1. A method for culturing stem cells of cynomolgus monkeys without a feeder layer is characterized by comprising the following steps:

digesting the pluripotent stem cells cultured on the feeder layer with collagenase;

cells obtained by basic cyclone digestion by a culture medium without a feeding layer are transferred to a culture dish coated with matrigel in advance and put into an incubator;

after being digested by ACCTRUSE, the mixture is transferred to a culture dish coated in advance; when the cell density reaches 80-90% of the area of the culture dish, the cells are digested by ACCTRUE enzyme and then transferred to the culture dish coated in advance;

after digestion of cells with accuse enzyme, the cells were digested according to 1: 5-1: the ratio of 10 was passed to previously coated dishes.

2. The method of claim 1, wherein the method comprises the steps of:

firstly, digesting pluripotent stem cells cultured in a feeder layer by using collagenase; firstly, 50ul of collagenase solution with the concentration of 10mg/ml is absorbed into 1ml of stem cell culture medium prepared in advance, the original culture medium in a culture dish is discarded after the collagenase solution is mixed evenly, and the culture medium containing the collagenase is added. Placing into incubator, and waiting for 10-15 min. Waiting for the edge of the clone to curl, the medium in the dish was discarded and the clone was removed from the dish with fresh medium. The medium with the clones mixed therein was transferred to a 15ml centrifuge tube and centrifuged at 1000rpm for 5 min.

Step two, taking out the centrifugal tube centrifuged in the step one, discarding the supernatant, carrying out rotary digestion on the cells by using a feeder-layer-free culture medium, transferring the cells to a culture dish coated with matrigel in advance, adding a cell adherence promoting factor CloneR (Stemcell), and then putting the cells into an incubator;

step three, when the cell density reaches 80% -90% of the area of the culture dish, abandoning the original culture medium, adding ACCTUSE (Gibco), placing the cell into an incubator for waiting for 5-10min, after the cell is digested into single cells, using a fresh culture medium to spin the cells in the culture dish, then transferring the digested cells into a 15ml centrifuge tube prepared to be clean in advance, centrifuging the cell at 1000rpm for 5min, removing the supernatant in the centrifuge tube, using the fresh culture medium to spin the cells at the bottom, adding a cell adherence promoting factor CloneR (Stemcell), and transferring the cells to the culture dish coated in advance.

3. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (substitute for L-glutamine) (Gibco,100X)0.5ml, Recombinant Human Nodal Protein (Recombinant Human Nodal Protein) (R & D system)5ug, chemically defined lipid concentrate (Gibco)0.5ml, Glutathione (Glutathione) (Sigma)1.94mg/L, LIF (leukemia inhibitory factor) (peptide) 0.4ul/ml-3 ul/ml.

4. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, bFGF (basic fibroblast growth factor) (proteintech)4ng/ml-20 ng/ml.

5. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (N-2 additive is based on the Bottenstein's N-1 formulation and is a chemically defined serum-free additive) (Thermo Fisher Scientific)250ul, B27 supplement (B-27 is an optimized serum-free supplement) (Thermo Fisher Scientific)500 ul.

6. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, bFGF (proteintech)4ng/ml-20ng/ml, LIF (peprotech)0.4ul/ml-3 ul/ml.

7. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant Human Nodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94 mg/L.

8. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TESRE8(Stemcell)47ml, TESRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant Human Nodal Protein (R & D system)5ug, chemically defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, Thiazovin (Thiazovin inhibitor) (Selleck)2.4 uM.

9. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5 ml.

10. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, KOSR (KnockOut serum replacement) (Thermo Fisher Scientific)500 ul.

11. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, LIF (peprotech)10 ng/ul.

12. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TESRE8(Stemcell)47ml, TESRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, IWR (IWR-1-end is a Wnt pathway inhibitor) (selelck).

13. The method of claim 1, wherein the 50ml feeder-free culture system comprises TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant Human Nodal Protein (R & D system)5ug, chemically defined restricted concentrate (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, and R-1(IWR-1-endo is a Wnt pathway inhibitor) (select).

14. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, ActivinA (activin A) (peprotech)10 ng/ml.

15. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant Human Nodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, Activin A (Activin A) (peprotech)10 ng/ml.

16. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, ActivinA (activin A) (peprotech)10ng/ml, IWR-1.

17. The method of claim 1, wherein the feeder-free culture system of 50ml comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant Human Nodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, Activin A (Activin A) (peprotech)10ng/ml, IWR-1.

Background

At present, the culture of pluripotent stem cells for culturing cynomolgus monkeys needs a feeder layer to provide partial nutrition, the feeder layer is made of 12.5-day mouse embryos, the embryos are taken out on 12.5 days after the mice of CF-1 strain are mated, after viscera, limbs and heads are removed, the remaining tissues are digested into single cells for culture, and the cells can be used as the feeder layer after proliferation. Therefore, a large amount of manpower and material resources are needed for preparing the feeding layer, and the feeding layer contains animal-derived components, so that the components of the culture medium are uncertain, the situation of unstable performance is easily caused, the results of the previous batch and the next batch are inconsistent, the quality of the culture medium is unstable and irregular, and the accuracy of the research result is directly influenced. In the culture of human pluripotent stem cells and mouse pluripotent stem cells, scientists have developed a new culture system to overcome the influence of feeder layers, and have the ability to culture human pluripotent stem cells and mouse pluripotent stem cells without feeder layers. However, until now, no culture system for culturing stem cells of cynomolgus monkeys without feeder layer has been developed, and therefore, a new method for culturing stem cells of cynomolgus monkeys without feeder layer is needed.

Through the above analysis, the problems and defects of the prior art are as follows: the preparation of the existing breeding layer needs a large amount of manpower and material resources, and the breeding layer contains animal-derived components, so that the components of the culture medium are uncertain, the condition of unstable performance is easily generated, the results of the previous batch and the next batch are inconsistent, the quality of the culture medium is unstable and irregular, and the accuracy of the research result is directly influenced.

The difficulty in solving the above problems and defects is: exploring a new culture method requires more manpower and material resources, and also requires a certain amount of reserve in the aspect of professional knowledge. It is necessary to search for the components and concentrations of the culture system, and therefore, a large amount of screening for the components of the culture system is required.

The significance of solving the problems and the defects is as follows: if the problem of the non-feeder culture of the cynomolgus monkey stem cells can be solved, the development of the non-human primate stem cells, such as the culture of organoid, can be facilitated. Can also provide a new reference for a culture system of the human pluripotent stem cells.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a feeder-free culture method for stem cells of cynomolgus monkeys.

The invention is realized in such a way that the method for culturing the stem cells of the cynomolgus monkey without the feeder layer comprises the following steps:

digesting the pluripotent stem cells cultured on the feeder layer with collagenase;

cells obtained by basic cyclone digestion by a culture medium without a feeding layer are transferred to a culture dish coated with matrigel in advance and put into an incubator;

after being digested by ACCTRUSE, the mixture is transferred to a culture dish coated in advance; when the cell density reaches 80-90% of the area of the culture dish, the cells are digested by ACCTRUE enzyme and then transferred to the culture dish coated in advance;

after digestion of cells with accuse enzyme, the cells were digested according to 1: 5-1: the ratio of 10 was passed to previously coated dishes.

Further, the method for culturing the stem cells of the cynomolgus monkey without a feeder layer comprises the following steps:

firstly, digesting pluripotent stem cells cultured in a feeder layer by using collagenase; firstly, 50ul of collagenase solution with the concentration of 10mg/ml is absorbed into 1ml of stem cell culture medium prepared in advance, the original culture medium in a culture dish is discarded after the collagenase solution is mixed evenly, and the culture medium containing the collagenase is added. Placing into incubator, and waiting for 10-15 min. Waiting for the edge of the clone to curl, the medium in the dish was discarded and the clone was removed from the dish with fresh medium. The medium with the clones mixed therein was transferred to a 15ml centrifuge tube and centrifuged at 1000rpm for 5 min.

Step two, taking out the centrifugal tube centrifuged in the step one, discarding the supernatant, carrying out rotary digestion on the cells by using a feeder-layer-free culture medium, transferring the cells to a culture dish coated with matrigel in advance, adding a cell adherence promoting factor CloneR (Stemcell), and then putting the cells into an incubator;

step three, when the cell density reaches 80% -90% of the area of the culture dish, abandoning the original culture medium, adding ACCTUSE (Gibco), placing the cell into an incubator for waiting for 5-10min, after the cell is digested into single cells, using a fresh culture medium to spin the cells in the culture dish, then transferring the digested cells into a 15ml centrifuge tube prepared to be clean in advance, centrifuging the cell at 1000rpm for 5min, removing the supernatant in the centrifuge tube, using the fresh culture medium to spin the cells at the bottom, adding a cell adherence promoting factor CloneR (Stemcell), and transferring the cells to the culture dish coated in advance.

Further, in step three, the cells were digested with ACCTRUE enzyme and transferred to pre-coated dishes in the ratio of (1: 5) - (1: 10).

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (substitute for L-glutamine) (Gibco,100X)0.5ml, Recombinant human Nodal Protein (Recombinant human Nodal Protein) (R & D system)5ug, chemically defined lipid concentrate (Gibco)0.5ml, Glutathione (Glutathione) (Sigma)1.94mg/L, LIF (leukemia inhibitory factor) (peptide) 0.4ul/ml-3 ul/ml.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, bFGF (basic fibroblast growth factor) (proteintech)4ng/ml-20 ng/ml.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (N-2 additive is based on the Bottenstein's N-1 formulation and is a chemically defined serum-free additive) (Thermo Fisher Scientific)250ul, B27 supplement (B-27 is an optimized serum-free supplement) (Thermo Fisher Scientific)500 ul.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, bFGF (proteintech)4ng/ml-20ng/ml, LIF (peprotech)0.4ul/ml-3 ul/ml.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, chemically defined lipid concentrate (Gibco)0.5ml, Glutathione (Sigma)1.94 mg/L.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TESRE8(Stemcell)47ml, TESRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, chemically defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, Thiazovin (Thiazovin inhibitor) (Selleck)2.4 uM.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5 ml.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, KOSR (KnockOut serum replacement) (Thermo Fisher Scientific)500 ul.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, LIF (peprotech)10 ng/ul.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TESRE8(Stemcell)47ml, TESRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, IWR (IWR-1-end is a Wnt pathway inhibitor) (selelck).

Further, the feeder-free culture system 50ml of the feeder-free culture method comprises TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant human nodal Protein (R & D system)5ug, chemically defined lipid concentrate (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, and IWR-1(IWR-1-end is a Wnt pathway inhibitor) (select).

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, ActivinA (activin A) (peprotech)10 ng/ml.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, Activin A (Activin A) (peprotech)10 ng/ml.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, ActivinA (activin A) (peprotech)10ng/ml, IWR-1.

Further, 50ml of the feeder-free culture system of the feeder-free culture method comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, Activin A (Activin A) (peprotech)10ng/ml, IWR-1.

By combining all the technical schemes, the invention has the advantages and positive effects that: according to the method for culturing the stem cells of the cynomolgus monkey without the feeder layer, provided by the invention, the pluripotent stem cells of the cynomolgus monkey can be cultured under the condition without the feeder layer by developing a new culture condition, the stem cells can be kept to have pluripotency for a long time, the stem cells with the pluripotency can express a plurality of pluripotency proteins, and the pluripotency of the stem cells of the cynomolgus monkey cultured without the feeder layer can be proved through immunofluorescence staining as shown in figure three. The invention develops a novel culture system without a feeder layer for the pluripotent stem cells of the cynomolgus monkey, which can maintain the pluripotency of the pluripotent stem cells of the cynomolgus monkey for a long time, does not need the preparation of a feeder layer, saves a large amount of manpower and material resources, has clear culture components, and can maintain the pluripotency of the stem cells for a long time. The method for culturing the cynomolgus monkey stem cells without the feeder layer has no batch detection influence, is beneficial to improving the accuracy of an experiment, and a culture system can be clinically used for reference.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow chart of a feeder-free culturing method for cynomolgus monkey stem cells according to an embodiment of the present invention.

FIG. 2 is a photograph of a cynomolgus monkey pluripotent stem cell cultured in a feeder-free layer in the bright field according to an embodiment of the present invention.

FIG. 3 is a schematic diagram of the immunofluorescence staining result of the feeding-layer-free culture of the cynomolgus monkey pluripotent stem cells provided by the embodiment of the invention.

FIG. 4 is a diagram illustrating the normal result of karyotype detection in the feeder-free culture of cynomolgus monkey pluripotent stem cells according to the present invention.

FIG. 5 is a schematic diagram of the three germ layer differentiation of the cynomolgus monkey pluripotent stem cell feeder-free culture provided in the example of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a feeder-free culture method for stem cells of cynomolgus monkeys, which is described in detail below with reference to the attached drawings.

As shown in fig. 1, the method for culturing cynomolgus monkey stem cells without feeder layer provided in the embodiment of the present invention comprises the following steps:

s101, digesting the pluripotent stem cells cultured in a feeder layer by using collagenase;

s102, cells which are subjected to basic cyclone digestion by a culture medium without a feeding layer are transferred to a culture dish coated with matrigel in advance and are placed in an incubator;

s103, when the cell density reaches 80% -90% of the area of the culture dish, the cells are digested by ACCTRUE enzyme and then transferred to the culture dish coated in advance according to the proportion of (1: 5) - (1: 10).

One of ordinary skill in the art can also perform the method for culturing cynomolgus monkey stem cells without feeder layer, and the method for culturing cynomolgus monkey stem cells without feeder layer provided by the present invention shown in fig. 1 is only one specific example.

The technical solution of the present invention is further described with reference to the following examples.

The method of the invention can solve the problem that the cynomolgus monkey pluripotent stem cells are cultured under the condition without a feeder layer by developing a new culture condition, and can keep the stem cells to have the pluripotency for a long time.

The method for culturing stem cells of cynomolgus monkeys without a feeder layer provided by the embodiment of the invention comprises the following steps: firstly, after the pluripotent stem cells cultured on a feeder layer are digested with collagenase, the cells obtained by the basic rotary digestion of the cells are transferred to a culture dish previously coated with matrigel, and the culture dish is placed in an incubator. When the cell density reached 80% -90% of the dish area, it was transferred to pre-coated dishes in the ratio of (1: 5) - (1: 10) after digestion with ACCTRUE enzyme.

The brightfield picture of the cynomolgus monkey pluripotent stem cell feeder-free culture provided by the embodiment of the invention is shown in fig. 2, and the schematic diagram of the immunofluorescence staining result of the cynomolgus monkey pluripotent stem cell feeder-free culture provided by the embodiment of the invention is shown in fig. 3.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (substitute for L-glutamine) (Gibco,100X)0.5ml, Recombinant human Nodal Protein (Recombinant human Nodal Protein) (R & D system)5ug, chemically defined lipid concentrate (Gibco)0.5ml, Glutathione (Glutathione) (Sigma)1.94mg/L, LIF (leukemia inhibitory factor) (peptide) 0.4ul/ml-3 ul/ml.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, bFGF (basic fibroblast growth factor) (proteintech)4ng/ml-20 ng/ml.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (N-2 additive is based on the Bottenstein's N-1 formulation and is a chemically defined serum-free additive) (Thermo Fisher Scientific)250ul, B27 supplement (B-27 is an optimized serum-free supplement) (Thermo Fisher Scientific)500 ul.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, bFGF (proteintech)4ng/ml-20ng/ml, LIF (peprotech)0.4ul/ml-3 ul/ml.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, chemically defined lipid concentrate (Gibco)0.5ml, Glutathione (Sigma)1.94 mg/L.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TESRE8(Stemcell)47ml, TESRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, chemically defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, Thiazovin (Thiazovin inhibitor) (Selleck)2.4 uM.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5 ml.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, KOSR (KnockOut serum replacement) (Thermo Fisher Scientific)500 ul.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, LIF (peprotech)10 ng/ul.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TESRE8(Stemcell)47ml, TESRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, IWR (IWR-1-end is a Wnt pathway inhibitor) (selelck).

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinan human nodal Protein (R & D system)5ug, chemically defined lipid concentrate (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, and IWR-1(IWR-1-end is a Wnt pathway inhibitor) (select).

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, ActivinA (activin A) (peprotech)10 ng/ml.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, Activin A (Activin A) (peprotech)10 ng/ml.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, N2supplement (Thermo Fisher Scientific)250ul, B27 supplement (Thermo Fisher Scientific)500ul, ActivinA (activin A) (peprotech)10ng/ml, IWR-1.

The feeder-free culture system 50ml of the feeder-free culture method of the present invention comprises: TeSRE8(Stemcell)47ml, TeSRE8 supplement (Stemcell)2ml, GlutaMAX (Gibco,100X)0.5ml, Recombinant HumanNodal Protein (R & D system)5ug, Chemicaly defined lipid concentrator (Gibco)0.5ml, Glutathione (Sigma)1.94mg/L, Activin A (Activin A) (peprotech)10ng/ml, IWR-1.

The invention develops a method for culturing stem cells of a cynomolgus monkey without a feeder layer, which can maintain the pluripotency of the pluripotent stem cells of the cynomolgus monkey for a long time. The preparation without a feeder layer saves a large amount of manpower and material resources, has definite culture components, and can maintain the pluripotency of the stem cells for a long time. The method for culturing the cynomolgus monkey stem cells without the feeder layer has no batch detection influence, is beneficial to improving the accuracy of an experiment, and a culture system can be clinically used for reference.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.