CN111088224B - Method for promoting directional differentiation of umbilical cord mesenchymal stem cells to chondroblasts - Google Patents
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Abstract
The invention provides a method for promoting directional differentiation of umbilical cord mesenchymal stem cells to chondroblasts, which comprises the following steps: taking out umbilical cord tissues, shearing, cleaning, adding tissue digestive juice for digestion, filtering and removing supernatant after digestion is finished to obtain umbilical cord mesenchymal stem cells; culturing the umbilical cord mesenchymal stem cells by using an umbilical cord mesenchymal stem cell selective culture medium; when the cell reaches 80-90% confluence degree, digesting the cell by using low-toxicity digestive juice, then performing cryopreservation by using umbilical cord mesenchymal stem cell cryopreservation liquid, and placing the frozen cell in liquid nitrogen; removing cells from liquid nitrogen, quickly thawing in a 37 ℃ water bath, centrifuging to remove supernatant, and adding a cell recovery solution to recover the cells; removing the resuscitation solution, and replacing the priming solution before induction for culture; digesting the cells with the low-toxicity digestive juice when the cells grow to 80-90% confluency; cells were seeded, cultured overnight, the supernatant removed, and cultured in chondrogenic induction medium in a hypoxic incubator with 2% oxygen concentration. Compared with the prior art, the method can stably and effectively promote the directional differentiation of the umbilical cord mesenchymal stem cells to the chondroblast in a shorter induction time.
Description
Technical Field
The invention belongs to the field of stem cells and regenerative medicine, and particularly relates to a method and a culture medium for promoting directional differentiation of umbilical cord mesenchymal stem cells to chondroblasts.
Background
The umbilical cord mesenchymal stem cells have the capability of multidirectional differentiation. The differentiation effect of the mesenchymal stem cells is mainly determined by the activity of the stem cells and induction conditions.
Mesenchymal stem cell chondrogenesis is a multi-step cellular event, in which mesenchymal stem cells of a specific subset are aggregated and then differentiated into cartilage.
The commonly used umbilical cord mesenchymal stem cell acquisition method comprises the following steps: walsh gel was minced and cultured adherently in complete medium (10% FBS in DMEM) for 16 days. The conventional cryopreservation method is as follows: after being subjected to program cooling in a complete culture medium containing 10% DMSO, the culture medium is frozen and stored in liquid nitrogen. The conventional resuscitation method is as follows: quickly thawed in a 37 ℃ water bath and cultured with complete medium. The conventional chondrogenic induction method is as follows: adding differentiation inducing culture medium after cell plate, adding 100 ug/ml sodium pyruvate, 10ng/ml TGF-beta 3, 100 ug M dexamethasone, 25 ug/ml vitamin C, 40 ug/ml proline, 1 XTS +1premix into high-sugar DMEM, and growing in fixed solid medium or spheroids. The culture medium was changed every 3 days, and cultured in a cell culture chamber at 37 ℃ for 20 days. Finally, the glycosaminoglycan in the cartilage tissue extracellular matrix can be stained blue with 1% toluidine blue.
The defects of the existing stem cell chondrogenic differentiation method are as follows: the required induction time is long; the supporting medium is used for assisting in chondrogenic differentiation, so that the method is complex and high in cost; although cell activity is detected before each induction, it is not guaranteed that cartilage differentiation can be completed in each batch of cells, and cell resources are wasted.
Disclosure of Invention
The present invention is directed to solve the above problems, and an object of the present invention is to provide a method for promoting the directional differentiation of umbilical cord mesenchymal stem cells into chondroblasts, which can stably and effectively promote the directional differentiation of umbilical cord mesenchymal stem cells into chondroblasts within a short induction time, and the obtained chondroblasts have high survival rate and high cell activity.
Therefore, the invention provides the following technical scheme.
A method for promoting directional differentiation of umbilical cord mesenchymal stem cells to chondroblasts comprises the following steps:
(1) taking out umbilical cord tissues, shearing, cleaning, adding tissue digestive juice for digestion, filtering and removing supernatant after digestion is finished to obtain umbilical cord mesenchymal stem cells;
(2) culturing the umbilical cord mesenchymal stem cells by using an umbilical cord mesenchymal stem cell selective culture medium;
(3) when the cell reaches 80-90% confluence degree, digesting the cell by using low-toxicity digestive juice, then performing cryopreservation by using umbilical cord mesenchymal stem cell cryopreservation liquid, and placing the frozen cell in liquid nitrogen;
(4) taking out cells from liquid nitrogen, quickly thawing in a water bath at 37 ℃, centrifuging to remove supernatant, and adding a cell recovery solution to recover the cells;
(5) removing the resuscitation solution, and replacing the priming solution before induction for culture;
(6) digesting the cells with the low-toxicity digestive juice when the cells grow to 80-90% confluency;
(7) cells were seeded, cultured overnight, the supernatant removed, and cultured in chondrogenic induction medium in a hypoxic incubator with 2% oxygen concentration.
Specifically, the step (1) is as follows: taking out umbilical cord tissue, cutting into pieces, cleaning, adding tissue digestive juice, and carrying out shake digestion at 37 ℃ for 30min for digestion, wherein the ratio of the weight of the umbilical cord tissue to the volume of the tissue digestive juice is 1: 1, adding a DMEM medium with a volume 3 times that of the digestion solution to stop digestion, centrifuging at 1000rpm for 5min, and removing the supernatant to obtain the umbilical cord mesenchymal stem cells.
Specifically, the step (2) is as follows: resuspending the cells in umbilical cord mesenchymal stem cell selective medium and adding 2 x 104/cm2Inoculating into culture flask, culturing at 37 deg.C for 13 days, and changing every 3 days.
Specifically, the step (3) is as follows: when the cells reach 80-90% confluence, digesting the cells with low-toxicity digestive juice, and adding 4 x 106-5*106And (3) freezing and storing the cells per ml, namely using umbilical cord mesenchymal stem cell freezing and storing the umbilical cord mesenchymal stem cells in a freezing and storing volume of 1ml by using a programmed cooling box, and finally placing the umbilical cord mesenchymal stem cells in liquid nitrogen.
Specifically, the step (5) is as follows: the resuscitating fluid was removed and the priming solution was replaced before induction for 2 days.
Specifically, the step (7) is as follows: according to 2 x 104Per cm2Inoculating the cells into a 24-well plate, and culturing overnight in an incubator at 37 ℃; the 24-well plate was removed, the supernatant was removed, and 1ml of the adult cartilage induction medium was added thereto, and the mixture was cultured in a hypoxic incubator with 2% oxygen concentration for 10 days, and the medium was changed every 3 days.
Preferably, the umbilical cord tissue is human umbilical cord tissue.
Preferably, the tissue digest comprises DMEM, collagenase type I at a final concentration of 3mg/ml and DNase at a final concentration of 0.3 mg/ml.
Preferably, the ratio of the weight of the umbilical cord tissue to the volume of the tissue digest is 1: 1.
preferably, the umbilical cord mesenchymal stem cell selective medium comprises: umbilical cord mesenchymal stem cells with 40% confluence degree are cultured by a serum-free culture medium for 2-3 days to obtain culture supernatant, centrifuging and filtering to obtain a product, and mixing the product with the serum-free culture medium of the mesenchymal stem cells in a ratio of 1: 1 are mixed by volume.
Preferably, the low toxicity digestive fluid comprises: DMEM, and 0.05% pancreatin, 0.3mg/ml DNase by volume of DMEM.
Preferably, the umbilical cord mesenchymal stem cell cryopreservation liquid comprises: DMEM, 25-35 mL/100mL of umbilical cord mesenchymal stem cell culture supernatant, 4-6 mL/100mL of glycerol, 4-6 g/100mL of polyvinylpyrrolidone (PVP), 0.5-1.5 g/100mL of polyvinyl alcohol (PVA), 1.5-2.5 g/100mL of trehalose, 1-2.5 g/100mL of hydroxyapatite nanoparticles, 0.5-1.5 mg/100mL of all-trans retinoic acid and 8-12 mL/100mL of serum substitute.
Preferably, the cell resuscitating fluid comprises: DMEM, and 25-35 mL/100mL of mesenchymal stem cell culture supernatant, 0.25-0.75 g/100mL of glucose, 0.5-1.5 mg/100mL of all-trans retinoic acid, and 0.5-1.5 mg/100mL of ascorbic acid in terms of volume of DMEM.
Preferably, the priming fluid comprises: DMEM, and 10% serum replacement, 1% diabody (penicillin streptomycin), 1mg/ml dimethyloxalylglycine by DMEM volume.
Preferably, the chondrogenic induction medium comprises: DMEM, and 1% diabody, 10% serum replacement, 1mM sodium pyruvate, 0.35mM proline, 0.284mM ascorbic acid, 0.1. mu.M dexamethasone, 10ng/ml TGF-. beta.3, 50mg/ml ITS, 1. mu.M IGF-1, 1. mu.M retinol receptor antagonist, based on the volume of DMEM.
In vitro study of mesenchymal stem cell differentiation into cartilage is carried out by inducing overexpression of specific genes, such as SOX9, by cytokines, thereby promoting differentiation into cartilage. The hypoxia environment is favorable for SOX9 gene expression of stem cells, also conforms to the simulated in vivo environment, promotes the differentiation of the cells into cartilage tissues, and can effectively promote the differentiation of mesenchymal stem cells to the chondrogenic direction by using a retinol receptor antagonist.
Compared with the prior art, the invention ensures the quality and quantity of the cells by designing a method and a system for cell separation, cryopreservation, resuscitation and simpler and more effective induction. By using the freezing solution, the resuscitation solution and the corresponding freezing and resuscitation methods, the survival rate and the cell activity of each tube of frozen cells are ensured, and the quality of each bottle of resuscitated cells can reach the same high-quality standard before induction. The cells are pretreated before induction, and a hypoxia environment and the chondrogenic induction culture medium prepared by the method are used for induction in the process, so that the chondrogenic induction condition can be effectively improved, and the time required by induction can be shortened.
Drawings
Fig. 1 is a photograph of tissue umbilical cord mesenchymal stem cells isolated and purified in example 1 for 13 days.
Fig. 2 is a photograph of tissue umbilical cord mesenchymal stem cells isolated and purified in example 2 for 13 days.
Fig. 3 is a photograph of tissue umbilical cord mesenchymal stem cells isolated and purified in example 3 for 13 days.
FIG. 4 is a photograph of chondrogenic staining obtained after applying the isolation, cryopreservation, resuscitation and induction methods of example 1.
FIG. 5 is a photograph of chondrogenic staining obtained after applying the isolation, cryopreservation, resuscitation and induction methods of example 2.
FIG. 6 is a photograph of chondrogenic staining obtained after applying the isolation, cryopreservation, resuscitation and induction methods of example 3.
Fig. 7 is a photograph of the tissue umbilical cord mesenchymal stem cells isolated and purified in comparative example 1 for 13 days.
FIG. 8 is a photograph of the tissue umbilical cord mesenchymal stem cells isolated and purified in comparative example 2 for 13 days.
FIG. 9 is a photograph of the tissue umbilical cord mesenchymal stem cells isolated and purified in comparative example 3 for 13 days.
FIG. 10 is a photograph of chondrogenic staining obtained after applying the separation, cryopreservation, resuscitation and induction methods of comparative example 1.
FIG. 11 is a photograph of chondrogenic staining obtained after applying the separation, cryopreservation, resuscitation and induction methods of comparative example 2.
FIG. 12 is a photograph of chondrogenic staining obtained after applying the separation, cryopreservation, resuscitation and induction methods of comparative example 3.
Detailed Description
The technical solution of the present invention will be further described with reference to the following specific examples, but the present invention is not limited to the following examples.
It is noted that the method for differentiating the umbilical cord mesenchymal stem cells into chondrogenic cells provided by the present invention is not used for the purpose of disease diagnosis and treatment.
Unless otherwise specified, all reagents used in the present invention are available commercially. For the sake of brevity, some of the technical operations are not described in detail, but it is understood that these operations are within the purview of one skilled in the art and may be implemented in accordance with the teachings set forth herein.
Promoting the directional differentiation of the umbilical cord mesenchymal stem cells to chondroblast, comprising the following steps:
1. taking out human umbilical cord tissue, shearing into 2-3cm long with scissors, and separating to obtain Wharton's jelly with forceps.
2. Place the Wharton's jelly in a 50ml centrifuge tube and cut it up with scissors.
3. Adding appropriate amount of tissue digestive juice (weight of umbilical cord tissue to volume of digestive juice is 1 g: 1.6ml), and digesting at 37 deg.C for 30min with shaking. The formula of the tissue digestive juice is as follows: DMEM (DMEM medium, purchased from Gibco, cat # 12491-015), collagenase type I at a final concentration of 3mg/ml, DNase at a final concentration of 0.3 mg/ml.
4. Taking out the centrifuge tube, adding DMEM medium with 3 times volume of the digestive juice to stop digestion, and centrifuging at 1000rpm for 5min
5. Removing supernatant, resuspending cells with umbilical cord mesenchymal stem cell selective medium, and following 2 × 104/cm2Inoculating into culture flask, culturing at 37 deg.C for 13 days, and changing every 3 days. The selective medium is prepared by the following steps: umbilical cord mesenchymal stem cells with 40% confluence degree are cultured for 2-3 days by using a serum-free culture medium (Youkang constant biotechnology (Beijing) Co., Ltd., mesenchymal stem cell serum-free culture medium, product No. NC0103), culture supernatant is centrifuged at 2400rpm for 10min, and the culture supernatant is filtered by a 0.4 mu m filter membrane and mixed with a fresh mesenchymal stem cell serum-free culture medium according to the ratio of 1: 1 volume mix)
6. When the cells reach 80-90% confluence, digesting the cells with low-toxicity digestive juice, and adding 4 x 106-5*106And (3) freezing and storing the cells per ml, using a special umbilical cord mesenchymal stem cell freezing and storing liquid with the freezing and storing volume of 1ml by using a program cooling box, and finally placing the liquid in liquid nitrogen. The formula of the low-toxicity digestive juice comprises the following components: 0.05% pancreatin, 0.3mg/ml dnase, DMEM balance. The formula of the umbilical cord mesenchymal stem cell frozen stock solution is as follows: 25-35 mL/100mL of umbilical cord mesenchymal stem cell culture supernatant, 4-6 mL/100mL of glycerol, 4-6 g/100mL of polyvinylpyrrolidone (PVP), 0.5-1.5 g/100mL of polyvinyl alcohol (PVA), 1.5-2.5 g/100mL of trehalose, 1-2.5 g/100mL of hydroxyapatite nanoparticles, 0.5-1.5 mg/100mL of all-trans-retinoic acid, 8-12 mL/100mL of serum substitute and the balance of DMEM.
7. And (3) cell recovery: taking out cells from liquid nitrogen, quickly thawing in a 37 ℃ water bath, centrifuging to remove supernatant, and adding cell recovery solution. The formula of the cell recovery liquid comprises: 25-35 mL/100mL of mesenchymal stem cell culture supernatant, 0.25-0.75 g/100mL of glucose, 0.5-1.5 mg/100mL of all-trans retinoic acid, 0.5-1.5 mg/100mL of ascorbic acid and the balance of DMEM, inoculating the mixture into a culture bottle, and culturing for 1 day at 37 ℃.
8. The resuscitating fluid was removed and the priming solution was replaced before induction for 2 days. The formula of the motivating liquid is as follows: 10% serum replacement, 1% diabody (penicillin streptomycin), 1mg/ml Dimethyloxalylglycine (DMOG), DMEM balance.
9. When the cells grew to 80-90% confluency, the cells were digested with the low toxicity digest described above, resuspended in fresh medium and counted.
10. According to 2 x 104Per cm2The cells were seeded in 24-well plates and cultured overnight in an incubator at 37 ℃.
11. The 24-well plate was removed, the supernatant was removed, and 1ml of the adult cartilage induction medium was added thereto, and the mixture was cultured in a hypoxic incubator with 2% oxygen concentration for 10 days, and the medium was changed every 3 days. The formula of the chondrogenic induction culture medium is as follows: DMEM, and 1% diabody, 10% serum replacement, 1mM sodium pyruvate, 0.35mM proline, 0.284mM ascorbic acid, 0.1. mu.M dexamethasone, 10ng/ml TGF-. beta.3, 50mg/ml ITS, 1. mu.M IGF-1, 1. mu.M retinol receptor antagonist, based on the volume of DMEM.
12. The 24-well plate was taken out, the supernatant was removed, and 500. mu.l of 4% (mass concentration) paraformaldehyde was added for treatment for 30 min.
13. The paraformaldehyde was removed, washed 3 times with PBS, and stained with 0.1% (by mass) toluidine blue solution for 30 min.
14. The staining solution was removed, washed 5 times with PBS and photographed under a microscope.
Examples
Examples 1-3 cartilage was isolated, cultured, passaged, cryopreserved and induced as described above. The results are shown in Table 1.
TABLE 1
| Example 1 | Example 2 | Example 3 | |
| Separating and culturing for 13 days | FIG. 1 shows a schematic view of a | FIG. 2 | FIG. 3 |
| Isolation culture 13 days cell count | 5*106An | 4.6*106An | 5.2*106An |
| Number of frozen cells | 5*106Per ml | 5*106Per ml | 5*106Per ml |
| Cell resuscitation count | 4.8*106An | 4.9*106An | 4.8*106An |
| Cell chondrogenesis induction for 10 days | FIG. 4 | FIG. 5 | FIG. 6 |
Wherein, FIGS. 1-3 are photographs of the tissue umbilical cord mesenchymal stem cells isolated and purified in examples 1-3 for 13 days, showing dense cells and good growth; FIGS. 4 to 6 are photographs of chondrogenic staining obtained after applying the separation, cryopreservation, resuscitation and induction methods of examples 1 to 3, showing deep blue precipitation of glycosaminoglycan from toluidine blue stained cells. Thus, examples 1 to 3 all effectively promote the differentiation of umbilical cord mesenchymal stem cells into chondroblasts.
Comparative example
Comparative examples 1-3 the following conventional isolation and purification culture cryopreservation induction method was used:
1. after cutting the Wharton's jelly, without digestion, the culture was carried out in fresh medium (DMEM, Gibco).
2. Cells were digested with 0.25% trypsin for passage.
3. The cells were frozen in programmed temperature-reducing cassettes using 10% DMSO 10% human serum, and finally placed in liquid nitrogen.
4. The cells were induced to chondrogenic culture in a conventional 37 ℃ incubator using an induction solution.
The results are shown in Table 2.
TABLE 2
| Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| Separating and culturing for 13 days | FIG. 7 | FIG. 8 | FIG. 9 |
| Isolation culture 13 days cell count | 1*106An | 1.4*106An | 1.2*106An |
| Number of frozen cells | 5*106Per ml | 5*106Per ml | 5*106Per ml |
| Cell resuscitation count | 2*106An | 2.5*106An | 2.1*106An |
| Cell chondrogenesis induction for 10 days | FIG. 10 shows a schematic view of a | FIG. 11 | FIG. 12 |
Wherein, FIGS. 7 to 9 are photographs of the tissue umbilical cord mesenchymal stem cells separated and purified in comparative examples 1 to 3 for 13 days, showing that the cells are sparse and grow poorly; FIGS. 10 to 12 are photographs of chondrogenic staining obtained after applying the separation, cryopreservation, resuscitation and induction methods of comparative examples 1 to 3, with only a few blue precipitates. As can be seen from this, comparative examples 1 to 3 have very low efficacy in promoting the directional differentiation of umbilical cord mesenchymal stem cells into chondrogenic cartilage.
Claims (2)
1. A method for promoting directional differentiation of umbilical cord mesenchymal stem cells to chondrogenesis is characterized by comprising the following steps:
(1) taking out umbilical cord tissues, shearing, cleaning, adding tissue digestive juice for digestion, filtering and removing supernatant after digestion is finished to obtain umbilical cord mesenchymal stem cells;
(2) culturing the umbilical cord mesenchymal stem cells by using an umbilical cord mesenchymal stem cell selective culture medium;
(3) when the cell reaches 80-90% confluence degree, digesting the cell by using low-toxicity digestive juice, then performing cryopreservation by using umbilical cord mesenchymal stem cell cryopreservation liquid, and placing the frozen cell in liquid nitrogen;
(4) taking out cells from liquid nitrogen, quickly thawing in a water bath at 37 ℃, centrifuging to remove supernatant, and adding a cell recovery solution to recover the cells;
(5) removing the resuscitation solution, and replacing the priming solution before induction for culture;
(6) digesting the cells with the low-toxicity digestive juice when the cells grow to 80-90% confluency;
(7) inoculating cells, culturing overnight, removing supernatant, adding chondrogenic induction culture medium, and culturing in a hypoxic incubator with 2% oxygen concentration;
the tissue digestive juice is: DMEM, collagenase type I at a final concentration of 3mg/ml and DNase at a final concentration of 0.3 mg/ml;
the ratio of the weight of the umbilical cord tissue to the volume of the tissue digestive fluid is 1: 1;
the selective culture medium for the umbilical cord mesenchymal stem cells is as follows: umbilical cord mesenchymal stem cells with 40% confluence degree are cultured by a serum-free culture medium for 2-3 days to obtain culture supernatant, centrifuging and filtering to obtain a product, and mixing the product with the serum-free culture medium of the mesenchymal stem cells in a ratio of 1: 1, mixing the volumes;
the low-toxicity digestive juice is: DMEM, and 0.05% pancreatin, 0.3mg/ml dnase by volume of DMEM;
the umbilical cord mesenchymal stem cell frozen stock solution is as follows: DMEM, 25-35 mL/100mL of umbilical cord mesenchymal stem cell culture supernatant, 4-6 mL/100mL of glycerol, 4-6 g/100mL of polyvinylpyrrolidone, 0.5-1.5 g/100mL of polyvinyl alcohol, 1.5-2.5 g/100mL of trehalose, 1-2.5 g/100mL of hydroxyapatite nanoparticles, 0.5-1.5 mg/100mL of all-trans retinoic acid and 8-12 mL/100mL of serum substitute in terms of volume of DMEM;
the cell recovery liquid is: DMEM, and 25-35 mL/100mL of mesenchymal stem cell culture supernatant, 0.25-0.75 g/100mL of glucose, 0.5-1.5 mg/100mL of all-trans retinoic acid, and 0.5-1.5 mg/100mL of ascorbic acid in terms of volume of DMEM;
the exciting liquid is: DMEM, and 10% serum replacement, 1% diabody, 1mg/ml dimethyloxalylglycine by DMEM volume;
the chondrogenic induction medium is: DMEM, and 1% diabody, 10% serum replacement, 1mM sodium pyruvate, 0.35mM proline, 0.284mM ascorbic acid, 0.1. mu.M dexamethasone, 10ng/ml TGF-. beta.3, 50mg/ml ITS, 1. mu.M IGF-1, 1. mu.M retinol receptor antagonist, based on the volume of DMEM.
2. The method of claim 1, wherein the umbilical cord tissue is human umbilical cord tissue.
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