Method for isolation and culture of human umbilical cord mesenchymal stem cells

Abstract

The present application belongs to the technical field of stem cells, and particularly relates to a method for separating and culturing human umbilical cord mesenchymal stem cells. The present application uses a low-concentration type II collagenase working solution to separate mesenchymal stem cells from a human umbilical cord, shortens the digestion time, reduces the production cost, and reduces cell damage. After being treated by the type II collagenase, the umbilical cord tissue block becomes loose, the tissue block is pre-cultured, and the mesenchymal stem cells proliferate and enrich in the tissue block. The mesenchymal stem cells are separated from the loose tissue block by mechanical blowing and are subjected to adherent culture, the culture time is short, the obtained cell amount is far more than that of the traditional adherent method, and the cell quality meets the standard requirements.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of stem cells, and particularly relates to a method for separating and culturing human umbilical cord mesenchymal stem cells. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are a kind of adult stem cells with self-renewal and multi-directional differentiation potential, which are first discovered in bone marrow and then obtained in umbilical cord, placenta, dental pulp and adipose tissue, etc. Due to its multi-directional differentiation and immune regulation function, MSCs are applied to clinical research of major diseases such as graft-versus-host disease, osteoarthritis, Crohn's disease, cirrhosis, systemic lupus erythematosus, etc. MSCs drugs have been marketed at home and abroad, and a number of MSCs drugs are in the clinical research stage. Because umbilical cord tissue is easy to obtain, the number of MSCs obtained by separation is large, and most of the MSCs drugs for clinical research are derived from umbilical cord mesenchymal stem cells.

[0003] At present, the main methods for separating 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 obtained cells are few. The enzyme digestion method can obtain more cells, but the economic cost of the enzyme digestion method is high, the digestion time is long, usually about 4-5 hours, and the digestion degree is not easy to control. If the digestion time is short, it is difficult to obtain enough cells, and if the digestion time is too long, the cells are easily damaged. In addition, because the liquid after enzyme digestion of the umbilical cord tissue is very viscous, it is not easy to separate the cells during centrifugation.

[0004] Therefore, Chinese patent CN109337867A discloses a method for separating umbilical cord mesenchymal stem cells, which mainly includes cutting the tissue block, then sequentially digesting the tissue block with trypsin and collagenase, and adhering and culturing the digested tissue block. This method can improve the efficiency of the tissue block adherent growth and crawling out of stem cells to a certain extent, but the sequential digestion with two enzymes makes the separation process more complex and increases the separation cost. Moreover, the subsequent adherent culture of the enzyme-treated tissue block also hinders the improvement of cell yield. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a method for separating and culturing human umbilical cord mesenchymal stem cells, which first digests the tissue block with low-concentration collagenase, then suspends and cultures the tissue block in a mesenchymal stem cell serum-free medium to preliminarily expand the umbilical cord mesenchymal stem cells in the loose tissue block, and finally separates and obtains the umbilical cord mesenchymal stem cells from the loose tissue block. The method can obtain a large number of cells with high viability and greatly reduces the separation cost.

[0006] The method for separating and culturing human umbilical cord mesenchymal stem cells comprises the following steps:

[0007] (1) Tissue processing: cut the umbilical cord into segments, put into a sterile disposable culture dish, cut the umbilical cord along the vein, remove the arteriovenous and adventitia of the umbilical cord, and obtain the Wharton's jelly fragments;

[0008] (2) Collagenase digestion: put the Wharton's jelly fragments prepared from a segment of umbilical cord into a sterile shaking tube, add type II collagenase working solution, and mix and digest in a shaking bed;

[0009] (3) Tissue block culture: after digestion is completed, add physiological saline flushing solution to the collagenase digestion solution for flushing, filter through a 100 μm filter screen, repeat flushing 2 times, then flush the umbilical cord fragments with DMEM / F12 medium 2 times, collect the fragments above the 100 μm filter screen, put the digested fragments into a cell culture bottle, add mesenchymal stem cell serum-free culture medium for culture, and the mesenchymal stem cells in the tissue block suspend to form colonies and grow;

[0010] (4) Cell separation and culture: after observing obvious mesenchymal stem cells in the loose tissue block, put the fragments obtained by digesting the umbilical cord tissue into a centrifuge tube, add physiological saline flushing solution, blow the umbilical cord fragments with a pipette, centrifuge and discard the supernatant, repeat centrifugation 2-3 times, separate the mesenchymal stem cells from the tissue, resuspend the cell pellet with mesenchymal stem cell serum-free culture medium, and transfer to a cell culture bottle for culture;

[0011] (5) Passage: when the cell fusion degree reaches 80-90%, harvest P0 cells for passage operation;

[0012] (6) Cryopreservation: when the cell fusion degree reaches 80-90%, harvest P1 cells, add mesenchymal stem cell cryopreservation solution, use a program-controlled cooling instrument or a program-controlled cooling box to reduce the temperature to -80°C at a rate of -1°C / min, and temporarily store in a -80°C ultra-low temperature freezer or transfer to liquid nitrogen gas phase for storage.

[0013] In step (1), the umbilical cord is cut into segments of 1-2 cm in length, preferably 1 cm.

[0014] In step (1), the bottom area of the fragments is 1 mm 2 -9 mm 2 .

[0015] In step (2), the concentration of the type II collagenase working solution is 10 U / mL-50 U / mL, and the amount added is 3 mL-5 mL.

[0016] In step (2), the digestion conditions are shaking at 37°C in a shaking bed for 1-2 h at a speed of 100 rpm-200 rpm.

[0017] In step (3), the culture conditions are 37°C, 5% CO2.

[0018] The cell density of the cell passage operation in step (5) is 4000 cells / cm 2 - 8000 cells / cm 2 , preferably 8000 cells / cm 2 .

[0019] The present application first uses low concentration collagenase to digest the tissue block, then suspends the tissue block in mesenchymal stem cell serum-free medium for culture, preliminarily expands the umbilical cord mesenchymal stem cells in the loose tissue block, and finally separates the umbilical cord mesenchymal stem cells from the loose tissue block. The method can obtain a large number of cells with high survival rate, and greatly reduces the separation cost.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] (1) The present application uses low concentration (10 U / mL-50 U / mL) type II collagenase working solution, reduces the amount of collagenase, and reduces the production cost; since the activity of different batches of collagenase is different, the working solution is prepared according to the enzyme activity as the concentration unit, which ensures the stability of the operation.

[0022] (2) The enzyme digestion time in the present application is short, which reduces the cell damage in the digestion process.

[0023] (3) After the treatment of type II collagenase, the umbilical cord tissue block becomes loose, the tissue block is pre-cultured first, and the mesenchymal stem cells are proliferated and enriched in the tissue block. After a certain time of enrichment, the mesenchymal stem cells are separated from the loose tissue block by mechanical blowing and adherent culture, the cell yield is high, the culture time is short, and several million cells can be separated and obtained per centimeter of umbilical cord after about 10 days of culture. BRIEF DESCRIPTION OF DRAWINGS

[0024] The drawings described herein are used to provide further understanding of the present application and form a part of the present application. In the drawings:

[0025] Figure 1 is a diagram of the growth of the umbilical cord mesenchymal stem cells of Example 1 in loose tissue blocks;

[0026] Figure 2 is a diagram of the growth morphology of the P0 generation umbilical cord mesenchymal stem cells of Example 1;

[0027] Figure 3 is a diagram of the growth morphology of the P1 generation umbilical cord mesenchymal stem cells of Example 1;

[0028] Figure 4 is a diagram of the flow cytometry detection results of the positive markers of the P1 generation umbilical cord mesenchymal stem cells of Example 1;

[0029] Figure 5 Figure of flow cytometry results of negative markers of P1 generation of umbilical cord mesenchymal stem cells of Example 1;

[0030] Figure 6 Figure of three-line differentiation results of P1 generation of umbilical cord mesenchymal stem cells of Example 1. DETAILED DESCRIPTION

[0031] The application will be further described below in conjunction with examples.

[0032] All raw materials used in the examples are commercially available, except for special instructions.

[0033] Example 1

[0034] A method for separating and culturing a human umbilical cord mesenchymal stem cell, comprising the following steps:

[0035] (1) Tissue processing: cut the umbilical cord into about 1 cm each, and put it into a sterile disposable culture dish. Cut the umbilical cord along the vein, remove the arteriovenous and perivascular membrane, obtain Wharton's jelly, and make fragments with a bottom area of 1 mm 2 -9mm 2 (length 1 mm-3 mm).

[0036] (2) Collagenase digestion: put the Wharton's jelly fragments obtained by preparing 1 cm long umbilical cord into a sterile 50 mL shaking tube, and add 3 mL of 25 U / mL type II collagenase working solution. Shake at 200 rpm and 37℃ for 1.5 h.

[0037] (3) Tissue culture: after digestion, add 30 mL of physiological saline flushing solution to the collagenase digestion solution, filter through a 100 μm filter screen, and repeat the flushing of the umbilical cord fragments twice with 30 mL of physiological saline flushing solution. Then flush the umbilical cord fragments twice with 5 mL of DMEM / F12 medium, and collect the fragments on the filter screen. Put the fragments obtained by digesting the Wharton's jelly of 1 cm umbilical cord tissue into a T75 bottle, add 15 mL of mesenchymal stem cell serum-free medium, and culture at 37℃ and 5% CO2. The mesenchymal stem cells in the tissue mass grow into colonies. Figure 1

[0038] (4) Cell separation and culture: after observing obvious mesenchymal stem cells in the loose tissue mass, put the fragments obtained by digesting the umbilical cord tissue into a centrifuge tube, add physiological saline flushing solution, blow the umbilical cord fragments with a pipette, centrifuge at 600g for 5 min, discard the supernatant, repeat the washing and centrifugation for 3 times, and 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 bottle for culture. ​

[0039] (5) Passage: when the cell fusion degree reaches 90%, the P0 generation cells are harvested, and the cells are inoculated at a density of 8000 cells / cm 2 for passage operation. The growth morphology of the P0 generation umbilical cord mesenchymal stem cells is shown in Figure 2 .

[0040] (6) Cryopreservation: when the cell fusion degree reaches 90%, the P1 generation cells are harvested. The mesenchymal stem cell cryopreservation solution is added, and the programmed cooling box is used to reduce the temperature to -80℃ at a rate of -1℃ / min. After the test results are qualified, it is transferred to the liquid nitrogen gas phase for cryopreservation. The growth morphology of the P1 generation umbilical cord mesenchymal stem cells is shown in Figure 3 , and the surface markers of the P1 generation umbilical cord mesenchymal stem cells are detected by flow cytometry.

[0041] The P1 generation cells are digested, centrifuged, resuspended in phosphate buffer solution to prepare a single cell suspension (concentration 1×10 6 cells / mL), and CD73, CD90, CD105, CD14, CD19, CD34, CD45, and HLA-DR are added respectively. Incubation, centrifugation, washing, and resuspension are performed, and flow cytometry is used to detect the surface markers.

[0042] The flow cytometry detection result graph of the positive markers of the P1 generation umbilical cord mesenchymal stem cells is shown in Figure 4 , and the flow cytometry detection result graph of the negative markers of the P1 generation umbilical cord mesenchymal stem cells is shown in Figure 5 .

[0043] The multilineage differentiation potential of the P1 umbilical cord mesenchymal stem cells is detected, and the harvested P1 umbilical cord mesenchymal stem cells are subjected to osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation experiments. The three-lineage differentiation result graph of the P1 generation umbilical cord mesenchymal stem cells is shown in Figure 6 .

[0044] Osteogenic induction: collect the P1 generation cells, inoculate them into a 6-well plate at a density of 6000 cells / cm 2 , and incubate them in a 37℃, 5% CO2 incubator. After 24 hours of culture, the original culture solution is discarded, and osteogenic induction differentiation solution (negative control: original culture solution) is added. The solution is replaced every 3 days, and after 21 days of culture, alizarin red staining is performed. The results are observed under a microscope, and the results are shown in Figure 6 a.

[0045] Adipogenic induction: collect the P1 generation cells, inoculate them into a 6-well plate at a density of 6000 cells / cm 2 , and incubate them in a 37℃, 5% CO2 incubator. After 24 hours of culture, the original culture solution is discarded, and adipogenic induction differentiation solution (negative control: original culture solution) is added. The solution is replaced every 3 days, and after 21 days of culture, oil red O staining is performed. The results are observed under a microscope, and the results are shown in Figure 6As shown in middle b.

[0046] Chondrogenic induction: collect P1 cells, inoculate at a density of 1.0 x 10 6 cells / mL into a 15 mL centrifuge tube, add chondrogenic induction differentiation premix (repeat 1 time), add chondrogenic induction differentiation medium after centrifugation, flick the centrifuge tube every 24 h, change the medium every 3 days, after 21 days of culture, paraffin embedding, sectioning, alizarin red staining, observe the results under a microscope, the results are shown in middle c. Figure 6

[0047] Example 2

[0048] The separation and culture method of the umbilical cord mesenchymal stem cells is the same as that of Example 1, except that the collagenase concentration in step 2 is 50 U / mL, and the digestion time is 1 h.

[0049] Example 3

[0050] The separation and culture method of the umbilical cord mesenchymal stem cells is the same as that of Example 1, except that the collagenase concentration in step 2 is 10 U / mL, and the digestion time is 2 h.

[0051] Example 4

[0052] The separation and culture method of the umbilical cord mesenchymal stem cells is the same as that of Example 1, except that the collagenase concentration in step 2 is 10 U / mL, and the digestion time is 2 h.

[0053] Example 5

[0054] The separation and culture method of the umbilical cord mesenchymal stem cells is the same as that of Example 1, except that the collagenase concentration in step 2 is 10 U / mL, and the digestion time is 2 h.

[0055] Comparative Example 1

[0056] This scheme uses high-concentration collagenase to directly culture the cells after enzyme digestion, without incubation after tissue block enzyme digestion.

[0057] (1) Tissue processing: cut the umbilical cord into about 1 cm each, and put it into a sterile disposable culture dish. Cut the umbilical cord along the vein, remove the arteriovenous and outer membrane of the umbilical cord, obtain the Wharton's jelly, and make fragments with a bottom area of 1 mm 2 -9mm 2 (length of 1 mm-3 mm).

[0058] ​(2) Collagenase digestion: put the Wharton's jelly fragments prepared from 1 cm long umbilical cord into a sterile 50 mL shaking tube, add 5 mL of 200 U / mL type II collagenase working solution. Shake at 200 rpm, 37°C for 1.5 h.

[0059] (3) After the digestion is completed, add DPBS to 45 mL, blow thoroughly, and centrifuge at 400g for 5 min. Discard the supernatant after centrifugation, add 5 mL of mesenchymal stem cell serum-free medium to each centrifuge tube, mix well by blowing, and inoculate into one T75 cell culture bottle, add 15 mL of medium, and place in a 37°C, 5% CO2 incubator for culture.

[0060] (4) Cell harvest: when the cell confluence reaches 90%, harvest the P0 cells.

[0061] The culture results of Examples 1-5 and Comparative Examples are shown in Table 1 below:

[0062] Table 1 Culture results table

[0063]

[0064] In summary, after treatment with type II collagenase, the umbilical cord tissue block becomes loose, the tissue block is pre-cultured, and the mesenchymal stem cells are proliferated and enriched in the tissue block. After a certain period of enrichment, the mesenchymal stem cells are separated from the loose tissue block by mechanical blowing and adherent culture, and the cell yield is higher and the culture time is shorter.

Claims

1. A method for isolating and culturing human umbilical cord mesenchymal stem cells, characterized by, The method comprises the following steps: (1) tissue processing: cutting the umbilical cord into segments, placing the segments into a sterile disposable culture dish, cutting the umbilical cord along the vein, removing the arteriovenous and adventitia of the umbilical cord, and obtaining Wharton's jelly fragments; (2) collagenase digestion: placing the Wharton's jelly fragments obtained from a segment of the umbilical cord into a sterile shaking tube, adding a working solution of type II collagenase, and mixing and digesting in a shaking bed; (3) tissue piece culture: after digestion is completed, adding a physiological saline flushing solution to the collagenase digestion solution for flushing, filtering with a filter screen, flushing the umbilical cord fragments with a culture medium, collecting the fragments on the filter screen, placing the digested fragments into a cell culture bottle, adding mesenchymal stem cell serum-free culture medium for culture, and allowing the mesenchymal stem cells in the tissue piece to suspend and grow into colonies; (4) cell separation and culture: after observing obvious mesenchymal stem cells in the loose tissue piece, placing the digested fragments of the umbilical cord tissue into a centrifuge tube, adding a physiological saline flushing solution, blowing the umbilical cord fragments apart with a pipette, centrifuging and discarding the supernatant, separating the mesenchymal stem cells from the tissue, resuspending the cell pellet with serum-free mesenchymal stem cell suspension, and transferring to a cell culture bottle for culture; (5) subculture: harvesting P0 cells and performing subculture; (6) cryopreservation: harvesting P1 cells, adding mesenchymal stem cell cryopreservation solution, and cryopreserving at a reduced temperature; The concentration of the type II collagenase working solution in step (2) is 10 U / mL-50 U / mL, and the amount added is 3 mL-5 mL; The digestion condition in step (2) is shaking at 37°C for 1 h-2 h; In step (1), the umbilical cord is cut into segments of 1 cm-2 cm in length; The bottom area of the fragments in step (1) is 1 mm 2 - 9 mm 2 .

2. The method of claim 1, wherein the human umbilical cord mesenchymal stem cells are isolated and cultured. The rotation speed for digestion in step (2) is 100 rpm-200 rpm.

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

4. The method for isolating and culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The cell density of the passaging operation in step (5) is 8000 cells / cm 2 .

5. The method for isolating and culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The temperature for cryopreservation at a reduced temperature in step (6) is -80°C.

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