METHOD FOR PROMOTING GROWTH OF PARIETAL DECIDUAL BASALIS-MESENCHYMAL STEM CELLS (PDB-MSCs)

Abstract

The present disclosure provides a method for promoting the growth of parietal decidual basalis-mesenchymal stem cells (PDB-MSCs), which comprises promoting the growth of parietal decidual basalis-mesenchymal stem cells (PDB-MSCs) via a human umbilical cord-mesenchymal stem cell (UC-MSC)-derived exosome. In the present disclosure, after the PDB-MSCs are co-cultivated with the human UC-MSC-derived exosome, the PDB-MSCs show strong cell proliferation ability, prominent cell shape, and desirable cell viability. That is, the human UC-MSC-derived exosome of the present disclosure can improve a quality of PDB-MSCs and effectively improve the ability of PDB-MSCs to secrete vascular endothelial growth factor (VEGF) and stem cell factor (SCF), so as to solve the problem that PDB-MSCs show decreased proliferation ability and poor cell viability after multiple passages, which effectively facilitates the large-scale cultivation and clinical practice of PDB-MSCs.

Claims

1. A method for promoting the growth of parietal decidual basalis-mesenchymal stem cells (PDB-MSCs), wherein the method comprises promoting the growth of parietal decidual basalis-mesenchymal stem cells (PDB-MSCs) via a human umbilical cord-mesenchymal stem cell (UC-MSC)-derived exosome.

2. The method according to claim 1, wherein the human UC-MSC-derived exosome is secreted by a P5 generation of human UC-MSCs.

3. The method according to claim 2, wherein a preparation method of the human UC-MSC-derived exosome comprises: S1. inoculating a P4 generation of human UC-MSCs into a culture flask at a concentration of 9,000 to 12,000 cells/cm.sup.2, adding 20 ml to 30 ml of a serum-free proliferation medium, and cultivating; S2. when confluency of the P4 generation of human UC-MSCs reaches 90%, collecting a culture supernatant to obtain a first supernatant; S3. centrifuging the first supernatant, and collecting a resulting supernatant to obtain a second supernatant; and S4. filtering the second supernatant to obtain a first filter residue, centrifuging the first filter residue, and collecting a precipitate at a bottom; and resuspending the precipitate with 0.8 ml to 1.5 ml of phosphate buffered saline (PBS), and filtering to obtain a second filter residue, which is the human UC-MSC-derived exosome.

4. The method according to claim 3, wherein a preparation method of the P4 generation of human UC-MSCs comprises: S11. washing an umbilical cord tissue with a tissue protection solution to remove blood on a surface, removing epidermal and vascular tissues, and taking out Wharton's jelly; and washing the Wharton's jelly, cutting the Wharton's jelly into pieces of 1 to 2 mm.sup.3, and inoculating and cultivating according to a tissue adhesion method; and S12. when primary cells grow to confluency of 70%, subculturing, and repeating the subculturing until the P4 generation is obtained.

5. The method according to claim 4, wherein in S12, after a generation of cells grow to confluency of greater than 80%, the cells are washed at least twice with PBS and then digested with trypsin for 4 min to 7 min, a medium is added to terminate the digestion, a resulting mixture is filtered and centrifuged, and a resulting precipitate is resuspended with a cell medium and subcultured, which is repeated until the P4 generation is obtained.

6. The method according to claim 5, wherein the trypsin is a 20% to 30% Tryple-EDTA enzyme.

7. The method according to claim 4, wherein in S11, the tissue protection solution is prepared from 0.5 ml to 3 ml of normal saline (NS), 20 μg to 30 μg of gentamicin sulfate, and 3 μg to 6 μg of amphotericin B.

8. A method for promoting the growth of PDB-MSCs, comprising the steps of: A. adding the human UC-MSC-derived exosome according to claim 1 to an MSC serum-free medium at a concentration of 10 to 30 μg/ml to obtain an exosome-containing cell medium; B. inoculating the PDB-MSCs in a cell culture plate at a concentration of 1.5×10.sup.3 to 2.5×10.sup.3 cells/well, and adding the exosome-containing cell medium; and C. incubating the cell culture plate in an incubator, with a temperature of 35° C. to 40° C., a CO.sub.2 concentration of 4% to 7%, and an O.sub.2 concentration of 1% to 3%.

9. A method for promoting the growth of PDB-MSCs, comprising the steps of: A. adding the human UC-MSC-derived exosome according to claim 2 to an MSC serum-free medium at a concentration of 10 to 30 μg/ml to obtain an exosome-containing cell medium; B. inoculating the PDB-MSCs in a cell culture plate at a concentration of 1.5×10.sup.3 to 2.5×10.sup.3 cells/well, and adding the exosome-containing cell medium; and C. incubating the cell culture plate in an incubator, with a temperature of 35° C. to 40° C., a CO.sub.2 concentration of 4% to 7%, and an O.sub.2 concentration of 1% to 3%.

10. A method for promoting the growth of PDB-MSCs, comprising the steps of: A. adding the human UC-MSC-derived exosome according to claim 3 to an MSC serum-free medium at a concentration of 10 to 30 μg/ml to obtain an exosome-containing cell medium; B. inoculating the PDB-MSCs in a cell culture plate at a concentration of 1.5×10.sup.3 to 2.5×10.sup.3 cells/well, and adding the exosome-containing cell medium; and C. incubating the cell culture plate in an incubator, with a temperature of 35° C. to 40° C., a CO.sub.2 concentration of 4% to 7%, and an O.sub.2 concentration of 1% to 3%.

11. A method for promoting the growth of PDB-MSCs, comprising the steps of: A. adding the human UC-MSC-derived exosome according to claim 4 to an MSC serum-free medium at a concentration of 10 to 30 μg/ml to obtain an exosome-containing cell medium; B. inoculating the PDB-MSCs in a cell culture plate at a concentration of 1.5×10.sup.3 to 2.5×10.sup.3 cells/well, and adding the exosome-containing cell medium; and C. incubating the cell culture plate in an incubator, with a temperature of 35° C. to 40° C., a CO.sub.2 concentration of 4% to 7%, and an O.sub.2 concentration of 1% to 3%.

12. A method for promoting the growth of PDB-MSCs, comprising the steps of: A. adding the human UC-MSC-derived exosome according to claim 5 to an MSC serum-free medium at a concentration of 10 to 30 μg/ml to obtain an exosome-containing cell medium; B. inoculating the PDB-MSCs in a cell culture plate at a concentration of 1.5×10.sup.3 to 2.5×10.sup.3 cells/well, and adding the exosome-containing cell medium; and C. incubating the cell culture plate in an incubator, with a temperature of 35° C. to 40° C., a CO.sub.2 concentration of 4% to 7%, and an O.sub.2 concentration of 1% to 3%.

13. A method for promoting the growth of PDB-MSCs, comprising the steps of: A. adding the human UC-MSC-derived exosome according to claim 6 to an MSC serum-free medium at a concentration of 10 to 30 μg/ml to obtain an exosome-containing cell medium; B. inoculating the PDB-MSCs in a cell culture plate at a concentration of 1.5×10.sup.3 to 2.5×10.sup.3 cells/well, and adding the exosome-containing cell medium; and C. incubating the cell culture plate in an incubator, with a temperature of 35° C. to 40° C., a CO.sub.2 concentration of 4% to 7%, and an O.sub.2 concentration of 1% to 3%.

14. A method for promoting the growth of PDB-MSCs, comprising the steps of: A. adding the human UC-MSC-derived exosome according to claim 7 to an MSC serum-free medium at a concentration of 10 to 30 μg/ml to obtain an exosome-containing cell medium; B. inoculating the PDB-MSCs in a cell culture plate at a concentration of 1.5×10.sup.3 to 2.5×10.sup.3 cells/well, and adding the exosome-containing cell medium; and C. incubating the cell culture plate in an incubator, with a temperature of 35° C. to 40° C., a CO.sub.2 concentration of 4% to 7%, and an O.sub.2 concentration of 1% to 3%.

15. The method for promoting the growth of PDB-MSCs according to claim 8, wherein in step B, a P12 generation of PDB-MSCs are inoculated in the cell culture plate.

16. The method for promoting the growth of PDB-MSCs according to claim 9, wherein in step B, a P12 generation of PDB-MSCs are inoculated in the cell culture plate.

17. The method for promoting the growth of PDB-MSCs according to claim 10, wherein in step B, a P12 generation of PDB-MSCs are inoculated in the cell culture plate.

18. The method for promoting the growth of PDB-MSCs according to claim 8, wherein in step A, the human UC-MSC-derived exosome is added to the MSC serum-free medium at a concentration of 20 μg/ml.

19. The method for promoting the growth of PDB-MSCs according to claim 9, wherein in step A, the human UC-MSC-derived exosome is added to the MSC serum-free medium at a concentration of 20 μg/ml.

20. The method for promoting the growth of PDB-MSCs according to claim 10, wherein in step A, the human UC-MSC-derived exosome is added to the MSC serum-free medium at a concentration of 20 μg/ml.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 shows the morphology of the P5 generation of human UC-MSCs in Example 1 of the present disclosure;

[0035] FIG. 2 is a scanning electron microscopy (SEM) image of the human UC-MSC-derived exosome in Example 1 of the present disclosure;

[0036] FIG. 3 shows the morphology of PDB-MSCs cultivated with the human UC-MSC-derived exosome at different concentrations in Example 2 of the present disclosure;

[0037] FIG. 4 shows cell growth curves of PDB-MSCs cultivated with the human UC-MSC-derived exosome at different concentrations in Example 2 of the present disclosure;

[0038] FIG. 5 is a schematic diagram illustrating a proportion of cells at each stage of the cell cycle in PDB-MSCs cultivated with the human UC-MSC-derived exosome at different concentrations in Example 3 of the present disclosure;

[0039] FIG. 6 is a histogram illustrating the secretion of VEGF after a P12 generation of PDB-MSCs grow to the logarithmic growth phase in the cell media with different concentrations of exosome in Example 4 of the present disclosure (PDB-MSCs are cultivated in media with different concentrations of exosome); and

[0040] FIG. 7 is a histogram illustrating the secretion of SCF after a P12 generation of PDB-MSCs grow to the logarithmic growth phase in the cell media with different concentrations of exosome in Example 4 of the present disclosure (PDB-MSCs are cultivated in media with different concentrations of exosome).

DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions, and advantages of the present disclosure more clear, the present disclosure will be further described in detail below with reference to the accompanying drawings. It should be noted that orientation terms such as “upper”, “lower”, “left”, “right”, “front”, “rear”, “inner”, and “outer” that appear or are about to appear in the present disclosure are only based on the accompanying drawings of the present disclosure, and do not specifically limit the present disclosure.

Example 1

[0042] A preparation method of a human UC-MSC-derived exosome was provided, including the following steps:

[0043] S11. A placental tissue delivered by caesarean section at term was collected in a hospital, and then transported to a laboratory in a refrigerated and sterile environment at 4° C. Before the collection, an informed consent form was signed by a customer. During the transportation, a tissue protection solution was used to protect the biological activity of the placental tissue and prevent the placental tissue from bacterial and fungal contamination. The tissue protection solution was prepared from 1 ml of NS, 25 μg of gentamicin sulfate, and 5 μg of amphotericin B.

[0044] S12. In the laboratory, the umbilical cord tissue was washed with the tissue protection solution to remove blood on a surface, epidermal and vascular tissues were removed, and Wharton's jelly was taken out, washed, cut into pieces of 1 to 2 mm.sup.3, and inoculated in a T75 culture flask and cultivated with a serum-free selective medium of well-known components according to a tissue adhesion method.

[0045] S13. After growing to confluency of 70% on day 14 of cultivation, primary cells were subcultured; and according to a conventional cell cultivation operation, the subculturing was conducted until a P4 generation was obtained (Subculturing: after a generation of cells grew to confluency of greater than 80%, the cells were washed twice with PBS and then digested with trypsin for 5 min, a medium was added to terminate the digestion, a resulting mixture was filtered and centrifuged, and a resulting precipitate was resuspended with a cell medium, counted to calculate a survival rate, and subcultured. The trypsin was a 25% Tryple-EDTA enzyme).

[0046] S14. UC-MSCs with well growth conditions in the P4 generation were collected and inoculated in a Corning T175 culture flask at a density of 10,000 cells/cm.sup.2, 25 ml of a serum-free proliferation medium was added, and then the cells were cultivated normally.

[0047] S15. When the cells grew to confluency of 90%, a culture supernatant was collected (a culture supernatant of human UC-MSCs in the P5 generation, as shown in FIG. 1) to obtain a first supernatant, which would be used to extract an exosome; and the MSCs were subcultured normally, and a cultivation generation was determined according to a cell growth state.

[0048] S16. The collected first supernatant was centrifuged at 3,000 rpm for 20 min to remove cell debris, and a resulting supernatant was collected to obtain a second supernatant.

[0049] S17. The second supernatant was filtered with a 0.22 μL filter membrane and centrifuged at 120,000 rpm for 120 min, a resulting supernatant was discarded, and a resulting precipitate (which was the human UC-MSC-derived exosome) was resuspended with 1 ml of PBS and filtered with a 0.22 μL filter membrane, which was temporarily stored at 4° C. and long-term stored at −80° C.

[0050] An SEM image of the human UC-MSC-derived exosome is shown in FIG. 2, and it can be seen that the human UC-MSC-derived exosome is in a shape of a saucer or a hemisphere with a concave side.

Example 2

[0051] A method for promoting the proliferation of PDB-MSCs was provided, including the following steps:

[0052] S21. Preparation of cell media with different concentrations of exosome: the human UC-MSC-derived exosome prepared in Example 1 was added at different concentrations (0 μg/ml, 10 μg/ml, 20 μg/ml, and 30 μg/ml) to different MSC serum-free media (Youkang Hengye Biotechnology (Beijing) Co., Ltd., Item NO.: NC0103).

[0053] S22. A P12 generation of PDB-MSCs were inoculated in a 96-well plate at a concentration of 2×10.sup.3 cells/well, and the exosome-containing cell media obtained in S21 were added separately.

[0054] S23. The cells were cultivated in an incubator with saturated humidity at 37° C., 5% CO.sub.2, and 1% O.sub.2.

[0055] Each experiment was repeated at least three times.

[0056] PDB-MSCs were collected every 24 h and counted. FIG. 3 shows the morphology of PDB-MSCs cultivated with the human UC-MSC-derived exosome at different concentrations in Example 2. FIG. 4 shows cell growth curves of PDB-MSCs cultivated with the human UC-MSC-derived exosome at different concentrations in Example 2.

[0057] It can be seen from FIG. 3 and FIG. 4 that the growth of PDB-MSCs is accelerated when the PDB-MSCs are cultivated in the cell media with different concentrations of exosome.

Example 3

[0058] A method for promoting the proliferation of PDB-MSCs was provided, including the following steps:

[0059] S21. Preparation of cell media with different concentrations of exosome: the human UC-MSC-derived exosome prepared in Example 1 was added at different concentrations (0 μg/ml, 10 μg/ml, 20 μg/ml, and 30 μg/ml) to different MSC serum-free media (Youkang Hengye Biotechnology (Beijing) Co., Ltd., Item NO.: NC0103).

[0060] S22. A P12 generation of PDB-MSCs were inoculated in a 96-well plate at a concentration of 4×10.sup.5 cells/well, and the exosome-containing cell media obtained in S21 were added separately.

[0061] S23. The cells were cultivated in an incubator with saturated humidity at 37° C., 5% CO.sub.2, and 1% O.sub.2.

[0062] Each experiment was repeated at least three times.

[0063] PDB-MSCs were collected after 48 h of cultivation and reacted with a cell cycle detection kit (Solarbio) for 30 min, and then G1, G2, and S phases of the cell cycle were tested by a flow cytometer (Beckman). FIG. 5 shows the flow cytometry results of cell cycle of PDB-MSCs cultivated with the human UC-MSC-derived exosome at different concentrations in Example 3.

[0064] It can be seen from FIG. 5 that a proportion of cells at the S phase of the cell cycle increases when PDB-MSCs are cultivated in the cell media with different concentrations of exosome, and a proportion of cells at the S phase in the group with an exosome content of 20 μg/ml is higher than that in other groups, indicating that the co-cultivation of PDB-MSCs with the human UC-MSC-derived exosome added to a medium can accelerate the DNA replication and proliferation of PDB-MSCs.

Example 4

[0065] A method for promoting the proliferation of PDB-MSCs was provided, including the following steps:

[0066] S21. Preparation of cell media with different concentrations of exosome: the human UC-MSC-derived exosome prepared in Example 1 was added at different concentrations (0 μg/ml, 10 μg/ml, 20 μg/ml, and 30 μg/ml) to different MSC serum-free media (Youkang Hengye Biotechnology (Beijing) Co., Ltd., Item NO.: NC0103).

[0067] S22. A P12 generation of PDB-MSCs were inoculated in a 96-well plate at a concentration of 2.5×10.sup.5 cells/well, and the exosome-containing cell media obtained in S21 were added separately.

[0068] S23. The cells were cultivated in an incubator with saturated humidity at 37° C., 5% CO.sub.2, and 1% O.sub.2.

[0069] Each experiment was repeated at least three times.

[0070] When a P12 generation of PDB-MSCs grew to the logarithmic growth phase, 1 ml of a culture supernatant was collected and centrifuged at 3,000 rpm for 20 min to obtain a cell supernatant, and the VEGF and SCF ELISA kits (Enzyme-linked Biotechnology Co., Ltd.) were respectively used to detect VEGF and SCF contents in the cell supernatant. FIG. 6 is a histogram illustrating the secretion of VEGF after the P12 generation of PDB-MSCs grow to the logarithmic growth phase in the cell media with different concentrations of exosome in Example 4; and FIG. 7 is a histogram illustrating the secretion of SCF after the P12 generation of PDB-MSCs grow to the logarithmic growth phase in the cell media with different concentrations of exosome in Example 4.

[0071] It can be seen from FIG. 6 and FIG. 7 that when the PDB-MSCs are cultivated in the cell media with different concentrations of exosome, the secretion of both VEGF and SCF in PDB-MSCs increases, indicating that the addition of the human UC-MSC-derived exosome can promote the secretion of both VEGF and SCF in PDB-MSCs.

[0072] The above are only preferred examples of the present disclosure, and are not intended to limit the claimed scope of the present disclosure. Therefore, equivalent changes made according to the claims of the present disclosure are still within the scope of the present disclosure.