Separation culture method and application of porcine endometrial mesenchymal stem cells

Abstract

The invention belongs to the technical field of cell biology and regenerative medicine, and particularly relates to a pig endometrial mesenchymal stem cell separation culture method and application, separation is carried out from uterus tissue of a large animal, namely, a pig, the steps are simple, the cell obtaining amount is large, the cultured endometrial mesenchymal stem cells have phenotypes of CD34-and CD11b-, and CD44 + and CD105 +, and the endometrial mesenchymal stem cells can be used for separating and culturing the endometrial mesenchymal stem cells. In vitro induced differentiation into bone cells, adipocytes and chondrocytes, during culture, collagen in extracellular matrix is decomposed, DNA is degraded, cell agglutination is reduced and effective digestion of tissues is promoted jointly through combined use of type II collagenase and DNase I, and a high-quality single-cell suspension can be obtained for subsequent centrifugation and culture; cells with intrauterine adhesion, skin and blood vessel injury and the like can be repaired, the repairing method is simple and easy to implement, regeneration and repairing can be performed by compositely implanting the material and a biological material into a focus part, and a new way is provided for cell transplantation treatment.

Description

Technical field:

[0001] The invention belongs to the technical field of cell biology and regenerative medicine, and specifically relates to a method for separating and culturing pig endometrial mesenchymal stem cells and an application thereof. Background technology:

[0002] The development of regenerative medicine and tissue engineering technology combines the theories and methods of multiple disciplines such as life science, engineering, computer science, and modern bioengineering technologies such as materials science, cell technology, and genetic engineering technology, providing new means and methods for tissue regeneration and repair, which can repair, replace, and enhance damaged, diseased, or defective tissues and organs in the human body. Stem cell therapy, a key component of regenerative medicine, is widely used in the medical field, and some product pipelines have entered the clinical stage. Stem cells have strong differentiation and regeneration capabilities and immunomodulatory effects. While becoming a research hotspot in regenerative medicine, they also face many challenges. Choosing a suitable cell source has always troubled researchers.

[0003] Mesenchymal stem cells (MSCs) are multipotent stem cells with multidirectional differentiation potential and self-renewal ability. They have the potential to regenerate various tissues, organs and cells. Related clinical studies have been widely carried out and have achieved many major breakthroughs. Endometrial mesenchymal stem cells (eMSCs) are cells located in the functional layer and basal layer of the endometrium. They are responsible for the regeneration and remodeling of the endometrium during the menstrual cycle. They have multipotent differentiation potential and self-renewal ability and can differentiate into a variety of cell types, including endothelial cells, smooth muscle cells and osteoblasts. eMSCs show great potential in regenerative medicine, especially in the treatment of endometrial damage and the promotion of endometrial regeneration. Studies have shown that eMSCs can promote angiogenesis and stromal cell proliferation by secreting growth factors or exosomes, while regulating inflammatory responses and inhibiting the regeneration of fibrotic endometrium. In the process of repairing endometrial damage, eMSCs can migrate to the damaged site, promote cell proliferation, angiogenesis, gland formation, and regeneration of epithelial and stromal components, thereby improving the thickness and integrity of the endometrium. eMSCs also show a regulatory effect on inflammatory responses, being able to reduce the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors, helping to create an environment more conducive to embryo implantation. In addition, eMSCs have also shown potential in the treatment of endometrial adhesions (Asherman syndrome). Asherman syndrome is a condition in which endometrial adhesions form due to infection or trauma, which reduces the available area for embryo implantation and leads to infertility. As local cells of the endometrium, eMSCs have a natural advantage in periodic regeneration and have become an ideal cell source for repairing damaged endometrium. In general, eMSCs have significant potential in endometrial repair and regeneration, providing new strategies and hope for the treatment of endometrial-related diseases and new therapeutic options for clinical applications. Summary of the invention:

[0004] The specific process of the pig endometrial mesenchymal stem cell isolation and culture method according to the present invention comprises the following steps:

[0005] (1) Obtaining tissue

[0006] Fresh porcine uterus (with ovaries) tissue was stored in ice-cold phosphate-buffered saline (PBS);

[0007] (2) Pretreatment of tissue

[0008] First, in a sterile room, the tissue was cut open, soaked and rinsed in pre-cooled sterile PBS (pH = 7.4) supplemented with magnesium chloride and calcium chloride;

[0009] Then, the outermost serosa layer was peeled off to separate the pig endometrium and myometrium;

[0010] Finally, the fragmented inner membrane layer was placed in an incubator at 37°C and digested with a basic medium supplemented with digestive enzymes for 2-3 hours, and pipetted every 15 minutes to obtain a cell suspension;

[0011] (3) Initial centrifugation

[0012] First, the cell suspension was filtered through 100 and 40 μm cell strainers, MSC (mesenchymal stem cell) complete medium was added to prevent further digestion, washed with PBS, and centrifuged at 1000 rpm for 5 min;

[0013] Then, the cells were treated twice with red blood cell lysis buffer and washed with PBS;

[0014] Finally, cells were cultured at 500 cell / cm 2 The clone density was inoculated in MSC complete medium and incubated at 37.0°C in a humidified atmosphere of 5% CO2. On the second day after cell inoculation, the cell adhesion was closely observed and the MSC complete medium was replaced every 2-3 days. The cells were P0 generation cells.

[0015] (4) Cultivation and subculture

[0016] First, when the cell density reaches 60%-80%, preheat MSC complete medium, 0.25% trypsin and PBS in a 37°C water bath, remove the MSC complete medium, and wash with PBS;

[0017] Then, trypsin was added for digestion. When the cells were observed to shrink and disperse under a microscope, an equal amount of MSC complete medium was immediately added to terminate the digestion. The cells were transferred to an EP tube and centrifuged at 1000 rpm for 5 minutes.

[0018] Finally, the supernatant was removed, and MSC complete medium was added to resuspend the cells. One-third of the culture medium was added to a new culture dish, and the mixture was gently shaken and placed in a CO2 incubator for culture. The cell status was observed after 12 hours. The cells were P1 generation cells.

[0019] (5) Counting, activity detection and culture

[0020] Take 100 μL of P1 cells, detect cell activity and count them by trypan blue, adjust the cell density, inoculate them into a six-well plate containing MSC complete medium, and culture them in a cell culture incubator. The cells after subculture are P2 cells, and so on. The inoculation concentration of subcultured cells is 0.5×10 4 / cm 2 -1.5×104 / cm 2 ;

[0021] (6) Induced differentiation

[0022] P2-P3 cells were added with different cell differentiation induction medium to induce differentiation into osteoblasts, chondrocytes, adipocytes, etc.

[0023] The concentrations of magnesium chloride and calcium chloride added to the sterile PBS in step (2) of the method for isolating and culturing pig endometrial mesenchymal stem cells of the present invention are 1 mM and 0.5 mM respectively;

[0024] The digestive enzyme contains 0.2% type II collagenase and 40 μg / mL type I deoxyribonuclease (DNase I). Type II collagenase and DNase I are used together. Type II collagenase is responsible for decomposing collagen in the extracellular matrix, and DNase I is responsible for degrading DNA and reducing cell aggregation. Together, they promote effective digestion of tissues, thereby obtaining high-quality single-cell suspensions.

[0025] Among them, most of the endometrium is endometrial cells, and the proportion of mesenchymal stem cells is relatively small. Type II collagenase separates cells by hydrolyzing proline in the intercellular matrix. It has a strong digestion effect on collagen and also has a good digestion effect on the intercellular matrix, but has little effect on the cells themselves. It can separate epithelial cells from collagen components without damage.

[0026] DNase I can act on DNA degraded during cell separation, digest DNA chains into oligodeoxynucleotides by randomly shearing them, reduce cell clumps, and help prepare single cell suspensions.

[0027] The MSC complete culture medium in steps (3) and (4) of the pig endometrial mesenchymal stem cell isolation and culture method of the present invention is an MSC basal culture medium containing 10% fetal bovine serum (FBS) and 1% double antibody, wherein the double antibody is 100U / mL penicillin and 100μg / mL streptomycin.

[0028] The culture conditions of the cell culture box in step (5) of the method for isolating and culturing pig endometrial mesenchymal stem cells of the present invention are: temperature 37.0° C., 5% CO 2 saturation.

[0029] The cell induced differentiation in step (6) of the method for isolating and culturing pig endometrial mesenchymal stem cells of the present invention includes three-lineage induced differentiation of mesenchymal stem cells, namely osteogenic differentiation, chondrogenic differentiation and adipogenic differentiation, and the three-lineage induced differentiation (adipogenic induction, osteogenic induction and chondrogenic induction) are all based on The steps of the adipose-derived mesenchymal stem cell osteogenic (GUXMD-90021), adipogenic (GUXMD-90031), and chondrogenic (GUXMD-90041) differentiation induction kits were followed to operate and verify the staining of calcium nodules, lipid droplets, and chondrogenic sphere sections; wherein, the osteogenic differentiation medium induced the endometrial mesenchymal stem cells to differentiate into osteocytes; the chondrogenic differentiation medium induced the endometrial mesenchymal stem cells to differentiate into chondrocytes; and the adipogenic differentiation medium induced the endometrial mesenchymal stem cells to differentiate into adipocytes.

[0030] The porcine endometrial mesenchymal stem cells involved in the present invention are identified by surface markers using a flow cytometer, and antibodies suitable for identifying the porcine endometrial mesenchymal stem cells are determined: CD44 (Abcam, ab95138), CD34 (Abcam, ab223930), CD105 (Abcam, ab53321), and CD11b (Abcam, ab34444).

[0031] The porcine endometrial mesenchymal stem cells involved in the present invention are used to repair cells of intrauterine adhesions, skin and vascular damage, including repairing premature ovarian failure and endometrial damage. The repair method is simple and easy. They can be combined with biological materials and implanted into the lesion site for regeneration and repair.

[0032] Compared with the prior art, the present invention separates endometrial mesenchymal stem cells from the uterine tissue of a large animal, such as a pig, with simple steps and a large amount of cells obtained. The cultured endometrial mesenchymal stem cells have the phenotypes of CD34- and CD11b-, as well as CD44+ and CD105+, and can be induced to differentiate into osteocytes, adipocytes, and chondrocytes in vitro, and can repair various tissue and organ injuries. The application of experimental research and clinical research and application provides a new approach for cell transplantation therapy. The present invention separates mesenchymal stem cells from the endometrial tissue of pigs, and culture and amplify the endometrial mesenchymal stem cells in a culture medium containing cell growth factors, and after inducing differentiation into various cells, has broad application prospects in tissue engineering and regenerative medicine. Description of the drawings:

[0033] Figure 1 This is a schematic diagram of the growth status of the P0 generation cells involved in the present invention after being cultured in a CO2 incubator for 5 days.

[0034] Figure 2 This is a schematic diagram of the growth status of the P2 generation cells involved in the present invention after being cultured in a CO2 incubator for 3 days, wherein A is 40× and B is 100×.

[0035] Figure 3 This is a schematic diagram of the results of the flow cytometer examination of cell surface specific markers (CD44) involved in the present invention.

[0036] Figure 4 This is a schematic diagram of the results of the flow cytometer examination of cell surface specific markers (CD11b) involved in the present invention.

[0037] Figure 5 This is a schematic diagram of the results of the flow cytometer examination of cell surface specific markers (CD105) involved in the present invention.

[0038] Figure 6 This is a schematic diagram of the results of the flow cytometer examination of cell surface specific markers (CD34) involved in the present invention.

[0039] Figure 7 The present invention relates to a control group of pig endometrial mesenchymal stem cells after 21 days of osteogenic induction, a schematic diagram of the cell morphology (A), and an Alizarin red-stained schematic diagram of a large number of bone nodules that appeared after osteogenic induction of pig endometrial mesenchymal stem cells (B).

[0040] Figure 8 The present invention relates to a control group of pig endometrial mesenchymal stem cells after 26 days of adipogenic induction, which is a schematic diagram of cell morphology (A), and a schematic diagram of Oil Red O staining showing that a large number of fat particles are contained in the cells (B).

[0041] Fig. 9 This is a schematic diagram showing the morphology of chondrocytes after immunohistochemical experiment 24 days after chondrogenic induction according to the present invention, wherein A is 50 μm and B is 25 μm. Specific implementation method:

[0042] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0043] Embodiment 1:

[0044] The specific process flow of a method for isolating and culturing porcine endometrial mesenchymal stem cells involved in this embodiment is as follows:

[0045] (1) Fresh porcine uterine (including ovary) tissue obtained from a local slaughterhouse was immediately placed in ice-cold PBS and transported to a sterile laboratory within 2 hours;

[0046] (2) First, use sterile saline to clean the blood stains on the tissue surface, sterilize and disinfect, rinse and place in a Ca-containing 2+ and Mg 2+ Soak in sterile PBS (pH = 7.4) for 6 hours and wash twice;

[0047] (3) Use scissors sterilized in an autoclave to cut the tissue into 5 cm pieces. 2 The small tissue pieces were placed again in a solution of Ca 2+ and Mg 2+Soak in sterile PBS (pH = 7.4) for 3 hours and wash twice;

[0048] (4) Cut the small tissue block in the middle to expose the endometrium. Use forceps to peel off the outermost serosal layer to obtain tissue containing the endometrium and myometrium. Because the matrix layer between the endometrium and myometrium is closely connected, use forceps and scissors to carefully remove the myometrium in the endometrium. Finally, cut the endometrium into 1 mm 2 The fragments were placed in MSC basal medium supplemented with 0.2% type II collagenase and 40 μg / mL DNase I, and digested at 150 rpm in a 37°C incubator for 2-3 hours, with inversion mixing every 15 minutes to obtain a cell suspension;

[0049] (5) Filter the cell suspension through 100 and 40 μm cell strainers, add MSC complete medium to prevent further digestion, centrifuge at 1000 g for 5 min, treat the cells twice with red blood cell lysis buffer, wash twice with PBS, and centrifuge at 500 cells / cm 2 The cells were inoculated at a density of 100 0.5 μg / mL in MSC complete medium and incubated at 37.0°C in a humidified atmosphere of 5% CO2. The next day after inoculation, the cell adhesion was closely observed and fresh MSC complete medium was replaced every 2-3 days.

[0050] (6) When the cells reached 85% confluence, they were digested with 1 mL of 0.25% trypsin for 2 minutes. When the cells were observed to shrink and disperse under a microscope, an equal amount of MSC complete medium was immediately added to terminate the digestion. The cells were transferred to an EP tube and centrifuged at 1500 rpm for 5 minutes.

[0051] Remove the supernatant, add MSC complete medium to resuspend the cells, take 1 / 3 and add it to a new culture dish, shake gently to mix;

[0052] Place in a CO2 incubator for culture, observe the cell status under an inverted microscope after 24 hours, discard if bacterial contamination is observed, continue culture if no contamination, and mark the position of single cells in the culture dish for continuous observation; after 2-4 days, remove part of the original MSC complete medium and use fresh MSC complete medium to replenish; continue culture, after 5-7 days, observe that the clones gradually extend to the surroundings, the clones gradually become larger, and the cell status gradually relaxes; after the cells reach 80-90% fusion, use trypsin to digest and centrifuge, resuspend to obtain P1 generation cells, and transfer to another new culture dish; in this way, the P5 generation cells still maintain a strong proliferation ability; among them, the P0 generation cells, such as Figure 1 As shown, the cells of the P2 generation showed a clone-like growth morphology. Figure 2As shown, the cells proliferate vigorously and have a long spindle-shaped growth morphology, similar to fibroblasts.

[0053] Embodiment 2:

[0054] The process flow for phenotypic identification of porcine endometrial mesenchymal stem cells involved in this embodiment is as follows:

[0055] (1) Take P2-P3 cells and inoculate them in 6cm 2 In a petri dish;

[0056] (2) When the cell density reaches 60-80%, add 1 mL of 0.25% trypsin for 2 minutes and centrifuge at 1500 rpm for 5 minutes;

[0057] (3) Wash once with PBS and wash twice with cell staining buffer containing BSA (bovine serum albumin);

[0058] (4) Prepare 5 EP tubes, one tube as a blank control, add FITC (fluorescein isothiocyanate)-labeled CD44 and CD11b, PE (thermoplastic resin)-labeled CD34 and CD105 to 4 EP tubes, 2 μL per tube, and incubate on ice at 4°C in the dark for 30 min;

[0059] (5) After washing twice with cell staining buffer, resuspend the cells with 200 μL of cell staining buffer and detect on a flow cytometer;

[0060] (6) The results are as follows Figure 3-6 As shown, the analysis showed that the subcultured porcine endometrial mesenchymal stem cells all expressed CD44 (97.4%) and CD105 (99.7%), with expression rates greater than 95%, which met the international standards for the phenotype of mesenchymal stem cells.

[0061] It does not express CD34 (0.54%) and CD11b (1.76%), and the expression rates are both less than 2%, which meets the international standards for mesenchymal stem cell phenotype;

[0062] The results showed that the isolated and cultured pig endometrial cells completely conform to the characteristics of mesenchymal stem cells and are endometrial mesenchymal stem cells.

[0063] Embodiment 3:

[0064] The process flow of differentiation of porcine endometrial mesenchymal stem cells into osteocytes involved in this embodiment is as follows:

[0065] (1) taking the porcine endometrial mesenchymal stem cells obtained in Example 1 and culturing them for the third generation;

[0066] (2) According to The adipose-derived mesenchymal stem cell osteogenic differentiation kit (GUXMD-90021) was used to induce porcine endometrial mesenchymal stem cells. After 24 days of induction culture, they were gently stained with Alizarin Red working solution. The culture plate was placed under a microscope to observe the osteogenic staining effect. The results were as follows: Figure 7 As shown, compared with the mesenchymal stem cells before induction, after staining with Alizarin Red working solution, the induced cells showed a large number of calcium nodules, indicating that porcine endometrial mesenchymal stem cells can differentiate into osteoblasts.

[0067] Embodiment 4:

[0068] The process flow of differentiation of porcine endometrial mesenchymal stem cells into adipocytes involved in this embodiment is as follows:

[0069] (1) taking the porcine endometrial mesenchymal stem cells obtained in Example 1 and culturing them for the third generation;

[0070] (2) According to The adipose-derived mesenchymal stem cell adipogenic differentiation kit (GUXMD-90031) was used to induce porcine endometrial mesenchymal stem cells. After 26 days of induction culture, they were gently stained with Oil Red O dye working solution. The culture plate was placed under a microscope to observe the adipogenic staining effect. The results were as follows: Figure 8 As shown, compared with the mesenchymal stem cells before induction, a large number of lipid droplets were visible in the induced cells after staining with Oil Red 0 working solution, indicating that porcine endometrial mesenchymal stem cells can differentiate into adipocytes.

[0071] Embodiment 5:

[0072] The process flow of differentiation of porcine endometrial mesenchymal stem cells into chondrocytes involved in this embodiment is as follows:

[0073] (1) taking the porcine endometrial mesenchymal stem cells obtained in Example 1 and culturing them for the third generation;

[0074] (2) According to Adipose-derived mesenchymal stem cell chondrogenic differentiation kit (GUXMD-90041) was used to induce porcine endometrial mesenchymal stem cells. After 24 days of induction culture, the chondrocytes were embedded and sliced, stained with alicin blue working solution, and scanned with a panoramic scanner to observe the adipogenic staining effect. The results are as follows Fig. 9 As shown, most of the induced porcine endometrial mesenchymal stem cells were stained with Alecin blue, indicating that Alecin blue can differentiate into chondrocytes.

Claims

1. A method for isolating and culturing porcine endometrial mesenchymal stem cells, characterized in that: The specific process includes the following steps: (1) Pretreatment First, in a sterile room, the pig uterus was cut open and soaked and rinsed with pre-cooled sterile PBS supplemented with magnesium chloride and calcium chloride; Then, the serosa layer is peeled off and the intima and muscular layers are separated; Finally, the fragmented inner membrane layer was placed in an incubator at 37°C and digested with a basic medium supplemented with digestive enzymes for 2-3 hours, and pipetted every 15 minutes to obtain a cell suspension; (2) Centrifugation First, the cell suspension was filtered through a cell strainer, MSC complete medium was added to prevent further digestion, washed with PBS, and centrifuged at 1000 rpm for 5 min; Then, the cells were treated twice with red blood cell lysis buffer and washed with PBS; Finally, the cells were seeded in MSC complete medium and incubated at 37.0°C in a humidified atmosphere of 5% CO2. The MSC complete medium was replaced every 2-3 days. The cells were P0 generation cells. (3) Cultivation and subculture First, preheat MSC complete medium, 0.25% trypsin, and PBS in a 37°C water bath. When the cell density reaches 60%-80%, remove the MSC complete medium and wash with PBS. Then, trypsin was added for digestion. When the cells began to shrink and disperse, an equal amount of MSC complete medium was added to terminate the digestion. The cells were transferred to an EP tube and centrifuged at 1000 rpm for 5 minutes. Finally, remove the supernatant, add MSC complete medium to resuspend the cells, take 1 / 3 and add it to a new culture dish, shake it gently to mix, and culture it in a CO2 incubator. The obtained cells are P1 cells; (4) Counting, activity detection and culture Take 100 μL of P1 cells, detect cell activity and count them, adjust cell density, inoculate them into MSC complete medium, culture them, and the cells after subculture again are P2 cells, and so on.

2. The method for isolating and culturing porcine endometrial mesenchymal stem cells according to claim 1, characterized in that: Adding P2-P3 cells to different cell differentiation induction culture media can induce them to differentiate into osteoblasts, chondrocytes and adipocytes.

3. The method for isolating and culturing porcine endometrial mesenchymal stem cells according to claim 1, characterized in that: The concentrations of magnesium chloride and calcium chloride added to the sterile PBS in step (1) are 1 mM and 0.5 mM, respectively; the digestive enzyme contains type II collagenase at a concentration of 0.2% and type I deoxyribonuclease at 40 μg / mL.

4. The method for isolating and culturing porcine endometrial mesenchymal stem cells and application according to claim 1, characterized in that: The MSC complete culture medium in steps (2) and (3) is a MSC basal culture medium containing 10% fetal bovine serum and 1% double antibody.

5. The method for isolating and culturing porcine endometrial mesenchymal stem cells and application according to claim 1, characterized in that: The culture conditions in step (3) are: temperature 37.0°C, 5% CO2 saturation.

6. The method for isolating and culturing porcine endometrial mesenchymal stem cells according to claim 1, characterized in that: The cell seeding concentration in step (4) is 0.5×10 4 / cm 2 -1.5×10 4 / cm 2 .

7. The method for isolating and culturing porcine endometrial mesenchymal stem cells and application according to claim 2, characterized in that: The cells are induced to differentiate into three lineages of mesenchymal stem cells including osteogenic differentiation, chondrogenic differentiation, and adipogenic differentiation.

8. The method for isolating and culturing porcine endometrial mesenchymal stem cells and application according to claim 4, characterized in that: The dual antibodies were 100 U / mL penicillin and 100 μg / mL streptomycin.

9. The method for isolating and culturing porcine endometrial mesenchymal stem cells according to any one of claims 1, 3-6 and 8, characterized in that: Through surface marker identification, antibodies suitable for identifying porcine endometrial mesenchymal stem cells were determined: CD44, CD34, CD105, CD11b.

10. An application of porcine endometrial mesenchymal stem cells, characterized in that: Repair intrauterine adhesions, damaged skin and vascular cells, including premature ovarian failure and endometrial damage.

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