Preparation method of matrigel and matrigel

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a preparation method of matrigel and the matrigel. The preparation method of the matrigel comprises the following steps: performing adherent culture or suspension culture on sarcoma cells by adopting a cell culture medium, then performing decellularization by using a decellularization solution, and performing dialysis and filtration to obtain the matrigel, the cell culture medium comprises a serum-free stem cell culture medium and the following components: 0.01-0.1 g / L of a wintercreeper extract, 0.04-0.12 g / L of a motherwort herb extract and 0.3-0.5 g / L of salvia miltiorrhiza freeze-dried powder. The invention also provides the matrigel obtained by the preparation method of the matrigel. The matrigel prepared by the preparation method of the matrigel provided by the invention has the advantages of high yield, no animal-derived components and the like, and can be used for related clinical tests of IPS cell culture.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a method for preparing a matrix glue and the matrix glue. Background Art

[0002] In the biomedical field, Matrigel is a commonly used research tool that plays an important role in cell biology research and drug screening. It can simulate the extracellular matrix environment in the body and provide cells with a three-dimensional spatial structure, thereby promoting cell growth and maintenance of function. Especially in tumor research, Matrigel can provide support similar to the tumor microenvironment, making the study of cell-to-cell interactions closer to the physiological state, greatly promoting the progress of disease model construction, cell proliferation and migration, etc.

[0003] At present, there are two main methods for preparing matrix glue: one is to extract it from animal tissues, and the other is to obtain it by decellularization through cell culture. Among them, the animal tissue extraction method is simple to operate and easy to achieve large-scale production, but the matrix glue obtained has risks such as large batch differences, unclear chemical composition, and potential pathogen contamination; while the matrix glue prepared by decellularization after cell culture can better control its composition, and the obtained matrix glue has good consistency, but the culture medium used contains animal-derived components such as fetal bovine serum, resulting in high raw material costs and poor biocompatibility of the obtained matrix glue, which is not conducive to clinical use. Summary of the invention

[0004] In order to overcome the problems of high raw material cost and the risk of residual animal-derived components in the matrix glue in the prior art, the present application provides a method for preparing the matrix glue and the matrix glue.

[0005] In a first aspect, the present application provides a method for preparing a matrix gel, using the following technical solution: A method for preparing matrix glue comprises the following steps: using cell culture medium to culture sarcoma cells in an adherent or suspended manner, then decellularizing with a decellularizing solution, and obtaining matrix glue through dialysis and filtration; The cell culture medium comprises a serum-free stem cell culture medium and the following components: 0.01-0.1 g / L of Euonymus fortunei extract, 0.04-0.12 g / L of Leonurus japonicus extract and 0.3-0.5 g / L of lyophilized salvia miltiorrhiza powder.

[0006] The present application provides a preparation method that can effectively improve the yield and biocompatibility of matrix glue. The method uses the cell culture medium composed of the above-mentioned specific components for cell culture, which can significantly promote the growth and differentiation of sarcoma cells, thereby making the yield of the prepared matrix glue higher and having better biological activity. In the present application, after the cells are cultured under the optimal growth environment, the non-specific components such as the cell membrane can be effectively removed by decellularization, avoiding the residual cell debris that may appear in the traditional method, and improving the purity of the final product matrix glue; finally, through the dialysis and filtration steps, small molecule impurities and other unnecessary components are further removed to ensure the quality of the obtained matrix glue. The component selection and concentration ratio of the culture medium of the present application have been carefully designed, which can not only support the growth of sarcoma cells well, but also effectively induce the formation of matrix glue. In summary, the method provided by the present application simplifies the production process of matrix glue, reduces costs, increases yield, and is suitable for large-scale industrial applications. In addition, the matrix glue obtained by the above method has excellent biocompatibility and low immunogenicity, and is suitable for a variety of biomedical research and clinical treatment purposes.

[0007] Optionally, the cell culture medium comprises the following components: 0.01-0.1 g / L of Euonymus fortunei extract, 0.06-0.10 g / L of Leonurus japonicus extract and 0.35-0.45 g / L of lyophilized Salvia miltiorrhiza powder.

[0008] In the cell culture medium provided by this application, the extract of Euonymus fortunei, the extract of Leonurus japonicus and the freeze-dried powder of Salvia miltiorrhiza contain growth factors or analogues, which can stimulate the proliferation and differentiation of cells, and help to obtain more cells when preparing matrix glue, thereby obtaining a higher yield of matrix glue; in addition, the extract of Leonurus japonicus contains a variety of trace elements, vitamins and minerals and other nutrients, which can provide necessary nutritional support for subsequent cell culture even if they remain in the culture medium, helping cells to grow better in the culture environment. This application has found through experimental exploration that further controlling the content of each component in the cell culture medium within the above range can produce a higher yield of matrix glue.

[0009] In some embodiments, the content of the motherwort extract can be 0.04-0.06 g / L, 0.04-0.08 g / L, 0.04-0.10 g / L, 0.04-0.12 g / L, 0.06-0.08 g / L, 0.06-0.10 g / L, 0.06-0.12 g / L, 0.08-0.10 g / L, 0.08-0.12 g / L, or 0.10-0.12 g / L.

[0010] In a specific embodiment, the content of the Leonurus japonicus extract can also be 0.04 g / L, 0.06 g / L, 0.08 g / L, 0.10 g / L or 0.12 g / L.

[0011] In some embodiments, the content of the Salvia miltiorrhiza freeze-dried powder can be 0.30-0.35 g / L, 0.30-0.40 g / L, 0.30-0.45 g / L, 0.30-0.50 g / L, 0.35-0.40 g / L, 0.35-0.45 g / L, 0.35-0.50 g / L, 0.40-0.45 g / L, 0.40-0.50 g / L or 0.45-0.50 g / L.

[0012] In a specific embodiment, the content of the Salvia miltiorrhiza freeze-dried powder can also be 0.30 g / L, 0.35 g / L, 0.40 g / L, 0.45 g / L or 0.50 g / L.

[0013] Optionally, the cell culture medium comprises the following components: 0.05 g / L of Euonymus fortunei extract, 0.08 g / L of Leonurus japonicus extract and 0.40 g / L of lyophilized Salvia miltiorrhiza powder.

[0014] Optionally, when suspension culture is used, the cell culture medium further comprises recombinant humanized collagen at a final concentration of 28-42 g / L.

[0015] In a specific embodiment, the final concentration of the recombinant humanized collagen is 35 g / L.

[0016] Optionally, the specific steps of the method for preparing the matrix gel are: resuspending sarcoma cells with cell culture medium, then placing them in a cell culture device, and culturing them at 37°C and 20-100rpm for 12-14 days; centrifuging at low speed to remove the supernatant and collecting the cells; adding cell fluid to the cells for resuspending, centrifuging at high speed, and dialyzing the collected supernatant to obtain the matrix gel.

[0017] Optionally, the operations of adding cell fluid for resuspension and centrifugation need to be repeated at least 3 times, and the supernatants obtained from the 3 centrifugations are combined and dialyzed.

[0018] Optionally, the rotation speed of the low-speed centrifugation is 1000-1200 rpm, and the rotation speed of the high-speed centrifugation is 10000-12000 rpm.

[0019] In a second aspect, the present application provides a matrix gel obtained by the method for preparing the matrix gel.

[0020] In a third aspect, the present application provides a method for preparing matrix gel and a method for using the matrix gel in cell culture.

[0021] In summary, this application has the following beneficial effects: 1. The present application provides a method for preparing matrix glue, which uses a cell culture medium prepared from Euonymus fortunei extract, Leonurus japonicus extract and lyophilized powder of Salvia miltiorrhiza to culture sarcoma cells, and obtains a high-yield matrix glue through post-processing such as decellularization, dialysis, and filtration. In addition, there are no residual animal-derived components in the matrix glue, and the matrix glue can be used in clinical trials related to IPS culture.

[0022] 2. In the cell culture medium used in the present application, the content of Leonurus japonicus extract is further controlled within the range of 0.06-0.10 g / L, and the content of Salvia miltiorrhiza lyophilized powder is controlled within the range of 0.35-0.45 g / L, so that the culture effect of the obtained cell culture medium is better, so that the yield of matrix gel can reach more than 320 mg, DETAILED DESCRIPTION

[0023] In the first aspect, the present application provides a cell culture medium, comprising a serum-free stem cell culture medium and the following components: 0.01-0.1 g / L of Euonymus fortunei extract, 0.04-0.12 g / L of Leonurus japonicus extract, and 0.3-0.5 g / L of lyophilized powder of Salvia miltiorrhiza. Further, the cell culture medium comprises the following components: 0.05 g / L of Euonymus fortunei extract, 0.08 g / L of Leonurus japonicus extract, and 0.40 g / L of lyophilized powder of Salvia miltiorrhiza. Further, the cell culture medium further comprises recombinant humanized collagen at a final concentration of 28-42 g / L.

[0024] In a second aspect, the present application provides a method for preparing matrix gel, using an adherent culture method, specifically comprising the following steps: (1) Resuspend the sarcoma cells in cell culture medium. The cell concentration after resuspension is (1-6)×10 5 cells / mL; then placed in a T175 cell culture flask / 3D shake flask, cultured at 37°C, 20-100 rpm for 12-14 days; the medium was changed once every 3-5 days; (2) After the culture is completed, low-speed centrifugation is performed at a speed of 1000-1200 rpm, and the supernatant is discarded to obtain the cell pellet; (3) Add decellularization solution to the cell pellet and resuspend it, and let it stand at 2-8°C overnight; then centrifuge at 10000-12000 rpm for 20-40 min, collect the supernatant, and repeat the above operation 3 times; (4) The supernatants obtained from three centrifugations were combined and dialyzed 3-5 times with a dialysate (pH 7.0, 10 mM Tris-HCl), each dialysis time for 3-4 hours; the dialyzed liquid was collected and filtered using a 0.22 μm syringe filter to obtain the matrix gel.

[0025] In the present application, the serum-free stem cell culture medium is a serum-free mesenchymal stem cell culture medium, purchased from Guangzhou Hucheng Technology Co., Ltd.; the Eucommia fortunei extract was purchased from Xi'an Senran Bioengineering Co., Ltd., and the Leonurus japonicus extract was purchased from Shaanxi Chenming Biotechnology Co., Ltd.; the Salvia miltiorrhiza lyophilized powder is Salvia miltiorrhiza for injection (lyophilized), purchased from Harbin Pharmaceutical Group Traditional Chinese Medicine Factory No. 2; the recombinant humanized collagen was purchased from Zhuhai Jibaikang Biotechnology Co., Ltd.; the decellularized fluid is a 2mol / L urea buffer (ratio of 2mol / L urea, 0.05mol / L Tris base, 150mmol / L NaCl); the remaining raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.

[0026] The present application is further described in detail below in combination with preparation examples, embodiments and performance testing experiments.

[0027] Preparation Example 1-9 Preparation Examples 1-9 each provide a cell culture medium.

[0028] The difference between the above preparation examples is that the content of each component in the cell culture medium is specifically shown in Table 1 below.

[0029] The preparation method of the cell culture medium provided in Preparation Example 1-9 comprises the following steps: Add the extract of Euonymus fortunei, the extract of Leonurus herb and the freeze-dried powder of Salvia miltiorrhiza to the serum-free stem cell culture medium according to the contents shown in Table 1, mix well, and obtain the cell culture medium.

[0030] Table 1 Content of each component in the cell culture medium of Preparation Examples 1-9 Preparation Example 10 Preparation Example 10 provides a cell culture medium.

[0031] The difference between the above preparation example and preparation example 3 is that the cell culture medium further includes 35 g / L recombinant humanized collagen.

[0032] Comparative Preparation Example 1 Comparative Preparation Example 1 provides a cell culture medium.

[0033] The difference between the comparative preparation example and preparation example 3 is that the content of the Euonymus fortunei extract in the cell culture medium is 0.

[0034] Comparative Preparation Example 2 Comparative Preparation Example 2 provides a cell culture medium.

[0035] The difference between the comparative preparation example and preparation example 3 is that the content of the motherwort extract in the cell culture medium is 0.

[0036] Comparative Preparation Example 3 Comparative Preparation Example 3 provides a cell culture medium.

[0037] The difference between the comparative preparation example and preparation example 3 is that the content of lyophilized Salvia miltiorrhiza powder in the cell culture medium is 0.

[0038] Comparative Preparation Example 4 Comparative Preparation Example 4 provides a cell culture medium.

[0039] The difference between the comparative preparation example and the preparation example 3 is that in the cell culture medium, the content of the Euonymus fortunei extract is 0.2 g / L, the content of the Leonurus japonicus extract is 0.15 g / L and the content of the Salvia miltiorrhiza lyophilized powder is 0.2 g / L.

[0040] Examples 1-9 Examples 1-9 respectively provide a method for preparing a matrix gel.

[0041] The difference between the above examples is that the cell culture medium used in the preparation method of the matrix gel is derived from Preparation Examples 1-9 respectively.

[0042] The preparation method of the matrix gel provided in Examples 1-9 adopts the method of adherent culture, and specifically comprises the following steps: (1) The sarcoma cells were resuspended using the cell culture medium provided in Preparation Examples 1-9. The cell concentration after resuspension was 3×10 5 cells / mL; then placed in a T175 cell culture flask and cultured at 37°C and 100 rpm for 14 days; the medium was changed once every 4 days; (2) After the culture is completed, low-speed centrifugation is performed at a speed of 1100 rpm, and the supernatant is discarded to obtain the cell pellet; (3) Add decellularization solution to the cell pellet and resuspend it, and let it stand at 5°C overnight; then centrifuge at 10,000 rpm for 30 min, collect the supernatant, and repeat the above operation 3 times; (4) The supernatants obtained from three centrifugations were combined and dialyzed three times with a dialysate (pH 7.0, 10 mM Tris-HCl), each time for 4 h. The dialyzed liquid was collected to obtain the matrix gel.

[0043] Example 10 Example 10 provides a method for preparing matrix gel. The method of adherent culture is adopted, which specifically includes the following steps: (1) The sarcoma cells were resuspended using the cell culture medium provided in Preparation Example 10. The cell concentration after resuspension was 3×10 5 cells / mL; then placed in a 3D cell shake flask and cultured at 37°C and 100 rpm for 14 days; the medium was changed once every 4 days; (2) After the culture is completed, the shaker arm is scraped with a cell scraper, and then the culture medium is centrifuged at a low speed of 1100 rpm, and the supernatant is discarded to obtain the cell pellet; (3) Add decellularization solution to the cell pellet and resuspend it, and let it stand at 5°C overnight; then centrifuge at 10,000 rpm for 30 min, collect the supernatant, and repeat the above operation 3 times; (4) The supernatants obtained from three centrifugations were combined and dialyzed three times with a dialysate (pH 7.0, 10 mM Tris-HCl), each time for 4 h. The dialyzed liquid was collected to obtain the matrix gel.

[0044] Comparative Examples 1-4 Comparative Examples 1-4 respectively provide a method for preparing a matrix gel.

[0045] The difference between the comparative example and Example 3 is that the cell culture medium used in the preparation method of the matrix gel is derived from comparative preparation examples 1-4 respectively.

[0046] Comparative Example 5 Comparative Example 5 provides a method for preparing matrix glue.

[0047] The difference between the comparative example and Example 3 is that the cell culture medium used in the preparation method of the matrix gel is 1640 culture medium containing 10% fetal bovine serum.

[0048] Performance testing The content of the matrix gel obtained in Examples 1-10 and Comparative Examples 1-5 was detected, and the total yield of each matrix gel was calculated. The results are shown in Table 2 below.

[0049] The protein content of the matrix gel obtained in each embodiment and preparation example was detected using the Solebow BCA protein kit, and the matrix gel yield was calculated based on the volume of the matrix gel. The calculation formula is as follows: Matrigel yield = Matrigel protein content × Matrigel volume.

[0050] Table 2 Matrigel yields obtained in Examples 1-10 and Comparative Examples 1-5 According to the test results in Table 2, the yield of matrix gel obtained by adherent culture in Examples 1-9 was 310.65-332.41 mg, and the yield of matrix gel obtained by suspension culture in Example 10 was 501.75 mg. Therefore, it is shown that the present application uses a serum-free stem cell culture medium containing Euonymus fortunei extract, Leonurus herb extract and Salvia miltiorrhiza lyophilized powder to culture sarcoma cells, and the yield of matrix gel obtained is high and there is no potential risk of residual animal-derived components.

[0051] The test results of Comparative Examples 1-3 show that when sarcoma cells are cultured in a serum-free stem cell culture medium containing any two of the Euonymus fortunei extract, the Leonurus japonicus extract and the lyophilized powder of Salvia miltiorrhiza, the yield of the obtained matrix gel is relatively low, which is only 247.11-265.23 mg.

[0052] From the test results of Comparative Example 4, it can be seen that when sarcoma cells were cultured with a serum-free stem cell culture medium containing Euonymus fortunei extract, Leonurus japonicus extract and Salvia miltiorrhiza lyophilized powder, and the contents of the three were adjusted to "Euonymus fortunei extract content of 0.2 g / L, Leonurus japonicus extract content of 0.15 g / L and Salvia miltiorrhiza lyophilized powder content of 0.2 g / L", the yield of matrix gel obtained was low, only 282.49 mg.

[0053] The test results of comparative example 5 show that when sarcoma cells are cultured in 1640 medium containing 10% fetal bovine serum, the yield of the obtained matrix gel is only 279.68 mg, and there is a risk of FBS residue in the matrix gel, which has poor biocompatibility and is not conducive to clinical use.

[0054] The test results of Examples 1-5 show that when the content of the Leonurus japonicus extract is controlled to 0.04 g / L or 0.12 g / L in Examples 1 and 5, the yield of the obtained matrix glue is 310.65-316.28 mg; when the content of the Leonurus japonicus extract is controlled to 0.06-0.10 g / L in Examples 2-4, the yield of the obtained matrix glue is 327.78-332.41 mg (>320 mg). Therefore, it is explained that the application further controls the content of the Leonurus japonicus extract within the range of 0.06-0.10 g / L, and the use effect of the obtained culture medium is higher, which can make the yield of the matrix glue higher.

[0055] The test results of Example 3 and Examples 6-9 show that when the content of Danshen freeze-dried powder is controlled to 0.30g / L or 0.50g / L in Example 6 and Example 9, the yield of the obtained matrigel is 317.10-319.26mg; when the content of Danshen freeze-dried powder is controlled to 0.35-0.45g / L in Example 3 and Examples 7-8, the yield of the obtained matrigel is 324.83-332.41mg (>320mg). Therefore, it is explained that the application further controls the content of Danshen freeze-dried powder within the range of 0.35-0.45g / L, and the use effect of the obtained cell culture medium is higher, which can make the yield of the matrigel higher.

[0056] Example 10: The sarcoma cells were suspended and cultured using a cell culture medium containing recombinant humanized collagen, and the yield of the obtained matrix gel was as high as 501.75 mg.

[0057] Plating and cell culture assays The matrix gel obtained in Examples 1-10 and Comparative Examples 1-5 was plated in a six-well plate, and then IPS cells were inoculated at a seeding volume of 1×10 5 cells / well, observed the cell growth, and performed cell digestion and counting 3 days later, and counted the total cell number and the number of living cells (Trypan blue staining method), and calculated the yield. The results are shown in Table 3 below.

[0058] Table 3 Cell culture test results of matrix gel obtained in Examples 1-10 and Comparative Examples 1-5 According to the test results in Table 2, after culturing IPS cells with the matrix gel obtained in Example 1-10, the number of viable cells after culture can reach 2.00×10 6 The cell viability can reach 85% or more. However, the number of viable cells after culture was only (0.89-1.81)×10 6 The cell viability is only 73-86%. Therefore, the matrix glue obtained by the preparation method of the matrix glue provided in the present application has a good cell culture effect and no infection risk, and can be used in clinical trials related to IPS culture.

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a matrix gel, characterized in that: The following steps are involved: The sarcoma cells are cultured adherently or suspended using a cell culture medium, and then decellularized using a decellularization solution, dialyzed, and filtered to obtain a matrix gel; The cell culture medium comprises a serum-free stem cell culture medium and the following components: 0.01-0.1 g / L of Euonymus fortunei extract, 0.04-0.12 g / L of Leonurus japonicus extract and 0.3-0.5 g / L of lyophilized salvia miltiorrhiza powder.

2. The method for preparing the matrix glue according to claim 1, characterized in that: The cell culture medium comprises the following components: 0.01-0.1 g / L of Euonymus fortunei extract, 0.06-0.10 g / L of Leonurus japonicus extract and 0.35-0.45 g / L of lyophilized powder of Salvia miltiorrhiza.

3. The method for preparing the matrix glue according to claim 1, characterized in that: The cell culture medium comprises the following components: 0.05 g / L of Euonymus fortunei extract, 0.08 g / L of Leonurus japonicus extract and 0.40 g / L of lyophilized powder of Salvia miltiorrhiza.

4. The method for preparing the matrix glue according to claim 1, characterized in that: When suspension culture is used, the cell culture medium also includes recombinant humanized collagen at a final concentration of 28-42 g / L.

5. The method for preparing the matrix gel according to claim 1, characterized in that: The specific steps of the preparation method of the matrix glue are: using cell culture medium to resuspend sarcoma cells, then placing them in a cell culture device, and culturing them at 37°C and a rotation speed of 20-100rpm for 12-14 days; centrifuging at a low speed to remove the supernatant and collecting the cells; adding cell fluid to the cells for resuspending, centrifuging at a high speed, and dialyzing the collected supernatant to obtain the matrix glue.

6. The method for preparing the matrix gel according to claim 5, characterized in that: The operations of adding cell solution for resuspension and centrifugation need to be repeated at least 3 times, and the supernatants obtained from the 3 centrifugations are combined and then dialyzed.

7. The method for preparing the matrix gel according to claim 5, characterized in that: The rotation speed of the low-speed centrifugation is 1000-1200 rpm, and the rotation speed of the high-speed centrifugation is 10000-12000 rpm.

8. A matrix gel obtained by the method for preparing the matrix gel according to any one of claims 1 to 7.

9. Use of the matrix gel obtained by the method for preparing the matrix gel according to any one of claims 1 to 7 in cell culture.

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