Protein hydrogels, methods of preparation, and uses thereof

Abstract

The present invention relates to novel protein hydrogels made based on low concentration components: Reagent A and Reagent B, a method for preparing the protein hydrogels, and their use for cell culture, including two-dimensional and three-dimensional cell cultures, of both healthy and neoplastic cells, cell lines and primary cells.

Description

[Technical Field]

[0001] The present invention relates to protein hydrogels, methods for their preparation and their use for cell culture, including two-dimensional and three-dimensional cell cultures, of both healthy and neoplastic cells, cell lines and primary cells, for migration and invasion assays in hydrogels in three-dimensional conditions, for performing angiogenesis assays, and for performing aortic sprouting assays. [Background technology]

[0002] Currently, there are many different types of media for cell culture in 3D environments, which can generally be divided into several types: protein hydrogels, synthetic hydrogels, scaffolds, hanging drops, etc.

[0003] The advantage of protein hydrogels is that they are closest to the physiological environment for cell growth. Currently, the most frequently used materials for cell culture in three-dimensional conditions are hydrogels, whose composition is composed of extracellular matrix (ECM) proteins. Other hydrogels, especially those made from synthetic peptides, are also available, but they are less commonly used. On the other hand, collagen and gelatin are used to coat culture surfaces in two-dimensional cell culture.

[0004] Collagen is also used in three-dimensional culture. Typically, a hydrogel crosslinked by pH change is used. At low pH, collagen, such as rat tail collagen, is dissolved. After a culture medium with a more neutral pH is added, the collagen gels and cultures can be grown on it. Gelatin is the product of partial hydrolysis of collagen fibers. Among the available gelatin species, two basic types can be identified: acidic types, in which hydrolysis occurs in an acidic environment, and alkaline types, in which hydrolysis occurs in an alkaline environment. Gelatins with different bloom values ​​are identified, depending on the value that determines the strength of gelation. The higher the bloom value, the stronger the gelation.

[0005] Hydrogels that enable three-dimensional cell culture and angiogenesis assays are now commercially available. One example of a product that allows for the performance of angiogenesis assays is Matrigel® (and its derivatives). Matrigel's main components are laminin, type IV collagen, proteoglycans, entactin, and growth factors extracted from the murine neoplasm, Engelbreth-Holm-Swarm (EHS) sarcoma. U.S. Pat. No. 4,829,000 discloses a composition for cell culture and a method for producing a bioactive extract. The use of collagen to perform angiogenesis assays is also known from Orci et al., "Vascular outgrowths from tissue explants embedded in fibrin or collagen gels: a simple in vitro model of angiogenesis," Cell Biology International Reports, Vol. 9, No. 10 (October 1985). However, the use of collagen has been described in the prior art as labor-intensive and providing poorly reproducible results. Hydrogels whose basic building material is gelatin, specifically methacrylated gelatin (FastLink GelMA), are also commercially available; an exemplary manufacturer of such hydrogels is Stemorgan Inc. However, these hydrogels use gelatin at concentrations of approximately 10%, which significantly exceeds the gelatin concentration range proposed in this invention. Moreover, the gelatin in GelMA is crosslinked with an initiator that generates free radicals under the influence of UV irradiation.

[0006] The objective of the present invention is to provide a hydrogel based on a low-concentration mixture, which solves the existing problems known from the prior art, reduces production costs, increases production efficiency, and is less toxic. Due to the components used, the novel hydrogel offers significantly higher reproducibility compared to hydrogels known from the prior art. This reproducibility stems from two fundamental features of the novel hydrogel. First, compared to hydrogels known from the prior art, the novel hydrogel is substantially free of growth factors. This is due to two reasons: first, gelatin production techniques dramatically reduce the viability of growth factors, and second, possible residual amounts of growth factors are inactivated during the gelatin cross-linking reaction with glutaraldehyde (GTA). The second basis for the reproducibility of the novel hydrogel is the reproducibility of the concentrations of its components.

[0007] In the prior art, DOILLON et al., "Three-dimensional Culture System as a Model for Studying Cancer Cell Invasion Capacity and Anticancer Drug Sensitivity," Anticancer Research, Vol. 24, pp. 2169-2178 (2004), discloses the use of collagen supplemented with fibrin as a component of a three-dimensional model for neoplastic cell culture. Additionally, Yamada and Even-Ram, "Cell migration in 3D matrix" (2005), disclose the use of three-dimensional cell cultures based on collagen and fibrin to test the potential of neoplastic cells to invade and migrate into hydrogels.

[0008] In the prior art, the use of three-dimensional endothelial cell cultures based on collagen and fibrin, which make it possible to carry out angiogenesis assays, is also known from MONTESANO et al., "Vascular outgrowths from tissue explants embedded in fibrin or collagen gels: a simple in vitro model of angiogenesis," Cell Biology International Reports, Vol. 9, No. 10 (October 1985). However, these hydrogels do not contain GTA.

[0009] GTA (glutaraldehyde) is one of the most frequently used chemical crosslinkers due to its highly effective stabilization of collagen materials, particularly through the reaction of aldehyde groups with free amino groups of lysine or hydroxylysine amino acid residues in polypeptide chains. However, its drawback in cell culture is its toxicity even at very low concentrations. As disclosed by Ou and Yang, "The micropatterning of glutaraldehyde (GA)-crosslinked gelatin and its application to cell culture," Lab on a Chip (2005), attempts have been made to solve this problem by wetting the hydrogel with 45% GTA and then washing the hydrogel with water to remove residues. However, even after an additional rinsing step, the GTA concentration used was too high, and the hydrogel produced according to the disclosed method has different qualities from the hydrogel of the present invention.

[0010] In contrast, Bigi et al., "Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking," Biomaterials, Vol. 22 (2001), pp. 763-768, show that hydrogel compositions containing GTA-crosslinked gelatin are characterized by good stability. Results shown for a range of 0.1% to 1% GTA indicate that the degree of crosslinking increases from 60% to 100%, with correspondingly different thermal and mechanical properties. Therefore, different GTA concentrations can be used to tailor the physicochemical properties of the films. However, the GTA concentration is high enough to render cell culture on hydrogels prepared in this manner ineffective.

[0011] Document No. CN105316285 discloses a method for producing a medium for three-dimensional cell culture, which involves dissolving collagen in acetic acid, dissolving chitosan in acetic acid, mixing them, drying them, adding GTA, leaving them to stand for 8 to 16 hours to crosslink, and purifying the resulting hydrogel.

[0012] Hydrogels made by cross-linking proteins with low concentrations of GTA do not currently exist in the prior art. By reducing the concentration and at the same time the ratio of GTA to the amount of lysine available for binding, hydrogels with better physicochemical quality and lower toxicity are obtained, which makes it possible to use the hydrogels of the present invention for two-dimensional and three-dimensional cell culture of both healthy and neoplastic cells, for migration and invasion assays in hydrogels in three-dimensional conditions, and for performing angiogenesis assays or aortic sprouting assays.

[0013] However, there remains a need to develop hydrogels with precisely selected qualities, such as density, hardness, and elasticity, so that they can be widely used in tests, for example, for performing angiogenesis assays. In the present invention, the precise selection of density and hardness parameters is achieved by modifying the gelatin or collagen and GTA concentrations. Elasticity is the result of modifying the above two parameters and the method of producing the hydrogel. The level of crosslinking determines the parameters of the final product, and only the technique described by the inventors can reduce the concentration to a level low enough to obtain the parameters of the hydrogels of the present invention.

[0014] Another object of the present invention is to provide a hydrogel for use in performing angiogenesis assays (angiogenesis assay, in vitro angiogenesis lumen formation assay, endothelial cell tube formation assay). The novel hydrogel can also be used in other cell cultures, such as neoplastic cell cultures, lab-on-a-chip cultures, plant and bacterial cell cultures, and flow cultures. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] U.S. Patent No. 4,829,000 [Non-patent literature]

[0016] [Non-Patent Document 1] Orci et al., "Vascular outgrowths from tissue explants embedded in fibrin or collagen gels: a simple in vitro model of angiogenesis," Cell Biology International Reports, Vol. 9, No. 10 (October 1985). [Non-patent document 2] DOILLON et al., "Three-dimensional Culture System as a Model for Studying Cancer Cell Invasion Capacity and Anticancer Drug Sensitivity," Anticancer Research, Vol. 24, pp. 2169-2178 (2004) [Non-patent document 3] Yamada and Even-Ram, "Cell migration in 3D matrix" (2005) [Non-patent document 4] MONTESANO et al., "Vascular outgrowths from tissue explants embedded in fibrin or collagen gels: a simple in vitro model of angiogenesis," Cell Biology International Reports, Vol. 9, No. 10 (October 1985). [Non-patent document 5] Ou and Yang, “The micro patterning of glutaraldehyde(GA)-crosslinked gelatin and its application to cell-culture”, Lab on a Chip (2005) [Non-patent document 6] Bigi et al., "Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking," Biomaterials, Vol. 22 (2001), pp. 763-768 Summary of the Invention

[0017] A further technical problem solved by the present invention is the elimination of the toxicity remaining after the reaction of GTA with gelatin. By removing the amount of GTA already remaining in the present invention, the resulting hydrogel is capable of growing even the most sensitive cells. Additionally, a very important feature of the present invention is its economical aspect. First, it is possible to react GTA with gelatin to produce hydrogels at such low concentrations. Moreover, the relatively inexpensive reagents used in the present invention significantly reduce costs while increasing the efficiency and cost-effectiveness of manufacturing, allowing the claimed products to function on a much larger scale.

[0018] A very important aspect is the fact that protein hydrogels produced in this manner are much more reproducible products for cell culture than products with a similar range of applications currently available on the market. The protein hydrogels of the present invention are the only products of this class that do not contain growth factors, significantly improving product reproducibility. The preparation of each commercially available component inactivates any residual growth factors that may be present therein, and these growth factors are further inactivated during the reaction with GTA, resulting in a product that is substantially free of or free of growth factors. The high reproducibility of the protein hydrogels of the present invention alone comes from the fact that they are synthesized from commercially available components, thereby allowing their concentrations to be very precisely selected and controlled in the final product.

[0019] The subject of the present invention is a protein hydrogel comprising reagent A, which is gelatin or collagen, reagent B, which is a crosslinking agent GTA (glutaraldehyde), and a solvent, characterized in that reagent A is present at a final concentration of 0.15% to 1.5% by weight, and the ratio of reagent A to reagent B is 0.375 to 4.5 mg to 0.01 to 0.15 mg in a portion of the hydrogel.

[0020] Preferably, the final concentration of reagent A is 0.25% to 1% by weight, and the ratio of reagent A to reagent B in one portion of the hydrogel is 0.625 to 3 mg to 0.0135 to 0.075 mg.

[0021] Particularly preferably, the final concentration of reagent A is 0.3% to 0.8% by weight, and the ratio of reagent A to reagent B in a portion of the hydrogel is 0.75 to 2.4 mg to 0.021 to 0.045 mg.

[0022] Preferably, the protein hydrogel of the present invention is characterized in that the gelatin is a gelatin with a Bloom value of at least 225, preferably a Bloom value of 300.

[0023] Preferably, the protein hydrogel of the present invention is characterized in that the solvent is an aqueous solution, more preferably selected from the group of dH2O, PBS, HBSS, and most preferably PBS.

[0024] Another subject of the present invention is a method for producing a protein hydrogel of the present invention, comprising the following steps: (a) adding an appropriate amount of reagent A, which is gelatin or collagen, to an aqueous solution, preferably selected from the group of dH2O, PBS, HBSS, and most preferably PBS; (b) heating the mixture of step (a) to dissolve the gel; (c) optionally initially stabilizing the gel; (d) preparing a crosslinker and GTA, Reagent B, by dissolving it in an aqueous solution and cooling; (e) adding Reagent B prepared in step (d) to the gel prepared in step (c); (f) optionally mixing the resulting mixture; (g) Cross-linking (h) optionally purifying the hydrogel of excess reagent B; wherein reagent A is present at a final concentration of 0.15% to 1.5% by weight, wherein the ratio of reagent A to reagent B in a portion of the hydrogel is 0.375 to 4.5 mg to 0.01 to 0.15 mg, and the initial stabilization of the gel occurs when the gel reaches a temperature of 0°C to 12°C for a duration of at least about 5 minutes; and wherein steps (d) to (g) are performed at a low temperature of about 0°C to about 12°C, and the duration of crosslinking in step (g) is at least 12 hours.

[0025] The addition of reagent B prepared in step (d) to the gel prepared in step (c) can be carried out by adding reagent B to the gel or by adding reagent B to an already gelled gel.

[0026] Preferably, the final concentration of reagent A is 0.25% to 1% by weight, and the ratio of reagent A to reagent B in one portion of the hydrogel is 0.625 to 3 mg to 0.0135 to 0.075 mg.

[0027] Particularly preferably, the concentration of reagent A is 0.3% to 0.8% by weight, and the ratio of reagent A to reagent B in a portion of the hydrogel is 0.75 to 2.4 mg to 0.021 to 0.045 mg.

[0028] Preferably, the duration of the initial stabilization is from 30 minutes to 48 hours, most preferably from 45 minutes to 24 hours.

[0029] Preferably, the duration of crosslinking is greater than 48 hours, most preferably greater than 72 hours.

[0030] If purification of the hydrogel is carried out in step (h), it is preferably carried out by rinsing with an aqueous solution, preferably an aqueous solution for cell culture, preferably PBS, or by adding a neutralizing reagent B, preferably L-lysine.

[0031] The optional purification of the hydrogel from excess reagent B in step (h) of the method is carried out with any substance capable of reacting with and inactivating the -CHO group. An example of such a substance is L-lysine, but also proteins containing the non-binding side chain -NH2 of lysine. This substance is used to neutralize toxic cross-linking substances, such as GTA, which contains two -CHO groups. This substance is used at a concentration that is a multiplication of the molar concentration of -CHO groups added when the hydrogel is produced. For example, when using 30 μL of 0.1% GTA, approximately 0.6 × 10 -3 mol of -CHO groups are present in such a volume. Using a 10x (10-fold) concentration of L-lysine means adding 10 times more L-lysine by mole than the -CHO groups added to form the hydrogel. L-lysine contains only one -NH group in the side chain, which can bond to the -CHO groups, and is typically added in a volume equal to the initial volume of the hydrogel.

[0032] Another subject of the present invention is the use of the protein hydrogels according to the invention for cell culture, preferably three-dimensional cell culture.

[0033] Yet another subject of the present invention is the use of a protein hydrogel produced by the method of the present invention for carrying out an angiogenesis assay, in which the duration of the initial stabilization in step (c) is between 10 and 90 minutes, preferably between 15 and 60 minutes, most preferably between 40 and 55 minutes, the duration of the crosslinking reaction is more than 60 hours, and the final concentration of reagent A is about 0.35 to 0.55% by weight, wherein such a ratio is maintained in a portion of the hydrogel such that 0.024 mg to 0.036 mg of reagent B for a mass of reagent A ranging from 0.875 to 1.375 mg.

[0034] Preferably, such a ratio is maintained in a portion of the hydrogel such that for a mass of reagent A in the range of 1 mg to 1.25 mg, reagent B is between 0.027 mg and 0.033 mg.

[0035] Particularly preferably, such a ratio is maintained in a portion of the hydrogel such that for every 1 mg of the mass of reagent A, there is 0.03 mg of reagent B.

[0036] GTA is added to gelatin at concentrations between 0.15% and 1.5% (i.e., up to 250 µL or 300 µL, respectively). Reducing the concentration of GTA to values ​​below 0.15 mg (i.e., 0.5% at 30 µL or 0.05% at 300 µL) not only reduces its toxicity (which then makes its removal easier), but most importantly, makes it possible to modify the elasticity and viscosity of the hydrogel, thus influencing the parameters of cell proliferation and offering completely new opportunities.

[0037] Terms used herein have meanings generally accepted in the art.

[0038] The term "low temperature" means a temperature within the range of about 0°C to about 12°C, preferably about 0°C to about 8°C, and particularly preferably above ice temperature, and can be used interchangeably with the expression "refrigerator temperature."

[0039] The term "about" is intended to indicate that a given numerical value has a defined value, however, there may be a 10% margin of error.

[0040] The term "aqueous solution" preferably means an aqueous solution for cell culture, preferably selected from the group dH2O, PBS, HBSS, particularly preferably PBS.

[0041] The terms "crosslinker" and "reagent B" refer to chemical compounds that function to link two or more protein chains. Protein chains are linked by amino acid side chains or amino acids at the ends of the proteins. Protein linkage is called crosslinking when the resulting protein network, also called a hydrogel, is formed. Exemplary protein crosslinking mechanisms are listed in Sung et al., "Evaluation of gelatin hydrogel crosslinked with various crosslinking agents as bioadhesives: In vitro study," Journal of Biomedical Materials Research (1999), where protein crosslinking can occur, for example, through -NH or -COOH functional groups. As disclosed in Bigi et al., the crosslinker is preferably pentane-1,5-dial (glutaraldehyde, GTA). GTA crosslinks gelatin or collagen by covalently linking -NH groups between proteins and also by linking these groups within a single protein, thereby stabilizing them.

[0042] The term "protein hydrogel portion / well" refers to an exemplary portion created with respect to the proportions described in the claims. Using two examples of "portions," 30 μL of GTA was added to 250 μL of gel (resulting in a target volume of 280 μL of hydrogel). Another example of a portion is a 300 μL gel portion, to the surface of which 300 μL of GTA was added. Here, the portion is limited to 300 μL of hydrogel because the GTA added to the surface of the gel does not mix with the gel itself and therefore does not increase the volume of the final hydrogel. In this manner, the applied reagent B diffuses into the gel, where the cross-linking reaction occurs, and the remaining excess is removed from the surface of the hydrogel by suction. For purposes of the present invention, the volumes of the hydrogel portions described above and disclosed in the embodiments are presented. However, this protection also covers smaller and larger amounts of reagents A and B, with respect to the proportions of reagents A and B as described herein.

[0043] "Reagent A" means gelatin or collagen, but also any other protein with the same or similar amino acid sequence as gelatin or collagen, obtained from an organism, and also a recombinant protein, i.e., a protein obtained by production in a genetically modified organism.

[0044] The subject matter of the present invention is illustrated in the embodiments and drawings. [Brief explanation of the drawings]

[0045] [Figure 1] Figure 1 shows tube-forming HUVEC endothelial cells on a hydrogel. This test is a model assay that illustrates blood vessel formation and allows for pro- and anti-angiogenic testing. [Figure 2] Figure 2 shows 4T1 cells cultured on hydrogels, which form three-dimensional spheroid structures. After long-term culture, cells are observed migrating between adjacent spheroids. Figure 2A shows 4T1 cells 14 days after seeding, and Figure 2B shows 4T1 cells 17 days after seeding. [Figure 3]Figure 3 shows a culture well half-filled with injected hydrogel and culture medium. Various types of cell growth and behavior are shown depending on the hydrogel. Figures A and C show growth and migration on a stiff, thick hydrogel, while Figures B and D show growth and migration on a soft, thin hydrogel. Figure A shows cell growth on the surface of the hydrogel. This is three-dimensional growth, but on the surface of the hydrogel. Figure B shows cell growth inside the hydrogel. Figure C shows migration on the surface of the hydrogel. Figure D shows two clusters of cells growing within the hydrogel and cells migrating between them within the hydrogel. DETAILED DESCRIPTION OF THE INVENTION

[0046] The following embodiments are provided to illustrate the present invention and are not intended to be limiting.

[0047] Example I - Protein Hydrogel Formation To produce 60 hydrogel portions at a concentration of 0.4%, 0.06 g of Bloom 300 gelatin type A was weighed and dissolved in 14.94 mL of PBS solution. Each hydrogel portion contained 0.001 g of gelatin. The solution was heated to 37 °C to dissolve the gel and then sterilized by filtration. Gels prepared in this manner were pipetted into a 48-well plate at 250 μL / well, placed in a refrigerator to cool, and then stabilized in the refrigerator for 45 minutes. The remaining 12 gel portions remained unused. A 0.1% GTA solution in dH2O was previously prepared by withdrawing 0.03 mg of GTA and replenishing it with 30 μL of dH2O, which was then cooled in the refrigerator for 30 minutes. 30 μL of the GTA solution was added to the cooled, stabilized, but ungelled, gels. The addition of GTA was performed on ice. Approximately 0.03 mg of GTA was present in each hydrogel portion. The plates containing the GTA-added gels were then placed in a refrigerator for 72 hours. The resulting hydrogels were then purified from excess GTA by neutralizing the hydrogels with L-lysine. L-lysine dissolved in PBS at a 10x concentration was used. The lysine was allowed to incubate with the hydrogels for 24 hours. The hydrogels prepared in this manner were ready for further use.

[0048] Example II - Protein Hydrogel Formation To produce 50 hydrogel portions at a concentration of 0.7%, 0.105 g of Bloom 300 gelatin type A was weighed and dissolved in 14.895 mL of PBS solution. Each hydrogel portion contained 0.0021 g of gelatin. The solution was heated to 37°C to dissolve the gel and then sterilized by filtration. The gel was pipetted into a 48-well plate at 300 μL / well. The remaining two gel portions remained unused. The plate prepared in this manner was placed in the refrigerator for 2 hours. A 0.02% GTA solution in dH2O was prepared first by withdrawing 0.06 mg of GTA and replenishing it with 300 μL of dH2O, which was then cooled in the refrigerator for a minimum of 30 minutes. 300 μL of the GTA solution was gently poured onto the surface of the gelled gel and placed in the refrigerator for 24 hours. The GTA addition was performed on ice. Approximately 0.06 mg of GTA was present in each hydrogel portion. The resulting hydrogel was then purified from excess GTA by rinsing with PBS three times. The hydrogel prepared in this manner was ready for further use.

[0049] Example III Endothelial (HUVEC) cells were seeded onto the hydrogel prepared in Example I at a density of 15,000 cells / well in a 48-well plate (depending on the specific cell line batch and cell division rate, the seeded cell density can vary from 5,000 to 50,000 cells / well in a 48-well plate). The endothelial cells were cultured in EGM™-2 BulletKit™ Lonza medium. The experimental result was the formation of tubes on the surface of the hydrogel by the endothelial cells (Figure 1).

[0050] Example IV Neoplastic 4T1 (mouse breast cancer) cells were seeded onto the hydrogel prepared in Example II at a density of 10,000 cells / well in a 48-well plate. The cells were cultured in RPMI + 10% FBS medium. The experimental result was the formation of spheroids by the neoplastic cells (Figure 2).

[0051] Example V (Comparative Example Using Prior Art Concentrations) The hydrogel formation assay was performed according to the method disclosed in the prior art (Bigi et al.). For this purpose, hydrogels were prepared with 5% Bloom 300 gelatin type A (by mass) and GTA at the mass concentrations listed in Tables 1-8. For the experiments shown in Tables 1, 2, 3, and 4, the protein hydrogels were dried for 24 hours (as described in Bigi et al.), whereas for Tables 5, 6, 7, and 8, the prepared protein hydrogels were crosslinked for 24 hours. Here, A means the hydrogel was not rinsed, B means it was rinsed five times with dH2O, and C means it was rinsed five times with PBS. The rinsing step is not described in the cited literature. After the experiment, the resulting cells were evaluated: 0 - no flat cells; probably all dead; 1 - a few flat cells present; 2 - many flat cells present. The following table shows the results obtained.

[0052] Seeding cell line: Panc_02 JPEG0007820150000001.jpg1457JPEG0007820150000002.jpg1357JPEG0007820150000003.jpg1357JPEG0007820150000004.jpg1357

[0053] Seeding cell line: HUVEC JPEG0007820150000005.jpg1357JPEG0007820150000006.jpg1357JPEG0007820150000007.jpg1357JPEG0007820150000008.jpg1357

[0054] The above data show that cells do not grow or do not grow flat on the more concentrated hydrogels of the prior art. In the case of the HUVEC cell line, this prevents the formation of blood vessels, i.e., prevents the performance of angiogenesis assays. In the case of the Panc_02 cell line, this means that cells can grow if the crosslinker is properly removed / neutralized, but this growth can be performed on a rigid medium such that the cells flatten, as is typical for standard 2D culture.

[0055] Example VI - Protein Hydrogel Formation with dH2O Solvent To produce 50 hydrogel portions (300 μL each) at a concentration of 0.3%, 0.045 g of Bloom 300 gelatin type A was weighed and dissolved in 14.955 mL of dH2O. Each hydrogel portion contained 0.0009 g of gelatin. The solution was heated to 37°C for 30 minutes to dissolve the gel and then sterilized by filtration. Gels prepared in this manner were pipetted into a 48-well plate at 300 μL / well. Two portions were left unpipetted for disposal. The 48-well plate with 48 gel portions was placed in a refrigerator to cool and then stabilized at refrigerator temperature for 23 hours. A 0.0035% (by mass) GTA solution in dH2O was previously prepared by withdrawing 0.0105 mg of GTA and replenishing it with 300 μL of dH2O. The temperature was then allowed to cool to refrigerator temperature by leaving the refrigerator for a minimum of 30 minutes. 300 μL of the cooled GTA solution was added to the surface of the cooled, stabilized, gelled gel. The GTA addition was performed on ice. The plate with the poured gel was then placed in a refrigerator for 72 hours. Approximately 0.0105 mg of GTA was present in each hydrogel portion. The resulting hydrogel was then purified from excess GTA by rinsing three times with PBS. The hydrogel prepared in this manner was ready for further use.

[0056] Example VII - Hydrogel formation for cell culture without the need to perform a step to remove toxic GTA residues To produce 50 hydrogel portions (250 μL each) at a concentration of 0.6%, 0.075 g of Bloom 300 gelatin type A was weighed and dissolved in 12.425 mL of PBS. Each hydrogel portion contained 0.0015 g of gelatin. The solution was heated to 37°C for 30 minutes to dissolve the gel and then sterilized by filtration. Gels prepared in this manner were pipetted into a 48-well plate at 250 μL / well. Two portions were left unpipetted for disposal. The 48-well plate with 48 gel portions was placed in a refrigerator to cool and then stabilized at refrigerator temperature for 60 minutes. A 0.06% (by mass) GTA solution in dH2O was previously prepared by withdrawing 0.018 mg of GTA and replenishing it with 30 μL of dH2O. The temperature was then allowed to cool to refrigerator temperature by leaving the refrigerator for a minimum of 30 minutes. To each of the cooled and stabilized, but not gelled, gels, 30 μL of cooled GTA solution was added. The addition of GTA was performed on ice. The plates with the GTA-added gels were then placed in a refrigerator for 72 hours. Approximately 0.018 mg of GTA was present in each hydrogel portion. The hydrogels prepared in this manner were ready for further use.

[0057] Example VIII - Formation of Protein Hydrogels with Type B Gelatin To produce 60 hydrogel portions (250 μL each) at a concentration of 0.4%, 0.06 g of type B gelatin was weighed and dissolved in 14.940 mL of PBS solution. Each hydrogel portion contained 0.001 g of gelatin. The solution was heated to 37°C for 30 minutes to dissolve the gel and then sterilized by filtration. Gels prepared in this manner were pipetted into a 48-well plate at 250 μL / well. Twelve portions were left unpipetted for disposal. The 48-well plate with 48 gel portions was placed in a refrigerator to cool and then stabilized at refrigerator temperature for 50 minutes. A 0.15% (by mass) GTA solution in dH2O was previously prepared by withdrawing 0.045 mg of GTA and replenishing it with 30 μL of dH2O, which was then cooled to refrigerator temperature. 30 μL of the cooled GTA solution was added to the cooled, partially stabilized, but ungelled, gel. The addition of GTA was performed on ice. Approximately 0.045 mg of GTA was present in each protein hydrogel portion. The plate containing the poured GTA gel was then placed in a refrigerator for 72 hours. The resulting protein hydrogel was then purified from excess GTA by rinsing it three times with PBS. The hydrogel prepared in this manner was ready for further use.

[0058] Example IX - Reproducibility of Angiogenesis To produce 50 hydrogel portions (250 μL each) at a concentration of 0.4%, 0.05 g of Bloom 300 gelatin type A was weighed and dissolved in 12.45 mL of PBS. Each hydrogel portion contained 0.001 g of gelatin. The solution was heated to 37°C for 30 minutes to dissolve the gel and then sterilized by filtration. Gels prepared in this manner were pipetted into a 48-well plate at 250 μL / well. Two portions were left unpipetted for disposal. The 48-well plate with 48 gel portions was placed in a refrigerator to cool and then stabilized at refrigerator temperature for 40 minutes. A 0.1% (by mass) GTA solution in dH2O was previously prepared by withdrawing 0.03 mg of GTA and replenishing it with 30 μL of dH2O, and the temperature was allowed to drop to refrigerator temperature by leaving it in the refrigerator for a minimum of 30 minutes. To each of the cooled, stabilized, but not gelled, gels, 30 μL of cooled GTA solution was added. The GTA addition was performed on ice. The plates containing the GTA-added gels were then placed in a refrigerator for 72 hours. Approximately 0.03 mg of GTA was present in each hydrogel portion. Protein hydrogels prepared in this manner were rinsed three times with PBS. Protein hydrogels were prepared in 10 separate production batches, each replicated four times.

[0059] Cells were seeded onto the protein hydrogel prepared in this manner, and after 10 hours of incubation, the cells were observed under a microscope. Table 9 below shows the assay results. A-cells form well-formed tubes B-cells begin to form lumens JPEG0007820150000009.jpg3941

[0060] The experiment shows that one of the features of the method of the present disclosure is the high reproducibility of the results. In all 40 trials, the angiogenesis assay returned positive results, with only two delays, which may be due to statistical error. The above results represent a significant improvement in the effectiveness of the angiogenesis assay compared to competing products.

[0061] The protein hydrogels that are the subject of the present invention make it possible to obtain culture media with precisely selected parameters, such as the density or hardness of the hydrogel, which influence the reproduction of the physiological conditions in which cells naturally grow, which in turn influences their behavior within the hydrogel, such as migration, ability to form spheroids, etc.

Claims

1. 1. A protein hydrogel comprising: Reagent A, which is gelatin; Reagent B, which is a crosslinker and is GTA; and a solvent, wherein Reagent A is present at a final concentration of 0.15% to 1.5% by weight, and the ratio of Reagent A to Reagent B is 0.375 to 4.5 mg to 0.01 to 0.15 mg in a portion of said hydrogel; and wherein said solvent is dH ２ 0 or PBS.

2. 2. The protein hydrogel of claim 1, wherein the final concentration of reagent A is 0.25% to 1% by weight, and the ratio of reagent A to reagent B in a portion of the hydrogel is 0.625 to 3 mg:0.0135 to 0.075 mg.

3. 3. The protein hydrogel of claim 2, wherein the final concentration of reagent A is 0.3% to 0.8% by weight, and the ratio of reagent A to reagent B in a portion of the hydrogel is 0.75 to 2.4 mg to 0.021 to 0.045 mg.

4. 4. The protein hydrogel according to claim 1, wherein the gelatin has a Bloom value of at least 225.

5. 5. The protein hydrogel of claim 4, wherein the gelatin is a gelatin with a Bloom value of 300.

6. 6. A method for producing the protein hydrogel of any one of claims 1 to 5, comprising: (a) adding an appropriate amount of reagent A, which is gelatin, to a solvent, said solvent being dH ２ 0 or PBS; (b) heating the mixture of step (a) to dissolve the gel; (c) initially stabilizing the gel; (d) preparing Reagent B, which is a crosslinker, GTA, by dissolving it in a solvent and cooling; (e) adding the reagent B prepared in step (d) to the gel prepared in step (c); (f) crosslinking; , including 1. A method according to claim 1, wherein reagent A is present at a final concentration of 0.15% to 1.5% by weight, wherein the ratio of reagent A to reagent B in a portion of the hydrogel is 0.375 to 4.5 mg to 0.01 to 0.15 mg, wherein the initial stabilization of the gel in step (c) occurs when the gel reaches a temperature of 0°C to 12°C for a duration of at least 5 minutes, and wherein steps (d) to (f) are carried out at a low temperature of 0°C to 12°C, and wherein in step (f) the duration of the crosslinking is at least 12 hours.

7. 7. The method of claim 6, further comprising mixing the mixture obtained in step (e).

8. 7. The method according to claim 6, characterized in that the hydrogel obtained in step (f) is purified from excess reagent B.

9. 7. The method of claim 6, wherein the final concentration of reagent A is 0.25% to 1% by weight, and the ratio of reagent A to reagent B in one portion of the hydrogel is 0.625 to 3 mg to 0.0135 to 0.075 mg.

10. 10. The method of claim 9, wherein the final concentration of reagent A is 0.3% to 0.8% by weight, and the ratio of reagent A to reagent B in one portion of the hydrogel is 0.75 to 2.4 mg to 0.021 to 0.045 mg.

11. 7. The method of claim 6, wherein the duration of the initial stabilization in step (c) is between 30 minutes and 48 hours.

12. 7. The method of claim 6, wherein the duration of the initial stabilization in step (c) is between 45 minutes and 24 hours.

13. 7. The method of claim 6, wherein the duration of the crosslinking is greater than 48 hours.

14. 7. The method of claim 6, wherein the duration of the crosslinking is greater than 72 hours.

15. 9. The method of claim 8, wherein the purification of the hydrogel of claim 8 is carried out by rinsing with a solvent or by neutralizing reagent B.

16. 16. The method of claim 15, wherein the solvent is PBS.

17. 16. The method of claim 15, wherein the neutralization of reagent B is achieved by adding L-lysine.

18. Use of the protein hydrogel according to any one of claims 1 to 5 for cell culture.

19. 19. The use according to claim 18 for three-dimensional cell culture.

20. 10. Use of the protein hydrogel produced by the method defined in claim 6 for performing an angiogenesis assay, wherein the duration of the initial stabilization in step (c) is 10 to 90 minutes, the duration of the cross-linking reaction at low temperature is more than 60 hours, and the final concentration of reagent A is 0.35 to 0.55% by weight, and wherein the ratio of reagent A to reagent B in one portion of the hydrogel is 0.875 to 1.375 mg to 0.024 to 0.036 mg.

21. 21. The use according to claim 20, wherein the duration of the initial stabilization in step (c) is from 15 to 60 minutes.

22. 21. The use according to claim 20, wherein the duration of the initial stabilization in step (c) is from 40 to 55 minutes.

23. 19. The use according to claim 18, wherein the ratio of reagent A to reagent B in one portion of the hydrogel is 1-1.25 mg to 0.027-0.033 mg.

24. 20. The use of claim 19, wherein the ratio of reagent A to reagent B in one portion of the hydrogel is 1 mg to 0.03 mg.

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