Patent Grant
12208179
The aspects of the disclosed embodiments concern a detergent-free decellularized ECM preparation method, a detergent-free decellularized ECM in a powder form and in a liquid form, a method of preparation of a primary bioink, the primary bioink, a method of preparation of a vascular bioink, the vascular bioink, a three dimensional structure comprising the primary bioink and/or the vascular bioink and a method of preparation of the three-dimensional structure.
Bioprinting enables an automated deposition of living cells together with other components for a development of a three-dimensional (3D) tissue construct. Bioink formulations are created from different sources, including synthetic as well as natural polymers such as collagen, gelatin, alginate, hyaluronic acid, fibrin and polyethylene glycol. It is commonly known that matrix materials used for bioprinting cannot represent the complexity of natural extracellular matrix (ECM), which constitutes a microenvironment for the cells and can modulate cellular processes, including migration, differentiation and other functions. Therefore the presence of ECMs in bioinks is considered beneficial for recreation of a microenvironment with cell-cell connections.
International patent application WO2017014582 reveals a bioink composition comprising 0.05-60×106/mL of cells, 0.1 to 10 w/v % of a cell carrier material, 0.01 to 1 w/v % of a viscosity enhancer, 1 to 30 v/v % of a lubricant and 0.1 to 10 w/v % of a structural material. The bioink composition may further comprise a tissue-derived component material. Preferably, the cell carrier material is gelatin or collagen, the viscosity enhancer is hyaluronic acid or dextran, the lubricant is glycerol and the structural material is fibrinogen or methacrylated gelatin (GelMa).
The literature comprises many publications regarding the issue of selecting an appropriate bioink composition with optimal properties for tissue engineering applications. Mohamed Ali et al. carried out works on the production of a bioink based on decellularized ECM (dECM) derived from a kidney [1]. A relatively low concentration (1-3%) dECM hydrogel was obtained employing a dissolution method, using 0.5M acetic acid and 0.1 mg/mL pepsin. Additionally, a process of methacrylation of the dECM was carried out with addition of a photoinitiator (Irgacure).
Subsequent research groups attempted to obtain a bioink using ECM adding methacrylated gelatin (GelMa) and a photoinitiator, i.e. LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) [2]. Others used the dECM hydrogel obtained with a relatively high concentration of pepsin with the addition of polycaprolactone (PCL) as a preserving synthetic agent [3].
Patent description KR20180125776 describes a bioink composition comprising a dECM powder and a hydrogel. The dECM powder can be selected form liver tissue, heart tissue, cartilage tissue, bone tissue, adipose tissue, muscle tissue, skin tissue, mucosal epithelial tissue, amniotic tissue, or corneal tissue. Preferably, the dECM powder has a particle size of 0.05 to 100 μm. The hydrogel may contain one or more selected from the group consisting of gelatin, hyaluronic acid, dextran, and collagen.
Falguni et al (2014) developed tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. Bioprinting method enabled reconstitution of the intrinsic cellular morphologies and functions. A higher-order assembly of the printed cellular constructs was observed with organized spatial patterns and tissue-specific gene expression. A key advantage of the methodology was the application of tissue specific ECM, providing crucial cues for cells engraftment, survival and long-term function [3].
Experiments involving the decellularization of an organ in order to obtain the dECM as a component of bioink have been studied by many research groups [4, 5, 7]. Various substances were used for decellularization, primarily Triton X-100 and/or dodecyl sulphate (SDS) detergents. A method of decellularization of liver is described in KR1020180011607A, wherein hepatic tissue is treated with a de-saturated solution containing a surfactant and a hyperactive solution. 0.5% of Triton X-100 (Triton X-100) may be used as the surfactant.
Mohamed Ali and colleagues constructed a photo-crosslinkable kidney comprising a ECM-derived bioink [1]. Porcine whole kidneys were decellularized through a perfusion method, dissolved in an acid solution, and chemically modified by methacrylation. The results showed that the bioprinted human kidney cells were highly viable and mature with time. Moreover, the bioprinted renal constructs exhibited the structural and functional characteristics of the native renal tissue. The tissue-specific ECM-derived bioink could enhance the cellular maturation and eventually tissue formation.
Mirmalek-Sani et al (2013) presented the decellularization process of porcine pancreas to create a scaffold for human stem cells and porcine pancreatic islets. Cellular material was effectively removed while preserving ECM proteins and the native vascular system. Moreover, demonstrated that the decellularized pancreas can support cellular adhesion and maintenance of cell functions [6].
The aim of the aspects of the disclosed embodiments is to provide a detergent-free dECM that could be used in bioprinting. Literature data provides no results on the residual content of detergents in the ECM obtained by decellularization or on the methods assaying their content. In the previously published procedures for decellularization of various tissues, the stage of removal of the detergents is relatively short. It is believed that the absence of the detergent in dECM substantially affects the quality of the dECM obtained. The procedure developed by the applicant allows for almost all of the detergent to be removed without the need of addition of other chemicals. The second aim of the aspects of the disclosed embodiments is to obtain a bioink of a proper consistency and viscosity, without the need of addition of viscosity enhancers.
In a first aspect, there is provided a detergent-free decellularized extracellular matrix (dECM) preparation method comprising the following steps:
Mechanical fragmentation of the organ enhances the removal of the detergent form the organ and results in a product with lower fat content, which improves the properties of the final product, i.e. increases viscosity and improves printability. Addition of DNAse is crucial for removal of the DNA of the organ of animal origin. If the resulting printed three-dimensional structure was comprising dECM with DNA, it could not be further used in transplantation experiments.
Preferably, the grinding step is followed by a step of checking the amount of octoxynol-9 in dECM powder, wherein preferably before dECM powder is checked for the presence of octoxynol-9, it is treated with collagenase, preferably at a concentration of at least 43,953 PZ/g dECM.
Preferably, the grinding step is followed by the following steps:
In a second aspect there is provided a detergent-free decellularized ECM in a powder form, obtainable by the method of preparation of a detergent-free decellularized extracellular matrix (dECM). Preferably, the dECM powder is sterile. If necessary the powder could be sterilized by radiation sterilization or ethylene oxide sterilization.
In a third aspect there is provided a detergent-free decellularized ECM in form of a solution, obtainable by the method of preparation of a detergent-free decellularized extracellular matrix (dECM).
In a fourth aspect there is provided a method of preparation of a primary bioink comprising the following steps:
Since the dECM powder is originally prepared by freeze-drying and is not dissolved afterwards, it retains the whole quaternary structure of ECM. Hence, use of dECM in the form of a paste, comprising both the dECM powder and the dECM solution, provides the primary bioink with a proper consistency and, since the dECM powder is not dissolved in the primary bioink, it retains the whole quaternary structure of ECM.
In a fifth aspect there is provided the primary bioink comprising a dECM paste and 1.46-7.32% (w/v) methacrylated gelatin, 0.15-1.10% (w/v) methacrylated hyaluronic acid, 5-10% (w/v) glycerol and a photoinitiator, preferably 0.03-0.17% (w/v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate, wherein the dECM paste comprises 5-50% (w/v), preferably 15-25% (w/v), of the dECM powder according to the second aspect of the disclosed embodiments, and 1-10% (w/v), preferably 8-10% (w/v), of the dECM solution according to the third aspect of the disclosed embodiments and wherein the viscosity of the primary bioink is at least 5 Pa·s, measured in a cone-plate system, at a constant shear rate of 21/s and a temperature of 37° C.
The use of dECM makes it possible to reproduce the extracellular conditions of the body, thus giving the bioprint the characteristics of native tissue, which stimulates cells to differentiate and improves their survival rate. Moreover, the extracellular matrix is necessary to obtain a proper viscosity of the bioink and to maintain a stable three-dimensional structure of the printed construct through the additional possibility of thermal cross-linking in the temperature range of 33 to 37° C.
Use of the photoinitiator enables cross-linking, which is non-toxic to cells, as compared to chemical cross-linking using chemicals, which are toxic to cells contained in the primary bioink. Cross-linking with the use of the photo-initiator and visible light minimizes cellular DNA damage as compared to thermal cross-linking. Both temperature and light have negative effects on cells, leading to DNA damage. However, when cross-linking with visible light, these changes are kept to a minimum.
Methacrylated gelatin (GelMa) is used for shaping of the printed construct. In addition, it brings the filaments together so as to prevent lobule delamination and improves cell and islet viability. GelMa is stable in higher temperatures as compared to gelatin, which is beneficial during thermal cross-linking.
Methacrylated hyaluronic acid (HAMA) helps to maintain the three-dimensional structure by cross-linking. Additionally, HAMA provides smoothness, silkiness, homogeneity of the printed filament and supports cell cultures. These features cannot be obtained by addition of hyaluronic acid, which is not methacrylated.
The use of glycerol improves cell and islet functionality. It also improves the lubricity of the bioink, enables formation of continuous filaments, improves the mixing of bioink components in a syringe or a mixer and reduces the pressure expenditure during printing.
Preferably, the primary bioink comprises at least one additive selected from: hyaluronic acid at a concentration of 0.001 to 0.100 mg/mL of the bioink, preferably, 0.007 mg/mL, laminin at a concentration of 0.005 to 0.100 mg/mL of the bioink, preferably, 0.084 mg/mL, collagen I at a concentration of 0.001 to 0.100 mg/mL of the bioink, preferably 0.041 mg/mL, collagen IV at a concentration of 0.005 to 0.175 mg/mL of the bioink, preferably, 0.122 mg/mL, fibronectin at a concentration of 3 to 300 μg/mL, preferably 100 μg/mL, human fibrinogen at a concentration of 10 to 100 mg/mL of the bioink, aprotinin at a concentration of 1 to 2 EPU/mL of the bioink, polysorbate at a concentration of 0.05 to 2 mg/mL of the bioink, human thrombin at a concentration of 5 to 55 mg/mL of the bioink, calcium chloride at a concentration of 20 to 60 mM/mL of the bioink; proangiogenic vitamins: vitamin A at a concentration of 1 nM-500 μM, preferably 100 μM, vitamin B1 at a concentration of 50-100 μM, preferably 100 μM, vitamin B3 at a concentration of 1 to 10 μM, preferably 10 μM, vitamin B12 at a concentration of 10 to 100 mg/mL of the bioink, vitamin D3 at a concentration of 0.1 to 10 nM, preferably 10 nM, growth factors supporting angiogenesis: VEGF at a concentration of 10 to 30 ng/mL of the bioink, preferably 30 ng/mL, FGF at a concentration of 10 to 20 ng/mL of the bioink, preferably 20 ng/mL, TGF-β at a concentration of 1 to 10 ng/mL of the bioink, preferably 20 ng/mL, interleukin (IL)-8 at a concentration of 0 to 100 ng/mL of the bioink, preferably 10 ng/mL, IL-17A at a concentration of 20 to 50 ng/mL of the bioink, preferably 20 ng/mL.
Commercial additives such as hyaluronic acid, collagen I and IV and laminin further improve the functionality of the printed three dimensional structure.
Vitamin A—ATRA (All Trans Retinoic Acid) as one of the metabolites of vitamin A has a proangiogenic effect—it improves the expression of the factors behind angiogenesis (e.g. cyclooxygenase-2 (COX-2), hypoxic-induced factor (HIF)-1, C-X-C, chemokine receptor (CXCR)-4, vascular endothelial growth factor (VEGF), angiotensin (Ang)-2, -4. Moreover, it has been demonstrated that ATRA reduces pro-MMP2 (pro-matrix metalloproteinase-2—type IV collagenase) activity.
Vitamin B1—benfotiamine (a thiamine derivative) inhibits apoptosis on the protein-dependent B-kinase pathway (PKB/Akt) and is responsible for inducing the proliferation of progenitor endothelial cells.
Vitamin B3—niacin, through its receptor, i.e. hydroxycarboxylic acid receptor 2 (GPR109A), enhances and promotes endothelial cell functions that support angiogenesis. Moreover, vitamin B3 is a precursor of NAD(+), which by way of response with a sirtuin mediator (SIRT), induces and supports vessel formation.
Vitamin B12 (cobalamin) induces the production of prostaglandins E1, prostacyclins and nitric oxide (NO). All of these substances have a favourable effect on the onset of angiogenesis.
Vitamin D3 is designed to stimulate angiogenesis in vitro. It induces increased expression of VEGF and pro-MMP2 activity. It also affects the function of ECFC (endothelial colony forming cells).
VEGF induces proliferation, migration, sporulation and formation of connections between endothelial cells, and, in addition, by inducing the production of various proteases, affects the degradation of extracellular matrix (ECM) and activates cell surface integrins of endothelial cells.
Fibroblast Growth Factor (FGF) increases endothelial cell migration and promotes capillary morphogenesis. It also increases endogenous VEGF production.
Transforming Growth Factor (TGF-β) promotes the formation of ECM (proteoglycans, fibronectin, collagen), regulates the proliferation of endothelial cells, their migration and formation of blood vessels. TGF-β mediates the interactions of endothelial cells and pericytes.
Interleukin (IL)-8 has a potent proangiogenic effect on endothelial cells by interacting with CXCR1 and CXCR2 receptors. It stimulates the formation of a microvascular network.
IL-17A—Induces angiogenesis, cell migration and cytoskeleton rearrangement.
Preferably, the primary bioink comprises one or more animal- or human-derived additives selected from endothelial cells at a density of 0.1-10×105/mL of the bioink, primary microvascular endothelial cells at a concentration of 0.1 to 10×105/mL of the bioink, animal- or human-derived α cells at a concentration of 3 to 9×106/mL of the bioink, animal- or human-derived β cells at a concentration of 1.1 to 3.4×107/mL of the bioink, animal- or human-derived pancreatic islets, preferably in the amount of 20,000 iEq/mL of the bioink.
Pancreatic islets are responsible for insulin production. Endothelial cells are added for a faster formation of a vascular network in the printed three-dimensional structure. Primary microvascular endothelial cells are used to support the formation and growth of microvessels in the bioprinted three-dimensional structure.
In a sixth aspect there is provided a method of preparation of a vascular bioink comprising the steps of:
a) optional preparation of a solution of microbiological gelatin supplemented with CMC comprising preparation of a 1-2% (w/v) solution of microbiological gelatin in a buffer solution, preferably PBS, by suspending microbiological gelatin in the buffer solution with agitation at a temperature between 50 and 65° C., preferably at 60° C., addition of a 2-5% (v/v) carboxymethyl cellulose (CMC) aqueous solution to obtain a final concentration of 0.2-1% (v/v) of CMC in the bioink and cooling the solution to a temperature equal or below 40° C.
b) preparation of a 5-10% (w/v) dECM solution by addition of dECM powder according to the second aspect of the disclosed embodiments, preferably sterilized by radiation, to (i) the solution of microbiological gelatin supplemented with CMC obtained in step a) or (ii) a buffer solution or (iii) a solution of cell medium with gentle agitation.
c) sonication of the obtained solution at a temperature not exceeding 37° C. for 0.5-2.0 hours
d) optional addition of at least one animal- or human-derived additive selected from: fibronectin at a concentration of 3 to 300 μg/mL, preferably 100 μg/mL, VEGF at a concentration of 10 to 30 ng/mL, preferably 30 ng/mL, FGF at a concentration of 10 to 20 ng/mL, preferably 20 ng/mL, PGE2 at a concentration between 100 and 300 nM, preferably 100 nM, endothelial cells at a density of between 0.1 and 10×107 cells/mL of the bioink, fibroblasts at a density of between 0.1 and 10×106 cells/mL of the bioink.
In a seventh aspect there is provided a method of preparation of a vascular bioink comprising the steps of:
a) optional preparation of a solution of microbiological gelatin supplemented with CMC comprising preparation of 1-5% (w/v) solution of microbiological gelatin in a buffer solution, preferably PBS, by suspending microbiological gelatin in the buffer solution with agitation at a temperature between 50 and 65° C., preferably at 60° C., addition of a 2-5% (v/v) aqueous CMC solution to obtain a final concentration of 0.2-2% (v/v) of CMC in the bioink and cooling the solution to a temperature equal or below 40° C.
b) preparation of a 2-10% (w/v) dECM solution by addition of dECM powder according to the second aspect of the disclosed embodiments, preferably sterilized by radiation, preferably sterilized by radiation, to (i) the solution of microbiological gelatin supplemented with CMC obtained in step a) or (ii) a buffer solution or (iii) a solution of cell medium to with gentle agitation.
c) boiling the mixture at 100° C. for 15-30 minutes
d) optional addition of at least one animal- or human-derived additive selected from: fibronectin at a concentration of 3 to 300 μg/mL, preferably 100 μg/mL, VEGF at a concentration of 10 to 30 ng/mL, preferably 30 ng/mL, FGF at a concentration of 10 to 20 ng/mL, preferably 20 ng/mL, PGE2 at a concentration between 100 and 300 nM, preferably 100 nM, endothelial cells at a density of between 0.1 and 10×107 cells/mL of the bioink, fibroblasts at a density of between 0.1 and 10×106 cells/mL of the bioink.
In an eighth aspect there is provided the vascular bioink comprising sonicated or boiled dECM solution according to the third aspect of the disclosed embodiments mentioned above at a concentration of 2-10% (w/v), preferably supplemented with microbiological gelatin at a concentration of 1 to 5% (w/v) and/or CMC at a concentration of 0.2 to 2% (v/v).
The sonicated or boiled dECM changes its physical and chemical properties with temperature changes. This component is designed to ensure proper viscosity of the bioink during printing at a relatively low temperature (15-20° C.) and to preserve the printed duct until cells infiltration as well as slow liquefaction at the culture temperature of 37° C.
Microbiological gelatin provides a desired consistency and improves cell survival rate. CMC increases viscosity and stabilises bioink consistency. Fibronectin promotes angiogenesis and depending on the dose, stimulates elongation of the vessels formed without affecting the proliferation rate.
Preferably, the vascular bioink comprises at least one animal- or human-derived additive selected from: fibronectin at a concentration of 3 to 300 μg/mL, preferably 100 μg/mL, VEGF at a concentration of 10 to 30 ng/mL, preferably 30 ng/mL, FGF at a concentration of 10 to 20 ng/mL, preferably 20 ng/mL, PGE2 at a concentration between 100 and 300 nM, preferably 100 nM, endothelial cells at a density of 0.1 and 10×107 cells/mL of the bioink, fibroblasts at a density of between 0.1 and 10×106 cells/mL of the bioink.
Endothelial cells produce blood vessels. Fibroblasts produce angiogenesis-inducing factors. VEGF induces proliferation, migration, sporulation and formation of connections between endothelial cells. Moreover, by inducing the production of various proteases, VEGF affects the degradation of the ECM and activates cell surface integrins of endothelial cells. FGF increases endothelial cell migration and promotes capillary morphogenesis. It also increases endogenous VEGF production. PGE2—prostaglandin E2, designed to induce migration, proliferation and formation of new vessels by activating (phosphorylation) FGF of the (R)-1 receptor.
In a ninth aspect there is provided a three-dimensional structure comprising at least three adjacent bioink layers, wherein a layer of the vascular bioink according to the eight aspect of the disclosed embodiments is arranged between two layers of the primary bioink according to the fifth aspect of the disclosed embodiments.
In a tenth aspect there is provided a method of preparation of a three-dimensional structure, wherein the primary bioink according to the fifth aspect of the disclosed embodiments and the vascular bioink according to the eight aspect of the disclosed embodiments are deposited layer by layer in a 3D-bioprinting process at a printing speed from 5 to 50 mm/s, pressure from 4 to 300 kPa and temperature from 4 to 37° C. and wherein during or after deposition the primary bioink is exposed to UV light and/or visible light, preferably of the wavelength form 365 to 405 nm, more preferably at 405 nm, for at least 5 seconds. Cross-linking at 405 nm is preferred, as it is not toxic for the cells contained in the three-dimensional structure.
The aspects of the disclosed embodiments enabled obtaining of a model of a lobule 27×17×2.5 mm in size. A lobule consisting of 5 layers was printed in 3-10 minutes. Additionally, a 3D model of a functional organ prototype 30×40×20 mm in size was obtained. The model consisted of 30 layers and was printed in 20 to 60 minutes. Also importantly, this is the first time that boiled or sonicated dECM use is reported. The aspects of the disclosed embodiments enable obtaining a construct in a short time due to the printing speed being properly correlated with the viscosity of bioink (up to 30 mm/s). A stable three-dimensional porous structure can be obtained (30 layers), which is preservable at a temperature of 37° C. for 20 days. In a preferred embodiment, the primary bioink is based on using a less toxic photoinitiator, i.e. LAP rather than Irgacure at a relatively low concentration. Moreover, a smaller amount of pepsin is used than found in the literature to obtain dECM solution.
A)-C)—SEM (scanning electron microscope) images; A) native tissue before decellularization; B) tissue after decellularization; C) construct printed from primary bioink.
D)-E) —TEM (transmission electron microscope) images; D) tissue after decellularization; E), F) constructs printed from the primary bioink with preserved collagen quaternary structure (visible collagen fibres).
1% (v/v) Triton X-100 solution with 0.1% (v/v) ammonia water in 1× concentrated PBS solution with 0.01% (w/v) streptomycin was prepared for removing cell structures from the pancreatic organ while leaving the extracellular matrix (scaffold). After harvesting, the tissue material was frozen at −80° C. Then, after thawing, the outer layer of fat tissue and the surrounding membranes were removed from the organ. The prepared pancreases were treated in two ways: cut into small pieces (about 1-1.5 cm) and mechanically ground (using an extrusion grinding method).
The fragmented tissue was placed in a bottle and suspended in a previously prepared solution of Triton X-100. The specimens were placed in an incubator at 4° C. at constant agitation of 150 rpm. Every 4 h to 12 h, the detergent was replaced until the cellular fraction was completely removed (3-5 days). The detergent was then washed out from the scaffold obtained. For this purpose, a solution of 1×PBS with 0.01% (w/v) streptomycin was used. The washing process was carried out for 72 h at 4° C. with continuous stirring at 150 rpm.
The next stage—decellularization—consisted in administering a deoxyribonuclease solution (0.0002% (w/v) DNAse in 1×PBS, supplemented with 0.12 mM calcium and magnesium ions). The scaffold was incubated in the abovementioned solution for 8 hours at 37° C. with stirring at 150 rpm. The last step involved washing again with 1×PBS solution with 0.01% (w/v) streptomycin at standard conditions (4° C.; 150 rpm; 72 h). In addition, washing out of the detergent using ammonia water at a concentration of 0.1% (v/v) in 1× concentrated PBS solution was also tested. Moreover, the effect of an increase in temperature to 20-24° C. on the washing step was studied.
After the end of the decellularization process, the scaffold obtained was frozen in liquid nitrogen and crushed into pieces of approx. 0.5 cm in size. The material was freeze-dried for 26 h at a temperature of −32° C. and 0.31 mbar (31 Pa) pressure. The final drying process lasted 10 minutes at 0.0010 mbar (0.1 Pa) pressure and temperature of −76° C. The crushed and dried scaffold was ground into powder using a cryogenic mill. The grinding procedure involved 3 cycles for 1 minute at 15 impacts per second.
In order to characterise the product obtained, i.e. dECM powder abbreviated as “dECM(p)”, powder grain size distribution in flow gradient was tested using a laser diffraction spectrometer Spraytec (Malvern, UK) equipped with an accessory inhalation chamber for studying inhalation sprays. In all the cases studied, the values of the parameters describing the analysed powder following aerosolization were comparable, indicating that there was no need to provide additional energy in the form of an increased air stream to break down the powder into individual particles. Table 1 presents the values of parameters describing the diameters of powder particles, where:
Dv(50) —median of the volume particle size distribution: the diameter of the particles that divides the cumulative volume distribution in half, in other words, all particles both smaller and larger than the median have the same volume (the particles below this diameter constitute 50% of the sample volume).
Dv(10) —the particles below this diameter constitute 10% of the sample volume.
Dv(90) —the particles below this diameter constitute 90% of the sample volume.
D[3][2]—the Sauter diameter is the diameter of a particle whose volume to surface ratio is the same as the ratio of the volume of all analysed particles to the surface of the total of all such particles.
D[4][3]—a diameter defined as the ratio of the sum of the fourth power of particle diameters to the sum of the third power of particle diameters.
The results of the measurements following the aerosolization of the dECM powder indicate that the powder was polydispersible. The median of the volume particle diameter distribution—Dv(50) at an air flow nominal for Cyclohaler type inhaler equaled 148.43±10.14 μm. At the same time, the smallest particles of the total volume not exceeding 10% of the total volume of the sample had a diameter of less than 28.23±1.48 μm (Dv(10)), while the diameter distinguishing the particles with total volume less than 90% of the total volume of the sample was 410.10±29.41 μm. Increasing the air flow rate fed to the inhaler to 200 and 270 dm3/min did not significantly affect the value of the median of the volume particle size distribution or the Dv(10) value. Only a slight increase in the size of the largest particles (Dv(90)) from approx. 410.1±29.41 μm to 498.3±62.7 μm could be observed. This resulted in an increase in the distribution span from 2.57±0.04 (for 100 dm3/min flow) to 3.3±0.4 (for 270 dm3/min flow).
Protein Characteristics of the Product:
Using the mass spectrometry technique, the ECM protein composition after decellularization of the porcine pancreas was determined. The results obtained clearly demonstrated the highest percentage of collagens in the samples tested, with collagen type 1 (COL1) of the alpha (A)-1 chain, the so-called COL1A1, showed the highest values compared to the other detected collagen types.
Also, significant amounts of type IV and type VI collagen were found. This shows that the decellularization protocol used allowed for preserving the types of collagen that have the highest level of integration with pancreatic islets and 13 cells. Type I and type IV collagen is the most effective in supporting the functionality and viability of pancreatic islets and they are commonly used as supplements in biomedical applications that are based on the functioning of pancreatic islet cells. Notably, collagen VI and IV are present on the extra secretory surface and basement membrane of pancreatic islets and they regulate fibronectin activity. The percentage content of any of other collagen types analysed (COL1A2, COL3A1, COL4A2, COL6A1, COL6A2, COL6A3 COL14A1) did not exceed 3.5% in all examined samples.
Final DNA Concentration
In order to determine the residual DNA concentration, three analyses were carried out:
The decellularization process has been successfully completed when the concentration of residual DNA did not exceed 50 ng of double-stranded DNA (dsDNA) per mg ECM of dry weight, and the molecules of the remaining DNA did not exceed 200 base pairs (bp). Also, the microscopic image of the scaffold obtained was evaluated for the presence of cell nuclei (haematoxylin & eosin staining).
The concentration of residual DNA in the dry matter was on average 0.077 ng/mg. In all examined samples the residual DNA content was lower than 0.15 ng/mg. The analysis was carried out using DNeasy Blood & Tissue Kit kits used to isolate residual DNA, and Quant-iT PicoGreen dsDNA Reagent and Kits to determine the concentration of the isolated genetic material.
No signal was found using agarose gel electrophoresis. All samples were below the detection level, which clearly demonstrates that ECM had no residual DNA in the form of particles larger than 200 bp.
Microscopic examination showed no genetic material, no cell nuclei were visible following the decellularization process.
Triton X-100 Residue and Effective Method for the Detection and Removal Thereof
For comparison, decellularization with 0.5% (w/v) SDS was conducted. The result, however, was not satisfactory due to the large quantities of detergent remaining in the final product. ECM powder showed high foaming level when attempts were made to dissolve it. This was not observed with the use of Triton X-100.
In order to verify the concentration of Triton X-100 remaining after the decellularization process, its residue in the final product was determined.
Preparation of the Samples for Analysis:
Given the white colour of the dissolved dECM, the samples were treated with three concentrations of collagenase: 4.3953 PZ activity units/g dECM(p), 43.953 PZ/g dECM(p) and 87.906 PZ/g dECM(p). Collagenase was prepared in a special solution containing 150 mL of Ringer's solution pH 7.2 to 7.4, 2.72 mL Hepes (1M), 1.125 mL NaBicarbonate (7.5%) and 1.05 mL CaCl2) (1M).
The samples were stirred for 24 h at 37° C. with a constant function of shaking at 1000 rpm. The solutions obtained were analysed for residual concentration of non-ionic detergent Triton X-100. Sample A was the result of treating dECM with a single collagenase concentration. Sample B was treated with 10-fold collagenase concentration. Sample C was treated with 20-fold collagenase concentration (A=6.977 μg Triton X-100/g dECM(p), B=40.475 μg Triton X-100/g dECM(p), C=39.325 μg Triton X-100/g dECM(p)).
Importantly, the highest concentration of the remaining Triton X-100 was found in the dECM solution treated with collagenase at a concentration of 43,953 PZ/g dECM. An increase in the concentration of collagenase did not result in a higher amount of Triton X-100 obtained, which indicated that the concentration of 43,953 PZ/g of dECM was sufficient for extraction of all the remaining Triton X-100 from the sample.
Previous published attempts to evaluate this detergent did not yield any tangible results for the following reasons:
Therefore, treating dECM with collagenase is hitherto the only method for evaluating the residue of detergents in biological material following decellularization. This is of importance where such material (dECM) was to be used in the bioprinting process with viable cells. This is crucial if such material was to be used for implantation in humans.
In the first step, the differences in fat composition in decellularized matrix in the function of the preparation of pancreas for decellularization were analysed. In the next step, the content of residual DNA, depending on the method of pancreatic preparation, collagen content and the content of residual detergent Triton X-100 were analysed.
The use of the mechanical extrusion grinding method allowed for significantly reducing the fat content in the extracellular matrix obtained. In the mechanical extrusion grinding method, the fat content was 6.24+/−0.07% (w/w) compared to 21.47+/−0.07% (w/w) of the fat content in the cutting method. The difference was statistically significant (p<0.001). Low fat content of obtained dECM significantly increased the viability of cells and pancreatic islets.
The content of residual DNA tested with Picogreen was significantly lower when using the mechanical extrusion grinding, namely 0.07+/−0.07 ng/mg compared to 0.13+/−0.06 ng/mg of tissue (p=0.027). This was in both cases well below the permissible 50 ng/mg value.
The use of the mechanical extrusion grinding method allowed for significantly reducing Triton X-100 content in the extracellular matrix obtained. In the mechanical extrusion grinding method, the detergent content was 3.79+/−2.33 μg/g as compared to 6.53+/−2.34 μg/g in the cutting method. The difference was statistically significant (p=0.008).
The content of collagens in the tested material resulting from the preparation method did not differ depending on the use of the cutting method and mechanical extrusion grinding.
The use of ammonia water to alkalize the environment for better washing out Triton did not result in improved washing out of Triton. It brought about, however, a change in the composition of the collagens obtained. Similarly, washing at 24° C. failed to improve washing out of Triton, while increasing the damage to collagen structures and resulting in obtaining higher results of DNA content, which could indicate the risk of infection of the material. Therefore, the optimal method was to wash the decellularized material in PBS at a temperature of 4° C. for 72 hours.
In order to obtain a dECM solution (dECM(r)), a dECM powder (dECM(p)) dissolution procedure has been established that used pepsin and hydrochloric acid (HCl).
The procedure for obtaining the dECM solution was divided into two parts:
(a) Dissolving of dECM.
Pepsin (at a concentration of 0-10 mg/mL, preferably 1 mg/mL) was dissolved in 50 ml of 0.01 M HCl, after which dECM(p) (0.5-5 g) was added. This method resulted in a dECM(r) concentration in the range of 1-10% (w/v). The prepared solution was placed on a magnetic stirrer, using the following stirring conditions: ambient temperature of approx. 25° C., dissolution time of 72 h, wherein the solution was agitated every hour for the first 8 h of stirring.
(b) Neutralisation of dECM(r).
Neutralization of 50 mL of dECM(r) was carried out on ice (the desired temperature of the dECM solution was 4 to 4.5° C.) to pH 7.2-7.4 using the following substances:
In order to identify the procedure appropriate for the preparation of dECM(r), analysis was carried out of solutions with a relatively high concentration of dECM(p) ground after radiation sterilization—10% (w/v) with varying pepsin content.
dECM solutions with varying pepsin content were prepared. The solution containing 1 mg/mL pepsin had relatively high homogeneity: a small span of viscosity values. A slight change in turbidity was observed with the temperature change. All analysed dissolution methods with varying pepsin content were used for the preparation of dECM(r), however, it was demonstrated that the amount of 1 mg/mL used was optimal.
(a) Conditions for Obtaining the Primary Bioink:
First, a paste was prepared containing an appropriate amount of neutralised dECM(r) and dECM(p) by thorough mixing with a sterile metal spatula. Since the dECM(p) was prepared by freeze-drying and was not dissolved afterwards, it retained the quaternary structure of ECM. The paste obtained was left at a temperature of 7-10° C. for at least 24 h. Directly before using the paste for bioink production, it was placed in a sterile syringe and mixed between syringes. At the same time, GelMa (10-20% (w/v)) and HAMA (1-3% (w/v)) solutions were prepared with LAP according to a commonly available procedure. The syringe containing the paste was attached with a connector to another syringe without the piston, which was moved upside down and stably arranged in the vertical position. Glycerol, culture medium, growth factors, vitamins, GelMa and HAMA solutions were successively added. The piston was then gently inserted, and the paste was mixed with the other reagents. After mixing, the prepared bioink was placed in the incubator for 5 minutes, islets and cells were added, then mixed again and introduced into a cartridge. In the next step, the filled cartridge was centrifuged for 2 minutes in 1500 rpm and reintroduced before printing for approx. 5 minutes to the incubator.
The compositions of the obtained primary bioinks were the following: 40-50% (v/v) of dECM(r), 2.763-27.692% (w/v) of dECM(p), 1.464-7.320% (w/v) of GelMa, 0.146-1.098% (w/v) of HAMA, 5.0-10.0% (w/v) of glycerol, 0.03-0.17% (w/v) of LAP, VEGF—30 ng/mL, FGF—20 ng/mL, TGF-β—10 ng/mL, IL-8—10 ng/mL, IL-17A—20 ng/mL, vitamin A—100 μM, vitamin B1—100 μM, vitamin B3—10 μM, vitamin D3—10 nM, pancreatic islets—20000 iEq/mL, endothelial cells—1×105/mL, primary microvascular endothelial cells—1×105/mL.
(b) Vascular Bioink
The process of vascular bioink production using sonication was divided into two steps:
The process of producing vascular bioink by boiling was divided into two steps:
Vascular bioink's bases thus prepared were supplemented with fibronectin, growth factors and endothelial cells.
The composition of the obtained sonicated vascular bioink was as follows: 5-10% (w/v), preferably 7.5% (w/v) of dECM(p), 0.2-1% (v/v) of CMC, 1—2% (w/v), preferably 1% (w/v) of microbiological gelatin, fibronectin—100 μg/mL, VEGF—30 ng/mL, FGF—20 ng/mL, PGE2—100 nM, 1.5×107/mL of endothelial cells and 3×106/mL of fibroblasts.
The composition of the obtained boiled vascular bioink was as follows: 2-10% (w/v), preferably 5% (w/v) of dECM(p), 0.2-2% (v/v) of CMC, 1-5% (w/v), preferably 1% (w/v) of microbiological gelatin, fibronectin—100 μg/mL, VEGF—30 ng/mL, FGF—20 ng/mL, PGE2—100 nM, 1.5×107/mL of endothelial cells and 3×106/mL of fibroblasts.
Alternatively, the vascular bioink consisted of 5-10% (w/v), preferably 5% (w/v) dECM(p) in a buffer solution or a cell medium.
The tests conducted served as a basis to determine the values of characteristic parameters, constituting factors limiting the possibility of using a particular system for printing a pancreas lobule model—viscosity value of more than 5 Pa·s. The influence of pepsin concentration on the properties of dECM hydrogel is presented below.
In order to identify the composition of the bioink with optimal properties, the viscosity of dECM solutions and pastes was tested using the MCR 72 rheometer (Anton Paar) following a specially developed procedure to represent the conditions existing during bioprinting: cone-plate system, constant shear rate of 21/s and the test temperature of 37° C. The results of system rheology testing taking into account the differences in samples by the type of powder used (MS—ground and sterilised, CS—cut and sterilised, MNS—ground, not sterilised, CNS—cut, not sterilised), and the concentration of components used are presented in
An increase in the concentration of dECM solutions results in an increase in viscosity [
The summary of the results shows that the use of the dECM paste was necessary to obtain a bioink base having a suitable consistency. All the systems from the summary have a viscosity within the range acceptable for use during printing. Moreover, it seems expedient to use for bioprinting a mixture of components with cells and islets using sterile powder, following sterilisation.
Addition of glycerol into the primary bioink (paste) caused a slight decrease in viscosity of the primary bioink, contrary to the literature data, reporting an increase of viscosity of bioinks upon addition of glycerol. Each of the agents added to the paste induced a change in viscosity. Adding substances supporting the maintenance of the construct or viability of cells and islets induces changes in the flowability of pastes that are insignificant to a point of being negligible. The paste from dECM constituted the basis for producing the primary bioink and for determination whether a particular bioink might be used for printing.
Cross-Linking of Printouts Using Cross-Linking Agents
The table below presents the differences in the composition of cross-linking agents used in the primary bioink [Table 6].
Cross-linkability tests of the systems as above have been conducted using light with a wavelength in the range of 365 to 405 nm with a positive outcome [Table 7].
The analysis of cross-linking results after the process or during the bioprinting process showed that both the use of 365 nm and 405 nm wavelength light achieved the intended effect, i.e. the change of hydrogel form from liquid to solid. However, since the bioink contains cells and microorganisms, only visible light may be used. Therefore, the most preferable method of cross-linking is to use light with a wavelength of 405 nm.
Adding supplementary chemical substances to the dECM paste resulted in smoothed topography of the filament surface. Moreover, an increase in the aeration of the bioink was identified when adding GelMa and HAMA, with this effect being the most potent with HAMA.
Thermal Gelation
The intensity of gelation process was tested using the identification of solution turbidity using specialised equipment over a wide range of temperatures and exposure time to the effect thereof.
Based on the kinetics of gelation at a constant temperature of 37° C., no significant changes in turbidity were observed when increasing the time of exposure to the temperature of 37° C. dECM(r) from ground sterile powder has the lowest absorbance value, while the cut non-sterile powder solution has the highest turbidity for all dECM(r) concentrations.
With the increase of the so-called driving force, i.e. glucose concentration, the time of delay and reaching the state of equilibrium decreases, with the corresponding increase in diffusivity. The data presented in Table 8 show that the diffusivity of the membranes obtained with the primary bioink was comparable to those obtained with 4% (w/v) alginate (Alg4).
In order to evaluate the usability of the bioink obtained, absorbency analysis of printed lobules was carried out using a specially prepared buffer imitating the internal condition of the body. For the first 15 minutes, a slight increase in the weight of the printed construct was observed, followed by the decrease and stabilization thereof at a specific level. In the next step, changes in weight over time of the printed lobule were observed in order to study the phenomenon of degradation in the SBF buffer environment [
Boiled dECM(r) has a much higher viscosity value than the sonicated one. However, due to proper stability of the vascular bioink after sonication, this method was determined to be more preferable for the printing of the vessel duct.
An increase in temperature in the range 25 to 37° C. and exposure time to the temperature of 37° C. produces a slight decrease in boiled and sonicated dECM concentration.
Viability tests were performed on fibroblasts (cell lines 3T3-L1 and HFF-1) and pancreatic islets. For this purpose, pancreatic cells/islets were subjected to pressure in the range from 15 kPa to 100 kPa using a needle with a diameter of 0.2 and 0.6 mm. The results of the tests conducted showed that shear forces induced during 3D bioprinting using extrusion method produce significant changes in cell and microorgan viability.
In order to obtain a viable and functional biological three-dimensional structure, the pressure and diameter of the needle needed to be matching the particular cell type. However, pressures of no more than 30 kPa were preferably applied.
Printouts using primary bioink were made using the following parameters: pressure: 4-100 kPa, printing speed: 5-40 mm/s, temperature: printhead—10-37° C.; printbed—4-37° C., needle diameter: 100 nm-1 mm Printouts using vascular bioink were made using the following parameters: pressure: 5-100 kPa, printing speed: 5-40 mm/s, temperature: printhead—10-37° C.; printbed—4-37° C., needle diameter: 100 nm-1 mm
Lobule
It took approx. 3 minutes to print a pancreatic lobule supplied with a vessel.
Vascularised Three-Dimensional Structure
It took approximately 30 minutes to print a prototype of a bionic pancreas supplied with a network of patent ducts. As in the case of the lobule, a loose arrangement of bioink filaments in the highly porous structure of the printed construct supplied with a network of patent ducts was observed.
The printed vascular system was evaluated using nuclear magnetic resonance imaging. The 3D reconstructions made show patent ducts with no the tendency to collapse or dissect.
An MTT assay on a fibroblast line (3T3) was performed in order to assess cytotoxicity of the primary bioink. The result is presented as % of control at maximum extract concentration [Table 14]. Exposure time to the extract was 24 h and cells of density 1×105/mL were plated. Both assays showed no cytotoxicity to the cell line tested.
In order to assess the effect of the individual components of the primary bioink on the vitality and functionality of pancreatic islets, a glucose stimulation test was performed.
Glycerol
Due to its properties, adding 5% (w/v) and 10% (w/v) glycerol to bioink improved the printability of the primary bioink. In order to assess its effect on pancreatic islet functionality, glycerol was added to culture medium at 5% or 10% concentration and the islets were incubated therein for 24 h [
Commercially Available Protein Supplements
The effect was tested of adding extracellular matrix proteins on the functionality and viability of pancreatic islets. For this purpose, a solution consisting of 0.007 mg/mL hyaluronic acid, 0.041 mg/mL collagen I, 0.122 mg/mL collagen IV and 0.084 mg/mL laminin was prepared, which was added to the culture medium. The experiment was carried out with two types of hyaluronic acid: high molecular weight or low molecular weight, which were added to the culture medium and the islets were incubated therein for 48 h [
GelMA
It was tested how the viability of islets may be affected by methacrylated gelatin which, as a component of the bioink, is to ensure proper cross-linking of the print. For this purpose, 7.8% v/v GelMa was added to the culture medium and the islets were incubated therein for 72 h [
HAMA
It was tested how the viability of islets may be affected by methacrylated hyaluronic acid, which, as a component of the bioink, is to ensure proper cross-linking of the print. For this purpose, 0.78% v/v HAMA was added to the culture medium and the islets were incubated therein for 48 h [
GelMA and HAMA
It was tested how the viability of islets may be affected by a mixture of methacrylated gelatin and methacrylated hyaluronic acid, which, as a component of the bioink, is to ensure proper cross-linking of the print. For this purpose, 4.68% v/v GelMa and 0.312% v/v HAMA (G3:2H) or 3.12% v/v GelMa and 0.468% v/v HAMA (G2:3H) were added to the culture medium and the islets were incubated therein for 72 h [
ECM Powder
It was tested how the ECM obtained by way of decellularization could affect the viability of the islets. For this purpose, 3.33% v/v cut or ground ECM during decellularization was added to the culture medium and the islets were incubated therein for 72 h [
Three bioinks were selected to assess the viability of pancreatic islets following 3D bioprinting: methacrylated gelatin, methacrylated hyaluronic acid, a mixture of methacrylated gelatin and methacrylated hyaluronic acid.
For this purpose, 7.8% v/v GelMa or 0.78% v/v HAMA or a mixture of 4.68% v/v GelMa and 0.312% v/v HAMA (MIX) were added to the primary bioink. After printing, the lobules with islets were incubated in culture medium for 24 h [
The islets in the lobule printed with the primary bioink, which contained an addition of GelMa, showed the highest level of insulin produced after the printing process, thus indicating a favourable effect of this component on the viability and functionality of pancreatic islets. Both the addition of HAMA and the mixture of GelMa and HAMA to the bioink induced a slight decrease in the levels of insulin produced by the islets compared to the control islets grown in the medium (that were not 3D bioprinted). Although the results for the bioink with the addition of GelMa alone showed the highest activity of the islets to the given glucose concentration, the structures printed were the least stable and they were the fastest to disintegrate in the culture medium. Therefore, the best solution was to use a mix of methacrylated gelatin and methacrylated hyaluronic acid for the bioprinting process. This combination allowed for preserving viable and functional pancreatic islets while maintaining proper bioprinting parameters and the stability of the printed model.
In order to visualize and confirm the preservation of the quaternary structure of ECM in the printed construct comprising the primary bioink, a visualization using electron microscopy of protein structures at individual stages of preparation of the dECM (in order to use it in bioprinting) was performed (
| Number | Date | Country | Kind |
|---|---|---|---|
| 19461559 | Jul 2019 | EP | regional |
| 19218191 | Dec 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/056856 | 7/21/2020 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2021/014359 | 1/28/2021 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20160279170 | Katane et al. | Sep 2016 | A1 |
| Number | Date | Country |
|---|---|---|
| 101628821 | Jun 2016 | KR |
| 20180011607 | Feb 2018 | KR |
| 20180125776 | Nov 2018 | KR |
| 2016126947 | Aug 2016 | WO |
| 2017014582 | Jan 2017 | WO |
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| Number | Date | Country | |
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| 20220280694 A1 | Sep 2022 | US |
