Use of self-assembling polypeptide derivative hydrogels in the construction of a platform for culturing pancreatic islets

Abstract

The present application relates to a kind of platform for three-dimensional organoid culture of islet, comprising: 0.15-0.3 w / v self-assembled polypeptide derivative hydrogel.The platform of the present application, the islet organoid of the method for preparing three-dimensional organoid of islet by the platform can maintain its cell integrity, not broken, and does not affect the secretion of insulin.Meanwhile the hydrogel can exist in mouse for a long time, lay a foundation for long-term enhancement of islet beta cell vitality and function.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to the application of a platform for culturing pancreatic islets using a self-assembled polypeptide derivative hydrogel. Background Technology

[0002] Type 1 and type 2 diabetes are caused by an absolute or relative deficiency of insulin. Beta cells within the pancreatic islets are responsible for insulin synthesis and secretion, which are crucial for maintaining glycemic homeostasis. Currently, islet cell transplantation is considered one of the important strategies for treating diabetes. However, this therapy still faces many challenges, most notably the low survival rate and functional impairment of transplanted beta cells, which significantly weakens their ability to synthesize and secrete insulin.

[0003] Recent studies have shown that the behavior and function of β cells are highly dependent on their microenvironment. Notably, pancreatic β cells are extremely sensitive to the mechanomechanical properties of their microenvironment. Traditional two-dimensional cell culture systems use rigid and static culture substrates, which differ significantly from the actual physiological microenvironment in vivo. This difference can lead to significant alterations in cell attachment morphology, insulin secretion, and other key cellular functions. Therefore, there is an urgent need to develop in vitro pancreatic islet cell culture models that can mimic the in vivo microenvironment to facilitate further research.

[0004] The main components of the extracellular matrix (ECM) of pancreatic tissue include type I, III, IV, and V collagen, as well as various laminin isoforms. Given the crucial role of the ECM in regulating cell behavior, hydrogels that mimic its physicochemical properties have become a commonly used matrix material for constructing cell spheroids in vitro. However, existing hydrogel systems have significant limitations: naturally derived hydrogels often suffer from complex compositions and large batch-to-batch variations, affecting experimental reproducibility; while synthetic hydrogels often face challenges such as high cost and insufficient biocompatibility. Therefore, a hydrogel culture platform that can effectively promote islet cell aggregation, maintain their long-term viability, and enhance their physiological functions (such as glucose-responsive insulin secretion) is still lacking. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention proposes a platform for three-dimensional organoid culture of pancreatic islets, comprising: 0.15-0.3 w / v% of a self-assembled polypeptide derivative hydrogel. Preferably, the concentration of the self-assembled polypeptide derivative hydrogel is 0.15-0.3 w / v%.

[0006] As described above, the self-assembled polypeptide derivative hydrogel has a structure of Biotin- D Phe-X; where: Biotin is the end-capping group; DPhe is the D-configuration amino acid Phe; and X is the active polypeptide sequence.

[0007] In the platform described above, the amino acid sequence of X is: YIGSRGD.

[0008] The platform described above further includes: a low-adhesion cell culture plate.

[0009] As described in any of the above platforms, the pancreatic organoids are obtained by dispersing human islet cells.

[0010] Application of the platform described above in three-dimensional organoid culture of pancreatic islets; wherein the concentration of the self-assembled polypeptide derivative hydrogel is 0.15-0.3 w / v.

[0011] A method for preparing a three-dimensional organoid of pancreatic islets includes: obtaining a dispersed suspension of islet cells; adding the islet cells to any of the platforms described above, and mixing the cells with the hydrogel; and culturing the mixture for a certain period of time.

[0012] In the preparation method described above, the specified time is 10-20 days; preferably, the specified time is 14 days.

[0013] In the preparation method described above, the number of cell clusters in each well of the cell culture plate is between 50 and 110.

[0014] A three-dimensional organoid of pancreatic islets prepared according to the method described above.

[0015] The pancreatic islets, as described above, are three-dimensional organoids that are cellular spheres.

[0016] The application of the three-dimensional pancreatic organoids prepared by the method described above in the preparation of insulin or glucagon.

[0017] A method for preparing insulin or glucagon includes: obtaining a three-dimensional organoid of the pancreas prepared according to the method described above; culturing the three-dimensional organoid of the pancreas for a certain period of time; and separating the insulin or glucagon secreted by the three-dimensional organoid of the pancreas.

[0018] The three-dimensional pancreatic organoids prepared by the method described above can be used in the preparation of drugs for treating diabetes or in the screening of drugs related to the treatment of diabetes.

[0019] The platform described in this application, and the method for preparing three-dimensional organoids of pancreatic islets using this platform, allow the cultured islet organoids to maintain cellular integrity, remain intact, and not affect insulin secretion. Furthermore, this hydrogel can persist long-term in mice, laying the foundation for long-term enhancement of pancreatic β-cell vitality and function. Attached Figure Description

[0020] The preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, wherein:

[0021] Figure 1 a- Figure 1 c illustrates the growth status of MIN6 cells cultured in cell culture plates or hydrogels of different concentrations according to an embodiment of this application; wherein...

[0022] Figure 1 a is an image of MIN6 cells and cell spheroids grown in a hydrogel for 1, 3, and 7 days according to an embodiment of the present invention;

[0023] Figure 1 b shows the statistical results of the cross-sectional area distribution of MIN6 cells and cell spheroids after 3 days of culture in a hydrogel (InGel) and on the hydrogel surface (onGel) according to an embodiment of the present invention;

[0024] Figure 1 c represents the statistical results of the cross-sectional area distribution of MIN6 cell spheroids cultured in gel (InGel) and on gel (onGel) for 7 days according to an embodiment of the present invention;

[0025] Figure 1 d is a representative image of MIN6 cells cultured on a cell plate or hydrogel at different dilutions according to an embodiment of the present invention;

[0026] Figure 1 e represents the cross-sectional integral distribution of a sphere after 3 days of cultivation according to an embodiment of the present invention;

[0027] Figure 1 f represents the cross-sectional integral distribution of the spheres in the hydrogel according to an embodiment of the present invention (dilution ratio of 2.5:1).

[0028] Figure 1 g shows a representative image of MIN6 cells cultured on a cell culture plate or in a hydrogel (dilution ratio of 2:1 or 3:1) according to an embodiment of the present invention;

[0029] Figure 2 a represents the results of live / dead staining of MIN6 cell spheroids in cell culture plates or hydrogels of different concentrations according to an embodiment of the present invention;

[0030] Figure 2 b shows the expression of insulin and Ki67 in MIN6 cell spheroids cultured for 14 days in cell culture plates or hydrogels of different concentrations according to an embodiment of the present invention.

[0031] Figure 3a represents the expression of insulin and Ki67 in MIN6 cell spheroids cultured for 1 day in cell culture plates or hydrogels of different concentrations according to an embodiment of the present invention.

[0032] Figure 3 b represents the statistical analysis of the Ki67 expression ratio of MIN6 cell spheroids cultured for 1 day in cell culture plates or hydrogels of different concentrations according to an embodiment of the present invention.

[0033] Figure 3 c represents the statistical analysis of green fluorescence of MIN6 cell spheroids cultured for 1 day in cell culture plates or hydrogels of different concentrations according to an embodiment of the present invention under the same laser intensity conditions;

[0034] Figure 4 a is a representative optical image of human pancreatic islet cells cultured under different gel conditions for 1 day or 3 days according to an embodiment of the present invention; scale bar = 100 micrometers;

[0035] Figure 4 b shows representative optical images of human islets cultured in hydrogels (dilution ratios of 2.5:1, 3:1, 5:1) for 1 or 3 days according to an embodiment of this application;

[0036] Figure 5 This invention illustrates the assessment of human islet viability and mortality in different gel matrices according to one embodiment of the present application; wherein,

[0037] Figure 5 a represents the live / dead staining results of human pancreatic islet cells according to an embodiment of the present invention; scale bar = 100 micrometers;

[0038] Figure 5 b represents the statistical results of human islet cell integrity according to an embodiment of the present invention, which counted more than 20 islets;

[0039] Figure 6 The image shows a confocal microscope image of human pancreatic islets, labeled with insulin and Ki67, and with the nuclei stained with DAPI, after 3 days of culture according to an embodiment of this application; scale bar = 40 micrometers;

[0040] Figure 7 This image shows cell spheroids in a hydrogel according to an embodiment of this application, on day 1, stained to show glucagon (…). Figure 7 a) / Smoatostatin ( Figure 7 b) and Ki67. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] In the following detailed description, reference can be made to the accompanying drawings, which form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to implement the technical solutions of the present application. It should be understood that other embodiments or modifications to the embodiments of the present application may also be utilized.

[0043] The term "organoid" as used in this article refers to a cluster of cells or tissues cultured in vitro that possesses functions similar to those in vivo. This application discloses a method for preparing human islet organoids and obtains human islet organoids, which can be used for research on the function and activity of human islets, and can also be used to prepare insulin, and even has the potential for use in organ transplantation.

[0044] The "three-dimensional organoid of pancreatic islets" mentioned in this article refers to a three-dimensional structure of pancreatic islet cells obtained from in vitro dispersion and proliferated / adhered into a spherical shape. This three-dimensional structure does not adhere to cell culture plates and possesses functions similar to those of pancreatic islets in animals, such as secreting insulin and glucagon. Furthermore, because this three-dimensional organoid closely resembles the natural morphology of pancreatic islets in animals and possesses their in vivo functions, it can be used for studies on pancreatic organ transplantation, insulin production, glucagon production, and the metabolism and function of pancreatic islets in animals.

[0045] The term "plate" as used in this article refers to cell culture plates, a common consumable in cell culture in this field. "Ordinary plates" (TC-treated) undergo TC (Tissue Culture) treatment, which makes the polystyrene surface positively charged or hydrophilic. When cells are cultured on ordinary plates, they can rapidly promote cell adhesion and form a uniform monolayer, making them suitable for routine culture of adherent cells. However, when culturing human pancreatic islet cells, they cannot form cell clusters, thus failing to develop organoid morphology.

[0046] "Ultra-Low-Attachment" (ULA) wells have a specially treated surface that results in extremely low cell adhesion, making them suitable for suspension culture. The cell clusters further assemble into tissue clusters, forming organoid morphology and maintaining cell function within the body, thus exhibiting organoid function.

[0047] The ordinary flat plate and low adhesion perforated plate mentioned in this application can be purchased directly from commercially available products, and this application does not limit them.

[0048] In conventional culture plates, a gel is added to the wells to provide support and prevent cells from settling and adhering due to gravity, thus preventing cell adhesion. The low-adhesion plate used in this application itself inhibits cell adhesion; therefore, the gel only serves as a soft support. In this case, it is not necessary to worry about whether the peptides can form a firm gel; it is sufficient to maintain its viscous liquid state and meet the appropriate stiffness for the growth of human pancreatic islet tissue.

[0049] Those skilled in the art should understand that pancreatic islets exist in organisms with a spherical structure, and maintaining their spatial structure is crucial for the functions of islet cells, such as insulin secretion. Low-adhesion surfaces can inhibit cell adhesion, keeping islets suspended in the culture medium and preventing them from spreading, deforming, or dying due to adhesion. However, in a scaffold-free environment, cells can only rely on their own secreted matrix, leading to unstable spherical morphology, uneven size distribution, and significant batch-to-batch variability. In addition, some cell lines (especially those highly dependent on adhesins or exogenous matrix) have difficulty spontaneously aggregating or maintaining a spherical shape in low-adhesion plates.

[0050] Therefore, a matrix is ​​needed to provide the microenvironment for cell culture, offering mechanical support, adhesion sites (such as YIGSR and RGD), and promoting stable organoid growth. The peptide hydrogel of this application is a material that meets these requirements.

[0051] The "Min6 cells" or "MIN6 cells" mentioned in this article are mouse pancreatic β-cell lines with typical epithelial-like morphology, mainly used for research on the mechanisms of diabetes and analysis of insulin secretion regulation. In this application, the MIN6 cells (BNCC358148, P4) were purchased from Beijing Kezhan Biotechnology Co., Ltd.

[0052] The "islet" cells used in this article were derived from a 52-year-old male, and the culture medium was provided by Tianjin First Central Hospital. All protocols were approved by the Medical Ethics Committee of Tianjin First Central Hospital (No.: 2017N074KY). In short, the donated islets were centrifuged at 1000 rpm for 1 min and then resuspended in 5 mL of culture medium. Next, the islets were pipetted several times until no cell clumps were observed. The cell suspension was then seeded into 24-gauge low-attachment cell culture plates. The culture medium was changed daily.

[0053] The terms "hydrogel," "peptide hydrogel," and "self-assembled peptide derivative hydrogel" used in this application have the same meaning and their structure is Biotin- D Phe-X; where: Biotin is the end-capping group; D Phe is the D-configuration amino acid Phe; X is the active polypeptide sequence. In this invention, the active polypeptide sequence is preferably YIGSRGD; in some embodiments, unless otherwise specified, the self-assembled polypeptide derivative hydrogel mentioned in this application is referred to as hydrogel; in some embodiments, Biotin- D Phe-X can be abbreviated as hydrogel, or Biotin-YIGSRGD, or Biotin- D YIGSRGD. The method for preparing the self-assembled polypeptide derivative hydrogel described in this invention is described in Chinese Patent 202110800384.5.

[0054] This application relates to a platform for three-dimensional organoid culture of pancreatic islets, comprising: 0.15-0.3 w / v of self-assembled polypeptide derivative hydrogel.

[0055] In some embodiments, the platform further includes: a low-adhesion cell culture plate.

[0056] In some embodiments, the islet organoid is obtained by dispersing human islet cells.

[0057] In some embodiments, this application relates to the application of the above-described platform in the three-dimensional organoid culture of pancreatic islets; wherein the concentration of the self-assembled polypeptide derivative hydrogel is 0.15-0.3 w / v.

[0058] This application relates to a method for preparing a three-dimensional organoid of the pancreatic islets, comprising: obtaining a dispersed suspension of islet cells; adding the islet cells to the aforementioned platform and mixing the cells with the hydrogel; and culturing the mixture for a certain period of time.

[0059] In some embodiments, the specified time period is 10-20 days; preferably, the specified time period is 14 days.

[0060] In some embodiments, the number of cell clusters in each well of the cell culture plate is between 50 and 110.

[0061] This application relates to a three-dimensional organoid of pancreatic islets prepared according to the method described above. In some embodiments, the three-dimensional organoid of the pancreatic islets is a cell spheroid.

[0062] Furthermore, this application relates to the use of the three-dimensional islet organoids prepared according to the method described above in the preparation of insulin or glucagon, and in the preparation of drugs for treating diabetes or in the screening of drugs related to the treatment of diabetes.

[0063] This application relates to a method for preparing insulin or glucagon, comprising: obtaining a three-dimensional organoid of the pancreas prepared according to the method described above; culturing the three-dimensional organoid of the pancreas for a certain period of time; and separating the insulin or glucagon secreted by the three-dimensional organoid of the pancreas.

[0064] The specific implementation methods of this application will be described below through specific embodiments. Those skilled in the art should understand that the following embodiments are only for illustrating the technical solutions and are not intended to limit the technical solutions of this application.

[0065] Example 1: Preparation of hydrogel

[0066] Weigh out Biotin- D 10 mg of F-YIGSRGD peptide derivative powder was placed in a glass vial, and 991.25 μL of 1×PBS (pH=7.4) and 8.75 μL of 1M Na2CO3 solution were added. The mixture was then thoroughly mixed by pipetting. The vial was heated with an alcohol lamp until the solution boiled and the peptide derivative powder was completely dissolved. After standing and cooling to room temperature, the self-assembled peptide derivative hydrogel stock solution was obtained, with a concentration of 10 mg / mL, expressed as 1 w / v.

[0067] Methods for preparing hydrogels of other concentrations include: taking the preparation of 1 mL of 0.2 w / v% hydrogel as an example, 200 μL of the hydrogel stock solution and 800 μL of the cell suspension are mixed evenly using a vortex mixer to obtain a 0.2% mucus. In some embodiments, the peptides at this concentration do not form a gel but are only a viscous liquid. The preparation of hydrogels of other volumes or concentrations can be carried out by referring to this method, and will not be elaborated further in this article.

[0068] Example 2: In-gel culture method for MIN6 cells

[0069] The MIN6 cell line was revived. When the MIN6 cells were in good condition, 37.5 μL of the self-assembled polypeptide derivative hydrogel obtained in Example 1 was vortexed and mixed thoroughly with 12.5 μL of MIN6 cell suspension containing 3000 cells. The mixture was then transferred to a 96-well plate and placed in a cell culture incubator. After incubating the cell-gel mixture in the incubator for 10 min, 100 μL of MIN6 cell culture medium was added to the surface. 50 mL of fresh complete culture medium was added every other day. The culture media used for MIN6 cell culture are shown in Table 1.

[0070] The growth of MIN6 cells in the gel is as follows: Figure 1 a, Figure 1 d- Figure 1 As shown in g. The results showed that after dilution to the stock solution concentration of 2.5:1, the cells rapidly expanded and formed spheroids in or above 0.71 w / v% gel (2.5:1). The cell spheroids gradually increased in size over 1–7 days, with the maximum average cross-sectional area reaching 3000 μm. 2 However, after dilution to the stock solution at a 5:1 ratio, the cross-sectional area of ​​the cell spheroids did not change significantly. In contrast, cells cultured on standard cell plates exhibited a stretched morphology, while cells embedded in a rigid gel (0.83 w / v%, 5:1) maintained a single-cell state. Furthermore, in the relatively rigid gel, spherical expansion was slower (0.75%, 3:1) (e.g. Figure 1 b- Figure 1 (As shown in c). This indicates that the 0.71 w / v% hydrogel is the optimal choice for MIN6 cell proliferation and spheroid formation.

[0071] OnGel and InGel refer to on-gel and in-gel cultures, respectively. Based on a comparison of cell cluster sizes on and in gel, in the first three days of cell culture, cell clusters grown faster on the gel surface than in the gel. Figure 1 As shown in b. However, by the seventh day of cultivation, there was no significant difference, as shown in b. Figure 1 As shown in c.

[0072] Given that the cell culture paradigm within a gel is similar to the physiological environment, this method was used in subsequent experiments.

[0073] Table 1 MIN6 cell culture medium

[0074]

[0075] Example 3: Cell live / dead staining protocol

[0076] Use Live&Dead TMThe cell spheroids obtained from Example 2 were incubated at 37°C in the dark for 10 minutes using the Viability / Cytotoxicity Assay Kit (1:3000; EVERBRIGHT® INC, L6023, USA). The samples were washed three times with PBS, and cell spheroid images were taken using a confocal microscope (CLSM, LSM900, Zeiss).

[0077] Shooting results as follows Figure 2 a- Figure 2 As shown in b. Because the live / dead cell staining exhibited a clear spherical cell structure, no significant cell death was observed within 7 days; only a small percentage of cells died at 14 days of culture (e.g., ...). Figure 2 (as shown in a). Therefore, the hydrogel showed no significant toxicity in MIN6 cells and was biocompatible.

[0078] After 14 days of culture, MIN6 cell spheroids could be directly observed as white dots in the culture plate, with a diameter of approximately 0.32 mm (e.g., ...). Figure 2 (As shown in b). The smallest object visible to the naked eye has a diameter of 0.1 mm. Therefore, these spheres are able to produce insulin and maintain good proliferative capacity.

[0079] The results showed that the polypeptide derivative hydrogel had no significant toxicity to MIN6 cells and exhibited good biocompatibility.

[0080] Example 4: Immunofluorescence staining protocol

[0081] Cells were stained using growth hormones (Insulin / Glucagon), Ki67 antibody, and DAPI. The specific steps were as follows: First, cell samples were fixed in 4% paraformaldehyde for 30 min, washed three times with PBS buffer, and then treated with 0.2% Triton X-100 to enhance cell membrane permeability. Next, they were blocked with 3% BSA at room temperature for 1 hour to reduce non-specific binding. Then, Insulin, Glucagon antibody, and Ki67 antibody were added, and the cells were incubated overnight at 4°C. The next day, the samples were washed three times with PBS buffer for 5 minutes each time, and then fluorescently labeled secondary antibody was added, followed by incubation in the dark for 2 hours. In some embodiments, the secondary antibody was mouse anti-human IgG. Finally, the cell nuclei were counterstained with DAPI, and the slides were mounted with anti-fluorescence quenching mounting medium. Images were then observed and acquired using a confocal microscope. Figure 3 a- Figure 3 As shown in c, etc.

[0082] The results showed that cell spheres cultured under gel conditions could increase the production of insulin and glucagon without significantly affecting cell proliferation.

[0083] Example 5: Preparation of human pancreatic islet organoids

[0084] Centrifuge at 1000 rpm to settle the islet clumps at the bottom of the centrifuge tube, and resuspend in 5 mL of culture medium. Trim the tip of a pipette with sterile scissors, attach it to the pipette, and gently pipette the cell clumps until the islets are evenly dispersed and free of obvious clumps. Then, transfer the human islets to 24-well low-adhesion plates (purchased from Corning, product number 3473), and change the medium daily for subsequent culture.

[0085] Digest with TrypLE Express for 8 minutes, then gently pipette the cell clumps until evenly dispersed. Add 5 mL of Advanced DMEM / F12 medium, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Resuspend the precipitated single cells in 2 mL of complete medium to obtain a cell suspension. Add the cell suspension to wells coated with Matrigel. In some embodiments, Matrigel can be purchased directly. In some embodiments, Matrigel is a commercial product from Corning (ref 356231). Since the impact of the culture medium involved in this embodiment on the technical solution of this application is negligible, and the culture medium is usually a commercially available product, the specific components of the culture medium are not limited in this embodiment.

[0086] The well-dispersed human islet cell clusters were spread in low-adhesion culture plates covered with hydrogels and collagen of different concentrations obtained in Example 1. The growth of the islet cells was observed and recorded using a microscope imaging system (AMEX1000).

[0087] In all the above experiments, the number of cell clusters in each well was ensured to be between 50 and 100, and the number of cell clusters in each parallel control group was kept consistent. Results are as follows: Figure 4 As shown.

[0088] The results showed that on the third day of culture, a large number of cells detached from both suspension and Matrigel cultures. The islet morphology was better maintained in Collagen and 2 mg / mL hydrogels after culture with different gels.

[0089] This embodiment further compares the cell survival status of human pancreatic islet cells under different culture methods, such as Figure 4 As shown.

[0090] Human pancreatic islet tissue typically disintegrates after approximately three days in suspension culture. To assess whether culturing human islets in various gel matrices could prolong the structural integrity of islet tissue, this application evaluated the state of human islets in suspension and in the presence of Matrigel, collagen, and hydrogels of varying concentrations (e.g., [missing information]). Figure 4 a, Figure 4 (as shown in b).

[0091] On the first day of culture, significant cell shedding was observed in the high-concentration Biotin-FYIGSRGD hydrogel (e.g. Figure 4 (As shown in b), while the morphology of pancreatic islets in other groups remained essentially unchanged. Therefore, this application focuses subsequent comparisons on the growth status of islets in low-concentration hydrogels (2 mg / mL, approximately 0.2 w / v%) and other groups. Here, a hydrogel ratio of 2:1 indicates a hydrogel concentration of approximately 0.67 w / v%, 2.5:1 indicates a hydrogel concentration of approximately 0.71 w / v%, 3:1 indicates a hydrogel concentration of approximately 0.75 w / v%, and 5:1 indicates a hydrogel concentration of approximately 0.83 w / v%.

[0092] On the third day of human pancreatic islet cell culture, some cells from the islets in suspension, Matrigel, and collagen began to detach. Representative images are shown below. Figure 5 As shown in figure a, the number of islets with poor morphology and cell detachment was counted. Figure 5 As shown in b. In contrast, the islet morphology in the Biotin-FYIGSRGD hydrogel was better preserved (e.g., ...). Figure 4 a, Figure 5 As shown in a), the number of intact islets in this hydrogel is approximately three times that observed in suspension culture (e.g., Figure 5 (as shown in b).

[0093] In summary, low-concentration Biotin-FYIGSRGD hydrogel effectively maintains the integrity of pancreatic islet tissue without affecting insulin secretion.

[0094] Example 5: Verification of pancreatic islet organoid function

[0095] This embodiment compares the expression of insulin in human pancreatic islet cell clusters in the presence of hydrogel and in a suspension state (low adhesion plate without peptide hydrogel addition); as well as the expression of insulin and somatostatin in MIN6 cell clusters under different concentrations of hydrogel.

[0096] like Figure 6 As shown, green fluorescence indicates insulin. It can be seen that green fluorescence can still be observed when human cell tissue clusters are cultured in gel, indicating that the gel can maintain insulin secretion from pancreatic islet cell clusters.

[0097] Compared to spheroids cultured on plates, MIN6 spheroids exhibited a higher intensity of green fluorescence in the cytoplasm (e.g., ...). Figure 3 a, Figure 3 (as shown in c), while the intensity of red fluorescence within the cell nucleus is similar (as shown in c). Figure 3 (as shown in b), indicating that hydrogels are suitable for insulin production. Furthermore, colloidal-cultured mouse islet cell spheroids produced more glucagon and growth hormone-inhibiting agents similar to those produced by plate-cultured spheroids (e.g., ...). Figure 7 (As shown). Therefore, the hydrogel formed visible spheres and promoted insulin secretion. This may be related to the 3D culture pattern, the composition of the matrix, and its stiffness.

[0098] As can be seen from the above embodiments, the present invention provides a pancreatic islet culture platform. By co-culturing pancreatic islet cells or human islet tissue with polypeptide derivative hydrogels, the activity of islet cells can be effectively improved, as well as insulin secretion and the integrity of human islets. The islet cell spheres and human islets obtained by the present invention can be used for drug screening or islet transplantation for the treatment of diabetes.

[0099] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. A platform for three-dimensional organoid culture of pancreatic islets, comprising: 0.15-0.7 w / v% of self-assembled peptide derivative hydrogel; preferably, the concentration of the self-assembled peptide derivative hydrogel is 0.15-0.3 w / v.

2. The platform according to claim 1, wherein the structure of the self-assembled polypeptide derivative hydrogel is Biotin- D Phe-X; where: Biotin is a capping group; D Phe is the D-configuration amino acid Phe; and, X represents the active polypeptide sequence.

3. The platform according to claim 2, wherein the amino acid sequence of X is: YIGSRGD.

4. The platform according to claim 1, further comprising: Low-adhesion cell culture plates.

5. The platform according to any one of claims 1-4, wherein the islet organoid is obtained by dispersion of human islet cells.

6. The application of the platform as described in any one of claims 1-4 in the three-dimensional organoid culture of pancreatic islets; wherein, The concentration of the self-assembled polypeptide derivative hydrogel is 0.15-0.3 w / v.

7. A method for preparing a three-dimensional organoid of the pancreas, comprising: Obtain a dispersed suspension of pancreatic islet cells; as well as The pancreatic islet cells are added to the platform according to any one of claims 1-5, and the cells are mixed with the hydrogel; as well as The mixture is incubated for a certain period of time.

8. The preparation method according to claim 7, wherein the certain time is 10-20 days; preferably, the certain time is 14 days.

9. The preparation method according to claim 8, wherein the number of cell clusters in each well of the cell culture plate is between 50 and 110.

10. A three-dimensional organoid of pancreatic islets prepared by the method according to claim 7.

11. The three-dimensional organoid of the pancreatic islet according to claim 10, wherein it is a cellular spheroid.

12. The application of the three-dimensional pancreatic organoid prepared by the method of claim 7 in the preparation of insulin or glucagon.

13. A method for preparing insulin or glucagon, comprising: Obtain a three-dimensional pancreatic islet organoid prepared according to the method of claim 7; as well as, The three-dimensional organoid structure of the pancreatic islets was cultured for a certain period of time; as well as, Insulin or glucagon secreted by the three-dimensional organoid of the pancreas is isolated.

14. The use of the three-dimensional pancreatic islet organoid prepared by the method of claim 7 in the preparation of drugs for treating diabetes or in the screening of drugs related to the treatment of diabetes.

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