阅读说明：本技术 一种提高人多功能干细胞体外悬浮培养的效率的方法 (Method for improving in-vitro suspension culture efficiency of human multifunctional stem cells ) 是由 段玉友 唐湘莲 吴海滨 陈洪林 于 2019-11-27 设计创作，主要内容包括：本发明属于细胞培养的技术领域,公开了一种提高人多功能干细胞体外悬浮培养的效率的方法。所述提高人多功能干细胞体外悬浮培养效率的方法：将人多功能干细胞用含聚乙烯醇或葡聚糖硫酸酯与聚乙烯醇的培养基进行三维培养；所述葡聚糖硫酸酯在培养基中的浓度为10～100ug/ml；所述聚乙烯醇在培养基中的浓度为0.1～1mg/ml。本发明的方法不仅能够减少细胞聚集体的大小且得到尺寸均一的聚集体,还能达到较高的细胞数量以满足体外扩增人多功能干细胞的需求,提高了人多功能干细胞的培养效率。(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.)

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: the human pluripotent stem cells are subjected to three-dimensional culture by using a culture medium containing polyvinyl alcohol or dextran sulfate and polyvinyl alcohol.

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 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.

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: when the culture medium containing glucan sulfate and polyvinyl alcohol is selected for three-dimensional culture, the concentration of the glucan sulfate in the culture medium is 100ug/ml, and the concentration of the polyvinyl alcohol in the culture medium is 1 mg/ml.

4. 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 molecular weight of dextran sulfate is 40000 kDA;

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

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 medium is mTeSR1 culture medium.

6. 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 also added with Y-27632.

7. 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.

8. The method for improving the efficiency of in vitro suspension culture of human pluripotent stem cells according to any one of claims 1 to 7, wherein the method comprises the following steps: the method specifically comprises the following steps:

s1 unicellular passaged human pluripotent stem cells: after hPSC passage, selecting cells with coverage rate of 70-80%, regular cloning edges and no differentiation, and preparing the cells into a single cell suspension;

s2 cell seeding: 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/ml LDS 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 changing solution: after culturing for 48h, changing the culture medium containing PVA once every 60 percent of culture medium;

s4 harvest cells: culturing for 5-6 days, and collecting cells.

9. The method for improving the efficiency of in vitro suspension culture of pluripotent human stem cells according to claim 8, wherein the method comprises the following steps: the culture apparatus in step S2 is a low-adhesion culture apparatus;

the single cell suspension in step S1 is specifically prepared by: selecting culture holes with coverage rate of 70-80% and regular cloning edges and without differentiated cells during hPSC passage, removing culture medium by suction, adding 1mL calcium-magnesium-free PBS to clean cells, removing culture medium by suction, adding 1mL GCDR, and returning CO₂Incubating in the incubator for 5-10 minutes, then sucking off GCDR in the hole, adding 1mL mTeSR1 culture medium, blowing down the cells and blowing into single cell suspension by a pipette;

in step S2, the single cell suspension is a cell suspension of 50 ten thousand single cells;

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 culture medium containing PVA is 0.1-1 mg/ml.

10. The method for improving the efficiency of in vitro suspension culture of pluripotent human stem cells according to claim 8, wherein the method comprises the following steps: and step S4, centrifuging after the culture is finished, removing the supernatant, adding PBS to wash the cell aggregate, centrifuging, removing the supernatant, adding trypsin, digesting at room temperature for 9-12 minutes, adding a serum-containing culture medium to stop the digestion, and obtaining the cell.

Technical Field

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

Background

Human embryonic stem cells (hescs) and human induced pluripotent stem cells (hipscs), collectively known as human pluripotent stem cells (hpscs), have great promise in cell therapy, tissue engineering and regenerative medicine, as well as in drug development and biotechnology applications. The conventional two-dimensional (2D) culture technology for cell expansion using plastic culture plates and heterologous media has limited cell expansion efficiency, and after long-term passage expansion, cells lose desiccation and differentiation capacity. In order to obtain high-quantity and high-quality human pluripotent stem cells, a bioreactor-based simulated in vivo three-dimensional (3D) culture technology is applied to carry out large-scale amplification on the human pluripotent stem cells. At present, compared with a microcarrier culture strategy, the strategy of inoculating in a bioreactor in a form of single cells and then amplifying in a form of cell aggregates has the advantages of cost saving, easy cell recovery and the like, but the strategy forms aggregates with larger size and nonuniform size. Due to the large diameter of large aggregates (> 300 μm), nutrients and oxygen are difficult to diffuse to the aggregate center resulting in cell differentiation or death.

Aggregate size control is a key parameter of the strategy of culturing cells in an aggregate mode, and the size of aggregates can be controlled by a physical method for controlling shearing force and a chemical method for adding small molecules and the like so as to obtain a more uniform cell aggregate product. Polysulfate compounds are widely used in the biopharmaceutical industry, and not only are they capable of reducing cell aggregation by modulating the charge on the cell surface, but also have an anti-apoptotic effect on cells. At the same time, Dextran Sulfate (DS) has been reported to reduce the cell aggregation of hPSCs without reducing their pluripotency. Lipsitiz YY et al [1 Lipsitiz YY, Tong PD, ZandstraPW.chemical ly controlled aggregation of pluratent stem cells.Biotechnol Bioeng 2018; 115(8): 2061-6.]It was reported that suspension culture of cells under 40000kDA (D40) and 100ug/ml DS conditions allowed the expansion of hPSCs aggregates in a small, uniform size with stable pluripotency and karyotype. Nogueira et al [ Nogueira, d.e.s., Rodrigues, c.a.v., Carvalho, m.s.et al.strategies for the expansion of human induced ploutient cells as aggregates in single-user vertical-Wheel^TMbioreactors.J Biol Eng 2019；13，74.]Reported the (2.3. + -. 0.2). times.10 obtained using a disposable vertical roller reactor in combination with 100ug/ml DS⁶cells/mL and harvest mean diameter 346 + -11 μm cell aggregates, which remain pluripotent, karyotype stable. Although DS can be usedThe cell aggregation is reduced by adjusting the charge on the cell surface, which is beneficial to obtaining high-quality small cell aggregates with uniform size and beneficial to recovery, but the cell expansion multiple cannot be improved, namely the cell number still cannot meet the requirement.

The invention combines DS (dextran sulfate) and PVA for use, improves the efficiency of stem cell in vitro suspension culture, controls the size of cell aggregates in an amplification strategy in the form of cell aggregates, improves the amplification times of the cells, and can maintain the pluripotency of the recovered cells and meet the requirement of in vitro amplification. The combined use of PVA and DS not only allows uniform sized aggregates to be obtained, but also allows higher cell numbers to be achieved to meet clinical needs.

Disclosure of Invention

Aiming at the defects of the technology, the invention aims to provide a method for improving the in-vitro suspension culture efficiency of human multifunctional stem cells. The PVA has the function of promoting the proliferation of the hPSCs, and the DS (dextran sulfate) and the PVA are further combined for use, so that the aggregate with uniform size can be obtained, the higher cell number can be achieved, and the efficiency of in-vitro suspension culture of the stem cells is improved. The method of the invention can realize large-scale in-vitro suspension culture of hPSC.

The purpose of the invention is realized by the following technical scheme:

a method for improving the efficiency of human pluripotent stem cell in-vitro suspension culture comprises the following steps: the human pluripotent stem cells are three-dimensionally cultured in a medium containing PVA (polyvinyl alcohol) or DS (dextran sulfate) and PVA (polyvinyl alcohol).

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

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

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

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

The medium was also supplemented with Y-27632 (Rocki).

After three-dimensional culture for 48h, the medium containing PVA is used for changing the medium, and the medium is changed once every day by 60 percent.

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

s1 single cell passaged human pluripotent stem cells (hpscs): after hPSC passage, selecting cells with coverage rate of 70-80%, regular cloning edges and no differentiation, and preparing the cells into a single cell suspension;

s2 cell seeding: sequentially adding a culture medium and Y-27632(Rocki) into a culture device, then adding PVA with the final concentration of 0.1-1mg/ml or PVA with the final concentration of 10-100ug/mLDS and O.1-1 mg/ml, inoculating the single cell suspension into the culture device, and shaking the culture device to uniformly suspend cells in a culture solution;

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

s4 harvest cells: culturing for 5-6 days, and collecting cells.

The culture apparatus in step S2 is a low-adhesion culture apparatus such as: commercial low adhesion orifice plate, low adhesion orifice plate prepared using conventional methods: the adhesive orifice plate prepared by the conventional method comprises the following specific steps: diluting Pluronic-F68 with DMEM/F12 medium to obtain diluted Pluronic-F68 (DMEM/F12: Pluronic-F68: 1 (volume ratio)); the diluted Pluronic-F68 was plated in a well plate, which was placed in CO₂And (5) incubation in an incubator.

The single cell suspension in step S1 is specifically prepared by: selecting culture holes with coverage rate of 70-80% and regular cloning edges and without differentiated cells during hPSC passage, removing culture medium by aspiration, adding 1mL of calcium-magnesium-free PBS to clean cells, removing cells by aspiration, adding 1mL of GCDR (cell dissociation reagent), and returning CO₂Incubate in the incubator for 5-10 minutes, then aspirate the GCDR in the well, add 1mL mTeSR1 media, blow the cells down and pipette into a single cell suspension.

The single cell suspension in step S2 was a cell suspension of 50 ten thousand single cells. The culture medium in the step S2 is a serum-free complete culture medium, preferably mTeSR1 culture medium.

Y-27632(Rocki) was added at 10 uM.

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

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

And step S4, centrifuging after the culture is finished, removing the supernatant, adding PBS to wash the cell aggregate, centrifuging, removing the supernatant, adding trypsin, digesting at room temperature for 9-12 minutes, adding a serum-containing culture medium to stop the digestion, and obtaining the cell. The centrifugation condition is 1500rpm centrifugation for 5 minutes; the trypsin is 0.25% trypsin.

The invention combines low concentration PVA with 40000kDA (D40) and 100ug/ml DS to use in vitro suspension culture hPSCs, the combination of the two can improve the efficiency of cell expansion, so that the harvested cell aggregate keeps small and uniform size, the quality of the cell aggregate is higher, the aggregate center does not lack nutrients and oxygen because of large size, and the recovered aggregate is easy to be digested into single cells for passage or clinical use.

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

Drawings

FIG. 1 is a graph representing cells obtained by 3D suspension culture +100ug/mL DS +1mg/mL PVA in example 1; a is an aggregate image cultured for 5 days, B is a comparison graph of the total number of cells cultured for 5 days, C is a comparison graph of the average diameter of the aggregate cultured for 5 days, and D is a statistical comparison graph of the distribution interval of the aggregate cultured for 5 days;

FIG. 2 is a graph representing cells obtained by 3D suspension culture +100ug/mL DS +0.1mg/mL PVA in example 2; a is an aggregate image cultured for 5 days, B is a comparison graph of the total number of cells cultured for 5 days, C is a comparison graph of the average diameter of the aggregate cultured for 5 days, and D is a statistical comparison graph of the distribution interval of the aggregate cultured for 5 days;

FIG. 3 is a graph representing the cells obtained by 3D suspension culture +10ug/mL DS +1mg/mL PVA in example 3; a is an aggregate image cultured for 5 days, B is a comparison graph of the total number of cells cultured for 5 days, C is a comparison graph of the average diameter of the aggregate cultured for 5 days, and D is a statistical comparison graph of the distribution interval of the aggregate cultured for 5 days;

FIG. 4 is a representation of cells obtained by 3D suspension culture of hPSC + dextran sulfate in comparative example 1, with no DS in the control group, 10ug/mL DS in the experimental group 1, and 100ug/mL DS in the experimental group 2; a is an aggregate image cultured for 5 days, B is a comparison graph of the total number of cells cultured for 5 days, C is a comparison graph of the average diameter of the aggregate cultured for 5 days, and D is a statistical comparison graph of the distribution interval of the aggregate cultured for 5 days; e is a comparison graph of the dry gene expression result detected by qPCR for 5 days;

FIG. 5 is a representation of cells obtained by 3D suspension culture of hPSC + polyvinyl alcohol (PVA) in comparative example 2, with no PVA added to the control group and 1mg/mL PVA added to the experimental group; a is an aggregate image cultured for 5 days, B is a comparison graph of the total number of cells cultured for 5 days, C is a comparison graph of the average diameter of the aggregate cultured for 5 days, and D is a statistical comparison graph of the distribution interval of the aggregate cultured for 5 days; and E is a comparison graph of the expression result of the dry gene detected by qPCR for 5 days.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto. Dextran sulfate DS: manufacturer: SIGMA, Mr (relative molecular mass) to 10000, concentration: storage concentration of 100 ug/ml: 100mg/ml, namely 500mgDS is fully dissolved in 5ml of sterile water for 4 degrees storage. DS is added into the culture medium when cells are inoculated, 60% of the culture medium is replaced when the cells are cultured for 48 hours, and the replaced culture medium does not need to be added with DS with the use concentration, namely DS only acts for 48 hours.

PVA: manufacturer: ALDRICH, Mw 31000 and 500000, and a degree of hydrolysis of 87% -89%.

The use concentration is as follows: 0.1mg/ml, 0.5mg/ml, 1 mg/ml. Storage concentration: 100mg/ml, namely 1g of PVA is fully dissolved in 1ml of sterile water for normal temperature storage. Low concentration PVA (0.1-1mg/ml) was added to the medium at the time of cell inoculation, 60% of the medium was changed at the time of cell culture for 48 hours, and PVA at the use concentration, i.e., PVA, was continuously added to the changed medium for the entire culture cycle.

Experimental materials: six orifice plates; mTeSR 1; GCDR; PBS; trypan blue; y-27632; 0.25% pancreatin; Pluronic-F68; PVA; and (5) a DS.