Isolated culture method of placenta decidua basalis stem cells

Abstract

The invention discloses an isolated culture method of placenta decidua basalis stem cells. The isolated culture method comprises the following steps: acquiring decidua basalis tissues from a placenta; a trypsin solution is added into the bottom demolding tissue for pre-digestion, and a pre-digested bottom demolding tissue is obtained; adding a mixed enzymatic hydrolysate of trypsin and collagenase into the pre-digested decidua basalis tissue, carrying out digestion treatment under a magnetic stirring condition, sieving, centrifuging, and discarding supernate to obtain decidua basalis stem cells; adding a complete culture medium into decidua basalis stem cells, blowing, beating and uniformly mixing, and inoculating the decidua basalis stem cells into a culture dish according to the inoculation density of 2000-5000 cells / cm < 2 > for culturing to obtain P0-generation cells; carrying out subculture on the P0 generation cells; according to the separation culture method, external pollution is effectively reduced through refined cleaning and stripping steps, and then a one-step digestion method is adopted and combined with a magnetic stirring technology, so that the cell separation efficiency and purity are remarkably improved; meanwhile, the optimal inoculation density is explored through practice, and the cell culture effect and yield are directly improved.

Description

Technical Field

[0001] The invention relates to the technical field of cell separation and culture, and in particular to a method for separating and culturing placenta decidua basalis stem cells. Background Art

[0002] Mesenchymal stem cells refer to a type of multipotent stem cells with multiple cell differentiation potentials. They are the most commonly used cells in clinical research and are widely used in the fields of tissue engineering research, wound repair, and terminal disease treatment. As research continues, people have discovered that there are a large number of trophoblasts and abundant extraembryonic mesoderm mesenchyme and blood vessels in the placenta, from which mesenchymal stem cells can be obtained. Human placental decidua basalis mesenchymal stem cells derived from the decidua basalis have advantages that bone marrow mesenchymal stem cells cannot match, such as sufficient sources, non-invasive acquisition, low immunogenicity, strong proliferation ability, and no effects of low serum and hypoxia. They play an important role in embryo implantation, immune tolerance establishment, and normal pregnancy maintenance during pregnancy. They are easy to collect and have no effect on the fetus and pregnant women after delivery. This meets ethical requirements. The cells are more primitive and have stronger proliferation ability. They are ideal seed cells for clinical application of stem cells.

[0003] At present, the method for isolating and extracting placental decidua basal mesenchymal stem cells mainly adopts the enzymatic digestion method. Although the enzymatic method can obtain a sufficient number of cells, the problems of low cell purity, long culture time, low activity and small number of primary placental decidua basal mesenchymal stem cells cannot be ignored. The entire decidua basal stem cell extraction process has cumbersome steps, the number of extracted cells fluctuates greatly, and the probability of contamination is greatly increased, which is not conducive to industrial production. At present, there is still a lack of a separation and culture method that can improve the purity, quantity, activity and culture time of placental decidua basal mesenchymal stem cells. Summary of the invention

[0004] The purpose of the present invention is to provide a method for isolating and culturing placental decidua basalis stem cells. The isolation and culture method of the present invention overcomes the problem of excessive miscellaneous cells in the isolation and culture process by improving and optimizing the isolation and culture process, and ensures that the placental decidua basalis-derived mesenchymal stem cells reach the quantity and quality of medical grade.

[0005] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0006] The first aspect of the present invention provides a method for isolating and culturing placental decidua basalis stem cells, comprising the following steps:

[0007] (a) cutting the basal decidua tissue from the cleaned placenta, and then soaking and rinsing the basal decidua tissue in PBS containing penicillin-streptomycin to obtain the basal decidua tissue;

[0008] (b) adding a trypsin solution to the basal decidua tissue for pre-digestion and washing, then removing the bulky tissue of the inner layer of the basal decidua tissue and washing, to obtain a pre-digested basal decidua tissue;

[0009] (c) chopping the pre-digested decidual tissue into pieces, adding a mixed enzymatic solution of trypsin and collagenase for digestion, sieving and centrifuging after digestion, discarding the supernatant, and obtaining the decidual stem cells, wherein the digestion is carried out under magnetic stirring at 36 to 38° C. for 25 to 40 minutes, and the magnetic stirring speed is 120 to 180 rpm;

[0010] (d) adding complete culture medium to the decidua basalis stem cells and mixing them by pipetting, then inoculating them into a culture dish for culture, and when the cells grow to a confluence of 80% to 90%, obtaining P0 cells, wherein the inoculation density is 2000 to 5000 cells / cm 2 ; After 36-60h of culture, the medium was completely replaced;

[0011] (e) The P0 cells were subcultured using serum-free stem cell complete medium. When the cells were subcultured to P1, surface antigen detection and chondrogenesis, osteoblastogenesis and adipogenesis induction were performed.

[0012] Preferably, in step (a), the concentration of penicillin-streptomycin in the PBS containing penicillin-streptomycin is 2% to 4%.

[0013] Preferably, in step (b), the trypsin concentration in the trypsin solution is 0.1% to 0.3%.

[0014] Preferably, in step (b), the pre-digestion temperature is 36-38° C. and the time is 10-20 min.

[0015] Preferably, in step (c), the volume ratio of the pre-digested demembraned tissue to the mixed enzymatic solution of trypsin and collagenase is 1:(8-10).

[0016] Preferably, in step (c), the concentration of trypsin in the mixed enzymatic solution of trypsin and collagenase is 0.1% to 0.3%, and the concentration of collagenase is 0.08% to 0.12%.

[0017] Preferably, in step (c), the magnetic rotor used for magnetic stirring is type A 8*15 mm. Preferably, in step (d), the inoculation density is 2000 to 5000 cells / cm 2 ; After 36 to 60 hours of culture, the medium was completely replaced.

[0018] Preferably, in step (c), the pore size of the filter used for sieving is 100 μm; the centrifugal force of the centrifuge is 500-700 g, and the centrifugation time is 5-15 min.

[0019] Preferably, in step (e), the subculture comprises: changing half of the medium at intervals of 2 to 4 days, and performing digestion and subculture when the confluence degree reaches 90%.

[0020] Compared with the prior art, the beneficial effects of the present invention include at least:

[0021] The method for isolating and culturing placental decidua basalis stem cells of the present invention is highly efficient and pollution-free, and can improve the yield of primary mesenchymal stem cells. Specifically, the present invention improves the traditional method of promoting the enzyme digestion process by an oscillating water bath, and adopts a method of adding magnetic stirring to make cell digestion more complete. At the same time, the inoculation density is optimized, and the optimal inoculation density is screened out, thereby overcoming the problem of excessive miscellaneous cells in the culture process, improving the purity, and increasing the yield and activity of the cells. The immune phenotype of the stem cells obtained by the separation and culture method of the present invention meets the identification standard of mesenchymal stem cells and has multidirectional differentiation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0023] Figure 1 This is a cell morphology observation diagram of cells extracted from the method for isolating placental decidua basalis stem cells described in Example 1 of the present invention under a microscope;

[0024] Figure 2 This is a cell morphology observation diagram of cells extracted by the method for isolating placental decidua basalis stem cells described in Example 2 of the present invention under a microscope;

[0025] Figure 3 This is a cell morphology observation diagram of cells extracted by the method for isolating placental decidua basalis stem cells described in Example 3 of the present invention under a microscope;

[0026] Figure 4 This is a cell morphology observation diagram of cells extracted by the method for isolating placental decidua basalis stem cells described in Example 4 of the present invention under a microscope;

[0027] Figure 5 The expression of cell surface markers of cells extracted by the method for isolating placental decidua basalis stem cells described in Example 1 of the present invention after subculture;

[0028] Figure 6 The expression of cell surface markers of cells extracted by the method for isolating placental decidua basalis stem cells described in Example 5 of the present invention after subculture;

[0029] Figure 7The method for isolating placental decidua basalis stem cells described in Example 1 of the present invention is used to extract cells and then culture them to determine the effects on the adipogenic, chondrogenic and osteogenic differentiation of the cells. DETAILED DESCRIPTION

[0030] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0031] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0032] Example 1

[0033] This embodiment is a method for isolating and culturing placental decidua basalis stem cells, comprising the following steps:

[0034] (a) With informed consent from the parturient, after the fetus was born at full term, the placental tissue was collected, and after passing the virus test, the dirt was washed away with physiological saline, and the placenta was immersed in a transport solution (DMEM, with a final concentration of 100 U / ml penicillin and 100 U / ml streptomycin) and transported to the laboratory at 4°C; after the surface of the vessel was disinfected, it was transferred to an ultra-clean bench, and the placenta was placed in PBS containing 3% penicillin and streptomycin and rinsed twice, and a 1 / 4 thick and 1 cm-sized piece was cut from the washed placenta. 2 4 pieces of basal decidua tissue were placed in a Φ10 cm sterile culture dish, and then soaked and rinsed 6 times with PBS containing 3% penicillin-streptomycin to obtain basal decidua tissue;

[0035] (b) adding trypsin solution to the basal decidua tissue for pre-digestion at 37°C for 15 min, rinsing with PBS containing 3% penicillin-streptomycin, then using forceps and sterile stainless steel tissue curved scissors to remove the bulky tissue of the inner layer of the basal decidua tissue and rinsing repeatedly with PBS containing 1% penicillin-streptomycin for 3 times to obtain pre-digested basal decidua tissue;

[0036] (c) Cut the pre-digested bottom demembraned tissue into small pieces (cut into individual pieces with a size of 1 mm). 3 The tissue blocks were cut into foamy minced meat), and then the pre-digested basal decidua tissue and the mixed enzymatic hydrolysate of trypsin and collagenase (0.2% trypsin, 0.1% collagenase concentration) were added to the minced pre-digested basal decidua tissue in a volume ratio of 1:10, and the mixed enzymatic hydrolysate of trypsin and collagenase was added to the minced pre-digested basal decidua tissue, and the mixture was digested for 30 minutes under magnetic stirring (speed of 150 rpm) at 37°C. After the digestion was completed, the mixture was sieved (100 μm) and centrifuged at 600 g for 10 minutes, and the supernatant was discarded to obtain basal decidua stem cells;

[0037] (d) Complete medium was added to the decidua basalis stem cells, and the cells were mixed by pipetting and counting, and then inoculated into culture dishes for culture at a density of 3000 cells / cm 2 The culture conditions were 37°C, 5% CO2, and saturated humidity. After 48 hours of culture, the medium was completely replaced. When the cells grew to a confluence of 80% to 90%, DMEM was used to wash the non-adherent cells to obtain P0 cells.

[0038] (e) Serum-free stem cell complete medium was added to the P0 cells for subculture, and half of the medium was replaced every 3 days. When the cell confluence reached 90%, the cells were digested to obtain the P1 generation; and the P1 generation cells were further subcultured.

[0039] The morphology of the P1 cells was observed under a microscope. Figure 1 shown.

[0040] The above P1 cells were inoculated into 24-well plates for chondrogenic, osteogenic and adipogenic induction analysis. The analysis method is as follows:

[0041] Osteogenic differentiation: The differentiation medium was DMEM medium containing 10 mol / L dexamethasone, 10 mmol / L β-glycerol phosphate, 0.05 mmol / L 2-phosphate ascorbic acid (all from Sigma) and 10% FBS. The medium was changed twice a week, and the cells were cultured for about 21 days and then stained for microscopic examination (rinsed with PBS once, fixed with 95% alcohol at room temperature for 30 minutes, and stained with 0.2% Alizarin Red S for 30 minutes).

[0042] Chondrogenic differentiation: The differentiation medium is DMEM medium containing 0.1umol / L dexamethasone, 50ug / ml L-ascorbic acid, 1mmol / L sodium pyruvate, 10ng / ml TGF-B, 50mg / ml ITS (all products of Sigma) and 10% FBS. The medium is changed twice a week. After 14 days of induction culture, staining and microscopic examination (rinsing with PBS once, fixing with 95% alcohol at room temperature for 30 minutes, and staining with 1% Alzheimer's blue for 30 minutes).

[0043] Adipogenic differentiation: The differentiation induction medium is a DMEM medium containing 10-6 mol / L dexamethasone, 10 μg / ml insulin, 0.5 mmol / L isobutylxanthine (IBMX), 200 μmol / L indomethacin (all products of Sigma) and 10% FBS. The medium is changed twice a week, and the cells are cultured for about 14 days (oil droplets can be seen in the cells under the microscope), and then stained for microscopic examination (washed with PBS twice, fixed with 75% alcohol at room temperature for 30 minutes, and stained with Oil Red O at room temperature for 30 minutes).

[0044] The above analysis results are as follows Figure 7 shown by Figure 7 It can be seen that:

[0045] Alizarin red staining showed that a large number of mineralized nodules stained orange-red were scattered among the dense cells; after staining with Oil Red O, the intracellular fat droplets were bright red, and the staining result was positive; the results of Alcian blue staining showed that the cell clusters were blue, which means that the isolated and cultured cells of the present invention can be induced to differentiate into osteocytes, chondrocytes and adipocytes under specific induction differentiation conditions, and have multidirectional differentiation potential, and the addition of rotors has no effect on the multidirectional differentiation potential of the cells. This shows that the isolation method of the placental decidua stem cells of the present invention is effective.

[0046] Example 2

[0047] This embodiment is a method for isolating and culturing placental decidua basalis stem cells. The method for isolating and culturing placental decidua basalis stem cells is basically the same as the method for isolating and culturing placental decidua basalis stem cells in Example 1, except that the inoculation density is 2000 cells / cm 2 .

[0048] The morphology of the P1 cells was observed under a microscope. Figure 2 shown.

[0049] Example 3

[0050] This embodiment is a method for isolating and culturing placental decidua basalis stem cells. The method for isolating and culturing placental decidua basalis stem cells is basically the same as the method for isolating and culturing placental decidua basalis stem cells in Example 1, except that the inoculation density is 4000 cells / cm 2 .

[0051] The morphology of the P1 cells was observed under a microscope. Figure 3 shown.

[0052] Example 4

[0053] This embodiment is a method for isolating and culturing placental decidua basalis stem cells. The method for isolating and culturing placental decidua basalis stem cells is basically the same as the method for isolating and culturing placental decidua basalis stem cells in Example 1, except that the inoculation density is 5000 cells / cm 2 .

[0054] The morphology of the P1 cells was observed under a microscope. Figure 4 shown.

[0055] Example 5

[0056] This embodiment is a method for isolating and culturing placental decidua basalis stem cells. The method for isolating and culturing placental decidua basalis stem cells is basically the same as the method for isolating and culturing placental decidua basalis stem cells in Example 1, except that step (c) is replaced by the following steps:

[0057] Cut the pre-digested tissue into small pieces (cut into individual pieces of 1 mm 3 The tissue blocks were cut into foamy minced meat), and then the mixed enzymatic hydrolysis solution of trypsin and collagenase (0.2% trypsin, 0.1% collagenase concentration) was added to the minced pre-digested decidua basal tissue in a volume ratio of 1:10, and the mixed enzymatic hydrolysis solution of trypsin and collagenase was added to the minced pre-digested decidua basal tissue and mixed, and the mixture was placed on a shaking water bath, and digested for 30 minutes at 37°C and 150 rpm. After the digestion was completed, the mixture was sieved (100 μm) and centrifuged at 600 g for 10 minutes, and the supernatant was discarded to obtain the decidua basal stem cells.

[0058] Experimental example

[0059] 1. This experimental example is the isolation and culture method of placental decidua mesenchymal stem cells, and the detection and analysis of the number and vitality of P1 cells;

[0060] The detection method is as follows: take the freshly separated cells, mix them thoroughly at a ratio of cell suspension: 0.08% trypan blue = 1:1 (v:v), take 20 μL of the cell mixture and add it to a cell counting plate, detect and calculate the cell viability and total number of cells.

[0061] The test results are shown in Table 1:

[0062] Table 1

[0063] Example Number of inoculation dishes Number of living cells Total cell number Cell viability Example 1 3 <![CDATA[12.23×10 5 ]]> <![CDATA[12.93×10 5 ]]> 94.62％ Example 2 3 <![CDATA[3.00×10 5 ]]> <![CDATA[3.25×10 5 ]]> 92.53％ Example 3 3 <![CDATA[6.84×10 5 ]]> <![CDATA[7.35×10 5 ]]> 93.15％ Example 4 3 <![CDATA[5.99×10 5 ]]> <![CDATA[6.78×10 5 ]]> 88.37％

[0064] From Table 1, we can see that:

[0065] The seeding density is 3000 cells / cm 2 , so that the separation and culture method of the present invention can obtain a higher number of living cells and cell viability.

[0066] 2. The number and viability of cells harvested in step (c) of Example 1 and Example 5 are shown in Table 2 below;

[0067] Table 2

[0068] Example The total number of cells obtained after digestion The number of viable cells after digestion Cell viability Example 1 <![CDATA[3.47×10 6 ]]> <![CDATA[3.14×10 6 ]]> 90.77％ Example 5 <![CDATA[2.58×10 6 ]]> <![CDATA[2.42×10 6 ]]> 94.02％

[0069] From Table 2, we can see that:

[0070] In the embodiment of the present invention, the original oscillating water bath process is replaced by magnetic stirring, and the number of cells and the number of living cells obtained by digestion counting are both higher than those in Example 5.

[0071] 3. Flow cytometry analysis of the P1 cells of Example 1 and Example 5:

[0072] Flow cytometry analysis of cell surface markers: suspend cells with PBS and filter cells with nylon mesh. Add antibodies and incubate in a 4°C refrigerator away from light for 30 minutes. Wash cells once with PBS by centrifugation. Add paraformaldehyde to suspend cells and test on the flow cytometer; the analysis results are as follows: Figure 5 , Figure 6 As shown, Figure 5 The cell analysis results of Example 1 are as follows: Figure 6 The results of cell analysis in Example 5 are as follows;

[0073] Depend on Figure 5 , Figure 6 It can be seen that:

[0074] The cells separated and cultured in Example 1 of the present invention have higher expression of CD44, CD105, and CD146; do not express CD34, CD45, and HLA-DR, which is consistent with the phenotypic characteristics of mesenchymal stem cells, and also shows that Example 1 has higher cell purity.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A method for isolating and culturing placental decidua basalis stem cells, characterized in that: The steps include: (a) cutting the basal decidua tissue from the cleaned placenta, and then soaking and rinsing the basal decidua tissue in PBS containing penicillin-streptomycin to obtain the basal decidua tissue; (b) adding a trypsin solution to the basal decidua tissue for pre-digestion and washing, then removing the bulky tissue of the inner layer of the basal decidua tissue and washing, to obtain a pre-digested basal decidua tissue; (c) chopping the pre-digested decidual tissue into pieces, adding a mixed enzymatic solution of trypsin and collagenase for digestion, sieving and centrifuging after digestion, discarding the supernatant, and obtaining the decidual stem cells, wherein the digestion is carried out under magnetic stirring at 36 to 38° C. for 25 to 40 minutes, and the magnetic stirring speed is 120 to 180 rpm; (d) adding complete culture medium to the decidua basalis stem cells and mixing them by pipetting, then inoculating them into a culture dish for culture, and when the cells grow to a confluence of 80% to 90%, obtaining P0 cells, wherein the inoculation density is 2000 to 5000 cells / cm 2 ; After 36-60h of culture, the medium was completely replaced; (e) The P0 cells were subcultured using serum-free stem cell complete medium. When the cells were subcultured to P1, surface antigen detection and chondrogenesis, osteoblastogenesis and adipogenesis induction were performed.

2. The method for isolating and culturing placenta decidua basalis stem cells according to claim 1, characterized in that: In the step (a), the concentration of penicillin-streptomycin in the PBS containing penicillin-streptomycin is 2% to 4%.

3. The method for isolating and culturing placenta decidua basalis stem cells according to claim 1, characterized in that: In the step (b), the trypsin concentration in the trypsin solution is 0.1% to 0.3%.

4. The method for isolating and culturing placenta decidua basalis stem cells according to claim 1, characterized in that: In the step (b), the pre-digestion temperature is 36-38° C. and the time is 10-20 min.

5. The method for isolating and culturing placenta decidua basalis stem cells according to claim 1, characterized in that: In the step (c), the volume ratio of the pre-digested bottom demembraned tissue to the mixed enzymatic solution of trypsin and collagenase is 1: (8-10).

6. The method for isolating and culturing placenta decidua basalis stem cells according to claim 1, characterized in that: In the step (c), the concentration of trypsin in the mixed enzymatic solution of trypsin and collagenase is 0.1% to 0.3%, and the concentration of collagenase is 0.08% to 0.12%.

7. The method for isolating and culturing placenta decidua basalis stem cells according to claim 1, characterized in that: In the step (c), the pore size of the filter used for sieving is 100 μm; the centrifugal force of the centrifuge is 500-700 g, and the centrifugation time is 5-15 min.

8. The method for isolating and culturing placenta decidua basalis stem cells according to claim 1, characterized in that: In the step (e), the subculture includes: changing half of the medium every 2 to 4 days, and performing digestion and subculture when the confluence degree reaches 90%.

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