Method for obtaining a decellularized extracellular matrix of the pancreas and the use of the decellularized pancreatic extracellular matrix as a component of bioink for bioprinting

Abstract

A method for obtaining decellularized extracellular matrix of the pancreas, that it comprises the following steps, in which: the pancreas is incubated in a disinfecting solution, advantageously containing betadine at a concentration of 2% (v / v), then in a solution with an antimicrobial agent, advantageously streptomycin at a concentration of 0.01% (w / v), and then frozen; the pancreas, warmed to a temperature of 4°C ± 2°C, is subjected to homogenization in a PBS solution and 5 mM EDTA-Na2 in a 3: 1 ratio, and the homogenized material is centrifuged, after which the hydrophobic fat phase is removed; the material is suspended in a solution of NaCl at a concentration of 0.5 M + 5 mM EDTA-Na2 (pH = 7.4), and then subjected to mixing, after which the material is filtered; the material is immersed in a PBS solution and incubated, then sieved, and the sieved material is mechanically minced; the material is immersed in a PBS solution with 0.01% (w / v) streptomycin and incubated, then sieved, and the sieved material is mechanically minced; the material is immersed in a PBS solution with 0.01% (w / v) streptomycin and incubated for 2 hours at 4°C ± 2°C, the step being repeated at least twice, and the sieved material is mechanically minced after each filtration; the obtained extracellular matrix is sieved, frozen, and then subjected to lyophilization and mechanical milling.

Description

[0001] Method for obtaining a decellularized extracellular matrix of the pancreas and the use of the decellularized pancreatic extracellular matrix as a component of bioink for bioprinting

[0002] The invention relates to a method for obtaining a decellularized extracellular matrix (dECM) from animal pancreatic tissue without the use of detergents. The procedure for obtaining dECM comprises removal of fat, tissue homogenization, rinsing with saline solutions, and purification of the fragmented tissue using washing solutions. This method allows decellularization without the use of harmful detergents, which may be detrimental and adversely affect the microstructure of the extracellular matrix (ECM). The invention also relates to the detergent-free method and dECM obtained by this method as a component of bioink intended for bioprinting. The invention finds application in 3D bioprinting, regenerative medicine, transplantology, cellular and tissue engineering, and biomaterials engineering, from basic research through to subsequent implementation.

[0003] Recent advances in tissue engineering enable the use of dECM as a component of scaffolds which, once populated with cells, serve as three-dimensional tissue models. The extracellular matrix (ECM) plays an important structural role and is also involved in the regulation of numerous cellular processes (Guo et al., 2024). ECM obtained through decellularization has been proposed in several strategies for tissue and organ reconstruction [Dussoyer et al., 2020; Uygun et al., 2010; Sicari B.M. et al., 2014; Thomas et al., 2010; Zhang X. et al., 2022; Kc, P., Hong et al., 2019], Despite the existence of known methods for pancreatic organ decellularization, they have limited applicability due to the increased lipid content of pancreatic tissue. Hydrogels derived from dECM can be obtained from many decellularized tissues; however, there is still a lack of methods that enable the complete elimination of detergents while simultaneously preserving the unique biochemical and functional profile, ultrastructural integrity, and higher levels of native components. At the same time, it is necessary to eliminate chemical contaminants that could disrupt cellular responses or reduce the biocompatibility of the material. Incorporation of a homogenization step into the decellularization protocol significantly improves lipid removal and the gelation capacity of the resulting dECM, which is capable of gelation at 37 °C both in vitro and in vivo and is cytocompatible with various cell types and pancreatic islet tissues in vitro [Ahn et al., 2017], The present invention provides a characterization of a novel yet simple and cost-effective protocol for decellularization and delipidization of animal pancreatic tissue to obtain acellular ECM and ECM hydrogel suitable for cell culture and transplantation applications. Fat removal is essential due to the potential occurrence of undesirable immune reactions, particularly in organ transplantation, regenerative medicine, and 3D model studies. Furthermore, in lipid-rich tissues, detergents are more difficult to remove, and residual traces may be toxic and inhibit cell proliferation and extracellular matrix production.

[0004] There are several methods for producing functional artificial organs for applications in tissue engineering and regenerative medicine. One of these methods is 3D bioprinting. Bioprinting enables the precise and controlled placement of living cells within biomaterials of synthetic origin as well as natural polymers such as collagen, gelatin, alginate, hyaluronic acid, fibrin, or polyethylene glycol, leading to the formation of three-dimensional tissue constructs [Ahn et al., 2017], dECM is a promising component of bioink. As a result of dECM solubilization, it can be converted into a hydrogel that undergoes crosslinking at physiological temperature due to collagen self-assembly mechanisms (Saldin et al., 2017). The hydrogel can be designed to mimic the native extracellular matrix, exhibit a degree of elasticity similar to natural tissue, and create an environment conducive to cell growth, differentiation, and proper function due to the presence of tissue-specific biochemical signatures of the extracellular matrix. The advancement of research on dECM-derived hydrogels opens the possibility of their commercial use as bioinks for 3D bioprinting [Ahn et al., 2017; Sackett et al., 2018], dECM is obtained through a decellularization process, which involves the removal of cells and other immunogenic material, particularly DNA, cell membranes, and DAMPs (Damage -Associated Molecular Patterns), essentially leaving only the non-immunogenic structural components of the ECM (such as collagen, elastin, laminin, etc.). Residual cell membranes or other immunogenic factors remaining due to incomplete tissue purification are recognized by the host immune system as damage signals. This triggers a cytokine cascade and the proliferation of inflammatory cells, resulting in a localized inflammatory response around the construct.

[0005] Inflammatory processes that may arise due to residual immunogenic factors or cell membrane remnants following incomplete decellularization are interpreted by the immune system as damage signals, leading to cytokine cascades and further proliferation of inflammatory cells, ultimately causing a localized inflammatory response around dECM constructs. This phenomenon is of significant clinical importance, as it may affect the success of graft integration and acceptance. Therefore, the decellularization method employed should ensure the most effective possible removal of cellular components while preserving the structure of the extracellular matrix [White et al., 2017],

[0006] The choice of decellularization method varies depending on the tissue and organ type due to tissuespecific factors such as size, thickness, shape, and the density of cells and extracellular matrix (ECM) [White et al., 2017], Therefore, different decellularization protocols are required depending on tissue architecture and ECM composition. The ECM of each tissue creates a unique, tissue -specific microenvironment for resident cells, providing structural support and biochemical signals essential for proper cell function within that tissue [Isaeva et al., 2022] . 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 their combination. Typical decellularization methods include a series of washes in chemical solutions containing various assisting agents. Commonly used agents to date include non-ionic detergents (Triton X-100), anionic detergents (sodium dodecyl sulfate - SDS, sodium deoxycholate - SD), enzymatic agents (trypsin, DNase I), and chelating agents such as ethylenediaminetetraacetic acid (EDTA). A solution may contain one or more compatible surfactants (e.g., anionic surfactants such as SDS and non-ionic surfactants such as Triton X-100). SDS and Triton X-100 are the most commonly used chemical agents in liver decellularization procedures. Surfactants act by disrupting the phospholipid cell membrane, thereby causing cell lysis [Isaeva et al., 2022], Ionic detergents have a more pronounced negative effect on tissue structure and are more difficult to remove using typical neutral solutions. However, detergents can be potentially cytotoxic and have the capacity to activate the immune system. Enzymes such as proteases, esterases, and nucleases are also used to promote DNA fragmentation and reduce the immunogenicity of the ECM construct [Moffat et al., 2022], Several studies have shown that despite repeated washing steps, it is not possible to completely remove all detergent residues from treated biological material [Caamano et al., 2009; Cebotari et al., 2010], Residual detergent can also alter the biophysical properties of proteins such as ECM elastin, affecting their mechanical strength and leading to subsequent structural degradation [Crapo et al., 2011; Herbert et al., 1972], Some protocols use biological load-reducing agents, including penicillin, streptomycin, peracetic acid, ethanol, or their combinations. After decellularization, all cellular elements and chemical residues must be thoroughly washed from the scaffold using a neutral solution, such as PBS. It is also necessary to consider the specific tissue type being decellularized, as the same decellularization protocol can produce different results in different tissues [Moffat et al., 2022] . The process of producing acellular, delipidized, lyophilized, and sterilized dECM has limitations. It involves multiple steps spanning several days. Each step requires strict control of conditions such as temperature, pH, and reagent concentrations to preserve the structural integrity and biochemical properties of pancreatic ECM [Chamberlain et al., 2025],

[0007] In the state of the art, there are known solutions regarding decellularization methods depending on tissue structure, ECM composition, and the type of organ subjected to the decellularization process.

[0008] In US2022305175A1, a detergent-free method for decellularizing the pancreas, liver, kidney, or muscle tissue is described. The method primarily relies on prolonged incubation of tissues in hypoosmotic solutions for periods ranging from 12 to 24, 36, or 48 hours, optionally with the addition of enzymes that digest DNA and / or other cellular materials. In some variants, the method further includes a step for enzyme deactivation followed by a second incubation in water. The process may be assisted by mechanical stirring and conducted at controlled temperatures ranging from 2 to 15°C or 25 to 40°C. After decellularization, the tissues can be further lyophilized, fragmented, and delipidized using proteases to obtain an ECM powder. The disclosed ECM contains less than 100 ng or 50 ng of DNA per mg of dry weight and well-preserved collagen. In some examples, the total collagen content ranged from 20 to 40 pg per mg of dry weight, while the glycosaminoglycan (GAG) content ranged from 2 to 10 pg per mg of dry weight. The document does not disclose specific washing parameters but describes delipidization with protease and tissue incubation with the enzyme. US 12208179B2 discloses the decellularization of the pancreas using Triton X-100 and the formulation of dECM-based bioinks for 3D bioprinting. The document describes a decellularization method comprising the following steps: a) Mechanical fragmentation of the organ (e.g., pancreas, liver, kidney, heart, skin, lungs, intestines, blood vessels, adipose tissue, placenta), b) Detergent-based decellularization: incubation of the organ tissue in a buffered detergent solution consisting of 1 x PBS with 0.5-1.5% octoxynol-9 (preferably 1% v / v) and an antimicrobial agent (0.01% w / v streptomycin) at 4°C with stirring for at least 72 hours, with solution replacement every 4-12 hours, c) First washing step: incubation in l x PBS with 0.01% w / v streptomycin for >72 hours at 4°C with stirring, replacing the solution every 4-12 hours, d) DNase treatment: incubation in a DNase solution at 0.0001-0.0003% w / v (preferably 0.0002% w / v) at a temperature suitable for the enzyme for >8 hours, e) Second washing step: reincubation in 1 x PBS with 0.01% w / v streptomycin for >72 hours at 4°C with stirring, replacing the solution every 4-12 hours, f) Freezing and fragmentation of the tissue, g) Lyophilization and milling to obtain a dECM powder with particle sizes of 25-500 pm. This document discloses detergent-based decellularization; however, it does not mention removal of the fat layer prior to or during the process.

[0009] CN104353115A discloses a set of three perfusion solutions and a method for decellularizing the pancreas. According to this document, perfusates are used to remove blood and loose cells (0.25 g EDTA and 10,000 U sodium heparin in 1 L PBS buffer, pH 7-8), lyse cells (10 mb Triton X-100 and 1 mb aqueous ammonia solution in 1 L deionized water, pH 7-8), and degrade residual DNA (0.01 g DNase I in 1 L deionized water, pH 7-8). The disclosed method results in a fully acellular yet well-preserved pancreatic scaffold (ECM). Detergents are washed out together with other chemical residues; however, the removal of fat tissue is not disclosed.

[0010] US 11077230B2 discloses atissue decellularization method using supercritical carbon dioxide combined with a decellularization medium, as well as methods for producing decellularized implants and the implants themselves. Tissues suitable for decellularization include endothelial, epithelial, interstitial, connective, or supportive tissues, covering organs such as the trachea, esophagus, larynx, colon, vascular tissue, bone, ligaments, tendons, urethra, bladder, skin, lungs, muscles, buccal tissue, pancreas, spleen, kidney, liver, lymphatic system, brain, osteotendinous junctions, synovial membrane, cartilage, and nervous tissue. The method comprises: a) placing tissue in a decellularization vessel; b) adding a decellularization medium containing at least one detergent and / or at least one enzyme; c) introducing supercritical CO2 into the vessel containing the tissue and medium; d) maintaining the tissue in contact with the supercritical CO2 and medium for at least 24 hours to achieve significant decellularization; e) washing the decellularized tissue with buffer solution. The vessel is heated to at least 28°C, and supercritical CO2 is maintained at pressures above 100 MPa. Detergents may include SDS, sodium deoxycholate, detergents containing hydrophilic polyoxyethylene - oxide and hydrophobic hydrocarbon fragments, or their combinations. Enzymes may include trypsin, lipase, cellulase, or their combinations. Fat removal is not disclosed; detergents, enzymes, and phosphate-buffered washes are used.

[0011] CN101850135A discloses stepwise perfusion decellularization ofthe liver via the vascular system, with control over solution composition and flow parameters adapted to ECM structure and vascular patency. Perfusates included: 1. slightly basic hypotonic solution (0.1% aqueous ammonia) or pure water; 2. ionic detergent solutions with gradient concentrations (1%, 0.5%, 0.25% SDS and 1% Triton X-100); 3. aqueous electrolyte solution (PDADMAC); 4. aqueous anticoagulant solution (heparin). Combined reagents may include perfusate 5 for sterilization (distilled water with penicillin, streptomycin, amphotericin B, fluconazole, and / or metronidazole). The method does not disclose fat tissue removal. Detergents and basic solutions are used, followed by intensive buffer washes to remove detergent residues.

[0012] EP3533475A1 discloses an ex vivo decellularization method for porcine, bovine, human, and equine pericardial tissue. The method includes mechanical removal of fat, three washes in PBS (without Ca2+and Mg2+) with protease inhibitors under continuous stirring, cell lysis via osmotic shock in hypotonic Tris-HCl buffer with protease inhibitors for 16 hours at 4°C, incubation in 1 % Triton X-100 for 24 hours at room temperature with stirring, multiple washes in 0.1% SDS, DNase I treatment, and final decontamination washes for 72 hours at 4°C with antibiotics and antifungals. The method is applicable for tissue-engineered heart valves and vascular constructs. Mechanical fat removal is mentioned, but no separate fat removal step is described.

[0013] WO2024152106A1 discloses a biosealing ECM composition from decellularized organs with preserved (ultra)structure, including laminins, collagen IV, VI, I, fibronectin, elastin, fibrillin, ECM 1, proteoglycans, glycoproteins, biglycan, histidine-rich glycoprotein, tenascin, and heparan sulfate proteoglycan 2, produced from SDS-decellularized bladder tissue. Other organs include liver, pancreas, kidney, or lung. Both detergent-based (SDS) and detergent-free decellularization are disclosed. The detergent-free method relies on freezing / thawing and extraction: tissue is washed in Milli-Q water for 4 hours at 37°C with stirring, incubated in 2M NaCl for 1 hour, then incubated 24 hours in Milli-Q water at 37°C, subjected to two cycles of liquid nitrogen freezing and thawing, fragmented, and incubated repeatedly in 2M NaCl and Milli-Q water. Final washes are performed with 70% ethanol and PBS with antibiotics. Mechanical fat removal is disclosed.

[0014] CN102933705A discloses a method for decellularizing adipose tissue and its use as a bioscaffbld for soft tissue reconstruction, cell / factor carriers, and as a component of hydrogels and composite biomaterials. The method includes three freeze-thaw cycles in Tris buffer (pH 8.0) with EDTA, incubation for 16 hours in an enzymatic solution containing trypsin and EDTA, 48-hour lipid extraction with 99.9% isopropanol, multiple washes in rinsing buffer, repeated enzymatic incubation, DNAse, RNase, and lipase treatment for 16 hours, followed by PBS / water washes, final lipid extraction with 99.9% isopropanol for 8 hours, three PBS washes, three washes in 70% ethanol, and storage in sterile PBS with antibiotics at 4°C. The method includes sequential buffer washes, enzymatic incubations (lipases, nucleases, proteases), and lipid extraction using polar / nonpolar solvents and alcohols. Fat removal is not explicitly described.

[0015] In the publication by Amish A. (“ Decellularized human pancreatic extracellular matrix-based physiomimetic microenvironment for human islet culture, ” 2023, Acta Biomaterialia, Volume 171, 261- 272, doi: 10.1016 / j.actbio.2023.09.034), a detergent-free method for decellularizing human pancreas is described. The method involves the use of deionized water, followed by freezing, lyophilization, and cryogenic milling, and then solubilization of the extracellular matrix (ECM) with pepsin in hydrochloric acid. This process lasts approximately 48 hours at room temperature, with subsequent neutralization of the solution to pH 7.4 at 4°C. The technique enables the removal of cells, lipids, and DNA while preserving approximately 33.3% of ECM proteins. According to the disclosure, the ECM biomaterial was used for microencapsulation of islets with alginate. The authors did not describe a step for fat layer removal, nor did they mention application in bioprinting.

[0016] In the publication by Sackett S.D. et al. (“Extracellular matrix scaffold and hydrogel derived from decellularized and delipidized human pancreas, ” 2018, Scientific Reports, volume 8, Article number: 10452, doi: 10.1038 / s41598-018-28857-1), a decellularization process was disclosed, including freezing the pancreatic tissue at -80 °C, and then thawing at 37 °C; washing with l x PBS for 30 minutes, and then incubating in 2.5 mM [sodium deoxy cholate / PBS] with shaking at room temperature for 24 hours. After the first step, the tissue was washed with water and the procedure was repeated with another 24 hours of incubation in deoxycholate. After 48 hours, the tissue was again washed with water and subjected to 72-hour washing in l x PBS with Pen / Strep, with daily replacement of the solution. The obtained decellularized pancreatic ECM was lyophilized and stored at -80 °C. The authors also disclosed a decellularization process through homogenization of the pancreas: the homogenate was centrifuged, and floating fat was removed from the surface. The sediment was resuspended in water and centrifuged again (4300 rpm, 5 min). Then the sediment was resuspended in 2.5 mM sodium deoxy cholate / PBS and incubated for 3 hours (RT, shaker). After this time, the homogenate was filtered through a sieve (Sigma, SI 145); all collected material was placed back into 2.5 mM sodium deoxy cholate / PBS and incubated for another 15 hours (RT, shaker). The ECM was again centrifuged, washed with water, and washed in l x PBS with Pen / Strep for 72 hours, rinsing with water every 24 hours, and fresh PBS + Pen / Strep was replaced each day (RT, shaker). The obtained decellularized pancreatic ECM was lyophilized and stored at -80 °C for future use. In the publication, fat removal was disclosed, but the process is multi-step and uses detergents.

[0017] In the publication by Bera A.K. et al. ( “Formulation of Dermal Tissue Matrix Bioink by a Facile Decellularization Method and Process Optimization for 3D Bioprinting toward Translation Research, ” 2022, Macromolecular Bioscience, Volume 22, Issue 8, doi: 10.1002 / mabi.202200109), a detergent-free, single-step decellularization method of goat skin tissue is disclosed, using hypotonic / hypertonic NaCl solutions. The method involves alternately treating the skin tissue with solutions of different sodium chloride concentrations (hypotonic / hypertonic). The publication discloses the use of the obtained ECM for bioink, but does not refer to the fat content in the final product.

[0018] The invention relates to a method for obtaining decellularized pancreatic extracellular matrix (dECM), which comprises the following steps: a pancreas is incubated in a disinfecting solution, preferably comprising 2% (v / v) Betadine, then in a solution with an antimicrobial agent, preferably streptomycin at 0.01% (w / v), and then frozen, the pancreas, warmed to a temperature of 4 °C ± 2 °C, is subjected to homogenization in a solution of PBS and 5 mM EDTA-Na2 in a ratio of 3: 1, and the homogenized material is centrifuged, after which the hydrophobic fat phase is removed,

[0019] Preferably, the homogenized material is centrifuged at 4000 rpm for 5 minutes at a temperature in the range of 2 °C to 8 °C, preferably the sample in the previous step is mixed at 150 rpm at a temperature in the range of 2 °C to 6 °C for 1-2 hours, the material is then suspended in a 0.5 M NaCl + 5 mM EDTA-Na2 solution (pH = 7.4), mixed, followed by filtration.

[0020] Preferably, the homogenized material is centrifuged at 4000 rpm for 5 minutes at a temperature in the range of 2 °C to 8 °C, and

[0021] Preferably the sample is mixed at 150 rpm at a temperature in the range of 2 °C to 6 °C for 1-2 hours,

[0022] The material is suspended in PBS and incubated, then filtered, and the filtered material is mechanically fragmented.

[0023] Preferably, the sample is mixed at 150 rpm at a temperature in the range of 2 °C to 6 °C for 16 hours.

[0024] The material is then suspended in PBS with 0.01% (w / v) streptomycin and incubated, then filtered, and the filtered material is mechanically fragmented.

[0025] Preferably, the sample is incubated for 2 hours, mixing at 150 rpm at 4 °C, and this step is repeated three times. The material is suspended in PBS with 0.01% (w / v) streptomycin and incubated for 2 hours at 4 °C ± 2 °C, this step being repeated at least twice, and the fdtered material is mechanically fragmented after each filtration.

[0026] Preferably, the sample is incubated for 2 hours, mixing at 150 rpm at 4 °C, and this step is repeated three times.

[0027] The obtained extracellular matrix is then filtered, frozen, and subjected to lyophilization and mechanical milling.

[0028] Preferably, before this step, the material is immersed in 70% ethanol and mixed at 150 rpm at a temperature in the range of 2 °C to 6 °C for 30 minutes.

[0029] Preferably, the material is sterilized by radiation using 25 kGy.

[0030] The invention also relates to the use of the decellularized matrix as a component of bioink intended for bioprinting.

[0031] The invention is illustrated in the Figures, where:

[0032] Fig. 1 shows the results of DNA content analysis obtained using a spectrophotometer.

[0033] Fig. 2 shows a graph illustrating collagen content depending on the variant of the decellularization method.

[0034] Fig. 3 shows the results of SDS-PAGE electrophoretic analysis of dECM samples conducted on polyacrylamide gels with concentrations of 10% (A) and 15% (B), where: 1) dECM obtained by the detergent method using Triton X-100, 2) Till.01, 3) Till.02.

[0035] Fig. 4 shows the dependence of the complex modulus on temperature for three batches: the detergent method using Triton X-100, Till.01, and Till.02.

[0036] Fig. 5 shows the phase transition temperature determined for the tested biomaterial compositions containing dECM from three batches: the detergent method using Triton X-100, Till.01, and Till.02.

[0037] Fig. 6 shows the variation of dynamic viscosity in atemperature gradient ranging from 40-5 °C for three batches: the detergent method using Triton X-100 (Triton), Till.01, and Till.02.

[0038] Fig. 7 shows the dependence of the storage modulus and loss modulus on stress for the tested materials not subjected to crosslinking from three batches: the detergent method using Triton X-100 (Triton), Till.01, and Till.02.

[0039] Fig. 8 shows the dependence of the storage modulus and loss modulus on strain for the tested materials not subjected to crosslinking from three batches: the detergent method using Triton X-100 (Triton), Till.01, and Till.02. Fig. 9 shows the dependence of the storage modulus and loss modulus on stress for the tested materials subjected to crosslinking for 30 seconds with light of 405 nm wavelength, 28.5 mW / cm2, from three batches: the detergent method using Triton X-100 (Triton), Till.01, and Till.02.

[0040] Fig. 10 shows the dependence of the storage modulus and loss modulus on stress and strain for the tested materials subjected to crosslinking for 30 seconds with light of 405 nm wavelength, 28.5 mW / cm2, from three batches: the detergent method using Triton X-100 (Triton), Till.01, and Till.02.

[0041] Fig. 11 shows photographs of the printed constructs for the MTT assay.

[0042] Fig. 12 shows the effect of 24- and 72-hour extracts obtained from dECM-based biomaterials on L-929 cell line. According to the guidelines of ISO 10993-5:2009(E), the continuous line indicates the 70% viability threshold.

[0043] Fig. 13 shows the results of the GSIS test for INS-1 P cells (rat) embedded in biomaterials containing dECM obtained using two methods: decellularization method using the detergent Triton X-100 (Triton X-100) and the detergent-free method (Till.02).

[0044] Examples of Implementation

[0045] Example 1: Preparation of detergent-free dECM

[0046] Pancreas decellularization procedure

[0047] Materials

[0048] Porcine pancreas was obtained from a local slaughterhouse. Visible fat was manually removed, and the tissue was rinsed in a 2% (v / v) Betadine solution, followed by a solution containing an antibiotic, preferably streptomycin (0.1 mg / ml) at 0.01% (w / v), after which the pancreas was frozen. Organs intended for the study were stored in zipper bags under freezing conditions at -20 °C ± 2 °C. Two days before the planned decellularization process, the appropriate number of organs, depending on their mass and the number of samples, were thawed at 4 °C ± 2 °C.

[0049] The detergent-free decellularization process of the pancreas was carried out for no less than 5 days. After thawing, the organs were mechanically fragmented in phosphate-buffered saline (l x PBS) with 5 mM EDTA-Na2 (pH = 7.4) in a 3: 1 ratio using a blender. The tissue material was fragmented four times in intervals of at least 5 seconds each. The fragmented and mixed material was placed in 500 mb centrifuge tubes and centrifuged at 4000 rpm for 5 minutes at 4 °C ± 2 °C in a centrifuge with a horizontal rotor. After centrifugation, a sample was obtained with a clearly separated hydrophobic phase (fat layer), which was manually removed. The material from the samples was then transferred to 2 L sterile glass bottles, with the content of one centrifuge tube transferred to one bottle.

[0050] The centrifuged material was then suspended in a 0.5 M NaCl + 5 mM EDTA-Na2 solution (pH = 7.4) to a volume of 1800 mb. The bottles containing the material were placed in an incubator with a mixing function at 150 rpm at 4 °C ± 2 °C for a period of 1-2 hours. After this time, the material from the bottles was sieved on a stainless steel sieve, and the obtained material was transferred back into bottles, reducing the number of bottles by half by combining the contents of two bottles into one. In the next step, the bottles were filled with PBS solution to 1800 mb and mixed at 150 rpm at 4 °C ± 2 °C for 16 hours. During the solution exchange, the pancreatic tissue material was mechanically fragmented using a blender.

[0051] In the next step, the material from the bottles was transferred onto a stainless steel sieve, sieved, and then transferred back into a bottle. The pancreatic tissue was placed in a previously prepared 0.5 M NaCl + 5 mM EDTA-Na2 solution (pH = 7.4) to a volume of 1800 mb. After 1 hour of incubation at 4 °C ± 2 °C with mixing at 150 rpm, the material was again transferred onto a stainless steel sieve and sieved. The obtained material was transferred back into a bottle, and the tissue was covered with previously prepared PBS solution to 1800 mb and incubated for 2 hours ± 1 hour at 150 rpm at 4 °C ± 2 °C. This step was repeated three times, with the material being cut with scissors at each decellularization solution exchange. During the last solution exchange, the material was mechanically fragmented using a blender.

[0052] Next, washing was performed using PBS solution supplemented with 0.01% (w / v) streptomycin, with mixing at 150 rpm at 4 °C ± 2 °C for 2 hours. This step was repeated three times, with the material cut with scissors at each solution exchange. During the last solution exchange, the material was mechanically fragmented using a blender.

[0053] The PBS solution used (PBSx l) is a lx phosphate-buffered saline solution, which in 1 L contains: 137 mM NaCl, 2.7 mM KC1, lOmMN HPIk, l.S mM KILPO^ pH = 7.4.

[0054] The next step was carried out in two variants: Till.01 and Till.02.

[0055] Variant Till.01

[0056] The material from the bottles was transferred onto a sieve and sieved. Then the material was transferred back into a bottle, and the tissue was covered with previously prepared PBS solution supplemented with 0.01% (w / v) streptomycin to a volume of 1800 mb and mixed for 2 hours at 4 °C ± 2 °C (150 rpm). This step was repeated three times, with the material cut with scissors at each solution exchange. During the last solution exchange, the material was mechanically fragmented using a blender.

[0057] Variant Till.02

[0058] The material from the bottles was transferred onto a stainless steel sieve and sieved. After draining, the material was transferred back into a bottle, and the tissue was covered with previously prepared 70% ethanol and mixed at 150 rpm at 4 °C ± 2 °C for 30 minutes. Then the material from the bottles was transferred onto a stainless steel sieve and sieved, after which the material was again transferred into a bottle, and the tissue was covered with previously prepared PBS solution supplemented with 0.01% streptomycin to a volume of 1800 mL and mixed at 150 rpm at 4 °C ± 2 °C for 2 hours. The PBS washing step was carried out twice. At each solution exchange, the tissue was cut with scissors. During the last solution exchange, the material was mechanically fragmented using a blender.

[0059] In the final step, common to both variants Till.01 and Till.02, the material was again transferred onto a stainless steel sieve and sieved. Then the material was transferred back into a bottle, and the tissue was washed with previously prepared PBS solution supplemented with 0.01% (w / v) streptomycin to a volume of 1800 mL and mixed at 150 rpm at 4 °C ± 2 °C for 2 hours. The PBS washing step was carried out twice. At each solution exchange, the tissue was cut with scissors.

[0060] After completion of the decellularization process, the material was prepared for lyophilization. The extracellular matrix of the pancreas, sieved on a stainless steel sieve, was placed on aluminum trays and stored at -80 °C ± 10 °C until lyophilization. Then the material was lyophilized. The obtained lyophilizates were fragmented in a cryogenic mill to obtain a powder with particle size smaller than 100 pm. The material was subjected to radiation sterilization (25 kGy).

[0061] Example 2

[0062] 2.1. Biochemical analysis of dECM powder a) Measurement of DNA content using a NanoDrop spectrophotometer DNA from powdered dECM was isolated using a commercial Qiamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions, i.e., the samples were digested using proteinase K and the digestion buffer. The samples were applied to columns, washed in steps, and the precipitated DNA was eluted. The DNA concentration was measured spectrophotometrically (NanoDrop, Thermo Scientific, USA). The average DNA content per mg of tissue was determined.

[0063] The results of the experiments conducted formed the basis for the development of the invention. Based on the DNA content, it was found that the detergent-free decellularization process is more than three times more effective than the detergent-based method. This indicates the removal of over 99.50% of the native tissue DNA, where the DNA content was estimated at 638.53 ng / mg. DNA analysis using a NanoDrop spectrophotometer confirmed the effectiveness of the dECM decellularization (Fig. 1). b) Measurement of fat content

[0064] The total fat content was determined using the Soxhlet extraction method. 500 mg of powdered pancreas (dry weight) and dECM (obtained by the detergent method with Triton X-100 and Till.02) were placed in cellulose thimbles, covered with cotton wool, and weighed. 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 was determined relative to the mass of the tested sample.

[0065] Results

[0066] The fat content in the native pancreas averaged 14.99% ± 0.71. The use of the detergent-free method (Till.02) reduced the percentage of fat to 3.04% ± 2.25. This parameter is crucial for the quality of the biomaterial, as excess lipids can negatively affect the process of scaffold colonization by cells, disturb their adhesion, and modify the mechanical and hydrophilic properties of the matrix. The reduced lipid content indicates a high level of purification of the biomaterial. Based on the results, it can be concluded that dECM obtained by the detergent-free method will have higher stability within the biomaterial, thereby optimizing the conditions for cell growth and function in 3D-printed structures. Furthermore, extracellular matrix with reduced fat content exhibits better rheological properties, such as increased viscosity, which improves the printability of the final product [Sackett et al., 2018], c) Measurement of sulfated glycosaminoglycans (sGAG)

[0067] The content of sulfated glycosaminoglycans (sGAG) in native tissues and in decellularized, cryomilled tissues was quantified using the Blyscan Sulfated Glycosaminoglycan (sGAG) Assay Kit (Biocolor, UK). 20-50 mg of dry tissue was weighed and placed in a 1.5 m tube containing 1 m of papain solution and incubated at 65 °C for 16 hours ± 8 hours, with occasional mixing. Portions of each sample were mixed with 1,9-dimethyhnethylene blue dye and the reagents from the kit. Absorbance at 565 nm was measured using a microplate reader and compared with standards prepared from bovine chondroitin sulfate to determine the absolute sGAG content.

[0068] Results

[0069] The average sGAG' s content in native pancreatic tissue was 3.49 ± 0.09 pg / mg of dry tissue. In contrast to dECM obtained using Triton X-100, dECM obtained by the detergent-free method had sGAG at a level of 0.23 pg / mg ± 0.14. According to Krasny et al. (2016), different decellularization methods lead to the loss of soluble dECM components in tissues. In our study, the detergent method with Triton X- 100 caused a loss of sGAG, whereas the detergent-free method allowed the preservation of a larger portion of these components, although their content was still lower than in native tissue.

[0070] The presence of sulfated glycosaminoglycans (sGAG) in dECM is important because it supports the mechanical properties of the biomaterial and the formation of a physiological cellular microenvironment, promoting cell adhesion, proliferation, and function. Therefore, preserving sGAG using the detergent-free method may contribute to higher bioactivity of the biomaterial (Mullen et al., 2010; Moria, 2019). d) Measurement of collagen content

[0071] The procedure for determining collagen content was carried out according to the manufacturer’s instructions (Abeam, UK) and is based on the alkaline hydrolysis of dECM samples to obtain free hydroxyproline. The released hydroxyproline is oxidized to form an intermediate compound, which in the subsequent reaction produces a brightly colored chromophore that can be easily quantified at OD 560 nm. Results are presented as multiples of the value obtained for native tissue. Results

[0072] The detergent-free decellularization method has a smaller impact on collagen content and confirms the preservation of this key component in dECM. Approximately a 47-fold increase in collagen content was observed in dECM compared to native tissue. This value results from the reduction in the total mass of the sample due to the removal of cellular components and other soluble proteins, while maintaining a high degree of collagen fiber preservation.

[0073] Higher collagen concentration in biomaterials leads to a significant improvement in the mechanical properties of the material, manifested as increased structural stability and durability of the resulting scaffolds. Such scaffolds maintain the desired shape more effectively after the 3D bioprinting process, which is critical for accurately reproducing the designed geometry. Moreover, the presence of collagen enhances the printability of the material, enabling the production of more stable and functional structures, which is highly relevant for applications in tissue engineering and regenerative medicine (Fig. 2). e) SDS-PAGE electrophoretic analysis

[0074] SDS-PAGE electrophoretic analysis of dECM samples performed on 10% and 15% polyacrylamide gels showed that materials obtained according to the Till.01 and Till.02 procedures are characterized by a clearly more preserved, richer, and more diverse protein profile compared to the reference material, i.e., dECM obtained using the detergent method with Triton X-100.

[0075] The presence of numerous bands across a wide range of molecular weights, along with the absence of clear signs of protein degradation, indicates better preservation of the native structure of extracellular matrix components, including structural, adhesive, and regulatory proteins. Such a protein profile, closer to the native state, supports the reconstruction of complex biochemical signals necessary for cell adhesion, proliferation, and differentiation.

[0076] Example 3 Preparation of dECM-based hydrogels as an example of dECM application dECM obtained by the detergent-free method can serve as a modifier of the functional properties of commonly used biomaterials. As a result, it finds applications in 3D cell printing technology and other tissue engineering techniques, contributing to improved functionality and biocompatibility of the produced constructs.

[0077] Preparation of dECM-based hydrogel using dECM lyophilizate dECM hydrogels were prepared using an acidic pepsin digestion method, employing dECM obtained by the detergent-free method (Till.01 and Till.02) as well as dECM obtained by the standard decellularization method using Triton X-100. Procedure: A 50 mL glass bottle with a plug, equipped with a mixing element, was filled quantitatively with 0.01 M HC1 solution. Then, pepsin was weighed and quantitatively transferred to the HC1 solution. The solution was stirred for 20 minutes (until the pepsin dissolved) at a speed of 500 rpm at room temperature. Subsequently, the weighed dECM powder was quantitatively added to the pepsin solution. The hydrogel was left on a magnetic stirrer for 72 hours at a stirring speed of 500 rpm, heated to 30°C. After 72 hours of continuous stirring, the hydrogels were neutralized stepwise by adding neutralizing agents to each hydrogel: PBSxl, PBSxlO, and 0.1 M NaOH, shaking the bottle contents after each portion of NaOH added. The neutralization step continued until a neutral pH was reached, within the range of 7.2-7.4.

[0078] Table 1. Parameters of the procedure for preparing hydrogels from dECM.

[0079] 3.1. Rheological analysis of the composite biomaterial containing dECM hydrogel

[0080] The rheological properties of the composite biomaterial containing dECM hydrogel were evaluated. For the analysis, 20% (w / v) methacrylated gelatin (DS=84%) with 0.5% (w / v) LAP and 5% (w / v) dECM hydrogel from each of the three variants (Triton X-100, Till.01, Till.02) were used. The components were mixed in equal volume ratios using a dual-syringe system. Rheological measurements were performed using a MCR 72 rheometer (Anton Paar, Poland). The following relationships were used to determine the desired rheological parameters: а) Dependence of the complex modulus on the temperature gradient in the range of 0-40°C - determination of the material gelation point.

[0081] Based on the complex modulus versus temperature curves for the three variants (Triton X-100, Till.01, Till.02) in two replicates, the phase transition temperature of the tested materials was determined (Fig. б).

[0082] The biomaterial containing dECM obtained via the detergent-free decellularization process exhibited the highest phase transition temperature among the analyzed samples compared to the reference material (Triton X-100). A different thermal profile was observed for the materials containing dECM from batches Till.01 and Till.02. For dECM from batch Till.01, no gelation was observed in the 0 - 40°C range, suggesting that the phase transition of this system can be controlled by additional stimuli, such as combined crosslinking mechanisms. The material maintained stable working viscosity at both room temperature and near-physiological conditions. In contrast, the material containing dECM from batch Till.02 exhibited the lowest phase transition temperature within the studied range and high reproducibility of measurements, with only minor temperature deviations compared to the other samples. Such a profile - featuring predictable and well-controlled gelation under mild thermal conditions - can be particularly advantageous for 3D bioprinting and tissue engineering applications, where both printing stability and reproducibility of material properties are critical.

[0083] Results

[0084] All samples demonstrated gelation temperatures below the optimal range, suggesting that the cell -laden material could be less sensitive to shear stresses despite the general principle that low gelation temperature typically requires higher extrusion pressures. The results indicate that, even with relatively low gelation temperatures, the detergent-free dECM biomaterials produced smooth and continuous filaments at low extrusion pressures. b) Dependence of dynamic viscosity on the temperature gradient in the range of 40-5°C at a constant shear rate of 50 / s is shown in Fig. 7.

[0085] Based on these data, a qualitative assessment of the tested materials was conducted. All variants showed the same overall trend - a decrease in viscosity with increasing temperature, which is advantageous for bioprinting applications. The lowest viscosity at relatively low temperatures was observed for the material containing dECM from batch Till.01, which may facilitate its handling, mixing with cell suspensions, and loading into printer cartridges under minimal shear stress applied to the cells. The other material variants, including the material enriched with dECM from batch Till.02, exhibited similar viscosity levels above 15 °C, indicating comparable conditions and stability of the printing process in the working temperature range. The highest viscosity at very low temperatures was observed for the material enriched with dECM obtained via Triton X-100 decellularization, which may limit its suitability for 3D bioprinting applications (e.g., increasing the risk of nozzle clogging and complicating precise dispensing).

[0086] These observations suggest that materials containing dECM from batches Till.01 and Till.02, due to more favorable dynamic viscosity values in temperature ranges relevant for biomaterial preparation and printing, can be better suited for 3D bioprinting and tissue engineering applications.

[0087] Results High viscosity of the processed biomaterial provides significant resistance to shear stress during extrusion-based printing, which requires the application of higher pressures to force the material through the nozzle. The addition of dECM from batch Till.01 reduces the viscosity of the reference material, resulting in the lowest dynamic viscosity among all tested biomaterials. c) Dependence of the complex modulus on shear stress and strain for non-crosslinked and crosslinked samples (crosslinked for 30 s using 405 nm light at 28.5 mW / cm2). The dependence of the storage modulus and loss modulus on shear stress for non-crosslinked materials is shown in Fig. 8. The dependence of the storage modulus and loss modulus on strain for non-crosslinked materials is presented in Fig. 9.

[0088] The results indicate that the reference material and the material enriched with detergent-free dECM reach approximately 100% strain at relatively low strain stress values. This indicates very low mechanical stiffness and high susceptibility to deformation under small loads, suggesting the dominance of the viscous component over the elastic component and a low degree of structural crosslinking. In contrast, the sample containing dECM obtained using Triton X-100 exhibited the highest resistance to shear stress, indicating greater stiffness but also a higher risk of nozzle clogging and increased mechanical stress on cells during extrusion.

[0089] In this context, a more balanced mechanical profile, observed for materials containing dECM from batches Till.01 and Till.02 (intermediate stiffness and better controlled deformability), appears advantageous for 3D bioprinting and tissue engineering applications, where it is crucial to maintain both precise shaping of structures and their subsequent mechanical integrity.

[0090] Results

[0091] The dependence of the storage modulus and loss modulus on shear stress for materials crosslinked for 30 s using 405 nm light at 28.5 mW / cm2is shown in Fig. 10. The dependence of the storage modulus and loss modulus on shear stress and strain for the same crosslinked materials is presented in Fig. 11.

[0092] Across the entire strain range, the sample enriched with Triton X-100 dECM exhibits a storage modulus higher than the loss modulus, indicating that elastic properties dominate over viscous properties for this material. The material enriched with dECM from batch Till.02 deformed under higher shear stress compared to the reference material. The material enriched with dECM from batch Till.01 exhibited a low degree of crosslinking, resulting in low resistance to stress and rapid deformation under applied forces.

[0093] 3.2 Bioprinting of dECM-based Biomaterials As part of the conducted study, two constructs of each of the three material variants were printed for two time points, with dimensions of 20 x 20 * 2 mm and 100% infill, according to the g-code: 20x20x2 _4szt _lh04 _ew 09 infl 00 G4S10Z60. geode .

[0094] The table summarizes the printing parameters for each material variant, where the designation “Triton X-100” refers to the biomaterial obtained using the detergent-based method with Triton X-100.

[0095] Printability of biomaterials based on GelMA 20% (w / v) + LAP 0.5% (w / v) (G20 / L05) with the addition of dECM hydrogels

[0096] All tested material variants demonstrated good printability within an acceptable range of printing parameters, i.e., extrusion temperature and pressure.

[0097] Effect of dECM hydrogel addition on printing parameters

[0098] During the conducted studies, the optimal printing parameters were determined for each tested material. The addition of dECM hydrogel to a 20% (w / v) methacrylated gelatin (GelMA) solution with 0.5% (w / v) LAP reduced its printing temperature, with the lowest optimal printing temperature observed for the material containing dECM from batch Till.02. Very predictable and stable printing was achieved using the GelMA 20% + LAP 0.5% material enriched with dECM hydrogel from batch Till.02.

[0099] Cytotoxicity assessment using the MTT assay

[0100] Cell viability was evaluated using an indirect MTT assay, which is based on the reduction of the yellow tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT] to purple formazan crystals by metabolically active cells. The mouse fibroblast cell line L-929 (ATCC, CCL-1, Manassas, VA, USA) was used. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and antibiotics (penicillin and streptomycin, Coming, Glendale, AZ, USA). Cultures were maintained under standard conditions (37°C, 5% CO2).

[0101] Printed and crosslinked biomaterials (containing dECM obtained by detergent-based Triton X-100 method and detergent-free Till.02 method) were fragmented, weighed (300-350 mg per sample) into 5 mL Eppendorf tubes, and covered with DMEM medium. Samples were incubated at 37°C for 24 and 72 hours. Extracts obtained from biomaterials at 24 h and 72 h were added to cells seeded on 96-well plates at a density of 1 x 104cells / well. Cells with extracts and control wells were incubated at 37°C in 5% CO2 for 24 hours.

[0102] After incubation, the medium was removed, and MTT solution (1 mg / mL, Sigma-Aldrich, USA) was added. The absorbance of the formed formazan was measured at 570 run (reference 650 run) using a microplate spectrophotometer (BioTek, Winooski, VT, USA). Cell viability was calculated relative to untreated control cells (negative control, K-) according to the formula:

[0103] OD of the sample refers to the absorbance of the sample measured at X = 570 run (mean of 6 repetitions) . OD of the negative control refers to the absorbance of the negative control measured at X = 570 nm (mean of 6 repetitions).

[0104] According to the applicable standard ISO 10993-5:2009(E), a material is considered non-cytotoxic if the cell viability is >70%.

[0105] Results

[0106] Cell viability

[0107] The viability of L-929 cells after incubation with extracts obtained from bioinks based on decellularized extracellular matrix (dECM), both obtained using Triton X-100 and the detergent-free method (Till.02), remained above 70% after 24 and 72 hours, indicating no significant cytotoxicity of the extracts. In the dECM samples prepared using Triton X-100, a decrease in cell viability was observed after 72 hours of incubation, suggesting that residual detergent may gradually leach from the biomaterial over time and negatively affect the cells. In contrast, extracts from detergent-free dECM showed no significant decrease in cell viability at any of the tested time points. The effect of 24- and 72-hour extracts from dECM-based biomaterials on L-929 cells is presented in Fig. 13, with the 70% viability threshold indicated according to ISO 10993-5:2009(E) guidelines.

[0108] Glucose-Stimulated Insulin Secretion (GSIS) Test

[0109] Three-dimensional constructs were prepared by casting the tested biomaterial enriched with INS- IE cells into specially designed 3D-printed molds made of biomedical resin. Volumes of 300 pL of the material with 53 pL of cell suspension (3.5 million cells) were cast in portions. The material was crosslinked after each layer using a UV-VIS lamp for 30 s with 405 nm light at 28.5 mW / cm2. The resulting constructs were analyzed for glucose-stimulated insulin secretion (GSIS). Insulin secretion was assessed in rat P-cells (INS-1E) embedded in the bioprinted constructs of the tested biomaterials (Triton X-100 and Till.02) at a density of 3.5 x 106cells per construct. Constructs were incubated in high-glucose solution (16.7 mM) for 1 hour. Supernatants were collected at 10-minute intervals, and insulin content was measured using an enzyme-linked immunosorbent assay (ELISA) (Rat / Mouse Insulin ELISA Kit, Merck, Germany) according to the manufacturer’s instructions. The obtained values were used to assess the ability of the cells to secrete insulin in response to high glucose stimulation.

[0110] Results

[0111] Insulin secretion by P-cells (INS- IE) embedded in biomaterials containing dECM obtained by classical decellularization with Triton X-100 and by the detergent-free method (Till.02) was compared using the GSIS test. Cell cultures were maintained for 7 days, and insulin secretion measurements were performed on days 1, 4, and 7.

[0112] On days 1 and 4, significantly higher insulin secretion levels were observed in the matrix with dECM obtained using Triton X-100 compared to the matrix obtained by the detergent-free method (Till.02) (p < 0.05). By day 7 of culture, insulin secretion levels in both groups were comparable, with mean insulin values and standard deviations being practically identical, indicating no statistically significant difference between the variants.

[0113] The dynamics of insulin secretion by P-cells embedded in dECM matrices obtained by Triton X-100 and detergent-free methods (Till.02) were analyzed. In the Triton X-100 group, no statistically significant changes in insulin secretion were observed between days 1, 4, and 7 (ANOVA, p = 0.279), indicating no significant improvement in endocrine function over the culture period. In contrast, the Till.02 matrices showed a significant increase in insulin secretion overtime (ANOVA, p = 0.0057), suggesting enhanced P-cell functionality with prolonged culture. These results indicate that the detergent-free method reduces the risk of residual cytotoxic compounds and creates a more favorable environment for stabilizing P-cell endocrine activity.

[0114] Glucose-stimulated insulin secretion (GSIS) in p-cells (INS-1E) embedded in biomaterials containing dECM obtained by classical decellularization with Triton X-100 and by the detergent-free method (Till.02) is presented in Fig. 14.

[0115] References

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Claims

Claims1. A method for obtaining decellularized extracellular matrix of the pancreas, characterized in that it comprises the following steps, in which: a) the pancreas is incubated in a disinfecting solution, preferably comprising betadine at a concentration of 2% (v / v), then in a solution with an antimicrobial agent, preferably streptomycin at a concentration of 0.01% (w / v), and then frozen, b) the pancreas, warmed to a temperature of 4°C ± 2°C, is homogenized in a PBS solution and 5 mM EDTA-Na2 in a 3 : 1 ratio, and the homogenized material is centrifuged, after which the hydrophobic fat phase is removed, c) the material from step b) is suspended in a solution of NaCl at a concentration of 0.5 M + 5 mM EDTA-Na2 (pH = 7.4), then mixed, and subsequently the material is filtered, d) the material from step c) is suspended in a PBS solution and incubated, then sieved, and the sieved material is mechanically minced, e) the material from step d) is suspended in a PBS solution with 0.01% (w / v) streptomycin and incubated, then sieved, and the sieved material is mechanically minced, f) the material from step e) is suspended in a PBS solution with 0.01% (w / v) streptomycin and incubated for 2 hours at 4°C ± 2°C, this step f) being repeated at least twice, and the sieved material is mechanically minced after each filtration, g) the obtained extracellular matrix from step f) is sieved, frozen, and subsequently subjected to lyophilization and mechanical grinding.

2. The method for obtaining decellularized extracellular matrix of the pancreas according to claim 1, characterized in that in step b) the homogenized material is centrifuged at a speed of 4000 rpm for 5 minutes at a temperature in the range of 2 °C to 8 °C.

3. The method for obtaining decellularized extracellular matrix of the pancreas according to claim1 or 2, characterized in that the sample in step b) is mixed at a speed of 150 rpm at a temperature in the range of 2°C to 6°C for 1-2 hours.

4. The method for obtaining decellularized extracellular matrix of the pancreas according to claims 1-3, characterized in that the sample in step c) is mixed at a speed of 150 rpm at a temperature in the range of 2°C to 6°C for 16 hours.

5. The method for obtaining decellularized extracellular matrix of the pancreas according to claims 1-4, characterized in that the sample in step d) and / or e) is incubated for 2 hours, mixing at a speed of 150 rpm at 4°C.

6. The method for obtaining decellularized extracellular matrix of the pancreas according to claims 1-5, characterized in that step d) and / or e) is repeated three times.

7. The method for obtaining decellularized extracellular matrix of the pancreas according to claims 1-6, characterized in that before the start of step f), the material is immersed in ethanol at a concentration of 70% and mixed at a speed of 150 rpm at a temperature in the range of 2°C to 6°C for 30 minutes.

8. The method for obtaining decellularized extracellular matrix of the pancreas according to claims 1-7, characterized in that the material obtained in step g) is subjected to radiation sterilization using 25 kGy of radiation.

9. Use of decellularized matrix obtained by the method according to claims 1-8 as a component of a bioink intended for bioprinting.

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