CN110872572B - Method for improving in-vitro suspension culture efficiency of human multifunctional stem cells - Google Patents

Abstract

The invention belongs to the technical field of cell culture, and discloses a method for improving the efficiency of in-vitro suspension culture of human multifunctional stem cells. The method for improving the in vitro suspension culture efficiency of the human pluripotent stem cells comprises the following steps: carrying out three-dimensional culture on the human pluripotent stem cells by using a culture medium containing polyvinyl alcohol or dextran sulfate and polyvinyl alcohol; the concentration of the dextran sulfate in the culture medium is 10-100 ug/ml; the concentration of the polyvinyl alcohol in the culture medium is 0.1-1 mg/ml. The method of the invention not only can reduce the size of the cell aggregate and obtain the aggregate with uniform size, but also can reach higher cell number to meet the requirement of in vitro amplification of the human pluripotent stem cells, thereby improving the culture efficiency of the human pluripotent stem cells.

Description

A method for improving the efficiency of suspension culture of human pluripotent stem cells in vitro

technical field

The invention belongs to the technical field of cell culture, and in particular relates to a method for improving the suspension culture efficiency of human pluripotent stem cells in vitro.

Background technique

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively referred to as human pluripotent stem cells (hPSCs), have great prospects in cell therapy, tissue engineering and regenerative medicine, as well as drug discovery and biotechnology applications. The traditional two-dimensional (2D) culture technology using plastic culture plates and heterologous medium for cell expansion has limited efficiency in expanding cells, and after long-term passage and expansion, cells will lose their stemness and differentiation ability. In order to obtain high-quantity and high-quality human pluripotent stem cells, the large-scale expansion of human pluripotent stem cells was carried out using a bioreactor-based three-dimensional environment (3D) culture technology that mimics the organism. At present, the strategy of expanding in the form of cell aggregates after seeding as single cells in a bioreactor has the advantages of cost saving and easy cell recovery compared with the microcarrier culture strategy, but this strategy forms a larger size and Agglomerates of uneven size. Due to the large diameter of large aggregates (>300μm), it is difficult for nutrients and oxygen to diffuse to the center of the aggregates, resulting in cell differentiation or death.

Aggregate size control is a key parameter in the strategy of culturing cells in the form of aggregates. The aggregate size can be controlled by physical methods such as controlling shear force and chemical methods such as adding small molecules to obtain more uniform cell aggregate products. Polysulfate compounds are widely used in the biopharmaceutical industry. They can not only reduce cell aggregation by regulating the charge on the cell surface, but also have anti-apoptotic effects on cells. At the same time, it has been reported that dextran sulfate (DS) can reduce the cell aggregation of hPSCs without reducing their pluripotency. Lipsitz YY et al [1Lipsitz YY, Tonge PD, ZandstraPW.Chemically controlled aggregation of pluripotent stem cells.BiotechnolBioeng 2018; 115(8):2061-6.] reported that suspension culture cells under the conditions of 40000kDA (D40) and 100ug/mlDS can The hPSCs aggregates are kept small and uniform in size for expansion, and the pluripotency and karyotype remain stable. Nogueira et al [Nogueira, DES, Rodrigues, CAV, Carvalho, MS et al. Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel ^TM bioreactors. J Biol Eng 2019; 13, 74.] reported the use of The disposable vertical roller reactor was combined with 100ug/mlDS to obtain (2.3±0.2)×10 ⁶ cells/mL cells and harvested cell aggregates with an average diameter of 346±11 μm, and their pluripotency and karyotype remained stable. Although the use of DS can reduce cell aggregation by adjusting the charge on the cell surface, which is conducive to obtaining high-quality small cell aggregates with uniform size and recovery, but the cell expansion factor has not been improved, that is, the number of cells still does not meet the requirements.

The present invention uses DS (dextran sulfate) in combination with PVA, improves the efficiency of stem cell suspension culture in vitro, controls the size of cell aggregates in the expansion strategy in the form of cell aggregates and improves the expansion of cells Multiples, the pluripotency of the recovered cells can be maintained to meet the needs of in vitro expansion. The combined use of PVA and DS can not only obtain aggregates with uniform size, but also achieve high cell numbers to meet clinical needs.

Contents of the invention

In view of the deficiencies of the above technologies, the purpose of the present invention is to provide a method for improving the efficiency of suspension culture of human pluripotent stem cells in vitro. The use of PVA in the present invention can promote the proliferation of hPSCs, and further use of DS (dextran sulfate) in combination with PVA can not only obtain aggregates with uniform size, but also achieve a higher number of cells, improving the stem cell Efficiency of in vitro suspension culture. The method of the present invention enables large-scale in vitro suspension culture of hPSCs.

The object of the present invention is achieved through the following technical solutions:

A method for improving the efficiency of suspension culture of human pluripotent stem cells in vitro, comprising the steps of: substituting human pluripotent stem cells with a culture medium containing PVA (polyvinyl alcohol) or DS (dextran sulfate) and PVA (polyvinyl alcohol) Perform three-dimensional culture.

The concentration of the DS (dextran sulfate) in the culture medium is 10-100ug/ml, and its molecular weight is 40000kDA (D40);

The concentration of the PVA in the culture medium is 0.1-1 mg/ml.

When the medium containing DS (dextran sulfate) and PVA (polyvinyl alcohol) is selected for three-dimensional culture, the concentration of DS (dextran sulfate) in the medium is preferably 100ug/ml, and the concentration of PVA in the medium is The concentration in is preferably 1 mg/ml.

The medium is a serum-free complete medium, preferably mTeSR1 medium.

Y-27632 (Rocki) was also added to the medium.

After 48 hours of three-dimensional culture, the medium containing PVA was used to change the medium, and the medium was changed once a day at 60% of the volume.

The method for improving the efficiency of in vitro suspension culture of human pluripotent stem cells specifically comprises the following steps:

S1 single-cell passaging of human pluripotent stem cells (hPSC): After hPSC passaging, select cells with a coverage rate of 70%-80%, clone cells with regular edges and no differentiation, and make them into a single-cell suspension;

S2 cell inoculation: add culture medium, Y-27632 (Rocki) successively in culture device, then add final concentration is 0.1～1mg/ml PVA or final concentration 10-100ug/mLDS and O.1～1mg/ml PVA, will The single cell suspension is inoculated into the culture device, and the culture device is shaken to suspend the cells evenly in the culture medium;

Change the medium of S3 cells: after culturing for 48 hours, replace the medium with PVA-containing medium, and change the medium once a day at 60% of the volume;

S4 Harvest the cells: culture for 5-6 days, and collect the cells.

The culture device described in step S2 is a low-adhesion culture device, such as: a commercial low-adhesion orifice plate, a low-adhesion orifice plate prepared by a conventional method: the adhesion orifice plate prepared by the conventional method Specific steps: using Dilute Pluronic-F68 in DMEM/F12 medium to obtain diluted Pluronic-F68 (DMEM/F12:Pluronic-F68=1:1 (volume ratio)); spread the diluted Pluronic-F68 in the well plate, place the well plate Incubate in a CO ₂ incubator.

The specific preparation steps of the single-cell suspension described in step S1: when hPSCs are subcultured, select culture wells with a coverage rate of 70%-80%, regular clone edges and no differentiated cells, and add 1 mL of calcium and magnesium-free PBS after aspirating and discarding the medium Wash the cells, add 1mL GCDR (cell dissociation reagent) after aspiration, put it back into the CO ₂ incubator and incubate for 5-10 minutes, then aspirate and discard the GCDR in the well, add 1mL mTeSR1 medium, blow the cells down and use a pipette Blow into a single cell suspension.

The single cell suspension in step S2 is a cell suspension of 500,000 single cells. The medium described in step S2 is a serum-free complete medium, preferably mTeSR1 medium.

The amount of Y-27632 (Rocki) added was 10uM.

The medium described in step S3 is a serum-free complete medium, preferably mTeSR1 medium.

The final concentration of PVA in the medium containing PVA is 0.1-1 mg/ml.

After the culture in step S4 is completed, centrifuge, discard the supernatant, add PBS to wash the cell aggregates, centrifuge, discard the supernatant, add trypsin, digest at room temperature for 9-12 minutes, add serum-containing medium to terminate the digestion, and obtain cells. The condition of the centrifugation is 1500 rpm for 5 minutes; the trypsin is 0.25% trypsin.

The present invention combines low-concentration PVA with 40000kDA (D40) and 100ug/ml DS to culture hPSCs in vitro. The combined use of the two can improve the efficiency of cell expansion, so that the harvested cell aggregates remain small and uniform Size, the quality of this cell aggregate is high, the center of the aggregate will not lack nutrients and oxygen due to its large size, and the recovered aggregate is easy to digest into single cells for passage or clinical use.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The existing technology uses DS to reduce cell aggregation by adjusting the charge on the cell surface, and obtain high-quality small cell aggregates with uniform size and easy recovery. However, the cell expansion factor of this technology has not been improved, that is, the number of cells still does not meet the requirements. . However, the present invention uses low-concentration PVA, 40000kDA (D40), and 100ug/ml DS, which can not only reduce the size of cell aggregates and obtain aggregates with uniform size, but also achieve a higher number of cells to meet the needs of in vitro amplification. Functional Stem Cell Requirements.

Description of drawings

Figure 1 is a characterization diagram of cells obtained in 3D suspension culture + 100ug/mL DS + 1mg/mL PVA in Example 1; A is the image of aggregates after 5 days of culture, B is the comparison chart of the total number of cells after 5 days of culture, and C is the image of the total number of cells after 5 days of culture A comparison chart of the average diameter of aggregates in one day, D is a statistical comparison chart of the distribution interval of aggregates after 5 days of cultivation;

Figure 2 is a characterization diagram of cells obtained in 3D suspension culture + 100ug/mL DS + 0.1mg/mL PVA in Example 2; A is the image of aggregates after 5 days of culture, B is the comparison chart of the total number of cells after 5 days of culture, and C is the image of the culture The comparison chart of the average diameter of the aggregates on the 5th day, D is the statistical comparison chart of the distribution interval of the aggregates after the 5th day of cultivation;

Figure 3 is a characterization diagram of cells obtained in 3D suspension culture + 10ug/mL DS + 1mg/mL PVA in Example 3; A is the image of aggregates after 5 days of culture, B is the comparison of the total number of cells after 5 days of culture, and C is the image of the total number of cells after 5 days of culture A comparison chart of the average diameter of aggregates in one day, D is a statistical comparison chart of the distribution interval of aggregates after 5 days of cultivation;

Figure 4 is a characterization diagram of cells obtained by 3D suspension culture hPSC+dextran sulfate in Comparative Example 1. The control group did not add DS, the experimental group 1 added 10ug/mL DS, and the experimental group 2 added 100ug/mL DS; A is the culture Aggregate images at 5 days, B is the comparison chart of the total number of cells at 5 days of culture, C is the comparison chart of the average diameter of aggregates at 5 days of culture, D is the statistical comparison chart of the distribution interval of aggregates at 5 days of culture; E is the qPCR detection stem of 5 days of culture Comparison chart of sex gene expression results;

Figure 5 is a characterization diagram of cells obtained by 3D suspension culture hPSC+polyvinyl alcohol (PVA) in Comparative Example 2. The control group did not add PVA, and the experimental group added 1 mg/mL PVA; A is the image of aggregates after 5 days of culture, and B is the image of culture The comparison chart of the total number of cells at 5 days, C is the comparison chart of the average diameter of aggregates at 5 days of culture, D is the statistical comparison chart of the distribution interval of aggregates at 5 days of culture; E is the comparison chart of the expression results of qPCR detection of stemness genes at 5 days of culture.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto. Dextran sulfate DS: Manufacturer: SIGMA, Mr (relative molecular mass) ~ 10000, use concentration: 100ug/ml storage concentration: 100mg/ml, that is, 500mg DS is fully dissolved in 5ml sterile water and stored at 4 degrees. DS was added to the medium when the cells were inoculated, and 60% of the medium was replaced when the cells were cultured for 48 hours. The replaced medium did not need to be added with the concentration of DS, that is, DS only acted for 48 hours.

PVA: Manufacturer: ALDRICH, Mw 31000-500000, degree of hydrolysis 87%-89%.

Use concentration: 0.1mg/ml, 0.5mg/ml, 1mg/ml. Storage concentration: 100mg/ml, that is, 1g of PVA is fully dissolved in 1ml of sterile water and stored at room temperature. Low-concentration PVA (0.1-1mg/ml) is added in the culture medium when cells are inoculated, and 60% of the culture medium is replaced when the cells are cultivated for 48 hours.

Experimental materials: six-well plate; mTeSR1; GCDR; PBS; trypan blue; Y-27632; 0.25% trypsin; Pluronic-F68; PVA; DS.

Example 1: 3D suspension culture+100ug/mL DS+1mg/mL PVA

A method for improving the efficiency of suspension culture of human pluripotent stem cells in vitro, comprising the following steps:

S1. Pre-spread Pluronic-F68 to prepare low-adhesion wells: pre-spread diluted Pluronic-F68 (DMEM/F12:Pluronic-F68=1:1) in a six-well plate at a volume of 2 mL/well, and place the well plate Return to the CO ₂ incubator and incubate for more than 1 hour;

S2. Single-cell passaging hPSC: select one well of hPSC in good condition (good condition: 4-5 days after hPSC passaging, the cell coverage rate reaches about 70%-80%, and the hPSC with regular edges and no differentiated cells is cloned), and suck After discarding the medium, add 1mL calcium and magnesium-free PBS to wash the cells, add 1mL GCDR after aspirating and discarding, put it back in the _CO2 incubator and incubate for 5-10 minutes, then aspirate and discard the GCDR in the well, add 1mL mTeSR1 medium, blow the cells down And use a pipette to blow into a single cell suspension;

S3. Cell inoculation: draw 20ul of single cell suspension, stain with trypan blue and count with a hemocytometer; take out the pre-coated Pluronic-F68 well plate, discard the Pluronic-F68 dilution, add 5mL mTeSR1 and add 10uM Y-27632 (Rocki) to improve the survival rate of single cell inoculation, in addition, add DS solution with a final concentration of 100ug/mL + 1mg/mL PVA solution in the well; draw the cell suspension containing 500,000 single cells and inoculate into the well , shake the well plate crosswise to suspend the cells evenly in the culture medium;

S4. Cell fluid replacement: After 48 hours, 60% of the fluid per well was changed every day, that is, 3 mL of mTeSR1 medium and PVA solution (1 mg/mL) of corresponding concentration was added, and DS solution was no longer added to the medium;

S5. Cell harvesting: when the culture reached the fifth day, the aggregates were photographed, and the diameter of the aggregates was counted; after that, the cell aggregates were collected, centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded, then 1 mL of PBS was added to wash the cell aggregates, and centrifuged at 1500rpm for 5 minutes. After discarding the supernatant, add 1 mL of 0.25% trypsin, digest at room temperature for 10 minutes, then add 1 mL of serum-containing medium to stop the digestion, and count with a hemocytometer after staining with trypan blue; sample 3 times from each well and count 3 times, and the final total number of cells is taken as average.

The experimental results are shown in Figure 1. In Figure 1, no PVA+DS was added to the control group, and 100ug/mL DS solution+1mg/mL PVA was added to the experimental group. Figure 1 is the characterization diagram of cells obtained in Example 1, 3D suspension culture + 100ug/mL DS + 1mg/mL PVA; A is the image of aggregates after 5 days of culture, B is the comparison chart of the total number of cells after 5 days of culture, and C is the image of the culture The comparison chart of the average diameter of the aggregates on the 5th day, D is the statistical comparison chart of the distribution interval of the aggregates after the 5th day of culture.

Example 2: 3D suspension culture+100ug/mL DS+0.1mg/mL PVA

A method for improving the efficiency of suspension culture of human pluripotent stem cells in vitro, comprising the following steps:

S1. Pre-spread Pluronic-F68 to prepare low-adhesion wells: pre-spread diluted Pluronic-F68 (DMEM/F12:Pluronic-F68=1:1) in a six-well plate at a volume of 2 mL/well, and place the well plate Return to the CO2 incubator and incubate for more than 1 hour;

S2. Single-cell passaging hPSC: Select a well of hPSC in good condition, add 1mL calcium and magnesium-free PBS to wash the cells after aspirating and discarding, add 1mL GCDR after aspirating and discarding, put it back into the CO2 incubator and incubate for 5-10 minutes, then aspirate Discard the GCDR in the well, add 1mL mTeSR1 medium, blow down the cells and blow them into a single-cell suspension with a pipette;

S3. Cell inoculation: draw 20ul of single cell suspension, stain with trypan blue and count with a hemocytometer; take out the pre-coated Pluronic-F68 well plate, discard the Pluronic-F68 dilution, add 5mL mTeSR1 and add 10uM Y-27632 (Rocki) to improve the survival rate of single cell inoculation, in addition, add DS solution + 0.1 mg/mL PVA solution with a final concentration of 100ug/mL in the well; draw the cell suspension containing 500,000 single cells and inoculate into well, shake the well plate criss-cross to make the cells evenly suspended in the culture medium;

S4. Cell medium change: at 48 hours, 60% of the medium was changed per well per day, that is, 3mL mTeSR1 medium and PVA solution of corresponding concentration were added, and DS solution was no longer added to the medium;

S5. Cell harvesting: when the culture reached the fifth day, the aggregates were photographed, and the diameter of the aggregates was counted; after that, the cell aggregates were collected, centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded, then 1 mL of PBS was added to wash the cell aggregates, and centrifuged at 1500rpm for 5 minutes. After discarding the supernatant, add 1 mL of 0.25% trypsin, digest at room temperature for 10 minutes, then add 1 mL of serum-containing medium to stop the digestion, and count with a hemocytometer after staining with trypan blue; sample 3 times from each well and count 3 times, and the final total number of cells is taken as average.

The experimental results are shown in Figure 2. The control group did not add PVA+DS, and the experimental group added 100ug/mL DS solution+0.1mg/mLPVA. Figure 2 is a characterization diagram of cells obtained in Example 2, 3D suspension culture + 100ug/mL DS + 0.1mg/mL PVA; A is the image of aggregates after 5 days of culture, B is the comparison chart of the total number of cells after 5 days of culture, and C is A comparison chart of the average diameter of aggregates after 5 days of culture, and D is a statistical comparison chart of the distribution interval of aggregates after 5 days of culture.

Example 3: 3D suspension culture+10ug/mL DS+1mg/mL PVA

A method for improving the efficiency of suspension culture of human pluripotent stem cells in vitro, comprising the following steps:

S1. Pre-spread Pluronic-F68 to prepare low-adhesion wells: pre-spread diluted Pluronic-F68 (DMEM/F12:Pluronic-F68=1:1) in a six-well plate at a volume of 2 mL/well, and place the well plate Return to the CO2 incubator and incubate for more than 1 hour;

S2. Single-cell passaging hPSC: Select a well of hPSC in good condition, add 1mL calcium and magnesium-free PBS to wash the cells after aspirating and discarding, add 1mL GCDR after aspirating and discarding, put it back into the CO2 incubator and incubate for 5-10 minutes, then aspirate Discard the GCDR in the well, add 1mL mTeSR1 medium, blow down the cells and blow them into a single-cell suspension with a pipette;

S3. Cell inoculation: draw 20ul of single cell suspension, stain with trypan blue and count with a hemocytometer; take out the pre-coated Pluronic-F68 well plate, discard the Pluronic-F68 dilution, add 5mL mTeSR1 and add 10uM Y-27632 (Rocki) to improve the survival rate of single cell seeding, in addition, add DS solution with a final concentration of 10ug/mL + 1mg/mL PVA solution in the well; draw the cell suspension containing 500,000 single cells and inoculate into the well , shake the well plate crosswise to suspend the cells evenly in the culture medium;

S4. Cell medium change: at 48 hours, 60% of the medium was changed per well per day, that is, 3mL mTeSR1 medium and PVA solution of corresponding concentration were added, and DS solution was no longer added to the medium;

S5. Cell harvesting: when the culture reached the fifth day, the aggregates were photographed, and the diameter of the aggregates was counted; after that, the cell aggregates were collected, centrifuged at 1500rpm for 5 minutes, and the supernatant was discarded, then 1 mL of PBS was added to wash the cell aggregates, and centrifuged at 1500rpm for 5 minutes. After discarding the supernatant, add 1 mL of 0.25% trypsin, digest at room temperature for 10 minutes, then add 1 mL of serum-containing medium to stop the digestion, and count with a hemocytometer after staining with trypan blue; sample 3 times from each well and count 3 times, and the final total number of cells is taken as average.

The experimental results are shown in Figure 3. The control group did not add PVA+DS, and the experimental group added 10ug/mL DS solution+1mg/mLPVA. Figure 3 is a characterization diagram of cells obtained in Example 3, 3D suspension culture + 10ug/mL DS + 1mg/mL PVA; A is the image of aggregates after 5 days of culture, B is the comparison chart of the total number of cells after 5 days of culture, and C is the image of the culture The comparison chart of the average diameter of the aggregates on the 5th day, D is the statistical comparison chart of the distribution interval of the aggregates after the 5th day of culture.

Comparative example 1: 3D suspension culture hPSC+dextran sulfate (DS)

Experimental materials: six-well plate; mTeSR1; GCDR; PBS; trypan blue; Y-27632; 0.25% trypsin; Pluronic-F68; DS;

Experimental procedure: 1. Prepare DS storage solution: Weigh 500mg DS into a 15mL centrifuge tube, add 5mL sterilized triple-distilled water, dissolve in a 37°C water bath, filter and sterilize with a 0.22um filter head, and store in a 4°C refrigerator for later use;

2. Pre-spread Pluronic-F68 to prepare low-adhesion wells: pre-spread diluted Pluronic-F68 (DMEM/F12:Pluronic-F68=1:1) in a six-well plate at a volume of 2mL/well, place the well plate Return to the CO ₂ incubator and incubate for more than 1 hour;

3. Single-cell passaging hPSC: Select a well of hPSC in good condition, add 1mL calcium and magnesium-free PBS to wash the cells after aspirating and discarding the medium, add 1mL GCDR after aspirating and discarding, put it back into the CO2 incubator and incubate for 5-10 minutes, then aspirate Discard the GCDR in the well, add 1mL mTeSR1 medium, blow down the cells and blow them into a single-cell suspension with a pipette;

4. Cell inoculation: draw 20ul of single cell suspension, stain with trypan blue and count with a hemocytometer; take out the pre-coated Pluronic-F68 well plate, discard the Pluronic-F68 dilution, add 5mL mTeSR1 and add 10uM Y-27632 (Rocki) to improve the survival rate of single cell seeding, in addition, add DS solutions with final concentrations of 10ug/mL and 100ug/mL to the 2 wells respectively; draw the cell suspension containing 500,000 single cells and inoculate into the wells , shake the well plate crosswise to suspend the cells evenly in the culture medium;

5. Cell medium change: 48 hours, change medium 60% per well per day, that is, 3mL mTeSR1 medium and add corresponding concentration of DS solution;

6. Cell harvesting: on the 5th day of culture, take pictures of the aggregates and count the diameter of the aggregates; then collect the cell aggregates, centrifuge at 1500rpm for 5 minutes, discard the supernatant, add 1mL PBS to wash the cell aggregates, and centrifuge at 1500rpm for 5 minutes. After discarding the supernatant, add 1 mL of 0.25% trypsin, digest at room temperature for 10 minutes, then add 1 mL of serum-containing medium to stop the digestion, and count with a hemocytometer after staining with trypan blue; sample 3 times from each well and count 3 times, and the final total number of cells is taken as Average number; RNA was extracted from the remaining cells after counting, reverse-transcribed into cDNA, and then qPCR was used to detect the expression level of stemness genes.

The experimental results are shown in Figure 4. Figure 4 is a characterization diagram of cells obtained by 3D suspension culture hPSC+dextran sulfate in Comparative Example 1. The control group did not add DS, the experimental group 1 added 10ug/mL DS, and the experimental group 2 added 100ug/mL DS; A is the culture Aggregate images at 5 days, B is the comparison chart of the total number of cells at 5 days of culture, C is the comparison chart of the average diameter of aggregates at 5 days of culture, D is the statistical comparison chart of the distribution interval of aggregates at 5 days of culture; E is the qPCR detection stem of 5 days of culture Comparison chart of sex gene expression results.

Comparative example 2: 3D suspension culture hPSC+polyvinyl alcohol (PVA)

Experimental materials: six-well plate; mTeSR1; GCDR; PBS; trypan blue; Y-27632; 0.25% trypsin; Pluronic-F68; PVA;

Experimental procedure: 1. Prepare PVA storage solution: Weigh 1g of PVA into a 15mL centrifuge tube, add 10mL of sterilized three-distilled water, autoclave, and store at room temperature for later use;

2. Pre-spread Pluronic-F68 to prepare low-adhesion wells: pre-spread diluted Pluronic-F68 (DMEM/F12:Pluronic-F68=1:1) in a six-well plate at a volume of 2mL/well, place the well plate Return to the CO2 incubator and incubate for more than 1 hour;

3. Single-cell passaging hPSC: Select a well of hPSC in good condition, add 1mL calcium and magnesium-free PBS to wash the cells after aspirating and discarding the medium, add 1mL GCDR after aspirating and discarding, put it back into the CO2 incubator and incubate for 5-10 minutes, then aspirate Discard the GCDR in the well, add 1mL mTeSR1 medium, blow down the cells and blow them into a single-cell suspension with a pipette;

4. Cell inoculation: draw 20ul of single cell suspension, stain with trypan blue and count with a hemocytometer; take out the pre-coated Pluronic-F68 well plate, discard the Pluronic-F68 dilution, add 5mL mTeSR1 and add 10uM Y-27632 (Rocki) to improve the survival rate of single cell seeding, in addition, add PVA solution with a final concentration of 1mg/mL in the well; draw the cell suspension containing 500,000 single cells and inoculate into the well, shake the well crosswise plate to suspend the cells evenly in the medium;

4. Cell medium change: 60% of the medium per well per day at 48 hours, that is, 3mL of mTeSR1 medium and the corresponding concentration of PVA solution;

5. Cell harvesting: When the culture reaches the fifth day, take pictures of the aggregates and count the diameter of the aggregates; then collect the cell aggregates, centrifuge at 1500rpm for 5 minutes, discard the supernatant, add 1mL PBS to wash the cell aggregates, and centrifuge at 1500rpm for 5 minutes. After discarding the supernatant, add 1 mL of 0.25% trypsin, digest at room temperature for 10 minutes, then add 1 mL of serum-containing medium to stop the digestion, and count with a hemocytometer after staining with trypan blue; sample 3 times from each well and count 3 times, and the final total number of cells is taken as Average number; RNA was extracted from the remaining cells after counting, reverse-transcribed into cDNA, and then qPCR was used to detect the expression level of stemness genes.

The experimental results are shown in Figure 5. Figure 5 is a characterization diagram of cells obtained by 3D suspension culture hPSC+polyvinyl alcohol (PVA) in Comparative Example 2. The control group did not add PVA, and the experimental group added 1 mg/mL PVA; A is the image of aggregates after 5 days of culture, and B is the image of culture The comparison chart of the total number of cells at 5 days, C is the comparison chart of the average diameter of aggregates at 5 days of culture, D is the statistical comparison chart of the distribution interval of aggregates at 5 days of culture; E is the comparison chart of the expression results of qPCR detection of stemness genes at 5 days of culture.

Claims (6)

1. A method for improving the efficiency of human multifunctional stem cell in-vitro suspension culture is characterized in that: the method comprises the following steps: carrying out three-dimensional culture on the human multifunctional stem cells by using a culture medium of dextran sulfate and polyvinyl alcohol;

the concentration of the dextran sulfate in the culture medium is 100ug/ml;

the concentration of the polyvinyl alcohol in the culture medium is 1mg/ml;

the molecular weight of dextran sulfate is 40000kDA;

the culture medium is a serum-free complete culture medium;

the culture medium is also added with Y-27632.

2. The method for improving the efficiency of in vitro suspension culture of pluripotent human stem cells according to claim 1, wherein the method comprises the following steps: the culture medium is mTeSR1 culture medium.

3. The method for improving the efficiency of in vitro suspension culture of pluripotent human stem cells according to claim 1, wherein the method comprises the following steps: after three-dimensional culture for 48h, the culture medium containing polyvinyl alcohol is used for changing the culture medium, and the culture medium is changed by 60 percent every day.

4. The method for increasing the efficiency of human pluripotent stem cell in vitro suspension culture according to any one of claims 1~3, wherein: the method specifically comprises the following steps:

s1, single-cell passage of human multifunctional stem cells: after the hPSC passage, selecting cells with coverage rate of 70-80 percent, and regular and undifferentiated cells of Long Bianyuan, and preparing the cells into a single cell suspension;

s2, cell inoculation: sequentially adding a culture medium and Y-27632 into a culture device, then adding PVA with the final concentration of 0.1-1mg/ml or 10-100ug/mLDS and 0.1-1mg/ml PVA, inoculating the single-cell suspension into the culture device, and shaking the culture device to uniformly suspend cells in a culture solution; PVA: polyvinyl alcohol, DS: dextran sulfate;

s3, cell exchange: after culturing for 48h, changing the culture medium containing PVA once every 60 percent of culture medium;

s4, cell harvesting: the cells were cultured for 5~6 days and collected.

5. The method for improving the efficiency of in vitro suspension culture of pluripotent human stem cells according to claim 4, wherein the method comprises the following steps: the culture device in the step S2 is a low-adhesion culture device;

the single cell suspension in step S1 is specifically prepared by the following steps: selecting culture holes with coverage rate of 70-80 percent and regular and undifferentiated cells of Long Bianyuan when the hPSC is subcultured, absorbing and discarding the culture medium, adding 1mL calcium-magnesium-free PBS to clean the cells, absorbing and discarding, adding 1mL GCDR, and returning CO ₂ Incubating in an incubator for 5-10 minutes, then sucking off GCDR in the hole, adding 1mL mTeSRR 1 culture medium, blowing down the cells and blowing into single cell suspension by a pipette;

the single cell suspension in the step S2 is 50 ten thousand single cell suspension;

the culture medium in the step S2 is a serum-free complete culture medium;

the culture medium in the step S3 is a serum-free complete culture medium; the final concentration of PVA in the PVA-containing medium is 0.1 to 1mg/ml.

6. The method for improving the efficiency of in vitro suspension culture of pluripotent human stem cells according to claim 4, wherein the method comprises the following steps: and S4, after the culture is completed in the step S4, centrifuging, removing the supernatant, adding PBS to clean the cell aggregate, centrifuging, removing the supernatant, adding trypsin, digesting at room temperature for 9 to 12 minutes, and adding a serum-containing culture medium to stop the digestion to obtain the cells.

Images