Patent Application
20240150720
The present invention relates to 3D cell cultures, which contain hepatic cells and are infected by a pathogen, methods for preparing such cell cultures and uses thereof.
A variety of pathogens transit or mature in the liver, in the case of infections by Plasmodium parasites, the causative agents of malaria, when an infected mosquito takes a blood meal from a mammalian, including human beings, sporozoites present in the salivary glands of the mosquito are inoculated into capillaries of the upper dermis from where they will reach the portal circulation. Subsequently, they travel to the liver where they invade hepatic cells. Here, the parasites undergo a process termed exoerythrocytic parasite development, in which the hepatic parasites replicate asexually and differentiate into merozoites. Upon completion of this replicative phase, 10 000-40 000 merozoites are eventually released into the blood stream, at which point they invade and replicate inside erythrocytes, initiating a new cycle of asexual replication and growth [Prudêncio, M., Rodriguez, A., & Mota, M. M. (2006). The silent path to thousands of merozoites: the Plasmodium liver stage. Nature Reviews. Microbiology, 4(November), 849-56]. When a single parasite is present inside an erythrocyte, it is termed an early trophozoite. The trophozoite grows and then begins to asexually replicate, a phenomenon known as schizogony. When schizonts are sufficiently mature, the erythrocytes rupture, releasing merozoites with a subsequent increase in the number of circulating malaria parasites. During this phase of infection, some parasites differentiate into gametocytes, both male and female. These are then taken up by mosquitoes during a blood meal and transform into male and female gametes. The union of male and female gametes forms diploid zygotes, which in turn become ookinetes. These ookinetes migrate to the midgut of the insect, pass through the gut wall and form the oocysts in the haemolymph. Meiotic division of the oocysts occurs, leading to maturation and rupture to release sporozoites, which then migrates to the salivary glands of the female Anopheles mosquito ready to continue the cycle of transmission back to man [Douglas, N. M., Simpson. J. a., Phyo, A. P., et al. (2013) Gametocyte dynamics and the role of drugs in reducing the transmission potential of Plasmodium vivax. Journal of Infectious Diseases, 208, 801-812; Swann, J., Corey, V., Scherer, C. a., et al, (2016) High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria. ACS Infectious Diseases, acsinfecdis.5b00143]. All mammalian-infective Plasmodium species transit and mature through the liver but P. vivax and P. ovale can generate latent hepatic forms—known as hypnozoites—, which can lead to disease relapses. Primaquine is currently the only marketed monotherapy drug for the latter indication thought to exert its effect by metabolic activation. For liver stage prophylaxis, atovaquone is used in combination with another partner drug.
The study of the liver stage of Plasmodium's life cycle has greatly benefited from the use of hepatic cell lines (e.g. HepG2, Huh7, HC04) and primary cultures of human hepatocytes [Prud{tilde over (e)}ncio, M., Mota, M. M., & Mendes, A. M. (2011), A toolbox to study liver stage malaria. Trends in Parasitology]. These cells have been mostly explored in 2D culture systems and combined with Plasmodium strains that constitutively express a reporter gene, either green fluorescent protein (GFP) or Luciferase (Luc), to follow and address specific features of the parasite's hepatic development. For example, the requirement of sporozoite cell traversal process towards the effective invasion of the final hepatocyte [Mota, M. M., Hafala, J. C. R., & Rodrigues, A. (2002) Migration through host cells activates Plasmodium sporozoites for infection, Nat Med, (9(11), 548. Risco-Castillo. V., Topçu, S., Marinach, C. et al. (2015) Malaria sporozoites traverse host cells within transient vacuoles. Cell Host and Microbe. 18, 593-603], development throughout liver infection inside transient vacuoles [Risco-Castillo, V., Topçu, S., Marinach, C. et al. (2015) Malaria sporozoites traverse host cells within transient vacuoles. Cell Host and Microbe, 18, 593-603] and the role and specific localization of key proteins involved in disrupting the parasitophorous vacuole during late liver-stage infection [Burda. P. C., Roeill, M. a., Schaffner, M., et at (2015) A Plasmodium phospholipase is involved in disruption of the liver stage parasitophorous vacuole membrane. PLoS Pathogens, 11, e1004760] have been addressed taking advantage of such models. In vitro hepatic models that allow P. falciparum and P. vivax development, the two most clinically relevant human-infective Plasmodium species, have also been developed [Chattopadhyay, R., Velmurugan, S., Chakiath, C., et al. (2010) Establishment of an in vitro Assay for Assessing the Effects of Drugs on the Liver Stages of Plasmodium vivax Malaria. PLoS ONE, 5(12), 1-8; Dumoulin. P. C., Trop, S. A., Ma, J., et al, (2015) Flow cytometry based detection and isolation of Plasmodium falciparum liver stages in vitro. PLoS ONE, 10, 1-2; March, S., Ng, S., Velmurugan, S., et at (2013) A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax. Cell Host and Microbe. 14(1), 104-115]. The relevance of the liver microenvironment's physicochemical features for Plasmodium infection and development has been shown in vitro by co-culturing primary hepatocytes and stromal cells. More specifically, hypoxia has been demonstrated to enhance the development of different Plasmodium species [Ng, S., March, S., Galstian, A., Hanson. K., et al. (2014) Hypoxia promotes liver stage malaria infection in primary human hepatocytes in vitro, 215-224]. Furthermore, these in vitro hepatic models have contributed to the development of high-throughput screening platforms, which can aid in the identification and development of anti-Plasmodium agents [Derbyshire. E. R., Prudbncio. M., Mota, M. M., et al. (2012) Liver-stage malaria parasites vulnerable to diverse chemical scaffolds. Proceedings of the National Academy of Sciences of the United States of America, 109(22), 8511-6; Swann, J., Corey, V., Scherer, C. A., et al. (2016) High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria. ACS Infectious Diseases, 2(4): 281-293.].
The previously established in vitro models of Plasmodium infection have proved their importance towards an increased understanding of the specific features of parasite biology. Nevertheless, few models and assays have addressed the Liver dormant forms of the parasite (hypnozoite), due not only to the difficulties inherent to obtaining P. vivax sporozoites to be used experimentally, but also to the lack of hepatic cell models that can be maintained in culture for long time periods with high viability and function. The available reports described in the literature are based on 2D cultures of primate primary hepatocytes infected with P. cynomoigi [Dembele, L., Gego, A., Zeeman, A. M., et al. (2011) Towards an in vitro model of plasmodium hypnozoites suitable for drug discovery. PLoS ONE, 6(3), 1-7; Voorberg-van der Wel, A., Zeeman, A. M., van Amsterdam, S. M., et al. (2013) Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms. PLoS ONE, 8(1)], where hypnozoites are distinguished from normal developing schizonts by their resistance to atovaquone. However, due to the limited time of primary hepatocytes cultures (up to two weeks when in collagen sandwich cultures), the reactivation capacity of hypnozoites was not assessed. One recent report contemplating an improved collagen sandwich system was able to maintain a co-culture of primary hepatocytes and a hepatoma cell line for 40 days after P. cynomolgi infection [Dembélé, L. Franetich, J., Lorthiois, A., et al. (2014) Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures. Nature Medicine, 20(3), 307-312]. With this model, the authors demonstrated the persistence of hypnozoites for more than one month in the hepatocytes and showed their activation towards normal development beyond three weeks after infection. However, this model's technical complexity decreases its throughput, compromising the applicability in drug screening settings.
Recent developments in human hepatic 3D cell models have demonstrated the ability to recapitulate many important hepatocyte features in stirred-tank bioreactors (STB), by generating cell spheroids of hepatic cell lines or freshly isolated human hepatocytes (Rebelo, S. P., Costa, R., Estrada, M., et al. (2014) HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism. Arch Toxicol; Tostões, R. M., Leite, S. B., Serra, M., et al. (2012) Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing. Hepatology, 55(4), 1227-1236; Rebelo, S., Costa, R., Sousa, M. F. O., at al. (2015) Establishing Liver Bioreactors for In Vitro Research. Protocols in In Vitro Hepatocyte Research, 1250, 1-390). Importantly, physiochemical parameters such as oxygen and pH, as well as the feeding regimen can be controlled in the STB, allowing the reproducibility and stability of hepatic phenotype in long-term cultures, as well as modeling specific characteristics of the liver (e.g, physiological periportal or perivenous oxygen concentrations). Moreover, the scalability of STB enables the production of large quantities of hepatic spheroids that can be used to feed high-throughput screening platforms [Rebelo, S. P., Costa, R. Estrada, M., et al. (2014) HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism, Arch Toxicol; Tostdes, R. M., Leite, S. B., Serra, M., et at (2012) Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing. Hepatology, 55(4), 1227-1236; Rebelo, S., Costa, R., Sousa, M. F. O., et at (2015) Establishing Liver Bioreactors for In Vitro Research. Protocols in In Vitro Hepatocyte Research, 1250, 1-390].
The present invention provides a 3D cell culture comprising cell aggregates, which contain hepatic cells, wherein the cell aggregates are infected by a pathogen.
The 3D cultures of the present invention have a good long-term stability and are therefore useful for drug screening and vaccine development.
In a particular embodiment of the present invention, the pathogen is a parasite.
In another specific embodiment of the present invention, the 3D cell culture is a mono-culture or a co-culture.
In another specific embodiment of the present invention, the hepatic cells are selected from a group of cell sources comprising primary human, murine and primate hepatocytes, cell lines such as HC-04, HepG2, HepaRG and/or Huh7, and hepatocyte-like cells derived from pluripotent or multipotent stem cells.
Yet, in a further specific embodiment of the present invention, the hepatic cells are selected from a group of cell lines comprising primary human and primate hepatocytes, HC-04, HepG2, HepaRG and/or Huh7.
In a specific embodiment, the 3D cell culture is a co-culture, which contains cells from at least one hepatic cell type (such as in particular primary human and primate hepatocytes, HC-04, HepG2, HepaRG and/or Huh7) and non-parenchymal cells such as endothelial, immune or stromal cells (Human Mesenchymal Stem Cells, macrophages, fibroblasts or stellate cells).
In a very specific embodiment, the 3D cell culture is a co-culture, which contains cells from at least one hepatic cell type (such as in particular primary human and primate hepatocytes, HC-04, HepG2, HepaRG and/or Huh7) and Human Mesenchymal Stem Cells.
In another particular embodiment, the 3D cell culture according to the invention contains cell aggregates having an average diameter in the range of 50 μm to 200 μm (by microscopy). The cell aggregates can be spheroids.
A further specific embodiment refers to a 3D cell culture, wherein the parasite is from the genus Plasmodium, preferably selected for a group comprising P. berghei, P. falciparum, P. vivax, P. ovale, P. cynomolgi, P. malariae and P. knowlesi. For infection of the cell aggregates, the sporozoites are put in contact with the cell aggregates.
In a further specific embodiment, the pathogen is a reporter strain such as e.g. a Plasmodium species expressing green fluorescent protein (GFP) or luciferase (Luc). Reporter strains allow a very easy detection and monitoring of the infection rate.
In another embodiment of the present invention, the 3D cell culture contains a cell culture medium, wherein culture medium is a mammalian cell culture medium (such as in particular DMEM supplemented or not with F12-supplement). In a preferred embodiment, the cell culture medium further contains up to 20% FBS concentration.
The choice of the mammalian culture medium depends on the cell line e.g. for HC-04 cell cultures DMEM supplemented with F12 is suitable, for HepG2 DMEM (without F12) is suitable. For HepaRG and primary hepatocytes the cell culture medium as described e.g in [Rebelo, S. P. et al, (2014). HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism. Arch Toxicol], [Tostdes, R. M., et at (2012). Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing. Hepatology, 55(4), 1227-1236], or [Rebelo, S., Costa, R., Sousa, M. F. Q., Brito, C., & Alves, P. M. (2015). Establishing Liver Bioreactors for In Vitro Research. Protocols in In Vitro Hepatocyte Research, 1250, 1-390] can be used.
Another embodiment of the invention relates to a 3D cell culture, wherein the 3D cell culture further contains soluble extracellular matrix (preferably laminin, fibronectin and/or collagen) and/or a biocompatible biomaterial (e.g. alginate, chitosan, polylactic acid).
Another embodiment of the invention relates to a 3D cell culture, wherein the 3D cell culture further contains soluble extracellular matrix, preferably laminin, fibronectin, and/or collagen.
In a specific embodiment of the present invention, the 3D cell culture is characterized by an infection rate of at least 0.01% (measured e.g. by fluorescence and luminescence; infection by parasites that do not express reporter genes can be assessed by a variety of methods, including immunofluorescence microscopy following staining with appropriate antibodies, and quantitative real-time PCR employing Plasmodium-specific primers and primers for appropriate housekeeping host genes [Prudencio, M., Mota, M. M., & Mendes. A. M. (2011). A toolbox to study liver stage malaria. Trends in Parasitology]).
Accordingly, a very specific embodiment refers to a 3D cell culture, wherein
Another very specific embodiment refers to a 3D cell culture, wherein
Preferably, such a 3D cell culture contains cell aggregates having an average diameter in the range of 50 μm to 200 μm (the corresponding cell aggregates are useful for long-term cultures). Such a cell culture may further contain soluble extracellular matrix (preferably laminin, fibronectin and/or collagen) or a biocompatible biomaterial (e.g. alginate, chitosan, polylactic acid).
For the infection, sporozoites are put in contact with the 3D cell aggregates.
The above described infected 3D cell cultures according to the invention are useful e.g. for drug screening and vaccine development. In particular, the cell cultures according to the present invention have following advantages: an improved long-term stability (the infected 3D cell cultures can be cultured up to 2/3 months), good culture functionality, and/or an improved infectivity.
The present invention further provides a multi-well plate containing a 3D cell culture of hepatic cells as described above. Multi-well plates are useful e.g. for high throughput screenings in drug or vaccine development.
In addition, the invention provides a method for the production of a 3D cell culture containing hepatic cells, comprising following steps:
The invention also provides a method for the production of a 3D cell culture containing hepatic cells, comprising following steps:
The 2D culture used for the inoculation is obtainable by different well known procedures (see e.g. Freshney RI: Culture of Animal Cells. 6th Ed. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010).
In a preferred embodiment of the method described above, the concentration of the single-cells in the in the cell medium is in range of 0.1×106 to 1×106 cell/mL. Furthermore, the agitation-based culture system is preferably a stirred-tank bioreactor or a spinner vessel. Preferably, the inoculation is also performed in the agitation-based culture system.
In another preferred embodiment of the described method, the inoculation (step a) and/or agitation (step b) and/or incubation (step c) is performed at a temperature in the range of 37° C.±2° C. in humidified atmosphere (up to 95% of relative humidity). 5%-10% of CO2 in air. In a specific embodiment, the agitation is performed for a time period of several weeks (for example 1-2 weeks) (wherein the culture medium is exchanged if needed, preferably every 2-3 days).
In another very important embodiment of this method, the 3D cell culture is centrifuged at up to 1800×g during the incubation (step c)). The centrifugation promotes cell-pathogen contact by increasing the local concentration of cells and pathogens in a certain layer within the culture medium due to their density. In this embodiment, the cell culture medium volume is preferably kept constant (which means that there is no or no significant reduction of the cell culture volume over time). Nevertheless, the culture medium can be exchanged if needed (preferably without changing the overall volume), preferably the culture medium is exchanged every 2-3 days. Most preferably, moderate acceleration and brake settings are used for this centrifugation procedure in order to avoid aggregate/spheroid fusion. According to the present invention, the conditions described above can be referred to as “static incubation conditions”.
Yet, in a specific embodiment of the present invention the cell culture is centrifuged at up to 1800×g during the incubation (step c)), wherein the cell culture volume is preferably kept at a constant level.
In another embodiment, the 3D cell culture is exposed to agitation during the incubation (step c)) (preferably in a spinner vessel or multiwell-plate). The agitation speed is preferably within a range of 110 to 40 rpm. Agitation is useful to promote cell-pathogen contact. Moreover, in such an embodiment the cell culture medium volume is preferably reduced during incubation to 10-75% of the starting volume. The reduction of the cell culture volume results in an increased concentration. This can further promote pathogen-cell contact. According to the present invention the conditions described above can be referred to as “dynamic conditions”. Under these conditions the incubation can e.g. be performed in approximately 2 h under continuous agitation, with tuning of the agitation speed within a range of 110 to 40 rpm). This is embodiment is particular suitable for large volume cell cultures and it can also be advantageous if the formation of large aggregates is desired.
Consequently, in a specific embodiment of the present invention the cell culture is exposed to agitation during the incubation (step c)), wherein agitation speed is preferably within a range of 110 to 40 rpm, and wherein the cell culture volume is preferably reduced to 10-75% of the starting volume.
In a further embodiment of the method for the production of a 3D cell culture according to the invention, the incubation is performed under static conditions, wherein 3D cell culture containing the cell aggregates together with the pathogen is exposed to centrifugation at up to 1800×g, or the incubation is performed under dynamic conditions, wherein the cell culture volume is reduced (preferably to 10-75% of the starting volume) and the cell culture is exposed to agitation (wherein agitation speed is preferably within a range of 110 to 40 rpm).
In another embodiment the cell culture medium volume is reduced during the infection to 50-75% of the starting volume under continuous agitation. This can be particular suitable if the formation of large aggregates is desired.
The present invention also relates to a 3D cell culture of hepatic cells obtainable with a method or the production of a 30 cell culture of hepatic cells as described above.
The invention also provides a screening method, comprising following steps:
The monitoring can be performed using different well-known techniques (such as e.g. fluorescence, luminescence, immunofluorescence and antigen detection).
The invention further relates to a use of a 3D cell culture according to the invention to determine a cytotoxic effect and/or metabolic properties of a compound contacted with the 3D cell culture and/or an effect of a compound contacted with the cell culture on the pathogen (preferably for drug screening purposes).
The 3D cultures and methods according to the present invention are suitable for the screening of novel anti-infective compounds e.g. because of the mature phenotype of the hepatocytes that can be achieved, the high infectivity that can be achieved (e.g. infection rate of up to 3% achievable for P. berghei cells) and the ease of the pathogen reporter system. In addition, it is possible to unveil the compound action point on the infection process by incubation of the compound at specific time periods (see
The invention further relates to a screening assay for an anti-parasitic drug and/or a vaccine.
The invention also relates to a kit for the screening for a drug (preferably an anti-parasitic drug) and/or a vaccine comprising a 3D cell culture according to the invention.
The production methods according to the present invention allow to produce such 3D cell cultures in large quantities, which is very useful e.g. for high-throughput screening.
In the context of the present invention, the term ‘3D cell culture’ or ‘3D culture’ refers to a cell culture comprising three dimensional cell aggregates (including in particular spheroids). In 3D cultures, the cells are attached to one another, thus allowing cell-to-cell interactions.
The term ‘2D cell culture’ or ‘20 culture’ refers to a two dimensional cell culture.
The term ‘cell aggregate’ refers to a 3D cell aggregate (in particular spheroids).
The term ‘co-culture’ refers to an in vitro cell culture containing at least two distinct cell types, wherein at least cell type is a hepatic cell type. Accordingly, a co-culture may for example contain cells from two (or more) different hepatic cell types or a co-culture may contain cells from one (or more) hepatic cell type(s) in combination with cells from at least one (or more) further non-hepatic cell type(s). The term ‘mono-culture’ refers to an in vitro cell culture containing only one (hepatic) cell type.
In view of a cell aggregate, the term ‘infected’ (or ‘infected aggregate’) means that at least one cell per cell aggregate is infected. In the context of the present invention, the infected cell is a hepatic cell.
In the context of the present invention, “hepatocyte-like cells derived from pluripotent or multipotent stem cells”, are undifferentiated cells that have the potential to differentiate into hepatic cells. “Pluripotent stem ceils” can differentiate into 3 germ layers, while “multipotent stem cells” refer to hepatic progenitor cells, that can only differentiate into tissue-specific cell types.
The term “single-cell suspension” refers to a suspension of cells that, basically comprises individual, non-aggregated cells.
Unless otherwise specified, all starting materials are obtained from commercial suppliers and used without further purifications. Unless otherwise specified, all temperatures are expressed in ° C. and all reactions are conducted at RT.
The invention will be illustrated (but not limited), by reference to the specific embodiments described in the following examples.
HepG2 spheroids were generated in stirred-tank systems. The culture conditions used for HepG2 spheroids are summarized in Table 1.
HepG2 cells formed spheroids with high cell viability (
HepaRG spheroids were generated in stirred-tank systems. The optimized 3D culture parameters are summarized in Table 2. Representative images of HepaRG spheroids and spheroid diameter along culture time are shown in
3D cultures of the HC-04 cell line were established based on the conditions implemented for HepG2 cells. When HC-04 cells were cultured in 10% FBS, variation of inoculum concentration and agitation rate had no beneficial effect on cell aggregation efficiency; HC-04 cells formed very few and non-compact spheroids (
The hepatic phenotype of HC-04 spheroids was characterized by immunofluorescence microscopy (
The 3D culture of cryopreserved primary human hepatocytes (PHH) was established based on the previously described strategy for hepatic cell lines, with the same cell inoculum concentration and increasing the initial agitation speed according to Table 4. PHH spheroids were compact after 6 days of culture and the 3D culture was maintained for up to two weeks in stirred-tank vessels (
A 3D co-culture of HC-04 and HepaRG cell lines was established based on of the aggregation conditions implemented for HC-04 cells. Cells were co-cultured in a ratio of 2 HC-04:1 HepaRG, in DMEM+F12 culture medium according to Table 3. The co-culture with HepaRG cells had a beneficial effect on cell aggregation, as compared to HC-04 mono-cultures, enabling the generation of spheroids with a FBS concentration of 10% (v/v). Variation of agitation rate from 50 to 80 rpm along two weeks of culture time led to the generation of compact spheroids (
For the co-culture of PHH with HepaRG cell line, a ratio of 9 PHH:1 HepaRG ratio at 2×105 cell/mL cell density was tested. The aggregation was efficient, with spheroids formed 3 days after inoculation and culture viability was maintained over the culture period (
For the infection of a large number of spheroids and maintenance in culture for long-term periods, the infection in dynamic conditions using spinner vessels was implemented. Several parameters were considered to establish the dynamic infection, such as the sporozoite and cell concentrations, cell-to-sporozoite ratio and culture volume and agitation during infection, with the aim of maximizing cell-to-sporozoite contact and minimizing the impact of shear stress on the viability of hepatic spheroids. The parameters and conditions used for implementation of infection in dynamic conditions using spinner vessels are summarized in Table 5.
The infection rate in dynamic conditions was assessed in 3D cultures of HepG2 and compared to static conditions using a cell-to-sporozoite ratio of 1:1, at 2.5×104 cell/well. Cell viability 48 h post-infection was high, indicating that the manipulation of culture parameters and resulting shear stress had no impact on spheroid integrity and viability (
Infection parameters including cell concentration, cell-to-sporozoite ratio, cell-to-sporozoite mode of contact and culture time of the spheroids were optimized. Sporozoites were obtained from the dissection of the salivary glands of infected Anopheles stephensi mosquitoes. Following mechanical disruption of salivary glands, the sporozoite suspension was kept on ice for up to 3 hours, until sporozoites were employed to inoculate the cells in culture.
For the implementation of standard infection conditions in spheroids, the effect of centrifugation and subsequent static culture in 96-well plates was assessed. All centrifugation speeds led to spheroid fusion except in the condition where the centrifugation speed was gradually reached and gradually decreased (equivalent to acceleration and braking profiles 5 in a Rotina420R, Hettich centrifuge),
Initially, a preliminary assay employing reporter lines of Plasmodium berghei, constitutively expressing luciferase (Pb-Luc) or GFP (Pb-GFP), was performed to optimize the range of cell-to-sporozoite ratios and mode of contact. Pb-Luc parasites enable measuring infection by luminescence readings of cell lysates following addition of the luciferin substrate. Pb-GFP parasites enable measuring infection flow cytometry analysis. Such analyses allow measuring the percentage of invaded cells (% GFP-positive cells) and the development of the parasite inside the hepatic cells (GFP intensity). The conditions tested and readouts employed are depicted in Table 6 and the results obtained are presented in
HepG2 spheroids presented higher infection rate when cell-to-sporozoite contact was promoted by centrifugation (
Therefore, the preferred procedure for infection was: (i) Distribution of spheroids from spinner vessel to 96 well plates for infection; (ii) Promotion of sporozoite-to-cell contact by centrifugation at 1800×g for 5 min with medium acceleration and braking; (iii) Maintenance of spheroids in 96-well plates, in static conditions, for 48 hours post-infection, for infection assessment.
Cell-to-sporozoite ratios of 1:2 and 1:1 were selected to proceed with the optimization of P. berghei infection. Aiming to maximize cell-to-sporozoite contact, cell density at infection was optimized to achieve the maximum coverage of the well surface. The results are presented in
The results showed that higher infection rates were obtained with hepatic spheroids generated by two weeks in culture (Table 6). Moreover, the infection rates could be maximized using 5×104 cell/well and a 1:2 cell-to-sporozoite ratio for both lines of P. berghei (150% and 80% relative to HepG2 2D cells, for Pb-Luc and Pb-GFP respectively;
Both analytical methods, assessment of luciferase activity or GFP fluorescence, were consistent in identifying the infection conditions leading to the highest infection rates (
Sporozoites were able to develop in HepG2 spheroids, presenting, in all the conditions employed, a development above 65% of that observed in 2D cultures (
Given the data obtained, an optimal strategy for P. berghei infection of HepG2 spheroids was implemented using: (i) spheroids from two-week cultures; (ii) a cell density of 5×104 cell/well; and (iii) a 1:2 cell-to-sporozoite ratio.
HC-04 cells were infected by both P. berghei parasite lines. In 2D cultures, the infection rate of HC-04 cells was approximately 79% and 47% of the one observed for HepG2 cells under 2D conditions for Pb-Luc and Pb-GFP, respectively (
The percentage of spheroids infected by Pb-GFP at 1:2 of cell-to-sporozoite ratio was quantified by fluorescence microscopy, for cell densities of 2.5×104 and 5×104 cell/well. In both conditions more than 55% of the spheroids were infected (
In addition to the implementation and optimization of P. berghei infection in 30, the characterization of parasite development was performed for both hepatic cell lines (HepG2 and HC-04). Parasite development observed 60 hours post-infection was characterized by quantification of GFP intensity. A comparable profile of development was observed for all the conditions tested (20 and 3D; HepG2 and HC-04) (
The characterization of HC-04 metabolic activity and its suitability to be used as an in vitro model for drug screening is scarce. This may represent a major limitation for anti-Plasmodium drug assessment in this model, given the importance of liver metabolic activity for the correct metabolization of some anti-Plasmodium drugs (e.g, primaquine). In order to overcome this limitation, strategies based on co-culture systems were considered. Here, HepaRG cell line was selected to pursue a co-culture strategy, since these cells have been previously described as a more accurate surrogate of liver function among the available human hepatic cell lines platforms [Rebelo, S. P., Costa, R., Estrada, M., et al. (2014) HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism. Arch Toxicol.]. Moreover, previous reports have shown that co-cultures of primary hepatocytes and HepaRG could extend hepatocyte integrity and fitness, as well as improve P. cynomolgi infection [Dembéié, L., Franetich, J., Lorthiois, A., et al, (2014) Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures. Nature Medicine, 20(3), 307-312].
It was assessed whether the co-culture of HC-04 and HepaRG would have an impact on P. berghei infection. A 3:1 ratio of HC-04 to HepaRG cells was tested. Infection of co-cultures and HC-04 monocultures were performed with Pb-GFP in the optimized conditions described above (two weeks spheroids, cell density 2.5×104 and 5×104 cell/well in a 1:2 ratio). The results are presented in Table 7.
The results indicate that co-culture did not influence the infection rate in the best condition identified for infection of HC-04 spheroids (1:2 cell-to-sporozoite ratio and cell density of 5×104 cell/well). Thus, this co-culture strategy constitutes a promising alternative to improve the metabolic capacity of the system, as compared to HC-04 monocultures.
The suitability of the platform presented in this invention for drug screening purposes of anti-infective agents was explored using one reference drug. Atovaquone (ATQ), requiring no metabolization to target the liver-stage Plasmodium infection.
HC-04 3D cultures were infected with Pb-Luc in the optimized conditions described above (cell density of 2.5×104 cell/well in a 1:2 ratio). The assessment of drug effect in the infection was performed by incubating the drug at a range of concentrations from 0.01 to 100 nM for 1 hour before incubation with the sporozoites and the readout was performed 48 hours after sporozoites addition, described as incubation regimen (A) in the detailed description of the invention section (
| Number | Date | Country | Kind |
|---|---|---|---|
| 17160277.4 | Mar 2017 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16492071 | Sep 2019 | US |
| Child | 18414253 | US |
