A porous microcarrier for culturing mesenchymal stem cells and its preparation method and application

Abstract

The present invention relates to the field of biotechnology, and specifically to a mesenchymal stem cell culture porous microcarrier and its preparation method and application, comprising the following steps: S1. stirring and mixing recombinant collagen, poloxamer 407, alginic acid and water to form a solution; S2. adding ethyl acetate solution to adjust the pH of the solution; S3. adding a pH stabilizer under stirring; S4. dropping into a calcium ion solution under stirring after cooling to form suspendable microspheres, filtering, centrifugally collecting the microspheres, purifying and washing until the solution is neutral, and obtaining the microcarrier. The mesenchymal stem cell culture microcarrier of the present invention has a temperature-sensitive property, can avoid the use of trypsin digestion, recover stem cells, reduce cell damage, improve the recovery rate of stem cells, and is conducive to large-scale stem cell culture applications.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a porous microcarrier for culturing mesenchymal stem cells and a preparation method and application thereof. Background Art

[0002] Cell culture is divided into two categories: adherent culture, also known as monolayer culture or plane culture, and suspension culture. Through cell culture, cell therapy products such as stem cells, immune cells and gene-modified cells for treating diseases can be produced, as well as cell-derived biological products such as viral vectors, vaccines, antibodies, peptide drugs, exosomes and microvesicles. Many cells from species such as humans, mammals and insects need to attach to the surface of materials to grow and proliferate. These cells can be called adherent cells or adherent cells. Containers such as tissue culture dishes, tissue culture flasks and cell factories allow adherent cells to attach to the surface of flat materials such as polystyrene and divide and proliferate. However, these culture containers can only provide a very limited growth surface for adherent cells, limiting the number of cells after amplification and culture. In addition, microcarriers prepared from natural polymers or / and synthetic polymers can provide a growth surface for adherent cells. Microcarriers used for cell culture are generally microspheres with a diameter of 100-300 microns or microparticles that are approximately spherical. They can be solid or have a porous structure.

[0003] Christian Weber reported that after using a variety of microcarriers to culture human mesenchymal stem cell lines, trypsin, accutase, collagenase or their mixed solutions were used to digest and recover the cells. The results showed that the efficiency of recovering cells from the dextran microcarriers Cytodex1 and Cytodex3 was extremely low, and only 5%-25% of the cells could be recovered from the microcarrier surface. We also found that it is difficult to separate mesenchymal stem cells using these proteases. Even if the enzyme treatment time is extended, the cell recovery rate is less than 50%.

[0004] In summary, although the various microcarriers currently on the market can support the expansion of adherent cells, they have one or more of the following problems: (1) The efficiency of recovering cells from microcarriers after cell expansion is low; (2) The residual substances in the microcarriers and the animal-derived substances in the microcarriers increase the safety risks of cell products or biological products produced by cells;

[0005] Hydrogels have been successfully used for stem cell culture and expansion due to their unique three-dimensional (3D) network structure and multifunctionality (porosity, permeability, mechanical properties), which can simulate the in vivo extracellular matrix environment (ECM) to regulate cell proliferation and differentiation. In the past few decades, a variety of hydrogel-based 3D microcarriers (MCs) have been developed to provide higher specific surface area for stem cell expansion to meet the rapidly growing clinical needs. However, as stem cell adhesion culture media, the structure, physicochemical properties and surface microenvironment of the material may affect cell adhesion, growth, proliferation and differentiation behavior. Most of the microcarriers currently on the market have complex processes, single functions and cannot be modified, such as microstructure, mechanical properties and mechanical properties. Therefore, it is crucial to design suitable microcarriers, select the best materials, the simplest processes and large-scale expansion technologies to meet expectations for quality, safety, efficacy and commercial feasibility. Summary of the invention

[0006] The purpose of the present invention is to provide a porous microcarrier for culturing mesenchymal stem cells and a preparation method and application thereof, so as to solve the technical problem that although the microcarrier can support the expansion of adherent cells in the prior art, the efficiency of recovering cells from the microcarrier after cell expansion is low.

[0007] The present invention discloses a method for preparing a porous microcarrier for culturing mesenchymal stem cells, comprising the following steps:

[0008] S1. Stir and mix the recombinant collagen, poloxamer 407, alginic acid and water to form a solution;

[0009] S2. Add ethyl acetate solution, stir homogenously, and adjust the pH of the solution;

[0010] S3. Adding a pH stabilizer under stirring;

[0011] S4. After cooling, add it dropwise to the calcium ion solution under stirring to form suspendable microspheres, filter, collect the microspheres by centrifugation, purify and wash until the solution is neutral, and dry. Reverse gelation can be achieved at 2-8 degrees, and it has fluidity.

[0012] Thermosensitive cell culture hydrogel microcarriers can avoid the use of trypsin digestion to recover stem cells, reduce cell damage, and increase the recovery rate of stem cells. It is conducive to large-scale stem cell culture applications.

[0013] Furthermore, in step S1, the recombinant collagen content is 5.8-6.2wt%, the poloxamer 407 content is 16.5-17.5wt%, the alginate content is 0.028-0.032wt%, and the rest is water.

[0014] Furthermore, the step S2 comprises the steps of adding ethyl acetate solution while maintaining stirring, wherein the volume ratio of the ethyl acetate solution to the solution obtained in step S1 is 1:1-4, and after forced homogenization and stirring, NaOH solution is added dropwise to the solution.

[0015] Furthermore, in step S2, the pH value of the solution is adjusted to 6.8-7.4.

[0016] Furthermore, in step S3, the pH stabilizer is added at a rate of 0.04 wt % per minute and the addition is completed within 50 to 60 minutes.

[0017] Furthermore, in step S3, the pH stabilizer contains borax and phosphate.

[0018] Furthermore, after the pH stabilizer is added in step S3, the concentration of borax+phosphate in the solution is 1 M, and the molar ratio of borax to phosphate is 1:8-12, so that the pH range of the solution is 6.5±1.

[0019] Furthermore, the cooling in step S4 is to cool the solution obtained in S3 to 2°C±2.

[0020] Furthermore, the calcium ion solution in step S4 is a calcium chloride solution with a mass concentration of 0.06%.

[0021] Furthermore, in step S4, the dropwise addition is performed by a microinjection pump, and the droplet diameter does not exceed 1 mm. The solubility and rate of the droplet addition and the droplet size affect the diameter and pore size of the porous microspheres.

[0022] A porous microcarrier for culturing mesenchymal stem cells is prepared by using the method.

[0023] Furthermore, the particle size of the suspendable microspheres is 200-600 um, and the pore size is 20-100 um.

[0024] Furthermore, the suspendable microspheres have memory temperature-sensitive properties.

[0025] Furthermore, the suspendable microspheres can form three-dimensional porous suspendable microspheres at 30-42 degrees.

[0026] Furthermore, the suspendable microspheres can achieve reverse gelation at 2 to 8 degrees and have fluidity.

[0027] The invention discloses an application of a porous microcarrier for culturing mesenchymal stem cells, and is used for recovering stem cells.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The mesenchymal stem cell culture microcarrier of the present invention has a temperature-sensitive property, which can avoid the use of trypsin digestion to recover stem cells, reduce cell damage, improve the quality of stem cell recovery, avoid protease digestion and high-speed centrifugation during the harvesting process, and can minimize the damage to stem cells to the greatest extent, ensuring the quality of stem cells. It is conducive to large-scale stem cell culture applications;

[0030] 2. The present invention solves the technical problem that the traditional emulsification method cannot prepare uniform microspheres, and provides the first process for stably preparing porous thermosensitive microspheres of uniform size, with alginate and calcium as the network skeleton, and recombinant collagen as a pore-forming agent that can provide nutrition at the same time;

[0031] 3. The present invention is suitable for cell culture and can effectively promote cell attachment and cell proliferation. The effective cell harvest rate of cell culture is more than 85%, and the cell harvest cell damage rate is less than 4%;

[0032] 4. The process of the present invention is simple and highly repeatable, and can obtain uniform spherical microcarriers of different particle sizes, which has great prospects for industrial application;

[0033] 5. Poloxamer 407 is a thermotropic material with emulsifying properties. It selects ethyl acetate as the solvent to form an emulsifying system, which has the phase separation emulsification effect for preparing microspheres. Borax + phosphate is added for stabilization to achieve emulsification phase separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only represent some embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0035] Figure 1 This is the SEM morphology image of Example 1 of the present invention.

[0036] Figure 2 This is the SEM morphology image of Example 2 of the present invention.

[0037] Figure 3 This is the SEM image of the microspheres prepared in Comparative Example 1 of the present invention.

[0038] Figure 4 The hUCMSCs distribution observation (40×) diagram of Example 2, wherein A: observation of collected cells for 7D under a fluorescence microscope; B: observation of the distribution of cells on microcarriers of Example 2 cultured for 7D under an inverted phase contrast microscope; C: observation of the distribution of cells on low-temperature phase-change microcarriers of Example 2 under an inverted phase contrast microscope (DAPI staining).

[0039] Figure 5 The graph shows the damage ratio of cells harvested under different conditions. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0041] Example 1

[0042] The method for preparing a porous microcarrier for culturing mesenchymal stem cells using the above-mentioned device in this embodiment comprises the following steps:

[0043] S1: 6wt% recombinant collagen, 17wt% poloxamer 407, 0.03wt% alginate and the rest water are mixed to obtain a solution.

[0044] The specific steps of S2 are: adding ethyl acetate solution under stirring at a ratio of 1:1, forcing homogenization and stirring to be uniform, and then dropping NaOH solution into the solution. The pH value is adjusted to 7.2.

[0045] S3 adds pH stabilizer borax + sodium dihydrogen phosphate under stirring conditions, the addition method is 0.04wt% per minute, and the addition is completed in 60 minutes. The concentration of borax + phosphate is 1M, and the molar ratio is 1:10, so that the pH range of the solution is 6.5±1.

[0046] S4 Keep the solution obtained in S3 at 2℃±2, and slowly add it to the calcium chloride solution with a mass concentration of 0.06% containing calcium ions. Use a micro-injection pump to add the solution evenly and slowly, with a droplet diameter not exceeding 1mm.

[0047] Filter and collect the microspheres by centrifugation, purify and wash three times until the solution is neutral, and slowly dry them at 35±2℃ with air blowing.

[0048] Example 2

[0049] In this embodiment, as a preferred embodiment of the present invention, a method for preparing a porous microcarrier for culturing mesenchymal stem cells comprises the following steps:

[0050] S1 recombinant collagen 6%, poloxamer 407 content 17wt%, alginate content 0.03wt%, and the rest water are mixed evenly to obtain a solution.

[0051] The specific steps of S2 are as follows: adding ethyl acetate solution under stirring at a ratio of 1:4, forcing homogenization and stirring to be uniform, and then dropping NaOH solution into the solution. The pH value is adjusted to 7.2.

[0052] S3 adds pH stabilizer borax + sodium dihydrogen phosphate under stirring conditions, the addition method is 0.04wt% per minute, and the addition is completed in 60 minutes. The concentration of borax + phosphate is 1M, and the molar ratio is 1:10, so that the pH range of the solution is 6.5±1.

[0053] S4 Keep the solution obtained in S3 at 2℃±2, and slowly add it to the calcium chloride solution with a mass concentration of 0.06% of the calcium ion solution at 35±2℃. Use a micro-injection pump to add the solution evenly and slowly, with a droplet diameter not exceeding 1mm.

[0054] Filter and centrifuge to collect the microspheres, purify and wash 3 times until the solution is neutral, and freeze-dry at -40±2℃.

[0055] Comparative Example 1

[0056] In this embodiment, as a comparative example of the present invention, a method for preparing a porous microcarrier for culturing mesenchymal stem cells is disclosed. The only change on the basis of Example 1 is that the alginate content is 1wt%, and the obtained particles are relatively large and have no micropores. This is not conducive to cell growth, and the thermosensitive phase transition cannot be achieved due to excessive cross-linking.

[0057] Comparative Example 2

[0058] In this embodiment, as a comparative example of the present invention, a method for preparing a porous microcarrier for culturing mesenchymal stem cells is disclosed. On the basis of Example 2, a calcium chloride solution containing a calcium ion solution with a mass concentration of 0.2% is used, and no particles are formed. Large cross-linked products are formed, which cannot be used in subsequent applications, and are failure cases.

[0059] Product Characterization and Testing:

[0060] 1. Characterization

[0061] The SEM image of the microsphere carrier obtained in Example 1 is shown in Figure 1 The SEM image of the microsphere carrier obtained in Example 2 is shown in Figure 2 It can be seen that the thermosensitive microsphere carrier product obtained in the present invention is a spherical structure with a particle size of about 200-600 μm, and has a porous feature with a pore size of about 20-100 um.

[0062] The SEM of the microsphere carrier obtained in Comparative Example 1 is as follows Figure 3 The particle uniformity is poor, and the particle size basically exceeds 600um, and there is adhesion and no porous characteristics.

[0063] 2. Cell adsorption and desorption test, cell desorption quality test

[0064] The experiment used the characteristics of human umbilical cord mesenchymal stem cells cultured and expanded on the two-dimensional well plate group, comparative example 1, example 1 and example 2. Human umbilical cord mesenchymal stem cells were subcultured, and the immune phenotype was identified by flow cytometry. hUCMSCs were cultured at 4×10 4 The cells were inoculated at a density of 2 g / L into a well plate, a roller bottle containing 2 g / L of the carrier of Comparative Example 1, a roller bottle containing 2 g / L of the carrier of Example 1, and a roller bottle containing 2 g / L of the carrier of Example 2, and cultured continuously for 7 days. During this period, samples were taken and the surface structure and cell attachment of the microcarriers were observed by scanning electron microscopy. The growth distribution of the cells on the microcarriers was observed by DAPI staining. Figure 4 .

[0065] Usually, cells need to be separated from growth supports, such as plates, microcarriers, etc., to obtain a pure cell suspension, and generally, cells need to be harvested by digestion with trypsin, etc. Examples 1 and 2 are a temperature-sensitive phase change material, so the cells can be directly harvested by cooling phase change as seeds for cell therapy, without the need for trypsin digestion to harvest the cells.

[0066] The cells on the two-dimensional well plate group and comparative example 1 were digested with 0.25% trypsin-EDTA at 37°C. After 3-4 min of digestion, the hUCMSCs on the two-dimensional well plate group and the cells fell off the plate and were collected by centrifugation at 500g; the cells on comparative example 1 were directly collected by centrifugation at 500g; and the cells on examples 1 and 2 were directly harvested by temperature phase change and low-speed centrifugation at 500g.

[0067] The expression of lactate dehydrogenase (LDH) in the supernatant of cells harvested in four conditions is shown in Figure 5 . The results showed that there were significant differences in the cell damage rates of cells harvested by different methods. The LDH release rate of the cells in the dish treated with trypsin and the cells in Comparative Example 1 was higher than that of the cells in Example 2 that were not treated with trypsin. That is, the cell damage caused by trypsin digestion was greater than that of the cells in the group that was not digested with trypsin. The above results show that the process of harvesting cells with trypsin will cause irreversible damage to the cells themselves, thereby affecting the cell quality.

[0068] When the cells are fully grown on the microcarriers after 7 days of culture, they are transferred to a low temperature of 2-4°C and placed. After 60 minutes, 10 μL of the cell suspension is taken for counting, which is the number of cells shed after temperature-sensitive cooling. Then, the cells that have not shed after cooling are collected by trypsin digestion, which is the number of cells harvested by trypsin.

[0069] The effective cell yield was calculated according to the following formula:

[0070] Total cell number = total number of cells detached by thermosensitive cooling + number of cells harvested by trypsin method;

[0071] Cell proliferation multiple = total cell harvest number / initial cell seeding number

[0072] Thermosensitive cell detachment rate = number of thermosensitive cooling detached cells / total number of cells harvested.

[0073] Effective cell detachment rate = thermosensitive cell detachment rate × (1-cell damage rate%)

[0074] The temperature-sensitive microcarriers obtained in Examples 1 to 2 were subjected to the above tests. The test results showed that the cell detachment rates of the flat plate group, comparative example 1, example 1 and example 2 were 98%, 48.8%, 89% and 93%, respectively; the harvested cell damage rates were 13.4%, 4.7%, 3.9% and 2.4%, respectively; the actual effective cell harvest rates were 84.8%, 46.5%, 85.5% and 90.7%, respectively; the cell proliferation multiples of the flat plate group, comparative example 1, example 1 and example 2 were 13.2, 6.6, 19.8 and 24.5, respectively.

[0075] The above are the implementation methods listed in this embodiment, but this embodiment is not limited to the above optional implementation methods. Those skilled in the art can arbitrarily combine the above methods to obtain other various implementation methods. Anyone can derive other various forms of implementation methods under the inspiration of this embodiment. The above specific implementation methods should not be understood as limiting the scope of protection of this embodiment. The scope of protection of this embodiment shall be based on the definition in the claims, and the description can be used to interpret the claims.

Claims

1. A method for preparing a porous microcarrier for culturing mesenchymal stem cells, characterized in that: The following steps are included: S1. Stir and mix the recombinant collagen, poloxamer 407, alginic acid and water to form a solution; S2. Add ethyl acetate solution, stir homogenously and adjust the pH of the solution; S3. adding a pH stabilizer under stirring, wherein the pH stabilizer comprises borax and phosphate; S4. After cooling, the mixture is added dropwise to the calcium ion solution under stirring to form suspendable microspheres, filtered, centrifuged to collect the microspheres, purified and washed until the solution is neutral, and dried; In step S1, the recombinant collagen content is 5.8-6.2wt%, the poloxamer 407 content is 16.5-17.5wt%, and the alginate content is 0.028-0.032wt%; The mass concentration of the calcium ion solution is 0.06%.

2. The method for preparing a porous microcarrier for culturing mesenchymal stem cells according to claim 1, characterized in that: In step S2, the pH value of the solution is adjusted to 6.8-7.

4.

3. The method for preparing a porous microcarrier for culturing mesenchymal stem cells according to claim 1, characterized in that: In step S3, the pH stabilizer is added at a rate of 0.04 wt % per minute and is added in 50 to 60 minutes.

4. The method for preparing a porous microcarrier for culturing mesenchymal stem cells according to claim 1, characterized in that: After the pH stabilizer is added in step S3, the concentration of borax+phosphate in the solution is 1 M, the molar ratio of borax to phosphate is 1:8-12, and the pH range of the solution is 6.5±1.

5. A porous microcarrier for culturing mesenchymal stem cells, characterized in that: The method for preparing a porous microcarrier for culturing mesenchymal stem cells according to any one of claims 1 to 4 is used to prepare the microcarrier.

6. The porous microcarrier for culturing mesenchymal stem cells according to claim 5, characterized in that: The particle size of the suspendable microspheres is 200-600 um, and the pore size is 20-100 um.

7. Use of a porous microcarrier for culturing mesenchymal stem cells prepared by the method for preparing a porous microcarrier for culturing mesenchymal stem cells according to any one of claims 1 to 4 or a porous microcarrier for culturing mesenchymal stem cells according to any one of claims 5 to 6, characterized in that: Used to recover stem cells.

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