PROTEIN HYDROGEL, PREPARATION METHOD AND USE THEREOF

Abstract

The invention relates to a new protein hydrogel created on the basis of low-concentrated components: reagents A and B, the method of hydrogel preparation and its use.

Claims

1-16. (canceled)

17. A protein hydrogel comprising: reagent A, being gelatin, reagent B, being a cross-linking agent, being GTA, and solvent, characterised in that reagent A is present in the final concentration from 0.15% wt. to 1.5% wt., with a ratio of reagent A to reagent B of 0.375-4.5 mg to 0.01-0.15 mg in one portion of the hydrogel, wherein solvent is dH.sub.2O or PBS.

18. The protein hydrogel according to claim 17, characterized in that the final concentration of reagent A is from 0.25% wt. to 1% wt., with a ratio of reagent A to reagent B of 0.625-3 mg to 0.0135-0.075 mg in one portion of the hydrogel.

19. The protein hydrogel according to claim 18, characterized in that the final concentration of reagent A is from 0.3% wt. to 0.8% wt., with a ratio of reagent A to reagent B of 0.75-2.4 mg to 0.021-0.045 mg in one portion of the hydrogel.

20. The protein hydrogel according to any one of the preceding claims, characterized in that gelatin is gelatin of the Bloom value of at least 225, preferably of the Bloom value of 300.

21. A method of producing the protein hydrogel as defined in claims 17-19, comprising the steps of: a) addition a suitable amount of reagent A, being gelatin, in an aqueous solution, selected from dH.sub.2O or PBS,; b) heating up the mixture of step a) to dissolve the gel; c) initially stabilising the gel; d) preparing reagent B, being a cross-linking agent, being GTA, by dissolving it in an aqueous solution and cooling it; e) adding reagent B, as prepared in step d), to the gel prepared in step c); f) optionally mixing the obtained mixture; g) cross-linking; h) optionally purifying the hydrogel of an excess of reagent B, characterised in that reagent A is present in the final concentration from 0.15% wt. to 1.5% wt., with a ratio of reagent A to reagent B of 0.375-4.5 mg to 0.01-0.15 mg in one portion of the hydrogel, the initial stabilisation of the gel takes place when the gel reaches the temperature 0° C.-12° C. and its duration is at least about 5 minutes; steps d)-g) are performed at reduced temperature from about 0° C. to about 12° C., in step g) the duration of cross-linking is at least 12 h.

22. The method according to claim 21, characterized in that the final concentration of reagent A is from 0.25% wt. to 1% wt., with a ratio of reagent A to reagent B of 0.625-3 mg to 0.0135-0.075 mg in one portion of the hydrogel.

23. The method according to claim 21, characterized in that the final concentration of reagent A is from 0.3% wt. to 0.8% wt., with a ratio of reagent A to reagent B of 0.75-2.4 mg to 0.021-0.045 mg in one portion of the hydrogel.

24. The method according to claim 21, characterized in that the duration of initial stabilisation is 30 minutes to 48 hours, most preferably 45 minutes to 24 hours.

25. The method according to claim 21, characterized in that the duration of cross-linking is above 48 hours, most preferably above 72 hours.

26. The method according to any one of the claims from 21, characterized in that if the purification of the hydrogel in step h) takes place, it takes place by means of rinsing with an aqueous solution, preferably an aqueous solution for cell cultures, preferably PBS, or by means of neutralising reagent B, preferably by adding L-lysine.

27. Use of the protein hydrogel as defined in claims 17-19 for cell cultures.

28. The use according to claim 27 for 3D cell cultures.

29. The use according to the protein hydrogel produced by the method as defined in claim 21, to perform an angiogenesis assay, with the duration of the initial stabilisation in step c) being from 10 to 90 minutes, preferably from 15 to 60 minutes, most preferably from 40 to 55 minutes, and the duration of the cross-linking reaction in the reduced temperature being above 60 hours, and the final concentration of reagent A being about 0.35-0.55% wt., with a ratio of reagent A to reagent B of 0.875-1.375 mg to 0.024-0.036 mg in one portion of the hydrogel.

30. The use according to claim 29, with a ratio of reagent A to reagent B of 1-1.25 mg to 0.027-0.033 mg in one portion of the hydrogel.

31. The use according to claim 30, wherein a proportion is maintained in one portion of the hydrogel that for the mass of reagent A in the amount of 1 mg falls 0.03 mg of reagent B.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The subject matter of the invention is shown in the embodiments and in the figures, where:

[0052] FIG. 1—shows tube-forming HUVEC endothelial cells on a hydrogel. This test is a model assay illustrating the formation of blood vessels. It enables pro- and anti-angiogenic tests.

[0053] FIG. 2—shows 4T1 cells cultured on a hydrogel, the cells forming three dimensional structures, spheroids. After long term culture, cell migrating between neighbouring spheroids are observed. FIG. 2A shows 4T1 cells 14 days after seeding and FIG. 2B 17 days after seeding.

[0054] FIG. 3—shows culture wells half-filled with hydrogel and with a culture medium poured into. Various kinds of cell growth and behaviour depending on the hydrogel are illustrated. FIGS. A and C show growth and migration on a hard and thick hydrogel, and FIGS. B and D show growth and migration on a soft and thin hydrogel. FIG. A shows cell growth on the surface of the hydrogel, this being a 3D growth but on the surface of the hydrogel. FIG. B shows cell growth into the inside of the hydrogel. FIG. C shows migration on the surface of the hydrogel. FIG. D shows two lumps, which have grown into the hydrogel, and cells migrating between them inside the hydrogel.

DETAILED DESCRIPTION

[0055] Embodiments are shown below, them being only an illustration of the invention and not of a limiting nature.

EXAMPLE I

Production of the Protein Hydrogel

[0056] To produce 60 hydrogel portions of a concentration of 0.4%, 0.06 g of type A Bloom 300 gelatin was weighed and dissolved in 14.94 ml of PBS solution. Each hydrogel portion contained 0.001 g of gelatin. The solution was heated at 37° C. to dissolve the gel and then sterilised by filtration. The gel prepared in this manner was pipetted into a 48-well plate at 250 μl per well and put into a fridge to cool and then stabilised for 45 minutes in the fridge. The remaining 12 gel portions remained unused. By taking 0.03 mg of GTA and supplementing to 30 μl of dH.sub.2O, 0.1% GTA solution in dH.sub.2O was prepared earlier and cooled for 30 min in the fridge. To a cool, stabilised, but not gelified gel, 30 μl of GTA solution was added. GTA addition took place on ice. In each hydrogel portion, there was about 0.03 mg of GTA. Then the plate with the gel with GTA added was put to the fridge for 72 h. After that time, the resulting hydrogel was purified of excess GTA by hydrogel neutralisation, by adding L-lysine. L-lysine of a concentration 10× dissolved in PBS was used. The lysine was incubated with hydrogel for 24 h. The hydrogel prepared in this manner is ready for further use.

EXAMPLE II

Production of the Protein Hydrogel

[0057] To produce 50 hydrogel portions of a concentration of 0.7%, 0.105 g of type A Bloom 300 gelatin was weighed and dissolved in 14.895 ml of PBS solution. Each hydrogel portion contained 0.0021 g of gelatin. The solution was heated at 37° C. to dissolve the gel and then sterilised by filtration. The gel was pipetted into a 48-well plate, 300 μl per well. The remaining 2 gel portions remained unused. The plate prepared in this manner was put to the fridge for 2 h. By taking 0.06 mg of GTA and supplementing to 300 μl of dH.sub.2O, 0.02% GTA solution in dH.sub.2O was prepared earlier and cooled for minimum 30 min in the fridge. Onto the surfaces of the gelified gel, 300 μl of GTA solution was poured gently and put into the fridge for 24 h. GTA addition took place on ice. In each hydrogel portion, there was about 0.06 mg of GTA. After that time, the resulting hydrogel was purified of excess GTA by rinsing the hydrogel 3 times with PBS. The hydrogel prepared in this manner is ready for further use.

EXAMPLE III

[0058] Onto the hydrogel prepared in Example I, endothelial (HUVEC) cells were seeded at density of 15 thousand cells/well of a 48-well plate (depending on the specific cell line batch and the number of cell divisions, the density of the seeded cells may vary from 5 to 50 thousand cells/well of a 48-well plate). The endothelial cells were cultured in EGMTM-2 BulletKit™ Lonza medium. The result of the experience were tubes formed on the surface of the hydrogel by the endothelial cells (FIG. 1).

EXAMPLE IV

[0059] Onto the hydrogel prepared in Example II, neoplastic 4T1 (murine mammary carcinoma) cells were seeded at density of 10 thousand cells/well of a 48-well plate. The cells were cultured in RPMI+10% FBS medium. The result of the experience were spheroids formed by the neoplastic cells (FIG. 2).

EXAMPLE V

a Comparative Example Using Prior Art Concentrations

[0060] Assays were performed, in which hydrogels were produced according to the methods disclosed in the prior art (Bigi et al.). To that end, a hydrogel of a composition 5% type A Bloom 300 gelatin (mass) and GTA of mass concentrations described in Tables 1-8 was prepared. In the experiments shown in Table 1, 2, 3 and 4, the protein hydrogel was dried 24 h (the drying was according to the description in publication Bigi et al.), whereas in Tables 5, 6, 7 and 8, the prepared protein hydrogel was cross-linked 24 h, with A meaning that the hydrogel was not rinsed, B meaning that it was rinsed 5× with dH.sub.2O and C meaning that it was rinsed 5× with PBS. The rinsing steps were not described in the cited publication. After the experiment, the resulting cells were evaluated: 0—no flattened cells; most probably all are dead; 1—a small number of flattened cells present; 2—a high number of flattened cells present. The Tables below show the obtained results.

Seeded cell line is: Panc_02

TABLE-US-00001 TABLE 1 GTA concentration [%] 0.1 0.125 0.25 0.5 A 0 0 0 0 B 0 0 0 0 C 1 1 0 0

TABLE-US-00002 TABLE 2 GTA concentration [%] 1 1.5 2 2.5 A 0 0 0 0 B 0 0 0 0 C 0 0 0 0

TABLE-US-00003 TABLE 5 GTA concentration [%] 0.1 0.125 0.25 0.5 A 0 0 0 0 B 0 0 0 0 C 1 1 0 0

TABLE-US-00004 TABLE 6 GTA concentration [%] 1 2 3 4 A 0 0 0 0 B 0 0 0 0 C 0 0 0 0
Seeded cell line is: HUVEC

TABLE-US-00005 TABLE 3 GTA concentration [%] 0.1 0.125 0.25 0.5 A 0 0 0 0 B 2 2 2 0 C 2 2 2 2

TABLE-US-00006 TABLE 4 GTA concentration [%] 1 1.5 2 2.5 A 0 0 0 0 B 0 0 0 0 C 0 0 0 0

TABLE-US-00007 TABLE 7 GTA concentration [%] 0.1 0.125 0.25 0.5 A 0 0 0 0 B 1 1 1 0 C 2 1 1 1

TABLE-US-00008 TABLE 8 GTA concentration [%] 1 1.5 2 2.5 A 0 0 0 0 B 0 0 0 0 C 1 1 1 1

[0061] The above data show that on the higher concentration hydrogels from the prior art, the cells do not grow or grow flattened. In the case of the HUVEC cell line, it prevents the formation of blood vessels, i.e. the performing of an angiogenesis assay. In the case of the Panc_02 cell line, this means that if the cross-linking agent has been properly removed/neutralised, the cells can grow but the growth is on a hard medium so that the cells flatten as is typical for a standard 2D culture.

EXAMPLE VI

Production of the Protein Hydrogel with dH.SUB.2.O Solvent

[0062] To produce 50 hydrogel portions (300 μl each) of a concentration of 0.3%, 0.045 g of type A Bloom 300 gelatin was weighed and dissolved in 14.955 ml of dH.sub.2O. 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 sterilised by filtration. The gel prepared in this manner was pipetted into a 48-well plate, at 300 μl per well. Two portions remained unpipetted for disposal. The 48-well plate with 48 gel portions was put into a fridge to cool and then to stabilise for 23 h in the fridge temperature. By taking 0.0105 mg of GTA and supplementing to 300 μl of dH.sub.2O, 0.0035% (mass) GTA solution in dH.sub.2O was prepared earlier and its temperature was reduced to the fridge temperature by allowing it to stand for minimum 30 minutes in the fridge. Onto the surface of a cool, stabilised and gelified gel, 300 μl of a cooled GTA solution was added. The additions of GTA were made on ice. Then the plate with the gel with GTA poured onto was put to the fridge for 72 h. In each hydrogel portion, there was about 0.0105 mg of GTA. After that time, the resulting hydrogel was purified of excess GTA by rinsing the hydrogel 3 times with PBS. The hydrogel prepared in this manner is ready for further use.

EXAMPLE VII

Hydrogel Production for a Cell Culture Without it Being Necessary to Carry Out the Step of Removing the Residues of Toxic GTA

[0063] To produce 50 hydrogel portions (250 μl each) of a concentration of 0.6%, 0.075 g of type A Bloom 300 gelatin 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 sterilised by filtration. The gel prepared in this manner was pipetted into a 48-well plate, at 250 μl per well. 2 portions remained unpipetted for disposal. The 48-well plate with 48 gel portions was put into a fridge to cool and then to stabilise for 60 minutes in the fridge temperature. By taking 0.018 mg of GTA and supplementing to 30 μl of dH.sub.2O, 0.06% (mass) GTA solution in dH.sub.2O was prepared earlier and its temperature was reduced to the fridge temperature by allowing it to stand for minimum 30 minutes in the fridge. To a cool, stabilised, but not gelified gel, 30 μl each of a cooled GTA solution was added. The addition of GTA was made on ice. Then the plate with the gel with GTA added was put to the fridge for 72 h. In each hydrogel portion, there was about 0.018 mg of GTA. The hydrogel prepared in this manner is ready for further use.

EXAMPLE VIII

Production of the Protein Hydrogel with Type B Gelatin

[0064] To produce 60 hydrogel portions (250 μl each) of 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 sterilised by filtration. The gel prepared in this manner was pipetted into a 48-well plate, at 250 μl per well. Twelve portions remained unpipetted for disposal. The 48-well plate with 48 gel portions was put into a fridge to cool and then to stabilise for 50 minutes in the fridge temperature. By taking 0.045 mg of GTA and supplementing to 30 μl of dH.sub.2O, 0.15% (mass) GTA solution in dH.sub.2O was prepared earlier and its temperature was reduced to the fridge temperature. To a cool and partially stabilised, but not gelified gel, 30 μl of a cooled GTA solution was added. GTA addition took place on ice. In each protein hydrogel portion, there was about 0.045 mg of GTA. Then the plate with the gel with GTA poured onto was put to the fridge for 72 h. After that time, the resulting protein hydrogel was purified of excess GTA by rinsing the hydrogel 3 times with PBS. The hydrogel prepared in this manner is ready for further use.

EXAMPLE IX

Reproducibility of Angiogenesis

[0065] To produce 50 hydrogel portions (250 μl each) of a concentration of 0.4%, 0.05 g of type A Bloom 300 gelatin 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 sterilised by filtration. The gel prepared in this manner was pipetted into a 48-well plate, at 250 μl per well. 2 portions remained unpipetted for disposal. The 48-well plate with 48 gel portions was put into a fridge to cool and then to stabilise for 40 minutes in the fridge temperature. By taking 0.03 mg of GTA and supplementing to 30 μl of dH.sub.2O, 0.1% (mass) GTA solution in dH.sub.2O was prepared earlier and its temperature was reduced to the fridge temperature by allowing it to stand for minimum 30 minutes in the fridge. To a cool, stabilised, but not gelified gel, 30 μl each of a cooled GTA solution was added. The additions of GTA were made on ice. Then the plate with the gel with GTA added was put to the fridge for 72 h. In each hydrogel portion, there was about 0.03 mg of GTA. The protein hydrogel prepared in this manner was rinsed 3× with PBS. The protein hydrogel was prepared in 10 separate production batches, in 4 replicates each time.

[0066] On the protein hydrogel prepared in this manner, cells were seeded and after 10 h of incubation the cells were observed under microscope. Table 9 below shows the assay results, where:

[0067] A—the cells form well-shaped tubes

[0068] B—the cells start to form tubes

TABLE-US-00009 TABLE 9 Production Replicates batch 1 2 3 4 1 A A A A 2 A A A A 3 A A A A 4 A A A B 5 A A A A 6 A A A A 7 A A A A 8 A A A A 9 A A A A 10 B A A A

[0069] The experiment shows that one of the characteristics of the method being the subject matter of the disclosure is a high reproducibility of results. In all 40 attempts, the angiogenesis assay returned a positive result and only twice was slightly delayed in time, which may be due to a statistical error. The above results represent a significant improvement in the effectiveness of the angiogenesis assay as compared to competitive products.

[0070] The protein hydrogel being the subject matter of the invention makes it possible to obtain a medium with precisely selected parameters, e.g. density or hardness of the hydrogel. These parameters have an influence on the reproduction of physiological conditions in which the cell grew naturally, which in turn affects their behaviours, such as: migration inside the hydrogel, ability to form spheroids, etc.