (fetal calf serum) (10% (v/v)). A volume of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) at a concentration of 10 mg/mL was added to obtain a modified William's E medium comprising concentration of GelMa of 5 g/100 mL, i.e., a final concentration of 5% GelMa and a final concentration of lithium phenyl-2,4,6-trimethylbenzoylphosphinate of 100 mg/mL, i.e., a final concentration of 0.1% LAP. Before use, the medium comprising 5% GelMa and 0.1% LAP was kept at 37° C., protected from light.
Inclusion in a GelMA matrix followed by culture of hepatocytes embedded in the collagen matrix: human hepatocytes were transferred to a suitable container such as an Eppendorf tube (1.5 or 2 mL) or a Falcon tube (15 mL) and centrifuged at 200 g for 2 min. The supernatant was removed and medium comprising 5% GelMa and 0.1% LAP was added to obtain a concentration of about 106 human hepatocytes per mL of GelMa matrix, i.e., of medium comprising 5% GelMa and 0.1% LAP. 100 μL of medium comprising 5% GelMa, 0.1% LAP and human hepatocytes were added to the wells of a 96-well plate. Alternatively, 300 μL of medium comprising 5% GelMa, 0.1% LAP and human hepatocytes were added to the wells of a 48-well plate. Polymerization of the GelMa matrix was induced by illumination for 30 seconds to 10 min with a 365, 405 nm or 530 nm LED, preferably by illumination for 60 seconds with a 405 nm LED. After polymerization of the GelMa matrix, a volume of the same modified William's E medium equal to the volume of GelMa matrix was added. The hepatocytes embedded in the GelMa matrix were cultured in the modified William's E medium as described above for at least 2 days at 37° C., 5% CO2, humidity 100%. The medium was changed every 48 h.
Results
Formation of PHH spheroids in 3D culture in GelMa matrix after a pre-culture in LAP
The size and morphology of the hepato-spheroids was assessed over two weeks of culture (
As with the PHH spheroids cultured in a collagen matrix, the PHH forming the spheroids cultured in a GelMa matrix were polarized, as could be observed notably with the exclusive localization of the drug-transporter MRP2 at the apico-lateral region and the apical/bile canalicular domain.
Proliferation of adult primary human hepatocytes in GelMa matrix after a pre-culture in LAP
Proliferation of the primary human hepatocytes in GelMa matrix was assessed through the analyses of cyclin D1 and albumin (Alb) expression over 30 days after seeding in 3D cultures established according to the method of the invention in a GelMa matrix (
Alb+/cyclin D1+ PHH could be detected from day 2, demonstrating that PHH were able to proliferate in 3D cultures established according to the method of the invention in a GelMa matrix. As shown on
| Number | Date | Country | Kind |
|---|---|---|---|
| 18305603.5 | May 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/062649 | 5/16/2019 | WO | 00 |
