PH response type polypeptide hydrogel and application

Abstract

The invention provides pH response type polypeptide hydrogel and application. The pH response type polypeptide hydrogel is formed by compounding a buffer salt solution, a polysaccharide aqueous solution and a polypeptide aqueous solution. The hydrogel is used for three-dimensional culture of cancer cell lines and stem cells, a hydrogel culture system can be formed only by suspending cells to be cultured in a mixed solution of a buffer salt solution and a polysaccharide aqueous solution, mixing polypeptide with a cell suspension and incubating; the tumor cell spheres or the stem cell network structure can be formed by adding a proper amount of universal complete culture medium suitable for cell growth, the operation is simple, the cost is low, and the culture success rate of the obtained tumor cell spheres or the stem cell network structure is 100%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of three-dimensional cell culture, and particularly relates to a pH-responsive polypeptide hydrogel and application thereof. BACKGROUND

[0002] At present, 3D culture research is widely applied in biomedical research, tissue engineering and other research fields. Three-dimensional porous nanoscaffolds can mimic the structure of the natural extracellular matrix and provide support for cell growth. Among many established 3D scaffolds, hydrogels show unique properties as extracellular matrix.

[0003] Self-assembling short peptide RADA16 is composed of short amino acid sequences, and when its aqueous solution is adjusted to pH = 7, a hydrogel composed of nanofiber network is formed, which is very similar to the structure of the natural extracellular matrix. The synthesis cost of this self-assembling material is low, and the hydrogel is prepared by adjusting the pH, which avoids the damage to cells or tissues caused by chemical cross-linking agents, ultraviolet light, etc., and also avoids the potential biological toxicity and immunogenicity caused by the introduction of chemical reactions. However, the aqueous solution of the material has obvious acidity and cannot be directly mixed with cell suspension to form a gel in situ, that is, it is difficult to achieve three-dimensional growth of cells embedded in RADA16 hydrogel. Therefore, the existing cell culture using RADA16 polypeptide hydrogel mainly adopts two ways, the first way is to prepare RADA16 polypeptide into hydrogel first and then inoculate cells on the surface of the hydrogel for culture, which is a typical two-dimensional culture mode, but this two-dimensional culture mode is quite different from the in-vivo culture environment, which affects cell growth and even changes in cell function. The other way is to use the commercially available RADA16 hydrogel product Pura Matrix (Corning), mix it with cells to form a gel, and then wash it with a large amount of culture medium until the system is neutral, and then culture the cells. This way requires a large amount of culture medium, so for expensive culture medium suitable for three-dimensional cell culture, the cost of cell culture will be greatly increased.

[0004] In summary, how to properly solve the biocompatibility and cost problem of RADA16 polypeptide in three-dimensional cell culture is a big problem for the wide application of this biomaterial. SUMMARY

[0005] Therefore, in view of the problems of complicated culture process, unstable results, high cost and other problems existing in the existing three-dimensional culture of tumor cells and stem cells, and the biocompatibility and cost problems existing in the existing RADA16 polypeptide culture system, the present application provides a pH-responsive polypeptide hydrogel and application thereof.

[0006] To achieve the above technical purposes, the technical scheme adopted by the present application is as follows: In a first aspect, the present invention provides a pH-responsive polypeptide hydrogel, wherein the pH-responsive polypeptide hydrogel is formed by compounding a buffer salt solution, a polysaccharide aqueous solution, and a polypeptide aqueous solution, and the pH of the pH-responsive polypeptide hydrogel is 7.0~7.4; wherein the buffer salt solution is selected from at least one of PBS, DPBS, HEPES, and TBS; the polysaccharide in the polysaccharide aqueous solution is selected from at least one of trehalose, sucrose, glucose, and mannose; and the polypeptide in the polypeptide aqueous solution is RADA16, with the amino acid sequence: AC-RADARADARADARADA-NH2 (SEQ ID NO.1), and the chemical formula: C 66 H 113 N 29 O 25 .

[0007] Preferably, the polysaccharide aqueous solution has a mass percentage concentration of 10-25%; and / or, the polypeptide aqueous solution has a concentration of 5-20 mg / mL.

[0008] Preferably, the polysaccharide aqueous solution has a mass percentage concentration of 10-20%; and / or, the polypeptide aqueous solution has a concentration of 6-15 mg / mL.

[0009] More preferably, the volume ratio of the buffer salt solution to the polysaccharide aqueous solution is 1:1 to 4; and the volume ratio of the sum of the buffer salt solution and the polysaccharide aqueous solution to the polypeptide aqueous solution is 1 to 4:1.

[0010] More preferably, the volume ratio of the buffer salt solution to the polysaccharide aqueous solution is 1:3~4; and the volume ratio of the sum of the buffer salt solution and the polysaccharide aqueous solution to the polypeptide aqueous solution is 1~2:1.

[0011] Secondly, the present invention provides the application of the pH-responsive polypeptide hydrogel described in the first aspect in three-dimensional cell culture, the application comprising: suspending the cultured cells in a mixed solution of the buffer salt solution and the polysaccharide aqueous solution, and then adding the polypeptide aqueous solution for three-dimensional cell culture.

[0012] The three-dimensional cell culture involves the growth, proliferation, and passage of cells in three dimensions to obtain cell spheroids or network-like cell structures.

[0013] Preferably, the concentration of the cells to be cultured in the mixed solution of buffered salt solution and polysaccharide aqueous solution is (2~10)×10⁻¹⁰. 5 per mL.

[0014] Preferably, the three-dimensional cell culture process includes: the system is first incubated at 37±1℃ until a hydrogel is formed, and then culture medium is added to continue culturing for 5~14 days.

[0015] Furthermore, the culture medium can be a general complete culture medium, or various growth factors can be flexibly added to the basic culture medium such as MEM, DMEM, 1640, F-12 as needed. Currently, common growth factors used for tumor cell and stem cell culture include: insulin, insulin-like growth factor, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, osteosarcoma-derived growth factor, growth hormone-releasing inhibitory factor, cortisol, nerve growth factor, thyroxine, etc.

[0016] More preferably, the static incubation time is 5 to 15 minutes.

[0017] Preferably, the cells suitable for the three-dimensional cell culture include tumor cells and stem cells.

[0018] Furthermore, the tumor cells include, but are not limited to: pancreatic cancer cells, gastric cancer cells, intestinal cancer cells, nasopharyngeal cancer cells, lung cancer cells, ovarian cancer cells, breast cancer cells, cervical cancer cells, liver cancer cells, and endometrial cancer cells; the stem cells are mesenchymal stem cells.

[0019] Compared with the prior art, the present invention has the following beneficial effects: Firstly, the three-dimensional culture results of the pH-responsive polypeptide hydrogel of this invention on cancer cell lines and stem cells show that both cancer cell lines and stem cells can stably proliferate and be passaged for a long time in the hydrogel, indicating that the hydrogel has good biocompatibility. Specifically, during the proliferation stage of cancer cells, relatively uniformly sized cell spheroids are formed, numerous and growing rapidly. During passage, the cell spheroids further expand and enlarge, becoming more compact. Stem cells ultimately form a stable, spindle-like network cell structure, proliferating rapidly and becoming more densely packed.

[0020] Secondly, the pH-responsive polypeptide hydrogel of the present invention also has a cell recovery function, specifically: when sterile ultrapure water is drawn with a pipette tip and the pH-responsive polypeptide hydrogel containing cells is gently blown 10-25 times, the hydrogel can be transformed back into a solution state, and the cells can be recovered for 3D culture again, or the recovered cells can be passaged, which is beneficial for the multiple recovery and reuse of certain precious cells or cells with utilization value.

[0021] Thirdly, the pH-responsive polypeptide hydrogel of this invention allows for three-dimensional culture of cancer cell lines and stem cells. The resulting tumor cell spheres facilitate a deeper understanding of the tumor microenvironment, enabling researchers to visualize cell-cell interactions, how tumor cells absorb nutrients, and tumor cell proliferation patterns. Furthermore, tumor cell spheres can reveal the tumor penetration (or lack thereof) of drugs and their inhibitory effects on metastasis, making them highly suitable for preclinical drug screening and validation. The resulting stem cell network structure facilitates intercellular communication and yields high-quality, homogeneous mesenchymal stem cells, achieving high cell yields and stable stem cell characteristics (steminess, multi-lineage differentiation capacity) to meet the demands of large-scale production. Moreover, large-scale production of mesenchymal stem cells can facilitate their transition from basic research to clinical applications, such as cell therapy, exosome preparation, and regenerative medicine.

[0022] Fourth, this invention is for three-dimensional culture of cancer cell lines and stem cells. It only requires suspending the cells to be cultured in a mixed solution of buffered salt solution and polysaccharide aqueous solution, then mixing the peptides with the cell suspension and incubating to form a hydrogel culture system. Adding an appropriate amount of general complete culture medium suitable for cell growth is sufficient. The operation is simple, the cost is low, and the success rate of obtaining tumor cell spheres or stem cell network structures is 100%. Attached Figure Description

[0023] Figure 1 The images show the state of the pancreatic cancer cell line PANC-1 in pH-responsive polypeptide hydrogel from day 1 to day 7 in Example 1 of this invention, where A to C correspond to cell morphology images on days 1, 4, and 7 of culture, respectively.

[0024] Figure 2 The images shown are of the pancreatic cancer cell line PANC-1 recovered after 7 days of culture in Example 1 of this invention at different magnifications, where A and B correspond to magnifications of 200 μm and 100 μm, respectively.

[0025] Figure 3 The images show the state of the pancreatic cancer cell line PANC-1 in Example 2 of this invention during passage culture in a pH-responsive polypeptide hydrogel from day 0 to day 5, where A to C correspond to cell morphology images on days 0, 3, and 5 of passage, respectively.

[0026] Figure 4 The images show a comparison of cell spheres formed by the pancreatic cancer cell line PANC-1 in Example 3 and Comparative Example 1 of the present invention. A and B correspond to the cell morphology images on day 7 of culture in Example 3 and Comparative Example 1, respectively.

[0027] Figure 5The figures show a comparison of the expansion and culture results of the mouse mesenchymal stem cell line C3H / 10T1 / 2 in Example 4 and Comparative Example 2 of the present invention. In the figures, A to C correspond to the cell morphology images on days 1, 4, and 7 of culture in Example 4, respectively; and D to F correspond to the cell morphology images on days 1, 4, and 7 of culture in Comparative Example 2, respectively. Detailed Implementation

[0028] In the description of this invention, it should be noted that the cell spherical structure generally refers to a spherical structure formed by the spontaneous aggregation of a single cell or multiple single cells under specific culture conditions.

[0029] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0030] This invention provides a pH-responsive polypeptide hydrogel, which is formed by combining a buffer salt solution, a polysaccharide aqueous solution, and a polypeptide aqueous solution. The pH of the pH-responsive polypeptide hydrogel is 7.0-7.4. The buffer salt solution is selected from at least one of PBS, DPBS, HEPES, and TBS. The polysaccharide in the polysaccharide aqueous solution is selected from at least one of trehalose, sucrose, glucose, and mannose. The polypeptide in the polypeptide aqueous solution is RADA16, with the amino acid sequence AC-RADARADARADARADA-NH2 and the chemical formula C. 66 H 113 N 29 O 25 .

[0031] The pH-responsive nature of the hydrogel of this invention means that its composition results in a pH of 7.0 to 7.4, allowing it to self-assemble into a hydrogel. Normally, cells generate metabolic waste during culture, affecting the pH of the culture system. However, once formed, the hydrogel of this invention is less affected by the pH of the environment, and its overall hydrogel structure can be stably maintained for at least 7-14 days, thus ensuring the stability of cell culture results.

[0032] In a specific embodiment of the present invention, the concentration of the buffer salt solution is 0.01~0.03 M; the mass concentration of the polysaccharide aqueous solution is 10~25%, preferably 10~20%, more preferably 15~20%; and the concentration of the polypeptide aqueous solution is 5~20 mg / mL, preferably 6~15 mg / mL, more preferably 6~10 mg / mL.

[0033] In a specific embodiment of the present invention, the volume ratio of buffer salt solution to polysaccharide aqueous solution is 1:1 to 4, preferably 1:3 to 4; the volume ratio of the sum of buffer salt solution and polysaccharide aqueous solution to polypeptide aqueous solution is 1 to 4:1, preferably 1 to 2:1, which helps to better form a hydrogel culture system.

[0034] In three-dimensional cell culture, the cells to be cultured are first suspended in a mixture of buffered salt solution and polysaccharide aqueous solution. Then, peptides are mixed with the cell suspension and incubated statically to form a hydrogel culture system. Adding an appropriate amount of general complete culture medium suitable for cell growth will form tumor cell spheres or stem cell network structures. During the static incubation process until hydrogel formation, a time of 5-15 minutes is ideal. Short incubation times are not conducive to the stable formation of the hydrogel culture system, while excessively long incubation times may affect cell viability.

[0035] This pH-responsive polypeptide hydrogel is currently suitable for use with the following tumor cell types: pancreatic cancer cells, gastric cancer cells, intestinal cancer cells, nasopharyngeal cancer cells, lung cancer cells, ovarian cancer cells, breast cancer cells, cervical cancer cells, liver cancer cells, and endometrial cancer cells. Specifically, it includes: pancreatic cancer AsPC-1, pancreatic cancer BxPC-3, pancreatic cancer Capan-1, pancreatic cancer Capan-2, and pancreatic cancer MIA. PaCa-2, Pancreatic Cancer PANC-1, Pancreatic Cancer Patu8988, Pancreatic Cancer T3M4, Gastric Cancer MGC803, Gastric Cancer OVCAR3, Gastric Cancer KATO-III, Gastric Cancer SGC-7901, Gastric Cancer BGC-803, Gastric Cancer AGS, Gastric Cancer MKN-45, Gastric Cancer 7A, Gastric Cancer 7B, Gastric Cancer HSC-38, Gastric Cancer HSC-66, Gastric Cancer SGC-7801, Colorectal Cancer SW480, Colorectal Cancer SW620, Colorectal Cancer SW1116, Colorectal Cancer LOVO, Colorectal Cancer HCT116, Colorectal Cancer HCT-15, Colorectal Cancer HT-29, Colorectal Cancer LS180, Colorectal Cancer LS174T, Colorectal Cancer NCI-H716, Colorectal Cancer Caco-2, Colorectal Cancer COLO205, Colorectal Cancer COLO320, Colorectal Cancer COLO320DM, Colorectal Cancer CT26.WT, DLD-1 (colorectal cancer), RKO (colorectal cancer), RKO-E6 (colorectal cancer), RKO-AS45-1 (colorectal cancer), T84 (colorectal cancer), CNE1 (nasopharyngeal carcinoma), CNE2 (nasopharyngeal carcinoma), HNE1 (nasopharyngeal carcinoma), HNE2 (nasopharyngeal carcinoma), HNE3 (nasopharyngeal carcinoma), HONE1 (nasopharyngeal carcinoma), 5-8F (nasopharyngeal carcinoma), 6-10B (nasopharyngeal carcinoma), C666-1 (nasopharyngeal carcinoma), HK-1 (nasopharyngeal carcinoma), A549 (lung cancer), HCC827 (lung cancer), H460 (lung cancer), H146 (lung cancer), A-427 (lung cancer), SPC-A1A (lung cancer), H838 (lung cancer), NCI-H1299 (lung cancer), 95-D (lung cancer), Calu-1 (lung cancer), Calu-3 (lung cancer), LTEP-a-2 (lung cancer), MSTO-211H (lung cancer), NCI-H1650 (lung cancer). Lung Cancer NCI-H292, Lung Cancer SK-MES-1, Lung Cancer T84, Lung Cancer NCI-H1395, Lung Cancer NCI-H1975, Lung Cancer NCI-H661, Lung Cancer NCI-H157, Lung Cancer NCI-H1688, Lung Cancer NCI-H1703, Lung Cancer NCI-H2087, Lung Cancer NCI-H209, Lung Cancer NCI-H2170, Lung Cancer NCI-H2227, Lung Cancer NCI-H226, Lung Cancer NCI-H23, Lung Cancer NCI-H358, Lung Cancer NCI-H446, Lung Cancer NCI-H520, Lung Cancer NCI-H524, Lung Cancer NCI-H596, Lung Cancer QG-56, Lung Cancer NCI-H441, Lung Cancer A549 / DDP, Lung Cancer DMS 153. Lung cancer NCI-H2228, Lung cancer NCI-H2347, Ovarian cancer Caov-3, Ovarian cancer PA-1, Ovarian cancer Skov-3, Breast cancer MCF7, Breast cancer T-47D, Breast cancer ZR-75-1, Breast cancer MDA-MB-231, Breast cancer SK-BR-3, Breast cancer MDA-MB-453, Breast cancer HCC1954, Cervical cancer HeLaHCC-94, Cervical cancer MEG-01, Cervical cancer MS751, Cervical cancer HCE-1, Liver cancer HepG2, Liver cancer SMMC-7721, Liver cancer Hep3B, Liver cancer J5, Endometrial cancer Ishikawa, Endometrial cancer ECC-1, Endometrial cancer Hec50co, Endometrial cancer KLE, Endometrial cancer RL95-2.

[0036] The pH-responsive polypeptide hydrogel is currently suitable for use with mesenchymal stem cells, which are derived from tissues such as bone marrow, umbilical cord blood, and placenta.

[0037] Furthermore, the polypeptide hydrogel of this invention possesses the characteristic that it can be reverted to a solution state by gently blowing on the formed polypeptide hydrogel 10-25 times using a pipette tip or other tool. This characteristic facilitates the recycling of cells.

[0038] In the description of this invention, it should be noted that cell spheres refer to spherical aggregates of tumor cells formed under three-dimensional culture conditions (such as hydrogel matrix), which are closer to the structure and microenvironment of tumor tissue in vivo and can be used to simulate the response of tumors to immune cells in vivo.

[0039] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0040] To facilitate understanding of the present invention, the embodiments will use PBS as the buffer salt and mannose or sucrose as the polysaccharide to prepare a pH-responsive polypeptide hydrogel for 3D culture of cancer cells and stem cells. The invention will be described more comprehensively and in detail with reference to the accompanying drawings. However, the scope of protection of the present invention is not limited to the following specific embodiments. Through the description of the following embodiments, those skilled in the art will readily understand that other buffer salts, polysaccharides, etc., used in the present invention for 3D culture of cancer cells and stem cells can also achieve the results claimed by the present invention.

[0041] Example 1 This embodiment provides a pH-responsive polypeptide hydrogel for 3D culture of the pancreatic cancer cell line PANC-1, as detailed below: 10 mg of RADA16 was weighed and dissolved in 1 ml of sterile ultrapure water to a concentration of 10 mg / ml, and stored at 4°C. 1.5 g of mannose was weighed and dissolved in 8.5 g of ultrapure water, stirred at room temperature until completely dissolved, to obtain a 15 wt% mannose solution. This solution was sterilized by 0.22 μm filtration in a biosafety cabinet and stored for later use. 1×PBS and the 15% mannose solution were mixed in a 1:1 ratio to obtain a 0.5×PBS / 7.5% mannose salt ion solution, which was stored for later use. After trypsin digestion and passage with PANC-1 cells, the cells were centrifuged and counted. The cells were resuspended in the salt ion solution to obtain a cell density of 1×10⁻⁶ cells / mL. 6 Cell suspension was prepared at a concentration of cells / ml. The resuspended cell suspension was mixed 1:1 with 10 mg / ml RADA16 peptide solution until the pH was 7.2. 30 μl of the hydrogel-cell mixture was dropped into a 24-well cell culture plate. The plate was carefully transferred to a 37°C CO2 incubator and incubated for 10 min to allow hydrogel formation. 1 ml of complete culture medium was added, according to the plate specifications. The cell culture plate was incubated at 37°C CO2 for 7 days, and cell growth was observed under a microscope. The complete culture medium was changed every 2-3 days during the culture period. Results are as follows: Figure 1As shown, the PANC-1 cell line began to form cell aggregates in the polypeptide hydrogel on the first day, and the tumor spheres gradually expanded and became denser with increasing culture time, indicating that the hydrogel not only promotes rapid growth and proliferation of tumor cells but also has good biocompatibility. After 7 days of culture, the RADA16 polypeptide hydrogel was gently pipetted 15-20 times until the hydrogel was completely degraded, and the cultured PANC-1 tumor spheres were recovered. The morphology of the recovered tumor spheres is shown in the figure. Figure 2 As shown, the tumor spheres recovered under different magnifications have relatively regular morphology, and the individual cell spheres become loose and their size increases.

[0042] Example 2 This embodiment provides a pH-responsive polypeptide hydrogel for 3D passage culture of pancreatic cancer cell line PANC-1 tumor spheres, as detailed below: Weigh 10 mg of RADA16 and dissolve it in 1 ml of sterile ultrapure water to a concentration of 10 mg / ml, then store at 4°C. Weigh 2 g of mannose and dissolve it in 8 g of ultrapure water, stirring at room temperature until completely dissolved to obtain a 20 wt% mannose solution. Sterilize the solution by 0.22 μm filtration in a biosafety cabinet and store for later use. Mix 1×PBS and 20% mannose solution at a 1:4 ratio to obtain a 0.25×PBS / 16% mannose salt ion solution and store for later use. Embed PANC-1 tumor spheres in the polypeptide hydrogel cultured for 7 days as described in Example 1. Gently pipette the culture medium into the hydrogel 15 times until the hydrogel is completely degraded. Centrifuge to collect the recovered tumor spheres, wash once with 1×PBS, centrifuge again, and resuspend the tumor spheres in the salt ion solution. The resuspended tumor spheres were mixed thoroughly with 10 mg / ml RADA16 peptide solution at a ratio of 2:1, resulting in a pH of 7.3. 30 μl of the hydrogel-cell mixture was dropped into a 24-well cell culture plate. The plate was carefully transferred to a 37°C CO2 incubator and incubated for 10 min to allow hydrogel formation. 1 ml of complete culture medium was added, according to the plate specifications. The cell culture plate was incubated at 37°C CO2 for 5 days, and cell growth was observed under a microscope. The complete culture medium was changed every 2–3 days during the culture period. Results are as follows: Figure 3 As shown, the morphological changes from day 0 to day 5 of passage are displayed. Initially, the structure of the PANC-1 tumor spheres became loose after degradation and recovery of the hydrogel. However, after re-inoculation and passage in the hydrogel, the structure of the tumor spheres became compact again, and the spheres became more regular and perfect in shape, with relatively uniform height. The edges of the cell spheres were sharp and smooth. As the culture time increased, the tumor spheres gradually expanded, enlarged, and became more compact.

[0043] Example 3 This embodiment provides a pH-responsive polypeptide hydrogel for 3D culture of the pancreatic cancer cell line PANC-1, as detailed below: 10 mg of RADA16 was weighed and dissolved in 1.66 ml of sterile ultrapure water to a concentration of 6 mg / ml, and stored at 4°C. 2 g of sucrose was weighed and dissolved in 8 g of ultrapure water at room temperature until completely dissolved, yielding a 20 wt% sucrose solution. This solution was sterilized by 0.22 μm filtration in a biosafety cabinet and stored for later use. 1×PBS and 20% sucrose solution were mixed at a 1:4 ratio to obtain a 0.25×PBS / 16% sucrose salt ion solution, which was also stored for later use. After trypsin digestion and passage, PANC-1 cells were centrifuged and counted. Resuspending the cells in the salt ion solution yielded a cell density of 6.67 × 10⁻⁶ cells / ml. 5 Cell suspension at 1 / ml. The resuspended cell suspension was mixed with 6 mg / ml RADA16 peptide solution at a 2:1 ratio until the pH was 7.4. 150 μl of the hydrogel-cell mixture was evenly spread into a 48-well cell culture plate (shaking to mix). The plate was carefully transferred to a 37°C CO2 incubator and incubated for 5 min to form a hydrogel. 500 μl of complete culture medium was added, according to the plate specifications. The cell culture plate was incubated at 37°C CO2 for 7 days, and cell growth was observed under a microscope. The complete culture medium was changed every 2-3 days during the culture period. The results after 7 days of culture are shown below. Figure 4 As shown in Figure A, the PANC-1 cell line began to form cell aggregates in the polypeptide hydrogel on the first day, and formed relatively regular and large tumor cell spheres on the 7th day.

[0044] Comparative Example 1 This comparative example uses the commercially available hydrogel product VitroGel Hydrogel Matrix (catalog number VHM01S-2ml) for 3D culture of the pancreatic cancer cell line PANC-1, as detailed below: After PANC-1 trypsin digestion and passage, cells were centrifuged and counted. Cells were resuspended in complete cell culture medium to obtain a cell density of 1 × 10⁶ cells / year. 6 Cell suspension was prepared at cells / ml. The resuspended cell suspension was mixed with VitroGel at a 1:2 ratio, and the cells were resuspended again. 150 μl of the hydrogel-cell mixture was spread evenly in a 48-well cell culture plate (shaking to mix). The plate was carefully transferred to a 37°C CO2 incubator and incubated for 15 min. Then, 500 μl of complete culture medium was added, according to the plate specifications. The cell culture plate was incubated at 37°C CO2 for 7 days, and cell growth was observed under a microscope. The complete culture medium was changed every 2-3 days during the culture period. The results after 7 days of culture are shown below. Figure 4As shown in Figure B, the PANC-1 cell line gradually expanded in VitroGel, forming larger tumor spheres. However, compared to the RADA16 peptide hydrogel culture system in Example 3, the tumor spheres in the peptide hydrogel were more uniform in size, while the tumor spheres formed in the commercially available VitroGel were uneven in size, and the number of large tumor spheres was relatively smaller.

[0045] Example 4 This embodiment provides a pH-responsive polypeptide hydrogel for 3D culture of the mouse mesenchymal stem cell line C3H / 10T1 / 2, as detailed below: 10 mg of RADA16 was dissolved in 1.66 ml of sterile ultrapure water to a concentration of 6 mg / ml and stored at 4°C. 2 g of sucrose was dissolved in 8 g of ultrapure water and stirred at room temperature until completely dissolved to obtain a 20 wt% sucrose solution. This solution was sterilized by 0.22 μm filtration in a biosafety cabinet and stored for later use. 1×PBS and 20% sucrose solution were mixed at a 1:4 ratio to obtain a 0.25×PBS / 16% sucrose salt ion solution, which was stored for later use. Cells were digested with C3H / 10T1 / 2 trypsin, passaged, centrifuged, and counted. Resuspending the cells in the salt ion solution yielded a cell density of 4×10⁻⁶ cells / mL. 5 Cell suspension was prepared at a concentration of cells / ml. The resuspended cells were mixed 1:1 with 6 mg / ml RADA16 peptide solution until the pH reached 7.2. 150 μl of the hydrogel-cell mixture was then evenly spread into a 48-well cell culture plate (shaking to mix). The plate was carefully transferred to a 37°C CO2 incubator and incubated for 5 min to allow hydrogel formation. 500 μl of complete culture medium was added, according to the plate size. The cell culture plate was incubated at 37°C CO2 for 7 days, and cell growth was observed under a microscope. The complete culture medium was changed every 2-3 days during the culture period. The culture results are shown below. Figure 5 As shown in Figures A through C, starting from day 1, the number of C3H / 10T1 / 2 cell lines gradually increased in the polypeptide hydrogel. As the culture time increased, the cell network continued to proliferate and became increasingly dense, eventually forming a dense cell network. This indicates that the hydrogel not only has the property of promoting the rapid growth and reproduction of stem cells, but also has good biocompatibility.

[0046] Comparative Example 2 This comparative example uses the commercially available hydrogel product PIC 5K-RGD (batch number 202501) for 3D culture of mouse mesenchymal stem cell line C3H / 10T1 / 2, as detailed below: 2.5 mg PIC 5K-RGD was dissolved in 0.5 ml PBS and incubated overnight at 4°C. After C3H / 10T1 / 2 trypsin digestion and passage, cells were centrifuged and counted. Cells were resuspended in complete cell culture medium to obtain a cell density of 4 × 10⁻⁶ cells / mL. 5Cell suspension at cells / ml. Resuspend the cells and mix thoroughly with PIC solution at a 1:1 ratio (operate on an ice box). Take 150 μl of the hydrogel-cell mixture and spread it evenly in a 48-well cell culture plate (shake well). Carefully transfer the culture plate to a 37°C CO2 incubator and incubate for 15 min to form a hydrogel. Add 500 μl of complete culture medium, the volume of which should match the plate size. Incubate the cell culture plate at 37°C CO2 for 7 days and observe cell growth under a microscope. Change the complete culture medium every 2-3 days during culture. Culture results are shown below. Figure 5 As shown in Figures D-F, the C3H / 10T1 / 2 cell line can also form a cell network in the PIC hydrogel. However, compared with the RADA16 peptide hydrogel in Example 4, the cell network of the C3H / 10T1 / 2 cell line in the peptide hydrogel proliferated significantly and became increasingly dense with the increase of culture time, and the network structure was dense and stable. In contrast, the PIC hydrogel showed good proliferation in the first 4 days, but no significant proliferation occurred after the 7th day of culture. This indicates that the culture environment of the PIC hydrogel cannot provide a long-term expansion culture condition for the stem cell line C3H / 10T1 / 2, while the culture conditions provided by the RADA16 hydrogel are stable and continuous.

[0047] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A pH-responsive polypeptide hydrogel, characterized in that, The pH-responsive polypeptide hydrogel is formed by compounding a buffer salt solution, a polysaccharide aqueous solution and a polypeptide aqueous solution, and the pH of the pH-responsive polypeptide hydrogel is 7.0-7.4; wherein the buffer salt solution is at least one selected from PBS, DPBS, HEPES and TBS; the polysaccharide in the polysaccharide aqueous solution is at least one selected from trehalose, sucrose, glucose and mannose; and the polypeptide in the polypeptide aqueous solution is RADA16, the amino acid sequence of which is AC-RADARADARADARADA-NH2, and the chemical formula of which is C 66 H 113 N 29 O 25 .

2. The pH-responsive polypeptide hydrogel of claim 1, wherein, The mass percentage concentration of the polysaccharide aqueous solution is 10-25%; and / or, the concentration of the polypeptide aqueous solution is 5-20 mg / mL.

3. The pH-responsive polypeptide hydrogel of claim 2, wherein, The mass percentage concentration of the polysaccharide aqueous solution is 10-20%; and / or, the concentration of the polypeptide aqueous solution is 6-15 mg / mL.

4. The pH-responsive polypeptide hydrogel of claim 2 or 3, wherein, The volume ratio of the buffer salt solution to the polysaccharide aqueous solution is 1:1-4; the volume ratio of the sum of the buffer salt solution and the polysaccharide aqueous solution to the polypeptide aqueous solution is 1-4:

1.

5. The pH-responsive polypeptide hydrogel of claim 4, wherein, The volume ratio of the buffer salt solution to the polysaccharide aqueous solution is 1:3-4; the volume ratio of the sum of the buffer salt solution and the polysaccharide aqueous solution to the polypeptide aqueous solution is 1-2:

1.

6. Use of the pH-responsive polypeptide hydrogel according to any one of claims 1 to 5 in three-dimensional cell culture, the use comprising: The mixed solution of the buffer salt solution and the polysaccharide aqueous solution is used to suspend the cells to be cultured, and then the polypeptide aqueous solution is added for three-dimensional culture of the cells.

7. Use according to claim 6, characterized in that, The concentration of the cells to be cultured in the mixed solution of the buffered saline solution and the aqueous polysaccharide solution is (2-10) x 10 5 cells / mL.

8. Use according to claim 6 or 7, characterized in that, The process of the three-dimensional culture of the cells comprises: first, static incubation of the system under the condition of 37±1℃ until a hydrogel is formed, and then addition of a culture medium for further culture for 5-14 days.

9. Use according to claim 8, characterized in that, The static incubation time is 5-15 min.

10. Use according to claim 9, characterized in that, The cells suitable for the three-dimensional culture include tumor cells and stem cells.

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