Preparation method of tissue-derived extracellular matrix hydrogel

Abstract

The present disclosure relates to a method for preparing an extracellular matrix hydrogel, the method comprising (a) decellularizing a tissue sample to form a decellularized tissue web; (b) depolymerizing the decellularized tissue web to form a depolymerized protein dispersion, the depolymerizing comprising mixing the decellularized tissue web with a depolymerizing solution comprising a chaotropic agent to form a depolymerized mixture; mechanically homogenizing the depolymerized mixture at a cooling temperature to form a homogenized mixture; separating the homogenized mixture into (a) a waste precipitate and (b) a supernatant comprising the depolymerized protein dispersion; (c) filtering the depolymerized protein dispersion to form a sol; and (d) gelling the sol to form the extracellular matrix hydrogel, the gelling comprising combining the sol with a gelling solution comprising a lyophilic agent; and separating a liquid phase from the extracellular matrix hydrogel during the formation of the extracellular matrix hydrogel.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a method of preparation of human or animal tissue-derived extracellular matrix hydrogel for applications in cell culture and organ bioprinting.

[0002] Cross Reference to Related Applications

[0003] This application claims priority to Ukrainian Patent Application No. a202403745, filed July 22, 2024, the entire contents of which are incorporated herein by reference in their entirety. BACKGROUND

[0004] Extracellular matrix (ECM) is a complex polymer network characterized by having an ill-defined physical state. In its liquid phase, ECM can facilitate diffusion of compounds while exhibiting varying degrees of structural dynamics, solid-like rigidity, and viscoelasticity. When used as a scaffold for developing organisms, ECM can transition between a tissue-organized solid structure and a disordered fluid state, which is a dynamic key to supporting morphogenesis.

[0005] Collagen, the main component of mammalian ECM, forms a polymer network through its triple helix structure. This polymer network has a transient stable region at temperatures ranging from 32 °C to 40 °C and at physiological pH, while it melts when exceeding a certain temperature. Reconstituting collagen in acid and then neutralizing it forms a sol-gel mixture with a critical gel point at about 35 °C. Other components such as fibronectin, elastin, and laminin are usually incorporated with polysaccharide fragments that contribute to the rigidity of the resulting network. Historically, collagen, basement membrane extract, and various natural, semi-synthetic, and synthetic hydrogels have been used as ECM models. Hydrogels are polymers that are kinetically trapped in a colloidal state, which have the advantage of being able to encapsulate both the kinetic and thermodynamic features of the extracellular matrix (ECM) tissue formation process.

[0006] Currently, researchers utilize three types of hydrogels to mimic natural tissue scaffolds. These include Engelbreth-Holm-Swarm (EHS) mouse basement membrane extract, synthetic polymers modified with collagen peptide residues, and pepsin-digested human-derived hydrogels. One common problem with these approaches is that they either do not include the initial extracellular matrix proteins or they disrupt these proteins. Furthermore, none of these options replicate the natural ECM, as EHS-derived hydrogels have non-native protein components compared to human tissues, synthetic polymers lack the complexity of natural tissues, and pepsin-digested human-derived hydrogels have altered primary structures of ECM proteins. There is a need for a sol-gel system extracted from human tissues that does not have modified native proteins and can replicate the structure, physicochemical properties, and biocompatibility of human extracellular matrix. Summary of the Invention

[0007] This disclosure relates to a method for preparing an extracellular matrix hydrogel. The method may include decellularizing a tissue sample to form a decellularized tissue mesh. Decellularization may include mixing the tissue sample with a washing solution to form a decellularized mixture, wherein the tissue sample is derived from a tumor with a fibrosis content ranging from about 40% to about 90%. Decellularization may include separating the decellularized mixture into cellular waste fluid and the decellularized tissue mesh. The method may include depolymerizing the decellularized tissue mesh to form a depolymerized protein dispersion. Depolymerization may include mixing the decellularized tissue mesh with a depolymerization solution containing a chaotropic agent and a surfactant to form a depolymerized mixture. Depolymerization may include mixing the decellularized tissue mesh with a depolymerization solution containing a chaotropic agent. Depolymerization may include mechanically homogenizing the depolymerized mixture at a cooling temperature to form a homogenized mixture. Depolymerization may include separating the homogenized mixture into (a) a waste precipitate and (b) a supernatant containing the depolymerized protein dispersion. In some embodiments, the waste precipitate may include a surfactant. The method may include filtering the depolymerized protein dispersion to form a sol. This method may include gelling a sol to form an extracellular matrix hydrogel. Gelation may include mixing the sol with a gelation solution containing a kosmotropic agent. Gelation may also include separating the liquid phase from the extracellular matrix hydrogel during the formation of the hydrogel.

[0008] This method may include sterilizing the sol during filtration. Sterilization may include maintaining the sol in the permeate loop while circulating the halogenated solvent in the permeate loop and collecting the sterilized sol in the permeate loop. Sterilization may include mixing the sol with the halogenated solvent. This method may include applying a vacuum to the sterilized sol. This method may include adding a hygroscopic agent to the gelation solution to adjust the stiffness of the extracellular matrix hydrogel. Tumors may include sarcoma, secondary liver cancer, liver fibrosis, osteosarcoma, chondrosarcoma, a mixture of a heart fibrosis, a lung tissue and lung fibrosis, mesenchymal tissues, and healthy organs and combinations thereof. Separating the homogenized mixture into waste precipitate and supernatant may include centrifugation. Filtration of the depolymerized protein dispersion may be performed at a temperature of about 4°C. This method may include using chloroform as a halogenation solvent. The halogenation solvent and the sol may be mixed at a concentration of about 0.4% v / v to about 3% v / v. The mixing of the halogenation solvent and the sol may continue for a period of about 3 hours to about 12 hours.

[0009] In some embodiments, mixing the tissue sample with the washing solution may include mixing the tissue sample and the washing solution at a speed ranging from about 25,000 rpm to about 75,000 rpm for at least about 30 minutes. The washing solution may contain about 0.34 M sodium chloride, about 1 mM ethylmaleimide, about 25 mM disodium ethylenediaminetetraacetate, and combinations thereof. Filtration of the depolymerized protein dispersion may be carried out at a flow rate of about 10 mL / min to about 70 mL / min and a temperature of about 0°C to about 8°C. In some embodiments, the decellularized tissue network may have glycosylated protein tails, wherein, by weight, the depolymerized protein dispersion derived from the decellularized tissue network may have at least about 95% of the amount of glycosylated protein tails compared to the decellularized tissue network. The method may include retaining at least about 95% of the glycosylated protein tails in the depolymerized protein dispersion relative to the decellularized tissue network. Surfactants may include at least one of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, Tween-20, Tween-80 (polysorbate-80), Triton X-100, and Pluronic F-127, and combinations thereof. Release agents may include at least one of sodium dodecyl sulfate, guanidine chloride, guanidine cyanosulfate, urea, guanidine, thiourea, calcium salts, nitrates, dithiothreitol, mercaptoethanol, tributylphosphine, sodium borohydride, lithium perchlorate, lithium acetate, magnesium chloride, and combinations thereof.

[0010] The method may include applying a vacuum at a temperature of about 0°C to about 8°C for about 1 hour to about 3 hours. The hygroscopic agent may include potassium citrate. The extracellular matrix hydrogel may have a stiffness of about 1 kPa to about 250 kPa. The method may also include mechanically homogenizing the depolymerized mixture at a temperature of about 4°C. Filtration may include at least one of double flow diafiltration, dialysis bag filtration, flow diafiltration, hollow fiber ultrafiltration, cartridge hollow fiber filtration, and cartridge tangential flow ultrafiltration. The yield of the depolymerized protein dispersion may be at least about 70 mg / mL (varying from 10 mg / mL in lipid-rich tissues to 110 mg / mL in heavily fibrotic tissues, depending on the initial value). The cooling temperature may be in the range of about 0°C to about 8°C.

[0011] The ECM hydrogels prepared by the disclosed method exhibit human primary hepatocyte adhesion values ​​ranging from about 1 kPa to about 10 kPa. The ECM hydrogels prepared by the disclosed method also exhibit FAK expression induction values ​​ranging from about 2 to about 10 times. The ECM hydrogels are non-toxic to mice at working concentrations. The ECM hydrogels can be incorporated into cells approximately 10 mm in size. 3 / day to approximately 200mm 3 The in situ tumor growth rate is within the range of / day. The ECM hydrogel formed by the method of this disclosure may include a filtration buffer used during filtration, comprising at least one of water, sodium dodecyl sulfate at a concentration ranging from about 0.5 vol% to about 2 vol%, and sodium chloride at a concentration ranging from about 0.1 M to about 4 M. Gelation may include raising the temperature of the sol from about 0°C to about 8°C to about 26°C to about 50°C for a period of time of at least about 1 minute.

[0012] In some embodiments, this disclosure relates to a method for preparing an extracellular matrix hydrogel. The method may include decellularizing a tissue sample to form a decellularized tissue network. Decellularization may include mixing the tissue sample with a washing solution to form a decellularized mixture, wherein the tissue sample is derived from a tumor with a fibrosis content ranging from about 40% to about 90%. Decellularization may include separating the decellularized mixture into cellular waste fluid and a decellularized tissue network. The method may include depolymerizing the decellularized tissue network to form a depolymerized protein dispersion. The method may include filtering the depolymerized protein dispersion through a tubular hollow fiber using a salt gradient to form a sol. The method may include gelling the sol to form the extracellular matrix hydrogel. Gelation may include (i) combining the sol with a gelling solution containing a lyophilizing agent; and (ii) separating the liquid phase from the extracellular matrix hydrogel during the formation of the extracellular matrix hydrogel.

[0013] In some embodiments, the method may include filtering the depolymerized protein dispersion through a tubular hollow fiber using a salt gradient to form a sol. The salt gradient used for filtration may include salts selected from sodium chloride, potassium chloride, zinc chloride, magnesium chloride, guanidine chloride, and combinations thereof. The salt gradient for filtration may include concentrations in the range of about 0.001 M to about 1 M. The tubular hollow fiber may include porous filaments in the range of about 5 kDa to about 200 kDa. In some embodiments, filtration may be performed at a flow rate of about 1 mL / min to about 1 L / min. Attached Figure Description

[0014] This disclosure is illustrated by way of example, not limitation, in the accompanying drawings, wherein the same reference numerals are used to refer to similar elements. It should be emphasized that the features may not be drawn to scale, and the dimensions of the features may be arbitrarily enlarged or reduced for clarity of explanation.

[0015] Figure 1 This is a flowchart illustrating the preparation of extracellular matrix hydrogels according to some embodiments of the present disclosure;

[0016] Figure 2 This is a flowchart illustrating the decellularization of tissue samples in a decellularization reactor according to some embodiments of the present disclosure;

[0017] Figure 3A These are photographs of stained tissue samples that have not undergone decellularization according to some embodiments of this disclosure;

[0018] Figure 3B These are photographs of stained, decellularized tissue reticulum according to some embodiments of this disclosure;

[0019] Figure 4 This is a flowchart illustrating the process of depolymerizing decellularized tissue networks to form depolymerized protein dispersions according to some embodiments of this disclosure;

[0020] Figure 5 This is a graph showing the change of guanidine hydrochloride concentration over time according to some embodiments of this disclosure;

[0021] Figure 6 This is a graph showing the relationship between guanidine hydrochloride and dialysis after 600 minutes according to some embodiments of this disclosure;

[0022] Figure 7 This is a flowchart of a percolation reactor setup for filtering depolymerized protein dispersions to form a sol, according to some embodiments of this disclosure.

[0023] Figure 8 This is a flowchart of a tangential flow filtration process according to some embodiments of this disclosure;

[0024] Figure 9A These are images of colloidal morphologies formed and fixed on a surface after a percolation process according to some embodiments of this disclosure;

[0025] Figure 9B It is a graph showing the average particle height (y-axis) and average diameter (x-axis) of small colloids formed according to some embodiments of this disclosure;

[0026] Figure 9C These are images of the highly corrected morphology of a gelled hSTS mixture fixed on a mica surface according to some embodiments of this disclosure;

[0027] Figure 9D This is a photograph showing the calculated D-band of fibers formed according to some embodiments of this disclosure;

[0028] Figure 10A This is a particle distribution diagram showing the number of particles according to some embodiments of this disclosure;

[0029] Figure 10B This is a particle distribution diagram based on intensity statistics according to some embodiments of this disclosure;

[0030] Figure 11 Various cell images according to some embodiments of this disclosure are shown;

[0031] Figure 12A In situ tumor progression in Balb / c mice with 4T1 subcutaneous tumors according to some embodiments of the present disclosure is shown;

[0032] Figure 12B The weight progression of Balb / c mice with 4T1 subcutaneous tumors according to some embodiments of this disclosure is shown;

[0033] Figure 13A Orthotopic tumor progression in SCID mice with MDA-MB-231 subcutaneous tumors according to some embodiments of this disclosure is shown; and

[0034] Figure 13B The weight progression of SCID mice with MDA-MB-231 subcutaneous tumors according to some embodiments of the present disclosure is shown. Detailed Implementation

[0035] This disclosure relates to methods for preparing extracellular matrix (ECM) hydrogels. The methods of this disclosure produce hydrogels that advantageously possess intact primary and secondary protein structures relative to the original tissue sample source. The intact primary and secondary protein structures allow the ECM hydrogels of this disclosure to advantageously achieve more accurate tissue modeling compared to known hydrogels. The methods of this disclosure produce ECM hydrogels with enhanced physiological properties, which allow for natural angiogenesis in artificial organs. The methods of this disclosure produce ECM hydrogels that are advantageously recognized by the host immune system, a feature not possessed by known hydrogels. Recognition by the host immune system increases the susceptibility of the hydrogels of this disclosure to cleavage by host matrix metalloproteinases, allowing them to be naturally cleared by the host body. Furthermore, human-derived hydrogels can increase cell adhesion in a manner different from animal-derived or synthetic polymers, thus promoting cells to present a more natural morphology and physiological state. These hydrogels can be used as matrices for assays based on complex cells, such as organ-on-a-chip or scaffold-based spherical cultures.

[0036] Figure 1A method for preparing ECM hydrogels disclosed herein is shown. For example... Figure 1 As shown, the method disclosed herein may include shredding and filtering tissue sample 105 to produce shredded tissue 110. The method may include decellularizing the shredded tissue 110 to form a decellularized tissue web 115. The method disclosed herein may include depolymerizing the decellularized tissue web 115 by mixing it with a depolymerization solution containing a dissociating agent, thereby forming a depolymerized mixture. The method may include mechanically homogenizing the depolymerized mixture to form a homogenized mixture, and then separating the homogenized mixture into a waste precipitate and a supernatant 120 containing depolymerized protein dispersions. Figure 1 As shown, the method may include filtering depolymerized protein dispersion 120, for example, by dual-flow filtration, to form sol 125. The method disclosed herein may include sterilizing sol 125 with a halogenated solvent such as chloroform. Sol 125 may be dispensed into vials 130 or any other known storage unit. In some embodiments, the method may include gelling sol 125 to form ECM hydrogel 135.

[0037] Harvesting and storing cancer-derived tissue

[0038] According to some embodiments, this disclosure relates to a method for preparing ECM hydrogels from tissue samples. The method of this disclosure may include collecting tissue samples from cancer-derived tissue. Cancer-derived tissue can be obtained from humans or animals. Compared to non-cancer tissue, cancer-derived tissue can have a longer delivery window range after collection and before processing, before it begins to degrade. The delivery window at +4°C is at most 2-10 hours.

[0039] Obtaining tissue samples from cancer-derived tissue can include excision, tearing, removal, cutting, and any other known method for obtaining such tissue. Under cold ischemic conditions, tissue samples can have a delivery window of approximately 4 hours to approximately 10 hours. In some embodiments, under cold ischemic conditions, tissue samples can have a delivery window of approximately 4 hours, or approximately 5 hours, or approximately 6 hours, or approximately 7 hours, or approximately 8 hours, or approximately 9 hours, or approximately 10 hours, where “approximately” includes plus or minus 0.5 hours. Cold ischemic conditions can include at least partial immersion in a solution, including phosphate buffer, Custodiol solution, histidine-tryptophan-ketoglutarate (HTK) solution, Dulbecco's Modified Eagle Medium (DMEM), antibiotic-containing Roswell Park Memorial Institute (RPMI) solution, heparin sulfate, etc.

[0040] The methods disclosed herein may include examining tissue samples for blood clots, lipid deposits, or other inclusions. Upon discovery of blood clots, lipid deposits, and / or other inclusions, the methods of this disclosure may include surgically removing them from the tissue sample. In some embodiments, the methods of this disclosure may include collecting tissue samples with a high fibrosis content, such that the tissue sample is advantageously enriched with ECM. For example, the method may include collecting tissue samples with a fibrosis content of about 40% to about 90% or higher by weight of cancer-derived tissue. The fibrosis content of the tissue sample may be about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% by weight of the tissue sample, where “about” includes plus or minus 5%. The measurements used in this particular work are based on area estimates from immunohistochemical sections.

[0041] In some embodiments, the method includes collecting tissue samples from tumors derived from humans or animals. Tumors may include sarcomas, secondary liver cancer, liver fibrosis, osteosarcoma, chondrosarcoma, mesenchymal tumors, carcinomas, mixtures of cardiac fibrosis, lung tissue and pulmonary fibrosis, mesenchymal tissue and healthy organs, human soft tissue sarcoma (HSTS), and combinations thereof. Tumors, such as HSTS, may advantageously include high levels of type I, III, and IV collagen, fibronectin, elastin, glycosylated laminin, non-glycosylated laminin, and combinations thereof. Tumors may be obtained from fresh tissue (e.g., human or animal) or frozen tissue (e.g., human or animal). For example, prior to collection, the method may include freezing the tumor to approximately 0°C, or approximately -10°C, or approximately -20°C, or approximately -30°C, or approximately -40°C, or approximately -50°C, or approximately -60°C, or approximately -70°C, or approximately -80°C, or a lower temperature, where “approximately” includes ±5°C. This method may include freezing the tumor in liquid nitrogen and then transferring it to a cryostat. In some embodiments, the tumor can be frozen for at least approximately six months without showing signs of degradation. The tumor may be shredded before freezing, which can advantageously enhance protein preservation.

[0042] Decellularizing tissue samples

[0043] According to some embodiments, methods for preparing ECM hydrogels may include decellularizing a tissue sample to form a decellularized tissue network and cellular waste. Decellularization may include shredding the tissue sample to form shredded tissue, and then decellularizing the shredded tissue to form a decellularized tissue network. The decellularized tissue network may include water, a mixture of proteins including collagen, and polysaccharides. Cellular waste may include removed cells. In some embodiments, residual cells that were not removed during decellularization can be detected within the decellularized tissue network. For example, residual cells may be detected by residual cell nuclei or lipid inclusions, which must be substantially removed to ensure a DNA-free and lipid-free colloid.

[0044] This method may include shredding a tissue sample to form shredded tissue with a size range of about 1 mm to about 10 mm or larger. For example, the shredded tissue may have dimensions of about 0.1 mm, or about 1 mm, or about 2 mm, or about 3 mm, or about 4 mm, or about 5 mm, or about 6 mm, or about 7 mm, or about 8 mm, or about 9 mm, or about 10 mm, where “about” includes plus or minus 0.5 mm. The size of the shredded tissue network may be measured in length, width, diameter, or any number of dimensions. This method may include allowing the shredded tissue to stand at a temperature of about 25°C or lower, which may facilitate the separation of the water-containing liquid portion from the solid portion containing proteins and polysaccharides. In some embodiments, the solid portion may be separated from the liquid portion. The solid portion may be weighed, and this weight is used to determine the protein yield using standard calculations.

[0045] The method disclosed herein may include mixing a tissue sample with a washing solution to form a decellularized mixture. If the tissue sample is shredded, the method may include combining the shredded tissue with a washing solution to form a decellularized mixture. The decellularized mixture may be separated into cellular waste fluid and a decellularized tissue network containing proteins and polysaccharides. Mixing the tissue sample or shredded tissue with the washing solution may be carried out in a batch reactor, a flow reactor, or a combination of both. Mixing the shredded tissue or shredded tissue with the washing solution may include maintaining a temperature in the range of about 0°C to about 35°C. For example, the temperature may be maintained at about 0°C, or about 5°C, or about 10°C, or about 15°C, or about 20°C, or about 25°C, or about 30°C, or about 35°C, wherein “about” includes ±2.5°C. The washing solution may include salts, including NaCl, KCl, LiCl, CsCl, CaCl2, other salts, and mixtures thereof. The washing solution may include salts with concentrations ranging from about 0.1 M to about 10 M or higher. For example, the washing solution may include salts with a concentration of about 0.1 M, or about 1 M, or about 2 M, or about 3 M, or about 4 M, or about 5 M, or about 6 M, or about 7 M, or about 8 M, or about 9 M, or about 10 M, where “about” includes ±0.5 M. Mixing the tissue sample or shredded tissue with the washing solution may be performed at a rotational speed of about 1,000 rpm to about 80,000 rpm or higher, and for at least about 5 minutes. For example, mixing may be performed at a rotational speed of about 1,000 rpm, or about 10,000 rpm, or about 20,000 rpm, or about 30,000 rpm, or about 40,000 rpm, or about 50,000 rpm, or about 60,000 rpm, or about 70,000 rpm, or about 80,000 rpm, where “about” includes ±5,000 rpm. The mixing can take place for a period of at least about 5 minutes, or at least about 10 minutes, or at least about 15 minutes, or at least about 20 minutes, or at least about 25 minutes, or at least about 30 minutes, or at least about 35 minutes, or at least about 40 minutes, or about 45 minutes, or about 50 minutes, or about 55 minutes, or about 6 minutes, where “about” includes plus or minus 2.5 minutes. In some embodiments, the mixing can take place for a period of about 1 hour to about 24 hours. For example, the mixing can take place for a period of about 1 hour, or about 4 hours, or about 8 hours, or about 12 hours, or about 16 hours, or about 20 hours, or about 24 hours, where “about” includes plus or minus 2 hours.

[0046] Decellularizing tissue samples may include mixing shredded tissue with more than one washing solution. For example, decellularization may include two, three, four, five, or more washing solutions. Each washing solution may also include a nonionic surfactant. Nonionic surfactants may include Triton X, Triton X-100, Tween, Tween 20, Tween 80, sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, polysorbate-80, Pluronic F-127, ethylenediamine, ethyl maleimide, and combinations thereof. Other reagents, such as disodium ethylenediaminetetraacetate (EDTA), may be included. Both nonionic and ionic surfactants may be included at concentrations from about 0.1 mM to about 1,000 mM or higher.

[0047] Decellularizing a tissue sample may include centrifuging a decellularized mixture to separate the decellularized mixture into cellular waste and a decellularized tissue web. Decellularizing a tissue sample may include filtering the decellularized mixture to separate the cellular waste and the decellularized tissue web. Decellularizing a tissue sample may include dynamically separating the components of the decellularized mixture. In some embodiments, decellularizing a tissue sample may be iterative. For example, the method disclosed herein may include multiple cycles of mixing shredded tissue with a washing solution to form a decellularized mixture, and then separating the decellularized mixture into cellular waste and a decellularized tissue web. The decellularized tissue web may be combined with an additional washing solution before further separation. In some embodiments, the tissue sample may be subjected to sonication, freeze-thaw cycles, and other known decellularization methods.

[0048] Figure 2 An embodiment is shown in which tissue sample 205 is decellularized to form a decellularized tissue network 215. For example... Figure 2 As shown, tissue sample 205 can be placed in mixer 240 for approximately five minutes to form a mixed tissue sample. The mixed tissue sample can be placed in filter 245 and rinsed with a 0.9% NaCl washing solution containing a protease inhibitor. After filtration and rinsing, shredded tissue 210 can be retained with a diameter of 2-3 mm. Shredded tissue 210 can be placed in decellularization reactor 250, which has a reagent inlet, pH meter, coolant inlet, coolant outlet, and filter outlet. After decellularization in decellularization reactor 250, the decellularized tissue net 215 can be separated from the reactor.

[0049] In some embodiments, the decellularized tissue network 215 may have dimensions ranging from about 0.1 mm to about 10 mm or greater. For example, the decellularized tissue network 215 may have dimensions of about 0.1 mm, or about 1 mm, or about 2 mm, or about 3 mm, or about 4 mm, or about 5 mm, or about 6 mm, or about 7 mm, or about 8 mm, or about 9 mm, or about 10 mm, where "about" includes plus or minus 0.5 mm. The dimensions of the decellularized tissue network 215 may be measured in length, width, diameter, or any number of dimensions.

[0050] After decellularizing the tissue sample, the method may include staining the decellularized tissue network 215 with a staining agent to confirm decellularization, such as... Figure 3A and Figure 3B As shown. Figure 3A It is a photograph of a stained tissue sample. Figure 3B This is a photograph of a stained, decellularized tissue network. The staining agent can include any known staining agent, including propidium iodide, which... Figure 1 This is used to show differences between tissue samples before and after decellularization. Staining agents may also include crystal violet, methylene blue, 7-AAD, acridine orange, calcein, carboxyfluorescein succinimide (CFSE), 4',6-diamidinyl-2-phenylindole (DAPI), Hoechst dye, trypan blue, tetrazolium salts, and combinations thereof.

[0051] Subsequent steps will largely depend on the structure of the decellularized tissue network. To aid in further decellularization, the tissue can be additionally pulverized using co-grinding with dry ice. After the remaining dry ice evaporates, the tissue network appears to be 0.1–0.7 mm in size.

[0052] Disaggregating decellularized tissue

[0053] like Figure 4 As shown, according to some embodiments, the preparation method of ECM hydrogel may include depolymerizing a decellularized tissue network 415 to form a depolymerized protein dispersion 460. Depolymerization may include mixing the decellularized tissue network 415 with a depolymerization solution to form a depolymerization mixture. At this stage, primarily highly insoluble structural proteins (collagen, fibronectin, and elastin) are present in the tissue along with encapsulated smaller proteins. The depolymerization solution may include a dissociation agent and a surfactant. Figure 4 As shown, depolymerization can occur in depolymerization reactor 455. (As indicated...) Figure 4As shown, the depolymerization reactor 455 may include various components, including a vacuum outlet, a homogenizer, a feed inlet, a temperature bar, a rotary stirrer, and a cooling platform. Within the depolymerization reactor 455, depolymerization according to this disclosure may include mechanically homogenizing the depolymerized mixture at a cooling temperature (e.g., less than 25°C) to form a homogenized mixture. Depolymerization may include separating the homogenized mixture into (a) a waste precipitate containing a surfactant and (b) a supernatant containing the depolymerized protein dispersion, which may be collected in a collection bottle containing the depolymerized protein dispersion 460. The method of this disclosure can advantageously form a depolymerized protein dispersion 460 having substantially retained glycosylated protein tails relative to a decellularized tissue network. The substantially retained glycosylated protein tails can provide a final ECM hydrogel with an increasingly complex microenvironment. In some embodiments, depolymerization can produce a depolymerized protein dispersion 460 having about 10%, about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 99% of glycosylated protein tails retained relative to the decellularized tissue network, where “about” includes plus or minus 5%. In some embodiments, retaining the glycosylated protein tails can ensure suitable viscosity (e.g., >10 mPa*s) and elastic modulus (e.g., in the range of 1 kPa to about 100 kPa) of the ECM hydrogel formed according to this disclosure. In some embodiments, having suitable viscosity and elastic modulus is necessary for cell-matrix recognition of the ECM hydrogel of this disclosure. The retained or removed glycosylated protein tails (i.e., glycosylation sites) can be determined by orbital trap LC-MS of the depolymerized protein dispersion after initial tissue network and / or after the tissue network has been dissolved by proteases and / or depolymerized. In some embodiments, gel electrophoresis can indicate the preservation or removal of glycosylated protein tails of the depolymerized protein dispersion relative to the tissue network. In some embodiments, acceptable glycosylated protein tail loss, based on the final protein molecular weight (MW) of the depolymerized protein dispersion, can be between about 20% and about 80%, depending on the specific details of the procedure.

[0054] By mixing the decellularized tissue network 415 with a depolymerization solution containing a dissociating agent, the polymeric proteins contained in the decellularized tissue network can be dispersed via the dissociating effect. The dissociating agent may include sodium dodecyl sulfate, guanidine chloride, guanidine cyanosulfate, urea, guanidine, thiourea, calcium salts, nitrates, dithiothreitol, mercaptoethanol, tributylphosphine, sodium borohydride, lithium perchlorate, lithium acetate, magnesium chloride, and combinations thereof. The dissociating agent may be contained in the depolymerization solution at a concentration of about 0.1 M to about 10 M or higher. In some embodiments, the solubility of the dissociating agent may affect the upper limit of the maximum concentration of the dissociating agent contained in the depolymerization solution. For example, the dissociating agent may be contained in the depolymerization solution at a concentration of about 0.1 M, or about 1 M, or about 2 M, or about 3 M, or about 4 M, or about 5 M, or about 6 M, or about 7 M, or about 8 M, or about 9 M, or about 10 M, where “about” includes ±0.5 M.

[0055] Depolymerization of the decellularized tissue network 415 to form a depolymerized protein dispersion 460 can be performed at temperatures ranging from about -10°C to about 25°C. For example, depolymerization can be performed at temperatures of about -10°C, or about -5°C, or about 0°C, or about 5°C, or about 10°C, or about 15°C, or about 20°C, or “about” includes ±2.5°C. The method of this disclosure can depolymerize the decellularized tissue network 415 while maintaining the structural integrity of the proteins, thereby preserving the structural integrity of the proteins found therein (e.g., glycosylated protein tails). In some embodiments, the method of this disclosure can depolymerize the decellularized tissue network 415 while maintaining temperatures below about 20°C, whereas known methods require temperatures above 25°C, resulting in a loss of protein structural integrity.

[0056] The pH of the depolymerization solution can be maintained within a specific range during the depolymerization of the decellularized tissue network 415 of this disclosure. The pH of the depolymerization solution can be maintained in the range of about 5 to about 10. For example, the pH of the depolymerization solution can be about 5, or about 6, or about 7, or about 8, or about 9, or about 10, where "about" includes plus or minus 0.5. For example, the pH of the depolymerization solution can have a range of about 6 to about 9.

[0057] The depolymerization solution may include a surfactant during depolymerization. Surfactants may include sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, Tween 20, Tween 80 (polysorbate-80), Triton X-100, Pluronic F-127, and combinations thereof. The surfactant may be included in the depolymerization solution at a concentration of about 0.1% to about 10% or higher of the depolymerization solution. For example, the surfactant may be included in the depolymerization solution at a concentration of about 0.1%, or about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10% of the depolymerization solution, where “about” includes plus or minus 0.5%. The maximum concentration of the surfactant is limited by the solubility of the surfactant in the depolymerization mixture. The depolymerization solution may also include buffers, including but not limited to Tris-HCl, phosphate-buffered saline, citrate buffer, etc. The depolymerization solution of this disclosure advantageously does not require protein digestive enzymes such as pepsin, trypsin, and chymotrypsin. The depolymerization solution may include, but does not need, a reducing agent, such as dithiothreitol, mercaptoethanol, PBu3, and NaBH4.

[0058] In some embodiments, depolymerization can be carried out in a depolymerization reactor 455 having various components. For example, depolymerization can be carried out in a depolymerization reactor 455 having one or more of the following: an upper head stirrer, a magnetic stirrer, a mechanical pulverizer, a rotary frame stirrer, a mechanical homogenizer, a high-pressure homogenizer, an ultrasonic homogenizer, a stator-rotor mechanism, a loading inlet for solid and liquid reagents, a temperature jacket, an ice bath device, a viscometer, a pH measuring rod, and a temperature rod. The depolymerization reactor 455 can have various sizes from 1 mL to about 10 L or more. The depolymerization reactor 455 can be connected to a pump (e.g., a peristaltic pump, a vacuum pump) that can facilitate the discharge of the reactor contents once depolymerization is complete. Depolymerization of this disclosure can be carried out using multiple homogenization devices, for example, by simultaneous homogenization via both a mechanical pulverizer and a stator-rotor mechanism.

[0059] The method disclosed herein may include separating a homogenized mixture into a waste precipitate and a supernatant 460 containing depolymerized protein dispersions. Separation may include any known form, including batch filtration, centrifugation, decantation, and flow filtration. Separation may include separating any surfactant from the supernatant. Figure 4 As shown, separation can provide a depolymerized protein dispersion that is essentially free of waste 475. Figure 4The diagram illustrates how the collected depolymerized protein dispersion 460 can be separated by centrifugation 470 while being cooled in an ice bath, and then decanted into another collection flask containing a substantially waste-free depolymerized protein dispersion 475. For example, the method may include separating the homogenized mixture into a supernatant and a waste precipitate containing protein aggregates. Separation can be carried out at temperatures from about -4°C to about 24°C. For example, separation can be carried out at temperatures from about -4°C, or about 0°C, or about 4°C, or about 8°C, or about 12°C, or about 16°C, or about 20°C, or about 24°C, where “about” includes ±2°C. Batch or flow separation can be carried out at flow rates from about 1 mL / min to about 1 L / min or higher. Separation can be carried out in batches or in a flow at rates of about 1 mL / min, or about 100 mL / min, or about 200 mL / min, or about 300 mL / min, or about 400 mL / min, or about 500 mL / min, or about 600 mL / min, or about 700 mL / min, or about 800 mL / min, or about 900 mL / min, or about 1,000 mL / min, where “about” includes ±50 mL / min. For example, separation can be carried out in a flow at a temperature of about 0°C to about 8°C at a rate of about 10 mL / min to about 70 mL / min.

[0060] According to some embodiments, the methods of this disclosure may include analyzing depolymerized protein dispersions formed by depolymerizing decellularized tissue networks. The analysis may include measuring yield, swelling consistency, protein hydrolysis level, and other measurements. These parameters may be measured using known methods, including but not limited to size exclusion chromatography, dynamic light scattering (DLS), atomic force microscopy, turbidimetry, ELISA, gel electrophoresis, Western blotting, size exclusion chromatography, 1H nuclear magnetic resonance spectroscopy (NMR), and 13C NMR. The yield of the depolymerized protein dispersion may be from about 5 mg / mL to about 200 mg / mL. For example, the yield of the depolymerized protein dispersion may be about 5 mg / mL, or about 20 mg / mL, or about 40 mg / mL, or about 60 mg / mL, or about 80 mg / mL, or about 100 mg / mL, or about 120 mg / mL, or about 140 mg / mL, or about 160 mg / mL, or about 180 mg / mL, or about 200 mg / mL, where “about” includes ±5 mg / mL. In some implementations, the yield can vary depending on the type of tumor tissue. For example, the yield may be about 10 mg / mL for lipid-rich tumor tissue, or about 110 mg / mL for heavily fibrotic tumor tissue.

[0061] Filtering the disaggregated protein dispersion to form a sol

[0062] According to some implementation methods, the preparation of ECM hydrogels may include filtering depolymerized protein dispersions to form a sol. Filtration may include batch filtration, flow filtration, two-flow percolation, dialysis bag filtration, hollow fiber ultrafiltration, tubular tangential flow ultrafiltration (TFF), tubular hollow fiber filtration, and combinations thereof. During filtration, denatured proteins found in the depolymerized protein dispersions can repolymerize and refold, thereby allowing sol formation. The sol contains collagen, fibronectin, elastin, and other structural proteins, which may depend on the specific tissue contents.

[0063] The filtration of this disclosure may include solvent displacement to form a stable sol. For example, a solvent containing one salt may be displaced with a solvent containing a salt of different concentrations or even a completely different salt. Displacing the solvent during filtration can prevent premature gelation, flocculation, and diffusion due to high viscosity. In some embodiments, the method of this disclosure may increase the filtration rate and be combined with online homogenization to prevent premature gelation, flocculation, and diffusion due to high viscosity. In addition to replacing one salt with another, solvent displacement may include changing the salt gradient, for example, from 1 M NaCl to 0.15 M NaCl in water. Solvent displacement may include replacing one solvent type with another, for example, from an aqueous solution containing NaCl to a culture medium. For example, the filtration of this disclosure may include solvent displacement from 6 M guanidine hydrochloride in water to 3.4 M NaCl in water, then to 0.15 M NaCl, and then to a culture medium. Another solvent displacement progression path may include solvent displacement from 6 M guanidine hydrochloride in water to 0.15 M NaCl in water, followed by purification of the 0.15 M NaCl in water, and then displacement to a culture medium. In other cases, solvent replacement may include different concentrations of NaCl, with surfactants, phosphate-buffered saline, Tris-HCl buffer, sodium citrate buffer, sodium acetate buffer, etc., specified herein. Any known salt or culture medium may be used within any known concentration range. In addition to solvent replacement, filtration of this disclosure may include the incorporation of stirring during the filtration process. For example, stirring may be incorporated into filtration performed in a dialysis bag.

[0064] Filtration can include filtering depolymerized protein dispersions through membranes having various pore sizes. For example, the membrane can have a pore size of about 1 kDa to about 200 kDa or greater. The membrane can have a pore size of about 1 kDa, or about 20 kDa, or about 40 kDa, or about 60 kDa, or about 80 kDa, or about 100 kDa, or about 120 kDa, or about 140 kDa, or about 160 kDa, or about 180 kDa, or about 200 kDa, where “about” includes ±10 kDa.

[0065] In some implementations, filtering depolymerized protein dispersions to form a sol can include centrifugal filtration, gravity filtration, bag filtration, reverse osmosis filtration, vacuum filtration, ultrafiltration, multilayer filtration, percolation, flow percolation, and combinations thereof. For example, as... Figure 7 As shown, filtration can include, but is not limited to, flow percolation. Percolation can include multiple dialysis bag membranes placed between manifolds. Figure 7 As shown, flow percolation may include transferring the depolymerized protein dispersion from a collection bottle to a sterile bag for further preservation. In some embodiments, a filtration unit may be provided between the collection bottle and the collection bag.

[0066] Filtration of depolymerized protein dispersions to form a sol may include sterilizing the sol during filtration. Sterilization of the sol during filtration can be performed in any manner known in the art, including heating, radiation treatment, steam treatment, electron beam treatment, mixing with a solvent, and combinations thereof. Mixing with a solvent may include mixing with a halogenated solvent. For example, sterilization may include circulating a halogenated solvent in a permeate loop such that it washes the sol, thereby removing unwanted contaminants. Halogenated solvents may include chloroform, dichloromethane, trichloroethylene, perchloroethylene, trichlorotrifluoroethane, 1,1,1-trichloroethane, and mixtures thereof. Based on the volume of water, the volume of the last solvent replaced, or the volume of the mixture of the sol and the halogenated solvent, the halogenated solvent may be circulated at a concentration ranging from about 0.1% v / v to about 10% v / v. For example, the halogenated solvent may be circulated at a concentration of about 0.1% v / v, or about 1% v / v, or about 2% v / v, or about 3% v / v, or about 4% v / v, or about 5% v / v, or about 6% v / v, or about 7% v / v, or about 8% v / v, or about 9% v / v, or about 10% v / v, where “about” includes ±0.5% v / v. Sol sterilization may include circulating the halogenated solvent in the permeate loop for a period of about 1 hour to about 24 hours. For example, the period may be about 1 hour, or about 2 hours, or about 4 hours, or about 6 hours, or about 8 hours, or about 10 hours, or about 12 hours, or about 14 hours, or about 16 hours, or about 18 hours, or about 20 hours, or about 22 hours, or about 24 hours, where “about” includes ±1 hour.

[0067] After separating the sol from the given solvent, the method may include applying approximately 760 Torr to approximately 1 x 10⁻⁶ tons of the sol at a temperature of approximately 4°C to approximately 24°C. -12 The vacuum period is approximately 0.1 hours to approximately 24 hours. It can be achieved from approximately 760 Torr to approximately 25 Torr (i.e., rough vacuum), and from approximately 25 Torr to approximately 1 × 10⁻⁶ Torr. -3 Torr (i.e., medium vacuum), approximately 1 × 10 -3 To about 1×10 -9 Torr (i.e., high vacuum), approximately 1×10 -9To about 1×10 -3 A vacuum is applied to the sol within the range of Tor (i.e., ultra-high vacuum). Vacuum application to the sol can be performed at temperatures of approximately -4°C, or approximately 0°C, or approximately 4°C, or approximately 8°C, or approximately 12°C, or approximately 16°C, or approximately 20°C, or approximately 24°C, where "approximately" includes ±2°C. Vacuum application can be performed for approximately 0.1 hours, or approximately 1 hour, or approximately 2 hours, or approximately 4 hours, or approximately 6 hours, or approximately 8 hours, or approximately 10 hours, or approximately 12 hours, or approximately 14 hours, or approximately 16 hours, or approximately 18 hours, or approximately 20 hours, or approximately 22 hours, or approximately 24 hours, where "approximately" includes ±1 hour.

[0068] In some implementation schemes, such as Figure 8 As shown, filtration can include, but is not limited to, hollow fiber tangential flow filtration. Perfiltration can include multiple dialysis bag membranes placed between manifolds. Figure 8 As shown, flow percolation may include transferring the depolymerized protein dispersion from collection bottle 891 to a sterile bag 893 for further preservation. In some embodiments, a filtration unit 892 may be disposed between the collection bottle and the collection bag. Figure 8 As shown, the filtration unit may include a hollow fiber filtration unit. Filtration unit 892 may include a permeate inlet, allowing a halogenated solvent (e.g., chloroform) to circulate through filtration unit 892 and exit through a permeate outlet. In some embodiments, filtration unit 892 may include a residual outlet and a residual inlet, allowing the sample used for filtration to enter and exit.

[0069] The filter unit 892 may have a pore size of about 1 kDa to about 200 kDa. For example, the filter unit 892 may have a pore size of about 1 kDa, or about 20 kDa, or about 40 kDa, or about 60 kDa, or about 80 kDa, or about 100 kDa, or about 120 kDa, or about 140 kDa, or about 160 kDa, or about 180 kDa, or about 200 kDa, where “about” includes ±10 kDa.

[0070] like Figure 8 As shown, collection bottle 891 may include a homogenizer rod, a conductivity meter, and an aliquot outlet for sampling the contents. Collection bottle 891 may include a cooling liquid inlet and outlet, allowing for temperature regulation of the contents of collection bottle 891. For example, the contents of collection bottle 891 may be cooled to approximately 25°C, or approximately 20°C, or approximately 15°C, or approximately 10°C, or approximately 5°C, or approximately 0°C, or approximately -5°C, or approximately -10°C, or approximately -15°C, or approximately -20°C, or a colder temperature, where "approximately" includes ±2.5°C.

[0071] In some embodiments, filtration may include filtering depolymerized protein dispersions to form a sol, and may include filtering depolymerized proteins using a tubular hollow fiber. Filtration may include using a salt gradient to filter depolymerized proteins using a tubular hollow fiber to form a sol. The salt gradient may include salts, including but not limited to lithium chloride, sodium chloride, potassium chloride, zinc chloride, magnesium chloride, guanidine chloride, and combinations thereof. The salt gradient may include salt concentrations ranging from about 0.001 M to about 1 M. For example, a salt gradient may include salt concentrations of about 0.001 M, or about 0.01 M, or about 0.1 M, or about 1 M, where "about" includes ±0.5 M. In some embodiments, the tubular hollow fiber may include porous filaments ranging from about 5 kDa to about 200 kDa. For example, the tubular hollow fiber may comprise porous filaments of about 5 kDa, or about 20 kDa, or about 40 kDa, or about 60 kDa, or about 80 kDa, or about 100 kDa, or about 120 kDa, or about 140 kDa, or about 160 kDa, or about 180 kDa, or about 200 kDa, where “about” includes ±10 kDa. Filtration can be performed at flow rates ranging from about 1 mL / min to about 1 L / min. For example, filtration can be carried out at a flow rate of about 1 mL / min, or about 50 mL / min, or about 100 mL / min, or about 150 mL / min, or about 200 mL / min, or about 250 mL / min, or about 300 mL / min, or about 350 mL / min, or about 400 mL / min, or about 450 mL / min, or about 500 mL / min, or about 550 mL / min, or about 600 mL / min, or about 650 mL / min, or about 700 mL / min, or about 750 mL / min, or about 800 mL / min, or about 850 mL / min, or about 900 mL / min, or about 950 mL / min, or about 1,000 mL / min, where “about” includes ±25 mL / min.

[0072] In some implementations, hollow fiber methods can allow for a larger surface-to-volume ratio during filtration; however, a significant drawback is rapid fouling. Some fouling can be irreversible, leading to a significant increase in production costs and a need for supply chain solutions for these devices. Faster filtration is required in the following situations: a) the depolymerized protein solution is too viscous; b) protein gelation occurs before the depolymerized mixture is completely removed; c) protein flocculation occurs when the GuaHCl concentration is below 1 M.

[0073] In some embodiments, all components used in the filtration step may be cooled to a temperature in the range of about 25°C to about -20°C or cooler. For example, each component in the filtration step may be cooled to a temperature of about 25°C, or about 20°C, or about 15°C, or about 10°C, or about 5°C, or about 0°C, or about -5°C, or about -10°C, or about -15°C, or about -20°C or cooler, where “about” includes ±2.5°C.

[0074] Gelling the sol to form an ECM hydrogel

[0075] According to some embodiments, methods of using ECM hydrogels may include gelling a sol to form an ECM hydrogel. Not removing the gel may include combining the sol with a gel solution. The gelling solution may include a hydrophilic agent and water. The hydrophilic agent may include inorganic or organic salts of ammonium, sodium salts, potassium salts, phosphates, polycarboxylate salts, succinates, citrates, or isocitrates. The hydrophilic agent may be present in a concentration range of about 0.01 M to about 0.15 M. For example, the hydrophilic agent may be present in concentrations of about 0.01 M, or 0.03 M, or 0.05 M, or 0.07 M, or 0.09 M, or 0.11 M, or 0.13 M, or 0.15 M.

[0076] Gelation may include raising the temperature of a sol from about 0°C to about 8°C to about 26°C to about 50°C for at least about 1 minute. For example, gelation may include raising the temperature of a sol from about 0°C to about 8°C to about 25°C, or about 30°C, or about 35°C, or about 40°C, or about 45°C, or about 50°C, wherein "about" includes ±2.5°C. Gelation may include raising the temperature of a sol from about 0°C to about 8°C to about 26°C to about 50°C for at least 1 minute, or at least about 5 minutes, or at least about 10 minutes, or at least about 15 minutes, or at least about 20 minutes, or at least about 25 minutes, or at least about 30 minutes, or at least about 35 minutes, or at least about 40 minutes, or at least about 45 minutes, or at least about 50 minutes, or at least about 55 minutes, or at least about 60 minutes, or more, wherein "about" includes ±5 minutes. Gelation may include increasing the temperature of the sol at a rate of about 1°C / min, or about 2°C / min, or about 3°C / min, or about 4°C / min, or about 5°C / min, or about 6°C / min, or about 7°C / min, or about 8°C / min, or about 9°C / min, or about 10°C / min, or about 11°C / min, or about 12°C / min, or about 13°C / min, or about 14°C / min, or about 15°C / min, or about 16°C / min, or about 17°C / min, or about 18°C / min, or about 19°C / min, or about 20°C / min, or about 21°C / min, or about 22°C / min, or about 23°C / min, or about 24°C / min, or about 25°C / min or higher, wherein “about” includes ±0.5°C / min.

[0077] In some embodiments, the ECM hydrogel formed by the methods of this disclosure can be preserved using shock preservation at temperatures from -100°C to about 0°C. Shock preservation may include immersing the ECM hydrogel in a solvent / dry ice mixture. For example, the solvent may include alcohols, such as isopropanol. Solvents may include any known solvents that are typically mixed with dry ice, including acetone, ethanol, methanol, water, ethylene glycol, alkanes, other alcohols, and mixtures thereof. The resulting liquid is then further transferred to a -80°C freezer. For example, the ECM hydrogel may be preserved using shock preservation at temperatures of about -100°C, or about -80°C, or about -60°C, or about -40°C, or about -20°C, or about 0°C, where "about" includes ±10°C. Similarly, gelation can be prevented by rapidly thawing the sol under stirring before it forms a gel from the sol. Rapid thawing involves placing a vial of the frozen sol in a water bath heated to +37°C and further vigorously mixing the sol. For example, to achieve rapid thawing of the sol or to raise its temperature from about -80°C to about 25°C, it can be done by stirring the sol as its temperature rises from about -80°C to about 25°C.

[0078] Methods for preparing ECM hydrogels may include gelling a sol to form an ECM hydrogel having an elastic modulus of about 1 kPa to about 250 kPa. In some embodiments, a thin ECM hydrogel may be formed by diluting the ECM sol with cell culture medium or buffer until a critical gelation concentration is reached and no gel forms. Biocompatibility and advantages of the ECM hydrogels of the present disclosure Examples

[0079] The method for preparing ECM hydrogels can produce ECM hydrogels with advantageous biocompatibility compared to known hydrogels. This method can form ECM hydrogels having an elastic modulus of about 1 kPa to about 100 kPa. For example, the method can form ECMs with elastic moduli of about 1 kPa, or about 2 kPa, or about 3 kPa, or about 4 kPa, or about 5 kPa, or about 6 kPa, or about 7 kPa, or about 8 kPa, or about 9 kPa, or about 10 kPa, where "about" includes plus or minus 0.5 kPa. In some embodiments, primary cell cultures can exhibit higher adhesion to the hydrogels of this disclosure compared to known hydrogels. For example, compared to known hydrogels, the adhesion of primary cell cultures to the hydrogels of this disclosure can be about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%, or more, where “about” includes plus or minus 5%. In some embodiments, the confluence of cell layers on the hydrogels of this disclosure can be higher than that of known hydrogels. For example, compared to known hydrogels, the confluence of cell layers on the hydrogels of this disclosure can be about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%, or more, where “about” includes plus or minus 5%. For example, in experiments with human hepatocytes isolated from liver donors, the cell layers showed approximately 40% higher confluence compared to known gels, and were more similar to human type I collagen coating.

[0080] The method disclosed herein can produce ECM hydrogels that induce immortalized cell lines (e.g., HepG2) to advantageously have FAK expression induction values ​​that are approximately 1 to approximately 20 times higher than those of known hydrogels. In some embodiments, the FAK expression induction value can be measured using RNA sequencing. For example, the method can prepare ECM hydrogels having FAK expression induction values ​​that are approximately 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 times higher than those of known hydrogels, where “approximately” includes plus or minus 1.

[0081] The ECM hydrogel of this disclosure does not induce cytotoxicity in immortalized cell lines. For example, the ECM hydrogel of this disclosure may not induce cytotoxicity in standard A549 and 4T1 cell lines. Cytotoxicity can be assessed by staining cells with propidium iodide, calcein AM, and DAPI, and by quantifying these images based on imaging data.

[0082] The method disclosed herein can prepare ECM hydrogels that are non-toxic to preclinical animals. For example, when the ECM hydrogel of this disclosure is injected into animals, no statistically significant weight loss, visual toxicity, or visual characteristics are observed in the animals (e.g., preclinical animals).

[0083] According to some embodiments, the ECM hydrogel of this disclosure can be used to develop xenograft models by injecting (e.g., orthotopic injection) cell lines (e.g., 4T1 cell line) into animals (e.g., nude mice) within the equivalent time of known hydrogels or less. The ECM hydrogel of this disclosure can be used to develop xenograft models by injecting (e.g., A549 cell line) cell lines (e.g., orthotopic injection) into animals (e.g., nude mice) within the equivalent time of known hydrogels or less. In some embodiments, the method of this disclosure can prepare cells with a diameter of approximately 1 mm. 3 / day to approximately 300mm 3 ECM hydrogels with in situ tumor growth rates ranging from 1 mm / day. For example, ECM hydrogels can have a thickness of approximately 1 mm. 3 / day, or approximately 20mm 3 / day, or approximately 40mm 3 / day, or approximately 60mm 3 / day, or approximately 80mm 3 / day, or approximately 100mm 3 / day, or approximately 120mm 3 / day, or approximately 140mm 3 / day, or approximately 160mm 3 / day, or approximately 180mm 3 / day, or approximately 200mm 3 / day, or approximately 220mm 3 / day, or approximately 240mm 3 / day, or approximately 260mm 3 / day, or approximately 280mm 3 / day, or approximately 300mm 3 The in situ tumor growth rate per day, where "approximately" includes ±10 mm. 3 / sky.

[0084] Depending on the degree of compression of the formed ECM hydrogel, cell invasion of the ECM hydrogel of this disclosure can be monitored using a vertical invasion assay. For this purpose, the gel is diluted 1:2 in a 96-well plate with cell culture medium and centrifuged. To generate an elastic modulus gradient, a protocol of 50-3000 g can be used. When the mixture is centrifuged at maximum rate, the most compressed gel state is formed. Cells can then be seeded on top and invade 0%-30% of the formed layer according to density differences. In some embodiments, similar analyses can be performed without a known hydrogel (e.g., Matrigel), as known hydrogels do not have a similar elastic gradient. The elastic gradient in human ECM may occur during centrifugation due to phase separation, which may be caused by water extrusion from the hydrogel. The compression of the hydrogel can range from about 50% to about 90% of the hydrogel volume, depending on the applied centrifugation intensity. Known hydrogels, such as Matrigel, cannot be stably compressed under similar centrifugation intensities applied to the ECM hydrogel of this disclosure.

[0085] Figure 5

[0086] Example 1: Decellularization of hSTS tissue

[0087] Thaw the tissue sample at room temperature until it is completely softened. Weigh and select 250 grams of tissue sample, and return any unused tissue sample to the freezer and freeze at -80°C. If not processing the tissue sample immediately, store it in a freezer at +4°C. Then cut the tissue sample into uniform slices of 2-3 cm and place them in a mixer. Grind the tissue sample using a large mixer until its size reaches 3-4 mm.

[0088] The ground tissue sample was placed in a beaker containing 4 liters of Wash Solution 1 (0.34 M NaCl, 1 mM ethylmaleimide, 25 mM EDTA-Na2). A top-driven stirrer was immersed in the beaker and stirred at 50,000 rpm for 1 hour. The resulting sediment was filtered through a 250 μm sieve.

[0089] The ground tissue sample was then transferred to a beaker containing 4 liters of washing solution 2 (1.3 M NaCl, 1 mM ethylmaleimide, 25 mM EDTA-Na2). A top-driven stirrer was immersed in the beaker and stirred at 50,000 rpm for 1 hour. The resulting sediment was filtered through a 250 μm sieve.

[0090] The tissue sample was ground to a size of 1 mm using an immersion stirrer. The ground tissue was placed in a beaker containing 4 L of washing solution 3 (1.3 M NaCl, 1% w / v Triton X-100, 1% w / v chloroform, 1 mM ethylmaleimide). A top-driven stirrer was immersed in the beaker and stirred at 50,000 rpm for 1 hour. The sediment was filtered through a 100 μm sieve.

[0091] The ground tissue sample was then placed in a beaker containing 2 liters of washing solution 4 (150 mM NaCl, 1% w / v Triton X-100, 1 mM ethyl maleimide, 1% w / v chloroform). A top-driven stirrer was immersed in the beaker and stirred overnight at 50,000 rpm. The resulting sediment was filtered through a 100 μm sieve. The sediment was washed in washing solution 5 (150 mM NaCl) until foam formation in the filtrate ceased.

[0092] Example 2: Depolymerization of decellularized hSTS tissue

[0093] Cool the swollen solution containing 6M guanidine hydrochloride, 0.05M Tris, and 1% Tween to +4°C. Cool the cooler to -20°C. Weigh 200-350g of protein into the reactor. Measure 2ml of the swollen solution per 1g of protein and pour it into the reactor. Start stirring in the reactor at 150rpm. The temperature inside the reactor reaches +4°C. Add the denatured protein to the reactor and mix it with the remaining components. Turn on the mechanical homogenizer at 0.1 power. Gradually increase the homogenizer speed until the temperature of the protein solution remains stable over time and does not exceed +4°C. Use a disperser to thoroughly homogenize the mixture overnight at +4°C to form a suspension.

[0094] Cool the centrifuge to +4°C. Pour the suspension into centrifuge flasks and centrifuge at 3000g for 30 minutes at +4°C. The supernatant contains the target product. All steps are performed on ice. Combine the supernatants from all flasks.

[0095] The swelling efficiency of the supernatant should be evaluated based on the following tests: determination of diquinoline carboxylic acid at acceptable protein concentrations of 8 to 110 mg / mL, precipitate volume (up to 20% of the total volume), atomic force microscopy, and dynamic light scattering assessment of colloid size (<300 μm is acceptable). Depolymerization of >5% of the protein should not be observed by gel electrophoresis or size exclusion chromatography.

[0096] Example 3: Flow Dialysis

[0097] The flow dialysis system used in this paper may include a manifold for the dialysis membrane, a collection bottle with cooling, permeate inlet and outlet, residual material inlet and outlet, and a second collection bottle with cooling. The concentration of GuaHCl is defined by measuring the permeate conductivity and converting it to concentration using a calibration curve. Figure 6 This is a graph of GuaHCl concentration versus time.

[0098] Several assumptions were derived from this data, including that the GuaHCl diffusion rate does not depend on the GuaHCl concentration, that the GuaHCl diffusion between the residual material and the permeate is in equilibrium, that the dialysis rate depends on the dialysis manifold surface area, and that the dialysis rate depends on the collection flask volume and the residual material displacement value. Based on these assumptions, a kinetic mathematical model was constructed. The equations used are as follows:

[0099] Where C0 = initial concentration of GuaHCl; S = total volume of dialysis manifold; t = time; v = permeate displacement rate; P = volume of collection flask; k = reaction rate constant; U(t) = function of decrease in concentration of GuaHCl in permeate; Z(t) = function of increase in concentration of GuaHCl in permeate; and e(t) = equilibrium function.

[0100] To determine the reaction rate constant, the concentration of guanidine in the residue was measured after a period of time. Then, for the above equation, different k values ​​were fitted, and the U(t) value at the time point when the guanidine concentration of the test sample was measured was determined. A U(t) value consistent with the empirically determined guanidine concentration was selected. If no U(t) value consistent with the empirically determined guanidine concentration, a k value with more significant figures was fitted again.

[0101] The flow dialysis conditions can be optimized based on the rate constants calculated above. Therefore, the permeate displacement flow rate can be determined as the reaction rate constant through fitting. To visualize the relationship between GuaHCl concentration and permeate displacement flow rate, a system was constructed as follows: Figure 6 The diagram shown. Figure 7 The graphs show that, after a dialysis interval of 600 minutes, the osmotic displacement rate range of 0 L / min to 0.05 L / min is the most effective in terms of dialysis rate and osmotic solution consumption.

[0102] High-speed mixing in the permeate loop may be required. Standard calculations for mixing include 1–3 volumes of reactor displacement per minute. If the mixing rate decreases or is delayed during this process, irreversible gel formation is observed. Hollow fiber ultrafiltration is also effective if a similar principle is introduced. This process may require a pore size of 1 mm. Rapid internal permeate flow may be required. However, a significant problem with this process is gelation within the fibers, which significantly reduces the efficiency of the process. Hollow fiber permeation is effective for hydrogels with low viscosity and low protein concentration.

[0103] Example 4: Percolation and cryopreservation of swollen protein mixtures

[0104] like Figure 7 As shown, cut a 21cm SnakeSkin. TM Cut a 10K MWCO dialysis tubing and attach the connector for the dialysis manifold 785 to each bag using two clamps on each side. Connect the bag with the dialysis manifold 785 to the dialysis unit. Purge the internal circuit of the dialysis unit with air and check the integrity of all connections.

[0105] After inflation, verify the alignment of the dialysis bags, ensuring all bags are evenly inflated with air. Compression or twisting should be avoided. Then, set the cooler to -5°C and turn it on. Pour 10 liters of cold dialysis solution (0.15M NaCl) into the external circuit of the dialysis apparatus, and then begin mixing in the external circuit. Allow the temperature in the external circuit to drop to +4°C. Then, as... Figure 7 As shown, the protein recovery container 780 of the dialysis apparatus is filled with a depolymerized protein solution.

[0106] like Figure 7 As shown, the internal pump is turned on at minimum power, and the protein solution is gradually added to the protein recovery container 780. The protein circulation rate is gradually increased to 5-10 solution volumes per minute. The volume of protein in the recovery container 780 should be 1 / 3 full. The conductivity sensor is placed in the protein recovery container 780 along with the disperser set to minimum power. Ten liters of dialysis solution are prepared and placed in a freezer at +4°C for further use.

[0107] The first dialysis cycle lasted 6 hours, and the protein solution conductivity indicated a conductivity less than 75 mS / cm. Thereafter, the dialysate in the external circuit was replaced with cold dialysate, and 10 liters of dialysate were prepared for the next use. The second dialysis cycle lasted overnight, and the protein conductivity indicated a conductivity less than 20 mS / cm. The dialysate in the external circuit was again replaced with cold dialysate, and another 10 liters of dialysate 2 (DMEM, 1% chloroform) were prepared for the next stage. The third dialysis cycle lasted 6 hours, and the protein conductivity indicated a conductivity of 16 mS / cm. The dialysate in the external circuit was again replaced with cold dialysate 2.

[0108] The fourth dialysis cycle continues overnight. Prepare aliquots (1 ml) of the protein solution for further quality control and freeze. The protein conductivity should be less than 16 mS / cm. Optionally, afterwards, connect a 0.45 μm in-line filter to the peristaltic pump. Figure 9A As shown, a pump with a filter is connected to the dialysis unit, and the protein solution is transferred to a sterile glass vial 790. 1% antibiotic-antifungal agent and 1% chloroform are added to the vial.

[0109] Bottle 790 containing the protein solution was moved to a shaker and kept at +4°C for 1 hour. Then, bottle 790 was connected to a membrane vacuum pump at 5 mBar and kept at +4°C for 1 hour without being removed from the shaker. Bottle 790 was then plugged, placed on ice, treated with a disinfectant solution, and transferred to a clean area for further dispensing. A work area with maximum cleanliness and sterility was prepared by treating all surfaces with a 70% ethanol solution. Subsequently, the automated dispenser used for filling underwent sterilization.

[0110] The bottles and their caps were also sterilized. For 2 and 4 mL packaging volumes, 5 mL low-temperature tubes were used, while for 10 and 20 mL packaging volumes, penicillin vials of the corresponding volume were used. Labels were affixed to each bottle for batch identification. The bottles were placed in racks and cooled in a refrigerator at +4°C.

[0111] A freezing solution is prepared in a polystyrene container containing 2 volumes of dry ice and 1 volume of isopropanol. Bottles containing the protein solution are stored on ice during use to prevent excessive water formation in an external reservoir. Before packaging, water droplets on the bottles containing the protein solution are wiped dry, treated with a disinfectant solution, the stopper is removed, and the bottles are connected to a dispenser. The dispensing volume is set on the dispenser.

[0112] Packaging is done in stages, one bottle rack per cycle. After removing the rack from the freezer, the protein mixture is quickly dispensed into the bottles. The bottles are plugged and placed on a table. When using penicillin bottles, a sealing machine is used. Each bottle is checked for a tight seal and correct labeling. Each bottle is then immersed in a freezing solution for shock freezing, and the process is repeated for the next rack. After the freezing solution containing the bottles has boiled, the bottles containing the packaged protein mixture are quickly removed using a large sieve with a handle and transferred to a freezer at -80°C. The protein mixture packaged and flash-frozen at -80°C is considered the final product.

[0113] Example 5: Immobilizing mica surface with ECM protein

[0114] Prepare a 0.0233 M solution of APTES in a sodium hydroxide methanol solution (I). Mix the solution and store it at +4°C. Prepare the working solution. Dilute the APTES solution (I) in methanol at a ratio of n:150, where n is the number of samples prepared. Place the mica sample in a tube containing the working solution, stir, and maintain at 40°C for 45 minutes. Remove the mica sample, wash it with methanol, and dry it. Add 20 μL of glutaraldehyde to the mica sample. After 1 minute, wash the sample with methanol. Cool the sample to 0°C. Add one drop of ECM protein dispersion to the mica sample, wipe off the residual solution with filter paper after 30 seconds, then rinse thoroughly with plenty of water and dry again.

[0115] AFM study of hSTS ECM sol (Scanning probe microscopy NanoScope IIIa Dimension 3000tm (Bruker Corp.)) showed that at +4 °C, the dominant particles were soft, elastic colloids with sizes ranging from 50 nm to 100 nm. Occasionally, particles with sizes >200 nm were observed (see [link to study]). Figure 9B ). Figure 9C The particle height (y-axis) and diameter (x-axis) of the small colloids are shown. After gelation at +37°C, long collagen fibers were observed, forming aggregates with a size of 0.5–2 micrometers (see [link to original text]). Figure 9D These fibers themselves are formed from characteristic bands (D-bands or D-cycles) of terminal collagen. For example... Figure 10A As shown, these bands have the characteristic length of collagen D-bands (61.75 ± 1.375 nm).

[0116] DLS studies of hSTS ECM sol (Zetasizer nano ZS) at +4°C showed that 10-30 nm particles dominated the solution (see [link to study]). Figure 10BThis is primarily related to AFM data. The mean shift from 75 nm to 20 nm may be attributed to the aggregation of sol particles on the mica surface. Larger particles of 250 nm and above are also present in the solution at +4 °C, though fewer in number than those of 250 nm, but with significantly higher scattering intensity (see [link to AFM data]). Figure 11 The concentration is significantly higher. This may be the reason why the mixture appears cloudy.

[0117] Data from other tissue sources are comparable to these findings. As a general phenomenon, the percolation of hECM sols occurs due to the formation of collagen fibers from colloids with a size of 10–50 nm. The critical viscosity (gelation) is achieved by forming a network of collagen fibers and aggregating these fibrous portions into particles of 0.5–2 micrometers.

[0118] Example 6: Coating a 96-well plate with hECM hydrogel

[0119] Remove 5 mL of the hECM hydrogel from the -80°C freezer. Place the hydrogel in an ice bath and mix carefully from time to time. When a small amount of ice remains in the mixture, vigorously stir the mixture with a 1 mL micropipette until homogeneous. The resulting solution can be stored at +4°C for 24 hours. Prepare solutions for thin-layer hydrogel coating by diluting with cold culture medium at a ratio of 1 / 20–1 / 10; and for thick-layer coating by diluting at a ratio of 1 / 4–1 / 2. While keeping the solution and wells cold, dispense the solution at 30 μL / well. Then centrifuge the mixture at +4°C at 2000 g for 10 minutes and solidify it in an incubator at +37°C. Remove the supernatant that forms. Seed cells on top of the layer.

[0120] HepG2 cells (ATCC) seeded on a thin gel surface exhibited the same phenotype as those seeded on Corning Methylgel. Cells on the coated surface and on the tissue culture plastic surface showed differences. Nevertheless, cells seeded within a thin layer of hydrogel exhibited similar colony formation rates. This demonstrates the unique properties of this material, acting not only as a solid carrier but also as a biological environment, which may lead to different cell characteristics compared to commercially available products.

[0121] To develop an allograph model, 4T1 cells were orthotopically seeded into the right fourth mammary fat pad using hECM. Cells were cultured in DMEM (4.5 g / L glucose) with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2. Cells were collected using 0.05% trypsin-EDTA solution, centrifuged, and resuspended in serum-free DMEM. Cell counts and viability were determined using a hemocytometer and trypan blue exclusion assay. DMEM was mixed with Corning's Matrigel or hECM at a 3:1 ratio to form 10 × 10⁶ cells. 6 The final cell suspension of / mL was stored on ice prior to injection.

[0122] To develop a xenograph model, 5M MDA-MB-231 cells were orthotopically seeded into the fourth right mammary fat pad of mice using hECM. Cells were cultured in RPMI (4.5 g / L glucose) at 37°C and 5% CO2 with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were collected using 0.05% trypsin-EDTA solution, centrifuged, and resuspended in serum-free DMEM. Cell counts and viability were determined using a hemocytometer and trypan blue exclusion assay. DMEM was mixed with either Corning's Matrigel or hECM at a 1:1 ratio in 50 × 10⁶ cells. 6 The final cell suspension of / mL was stored on ice prior to injection.

[0123] In summary, similar to the thick-coating experiment, mice showed no signs of toxicity and their growth rate was similar to that of Corning. The product's growth rate. However, there are significant phenotypic changes tending towards more matrix-adherent cell structures. Figure 10 shows the hECM hydrogel prepared using the methods of this disclosure with Corning. Images and photographs comparing products (BME). Figure 12A The results showed that, compared with thick hydrogels, HepG2 cells had a higher adhesion rate on thin-coated surfaces, resulting in enhanced differentiation (calcein AM, propidium iodide staining), and the colony formation results also showed differences. Figure 13A and 12B The study illustrates in situ tumor progression in mice and the body weight of Balb / c mice bearing 4T1 subcutaneous tumors, while the hECM hydrogel of this disclosure exhibits similar properties to the BME hydrogel. Similarly, ​ and 13BThe study shows the progression of in situ tumors in mice and the body weight of SCID mice bearing MDA-MB-231 subcutaneous tumors, while the hECM hydrogel of this disclosure exhibits similar properties to the BME hydrogel.

[0124] References to "one embodiment" or "implementation" in the specification mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. The phrases "in one embodiment," "in some embodiments," "in one instance," "in some instances," "in one case," "in some cases," "in one implementation," or "in some implementations" appearing in various places in the specification do not necessarily refer to the same embodiment or implementation.

[0125] Finally, the foregoing description of embodiments of this disclosure is intended to be illustrative and descriptive. It is not intended to be exhaustive or to limit this disclosure to the exact forms disclosed. Many modifications and variations are possible in accordance with the foregoing teachings. The scope of this disclosure is intended to be limited not by this detailed description, but by the claims of this application. As will be understood by those skilled in the art, this disclosure may be practiced in other specific forms without departing from the spirit or essential characteristics of this disclosure. Therefore, this disclosure is intended to illustrate, and not limit, the scope of the disclosure as set forth in the appended claims.

Claims

1. A method for preparing an extracellular matrix hydrogel, the method comprising: (a) Decellularizing a tissue sample to form a decellularized tissue network, wherein the decellularization includes: (i) The tissue sample is mixed with a washing solution to form a decellularized mixture, wherein the tissue sample is derived from a tumor with a fibrosis content ranging from about 40% to about 90%; and (ii) Separating the decellularized mixture into cellular waste fluid and decellularized tissue network; (b) Depolymerizing the decellularized tissue network to form a depolymerized protein dispersion, the depolymerization comprising: (i) The decellularized tissue network is mixed with a depolymerization solution containing a dissociation agent to form a depolymerization mixture; (ii) Mechanically homogenize the depolymerized mixture at a cooling temperature to form a homogenized mixture; (iii) Separating the homogenized mixture into (a) a waste precipitate and (b) a supernatant containing the depolymerized protein dispersion; (c) Filtering the depolymerized protein dispersion to form a sol; and (d) gelling the sol to form the extracellular matrix hydrogel, the gelation comprising: (i) Combining the sol with a gelling solution containing a lyophilizing agent; and (ii) Separating the liquid phase from the extracellular matrix hydrogel during the formation of the extracellular matrix hydrogel.

2. The method for preparing extracellular matrix hydrogel according to claim 1, further comprising: The sol is sterilized during the filtration process.

3. The method for preparing extracellular matrix hydrogels according to claim 2, wherein the sterilization comprises mixing the sol with a halogenated solvent.

4. The method for preparing extracellular matrix hydrogel according to claim 2, further comprising: Apply a vacuum to the sterilized sol.

5. The method for preparing extracellular matrix hydrogel according to claim 1, further comprising: A hygroscopic agent is added to the gelation solution to adjust the stiffness of the hydrogel.

6. The method for preparing extracellular matrix hydrogel according to claim 1, wherein, The tumors include sarcomas, secondary liver cancer, liver fibrosis, osteosarcoma, chondrosarcoma, cardiac fibrosis, lung tissue and mixtures of pulmonary fibrosis, mesenchymal tissue and healthy organs and combinations thereof.

7. The method for preparing extracellular matrix hydrogels according to claim 1, comprising at least one of the following: Mixing the tissue sample with the washing solution includes mixing the tissue sample and the washing solution at a speed ranging from about 25,000 rpm to about 75,000 rpm for at least about 30 minutes. The washing solution comprises approximately 0.34 M sodium chloride, approximately 1 mM ethylmaleimide, approximately 25 mM disodium ethylenediaminetetraacetate, and combinations thereof, and The depolymerized protein dispersion was filtered at a flow rate of about 10 mL / min to about 70 mL / min at a temperature of about 0°C to about 8°C.

8. The method for preparing extracellular matrix hydrogel according to claim 1, The decellularized tissue network contains proteins with glycosylated protein tails, and in, Based on mass, the depolymerized protein dispersion contains proteins having at least about 95% of the amount of glycosylated protein tails compared to the decellularized tissue network.

9. The method for preparing extracellular matrix hydrogels according to claim 1, wherein the dissociation agent comprises at least one of sodium dodecyl sulfate, guanidine chloride, guanidine cyanosulfate, urea, guanidine, thiourea, calcium salt, nitrate, dithiothreitol, mercaptoethanol, tributylphosphine, sodium borohydride, lithium perchlorate, lithium acetate, magnesium chloride, and combinations thereof.

10. The method for preparing extracellular matrix hydrogel according to claim 1, wherein, Separating the homogenized mixture into waste precipitate and supernatant includes centrifugation.

11. The method for preparing an extracellular matrix hydrogel according to claim 1, wherein the filtration is performed at a temperature of about 4°C.

12. The method for preparing extracellular matrix hydrogel according to claim 3, comprising at least one of the following: The halogenated solvent is chloroform; The halogenated solvent is mixed with the sol at a concentration of about 0.4% v / v to about 3% v / v, and The halogenated solvent is mixed with the sol for a period of about 3 hours to about 12 hours.

13. The method for preparing extracellular matrix hydrogel according to claim 4, further comprising: Apply a vacuum for about 1 hour to about 3 hours at a temperature of about 0°C to about 8°C.

14. The method for preparing extracellular matrix hydrogel according to claim 4, comprising at least one of the following: The hygroscopic agent includes potassium citrate, and The extracellular matrix hydrogel has a stiffness of about 1 kPa to about 250 kPa.

15. The method for preparing an extracellular matrix hydrogel according to claim 1, wherein the extracellular matrix hydrogel comprises at least one of the following: Adhesion values ​​of human primary hepatocytes from approximately 1 kPa to approximately 10 kPa; Approximately 2 to approximately 10 times the FAK expression induction value; The extracellular matrix hydrogel exhibited a toxicity rate of less than approximately 1% in mice; and At approximately 10mm 3 / day to approximately 200mm 3 In situ tumor growth rate within a range of / day.

16. The method for preparing extracellular matrix hydrogel according to claim 1, further comprising: The depolymerized mixture was mechanically homogenized at a temperature of approximately 4°C.

17. The method for preparing extracellular matrix hydrogels according to claim 1, comprising at least one of the following: Filtration includes at least one of the following: dual-flow percolation, dialysis bag filtration, flow percolation, hollow fiber ultrafiltration, cartridge hollow fiber filtration, and cartridge tangential flow ultrafiltration. The yield of the depolymerized protein dispersion is at least about 70 mg / mL (which can vary from 10 mg / mL in lipid-rich tissues to 110 mg / mL in heavily fibrotic tissues, depending on the initial value). The cooling temperature is from about 0°C to about 8°C.

18. The method for preparing extracellular matrix hydrogel according to claim 1, further comprising: Tissue samples were collected from the tumor.

19. The method for preparing extracellular matrix hydrogel according to claim 1, wherein, The filtration buffer used during the filtration process includes at least one of water, sodium dodecyl sulfate at a concentration ranging from about 0.5 vol% to about 2 vol%, and sodium chloride at a concentration ranging from about 0.1 M to about 4 M.

20. The method for preparing extracellular matrix hydrogel according to claim 1, The filtering of the depolymerized protein dispersion is carried out at a temperature of about 0°C to about 8°C, and The gelation process also includes raising the temperature of the sol from about 0°C to about 8°C to about 26°C to about 50°C for a period of time of at least about 1 minute.

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