Method for separating and culturing human umbilical cord mesenchymal stem cells

Abstract

The invention belongs to the technical field of stem cells, and particularly relates to a method for separating and culturing human umbilical cord mesenchymal stem cells. According to the method, the mesenchymal stem cells are separated from the human umbilical cord by adopting the low-concentration type II collagenase working solution, so that the digestion time is shortened, the production cost is reduced, and the cell damage is reduced. After treatment with type II collagenase, umbilical cord tissue blocks become loose, the tissue blocks are pre-cultured, and the mesenchymal stem cells are proliferated and enriched in the tissue blocks. The mesenchymal stem cells are separated from loose tissue blocks through mechanical blowing and are subjected to adherent culture, the culture time is short, the cell obtaining amount far exceeds that of a traditional adherent method, and the cell quality meets the standard requirement.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cells, and particularly relates to a method for isolating and culturing human umbilical cord mesenchymal stem cells. Background Art

[0002] Mesenchymal Stem Cells (MSCs) are a type of adult stem cells with self-renewal and multi-directional differentiation potential. They were first discovered in bone marrow and later obtained from umbilical cord, placenta, dental pulp, adipose tissue, etc. Due to their multi-directional differentiation and immunomodulatory functions, MSCs have been applied to clinical studies of various major diseases such as graft-versus-host disease, osteoarthritis, Crohn's disease, liver cirrhosis, and systemic lupus erythematosus. There are already MSCs drugs on the market at home and abroad, and many MSCs drugs are in the clinical research stage. Since umbilical cord tissue is easily obtained and the number of MSCs isolated is large, most of the MSCs drugs in clinical research are derived from umbilical cord mesenchymal stem cells.

[0003] Currently, the main methods for isolating umbilical cord mesenchymal stem cells are the adherent method and the enzyme digestion method. The adherent method is simple to operate, but the culture time is long, usually about 14 - 20 days, and the number of cells obtained is small. The enzyme digestion method can obtain more cells, but the economic cost of the enzyme digestion method is high, the digestion time is long, generally about 4 - 5 hours, and the digestion degree is not easy to control. If the digestion time is short, enough cells cannot be obtained, and if the digestion time is too long, the cells are easily damaged. Moreover, since the liquid after enzyme digestion of umbilical cord tissue is very viscous, it is not easy to separate the cells during centrifugation.

[0004] Based on this, Chinese Patent CN109337867A discloses a method for isolating umbilical cord mesenchymal stem cells. The method mainly includes cutting the tissue block into pieces, and then successively digesting the tissue block with trypsin and collagenase, and culturing the digested tissue block by adhesion, etc. Although this method can improve the efficiency of stem cells crawling out from the adherent tissue block to a certain extent, however, digesting with two enzymes successively makes the separation process more complex and increases the separation cost. And subsequent culturing is still by adhesion of the tissue block after enzyme treatment, which also hinders the improvement of cell yield. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for isolating and culturing human umbilical cord mesenchymal stem cells. First, digest the tissue block with low-concentration collagenase, then suspend and culture the tissue block in a serum-free medium for mesenchymal stem cells to preliminarily amplify the umbilical cord mesenchymal stem cells in the loose tissue block, and finally isolate and obtain umbilical cord mesenchymal stem cells from the loose tissue block. The method of the present invention can obtain a large number of cells with high viability and greatly reduce the separation cost.

[0006] The method for isolating and culturing human umbilical cord mesenchymal stem cells described in the present invention includes the following steps: (1) Tissue processing: Cut the umbilical cord into segments, place them in a disposable sterile petri dish, cut the umbilical cord along the vein, remove the arteries, veins, and the outer membrane of the umbilical cord to obtain Wharton's jelly fragments; (2) Collagenase digestion: Put the Wharton's jelly fragments obtained from one segment of the umbilical cord into a sterile shaking tube, add the type II collagenase working solution, and mix and digest in a shaking incubator; (3) Tissue block culture: After digestion, add physiological saline rinse to the collagenase digestion solution for rinsing, filter through a 100 μm filter, repeat rinsing 2 times, then rinse the umbilical cord fragments 2 times with DMEM / F12 medium, collect the fragments on the 100 μm filter, put the digested fragments into a cell culture flask, add serum-free mesenchymal stem cell medium for culture, and the mesenchymal stem cells in the tissue block grow suspended as colonies; (4) Cell isolation and culture: After obvious mesenchymal stem cells are observed in the loose tissue blocks, put the fragments obtained by digesting the umbilical cord tissue into a centrifuge tube, add physiological saline rinse, disperse the umbilical cord fragments with a pipette, centrifuge and discard the supernatant, repeat centrifugation 2 - 3 times, isolate the mesenchymal stem cells from the tissue, resuspend the cell pellet with serum-free mesenchymal stem cell medium, and transfer to a cell culture flask for culture; (5) Passage: When the cell confluence reaches 80 - 90%, harvest the P0 generation cells and perform passage operation; (6) Cryopreservation: When the cell confluence reaches 80 - 90%, harvest the P1 generation cells, add mesenchymal stem cell cryopreservation solution, and use a programmable freezer or a programmable freezing box to cool at a rate of -1°C / min to -80°C, and temporarily store in a -80°C ultra-low temperature refrigerator or transfer to the vapor phase of liquid nitrogen for cryopreservation.

[0007] In step (1), cutting the umbilical cord into segments means cutting the umbilical cord into segments 1 - 2 cm long, preferably 1 cm.

[0008] The bottom area of the fragments in step (1) is 1 mm 2 - 9 mm 2 .

[0009] In step (2), the concentration of the type II collagenase working solution is 10 U / mL - 50 U / mL, and the added amount is 3 mL - 5 mL.

[0010] The digestion conditions in step (2) are shaking in a 37°C shaking incubator for 1 h - 2 h, and the rotation speed is 100 rpm - 200 rpm.

[0011] The culture conditions in step (3) are 37°C and 5% CO2.

[0012] The cell density for the passage operation in step (5) is 4000 cells / cm 2-8000 cells / cm 2 , preferably 8000 cells / cm 2 .

[0013] The present invention first uses low-concentration collagenase to digest tissue blocks, and then suspends and cultures the tissue blocks in a serum-free medium for mesenchymal stem cells, enabling the primary expansion of umbilical cord mesenchymal stem cells in the loose tissue blocks. Finally, umbilical cord mesenchymal stem cells are isolated from the loose tissue blocks. The cells obtained by this method are large in quantity, high in viability, and the isolation cost is greatly reduced.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses a type II collagenase working solution with a low concentration (10 U / mL - 50 U / mL), reducing the dosage of collagenase and lowering the production cost. In view of the different activities of collagenase in different batches, the working solution is prepared with enzyme activity as the concentration unit, ensuring the stability of the operation.

[0015] (2) In the present invention, the enzyme digestion time is short, reducing cell damage during the digestion process.

[0016] (3) After treatment with type II collagenase in the present invention, the umbilical cord tissue blocks become loose. First, the tissue blocks are pre-cultured, and mesenchymal stem cells proliferate and enrich in the tissue blocks. After a certain period of enrichment, mesenchymal stem cells are isolated from the loose tissue blocks by mechanical dispersion and then subjected to adherent culture. The cell yield is high, the culture time is short, and millions of cells can be isolated from each centimeter of umbilical cord after about 10 days of culture. Description of the Drawings

[0017] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. In the drawings: Figure 1 is a diagram showing the growth of umbilical cord mesenchymal stem cells in loose tissue blocks in Example 1; Figure 2 is a diagram showing the growth morphology of P0-generation umbilical cord mesenchymal stem cells in Example 1; Figure 3 is a diagram showing the growth morphology of P1-generation umbilical cord mesenchymal stem cells in Example 1; Figure 4 is a diagram showing the flow cytometry detection results of positive markers of P1-generation umbilical cord mesenchymal stem cells in Example 1; Figure 5 is a diagram showing the flow cytometry detection results of negative markers of P1-generation umbilical cord mesenchymal stem cells in Example 1; Figure 6 is a diagram showing the trilineage differentiation results of P1-generation umbilical cord mesenchymal stem cells in Example 1. Detailed Embodiments

[0018] The present invention will be further described below in conjunction with embodiments.

[0019] All raw materials used in the embodiments are commercially available unless otherwise specified.

[0020] Embodiment 1 A method for the isolation and culture of human umbilical cord mesenchymal stem cells, comprising the following steps: (1) Tissue treatment: Cut the umbilical cord into sections about 1 cm long and place them in a disposable sterile culture dish. Cut the umbilical cord along the vein, remove the arteries and veins and the outer membrane of the umbilical cord to obtain Wharton's jelly, and make fragments with a bottom area of 1 mm 2 - 9 mm 2 (side length 1 mm - 3 mm).

[0021] (2) Collagenase digestion: Put the Wharton's jelly fragments prepared from 1 cm long umbilical cord into a sterile 50 mL shaking tube, and add 3 mL of type II collagenase working solution at 25 U / mL. Place it in a shaking incubator and shake at 200 rpm and 37 °C for 1.5 h.

[0022] (3) Tissue block culture: After digestion, add 30 mL of physiological saline rinsing solution to the collagenase digestion solution, filter through a 100 μm filter, and repeat rinsing the umbilical cord fragments 2 times with 30 mL of physiological saline rinsing solution. Then rinse the umbilical cord fragments 2 times with 5 mL of DMEM / F12 medium, and collect the fragments on the filter. Put the fragments obtained by digesting the Wharton's jelly of 1 cm umbilical cord tissue into 1 T75 flask, add 15 mL of mesenchymal stem cell serum-free medium, and culture at 37 °C and 5% CO2. The mesenchymal stem cells in the tissue block grow suspended in colonies. The growth of umbilical cord mesenchymal stem cells in loose tissue blocks is shown in Figure 1 as follows.

[0023] (4) Cell isolation and culture: After obvious mesenchymal stem cells are observed in the loose tissue block, put the fragments obtained by digesting the umbilical cord tissue into a centrifuge tube, add physiological saline rinsing solution, disperse the umbilical cord fragments with a pipette, centrifuge at 600 g for 5 min, discard the supernatant, and repeat washing and centrifuging 3 times to separate the mesenchymal stem cells from the tissue. Resuspend the cell pellet with mesenchymal stem cell serum-free medium and transfer it to a cell culture flask for culture.

[0024] (5) Passage: When the cell confluence reaches 90%, harvest the P0 generation cells and perform passage operation at 8000 cells / cm 2 . The growth morphology of P0 generation umbilical cord mesenchymal stem cells is shown in Figure 2 as follows.

[0025] (6) Cryopreservation: When the cell confluence reaches 90%, harvest the P1 generation cells. Add mesenchymal stem cell cryopreservation solution and use a programmable cooling box to cool down to -80°C at a rate of -1°C / min. Transfer to the vapor phase of liquid nitrogen for cryopreservation after the test results are qualified. The growth morphology diagram of P1 generation umbilical cord mesenchymal stem cells is as Figure 3 shown, and the surface markers of P1 generation umbilical cord mesenchymal stem cells are detected by flow cytometry.

[0026] The P1 generation cells are digested, centrifuged, and resuspended in phosphate buffer to prepare a single-cell suspension (concentration 1×10 6 cells / mL). Add CD73, CD90, CD105, CD14, CD19, CD34, CD45, HLA-DR respectively, incubate, centrifuge, wash, and resuspend, and then detect the surface markers by flow cytometry.

[0027] The flow cytometry detection result diagram of positive markers of P1 generation umbilical cord mesenchymal stem cells is as Figure 4 shown; the flow cytometry detection result diagram of negative markers of P1 generation umbilical cord mesenchymal stem cells is as Figure 5 shown.

[0028] Detect the multi-directional differentiation potential of P1 umbilical cord mesenchymal stem cells. Take the harvested P1 umbilical cord mesenchymal stem cells for osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation experiments. The three-line differentiation result diagram of P1 generation umbilical cord mesenchymal stem cells is as Figure 6 shown.

[0029] Osteogenic induction: Collect P1 generation cells and inoculate them into a 6-well plate at a density of 6000 cells / cm 2 2. Incubate statically in an incubator at 37°C and 5% CO2 for 24 hours, discard the original culture medium, add osteogenic induction differentiation medium (add the original culture medium for the negative control), change the medium once every 3 days, and after 21 days of culture, perform alizarin red staining and observe the results under a microscope. The results are as Figure 6 shown in a of

[0030] Adipogenic induction: Collect P1 generation cells and inoculate them into a 6-well plate at a density of 6000 cells / cm 2 2. Incubate statically in an incubator at 37°C and 5% CO2 for 24 hours, discard the original culture medium, add adipogenic induction differentiation medium (add the original culture medium for the negative control), change the medium once every 3 days, and after 21 days of culture, perform oil red O staining and observe the results under a microscope. The results are as Figure 6 shown in b of

[0031] Chondrogenic induction: Collect P1 generation cells and inoculate them at a density of 1.0×10 6Cells were inoculated into a 15 mL centrifuge tube at a density of Figure 6 as shown in c in

[0032] Example 2 The method for isolating and culturing the umbilical cord mesenchymal stem cells was the same as that in Example 1, except that in step 2, the collagenase concentration was 50 U / mL and the digestion time was 1 h.

[0033] Example 3 The method for isolating and culturing the umbilical cord mesenchymal stem cells was the same as that in Example 1, except that in step 2, the collagenase concentration was 10 U / mL and the digestion time was 2 h.

[0034] Example 4 The method for isolating and culturing the umbilical cord mesenchymal stem cells was the same as that in Example 1, except that in step 2, the collagenase dosage was 4 mL and the digestion time was 2 h. The shaker speed was 100 rpm.

[0035] Example 5 The method for isolating and culturing the umbilical cord mesenchymal stem cells was the same as that in Example 1, except that in step 2, the collagenase dosage was 5 mL and the digestion time was 2 h. The shaker speed was 150 rpm.

[0036] Comparative Example 1 In this scheme, a high-concentration collagenase was used, and the cells after enzyme digestion were directly cultured without incubation after tissue block enzyme digestion.

[0037] (1) Tissue treatment: The umbilical cord was cut into sections about 1 cm long and placed in a disposable sterile culture dish. The umbilical cord was cut along the vein, and the arteries, veins, and outer membrane of the umbilical cord were removed to obtain Wharton's jelly, which was made into fragments with a bottom area of 1 mm 2 -9 mm 2 (side length 1 mm - 3 mm).

[0038] (2) Collagenase digestion: The Wharton's jelly fragments prepared from 1 cm long umbilical cord were put into a sterile 50 mL shaking tube, and 5 mL of type II collagenase working solution at 200 U / mL was added. It was shaken in a shaker at 200 rpm and 37 °C for 1.5 h.

[0039] After digestion, add DPBS to 45 mL, pipette thoroughly, and centrifuge at 400 g for 5 min. After centrifugation, discard the supernatant. Add 5 mL of mesenchymal stem cell serum-free medium to each centrifuge tube, pipette to mix well, and inoculate into 1 T75 cell culture flask. Supplement with 15 mL of medium and place in an incubator at 37 °C and 5% CO2 for culture.

[0040] (4)Cell harvesting: When the cell confluence reaches 90%, harvest the P0 generation cells.

[0041] The culture results of Examples 1-5 and the comparative example are shown in Table 1 below: Table 1 Culture result table

[0042] In summary, after treatment with type II collagenase, the umbilical cord tissue blocks become loose. After pre-culturing the tissue blocks, mesenchymal stem cells proliferate and enrich in the tissue blocks. After a certain period of enrichment, mesenchymal stem cells are isolated from the loose tissue blocks by mechanical dispersion and subjected to adherent culture, resulting in a higher cell yield and a shorter culture time.

Claims

1. A method for isolating and culturing human umbilical cord mesenchymal stem cells, characterized in that, It includes the following steps: (1) Tissue processing: Cut the umbilical cord into segments, place them in a disposable sterile petri dish, cut the umbilical cord along the vein, remove the arteries, veins and the outer membrane of the umbilical cord to obtain Wharton's jelly fragments; (2) Collagenase digestion: Put the Wharton's jelly fragments obtained from a segment of umbilical cord into a sterile shaking tube, add the type II collagenase working solution, and mix and digest in a shaking incubator; (3) Tissue block culture: After digestion is completed, add physiological saline rinse solution to the collagenase digestion solution for rinsing, filter through a sieve, then rinse the umbilical cord fragments with the culture medium, collect the fragments on the sieve, put the digested fragments into a cell culture flask, add serum-free mesenchymal stem cell medium for culture, and the mesenchymal stem cells in the tissue block grow in suspension as colonies; (4) Cell isolation and culture: After obvious mesenchymal stem cells are observed in the loose tissue block, put the fragments obtained by digesting the umbilical cord tissue into a centrifuge tube, add physiological saline rinse solution, disperse the umbilical cord fragments with a pipette, centrifuge and discard the supernatant, isolate the mesenchymal stem cells from the tissue, resuspend the cell pellet with serum-free mesenchymal stem cells, and transfer to a cell culture flask for culture; (5) Subculture: Harvest the P0 generation cells and perform subculture operations; (6) Cryopreservation: Harvest the P1 generation cells, add the mesenchymal stem cell cryopreservation solution, and cool down for cryopreservation.

2. The method for separating and culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step (1), cutting the umbilical cord into segments means cutting the umbilical cord into segments with a length of 1 cm - 2 cm.

3. The method for isolating and culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The bottom area of the debris described in step (1) is 1 mm 2 -9 mm 2 .

4. The method for separating and culturing human umbilical cord mesenchymal stem cells according to claim 1, wherein, In step (2), the concentration of the type II collagenase working solution is 10 U / mL - 50 U / mL, and the added amount is 3 mL - 5 mL.

5. The method for separating and culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The digestion conditions in step (2) are shaking in a shaking incubator at 37°C for 1 h - 2 h, and the rotation speed is 100 rpm - 200 rpm.

6. The method for isolating and culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The culture conditions in step (3) are 37°C and 5% CO2.

7. The method for the isolation and culture of human umbilical cord mesenchymal stem cells according to claim 1, wherein, The cell density for the subculture operation described in step (5) is 8000 cells / cm 2 .

8. The method for separating and culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The temperature for cooling and cryopreservation in step (6) is -80°C.

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