CN109593706B

CN109593706B - Culture medium and method for culturing endometrial stem cells

Info

Publication number: CN109593706B
Application number: CN201811354095.1A
Authority: CN
Inventors: 李文东; 宋云庆; 卢瑞珊; 李捷
Original assignee: Guangdong Huaxia Health Life Science Co ltd
Current assignee: Guangdong Huaxia Health Life Science Co ltd
Priority date: 2018-11-14
Filing date: 2018-11-14
Publication date: 2021-11-12
Anticipated expiration: 2038-11-14
Also published as: CN109593706A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a culture medium and a method for culturing endometrial stem cells. The invention provides a culture medium, comprising: serum-free medium, platelet lysate, N-acetylcysteine, galacturonic acid, and antibiotics. The invention provides a culture medium and a culture method of endometrial stem cells, which are used for solving the technical defects that the conventional culture medium culture can cause low proliferation efficiency, poor cell quality and easy differentiation of endometrial stem cells.

Description

Culture medium and method for culturing endometrial stem cells

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a culture medium and a method for culturing endometrial stem cells.

Background

Stem cells refer to a class of early undifferentiated cells with high proliferative potential, self-renewal capacity, and high differentiation capacity. Mesenchymal stem cells are currently the most interesting stem cell members, with high self-renewal capacity and multipotentiality. The human uterus is a tissue with high regeneration and differentiation capacity, the endometrium contains stem cells with high proliferation capacity, and scientists can separate the stem cells from menstrual blood of healthy women, and the stem cells are named as endometrial stem cells and are also called as menstrual blood source stem cells.

Endometrial stem cells (EDSCs) refer to undifferentiated endometrial cells that have the ability to self-renew, differentiate in multiple directions, and proliferate indefinitely, including minute quantities of endometrial epithelial cells, endometrial stromal cells, and most endometrial vascular endothelial cells. Because the endometrial stem cells have convenient material acquisition and are not limited by moral and legal problems, the endometrial stem cells have wide application prospects in the aspects of experimental research and clinical application. Preclinical studies and clinical trials of endometrial stem cells for the treatment of disease have been extensively studied, such as the treatment of myocardial infarction, stroke, diabetes, liver fibrosis, pulmonary fibrosis, premature ovarian failure, chronic ischemic pain, muscle atrophy, osteoarthritis.

The major current methods for separating endometrial stem cells are Ficoll separation, which is a mononuclear cell separated from menstrual blood, and cell natural sedimentation adherence, which contains various types of cells: human peripheral blood mononuclear cells, vascular endothelial cells, mesenchymal stem cells, endometrial epithelial cells, and the like, and thus endometrial stem cells can be obtained by a Ficoll density gradient centrifugation method. The method adopts a cell natural adherence method, and the purity of the cells obtained by the method is low, thus being not beneficial to the subsequent purification of the endometrial stem cells. In addition, the existing endometrial stem cells are directly inoculated after being separated, and the direct inoculation mode can influence the adherence of the endometrial stem cells; and the culture systems are common culture systems, so that the proliferation of the endometrial stem cells is slow, and the endometrial stem cells are differentiated due to overlong culture time.

In conclusion, the traditional stem cell culture medium is not suitable for culturing the endometrial stem cells, and the traditional stem cell culture medium can cause low proliferation efficiency and poor cell quality of the endometrial stem cells; after the endometrial stem cells proliferate for a limited number of generations, differentiation occurs; can not meet the clinical cell dosage requirement.

Disclosure of Invention

In view of the above, the present invention aims to provide a novel culture medium suitable for endometrial stem cell purification and large-scale expansion.

Another object of the present invention is to provide a method for isolating and culturing endometrial stem cells.

The invention provides a culture medium, comprising: serum-free medium, platelet lysate, N-acetylcysteine, galacturonic acid, and antibiotics.

Preferably, the platelet lysate is present in the culture medium in an amount of 10-20 wt.%.

Preferably, the platelet lysate is obtained by subjecting platelets to ultrasonic treatment.

Preferably, the content of the N-acetylcysteine in the culture medium is 2-5 wt.%.

Preferably, the content of galacturonic acid in the culture medium is 5-10 wt.%.

Preferably, the antibiotic is specifically streptomycin; the content of the antibiotic in the culture medium is 1 x-2 x. The working concentration of penicillin-streptomycin is 1X to 2X (1-2 mL of penicillin-streptomycin per 100mL of the medium).

Preferably, the serum-free medium is human mesenchymal stem cell serum-free medium.

The antibiotic contained in the culture medium can inhibit microorganisms in menstrual blood.

The invention also provides a culture method of the endometrial stem cells, the endometrial stem cells are cultured after being resuspended in a culture dish by using the culture medium, and the fresh culture medium is replaced every 2-4 days.

More preferably, the endometrial stem cells are obtained by collecting menstrual Blood of a human on the next and third days, separating a leukocyte layer by FFicoll density gradient centrifugation, and lysing erythrocytes of the menstrual Blood with an erythrocyte lysate (Red Blood Cell Lysis Buffer). The invention adopts erythrocyte lysate, which can effectively avoid the influence of erythrocyte on the adherence of endometrial stem cells.

Wherein, the menstrual blood of the first day can wash the fine fungi in the vagina, and the menstrual blood of 2-3 days in this period has better quality, and can also reduce pollution simultaneously.

Preferably, the culture dish is subjected to infiltration and incubation treatment of the human mesenchymal stem cell adherence promoting reagent in advance.

The culture dish is treated by the human mesenchymal stem cell adherence promoting reagent, so that the wall growth promotion of the intimal epithelial cells and the vascular endothelial cells of menstrual blood is promoted, the pollution of the intimal epithelial cells and the vascular endothelial cells of the menstrual blood is avoided, and the cell purification effect is achieved.

Preferably, the density of the resuspended endometrial stem cells is 8X 10⁴cells/mL～1×10⁵cells/mL。

Compared with the prior art, the culture medium provided by the invention adopts the platelet lysate which contains natural nutrient components, and the platelet lysate is the basic components of the platelets after the platelets are subjected to ultrasonic lysis, so that the substances contained in the platelets are split into the basic components and are more easily utilized by cells. The combined use of the platelet lysate, the N-acetylcysteine and the galacturonic acid is selected, so that on one hand, the culture medium is beneficial to the growth of the mesenchymal stem cells, and can comprehensively supplement nutrient components required by the growth of the mesenchymal stem cells, so that the mesenchymal stem cells can be rapidly divided and added with value in vitro; on the other hand, the culture medium can effectively avoid differentiation of the mesenchymal stem cells in the in vitro culture process, can also ensure that the mature mesenchymal stem cells of the endometrial stem cells can keep the original dryness, can be rapidly divided and added with value, has high cell purity, uniform quality and sufficient quantity, and overcomes the defect that the mesenchymal stem cells with higher differentiation degree are easy to differentiate in the in vitro culture.

In addition, the invention also provides a culture method of the endometrial stem cells, the culture method of the invention adopts a culture dish which is subjected to infiltration and incubation treatment by the human mesenchymal stem cell adherence promoting reagent in advance, so that adherence of endothelial cells and epithelial cells of menstrual blood can be further limited, and a cell purification effect is achieved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the results of detecting surface antigens of group 4 cultured endometrial stem cells provided in example 3 of the present invention;

FIG. 2 shows the cell proliferation tendencies of the endometrial stem cells cultured in groups 1-4 provided in example 4 of the present invention;

FIG. 3 shows the cell proliferation tendency of the endometrial stem cells cultured in group 4, patent 1 and patent 2 provided in example 4 of the present invention;

FIG. 4 is a graph showing the results of osteogenesis induced by endometrial stem cells of group 4, patent 1 and patent 2 provided in example 5 of the present invention;

FIG. 5 is a graph showing the results of osteogenesis induced by endometrial stem cells cultured in group 1 according to example 5 of the present invention;

FIG. 6 is a graph showing the results of osteogenesis induced by endometrial stem cells cultured in group 2 according to example 5 of the present invention;

FIG. 7 is a graph showing the results of osteogenesis induced by endometrial stem cells cultured in group 3 provided in example 5 of the present invention;

FIG. 8 is a graph showing the results of osteogenesis induced by group 4 cultured endometrial stem cells according to example 5 of the present invention.

Detailed Description

The invention provides a culture medium and a culture method of endometrial stem cells, which are used for solving the technical defects that the conventional culture medium culture can cause low proliferation efficiency, poor cell quality and easy differentiation of endometrial stem cells.

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The raw materials used in the following examples are all commercially available or self-made.

Example 1

The embodiment of the invention provides a preparation method of a part of reagents, which comprises the following steps:

1. the preparation method of the human mesenchymal stem cell adherence promoting reagent comprises the following steps: and (3) uniformly mixing the human mesenchymal stem cell adherence promoting reagent with a 1640 basic culture medium according to the volume ratio (v: v) of 1:99 to obtain the human mesenchymal stem cell adherence promoting reagent, and keeping the human mesenchymal stem cell adherence promoting reagent at the temperature of 4 ℃ for later use.

2. The preparation method of the platelet lysate comprises the following steps: taking 100mL of human peripheral blood at 4 ℃, centrifuging at 2000rpm for 15min, and transferring the upper plasma layer into a new centrifuge tube; the plasma was centrifuged at 3000rpm for 20min, leaving the bottom 20mL of plasma and platelets, and the upper plasma was aspirated. And (3) after the mixture is blown and beaten uniformly by a Pasteur pipette, carrying out ultrasonic lysis in an ultrasonic instrument for 10min to obtain a platelet lysate after the lysis is finished, and transferring the platelet lysate to an environment at 4 ℃ for later use.

3. The preparation method of the culture medium of group 1 comprises the following steps: MSC

XF Medium (purchased from BI Inc.: 05-200-1A) and 2 XQing-streptomycin were mixed well to give group 1.

4. The preparation method of the culture medium of group 2 comprises the following steps: MSC

XF Medium (purchased from BI Inc. Cat. 05-200-1A), 10 wt.% platelet lysate and penicillin were mixed well to give group 2.

5. The preparation method of the culture medium of group 3 comprises the following steps: MSC

XF Medium (purchased from BI Inc. Cat. 05-200-1A), 5 wt.% N-acetylcysteine, 5 wt.% galacturonic acid, and penicillin were mixed together to give group 3.

6. The preparation method of the culture medium of group 4 comprises the following steps: MSC

XF Medium (purchased from BI Inc. Cat. 05-200-1A), 10 wt.% platelet lysate, 5 wt.% N-acetylcysteine, 5 wt.% galacturonic acid, and streptomycin were mixed together to give group 4.

Example 2

The embodiment of the invention provides a method for culturing endometrial stem cells, which comprises the following steps:

1. adding the human mesenchymal stem cell adherence promoting reagent prepared in the example 1 into a culture dish, enabling the human mesenchymal stem cell adherence promoting reagent to infiltrate the bottom of the dish, placing the dish at 37 ℃ for incubation, and after 1h, removing the human mesenchymal stem cell adherence promoting reagent by suction.

2. Collecting menstrual blood of the person from the second day to the third day.

3. According to the reference documents (Zhouyanfan, populus, Huxiang, and the like; separation, culture and identification of menstrual Blood-derived endometrial mesenchymal stem cells [ J ], Chinese tissue engineering research and clinical rehabilitation; 2010, 14 (32): 5952-5956.), the menstrual Blood in the step 2 is separated into a white membrane layer by a Ficoll density gradient centrifugation method, Red Blood Cell lysate (Red Blood Cell Lysis Buffer) is used for lysing Red Blood cells of the menstrual Blood, the Red Blood Cell lysate is centrifuged at 1500rpm for 5min, the Red Blood Cell lysate is discarded, a 1640-based culture medium is used for resuspension, the Red Blood Cell lysate is centrifuged at 1500rpm for 5min, and the supernatant is discarded to obtain a Cell precipitate.

4. Resuspending the cell pellet of step 3 with the media of groups 1-4 prepared in example 1, respectively, adjusted to a density of 10⁵cells/mL, then, inoculated into the step 1 incubation Petri dish.

5. Transfer the Petri dish of step 4 to 37 ℃ with 5% CO₂Culturing in an environment with 95% humidity, and changing the culture solution after 24 h.

6. The culture medium of the corresponding group 1-group 4 is replaced once every 3 days, and cell subculture is carried out until the cell fusion rate reaches about 80%.

Example 3

The embodiment of the invention provides surface antigen detection of endometrial stem cells, which comprises the following specific steps:

taking endometrium stem cell, and making into 1 × 10⁶cell suspension of cells/mL, respectively taking 5 muL of monoclonal antibodies of anti-human CD45, CD14, CD29, CD90, CD105 and HLA-DR, adding 500 muL of cell suspension cultured by group 4, incubating for 20min in dark place at room temperature, simultaneously establishing blank isotype control, centrifuging for 5min at 1500r/min, discarding supernatant, washing for 2 times by PBS containing 10% FBS, resuspending by 500 muL of PBS, detecting on a machine, respectively obtaining endometrial stem cells of two patents according to methods of domestic patents CN105586308A (a stem cell culture medium and a method for culturing the endometrial stem cells, which are marked as patent 1) and CN106801034A (a large-scale preparation method of the endometrial stem cells and application thereof, which are marked as patent 2), and detecting surface antigens of patent 1 and patent 2 by adopting the detection method, wherein the results are shown in Table 1 and figure 1.

TABLE 1

Name of surface antigen	CD29	CD90	CD105	CD14	CD45	HLA-DR
							Group
4	97.5％	98.6％	95.4％	1.1％	0.8％	0.3％
							Patent
1	98.4％	94.7％	91.7％	1.3％	2.8％	2.6％
							Patent
2	96.9％	97.2％	93.8％	1.5％	5.9％	4.8％
							Group
1	47.5％	57.6％	72.8％	0.8％	5.4％	0.1％
							Group
2	95.2％	93.8％	96.7％	1.6％	1.3％	0.0％
							Group
3	96.8％	95.3％	98.1％	2.5％	5.7％	3.3％

As is clear from the results of table 1 and fig. 1, the culture medium of group 4, compared with the culture medium of patent 1 and patent 2, gave endometrial stem cells having high purity. The culture medium of group 1 has no effective nutrient components, and the endometrial stem cells have an automatic differentiation phenomenon when being cultured in vitro; the cell proliferation rate of the culture medium of group 2 is slow, and partial cells have enlarged volume and deformed shape; the cell proliferation rate of the culture medium of group 3 is slow, and part of cells have enlarged volume and deformed shape, which shows that the platelet lysate, N-acetylcysteine and galacturonic acid have the function of synergistically promoting the proliferation of endometrial stem cells.

Example 4

The embodiment of the invention provides a method for detecting a growth curve of endometrial stem cells, which comprises the following steps:

re-suspending and inoculating the group 1-group 4 obtained by the culture in the step 2 and the P2-P3 generation endometrial stem cells of the patent 1 and the patent 2 in the step 3 into a 6-well plate, wherein the cell inoculation density is 5 multiplied by 10⁴cells/mL, 2 mL/well. The total number of cells was counted by taking one well cell every 2 days, and the growth curves of the cells of groups 1 to 4 and patent 1 and patent 2 were recorded, and the results are shown in table 2 and fig. 2 to 3.

TABLE 2

Denotes P <0.05, significant difference compared to group 1.

Indicates P <0.001, a very significant difference compared to group 1.

As can be seen from table 2 and fig. 2, the cell proliferation curves of the endometrial stem cells cultured in group 1 were gentle, and no significant increase in number occurred throughout the entire culture period; the cell latency of the endometrial stem cells cultured in the

groups

2 and 3 is longer, obvious logarithmic growth begins to appear on the 9 th day, but the cell number is still small; group 4 cultured endometrial stem cells started to proliferate significantly after 6 days of incubation and entered the plateau phase at 15 days. The platelet lysate, the N-acetylcysteine and 5-10 wt.% galacturonic acid have obvious synergistic promotion effect on division and proliferation of the endometrial stem cells.

As can be seen from fig. 3, the cell proliferation tendency of the endometrial stem cells cultured in group 4 is better than that of the endometrial stem cells cultured in patent 1 and patent 2, which shows that the endometrial stem cells can be promoted to proliferate by the culture medium culture of the present patent.

Example 5

The embodiment of the invention provides an osteogenesis adipogenesis detection method of endometrial stem cells, which comprises the following steps:

reference documents: research on the culture and identification of menstrual blood source endometrial stem cells and the potential for in vitro differentiation, Yanyan, Dongjianhexi, sauropus and the like, Chinese cytobiology reports [ J ].2014, 36 (7): 892-899, the differentiation of the endometrial stem cells into osteogenic fat cells induced by this reference for groups 1-4, patent 1 and patent 2 is shown in FIGS. 4-8.

As can be seen from FIG. 4, FIG. 4-A shows that the endometrial stem cells cultured in patent 2 do not exhibit the phenomenon of fat formation, which indicates that the cells have differentiated during the in vitro culture process, and the differentiation capacity of the cells is lost, so that no fat drop is formed during the in vitro induction of fat formation; FIG. 4-B shows the cells cultured in patent 1, wherein lipid droplets are visible, but some of the cells still do not contain lipid droplets, indicating that some of the cells have differentiated; FIG. 4-C shows that the cells cultured in group 4 of the present invention showed large-area "lipid droplets", indicating that the cultured endometrial stem cells did not differentiate and maintained good dryness when cultured in vitro.

FIG. 5-A shows the osteogenesis results of the endometrial stem cells cultured in the culture medium of group 1, FIG. 5-B shows the adipogenic results, and as can be seen from FIG. 5, the osteogenesis and adipogenic phenomena do not appear in the culture medium of group 1, which indicates that the endometrial stem cells have differentiated and lost the differentiation capability of the cells during the in vitro culture process; FIG. 6-A shows the osteogenic results of the endometrial stem cells cultured in the culture medium of group 2, and FIG. 6-B shows the adipogenic results, and as can be seen from FIG. 6, most of the endometrial stem cells cultured in group 2 have no osteogenic adipogenic phenomenon, and a small part of the endometrial stem cells have the capacity of differentiating into osteogenic adipogenic substances, which indicates that most of the endometrial stem cells cultured in group 2 have differentiated during the in vitro culture process and lose the differentiation capacity of the cells; FIG. 7-A shows the osteogenesis results of the endometrial stem cells cultured in the culture medium of group 2, and FIG. 7-B shows the adipogenic results, and as can be seen from FIG. 7, most of the endometrial stem cells cultured in group 3 have no osteogenesis phenomenon, and a small part of the endometrial stem cells have the capacity of differentiating into osteogenesis, which indicates that most of the endometrial stem cells cultured in group 3 have differentiated during the in vitro culture process and lose the differentiation capacity of the cells; fig. 8-a shows the osteogenesis results of the endometrial stem cells cultured in the culture medium of group 4, fig. 8-B shows the adipogenic results, and fig. 8 shows that most of the endometrial stem cells cultured in group 4 have osteogenesis and adipogenic phenomena, which indicates that most of the endometrial stem cells cultured in group 4 are undifferentiated in the in vitro culture process, have differentiation capacity of the stem cells, and indicate that the culture medium of group 4 can maintain the dryness of the endometrial stem cells.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A culture medium, comprising: serum-free medium, platelet lysate, N-acetylcysteine, galacturonic acid, and antibiotics; the platelet lysate is present in the culture medium in an amount of 10 wt.%; the content of the N-acetylcysteine in the culture medium is 5 wt.%; the content of galacturonic acid in the culture medium is 5 wt.%; the antibiotic is specifically penicillin-streptomycin; the content of the antibiotic in the culture medium is 1 x-2 x.

2. The culture medium of claim 1, wherein the platelet lysate is obtained by sonicating platelets.

3. The culture medium according to claim 1, wherein the serum-free culture medium is specifically human mesenchymal stem cell serum-free culture medium.

4. A method for culturing endometrial stem cells, which is characterized in that the endometrial stem cells are cultured by the culture medium of any one of claims 1 to 3 after being resuspended in a culture dish, and fresh culture medium of any one of claims 1 to 3 is replaced every 2 to 4 days.

5. The culture method according to claim 4, wherein the culture dish is previously subjected to infiltration and incubation with a human mesenchymal stem cell adherence-promoting agent.

6. The culture method according to claim 4, wherein the density of the resuspended endometrial stem cells is 8 x 10⁴cells/mL～1×10⁵cells/mL。