Modification buffer solution and cell culture replenishing solution for alginic acid microcarrier stem cell culture, combination of modification buffer solution and cell culture replenishing solution and culture method

Abstract

The invention relates to the technical field of biology, and provides a modification buffer solution and a cell culture replenishing solution for alginic acid microcarrier stem cell culture, and a combination and a culture method thereof. Specifically, the invention provides an alginic acid microcarrier modification buffer solution, a cell culture replenishing solution, a complete medium for stem cell culture, a liquid composition, a method for modifying an alginic acid microcarrier and a method for culturing stem cells by using the alginic acid microcarrier.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, the present application relates to a modified buffer for alginic acid microcarrier stem cell culture, a cell culture supplement, a combination thereof and a culture method. BACKGROUND

[0002] Three-dimensional cell culture refers to a cell culture method that simulates the specific microstructure of tissues and organs by culturing cells in micro devices containing three-dimensional structures. Unlike two-dimensional cell culture, which allows cells to grow on polyester or glass planes, three-dimensional cell culture promotes cell differentiation and tissue growth by using micro-assembly structures and adjusting complex environmental parameters, which can better simulate in vivo cell behavior and tissue (morphology and physiology).

[0003] Alginic acid microcarriers are tiny particles made of alginic acid polymers. Alginic acid is a natural anionic and hydrophilic polysaccharide derived from brown algae, which has similar biophysical properties to the extracellular matrix (ECM).

[0004] Methods for culturing cells using alginic acid microcarriers have been reported in the prior art. The existing technical solution is to pre-wet the alginic acid microcarriers and then inoculate the stem cells. The stem cells are allowed to adhere to the alginic acid microcarriers by standing for a long time, and then the cells are cultured and proliferated at a low stirring speed. However, the existing method can allow stem cells to adhere to alginic acid microcarriers and proliferate in a suspension environment in the reactor, but it cannot achieve efficient proliferation and harvesting of stem cells adhering to alginic acid microcarriers in a suspension environment in the reactor, which is mainly due to the following reasons:

[0005] (1) In the existing method, the general buffer used for alginic acid microcarriers is physiological saline, which does not have the function of modifying the surface of alginic acid microcarriers. However, the surface of alginic acid microcarriers carries a negative charge, and the surface of stem cells also carries a negative charge, which makes the adhesion efficiency of stem cells on alginic acid microcarriers low, resulting in slow proliferation of stem cells on alginic acid microcarriers.

[0006] (2) In the existing method, alginic acid microcarriers are in a state of stirring and standing during cell culture. The culture medium used for cells is generally a general flat culture medium, which contains animal-derived components, and there is a risk of batch-to-batch variation and pathogen contamination during culture. In addition, the rich carboxyl functional groups in alginic acid microcarriers consume cationic nutrients in the culture medium, resulting in low migration and proliferation efficiency of stem cells on alginic acid microcarriers.

[0007] Therefore, there is an urgent need in the art for a cell culture method that can improve the proliferation of cells in alginic acid microcarriers. SUMMARY

[0008] In view of the problems in the prior art, the present application provides the following technical solutions.

[0009] In a first aspect, the present application provides an alginate microcarrier modification buffer, wherein 100 mL of the modification buffer comprises: 1.9-2.1 mL of human platelet lysate, 300-1000 μg of calcium gluconate, 2-10 mg of zinc sulfate, 10-30 mg of potassium chloride, 1-3 mg of polylysine, 2-10 mg of magnesium sulfate, and 100-200 μg of human apolipoprotein.

[0010] In a second aspect, the present application provides a cell culture supplement, wherein 100 mL of the cell culture supplement comprises: 200-400 mg of dextrose, 2-6 mg of L-glutamine, 1.0-3.0 mg of L-leucine, 1.0-3.0 mg of L-lysine salt, 1.0-3.0 mg of L-valine, 20-30 μg of basic fibroblast growth factor, 100-200 μg of epidermal growth factor, 20-30 μg of insulin-like growth factor, 5-15 μg of platelet-derived growth factor, 20-30 μg of hepatocyte growth factor, 20-30 μg of transforming growth factor-β1, 1.0-3.0 mg of fibrin, 3.0-6.0 μg of ascorbic acid phosphate, and 1.0-3.0 μg of glutathione.

[0011] In a third aspect, the present application provides a complete culture medium for stem cell culture, which is prepared by mixing the cell culture supplement of the second aspect with a base medium at a volume ratio of 2:1-4:1.

[0012] In a fourth aspect, the present application provides a liquid combination comprising the alginate microcarrier modification buffer of the first aspect and the cell culture supplement of the second aspect in separate configurations.

[0013] In a fifth aspect, the present application provides a method for modifying alginate microcarriers, comprising modifying the alginate microcarriers using the alginate microcarrier modification buffer of the first aspect, wherein a suspension of alginate microcarriers is prepared by mixing 20-80 mL, for example 50 mL, of the alginate microcarrier modification buffer with 200 mg of alginate microcarriers, and then stirring the suspension of alginate microcarriers at room temperature for a period of time.

[0014] In a sixth aspect, the present application provides a method for culturing stem cells using alginate microcarriers, comprising: culturing stem cells using the complete culture medium of the third aspect in the presence of alginate microcarriers, wherein the alginate microcarriers are modified alginate microcarriers obtained by the method of the fifth aspect.

[0015] The beneficial effects of the present application are:

[0016] (1) The alginate microcarriers modified by the modification buffer provided by the present application have better cell affinity, and thus can be more effectively used in microcarrier large-scale expansion culture; and the aggregation of the alginate microcarriers modified in this way is greatly reduced, thereby increasing the effective adhesion area of cells on / in alginate microcarriers.

[0017] (2) The culture supplement provided by the present application can directly or indirectly increase the adhesion efficiency, uniformity, and growth and proliferation efficiency of stem cells when used in microcarrier large-scale expansion culture, thereby reducing the consumption of culture medium and other resources. And the culture supplement has no animal-derived components, thereby reducing the batch-to-batch variation and pathogen contamination risk in the culture process. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The cell state diagram under different culture methods and incubation times is shown. DETAILED DESCRIPTION

[0019] The following description is merely illustrative of the application and is not intended to limit the scope of the application. The scope of the application is defined by the appended claims. And those skilled in the art understand that modifications can be made to the technical solutions of the present application without departing from the spirit and essence of the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before the present application is described in detail, the following definitions are provided to better understand the present application.

[0021] In the case of providing a numerical range, such as a concentration range, a percentage range, or a ratio range, it should be understood that, unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of the range, to the tenth of the lower limit unit, and any other stated or intervening value within the range is included within the subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and such embodiments are also included within the subject matter, subject to any specific excluded limit values in the range. In the case where the range includes one or two limit values, the range excluding any one or both of those included limit values is also included in the subject matter.

[0022] In the context of the present application, many embodiments use the expressions "comprising", "including" or "consisting essentially of". The expressions "comprising", "including" or "consisting essentially of" can be understood in the broadest sense as open-ended expressions, meaning that, in addition to the elements, components, components, method steps specifically recited, other elements, components, components, method steps can also be included. In addition, in this document, the expressions "comprising", "including" or "consisting essentially of" can also be understood in some cases as closed expressions, meaning that only the elements, components, components, method steps specifically recited are included, and no other elements, components, components, method steps are included. At this point, the expression is equivalent to the expression "consisting of".

[0023] In order to better understand the present teachings and not to limit the scope of the present teachings, unless otherwise indicated, all numbers expressing quantities, percentages or ratios and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of significant digits and by applying ordinary rounding techniques.

[0024] In this document, alginate microcarriers refer to porous microcarriers prepared from alginate that can be used for three-dimensional cell culture.

[0025] In the first aspect, the present application provides an alginate microcarrier modification buffer, wherein 100 mL of the modification buffer comprises: 1.9-2.1 mL of human platelet lysate, 300-1000 μg of calcium gluconate, 2-10 mg of zinc sulfate, 10-30 mg of potassium chloride, 1-3 mg of polylysine, 2-10 mg of magnesium sulfate, and 100-200 μg of human apolipoprotein.

[0026] The modification buffer used in the present application adds alginate microcarrier adhesion promoting factors and charge modification factors compared to the general buffer in the prior art, so as to improve the adhesion efficiency and uniformity of cells during alginate microcarrier culture. More specifically, the human platelet lysate and human apolipoprotein in the modification buffer can promote cell adhesion, and calcium gluconate, zinc sulfate, potassium chloride, polylysine and magnesium sulfate can modify the surface charge of alginate microcarriers to reduce alginate microcarrier aggregation.

[0027] In one embodiment, wherein the modified buffer solution comprises: 2 mL human platelet lysate, 600 μg calcium gluconate, 8 mg zinc sulfate, 22 mg potassium chloride, 2 mg polylysine, 3 mg magnesium sulfate, 180 μg human apolipoprotein per 100 mL of the modified buffer solution.

[0028] In addition, the alginate microcarrier modified buffer solution can be prepared from normal saline with a concentration of sodium chloride of 0.85% to 0.9% or other suitable buffer solution for stem cell culture.

[0029] In a second aspect, the present application provides a cell culture supplement, wherein the cell culture supplement comprises: 200 to 400 mg dextrose, 2 to 6 mg L-glutamine, 1.0 to 3.0 mg L-leucine, 1.0 to 3.0 mg L-lysine salt, 1.0 to 3.0 mg L-valine, 20 to 30 μg basic fibroblast growth factor, 100 to 200 μg epidermal growth factor, 20 to 30 μg insulin-like growth factor, 5 to 15 μg platelet-derived growth factor, 20 to 30 μg hepatocyte growth factor, 20 to 30 μg transforming growth factor-β1, 1.0 to 3.0 mg fibrin, 3.0 to 6.0 μg ascorbic acid phosphate, 1.0 to 3.0 μg glutathione per 100 mL of the cell culture supplement.

[0030] The culture supplement provided by the present application can increase the proliferation efficiency of cells in the alginate microcarrier suspension culture environment, and has no animal-derived ingredients, thereby reducing the batch-to-batch variation and pathogen contamination risk in the culture process. More specifically, the dextrose, L-glutamine, L-leucine, L-lysine salt, L-valine of the culture supplement can supplement the nutritional ingredients consumed by the culture medium, the basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, hepatocyte growth factor, transforming growth factor-β1 and fibrin can promote cell proliferation and migration, and ascorbic acid phosphate and glutathione can slow down cell aging.

[0031] In one embodiment, wherein the cell culture supplement comprises: 350 mg dextrose, 2.5 mg L-glutamine, 1.5 mg L-leucine, 1.5 mg L-lysine hydrochloride, 1.5 mg L-valine, 25 μg basic fibroblast growth factor, 150 μg epidermal growth factor, 25 μg insulin-like growth factor, 10 μg platelet-derived growth factor, 25 μg hepatocyte growth factor, 26 μg transforming growth factor-β1, 3 mg fibrin, 3.5 μg ascorbic acid phosphate, 1.1 μg glutathione per 100 mL of the cell culture supplement.

[0032] The cell culture supplement can be prepared from PBS buffer or other suitable buffer for stem cell culture.

[0033] In a third aspect, the present application provides a complete medium for stem cell culture, which is prepared from the base medium and the cell culture supplement of the second aspect at a volume ratio of 2: 1-4: 1.

[0034] In one embodiment, the base medium is a basal Eagle's medium, a DMEM cell culture medium, or a 1640 medium.

[0035] In a preferred embodiment, the complete medium is prepared from the base medium and the cell culture supplement of the second aspect at a volume ratio of 4: 1.

[0036] In a fourth aspect, the present application provides a liquid combination comprising the alginate microcarrier modification buffer of the first aspect and the cell culture supplement of the second aspect separately configured.

[0037] In a fifth aspect, the present application provides a method for modifying alginate microcarriers, comprising modifying the alginate microcarriers using the alginate microcarrier modification buffer of the first aspect, wherein an alginate microcarrier suspension is prepared by 20-80 mL, for example 50 mL, of the alginate microcarrier modification buffer per 200 mg of alginate microcarriers, and then the alginate microcarrier suspension is stirred at room temperature for a period of time.

[0038] After stirring, the alginate microcarrier suspension can be allowed to stand to precipitate the alginate microcarriers, and then the modification buffer is removed to obtain the modified alginate microcarriers.

[0039] In a preferred embodiment, the alginate microcarrier suspension is stirred at 40-100 rpm, for example 60 rpm, at room temperature for 24 hours, and then allowed to stand for at least 15 minutes to completely precipitate the alginate microcarriers, and then the modification buffer is removed to obtain the modified alginate microcarriers.

[0040] In a sixth aspect, the present application provides a method for culturing stem cells using alginate microcarriers, comprising culturing stem cells using the complete medium of the third aspect in the presence of alginate microcarriers, which are modified alginate microcarriers obtained by the method of the fifth aspect.

[0041] In one embodiment, the stem cells are subjected to variable speed and cycle agitation culture, which is agitation at 25-35 rpm (e.g. 30 rpm) for 3-5 minutes (e.g. 4 minutes), followed by 45-55 minutes (e.g. 50 minutes) of resting, which is repeated for 22-26 hours (e.g. 23 hours, 24 hours or 25 hours), followed by constant speed agitation at 40-50 rpm (e.g. 45 rpm) until the end of the culture.

[0042] In one preferred embodiment, the modified alginate microcarriers are resuspended at a ratio of 50-60 mL (e.g. 50 mL) complete medium per 200 mg of modified alginate microcarriers, and then inoculated with a seeding amount of 1.40 x 10 6 -1.60 x 10 6 6 cells per 200 mg of alginate microcarriers.

[0043] In one preferred embodiment, the culture is supplemented with the culture supplement at a ratio of 50-60 mL (e.g. 50 mL) complete medium per 200 mg of modified alginate microcarriers to the culture reactor after 48 hours of culture.

[0044] In one embodiment, the cell culture method further comprises a microcarrier lysis step using an alginate microcarrier lysis solution comprising 1-5 mM EDTA 2Na and 0.1-0.3% trypsin by volume.

[0045] In one preferred embodiment, the microcarrier lysis step specifically comprises centrifuging the culture product and resuspending with physiological saline, followed by the addition of the microcarrier lysis solution, and then agitation at 40-60 rpm at 37°C for at least 10 minutes for microcarrier lysis treatment.

[0046] In one preferred embodiment, the culture conditions are 37°C and 5% carbon dioxide concentration.

[0047] In one embodiment, the stem cells are embryonic stem cells or adult stem cells.

[0048] In one preferred embodiment, the stem cells are mesenchymal stem cells, neural stem cells, hematopoietic stem cells, cardiac stem cells, adipose stem cells.

[0049] In one more preferred embodiment, the stem cells are adipose mesenchymal stem cells, skin mesenchymal stem cells, bone marrow mesenchymal stem cells or dental pulp mesenchymal stem cells.

[0050] ​In the present text, the stem cells can be commercially available or derived from a human embryo that has not undergone in vivo development for up to 14 days after fertilization or from a stem cell that has been edited.

[0051] Examples

[0052] Embodiments of the present application will be described in detail below with reference to examples. Those skilled in the art will appreciate that the following examples are illustrative only and should not be viewed as limiting the scope of the present application. Unless specific techniques or conditions are mentioned in the examples, techniques or conditions described in the literature or according to the product manual are used. Unless the manufacturer is specified, all reagents or instruments used are conventional products that can be commercially available.

[0053] Materials and methods

[0054] The main reagents and materials used in the examples and their sources are as follows:

[0055] DMEM medium, L-leucine, L-lysine hydrochloride, L-valine, PBS, trypsin, basic fibroblast growth factor, inner epidermal growth factor, insulin-like growth factor, hepatocyte growth factor, platelet-derived growth factor, purchased from Gibco company;

[0056] Transforming growth factor-β1, purchased from AbMole company;

[0057] Fetal bovine serum, EDTA·2Na, human apolipoprotein, fibrin, dextrose, calcium gluconate, zinc sulfate, magnesium sulfate, potassium chloride, polylysine and ascorbic acid phosphate, purchased from Sigma company;

[0058] Glutathione, purchased from Thermo company;

[0059] Platelet lysate, purchased from Sartorius company;

[0060] Physiological saline, purchased from Kelun Pharmaceutical;

[0061] 125 mL stirring suspension culture reactor, purchased from Huageng Biological;

[0062] 50 mL centrifuge tube, T175 culture bottle, pipette, 500 mL 0.22 μm filter bottle, purchased from Corning company;

[0063] Human mesenchymal stem cell analysis kit, purchased from BD company;

[0064] Flow analyzer, flow analysis software, purchased from Agilent company;

[0065] Alginic acid microcarrier, provided by Dalian University of Technology.

[0066] The alginate microcarrier modification buffer solution is prepared as follows: 2 mL of human platelet lysate, 600 μg of calcium gluconate, 8 mg of zinc sulfate, 22 mg of potassium chloride, 2 mg of polylysine, 3 mg of magnesium sulfate, and 180 μg of human apolipoprotein are added to 98 mL of normal saline.

[0067] The cell culture supplement solution is prepared as follows: 10 mL of deionized water, 350 mg of dextrose, 2.5 mg of L-glutamine, 1.5 mg of L-leucine, 1.5 mg of L-lysine hydrochloride, 1.5 mg of L-valine, 25 μg of basic fibroblast growth factor, 150 μg of internal epidermal growth factor, 25 μg of insulin-like growth factor, 10 μg of platelet-derived growth factor, 25 μg of hepatocyte growth factor, 26 μg of transforming growth factor-β1, 3 mg of fibrin, 3.5 μg of ascorbic acid phosphate, and 1.1 μg of glutathione are added to 90 mL of PBS.

[0068] The general buffer solution is normal saline.

[0069] The general culture medium is prepared as follows: 100 mL of fetal bovine serum is added to 900 mL of basal medium.

[0070] The alginate microcarrier mesenchymal stem cell complete culture medium (hereinafter referred to as the complete culture medium) is prepared as follows: the basal medium and the culture supplement solution are mixed at a volume ratio of 4:1.

[0071] The alginate microcarrier lysate is prepared as follows: trypsin is diluted to a volume fraction of 0.125% with normal saline, and 0.5 M EDTA·2Na is dissolved in the diluted trypsin to prepare the alginate microcarrier lysate.

[0072] The reagents are sterilized by filtering them through a 500 mL 0.22 μm filter bottle. After sterilization, the reagents are ready for use.

[0073] The mesenchymal stem cell sample is obtained from the same line of mesenchymal stem cells, and the umbilical cord sample from which the mesenchymal stem cells are derived is provided by Shenzhen People's Hospital. The line is established in a traditional flat culture medium system using the adherent method, and is passaged and expanded to the P1 passage, then harvested and stored in a gas-phase liquid nitrogen tank.

[0074] Example 1

[0075] 500 mL of the complete culture medium is prepared as described above, and then the cell culture is performed by the following steps:

[0076] 1. Take 200 mg alginate microcarrier and 50 mL of modified buffer to make alginate microcarrier suspension and add to 125 mL stirred suspension culture flask.

[0077] 2. Put the flask into 25 °C environment with 60 rpm stirring for 24 hours, then let it stand for 15 minutes to make alginate microcarrier completely precipitate, then remove the modified buffer for standby.

[0078] 3. Take the frozen tube containing mesenchymal stem cells out of the gas phase liquid nitrogen tank and put it into a 37 °C water bath for 2 minutes to thaw and recover.

[0079] 4. Collect the thawed and recovered mesenchymal stem cell suspension into a centrifuge tube, add 30 mL of normal saline, centrifuge at 500 g for 5 minutes, discard the supernatant after centrifugation, and wash twice.

[0080] 5. Resuspend the obtained mesenchymal stem cells with complete medium, then take a sample to detect the viable cell density of the mesenchymal stem cell suspension.

[0081] 6. Seed the cells into a T175 culture flask at a density of 6000 viable cells per square centimeter, then add complete medium, and cultivate in a 37 °C and 5% carbon dioxide concentration incubator until the confluence reaches 80%.

[0082] 7. Perform harvest digestion with 0.125% trypsin, and terminate digestion with 2 times the volume of trypsin with complete medium.

[0083] 8. Collect the mesenchymal stem cell suspension and wash it twice with normal saline, then resuspend it with complete medium, and then take a sample to detect the viable cell density of the mesenchymal stem cell suspension.

[0084] 9. Take 50 mL of base medium to resuspend the modified alginate microcarrier, then let it stand for 15 minutes until the alginate microcarrier completely precipitates, and remove the supernatant.

[0085] 10. Take 50 mL of complete medium to resuspend the washed alginate microcarrier.

[0086] 11. Seed 1,500,000 mesenchymal stem cells into a stirred suspension culture flask containing alginate microcarriers resuspended with complete medium, and put it into a 37 °C and 5% carbon dioxide concentration incubator.

[0087] 12. Stir at 35 rpm for 5 minutes, then stand for 45 minutes, cycle the stirring and standing steps, repeat for 24 times, then continuously stir at 40 rpm until the end of cultivation.

[0088] 13. After 48 hours of cultivation, supplement 50 mL of the culture supplement to the stirred suspension culture flask.

[0089] 14. After the culture is completed, centrifuge the culture-treated product at 500g for 5 minutes to separate the centrifugal substrate and the centrifugal supernatant; resuspend the centrifugal substrate with physiological saline and add the microcarrier lysis solution, the volume ratio of the resuspension solution to the microcarrier lysis solution being 1:1; perform the microcarrier lysis treatment by stirring at 40 rpm at 37°C for 10 minutes; terminate the microcarrier lysis with the centrifugal supernatant, the volume ratio of the centrifugal supernatant to the microcarrier lysis solution being 1:1; centrifuge the digested cell suspension at 500g and collect the cells, resuspend the cells with physiological saline, and then sample to detect the viable cell density and surface markers. The viable cell density is detected using a cell counter, and the human mesenchymal stem cell surface markers are detected using flow cytometry.

[0090] Example 2

[0091] Prepare 500 ml of complete medium as described above, and then perform mesenchymal stem cell culture in the same cell culture steps as in Example 1, except that the general buffer is used instead of the modified buffer.

[0092] Example 3

[0093] Prepare 500 ml of general medium as described above, and then perform mesenchymal stem cell culture in the same cell culture steps as in Example 1, except that the general medium is used instead of the complete medium.

[0094] Example 4

[0095] Prepare 500 ml of general medium as described above, and then perform mesenchymal stem cell culture in the same cell culture steps as in Example 1, except that the general buffer is used instead of the modified buffer and the general medium is used instead of the complete medium.

[0096] Experimental results

[0097] The experimental results of each example are shown in Figure 1 , Table 1 and Table 2.

[0098] Figure 1 is a cell state diagram at different times of incubation using different culture methods, from which it can be seen that the use of the modified buffer and the culture supplement of the present application alone (Examples 2 and 3) can improve the adhesion and proliferation of cells on the alginate microcarriers compared to the use of the general buffer and the general medium (Example 4), and the simultaneous use of the modified buffer and the culture supplement of the present application (Example 1) can more obviously improve the adhesion and proliferation of cells.

[0099] Table 1 shows the expansion fold and the viable cell rate of the cells after different culture methods, wherein the expansion fold is the ratio of the total viable cell number obtained after culture to the inoculation amount, and the viable cell rate is the ratio of the total viable cell number obtained after culture to the total cell number. As can be seen from Table 1, the use of the modified buffer or the culture supplement of the present application alone (Examples 2 and 3) can improve the expansion fold of the cells on the alginate microcarrier compared to the use of the general buffer and the general medium (Example 4), but has no significant effect on the viable cell rate, while the use of the modified buffer and the culture supplement of the present application simultaneously can significantly improve the expansion fold and the viable cell rate of the cells.

[0100] Table 2 is the flow cytometry analysis results of the cell markers after different culture methods, wherein the target cells are the cells positive for CD90, CD73 and CD105 and negative for CD34, CD11b, CD19, CD45 and HLA-DR, i.e. the undifferentiated mesenchymal stem cells. In the flow cytometry analysis, the cell population within the event gate in the FSC-H and SSC-H cell scatter diagram meets the positive expression rate greater than 95% and the negative expression rate less than 2% as the target cell population, i.e. the undifferentiated mesenchymal stem cell population. The higher the proportion of the target cell population, i.e. the more mesenchymal stem cells in the detected cells; the higher the positive expression rate and the lower the negative expression rate of the target cell population, i.e. the fewer impurities in the target cell population. The positive expression rates of the markers in the target cell population are listed in Table 2, which indicate whether the positive expression of the sorted cells is qualified. As can be seen from Table 2, the use of the modified buffer and the culture supplement of the present application (Example 1) can increase the undifferentiated target cell population of the cultured cells, and compared to the use of the general medium, can make the cells proliferate stably and reduce the occurrence of cell differentiation or cell apoptosis.

[0101] The above results show that the modified buffer and the culture supplement of the present application can effectively improve the cell culture results, and have a synergistic effect, and the use of both can effectively improve the expansion fold and the viable cell rate of the cells, and reduce the occurrence of cell differentiation or cell apoptosis.

[0102] Table 1

[0103]

[0104]

[0105] Table 2

[0106]

Claims

1. An alginate microcarrier modification buffer, wherein each 100 mL of the modification buffer comprises: 1.9–2.1 mL of human platelet lysis buffer, 300–1000 μg of calcium gluconate, 2–10 mg of zinc sulfate, 10–30 mg of potassium chloride, 1–3 mg of polylysine, 2–10 mg of magnesium sulfate, and 100–200 μg of human apolipoprotein.

2. The alginate microcarrier modification buffer according to claim 1, wherein each 100 mL of the modification buffer comprises: 2 mL of human platelet lysis buffer, 600 μg of calcium gluconate, 8 mg of zinc sulfate, 22 mg of potassium chloride, 2 mg of polylysine, 3 mg of magnesium sulfate, and 180 μg of human apolipoprotein.

3. A cell culture supplement, wherein each 100 mL of the cell culture supplement comprises: 200–400 mg dextran glucose, 2–6 mg L-glutamine, 1.0–3.0 mg L-leucine, 1.0–3.0 mg L-lysine, 1.0–3.0 mg L-valine, 20–30 μg basic fibroblast growth factor, 100–200 μg endothelial growth factor, 20–30 μg insulin-like growth factor, 5–15 μg platelet-derived growth factor, 20–30 μg hepatocyte growth factor, 20–30 μg transforming growth factor-β1, 1.0–3.0 mg fibrin, 3.0–6.0 μg ascorbate phosphate, and 1.0–3.0 μg glutathione.

4. The cell culture supplement according to claim 3, wherein each 100 mL of the cell culture supplement comprises: 350 mg dextran glucose, 2.5 mg L-glutamine, 1.5 mg L-leucine, 1.5 mg L-lysine hydrochloride, 1.5 mg L-valine, 25 μg basic fibroblast growth factor, 150 μg endothelial growth factor, 25 μg insulin-like growth factor, 10 μg platelet-derived growth factor, 25 μg hepatocyte growth factor, 26 μg transforming growth factor-β1, 3 mg fibrin, 3.5 μg ascorbate phosphate, and 1.1 μg glutathione.

5. A complete culture medium for stem cell culture, comprising a basal culture medium and a cell culture supplement according to claim 3 or 4 in a volume ratio of 2:1 to 4:

1.

6. The complete culture medium according to claim 5, wherein the basal culture medium is basic Eagle medium, DMEM cell medium, or 1640 medium.

7. A liquid combination comprising separately configured alginate microcarrier modification buffer according to claim 1 or 2 and cell culture supplement according to claim 3 or 4.

8. A method for modifying alginate microcarriers, comprising: The alginate microcarriers are modified using the alginate microcarrier modification buffer according to claim 1 or 2, wherein an alginate microcarrier suspension is prepared by using 20-80 mL, for example, 50 mL, of the alginate microcarrier modification buffer per 200 mg of alginate microcarrier, and then the alginate microcarrier suspension is stirred at room temperature for a period of time.

9. A method for culturing stem cells using alginate microcarriers, comprising: Stem cells were cultured in the presence of alginate microcarriers using the complete culture medium according to claim 5, wherein the alginate microcarriers are alginate microcarriers modified by the method of claim 8.

10. The method according to claim 9, wherein the stem cells are subjected to variable speed circulating stirring culture, wherein the variable speed circulating stirring culture is performed by stirring at 25-35 rpm for 3-5 minutes, then letting stand for 45-55 minutes, and the above process is repeated for 22-26 hours, for example 24 hours, and then stirring at a constant speed of 40-50 rpm, for example 40 rpm, until the culture is completed.

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