Preparation methods for islet extracellular matrix scaffold and biomimetic islet organoid thereof, and use thereof

Abstract

Provided are preparation methods for an islet extracellular matrix scaffold and a biomimetic islet organoid. The scaffold is a gel, and the gel contains 3-5 mg / mL Collagen I, 3-5 mg / mL Collagen IV, and 0.8-1.2 mg / mL Laminin. For the scaffold, Collagen I, Collagen IV, and Laminin are simultaneously selected as extracellular matrix components, which have relatively strong interactions with islet cells, and effectively promote the islet cells and enhance insulin secretion function stimulated by glucose after binding to the islet cells. Collagen VI can also be used. Collagen VI can act on the islet cells to effectively enhance the survival of the islet cells and the insulin secretion function stimulated by glucose. The gel can form a relatively intact network structure without needing to use a cross-linking agent, thereby enhancing the support capacity for the islet organoid.

Description

Islet cell extracellular matrix scaffold and preparation method and application of biomimetic islet organoid thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological materials, and more specifically relates to an islet cell extracellular matrix scaffold and a preparation method and application of a biomimetic islet organoid thereof. BACKGROUND

[0002] As an islet cell extracellular matrix scaffold, the hydrogel can provide structural support and a cell-attached surface. By wrapping islet cells in the hydrogel, a biomimetic islet structure can be constructed to replace or assist the function of human islets. Islet cell transplantation: the hydrogel wrapped with islet cells is transplanted into the body of a diabetic patient, and the islet hormone in the cells is released at a suitable location to restore the function of the pancreas of the diabetic patient. The hydrogel islet organoid is a biomimetic islet structure constructed using a hydrogel as a carrier. The preparation method is as follows: 1. preparing a hydrogel carrier: selecting a suitable hydrogel as a carrier material, such as collagen, gelatin, sodium alginate, etc.; 2. constructing an islet cell environment: culturing islet cells in the hydrogel so that they are uniformly distributed in the gel; and 3. islet organoid wrapping: mixing the islet organoid with the hydrogel so that the islet organoid is completely wrapped by the hydrogel.

[0003] CN116622618A discloses a vascularized islet organoid and a preparation method and application thereof, and the gel used is Col-Gelma hydrogel; the preparation process of the Col-Gelma hydrogel solution is as follows: 2-5 mg / mL type I collagen solution is added with a photo-crosslinking agent LAP, and after complete dissolution, a weighed Gelma is added, dissolved completely in a 35-38℃ water bath, and filtered with a filter to obtain the solution.

[0004] In the research of the present application, it is found that the use of only type I collagen cannot achieve more excellent culture effect. And most of the hydrogels need to use a crosslinking agent.

[0005] The defects of the prior art are as follows: 1. the extracellular matrix components are insufficient or some protein components that cannot effectively interact with islet cells are used; 2. the prepared mechanical properties are poor and are not convenient to use; and 3. the prepared cell matrix components cannot effectively maintain the activity and function of islet cells. SUMMARY

[0006] The main purpose of the present application is to provide an islet cell extracellular matrix scaffold, which simultaneously selects Collagen I, Collagen IV and Laminin as extracellular matrix components, these components are the main components of the basement membrane of in vivo islets, have strong interaction with islet cells, and effectively promote islet cells and enhance glucose-stimulated insulin secretion function after combining with islet cells.

[0007] In the preferred solution, Collagen VI is additionally used, which is a closely related component found through in-depth analysis of the acellular amniotic membrane and the acellular mouse pancreas, and through research, it is found that Collagen VI can act on islet cells, and can effectively improve the survival and glucose-stimulated insulin secretion function of islet cells.

[0008] Meanwhile, the gel of the present application can form a relatively complete network structure without using a crosslinking agent, thereby improving the support force for the islet organoid.

[0009] Meanwhile, the present application also discloses a preparation method and application of the bionic islet organoid.

[0010] According to a first aspect of the present application, an islet extracellular matrix scaffold is provided, the scaffold is a gel, and the gel contains 3-5 mg / ml Collagen I, 3-5 mg / ml Collagen IV and 0.8-1.2 mg / ml Laminin.

[0011] In the islet extracellular matrix scaffold described above, 3-5 mg / ml Collagen VI is further included.

[0012] In the islet extracellular matrix scaffold described above, the gel contains 4 mg / ml Collagen I, 4 mg / ml Collagen IV, 4 mg / ml Collagen VI and 1 mg / ml Laminin.

[0013] In the islet extracellular matrix scaffold described above, the matrix of the gel is 10x DMEM culture medium.

[0014] Meanwhile, the present application also discloses a preparation method of the islet extracellular matrix scaffold as described above, Collagen I, Collagen IV and Laminin are added to the matrix, the pH is adjusted to neutral, and incubation is performed to gelate.

[0015] In the preparation method described above, Collagen I, Collagen IV, Collagen VI and Laminin are added to the matrix, the pH is adjusted to neutral, and incubation is performed to gelate.

[0016] In the preparation method described above, the matrix is 10x DMEM culture medium.

[0017] In addition, the present application also discloses a preparation method of a bionic islet organoid, the islet organoid is embedded in the islet extracellular matrix scaffold as described above to form the bionic islet organoid.

[0018] The pancreatic islet organoid described in the present application refers to the pancreatic islet organoid obtained by various different technical routes in the art, such as CN118064353A, a method for establishing a pancreatic islet organoid by inducing differentiation of human induced pluripotent stem cells, CN118048293A, a vascularized pancreatic islet organoid and a construction method and application thereof, and CN114561341B, a three-dimensional pancreatic islet organoid based on iPSC and a construction method thereof.

[0019] More specifically, the specific steps are as follows: neutralize and centrifuge the culture medium added with Collagen I, Collagen IV and Laminin, or the culture medium added with Collagen I, Collagen IV, Collagen VI and Laminin, add the organoid to the centrifuged culture medium, add about 10-20 pancreatic islet organoids to about 100 μL of the culture medium, then add to the syringe for incubation, so that the hydrogel is gelled, then inject the gelled hydrogel according to the required amount to obtain the biomimetic pancreatic islet organoid.

[0020] In the embodiments of the present application, the pancreatic islet organoid prepared by the team is used for loading.

[0021] In addition, a biomimetic pancreatic islet organoid comprises the pancreatic islet extracellular matrix scaffold and the pancreatic islet organoid embedded in the pancreatic islet extracellular matrix scaffold.

[0022] Finally, the present application also discloses a use of the pancreatic islet extracellular matrix scaffold for preparing a biomimetic pancreatic islet organoid.

[0023] The above technical solutions of the present application have at least one of the following advantages or beneficial effects:

[0024] The present application simultaneously selects Collagen I, Collagen IV and Laminin as extracellular matrix components, which are the main components of the basement membrane of the in vivo islet, have strong interaction with islet cells, and effectively promote the islet cells and enhance the glucose-stimulated insulin secretion function after binding with the islet cells.

[0025] In the preferred scheme, Collagen VI is additionally used, which is a closely related component found through in-depth analysis of decellularized amniotic membrane and decellularized mouse pancreas. Through research, it is found that Collagen VI can interact with islet cells, and can effectively improve the survival and glucose-stimulated insulin secretion function of islet cells.

[0026] Meanwhile, the gel of the present invention can form a relatively complete network structure without the use of cross-linking agents, thereby improving the support for pancreatic organoids. Attached Figure Description

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0028] Figure 1 is a schematic diagram of step 1 of embodiment 1 of the present invention;

[0029] Figure 2 shows the relationship between the amount of cells added to each microwell and the size of the cell spheres in Example 1 of the present invention;

[0030] Figure 3 is a graph showing the relationship between cell number and cell spheroid size in Example 1 of the present invention;

[0031] Figure 4 is a schematic diagram of the induction steps in Embodiment 1 of the present invention;

[0032] Figure 5 shows photographs of pancreatic islet organoids induced by different matrix gels in Example 1 of the present invention;

[0033] Figure 6 shows the cell viability test results of pancreatic islet organoids induced by different matrix gels in Example 1 of the present invention;

[0034] Figure 7 is an operation flowchart of Embodiment 3 of the present invention;

[0035] Figure 8 is an active photograph of the pancreatic islet organoid of Example 3 of the present invention;

[0036] Figure 9 is an in vivo imaging photograph of a mouse after pancreatic islet organoid transplantation according to Example 3 of the present invention. Detailed Implementation

[0037] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0038] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0039] Example 1

[0040] Preparation of pancreatic organoids

[0041] Step 1: Digest induced pluripotent stem cells into individual cells, measure the cell concentration, and then add the corresponding amount of cells to each well of each ultra-low adhesion 96-well plate (Corning, 7007) according to the required organ diameter. After 2 days, cell spheres of uniform size can be formed.

[0042] Referring to FIGS. 1 to 3, FIG. 1 is a schematic diagram of step 1;

[0043] FIG. 2 is a relationship between the amount of cells added in each microwell and the size of the cell ball; the number of cells that can be added in each microwell ranges from 100 to 10,000; the upper column is the size of the cell ball in the microwell on the first day, and the lower column is the size of the cell ball in the microwell on the second day;

[0044] FIG. 3 is a graph of the relationship between the number of cells and the size of the cell ball;

[0045] Step 2: The cell ball is then embedded in Matrigel (BME2) or Matrigel-decellularized amniotic membrane hydrogel (BME2 / dAM), and after the Matrigel solidifies, it is used for step 3;

[0046] The Matrigel-decellularized amniotic membrane hydrogel is composed of Matrigel and decellularized amniotic membrane powder; the weight-volume ratio of the decellularized amniotic membrane powder and the Matrigel is 1:1;

[0047] The preparation method of the decellularized amniotic membrane powder is as follows: the human amniotic membrane is obtained from Guangzhou Maternal and Child Health Hospital, and the amniotic membrane of a healthy full-term cesarean section fetus is obtained with the consent of the mother and her family and the signing of an informed consent form. The placenta of a healthy newborn is taken under sterile conditions, washed with 1% gentamicin PBS buffer, and the amniotic membrane is separated, cut into about 6mmx6mm tissue blocks, placed in a culture dish, treated with 1% SDS for 2h, then treated with 1% Triton X-100 for 30min, and then washed with PBS for 72h. After decellularization, it is freeze-dried, ground into powder with liquid nitrogen, and then mixed with Matrigel (1:1) (wv) to serve as an iPSC differentiation support.

[0048] According to 1ml of Matrigel containing 1x10 6 The proportion of the pluripotent stem cell package is mixed with the cell ball and Matrigel to obtain a cell hydrogel, and 40μL of the cell hydrogel suspension is dropped onto a culture plate warmed at 37℃, inverted and placed in a CO2 incubator for 30min, and after the Matrigel solidifies, a dome-shaped peak group is formed on the culture plate.

[0049] Step 3: Differentiation;

[0050] The differentiation is carried out in a basic culture medium, and the solidified Matrigel is added to the basic culture medium, and different growth factors and other raw materials are contained in the basic culture medium in different differentiation steps, so as to obtain a designed differentiation product;

[0051] Specifically, the entire induction process is divided into 25 days;

[0052] Days 1-3, use basal medium containing 100 ng / ml Activin A, 3 uM CHIR99021, Activin A and CHIR99021 molecules regulate Wnt and Nodal / TGF signals to promote iPSC differentiation into definitive endoderm;

[0053] Days 4-5, use basal medium containing 50 ng / ml KGF, 50 ng / ml FGF2, KGF and FGF2 growth factors regulate FGF signals to differentiate definitive endoderm into primitive gut tube;

[0054] Days 6-7, use basal medium containing 2 uM RA, 2 uM LDN193189, RA and LDN193189 regulate RA and BMP signals to promote primitive gut tube differentiation into posterior foregut;

[0055] Days 8-14, use basal medium containing 2 uM RA, 10 uM Nicotinamide, 50 ng / ml EGF and 5 mg / ml Vitamin C, RA, Nicotinamide, EGF and Vitamin C mainly regulate EGF and SHH signals to form pancreatic progenitor cells from posterior foregut;

[0056] Days 15-21, use basal medium containing 1 uM DAPT, 10 ug / ml Heparin, 1 uM T3, 10 uM A83-01, 50 ng / ml Exendin4, DAPT, Heparin, T3, A83-01, Exendin4 molecules regulate Notch, TGF, Thyroid to promote pancreatic progenitor cells to form pancreatic islet organoids;

[0057] The pancreatic islet organoids induced by the above process are mainly composed of four cell types: ɑ cells (35-40%), β cells (50%), δ cells (10-15%), and γ cells. Among them, ɑ cells produce glucagon, β cells produce insulin, δ cells produce somatostatin, and γ cells produce pancreatic polypeptide. These cell types collectively coordinate and regulate blood glucose levels and appetite.

[0058] Figure 4 further illustrates the steps of the induction of the present application;

[0059] Referring to Figures 5 and 6, Figure 5 is a photograph of pancreatic islet organoids induced by different Matrigel;

[0060] Figure 6 is the cell activity test results of pancreatic islet organoids induced by different Matrigel;

[0061] wherein, aIO is without decellularized amniotic membrane hydrogel embedding differentiated pancreatic islet organoids; dIO is with decellularized amniotic membrane hydrogel embedding differentiated pancreatic islet organoids;

[0062] More specifically, dIO-1, dIO-2, dIO-3 represent the addition of decellularized amniotic membrane 10 ug, 20 ug, 30 ug, respectively;

[0063] As can be seen from FIG. 5 and FIG. 6, even if only Matrigel is used to embed cell spheres, the cell activity is increased by more than 1 times compared with traditional technology; if Matrigel hydrogel with decellularized amniotic membrane is used to embed cell spheres, the cell activity can be increased by more than 2 times.

[0064] Example 2

[0065] Preparation of islet organoid ECM scaffold

[0066] Preparation of ECM scaffold 1:

[0067] Under sterile conditions, using 10x DMEM medium, Collagen I, Collagen IV, Laminin (laminin) are added to the medium, so that the final concentration of each component in the medium is 4 mg / ml Collagen I, 4 mg / ml Collagen IV and 1 mg / ml Laminin. Using 1N NaOH, the pH of the mixture is quickly neutralized to 7.4 and centrifuged. Subsequently, the mixed solution is aliquoted into special syringes, and then incubated at 37°C for 1 hour to initiate gelation. After gelation, the gel seed is transferred from the syringe to the culture plate to obtain ECM scaffold 1.

[0068] Preparation of ECM scaffold 2:

[0069] Under sterile conditions, using 10x DMEM medium, Collagen I, Collagen IV, Collagen VI, Laminin (laminin) are added to the medium, so that the final concentration of each component in the medium is 4 mg / ml Collagen I, 4 mg / ml Collagen IV, 4 mg / ml Collagen VI and 1 mg / ml Laminin. Using 1N NaOH, the pH of the mixture is quickly neutralized to 7.4 and centrifuged. Subsequently, the mixed solution is aliquoted into special syringes, and then incubated at 37°C for 1 hour to initiate gelation. After gelation, the gel seed is transferred from the syringe to the culture plate to obtain ECM scaffold 2.

[0070] Example 3

[0071] Preparation of biomimetic islet organoids

[0072] The preparation process of the biomimetic islet organoids is shown in Figure 7.

[0073] Preparation of the biomimetic islet organoids bIO:

[0074] Under sterile conditions, using 10x DMEM medium, Collagen I, Collagen IV, Laminin (laminin) were added to the medium, finally the concentration of each component in the medium was 4 mg / ml Collagen I, 4 mg / ml Collagen IV and 1 mg / ml Laminin. Using 1N NaOH, the pH of the mixture was quickly neutralized to 7.4 and centrifuged. Subsequently, islet organoids were added according to the ratio of 15 islet organoids in 100 μL mixture, then the mixed solution was aliquoted into special syringes, then incubated at 37°C for 1 hour to initiate gelation. After gelation, the gel seed was transferred from the syringe to the culture plate to obtain the biomimetic islet organoids bIO.

[0075] Preparation of the biomimetic islet organoids bIO-ColVI:

[0076] Under sterile conditions, using 10x DMEM medium, Collagen I, Collagen IV, Collagen VI, Laminin (laminin) were added to the medium, finally the concentration of each component in the medium was 4 mg / ml Collagen I, 4 mg / ml Collagen IV, 4 mg / ml Collagen VI and 1 mg / ml Laminin. Using 1N NaOH, the pH of the mixture was quickly neutralized to 7.4 and centrifuged. Subsequently, islet organoids were added according to the ratio of 15 islet organoids in 100 μL mixture, then the mixed solution was aliquoted into special syringes, then incubated at 37°C for 1 hour to initiate gelation. After gelation, the gel seed was transferred from the syringe to the culture plate to obtain the biomimetic islet organoids bIO-ColVI.

[0077] Referring to Figure 8, which is a photograph of the above-mentioned biomimetic islet organoids or single islet organoids after being cultured in rpm1640 medium for 7 days, the photographs of the single islet organoids (IO), the biomimetic islet organoids bIO, and the biomimetic islet organoids bIO-ColVI are shown from left to right; it can be seen that the islet organoids are embedded in the biomimetic extracellular matrix hydrogel and have stronger cell activity than the single islet organoids, and the Collagen VI in the bIO-ColVI can enhance the protection of the biomimetic extracellular matrix hydrogel on the islet cells.

[0078] Bionic islet organoid pancreatic islets (bIO, bIO-Collagen VI) and islet organoids alone were transplanted into STZ-induced diabetic mice; as shown by live imaging in Figure 9, the bionic islet cell matrix component helps the islet cells survive in vivo, and Collagen VI can enhance the effect of the bionic extracellular matrix scaffold.

Claims

1. A pancreatic islet cell extracellular matrix scaffold, characterized in that, The scaffold is a gel containing 3–5 mg / ml Collagen I, 3–5 mg / ml Collagen IV, and 0.8–1.2 mg / ml Laminin.

2. The pancreatic islet cell extracellular matrix scaffold according to claim 1, characterized in that, It also includes 3-5 mg / ml Collagen VI.

3. The pancreatic islet cell extracellular matrix scaffold according to claim 1, characterized in that, The gel contains 4 mg / ml Collagen I, 4 mg / ml Collagen IV, 4 mg / ml Collagen VI and 1 mg / ml Laminin.

4. The pancreatic islet cell extracellular matrix scaffold according to claim 1, characterized in that, The gel matrix is ​​10x DMEM medium.

5. A method for preparing an extracellular matrix scaffold for pancreatic islet cells as described in any one of claims 1 to 4, characterized in that, Collagen I, Collagen IV, and Laminin were added to the matrix, and the pH was adjusted to neutral. The mixture was then incubated to allow it to gel.

6. The preparation method according to claim 5, characterized in that, Collagen I, Collagen IV, Collagen VI, and Laminin were added to the matrix, and the pH was adjusted to neutral. The mixture was then incubated to allow it to gel.

7. The preparation method according to claim 5 or 6, characterized in that, The substrate is 10x DMEM medium.

8. A method for preparing a biomimetic pancreatic islet organoid, characterized in that, Pancreatic islet organoids are constructed by embedding pancreatic islet cell extracellular matrix scaffolds as described in any one of claims 1 to 4.

9. A biomimetic pancreatic islet organoid, characterized in that, Includes the islet extracellular matrix scaffold as described in any one of claims 1 to 4, and islet organoids embedded within the islet extracellular matrix scaffold.

10. Use of the pancreatic islet organoid fabricated using the pancreatic islet extracellular matrix scaffold as described in any one of claims 1 to 4.

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