Method for decellularization of animal-derived tissue and bioink containing animal-derived decellularized tissue

Abstract

The present invention relates to a method for decellularizing animal tissue, comprising the steps of: mechanically homogenizing animal-derived tissue; incubating the homogenized tissue from step a) in a solution of trypsin and EDTA; applying the filtered tissue to incubation in a PBS buffer solution having a pH in the range of 7.2–7.6 containing a 4–2% concentration of non-ionic detergent and a 0.2–0.08% concentration of NH4OH; washing the tissue with a PBS buffer solution containing an antimicrobial agent; incubating in a deoxyribonuclease solution having a concentration of 0.001 to 0.003% (w / v); and washing the obtained decellularized tissue with a PBS buffer solution containing an antimicrobial agent, wherein the non-ionic detergent is a compound having a molecular weight of 596 g / mol, and the pH containing a 1.5–0.5% concentration of non-ionic detergent and a 0.2–0.08% concentration of NH4OH A bioink containing decellularized tissue of animal origin, comprising an additional step of incubating tissue in a PBS buffer solution of pH 7.2-7.6, said incubation being performed for 36-48 hours at a temperature below room temperature, and a step of incubating tissue in a PBS buffer solution of pH 7.2-7.6 containing a non-ionic detergent at a concentration of 0.75-0.25% and NH4OH at a concentration of 0.2-0.08%, said incubation being performed for 36-48 hours at a temperature below room temperature.

Description

Technology Field

[0001] The present invention relates to a method for obtaining an extracellular matrix from liver tissue. A method for decellularizing the liver comprises the steps of homogenizing the tissue, treating the tissue with an enzyme and a surfactant, and washing the tissue with a washing solution. This method removes almost all detergent from the decellularized matrix without the need to add other chemicals. In a second aspect, the present invention relates to a bioink composition based on a decellularized extracellular matrix having suitable consistency and viscosity. This patent application relates to the field of tissue engineering. Background Technology

[0002] Liver disease causes approximately 2 million deaths worldwide annually, accounting for 3.5% of total deaths (Asrani et al., 2019). Liver transplantation is widely considered the only effective treatment for end-stage failure of the organ (Dutkowski et al., 2011).

[0003] Previous advancements in tissue engineering have made it possible to develop therapeutic solutions for patients with liver failure by producing living tissue pieces used to regenerate damaged organs or functional liver substitutes that can be transplanted into humans. These capabilities highlight the need to develop methods for producing artificial tissues that possess both short-term and long-term utility. Artificial organs—organoids—can also be used in toxicology or virology studies as models to evaluate drug safety (Chu and Lamers, 2024). Existing in vitro liver models are often insufficient to fully reflect the organ's natural characteristics and overall pathophysiology. Therefore, it is necessary to construct in vitro liver models that possess characteristics closer to those of the human liver. The more widespread use of organoids in biomedical research will also limit the use of model animals (McGill and Jaeschke, 2019).

[0004] The major components of an organ are the extracellular matrix (ECM) and the cells embedded within it. In a healthy liver, the extracellular matrix constitutes less than 3% of the organ and is generally localized to Glisson's capsule, the portal tract, and the central vein (Bedossa et al., 2003). As a complex network of hydrated proteins and polysaccharides, it not only provides physical support to cells but, above all, serves as a reservoir for cytokines, growth factors, and bioactive peptides (McQuitty et al., 2020), thereby participating in processes of morphogenesis, cell growth and differentiation, tissue repair, maintenance of tissue homeostasis, and stimulation of the expression of liver-specific functions (Arriazu et al., 2014).

[0005] Recent advances in tissue engineering make it possible to use ECM as a scaffold that functions as a three-dimensional tissue model when filled with cells. One of the major challenges in obtaining artificial livers is maintaining the natural functional properties of these organs in vitro. Several methods exist for obtaining functional artificial organs for application in tissue engineering and regenerative medicine. One of them is 3D bioprinting. Bioprinting enables the automatic embedding of living cells into materials of synthetic origin as well as natural polymers such as collagen, gelatin, alginate, hyaluronic acid, fibrin, and polyethylene glycol, thereby creating three-dimensional tissue structures. Decellularized ECM (dECM) is a promising component of bioinks. Upon solubilization, dECM can be converted into a hydrogel—a highly absorbent material that cross-links at physiological temperatures due to the self-organization mechanism of collagen (Saldin et al., 2017). Hydrogels mimic natural extracellular matrix, possess elasticity similar to natural tissues, and can be engineered to create an environment that promotes cell growth, differentiation, and proper function due to the presence of tissue-specific biochemical signatures of the extracellular matrix. Hydrogels are obtained by the enzymatic digestion of the extracellular matrix in an acidic environment. The digestion solution is typically maintained at room temperature under constant stirring for a specific period. Enzymes such as pepsin, trypsin, or combinations thereof are used. Subsequently, cells are embedded in the structure to produce bioinks that mimic functional tissues. The development of ECM-derived hydrogels opens up the possibility for these hydrogels to be used commercially for cell encapsulation, as in vivo injection materials, or as bioinks for 3D bioprinting applications.

[0006] dECM is obtained by decellularization, which involves the removal of cells and other immunogenic substances, leaving essentially only non-immunogenic structural materials of the extracellular matrix (collagen, elastin, laminin, etc.). Since inflammation caused by other immunogenic factors can negatively affect the success of graft acceptance, the decellularization method must ensure that as many cells as possible are removed from the tissue.

[0007] Different tissue or organ types require different decellularization protocols depending on their structure and ECM composition. The desired decellularization process begins with cell lysis and the separation of nuclear and cytoplasmic components from the ECM using physical, enzymatic, chemical methods, or a combination thereof. Typical decellularization methods involve a series of tissue washes in chemical solutions containing various substances to assist the process. To date, the most commonly used have been non-ionic detergents (Triton X-100), anionic detergents (sodium dodecyl sulfate-SDS, sodium deoxycholate-SD), enzymatic agents (trypsin, DNase I), or chelating agents such as ethylenediaminetetraacetic acid (EDTA). These solutions may contain one or more compatible surfactants (e.g., anionic surfactants such as SDS and non-ionic surfactants such as Triton X-100). Sodium dodecyl sulfate and Triton X-100 are the most commonly used chemicals in liver decellularization procedures (Pan et al., 2014; Mazza et al., 2015). Surfactants act by disrupting phospholipid cell membranes, thereby causing cell lysis (Parsi, 2015). Generally, organ perfusion or a mixture of organ fragments in a solution is used to allow detergents and enzymes to penetrate deep into the tissue. The substrate for cell disruption is an aqueous solution of at least one detergent, or alternatively, said detergent dissolved in a buffer solution (e.g., PBS-phosphate-buffered saline) to achieve osmotic compatibility with the cells. Non-ionic detergents are preferred due to their milder effect on tissue structure. They are considered relatively non-denaturing because they affect lipid-lipid and lipid-protein interactions rather than protein-protein interactions (Seddon et al., 2004). Ionic detergents have a greater negative impact on tissue structure and are also more difficult to remove with typical neutral solutions.Enzymes such as proteases, esterases, or nucleases are also used. Some procedures use biological load-reducing agents including penicillin, streptomycin, peracetic acid, ethanol, or combinations thereof. After decellularization, all cellular elements and residual chemicals must be thoroughly washed from the scaffold with a neutral solution such as PBS.

[0008] Decellularized tissue can be treated with stabilizing compounds such as glutaraldehyde to cross-link and stabilize the components of the decellularized tissue. Decellularized materials must undergo sterilization treatment such as radiation.

[0009] To date, the use of Triton X-100 and SDS in liver decellularization has been described several times. Triton X-100, one of the most commonly used detergents for this purpose, was added to the European Chemicals Agency (ECHA) Candidate List as a substance of concern due to its degradation into an endocrine disruptor (ECHA, 2022).

[0010] There are several protocols describing liver decellularization, including a U.S. patent application in which tissue undergoes multiple freeze-thaw cycles and is washed with PBS solution US20230016722A1 This is included. Next, the tissue is incubated with a detergent such as SDS, SDC, or Triton X-100. Then, the tissue may be treated with a DNase solution with streptomycin. The literature does not disclose the use of Tergitol for hepatic decellularization.

[0011] Another U.S. patent holder US11648335B2The patent discloses a method for decellularizing liver tissue comprising perfusing the tissue with an ionic or non-ionic detergent and then washing it with a buffer solution. Additionally, treating the tissue with a solution of DNase and Betadine is provided. This patent does not disclose the use of Tergitol for decellularization.

[0012] EP3027235A1 This is a European patent application disclosing a method for decellularizing liver tissue by treating the tissue with a solution that is hypertonic or hypotonic with respect to the interior of the cell. Such solutions also contain emulsifiers or surfactants. Additionally, such solutions may contain trypsin, protease inhibitors such as EDTA, and antibiotics. This document does not disclose the use of Tergitol.

[0013] A study by Hussein et al. (2024) is a review comparing 25 liver decellularization methods of various origins, but none of the cited papers mention the use of Tergitol.

[0014] To date, Tergitol has been used for the successful decellularization of porcine aortic valves (Faggioli et al., 2022), porcine pulmonary artery valves (Tondato et al., 2023), porcine pericardium (Todesco et al., 2022) and small intestinal submucosal tissue (Casarin et al., 2022).

[0015] EP3095469A1 It describes the use of Tergitol for decellularization, but does not disclose a method for decellularization of liver tissue.

[0016] On the other hand, patents WO2011057174A1 This application discloses that an exemplary detergent used for the decellularization of liver tissue may be Tergitol. This application does not disclose the use of Tergitol at stepwise gradient concentrations.

[0017] Tergitol also filed EP3328992 It was mentioned in [source]. It was used for liver decellularization in combination with other selective decellularization reagents. For example, an enzyme containing trypsin with EDTA was claimed. Samples were washed with PBS, but again, the method for preparing the decellularized scaffold does not reveal the use of Tergitol at stepwise concentration gradients for the decellularization of porcine liver.

[0018] also, WO2023154873A3 The document discloses a method for obtaining extracellular matrix isolated from amphibians. This process involves washing a tissue sample with a detergent followed by a buffer solution, and incubating the sample with at least one protease and nuclease to obtain the isolated ECM. An example of a buffer solution may be PBS. The detergent used may be Tergitol. Streptomycin is listed as an exemplary antibiotic, and the sample enzymes may be trypsin and DNase. The document does not disclose a method for decellularizing liver tissue.

[0019] On the other hand, an international patent application describing a bioink composition for 3D printing WO2023235832A1 In this study, a hyaluronic acid derivative was used as a cleavable polymer precursor, and methacrylated gelatin was presented as a non-cleavable polymer precursor. LAP was used as an exemplary photoinitiator. These bioinks do not contain decellularized dECM, and sonicated bioinks are not used.

[0020] The solution described below CN115634316B In this case, acetic acid and pepsin are added to powdered dECM obtained from dental follicle tissue, followed by the addition of NaOH. The dECM thus produced and dissolved is mixed with GelMA.

[0021] EP4003439A1 This is a European patent application describing a method for manufacturing bioink.

[0022] The first step in the manufacture of the bioink is to obtain a solution of dECM. This procedure involves dissolving dECM powder in pepsin and hydrochloric acid. The solution is neutralized with NaOH and PBS. Then, the dECM powder and the solution are mixed to produce a paste, which is subsequently mixed with GelMA, HAMA, LAP, glycerol, growth factors, vitamins, cells, and islets of Langerhans. This solution provides dECM sonication. In relation to the described application, there are differences in the proportions of the bioink components, one of which is glycerol, which is not present in the tested composition. HepG2 cells were also not used.

[0023] EP4175688A1 is another European patent application disclosing a composition of a biocompatible hydrogel that may contain dECM, GelMA, HAMA, and LAP. This document does not disclose instructions for a decellularization method and a method for producing a bioink using the obtained dECM.

[0024] on the other side, CN109054496A A Chinese patent application discloses the manufacture of a bioink composed of a cell-free substrate hydrogel, a crosslinking curing agent, and a bioactive molecule. As crosslinking curing agents, PEG polyether esters, collagen, gelatin, chitosan, agar, hyaluronic acid, or products of the above materials modified with unsaturated double bonds are listed. The method according to this invention does not produce an ultrasonically treated hydrogel.

[0025] The subject of the present invention is a method for enzyme-detergent decellularization of liver, particularly porcine liver tissue, using Tergitol 15-S-9, a less toxic and biodegradable non-ionic detergent having a molecular weight of 596 g / mol, in a stepwise concentration gradient.

[0026]

[0027] Tergitol 15-S-9 structural formula.

[0028] The object of the present invention is to develop a composition for a biomaterial containing dECM obtained by decellularizing animal liver and enriching it with other factors that improve its functional properties. Additionally, the goal was to develop a method for producing a biomaterial having unique properties in a manner that satisfies the following conditions:

[0029] - Ensuring optimal rheological properties of the developed liver bioink: viscosity in the range of 130–1700 mPa-s, storage modulus (G') 2–130 Pa, and loss modulus (G") 2–15 Pa

[0030] - Guaranteeing optimal mechanical parameters for objects printed with the developed bioink: mechanical strength in the range of 50-650 kPa, elasticity of 100-200 kPa

[0031] - Ensuring adequate fiber integrity to enable continuous printing from 2-3 ml of material while maintaining printing parameters safe for cell materials: temperature 5-35℃, pressure 5-40 kPa, printing speed 5-45 mm / s.

[0032] The present invention has the following steps:

[0033] a) Mechanical homogenization step of tissues of animal origin,

[0034] b) The homogenized tissue from step a) was treated in a solution of trypsin at a concentration of 0.005-0.5% and EDTA at a concentration of 0.002-0.2% for 1-3 hours 37 ℃ The incubation stage,

[0035] c) a step of removing trypsin and EDTA solutions and incubating the filtered tissue in a PBS buffer solution of pH 7.2-7.6 containing 4-2% non-ionic detergent and 0.2-0.08% NH4OH, wherein the incubation is performed for 24-48 hours at a temperature below room temperature.

[0036] d) a step of washing the tissue with a PBS buffer solution containing an antimicrobial agent, wherein the washing is performed at a temperature below room temperature for at least 72 hours,

[0037] e) 37 for 8 hours in a 0.001 to 0.003% (w / v) deoxyribonuclease solution ℃ The incubation stage,

[0038] f) a step of washing the decellularized tissue obtained in steps a) to e) with a PBS buffer solution containing an antimicrobial agent, wherein the washing is performed at a temperature below room temperature for at least 72 hours.

[0039] Includes,

[0040] Here,

[0041] - The above non-ionic detergent is a compound having a molecular weight of 596 g / mol of the following chemical formula:

[0042]

[0043] - Following step c) above, the following additional steps:

[0044] c1) a step of incubating tissue in a PBS buffer solution having a pH in the range of 7.2–7.6 containing 1.5–0.5% concentration of non-ionic detergent and 0.2–0.08% concentration of NH4OH, wherein the incubation is performed for 36–48 hours at a temperature below room temperature, and

[0045] c2) a step of incubating tissue in a PBS buffer solution having a pH in the range of 7.2-7.6 containing a non-ionic detergent at a concentration of 0.75-0.25% and NH4OH at a concentration of 0.2-0.08%, wherein the incubation is performed for 36-48 hours at a temperature below room temperature.

[0046] It follows.

[0047] In a favorable way, the tissue of animal origin is liver tissue.

[0048] Advantageously, during incubation in steps c), c1), and c2), the PBS buffer solution containing non-ionic detergent and NH4OH is exchanged three times.

[0049] Advantageously, steps c), c1), and c2) are performed at 4°C.

[0050] Advantageously, the antimicrobial agent is streptomycin at a concentration of 0.01% (w / v).

[0051] Advantageously, during washing in step d), the PBS buffer solution containing the antimicrobial agent is exchanged 8 times.

[0052] Advantageously, during washing in step f), the PBS buffer solution containing the antimicrobial agent is exchanged 5 times.

[0053] In a second aspect, the present invention relates to a bioink containing decellularized tissue of animal origin at a concentration of 0.5-10% (w / v), preferably 5% (w / v).

[0054] Advantageously, the bioink comprises 0.5-2% (w / v) of HAMA solution, 5-20% (w / v) of GELMA solution, and 0.15-0.5% (w / v) of LAP solution. Brief explanation of the drawing

[0055] The present invention is illustrated in the drawings, wherein: Figure 1 shows the appearance of the liver before and after decellularization. Figure 2 illustrates the electrophoretic separation of DNA on a 1% agarose gel. No. 12 is DNA isolated from the ECM obtained by decellularization in a Tergitol gradient. Figure 3 shows cells after exposure to bioink extracts prepared with 10 different variants. K- (negative control), K+ (positive control). Figure 4 shows the temperature dependence of the elastic modulus for liver bioink. Figure 5 shows the results of the viscosity test of the liver bioink material. Figure 6 shows the dependence of storage modulus and loss modulus of liver bioink on shear stress. Figure 7 is a schematic diagram of a g-code file. template.gcode Figure 8 is a schematic diagram of a fiber breakdown test platform. Figure 9 shows the fiber diffusion rate Dfr It is a percentage-fiber fusion test. Fig. 10 shows printability Pr- It is a fiber fusion test. Figure 11 is a photograph of the structure in a fiber collapse test. Figure 12 is a fiber decay rate test of Cf-fiber decay. Specific details for implementing the invention

[0056] Examples

[0057] Example 1 Decellularized liver ECM

[0058] ingredient

[0059] Pork livers were obtained from a local slaughterhouse and stored at -20℃. The trachea was frozen in advance, thawed, washed with Betadine solution (2% v / v), and then [treated] with streptomycin solution (0.1 mg * ml) dissolved in PBS. -1 It was cleaned with ). After cleaning, the organ was fragmented using scissors while visually removing visible fat, blood vessels, and other contaminants.

[0060] Quantitative results are presented as mean ± standard deviation. All analyses were performed in triplicate.

[0061] Tergitol with a molecular weight of 596 g / mol TM 15-S-9, CAS: 84133-50-6

[0062] Method for decellularizing liver tissue

[0063] Activities specified to be performed at temperatures below room temperature are preferably performed at a temperature of 4°C.

[0064] The fragmented organs were mechanically fragmented using a jug blender. The fragmented and mixed material was weighed and placed in a bottle, and incubated for 1.5 hours at 37°C in a trypsin-EDTA solution (0.05% (w / v) trypsin (Gibco, USA) and 0.02% (w / v) EDTA (Sigma Aldrich, USA)) with an antimicrobial agent, preferably at a concentration of 0.01% (w / v). After this time, the material from the bottle was transferred to a stainless steel sieve and filtered.

[0065] The unfiltered material was poured back into the bottle. The minced liver tissue was immersed in a 3% solution of Tergitol 15.S.9 (Sigma-Aldrich, USA), a non-ionic detergent, in PBS solution of pH 7.4 and 0.1% NH4OH. The bottle containing the material was placed in an incubator with a stirring function at 4°C at 150 rpm for 24–48 hours, preferably 17–19 hours. After this time, the decellularization solution was replaced 1–3 times per day for 12 days. After replacing the 3% Tergitol solution three times, the detergent concentration was changed to 1% Tergitol + 0.1% NH4OH in PBS. After replacing the 1% solution four times, the next four replacements were performed using 0.5% + 0.1% NH4OH in PBS. At each replacement of the decellularization solution, the material was fragmented with scissors. Tissue purification is performed using a neutral wash buffer, namely streptomycin (0.1 mg * ml) -1 Ca with ) added 2+ and Mg 2+ The procedure was performed using PBS without [unclear]. After changing the washing solution 9 times, the decellularized tissue was ground using a blender. Then, to remove DNA fragments dispersed after detergent treatment, Ca [unclear] was mixed at a speed of 150 rpm at a temperature optimal for DNase efficiency for 8 hours. 2+ and Mg 2+Washing was performed with a solution of nuclease-DNase I dissolved in PBS to which was added. After incubating the material in the DNase I solution for 8 hours, the material was filtered through a stainless steel strainer and returned to the bottle. Then, the tissue was treated with streptomycin (0.1 mg * ml -1 Washing was performed again with PBS containing ) three times a day at 3-hour intervals for an additional 2 days. The single decellularization process takes 12 days (the procedure diagram is provided in Table 1).

[0066] Table 1. Table showing an overview of the decellularization procedure (12th)

[0067] Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 -Material Processing- Incubation with trypsin (0.05%) and EDTA (0.02%) in PBS (37℃, 1.5h)- Incubation in 3% Tergitol solution in PBS (4℃) - 3x washing in 3% Tergitol solution in PBS (4℃) - 3x washing in 1% Tergitol solution in PBS (4℃) - 1x washing in 1% Tergitol solution in PBS (4℃) - 2x washing in 0.5% Tergitol solution in PBS (4℃) - 2x washing in 0.5% Tergitol solution in PBS (4℃) - 1x washing in PBS containing streptomycin (4℃) - Wash 1x in PBS containing streptomycin (4℃) - Wash 1x in PBS containing streptomycin (4℃) Day 8 Day 9 10th day Day 11 Day 12 - Wash 3 times in PBS containing streptomycin (4℃) - Wash 3 times in PBS containing streptomycin (4℃) - Incubation with DNAase (10 mg / l) in PBS (37℃, 8 h). - 1x washing in PBS + strep. (4℃) - 3x cleaning in PBS + strep (4℃) - 1x washing in PBS + strep. (4℃) - Pooling - Sedimentation of material in liquid nitrogen (preparation for freeze-drying)

[0068] After decellularization, the material was prepared for freeze-drying. A small amount of tissue was crushed against the wall of a mortar cooled with liquid nitrogen. The dECM was crushed into small fragments and stored at -80°C until freeze-drying. Then, the material was freeze-dried. The obtained freeze-dried material was ground in a cryogenic grinder to obtain a powder having a particle diameter of less than 100 μm. The material was subjected to radiation sterilization (25 kGy).

[0069] DNA from powdered dECM was isolated using the Qiamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. Briefly, the samples were digested with proteinase K and digestion buffer. The samples were applied to a column for DNA uptake. After a washing step, the DNA was precipitated and eluted. DNA concentration was measured by spectrophotometry (Nanodrop, Thermo Scientific, USA). The average DNA content per mg of tissue was determined. Electrophoresis was performed on a 1% agarose gel to separate and determine the size of DNA fragments in dECM.

[0070] The results of the experiment formed the basis of the present invention. It was shown that after the decellularization process, the color of the tissue changed from the initial red-pink color of natural liver tissue to white. Simultaneously, the removal of cellular components was detected based on DNA content. The DNA concentration, or step gradient, in the decellularized powdered tissue using three different concentrations of Tergitol was 13.73 ng / mg ± 2.76. This indicates that more than 99% of the natural tissue DNA was removed, and the DNA content of the natural tissue was estimated to be 4646.2 ± 339.04 ng / mg. DNA analysis by agarose gel electrophoresis (Fig. 3) confirmed the removal of DNA to levels below a detectable threshold. Crapo et al. proposed a minimum standard for DNA residue that the authors believe would be sufficient to confirm the effect of the decellularization process. The proposed criteria suggest that cell-free dECM should contain less than 50 ng of dsDNA per mg of dECM (dry weight) and that the fragments should be smaller than 200 pz (Crapo et al., 2011). According to the results, the reproducibility of this technique can be observed as it adheres to established DNA detection limits.

[0071] fat measurement

[0072] Soxhlet extraction was used to measure the free fat content. 0.5 g of liver powder (dry weight) was placed in a cellulose thimble, covered with cotton wool, and weighed. The lipids were extracted with 70 ml of n-hexane for 4 hours with a cycle time of approximately 10 minutes. The weight content of the extracted lipids relative to the weight of the test sample was determined.

[0073] result

[0074] The fat content of the natural organ averaged 18.03% ± 7.78 and showed significant variability. The method described above reduced the percentage of fat to 1.46% ± 1.16. An extracellular matrix with a lower fat content improves the characteristics of the final product, namely increasing viscosity and improving printability.

[0075] Measurement of sulfated glycosaminoglycan (sGAG) content

[0076] The content of sulfated glycosaminoglycans (sGAGs) in natural cryogenic-ground tissues and acellular tissues was quantified using the Blyscan GAG Assay Kit (Biocolor, UK). Briefly, 20–50 mg of dried tissue was weighed, placed in a 1.5-ml tube containing 1 ml of papain solution, and incubated at 65°C for 16 hours with intermittent stirring. A portion of each sample was mixed with 1,9-dimethylmethylene blue dye and the reagents of the kit. Absorbance at 565 nm was measured using a microplate reader and compared to a standard prepared with sochondroitin sulfate to determine the absolute sGAG content.

[0077] result

[0078] Glycosaminoglycans were present in all natural tissue samples, but their content decreased dramatically after decellularization. In the liver, sGAG content decreased from 7.91 µg / mg ± 1.07 to 0.47 µg / mg ± 0.12. Decellularization or matrisome reinforcement methods result in a significant loss of soluble molecules associated with the extracellular matrix in various organs (Krasny et al., 2016). The effect of sGAG deficiency on the durability of transplanted decellularized scaffolds requires further study.

[0079] Measurement of collagen content

[0080] The quantitative determination of collagen was performed using a commercially available fluorescence assay (Collagen Assay Kit, Sigma-Aldrich, USA). A sample of powdered substrate dissolved in 0.5 M acetic acid was applied to a 96-well plate. Then, acid-soluble collagen was enzymatically digested into peptides. Subsequently, peptides containing glycine at the N-terminus reacted with a dye to form a fluorescent complex. The fluorescence intensity of the product, measured at λex / em = 375 / 465 nm, was directly proportional to the concentration of collagen in the sample.

[0081] result

[0082] The decellularization method has a smaller effect on collagen content, and the preservation of these key components was confirmed in dECM. Approximately 10-fold concentration of collagen was observed in the final material compared to natural tissue (654.3 ug / mg ± 74 vs. 58.6 ± 38.6).

[0083] Study of cell viability in contact with dECM and bioinks produced based on these components

[0084] The MTT assay was used to evaluate cell viability, which is based on the reduction of yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-MTT) to purple formazan crystals by metabolically active cells. L-929 cell line (ATCC) cultured in DMEM containing 10% FBS and 1% antibiotics ® , cat no: CCL-1 TMFresh bioinks of 10 variants were placed into inserts in the culture medium according to the principle of 0.1 g of cross-linked bioink per 1 ml of DMEM. The extracts were incubated in complete culture medium at 37°C for 24 hours. The extracts from beneath the inserts were applied to the cells. The plates were incubated at intervals of 24, 48, and 72 hours. Then, the MTT solution was removed and 100 μl of DMSO was added. The plates were shaken until the crystals were completely dissolved. Absorbance was measured at 570 nm and 650 nm. The positive control was a culture incubated with 0.1% Triton X-100.

[0085] result

[0086] Cell viability for L-929 cells was greater than 70% for all bioink extracts and at all time points (24, 48, and 72 hours), which means that the obtained dECM-based bioink is capable of maintaining high cell viability. Figure 3 shows L-929 lineage cells after exposure to biomaterial extracts.

[0087] Detergent content

[0088] The residual detergent content, i.e., Tergitol, was determined using a UHPLC / HPLC Ultimate 3000RS system (Thermo Scientific) equipped with a Corona CAD corona discharge detector. Separation was achieved on an Acclaim C8 120A column (150 x 4.6 mm; 3 μm) (Dionex) at a column temperature of 30°C. Mobile phase A was 0.1% HCOOH, and mobile phase B was methanol.

[0089] result

[0090] The concentration of non-ionic detergent in the final product was low, less than 5 ng / mg. This was achieved by repeatedly washing the tissue in a neutral PBS solution. Residual detergent content is important when these materials (dECM) are to be used in the bioprinting process with living cells. Although Tergitol is considered less toxic, it is still important to minimize detergent residue in the final product as much as possible.

[0091] The efficiency of the decellularization method is 0.22% ± 0.096, which is attributed to the highly cellular nature of liver tissue. After the process is completed, the decellularized tissue section can reduce its weight by more than 99%.

[0092] Example 2 Bioink

[0093] Preparation of bioink based on decellularized liver ECM

[0094] Preparation of 8 ml of liver dECM hydrogel 6.5% (w / v) - not sonicated.

[0095] 195.2 mg of pepsin was transferred to 30 ml of 0.1 M HCl solution. The solution was stirred at 500 rpm on a magnetic stirrer at room temperature until the enzyme was dissolved (approximately 20 minutes). Next, 1951.2 mg of powdered decellularized liver tissue was transferred to the pepsin solution. The solution was placed in a metal bath at 23°C on a magnetic stirrer at 500 rpm for 48 hours. After 48 hours of continuous stirring, the resulting hydrogel was neutralized. To do this, 3.33 ml of PBS x 10, 0.7 ml of 1 x PBS, and 0.6 ml of 5 M NaOH were added to the hydrogel. The pH of the solution was stabilized to 7.4 with NaOH. The solution after neutralization was used to produce liver bioink and sealing bioink.

[0096] Preparation of dECM hydrogel 5% (w / v)

[0097] 500.4 mg of liver dECM was weighed into a sterile Falcon. Then, 10 ml of PBS x 1 was added to the weighed portion. The Falcon containing the suspension was placed in a thermoblock at 23°C and 500 rpm. Next, the Falcon containing the hydrogel was placed in an ice bath and sonicated for 5 minutes at approximately 22°C and 60% amplitude. Before use, the hydrogel was heated in a thermoblock at 37°C with stirring at 800 rpm to liquefy and degas the hydrogel.

[0098] Preparation of 15 ml of GELMA 20% (w / v) + LAP 0.38% (w / v) solution

[0099] 78.1 mg of LAP was weighed into 20.0 ml of PBS x 1. The solution was stirred at 500 rpm at 50°C for about 15 minutes until the photoinitiator dissolved. Then, 4.0021 g of lyophilized GELMA was added to the solution. The solution was placed in a water bath (50°C) until completely dissolved, and the stirring speed was adjusted according to the degree of foaming (100-200 rpm). The prepared solution was filtered using a syringe filter with a pore diameter of 0.22 μm. The filtered solution was stored in a refrigerator protected from light.

[0100] Preparation of 10.0 ml of HAMA 2% (w / v) solution

[0101] A portion of 201.8 mg of lyophilized HAMA was weighed into a 10.0 ml PBS x 1 Falcon. The solution was shaken in a thermoblock at 1000 rpm and 8°C for 1 hour. After complete dissolution, the pH was stabilized to 7.35 with NaOH and HCl. Then, the solution was filtered using a syringe filter (0.22 μm). The filtered solution was stored in a refrigerator.

[0102] Preparation of 8 ml of GELMA 20% (w / v) + LAP 0.6% (w / v) solution

[0103] 60.7 mg of LAP was added to 10 ml of PBS x 1. The solution was transferred to a thermoblock (until the photoinitiator dissolved) and heated at 500 rpm and 50 for approximately 15 minutes. ℃The mixture was stirred. Next, 2.0016 g of freeze-dried gelma was added to the solution. The mixture was heated to a thermoblock (50°C) until the entire solution was completely dissolved, and the stirring speed was selected based on the degree of foaming (300-500 rpm). After the gelma was completely dissolved, the solution was filtered using a syringe filter (0.22 μm). The filtered solution was stored in a refrigerator protected from light.

[0104] Preparation of 10 ml of HAMA 2% (w / v) + LAP 0.5% (w / v) solution

[0105] 50.6 mg of LAP was added to 10.0 ml of PBS x 1. The solution was transferred to a thermoblock and mixed at 500 rpm and 50°C for 15 minutes (until the photoinitiator was dissolved). Then, 201.8 mg of lyophilized HAMA was added to the solution and shaken in a thermoblock at 1000 rpm and 8°C for 1 hour. After complete dissolution, the pH was adjusted to 7.3 using NaOH and HCl. Next, the solution was filtered using a syringe filter (0.22 μm). The filtered solution was stored in a refrigerator.

[0106] Preparation of cell-added liver bioink

[0107] 2 ml of unsonicated hydrogel was taken using a syringe fitted with a needle. A syringe without a Luer-lock adapter and plunger was attached to the syringe containing the hydrogel, and 2.5 ml of HAMA solution and 5.0 ml of GELMA + LAP 0.38% solution were added. The entire mixture was mixed between syringes and transferred to a thermoblock set to 12°C. This procedure was repeated the next day. Then, 150–180 µl of HepG2 cell suspension (2–12 million cells per ml of bioink) was added to the above portion of the bioink and mixed between syringes. This bioink can be transferred to a sterile cartridge and placed on a printer head.

[0108] Cells were counted using an automated cell counter. Then, 2 to 12 million cells per ml of bioink were suspended in the culture medium and filled to 0.2 mL.

[0109] Preparation of liver sealing bioink

[0110] 4.5 ml of liver hydrogel was placed in a syringe, and then 2.7 ml of HAMA 2% (w / v) + LAP 0.5% (w / v) solution and 1.8 ml of GELMA 20% (w / v) + LAP 0.6% solution were added to it. The entire mixture was combined to obtain a homogeneous mixture of the components. Until use, the bioink was stored at room temperature in a light-protected environment.

[0111] Preferably, the bioink composition contains decellularized tissue of animal origin at a concentration of 0.5-10% (w / v), preferably 5% (w / v), and additionally contains a 0.5-2% (w / v) HAMA solution, a 5-20% (w / v) GELMA solution, and a 0.15-0.5% (w / v) LAP solution.

[0112] printability

[0113] The printability test bioprinter It was performed using [the tool / tool]. The printing parameters using liver biomaterial are Table 2It is included in [the document]. The applied parameters enabled the acquisition of uniform and dense fibers. The evaluation of the printability of the given material was performed according to a procedure specifically developed based on a literature review (Ahasan Habib, Venkatachalem Sathish, Sanku Mallik, Bashir Khoda, 3D Printability of Alginate-Carboxymethyl Cellulose Hydrogel Materials (Basel) 2018 Mar 20;11(3):454. doi: 10.3390 / ma11030454). As part of the work performed, a number of tests were conducted, in which the following parameters were evaluated: material diffusion rate—percentage of diffusion rate, printability, decay coefficient of the printed fiber, and fiber continuity and smoothness.

[0114] Table 2 . Printing parameters in printability tests of pre-crosslinked materials

[0115] deformed body Pressure range [kPa] Temperature [℃] Speed ​​[mm / s] Liver bioink 5-45 5-35 10-35

[0116] The presented technology involves extrusion printing, which requires appropriate viscosity of the material to obtain uniform and cohesive filaments to maintain print resolution. The printing temperature can vary from 5°C to 37°C depending on the material used. For extrusion printing, the printing pressure is in the range of 5 to 200 kPa. The printing speed is maintained in the range of 1 to 50 mm / s. During printing, a needle with a diameter of 50 to 900 μm is used. The 3D model designed for printing can feature any geometry tailored to the needs of the experiment as well as a fill level in the range of 5% to 100%, thereby obtaining an openwork print.

[0117] Fiber fusion test

[0118] For fiber fusion testing, an appropriate g-code is template.gcode A was prepared, assuming the sequential printing of two layers using the tested material without using external ramp crosslinking between them. Prints were fabricated using a bioprinter. The prints followed a pattern of 0°–90° alignment, which reflects a 2D effect and increases the distance between fibers. The distance between fibers ranged from 1–5 mm in increments of 1 mm. The template pattern is illustrated in Fig. 7. The printing speed, needle diameter, and extrusion width used in the test were, respectively 10 mm / s, 25G (0.250 mm) and 0.3 mm During the test, the material was administered at an appropriate pressure and temperature range tailored to the material being tested. The prints were crosslinked using an external UV-Vis lamp (Polbionica, Warsaw) with the following parameters: Wavelength 405 nm, time 30 sec, output 28.5 mW / cm² 2 After printing, microscopic images were taken. The images were analyzed using AxioVision software. Based on the results, two parameters described by the following equation, namely, the diffusivity Df r Percentage of (material diffusion rate) and printability Pr ...was determined. The pore diffusion rate without material diffusion is 0 (i.e., At = Aa), and for the ideal reproduction of the model, the printability is 1.

[0119]

[0120]

[0121] A t - Theoretical pore area,

[0122] A a - Actual porous area,

[0123] L - Actual pore circumference.

[0124] Fiber breakdown test

[0125] To determine the degree of material collapse, collapse at the center of the suspended fibers was analyzed. To conduct the experiment, a special platform consisting of seven pillars spaced apart by known distances of 1, 2, 3, 4, 5, and 6 mm was designed. The dimensions of the five pillars placed inside the structure were 2 × 10 × 6 mm. 3 And, the dimensions of the two edge posts are 5 × 10 × 6 mm 3 is. The platform design is illustrated in Fig. 8. A single fiber of the tested material is g-coded MRoctest1.gcode After depositing it on the platform according to [the method], a photograph of the printout was taken immediately. The photograph was analyzed using AxioVision software. During the test, temperature and pressure conditions were adjusted according to the material being tested. The printout 25 G (0.250 mm) Using a needle 10 mm / s It was produced at a speed of . Collapse area coefficient Cf is the suspended fiber with respect to the theoretical area breakdown It is a percentage of the actual area after.

[0126]

[0127] A a c - Theoretical area under the curve,

[0128] A t c - Actual area under the curve.

[0129] result:

[0130] Fiber fusion test

[0131] ● For each material variant, 3 attempts at fiber fusion testing ...was performed. Next, microscopic images of the obtained structures were taken. Based on the measurements, the fiber diffusivity Dfr Percentage and printability Pr was calculated, and the result is Figures 9 and 10This is presented in the graph. The liver bioink features continuous and dense fibers, which enabled the production of prints with excellent resolution, and

[0132] ● Using the tested material, it was not possible to print a 1x1 pore, and

[0133] ● For each material variant, not only was a printability greater than 0.8 obtained, but a diffusion rate percentage of less than 75% was obtained (excluding pores of size 1x1), and

[0134] ● The diffusion rate decreases as pore size increases, and

[0135] ● The printability for each pore size, excluding 1x1 pores, is 0.88-0.98 for all materials.

[0136] Fiber breakdown test

[0137] For each material variant, 3 fibers breakdown The test was performed. Then, a photograph of the obtained fiber printed on the platform was taken ( Fig. 11 Based on the obtained measurements, the fiber breakdown coefficient Cf was calculated, and the result is Fig. 12 It appears in.

[0138] The fiber breakdown coefficient shows a value of over 80%.

[0139] Rheology:

[0140] To perform rheological studies, 4.5 ml of liver bioink was prepared and placed in a thermoblock at 35°C (at least 15 minutes before starting the measurement).

[0141] Rheological measurements were performed using an MCR 72 instrument (Anton Paar, Poland). The following relationship was used to determine the desired rheological parameters:

[0142] ● Dependence of complex elastic modulus on temperature gradient in the 40-0℃ range - Gelation point of material.

[0143] ● Dependence of complex elastic modulus on shear stress and strain.

[0144] ● Change in viscosity values ​​during measurements at a constant temperature of 20°C at a shear rate of 100 / s. Detailed measurement conditions are presented in Table 3:

[0145] Table 3 Measurement parameters

[0146] parameters Gelation point Complex elasticity viscosity Temperature [℃]: 40-0 20 20 Shear rate [1 / s] - - 100 Frequency [Hz] 1 0.01-10 - Amplitude [%] 30 10 - Spindle [-] CP50 Gap (mm) 0.112 Sample volume [ml] 0.49

[0147] Gelation point

[0148] Figures 4 and 5 illustrate the dependence of storage modulus and loss modulus on a temperature gradient for liver bioink.

[0149] The gelation temperature of the liver bioink was determined. The results are shown in Figure 4, and the average values ​​of the gelation temperatures of the materials are listed in Table 4.

[0150] Table 4 Gelation temperature of liver bioink.

[0151] repeat I II III IV T [℃] 18.81 19.25 18.81 -

[0152] Liver bioink has a gelation temperature in the range of 18.81-19.25℃.

[0153] ● Viscosity

[0154] Figure 5 shows the results of a viscosity test of a material at a constant temperature of 20°C at a constant shear rate of 100 / s.

[0155] Table 5 shows the average viscosity values ​​of the tested materials.

[0156] Table 5 Average viscosity value of the tested material

[0157] repeat I II III η [mPas] 225.23 (±78.56) 235.69 (±80.4) 178.56 (±58.25)

[0158] During the test, the viscosity of all materials increases over time.

[0159] ● Complex modulus

[0160] Figure 6 illustrates the dependence of storage modulus and loss modulus on shear stress for the tested material.

[0161] All tested materials are characterized by a storage modulus G' having a value higher than the loss modulus G', indicating the dominance of elastic properties over the viscous properties of the materials. In both cases of the tested materials, no points of sample deformation were identified under the influence of applied shear stress.

[0162] References

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[0167] Casarin, M., Fortunato, TM, Imran, S., Todesco, M., Sandrin, D., Borile, G., Toniolo, I., Marchesan, M., Gerosa, G., Bagno, A., Romanato, F., Carniel, EL, Morlacco, A., & Dal Moro, F. (2022). Porcine Small Intestinal Submucosa (SIS) as a Suitable Scaffold for the Creation of a Tissue-Engineered Urinary Conduit: Decellularization, Biomechanical and Biocompatibility Characterization Using New Approaches. International journal of molecular sciences, 23(5), 2826.

[0168] Chu, JTS, Lamers, MM Organoids in virology. npj Viruses 2, 5 (2024).

[0169] Dutkowski, P., Oberkofler, CE, Bechir, M., Mullhaupt, B., Geier, A., Raptis, DA, & Clavien, PA (2011). The model for end-stage liver disease allocation system for liver transplantation saves lives but increases morbidity and cost: a prospective outcome analysis. Liver transplantation: official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, 17(6), 674-684.

[0170] European Chemical Agency. Inclusion of Substances of Very High Concerns in the Candidate List 2021. [(Accessed February 21, 2022)]. Available online: https: / / echa.europa.eu / it / candidate-list-tablec

[0171] Faggioli, M.; Moro, A.; Butt, S.; Todesco, M.; Sandrin, D.; Borile, G.; Bagno, A.; Fabozzo, A.; Romanato, F.; Marchesan, M.; et al. A New Decellularization Protocol of Porcine Aortic Valves Using Tergitol to Characterize the Scaffold with the Biocompatibility Profile Using Human Bone Marrow Mesenchymal Stem Cells. Polymers 2022, 14, 1226.

[0172] Hussein, KH, Ahmadzada, B., Correa, JC, Sultan, A., Wilken, S., Amiot, B., & Nyberg, SL (2024). Liver tissue engineering using decellularized scaffolds: Current progress, challenges, and opportunities. Bioactive materials, 40, 280-305.

[0173] Mazza, G., Rombouts, K., Rennie Hall, A. et al. Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation. Sci Rep 5, 13079 (2015).

[0174] McGill MR, Jaeschke H. Animal models of drug-induced liver injury. Biochim Biophys Acta Mol Basis Dis. 2019 May 1;1865(5):1031-1039.

[0175] McQuitty CE, Williams R, Chokshi S and Urbani L (2020) Immunomodulatory Role of the Extracellular Matrix Within the Liver Disease Microenvironment. Front. Immunol. 11:574276.

[0176] Orman ES, Barritt AS 4th, Wheeler SB, Hayashi PH. Declining liver utilization for transplantation in the United States and the impact of donation after cardiac death. Liver Transpl. 2013 Jan;19(1):59-68.

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Claims

Claim 1 A method for decellularizing animal-derived tissue, comprising the following steps: g) a step of mechanically homogenizing the tissue of animal origin; h) a step of incubating the homogenized tissue from step a) at 37°C for 1-3 hours in a solution of 0.005-0.5% trypsin and 0.002-0.2% EDTA; i) a step of removing the solution of trypsin and EDTA and applying the filtered tissue to incubation in a PBS buffer solution of pH 7.2-7.6 containing 4-2% non-ionic detergent and 0.2-0.08% NH4OH, wherein the incubation is performed for 24-48 hours at a temperature below room temperature; j) a step of washing the tissue with a PBS buffer solution containing an antimicrobial agent, wherein the washing is performed for at least 72 hours at a temperature below room temperature; and k) 0.001 to 0.003% (w / v) The method comprises the step of incubating at 37°C for 8 hours in a deoxyribonuclease solution of a certain concentration, i) washing the decellularized tissue obtained in steps a) to e) with a PBS buffer solution containing an antimicrobial agent, wherein the washing is performed at a temperature below room temperature for at least 72 hours, and - the non-ionic detergent is a compound having a molecular weight of 596 g / mol of the following chemical formula: - A method for decellularizing animal-derived tissue, characterized by the following additional steps following step c): c1) incubating the tissue in a PBS buffer solution having a pH in the range of 7.2-7.6 containing a non-ionic detergent at a concentration of 1.5-0.5% and NH4OH at a concentration of 0.2-0.08%, wherein the incubation is performed for 36-48 hours at a temperature below room temperature, and c2) incubating the tissue in a PBS buffer solution having a pH in the range of 7.2-7.6 containing a non-ionic detergent at a concentration of 0.75-0.25% and NH4OH at a concentration of 0.2-0.08%, wherein the incubation is performed for 36-48 hours at a temperature below room temperature. Claim 2 A method for decellularizing animal-derived tissue according to claim 1, characterized in that the animal-derived tissue is liver tissue. Claim 3 A method for decellularizing animal-derived tissue according to claim 1, characterized in that, during incubation in steps c), c1), and c2), the PBS buffer solution containing the non-ionic detergent and NH4OH is exchanged three times. Claim 4 A method for decellularizing animal-derived tissue according to claim 1, wherein the incubation in steps c), c1), and c2) is performed at a temperature of 4°C. Claim 5 A method for decellularizing animal-derived tissue according to claim 1, characterized in that the antimicrobial agent is streptomycin at a concentration of 0.01% (w / v). Claim 6 A method for decellularizing animal-derived tissue according to claim 1, characterized in that, during the washing in step d), the PBS buffer solution containing the antimicrobial agent is exchanged eight times. Claim 7 A method for decellularizing animal-derived tissue according to claim 1, characterized in that, during the washing in step f), the PBS buffer solution containing the antimicrobial agent is exchanged five times. Claim 8 A bioink characterized by containing decellularized animal-derived tissue obtained by the method of claim 1 at a concentration of 0.5-10% (w / v), preferably 5% (w / v). Claim 9 A bioink according to claim 8, further comprising 0.5-2% (w / v) of HAMA solution, 5-20% (w / v) of GELMA solution, and 0.15-0.5% (w / v) of LAP solution.

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