Culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells

Abstract

The invention relates to the technical field of cell biology, in particular to a culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells, which comprises the following steps: cutting Wharton's jelly tissues in an umbilical cord, placing the Wharton's jelly tissues in a centrifugal tube, cutting the Wharton's jelly tissues into fragments, digesting and centrifuging the cut-up tissues, adding a low-sugar DMEM culture medium, and culturing the low-sugar DMEM culture medium to obtain the umbilical cord mesenchymal stem cells. Calcitonin with the final concentration being 20 mol, cordycepin with the final concentration being 10 mol and NaCl with the final concentration being 2% are added into a low-sugar DMEM culture medium, when the umbilical cord mesenchymal stem cells grow to 80% of the bottom area of a culture bottle, digestion and centrifugation are conducted, the low-sugar DMEM culture medium is added, and NaCl with the final concentration being 2% is added into the low-sugar DMEM culture medium. Therefore, when the umbilical cord mesenchymal stem cells are proliferated, the cell Ca < 2 + > concentration and the channel transmembrane transport efficiency can be reduced, and the expression quantity of cAMP can be reduced, so that the asymmetric division of the umbilical cord mesenchymal stem cells is improved.

Description

Technical Field

[0001] The invention relates to the technical field of cell biology, and in particular to a culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells. Background Art

[0002] Stem cells are a group of cells with high self-renewal and multidirectional differentiation potentials, which can replenish aging, damaged or dead cells in mature organs. They have been increasingly widely used in the research of a variety of terminal and refractory diseases.

[0003] Mesenchymal stem cells have the advantages of easy separation, high proliferation potential and genetic stability. They can also migrate to damaged tissues to fight inflammation, affect the microenvironment, promote angiogenesis, resist fibrosis, resist apoptosis, and release cytokines to participate in the repair process and tissue regeneration. Mesenchymal stem cells are easy to obtain and separate and culture. Under appropriate conditions, they can differentiate into various tissue cells such as bones, skin, blood vessels and nerves. They are currently widely used in many fields such as tissue repair and organ reconstruction. However, mesenchymal stem cells will undergo symmetrical division and asymmetrical division during the culture process. Asymmetrical division will split into two stem cells of different sizes, and larger stem cells still have the potential for multidirectional differentiation, but smaller stem cells basically lose the ability to differentiate in multiple directions and become unipotent stem cells or progenitor cells. Increasing the incidence of asymmetric division during the culture of mesenchymal stem cells, thereby promoting stem cell differentiation in a specific direction, and improving the damage repair effect of mesenchymal stem cells on specific targeted organs has important clinical significance for improving the effect of subsequent research and clinical transformation and application of mesenchymal stem cells.

[0004] How to reduce cell Ca during the proliferation of umbilical cord mesenchymal stem cells 2+ Concentration and channel transmembrane transport efficiency, reducing the expression of cAMP, and thus improving the asymmetric division of umbilical cord mesenchymal stem cells has become a technical problem that needs to be broken through.

[0005] In summary, the prior art obviously has inconveniences and defects in practical use, so it is necessary to improve it. Summary of the invention

[0006] In view of the above-mentioned defects, the purpose of the present invention is to provide a culture method for promoting the asymmetric division of umbilical cord mesenchymal stem cells, which has a simple structure and is easy to use. It can effectively improve the accuracy of positioning the advance small catheter, improve the ability of the advance small catheter to withstand the low relaxation stress of the surrounding rock and the bending and shear resistance, and reduce the safety risks of tunnel construction.

[0007] In order to achieve the above object, the present invention provides a culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells, comprising the following steps: Step 1 Umbilical Cord Tissue Processing Preheat a low-glucose DMEM culture medium bottle, cut and clean the umbilical cord, cut the umbilical cord with a dissecting tool, remove the blood vessels, cut the Wharton's jelly tissue in the umbilical cord, place it in a centrifuge tube and cut it into pieces.

[0008] Step 2: Extraction of umbilical cord mesenchymal stem cells The chopped tissue was digested, and the suspended tissue was centrifuged to precipitate cells, the supernatant was discarded, and low-glucose DMEM medium was added. The cells were inoculated into the culture flask, and calcitonin with a final concentration of 20 mol, cordyceps sinensis with a final concentration of 10 mol, and NaCl with a final concentration of 2% were added to the low-glucose DMEM medium. 5% CO2 was passed through the medium for constant temperature culture, and new low-glucose DMEM medium was replaced every 3 to 4 days.

[0009] Step 3: Subculture of umbilical cord mesenchymal stem cells When the umbilical cord mesenchymal stem cells grow to 80% of the bottom area of ​​the culture bottle, discard the original culture medium in a sterile environment, wash with pre-cooled PBS solution, add trypsin digestion solution for digestion, and when 20% of the cells slide down the wall of the culture bottle, add 10% fetal bovine serum low-glucose DMEM culture medium to terminate digestion, pipette the cell suspension into a centrifuge tube for centrifugation, discard the supernatant, add low-glucose DMEM culture medium, and blow into single cells, inoculate the cells into a new culture bottle, add NaCl with a final concentration of 2% to the low-glucose DMEM culture medium, and replace it with new low-glucose DMEM culture medium regularly.

[0010] According to the culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells of the present invention, the new low-sugar DMEM culture medium is a low-sugar DMEM culture medium containing 20 mol calcitonin, 10 mol cordycepin, and 2% NaCl.

[0011] According to the culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells of the present invention, in the step 2, during the digestion treatment, pancreatic digestion solution is added to the chopped tissue and incubated in a 37°C water bath for 10 to 20 min, and then a digestion stop solution of the same volume as the digestion solution is added.

[0012] According to the culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells of the present invention, in step 1, when cleaning the umbilical cord, the umbilical cord is first cut and then repeatedly rinsed with sterile saline for 3 to 5 times to wash out the blood clots in the blood vessels, then rinsed with 75% ethanol, and then quickly rinsed with saline, and finally washed with 1× double antibody solution and rinsed with saline.

[0013] According to the culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells of the present invention, the dissection tools include scissors, ophthalmic scissors, tweezers and hemostatic forceps, all of which need to be sterilized by high-pressure steam before use.

[0014] According to the culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells of the present invention, in step 2, the centrifugation condition is 1500 rpm for 10 min, and in step 3, the centrifugation condition is 2000 rpm for 10 min.

[0015] According to the culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells of the present invention, in the step three, during the primary subculture and P2-P3 subculture of the umbilical cord mesenchymal stem cells, new low-glucose DMEM culture medium is replaced every 2 to 3 days, and during the P3-P4 subculture of the umbilical cord mesenchymal stem cells, new low-glucose DMEM culture medium is replaced every day.

[0016] The purpose of the present invention is to provide a culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells by adding calcitonin, cordycepin, NaCl, etc. to reduce cell Ca 2+ concentration, thereby reducing Ca 2+ The transmembrane transport efficiency of the channel is improved, the expression of cAMP is reduced, and the asymmetric division of umbilical cord mesenchymal stem cells is greatly improved during the proliferation of umbilical cord mesenchymal stem cells. In summary, the beneficial effects of the present invention are: it can reduce the expression of cAMP during the proliferation of umbilical cord mesenchymal stem cells, and the expression of cAMP is reduced. 2+ concentration and channel transmembrane transport efficiency, reducing the expression of cAMP, thereby improving the asymmetric division of umbilical cord mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the proliferation of umbilical cord mesenchymal stem cells cultured using two culture methods using MTT assay; Figure 2 The morphological images of two groups of umbilical cord mesenchymal stem cells P3-P4 under a microscope; Figure 3 This is a diagram showing the ratio of asymmetric division of two groups of mesenchymal stem cells; DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] In the description of the present invention, it should be noted that the terms "upper", "lower", "inside", "outside", "top / bottom" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0020] In the description of the present invention, it should be noted that, unless otherwise clearly stipulated and limited, the terms "installed", "provided with", "mounted / connected", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection. It can be a mechanical connection or an electrical connection. It can be a direct connection or an indirect connection through an intermediate medium. It can be the internal connection of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0021] The present invention provides a culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells, comprising the following steps: Step 1 Umbilical Cord Tissue Processing Place the low-glucose DMEM medium bottle in a 37° constant temperature water bath to preheat. Use dissecting tools (scissors) under sterile conditions to cut the umbilical cord into a length of about 5 cm. First, rinse repeatedly with sterile saline for 3 to 5 times to wash out the blood clots in the blood vessels. Then rinse with 75% ethanol, and then quickly rinse with saline. Finally, wash with 1× double antibody solution and rinse with saline. Cut the umbilical cord longitudinally and remove the blood vessels with tweezers. Use dissecting tools (ophthalmic scissors) to cut the Wharton's jelly tissue in the umbilical cord. Add the retained internal Wharton's jelly tissue to a 50 mL centrifuge tube and cut the internal tissue into about 1 mm 3 of fragments.

[0022] The dissection tools include scissors, ophthalmic scissors, forceps and hemostats, all of which need to be sterilized by high-pressure steam (120°C, 20 min) before use. The umbilical cord tissue of the present invention was collected in Weifang City, Shandong Province.

[0023] Step 2: Extraction of umbilical cord mesenchymal stem cells Add an equal volume of trypsin digestion solution to the chopped tissue. Incubate in a 37℃ water bath for 10-20 min. Add an equal volume of digestion stop solution to the digestion solution. Mix well to avoid the generation of air bubbles. Centrifuge the suspended tissue at 1500rpm for 10 min to precipitate the cells. Discard the supernatant and add an appropriate amount of low-glucose DMEM medium to a final volume of about 25 mL. Inoculate cells. Inoculate one 75 cm 2 Culture flask. Add calcitonin with a final concentration of 20 mol, cordycepin with a final concentration of 10 mol, and NaCl with a final concentration of 2% to the low-glucose DMEM medium, place in a 37°C constant temperature incubator, and culture under 5% CO2 gas phase conditions. Replace the low-glucose DMEM medium containing 20 mol calcitonin, 10 mol cordycepin, and 2% NaCl every 3 to 4 days.

[0024] Step 3: Primary subculture of umbilical cord mesenchymal stem cells When the umbilical cord mesenchymal stem cells grow to 75 cm 2 When the cells covered 80% of the bottom area of ​​the culture flask, discard the original culture medium in a sterile environment, wash twice with 8 mL of pre-cooled PBS solution, add 5 mL of trypsin digestion solution for digestion, and when about 20% of the cells slid down the wall of the culture flask, add 10% fetal bovine serum low-glucose DMEM culture medium to terminate the digestion, use an electric pipette to blow all the cells off, pipette the cell suspension into a 50 mL centrifuge tube, centrifuge at 2000 rpm for 10 min, discard the supernatant, add low-glucose DMEM culture medium, blow into single cells, and inoculate the cells into a new 75 cm 2 Place the cells in the culture flask and add NaCl at a final concentration of 2% to the low-glucose DMEM medium. Replace the medium with new low-glucose DMEM medium containing 20 mol calcitonin, 10 mol cordycepin and 2% NaCl every 2 to 3 days.

[0025] Step 4: P2-P3 subculture of umbilical cord mesenchymal stem cells When the umbilical cord mesenchymal stem cells grow to 75 cm 2 When the cells covered 80% of the bottom area of ​​the culture flask, discard the original culture medium in a sterile environment, wash twice with 8 mL of pre-cooled PBS solution, add 5 mL of trypsin digestion solution for digestion, and when about 20% of the cells slid down the wall of the culture flask, add 10% fetal bovine serum low-glucose DMEM culture medium to terminate the digestion, use an electric pipette to blow all the cells off, pipette the cell suspension into a 50 mL centrifuge tube, centrifuge at 2000 rpm for 10 min, discard the supernatant, add low-glucose DMEM culture medium, blow into single cells, and inoculate the cells into a new 175 cm 2 In the culture flask, add NaCl with a final concentration of 2%, calcitonin with a final concentration of 30 mol, and cordycepin with a final concentration of 20 mol to the low-glucose DMEM medium.

[0026] Step 5: P3-P4 subculture of umbilical cord mesenchymal stem cells When the umbilical cord mesenchymal stem cells grow to 175 cm 2 When the cells covered 80% of the bottom area of ​​the culture flask, discard the original culture medium in a sterile environment, wash twice with 8 mL of pre-cooled PBS solution, add 5 mL of trypsin digestion solution for digestion, and when about 20% of the cells slid down the wall of the culture flask, add 10% fetal bovine serum low-glucose DMEM culture medium to terminate the digestion, use an electric pipette to blow all the cells off, pipette the cell suspension into a 50 mL centrifuge tube, centrifuge at 2000 rpm for 10 min, discard the supernatant, add new low-glucose DMEM culture medium, blow into single cells, and inoculate the cells into a new 175 cm2 In the culture flask, the new low-glucose DMEM medium containing 20 mol calcitonin, 10 mol cordycepin, and 2% NaCl was replaced every day.

[0027] See also Figures 1 to 3 ( Figure 2 In the figure, the left side shows P3-P4 umbilical cord mesenchymal stem cells cultured by conventional culture method, and the right side shows P3-P4 umbilical cord mesenchymal stem cells cultured by the method of promoting asymmetric division of mesenchymal stem cells in the present invention). The conventional umbilical cord mesenchymal stem cell culture method is used as a comparative experiment to further illustrate the effect of the present invention (the conventional umbilical cord mesenchymal stem cell culture method is a mature prior art in the field, and its specific method and principle are not repeated here). The cultured umbilical cord mesenchymal stem cells are compared, and the following results are obtained: The MTT method was used to detect and compare the proliferation of umbilical cord mesenchymal stem cells cultured by the conventional culture method and the culture method of the present invention. The proliferation of P1-P2 umbilical cord mesenchymal stem cells cultured by the conventional culture method was (20.54±2.98), while the proliferation of P2-P3 umbilical cord mesenchymal stem cells cultured by the culture method for improving asymmetric division of the present invention was (19.76±3.32); the proliferation of P2-P3 umbilical cord mesenchymal stem cells cultured by the conventional culture method was (31.61±3.45), while the proliferation of P2-P3 umbilical cord mesenchymal stem cells cultured by the culture method for improving asymmetric division of the present invention was (29.64±4.14); the proliferation of P3-P4 umbilical cord mesenchymal stem cells cultured by the conventional culture method was (40.74±4.15), while the proliferation of P3-P4 umbilical cord mesenchymal stem cells cultured by the culture method for improving asymmetric division of the present invention was (43.26±4.63).

[0028] Mesenchymal stem cells will undergo symmetrical and asymmetrical divisions during the culture process. Symmetrical division will split into two larger stem cells of the same size, both of which have the ability of multidirectional differentiation and the characteristics of unlimited proliferation. Asymmetric division will split into two stem cells of different sizes, of which the larger one still has the ability of multidirectional differentiation and the characteristics of unlimited proliferation, while the smaller one basically loses the ability of multidirectional differentiation and the characteristics of unlimited proliferation and becomes a fibroblast or progenitor cell. The progenitor cell will carry out targeted repair of the target organ in the body.

[0029] Under an inverted microscope, 5 fields of view were selected for each group of cells to statistically analyze the asymmetric division ratios of the two groups of umbilical cord mesenchymal stem cells. The results showed that the asymmetric division ratio of the umbilical cord mesenchymal stem cells of the P1-P2 generations cultured by the conventional culture method was (56.15±3.71)%, while the asymmetric division ratio of the umbilical cord mesenchymal stem cells cultured by the culture method for improving asymmetric division of the present invention was (74.69±4.46)%; the asymmetric division ratio of the umbilical cord mesenchymal stem cells of the P2-P3 generations cultured by the conventional culture method was (74.69±4.46)%. The asymmetric division ratio of P2-P3 umbilical cord mesenchymal stem cells cultured by the conventional culture method was (44.71±3.92)%, while the asymmetric division ratio of P2-P3 umbilical cord mesenchymal stem cells cultured by the culture method for improving asymmetric division in the present invention was (79.62±5.45)%; the asymmetric division ratio of P3-P4 umbilical cord mesenchymal stem cells cultured by the conventional culture method was (39.69±4.26)%, while the asymmetric division ratio of P3-P4 umbilical cord mesenchymal stem cells cultured by the culture method for improving symmetric division in the present invention was (86.26±6.71)%.

[0030] In summary, the present invention adds different concentrations of calcitonin, cordycepin, NaCl, etc., while promoting the proliferation of umbilical cord mesenchymal stem cells, reduces the expression of cAMP to 68.31% of the normal level, and increases the asymmetric division ratio of P1-P2 umbilical cord mesenchymal stem cells cultured in ordinary low-glucose DMEM medium from 56.15% to 74.69%, the asymmetric division ratio of P2-P3 umbilical cord mesenchymal stem cells from 44.71% to 79.62%, and the asymmetric division ratio of P3-P4 umbilical cord mesenchymal stem cells from 39.69% to 86.26%, thereby promoting and improving the multidirectional differentiation potential of umbilical cord mesenchymal stem cells.

[0031] The present invention provides a culture method for promoting asymmetric division of umbilical cord mesenchymal stem cells, by adding calcitonin, cordycepin, NaCl, etc., to reduce cell Ca 2+ concentration, thereby reducing Ca 2+ The transmembrane transport efficiency of the channel is improved, the expression of cAMP is reduced, and the asymmetric division of umbilical cord mesenchymal stem cells is greatly improved during the proliferation of umbilical cord mesenchymal stem cells. In summary, the beneficial effects of the present invention are: it can reduce the expression of cAMP during the proliferation of umbilical cord mesenchymal stem cells, and the expression of cAMP is reduced. 2+ concentration and channel transmembrane transport efficiency, reducing the expression of cAMP, thereby improving the asymmetric division of umbilical cord mesenchymal stem cells.

[0032] Of course, the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art may make various corresponding changes and modifications based on the present invention, but these corresponding changes and modifications should all fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for promoting asymmetric division of umbilical cord mesenchymal stem cells, characterized in that: The steps include: Step 1 Umbilical Cord Tissue Processing Preheat a low-glucose DMEM medium bottle, cut and clean the umbilical cord, cut the umbilical cord with a dissecting tool, remove the blood vessels, cut the Wharton's jelly tissue in the umbilical cord, place it in a centrifuge tube and cut it into pieces; Step 2: Extraction of umbilical cord mesenchymal stem cells Digest the chopped tissues, centrifuge the suspended tissues to precipitate cells, discard the supernatant, add low-glucose DMEM medium, inoculate cells into culture bottles, add calcitonin with a final concentration of 20 mol, cordycepin with a final concentration of 10 mol, and NaCl with a final concentration of 2% to the low-glucose DMEM medium, and culture at a constant temperature of 5% CO2. Replace the low-glucose DMEM medium every 3 to 4 days; Step 3: Subculture of umbilical cord mesenchymal stem cells When the umbilical cord mesenchymal stem cells grow to 80% of the bottom area of ​​the culture bottle, discard the original culture medium in a sterile environment, wash with pre-cooled PBS solution, add trypsin digestion solution for digestion, and when 20% of the cells slide down the wall of the culture bottle, add 10% fetal bovine serum low-glucose DMEM culture medium to terminate digestion, pipette the cell suspension into a centrifuge tube for centrifugation, discard the supernatant, add low-glucose DMEM culture medium, and blow into single cells, inoculate the cells into a new culture bottle, add NaCl with a final concentration of 2% to the low-glucose DMEM culture medium, and replace it with new low-glucose DMEM culture medium regularly.

2. The method for promoting asymmetric division of umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The new low-glucose DMEM medium is a low-glucose DMEM medium containing 20 mol calcitonin, 10 mol cordycepin, and 2% NaCl.

3. The method for culturing umbilical cord mesenchymal stem cells for promoting asymmetric division according to claim 1, characterized in that: In the step 2, during the digestion treatment, pancreatic digestion solution is added to the chopped tissue and incubated in a 37° C. water bath for 10 to 20 min, and then a digestion stop solution having the same volume as the digestion solution is added.

4. The method for culturing umbilical cord mesenchymal stem cells for promoting asymmetric division according to claim 1, characterized in that: In step 1, when cleaning the umbilical cord, first cut the umbilical cord and rinse it repeatedly with sterile saline for 3 to 5 times to wash out the blood clots in the blood vessels, then rinse it with 75% ethanol, then quickly rinse it with saline, and finally wash it with 1× double antibody solution and rinse with saline.

5. The method for culturing umbilical cord mesenchymal stem cells for promoting asymmetric division according to claim 1, characterized in that: The dissection tools include scissors, ophthalmic scissors, tweezers and hemostats, all of which need to be sterilized by high-pressure steam before use.

6. The method for culturing umbilical cord mesenchymal stem cells for promoting asymmetric division according to claim 1, characterized in that: In the step 2, the centrifugation condition is 1500 rpm for 10 min, and in the step 3, the centrifugation condition is 2000 rpm for 10 min.

7. The method for culturing umbilical cord mesenchymal stem cells for promoting asymmetric division according to claim 1, characterized in that: In the step 3, during the primary subculture and P2-P3 subculture of the umbilical cord mesenchymal stem cells, the low-glucose DMEM medium is replaced every 2-3 days, and during the P3-P4 subculture of the umbilical cord mesenchymal stem cells, the low-glucose DMEM medium is replaced every day.

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