Efficient isolated culture method and application of primary human umbilical cord mesenchymal stem cells

Abstract

The invention discloses an efficient isolated culture method of primary human umbilical cord mesenchymal stem cells, which comprises the following steps: cutting umbilical cord tissues into tissue blocks with proper sizes, and then adding the tissue blocks into a primary complete medium for primary culture and secondary culture in sequence. A secondary cell culture method is adopted, a certain number of primary cells can still be harvested after umbilical cord tissue blocks abandoned after primary culture is finished are cultured again, and the number of primary seed cells is increased. Besides, the umbilical cord tissue is cut into tissue blocks with the volume of 9-25mm < 3 >, so that primary cells can climb out of the edges of the tissue blocks to form colonies, and relatively more primary human umbilical cord mesenchymal stem cells can be harvested in a shorter time. According to the method, more primary human umbilical cord mesenchymal stem cells can be obtained by utilizing umbilical cord tissue blocks to the maximum extent, the cell yield is increased, the operation steps are few, simple, more efficient and safer, the primary separation and culture process of the human umbilical cord mesenchymal stem cells is further optimized, and industrialization of the human umbilical cord mesenchymal stem cells is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of mesenchymal stem cell culture, and in particular to a high-efficiency separation and culture method for primary human umbilical cord mesenchymal stem cells and its application. Background Art

[0002] Primary human umbilical cord mesenchymal stem cells (HUMSCs) are mesenchymal stem cells isolated and cultured from neonatal umbilical cord tissue fragments. They possess the potential for self-renewal and multidirectional differentiation. Expanded and cultured HUMSCs are widely used in preclinical and clinical research due to their biological properties, including support for hematopoiesis, immune regulation, and tissue repair. Primary HUMSCs can be obtained by isolating HUMSCs and then culturing them. However, the current culture efficiency of primary HUMSCs is low, and the culture process is complex. Therefore, it is crucial to develop a method for culturing HUMSCs that is efficient and safe, with a high yield, a few simple steps, and a low production rate.

[0003] Chinese invention patent application CN109593709A discloses a method for extracting, culturing and subculturing primary mesenchymal stem cells. During primary culture, the time for the first fluid change is extended to 72 hours, and a complete fluid change method is adopted. Thereafter, the fluid is completely changed every 2-3 days. A large number of colonies can be seen under the microscope on the 7th to 10th day of culture. The invention shortens the primary culture time and retains the cell growth activity to the greatest extent by adjusting the trypsin digestion concentration, dosage and time. However, the cell source range is narrow for bone marrow mesenchymal stem cells. Chinese invention patent application CN118291377A discloses a method for isolating and culturing primary umbilical cord mesenchymal stem cells. The suspension culture method can obtain primary umbilical cord mesenchymal stem cells with high viability and high proliferation ability from a serum-free and serum-free substitute culture medium with clear components in 4-8 days. However, collagenase digestion is required before culture, and the process steps are cumbersome, and there is a possibility of obtaining other miscellaneous cells. Summary of the Invention

[0004] In order to develop a method for culturing primary human umbilical cord mesenchymal stem cells with high cell yield, few and simple operation steps, high efficiency and safety, the first aspect of the present invention provides a method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells, comprising the following steps:

[0005] Initial cultivation:

[0006] S1 pre-processed umbilical cord tissue;

[0007] S2 cuts the umbilical cord tissue into small pieces to obtain umbilical cord tissue blocks;

[0008] S3 Add primary complete culture medium to the umbilical cord tissue pieces and inoculate them according to weight;

[0009] S4 was placed in an incubator and cultured for 9-11 days;

[0010] S5 Cell medium change: Perform the first medium change after culturing for 4 days. Aspirate and discard the supernatant of the primary complete medium, then add the primary complete medium again. Change the medium every other day thereafter. Observe the cell growth status under a microscope before each medium change. When the cell confluence reaches 80-85% and more than 50% of the colonies are formed, collect the primary cells of the initial culture.

[0011] S6: Transfer the umbilical cord tissue pieces, discard the cell culture supernatant, then wash the primary cultured cells, add dissociation solution respectively, observe the cell digestion under a microscope, and terminate the digestion when the cells become round and fall off in large pieces;

[0012] S7 collected the cell suspension and calculated the number of cells obtained from the initial culture;

[0013] Secondary culture:

[0014] S8: After the initial culture, the umbilical cord tissue pieces are removed and transferred to a new culture bottle, and new primary complete culture medium is added for secondary culture;

[0015] S9 was placed in an incubator and cultured for 4-6 days;

[0016] S10 cell culture medium change: Change the medium every other day, and observe the cell growth status under a microscope before each change. When the cell confluence reaches 80-85% and more than 50% of the colonies are formed, collect the primary cells for secondary culture;

[0017] S11: Aspirate and discard the umbilical cord tissue pieces and cell culture supernatant, then wash the secondary cultured cells, add dissociation solution, and observe the cell digestion under a microscope. When the cells become round and fall off in large pieces, digestion is terminated.

[0018] S12: Collect the cell suspension of the secondary culture and calculate the number of cells obtained from the secondary culture.

[0019] As an embodiment, the pretreatment of the umbilical cord tissue in step S1 is specifically as follows: the umbilical cord sample collected clinically is rinsed with physiological saline to remove residual blood, and then disinfected with 75 vol% alcohol for 20 seconds, and then the umbilical cord is cut into 3 sections and rinsed with physiological saline twice.

[0020] As an embodiment, the inoculation culture method in step S3 is flat culture or suspension culture.

[0021] As an embodiment, the flat culture is specifically to inoculate the umbilical cord tissue block into a culture bottle, let it stand in an incubator for 6 hours, add primary complete culture medium, take care to avoid blowing up the umbilical cord tissue block, and then continue to culture in the incubator for 9-11 days.

[0022] As an embodiment, the suspension culture is specifically performed by adding umbilical cord tissue pieces into primary complete culture medium, mixing them evenly and then inoculating them into a culture bottle.

[0023] As an embodiment, the culture flask is a T75 culture flask.

[0024] As an embodiment, the volume of the umbilical cord tissue block is 9-25 mm 3 .

[0025] As an embodiment, the mass-to-volume ratio of the umbilical cord tissue block to the primary complete culture medium in step S3 and step S8 is 1 g: (14-18) mL.

[0026] As an embodiment, the primary complete culture medium includes a solute and a solvent, the solute includes 5-15 vol% fetal bovine serum, 40-60 ng / mL epidermal growth factor EGF, 5-15 ng / mL basic fibroblast growth factor bFGF, and the solvent is DMEM / F-12 culture medium.

[0027] As an embodiment, the solute includes 10 vol% fetal bovine serum, 50 ng / mL epidermal growth factor EGF, and 10 ng / mL basic fibroblast growth factor bFGF.

[0028] As an embodiment, the DMEM / F-12 culture medium includes 150-155ug / L L-glutamine, 105-115ug / L sodium pyruvate, 3.5-3.6g / L HEPES, 2.3-2.5g / L sodium bicarbonate and DMEM / F-12 basal culture medium.

[0029] As an embodiment, the DMEM / F-12 culture medium includes 151ug / L L-glutamine, 110ug / L sodium pyruvate, 3.58g / L HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 2.438g / L sodium bicarbonate and DMEM / F-12 basal culture medium.

[0030] As an embodiment, the preparation method of the DMEM / F-12 medium is to add 151ug L-glutamine, 110ug sodium pyruvate, 3.58g HEPES and 2.438g sodium bicarbonate to 1000mL DMEM / F12 basal medium to obtain DMEM / F-12 medium.

[0031] As an embodiment, the DMEM / F12 basal culture medium is purchased from Gibco, catalog number 12500-096.

[0032] As an embodiment, the environmental conditions of the incubator are 37° C. and 1-5 vol% CO 2 .

[0033] As an embodiment, the environmental conditions of the incubator are 37° C. and 5 vol% CO 2 .

[0034] As an embodiment, the dissociation solution is recombinant trypsin, and the digestion is terminated by adding fetal bovine serum.

[0035] As an embodiment, step S6 specifically comprises: transferring the umbilical cord tissue block, aspirating the cell culture supernatant, then washing the primary cultured cells twice with physiological saline, adding 1 mL of 0.125 vol% recombinant trypsin respectively, observing the cell digestion under a microscope, and adding 250 uL of fetal bovine serum to terminate the digestion when the cells become round and fall off in large pieces.

[0036] As an embodiment, the step S11 is specifically as follows: aspirating and discarding the umbilical cord tissue block and cell culture supernatant, then washing the secondary cultured cells twice with physiological saline, adding 1 mL of 0.125 vol% recombinant trypsin respectively, observing the cell digestion under a microscope, and adding 250 uL of fetal bovine serum to terminate the digestion when the cells become round and fall off in large pieces.

[0037] As an embodiment, the trypan blue staining method is used to detect the cell number in step S7 and step S12.

[0038] As an embodiment, the steps S7 and S12 are specifically as follows: using 10 mL of the compound electrolyte solution to blow off the cells from the surface of each culture flask and collect them; after mixing, a cell suspension is obtained, 50 uL of the cell suspension is taken as a cell counting sample, and three replicates are performed. 50 uL of 0.4 wt% trypan blue solution is added to each sample, and after thorough mixing, 20 uL is taken and added to a cell counting plate, and then the cells are counted using a Countstar cell counter to calculate the number of harvested cells.

[0039] The second aspect of the present invention provides an application of a method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells, which is applied to the culture of human umbilical cord mesenchymal stem cells.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The highly efficient isolation and culture method of primary human umbilical cord mesenchymal stem cells of the present invention adopts a suspension culture method, which can harvest a larger number of primary human umbilical cord mesenchymal stem cells compared to the traditional flat culture method.

[0042] (2) The efficient isolation and culture method of primary human umbilical cord mesenchymal stem cells described in the present invention adopts a secondary cell culture method. After the umbilical cord tissue pieces discarded after the initial culture are cultured again, a certain number of primary cells can still be obtained, thereby increasing the number of primary seed cells.

[0043] (3) The efficient isolation and culture method of primary human umbilical cord mesenchymal stem cells of the present invention is to cut the umbilical cord into a volume of 9-25 mm 3 The tissue block is more conducive to the primary cells crawling out of the umbilical cord tissue block to form colonies, and then relatively more primary umbilical cord mesenchymal stem cells can be harvested in a shorter time.

[0044] (4) The efficient isolation and culture method of primary human umbilical cord mesenchymal stem cells described in the present invention uses a mass-to-volume ratio of 1 g: (14-18) mL for the umbilical cord tissue block and the primary complete culture medium, which is more conducive to colony formation during the initial culture and shortens the culture time.

[0045] (5) The efficient isolation and culture method of primary human umbilical cord mesenchymal stem cells described in the present invention can maximize the use of umbilical cord tissue blocks to obtain more primary cells, thereby improving the cell yield. In addition, the operation steps are few and simple, efficient, and safe, which is more conducive to the industrialization of the isolation and culture process. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A bar graph showing the number of primary human umbilical cord mesenchymal stem cells obtained in Comparative Example 1 and Comparative Example 2;

[0047] Figure 2 This is a bar graph showing the number of primary human umbilical cord mesenchymal stem cells obtained in Example 1 and Comparative Example 1;

[0048] Figure 3 This is a bar graph showing the number of primary human umbilical cord mesenchymal stem cells obtained in Example 2 and Comparative Examples 3 and 4;

[0049] Figure 4 This is a bar chart showing the number of primary human umbilical cord mesenchymal stem cells obtained in Example 3 and Comparative Examples 5 and 6. DETAILED DESCRIPTION

[0050] In this experiment, Example 1, Comparative Example 1 and Comparative Example 2 used umbilical cords from the same source; Example 2, Comparative Example 3 and Comparative Example 4 used umbilical cords from the same source; Example 3, Comparative Example 5 and Comparative Example 6 used umbilical cords from the same source, and Examples 4-6 used umbilical cords from the same source.

[0051] Example 1

[0052] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells comprises the following steps:

[0053] Initial cultivation:

[0054] S1 pre-processed umbilical cord tissue;

[0055] S2 cuts the umbilical cord tissue into small pieces to obtain umbilical cord tissue blocks;

[0056] S3 Add primary complete culture medium to the umbilical cord tissue pieces and inoculate them according to weight;

[0057] S4 was placed in an incubator and cultured for 9-11 days;

[0058] S5 Cell medium change: Perform the first medium change after culturing for 4 days. Aspirate and discard the supernatant of the primary complete medium, then add the primary complete medium again. Change the medium every other day thereafter. Observe the cell growth status under a microscope before each medium change. When the cell confluence reaches 80-85% and more than 50% of the colonies are formed, collect the primary cells of the initial culture.

[0059] S6: Transfer the umbilical cord tissue pieces, discard the cell culture supernatant, then wash the primary cultured cells, add dissociation solution respectively, observe the cell digestion under a microscope, and terminate the digestion when the cells become round and fall off in large pieces;

[0060] S7 collected the cell suspension and calculated the number of cells obtained from the initial culture;

[0061] Secondary culture:

[0062] S8: After the initial culture, the umbilical cord tissue pieces are removed and transferred to a new culture bottle, and new primary complete culture medium is added for secondary culture;

[0063] S9 was placed in an incubator and cultured for 4-6 days;

[0064] S10 cell culture medium change: Change the medium every other day, and observe the cell growth status under a microscope before each change. When the cell confluence reaches 80-85% and more than 50% of the colonies are formed, collect the primary cells for secondary culture;

[0065] S11: Aspirate and discard the umbilical cord tissue pieces and cell culture supernatant, then wash the secondary cultured cells, add dissociation solution, and observe the cell digestion under a microscope. When the cells become round and fall off in large pieces, digestion is terminated.

[0066] S12: Collect the cell suspension of the secondary culture and calculate the number of cells obtained from the secondary culture.

[0067] The umbilical cord tissue pretreatment in step S1 is specifically as follows: the umbilical cord sample collected clinically is rinsed with physiological saline to remove residual blood, and then disinfected with 75 vol% alcohol for 20 seconds, and then the umbilical cord is cut into 3 sections and rinsed with physiological saline twice.

[0068] The inoculation culture method in step S3 is flat culture, specifically, the umbilical cord tissue block is inoculated into a culture bottle, and after standing in an incubator for 6 hours, 14 mL of primary complete culture medium is added, and care is taken to avoid blowing up the umbilical cord tissue block, and then the culture is continued in the incubator for 9-11 days.

[0069] The culture flask is a T75 culture flask.

[0070] The volume of the umbilical cord tissue block is 9-25 mm 3 .

[0071] The mass-to-volume ratio of the umbilical cord tissue block to the primary complete culture medium in step S3 and step S8 is 1 g:14 mL.

[0072] The primary complete culture medium comprises a solute and a solvent, wherein the solute comprises 10 vol% fetal bovine serum, 50 ng / mL epidermal growth factor (EGF), and 10 ng / mL basic fibroblast growth factor (bFGF), and the solvent is a DMEM / F-12 culture medium. The solute and the solvent are mixed to obtain the primary complete culture medium.

[0073] Fetal bovine serum was purchased from ExCell, catalog number FND500; epidermal growth factor (EGF) was purchased from PeproTect, catalog number AF-100-15-1000; basic fibroblast growth factor (bFGF) was purchased from PeproTect, catalog number AF-100-18B-1000.

[0074] The DMEM / F-12 culture medium includes 151 ug / L L-glutamine, 110 ug / L sodium pyruvate, 3.58 g / L HEPES, 2.438 g / L sodium bicarbonate and DMEM / F-12 basal culture medium.

[0075] The DMEM / F-12 culture medium is prepared by adding 151 ug L-glutamine, 110 ug sodium pyruvate, 3.58 g HEPES and 2.438 g sodium bicarbonate to 1000 mL of DMEM / F12 basal culture medium to obtain a DMEM / F-12 culture medium.

[0076] The DMEM / F12 basal medium was purchased from Gibco, catalog number 12500-096. L-glutamine was purchased from Gibco, catalog number 21051-024. HEPES was purchased from Sigma, catalog number H4034.

[0077] The environmental conditions of the incubator were 37° C. and 5 vol% CO 2 .

[0078] Step S6 specifically includes: transferring the umbilical cord tissue block, aspirating and discarding the cell culture supernatant, then washing the primary cultured cells twice with 30 mL of normal saline, adding 1 mL of 0.125 vol% recombinant trypsin to each wash, observing the cell digestion under a microscope, and adding 250 μL of fetal bovine serum to terminate the digestion when the cells become round and fall off in large pieces.

[0079] Step S7 and step S12 are specifically as follows: using 10 mL of the compound electrolyte solution to blow off the cells from the surface of each culture flask and collect them; after mixing, a cell suspension is obtained, 50 uL of the cell suspension is taken as a cell counting sample, and three replicates are performed. 50 uL of a 0.4 wt% trypan blue aqueous solution is added to each sample, and then after thorough mixing, 20 uL is taken and added to a cell counting plate, and the cells are counted using a Countstar cell counter to calculate the number of harvested cells.

[0080] The compound electrolyte solution was purchased from Shanghai Better, product number: BoMili A A6E2543.

[0081] Step S12 specifically includes: aspirating and discarding the supernatant of the umbilical cord tissue block and the primary complete culture medium, then washing the secondary cultured cells twice with physiological saline, adding 1 mL of 0.125 vol% recombinant trypsin to each of the cells, observing the cell digestion under a microscope, and adding 250 uL of fetal bovine serum to terminate the digestion when the cells become round and fall off in large pieces.

[0082] Comparative Example 1

[0083] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells comprises the following steps:

[0084] Initial cultivation:

[0085] S1 pre-processed umbilical cord tissue;

[0086] S2 cuts the umbilical cord tissue into small pieces to obtain umbilical cord tissue blocks;

[0087] S3 Add primary complete culture medium to the umbilical cord tissue pieces and inoculate them according to weight;

[0088] S4 was placed in an incubator and cultured for 9-11 days;

[0089] S5 Cell medium change: Perform the first medium change after culturing for 4 days. Aspirate and discard the supernatant of the primary complete medium, then add the primary complete medium again. Change the medium every other day thereafter. Observe the cell growth status under a microscope before each medium change. When the cell confluence reaches 80-85% and more than 50% of the colonies are formed, collect the primary cells of the initial culture.

[0090] S6: Aspirate and discard the supernatant of the umbilical cord tissue pieces and the primary complete culture medium, then wash the primary cultured cells, add dissociation solution respectively, and observe the cell digestion under a microscope. When the cells become round and fall off in large pieces, the digestion is terminated.

[0091] S7: Collect the primary cultured cells to form a cell suspension, and calculate the number of primary cultured cells.

[0092] The umbilical cord tissue pretreatment in step S1 is specifically as follows: the umbilical cord sample collected clinically is rinsed with physiological saline to remove residual blood, and then disinfected with 75 vol% alcohol for 20 seconds, and then the umbilical cord is cut into 3 sections and rinsed with physiological saline twice.

[0093] The inoculation culture method in step S3 is flat culture, specifically, the umbilical cord tissue block is inoculated into a culture bottle, and after standing in an incubator for 6 hours, 14 mL of primary complete culture medium is added, and care is taken to avoid blowing up the umbilical cord tissue block, and then the culture is continued in the incubator for 9-11 days.

[0094] The culture flask is a T75 culture flask.

[0095] The volume of the umbilical cord tissue block is 9-25 mm 3 .

[0096] The mass-to-volume ratio of the umbilical cord tissue block to the primary complete culture medium in step S3 and step S8 is 1 g:14 mL.

[0097] The primary complete culture medium comprises a solute and a solvent, wherein the solute comprises 10 vol% fetal bovine serum, 50 ng / mL epidermal growth factor (EGF), and 10 ng / mL basic fibroblast growth factor (bFGF), and the solvent is a DMEM / F-12 culture medium. The solute and the solvent are mixed to obtain the primary complete culture medium.

[0098] The DMEM / F-12 culture medium includes 151 ug / L L-glutamine, 110 ug / L sodium pyruvate, 3.58 g / L HEPES, and 2.438 g / L sodium bicarbonate.

[0099] The DMEM / F-12 culture medium is prepared by adding 151 ug L-glutamine, 110 ug sodium pyruvate, 3.58 g HEPES and 2.438 g sodium bicarbonate to 1000 mL of DMEM / F12 basal culture medium to obtain a DMEM / F-12 culture medium.

[0100] The DMEM / F12 basal culture medium was purchased from Gibco, catalog number 12500-096.

[0101] The environmental conditions of the incubator were 37° C. and 5 vol% CO 2 .

[0102] Step S6 specifically includes: aspirating and discarding the supernatant of the umbilical cord tissue block and the primary complete culture medium, then washing the primary cultured cells twice with 30 mL of normal saline, adding 1 mL of 0.125 vol% recombinant trypsin to each of the washes, observing the cell digestion under a microscope, and adding 250 uL of fetal bovine serum to terminate the digestion when the cells become round and fall off in large pieces.

[0103] The step S7 specifically comprises: blowing off the cells from the surface of each culture flask with 10 mL of the compound electrolyte solution and collecting them; mixing to obtain a cell suspension, taking 50 uL of the cell suspension as a cell counting sample, repeating three times, adding 50 uL of 0.4 wt% trypan blue aqueous solution to each sample, and then mixing thoroughly and taking 20 uL to add to a cell counting plate, using a Countstar cell counter to count the cells and calculate the number of harvested cells.

[0104] The compound electrolyte solution was purchased from Shanghai Better, product number: BoMili A A6E2543.

[0105] Comparative Example 2

[0106] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells, the specific implementation method is the same as that of Comparative Example 1, except that the inoculation and culture method in step S4 is suspension culture, specifically, adding umbilical cord tissue pieces to primary complete culture medium, mixing and then inoculating into a culture bottle.

[0107] Example 2

[0108] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells is described. The specific implementation method is the same as that of Example 1, except that the inoculation and culture method in step S3 is suspension culture, specifically, adding umbilical cord tissue pieces to primary complete culture medium, mixing, and then inoculating in a culture flask.

[0109] The volume of the umbilical cord tissue block is 9-25 mm 3 .

[0110] Comparative Example 3

[0111] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells, the specific implementation method is the same as that of Example 2, except that the volume of the umbilical cord tissue block is less than 9 mm 3 .

[0112] Comparative Example 4

[0113] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells, the specific implementation method is the same as that of Example 2, except that the volume of the umbilical cord tissue block is 25-100 mm 3 .

[0114] Example 3

[0115] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells is provided. The specific implementation is the same as in Example 1, except that in steps S3 and S8, the mass volume ratio of the umbilical cord tissue block to the primary complete culture medium is 1 g:14 mL.

[0116] The inoculation culture method in step S3 is suspension culture, specifically adding the umbilical cord tissue pieces into the primary complete culture medium, mixing them evenly, and then inoculating them into a culture bottle.

[0117] Comparative Example 5

[0118] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells is provided. The specific implementation method is the same as that of Example 3, except that the mass-to-volume ratio of the umbilical cord tissue block to the primary complete culture medium in steps S3 and S8 is 1 g:10 mL.

[0119] Comparative Example 6

[0120] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells is provided. The specific implementation method is the same as that of Example 3, except that in steps S3 and S8, the mass volume ratio of the umbilical cord tissue block to the primary complete culture medium is 1 g:18 mL.

[0121] Example 4

[0122] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells, the specific implementation method is the same as that of Example 1, except that the volume of the umbilical cord tissue block is less than 9 mm 3 In step S3 and step S8, the mass volume ratio of the umbilical cord tissue block and the primary complete culture medium is 1 g:14 mL.

[0123] Example 5

[0124] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells, the specific implementation method is the same as that of Example 1, except that the inoculation culture method in step S4 is suspension culture, and the volume of the umbilical cord tissue block is 9-25 mm 3 In step S3 and step S8, the mass volume ratio of the umbilical cord tissue block and the primary complete culture medium is 1 g:14 mL.

[0125] Example 6

[0126] A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells, the specific implementation method is the same as that of Example 5, and the volume of the umbilical cord tissue block is less than 9 mm 3 In step S3 and step S8, the mass volume ratio of the umbilical cord tissue block and the primary complete culture medium is 1 g:14 mL.

[0127] Performance test (I) Number of primary cells: Example 1 Number of primary cells (cells / g) = (number of primary cells in primary culture + number of primary cells in secondary culture) ÷ weight of umbilical cord tissue block.

[0128] Comparative Example 1: Number of primary cells (cells / g) = (number of primary cells in initial culture) / weight of umbilical cord tissue. Comparative Example 2: Number of primary cells (cells / g) = (number of primary cells in initial culture) / weight of umbilical cord tissue.

[0129] The test results of Example 1, Comparative Example 1 and Comparative Example 2 are shown in Figure 1 and Figure 2 .

[0130] Depend on Figure 1 It can be seen that: Comparative Example 2 adopts suspension culture method (1.88±0.81×10 6 / g umbilical cord tissue block) compared with the comparative example 1 flat culture method (1.56±0.47×10 6 The results indicate that the suspension culture method can be used to culture primary umbilical cord mesenchymal stem cells and has advantages over the traditional flat culture method.

[0131] Depend on Figure 2 It can be seen that the number of primary cells obtained per gram of umbilical cord by secondary culture in Example 1 (2.35±0.85×10 6 / g umbilical cord tissue block) was about the same as that of the single culture of Example 1 (1.56±0.47×10 6 This indicates that after the traditional flat culture method, the discarded umbilical cord tissue block can still be cultured again to obtain a certain number of primary cells, which is about 15-65% of the single culture.

[0132] The number of primary cells (cells / g) of Examples 2-3 and Comparative Examples 3-6 = (the number of primary cells in the initial culture + the number of primary cells in the secondary culture) ÷ the weight of the umbilical cord tissue block, and the number of primary umbilical cord mesenchymal stem cells that can be obtained per gram of umbilical cord tissue block is calculated. The test results of Example 2, Comparative Example 3 and Comparative Example 4 are shown in Figure 3 The test results of Example 3, Comparative Example 5 and Comparative Example 6 are shown in Figure 4 .

[0133] like Figure 3 As shown in Example 2, 1.50±0.18×10 6 cells / g umbilical cord tissue, and comparative examples 3 and 4 can obtain 1.41±0.28×10 cells / g umbilical cord tissue after 16.67±1.53 days and 16.33±1.15 days of primary culture, respectively.6 and 8.81±4.84×10 5 cells / g umbilical cord tissue block, indicating that the primary culture period of Example 2 was the shortest, but the obtained primary human umbilical cord mesenchymal stem cells were the largest.

[0134] In summary, when the umbilical cord is cut to a volume of 9-25mm 3 When the tissue blocks are large, it is more conducive for primary cells to crawl out from the edge of the tissue to form colonies, and then relatively more primary human umbilical cord mesenchymal stem cells can be harvested in a shorter time.

[0135] like Figure 4 As shown in the figure, the number of primary cells obtained in Example 3 and Comparative Example 6 is similar, which is 1.34±0.81×10 6 and 1.33±7.11×10 6 The umbilical cord tissue pieces / g were more than the 9.91±4.21×10 obtained in Comparative Example 5. 5 / g umbilical cord tissue pieces.

[0136] In summary, a smaller amount of liquid added to the umbilical cord tissue block for the first time and the volume of liquid added during the transfer of the secondary cultured tissue block will affect the primary cells crawling out from the edge of the tissue block, but adding more culture medium will not increase the number of primary cells that can be harvested. Therefore, adding 14 mL of primary complete culture medium to the umbilical cord tissue block is most appropriate.

[0137] The number of primary cells in Example 4-6 (cells / g) = (number of primary cells in primary culture + number of primary cells in secondary culture) ÷ weight of umbilical cord tissue block, and the number of primary umbilical cord mesenchymal stem cells that can be obtained per gram of umbilical cord tissue block is calculated.

[0138] Example 5 After 14 days of primary culture, 2.26×10 6 cells / g umbilical cord tissue block, Example 4 and Example 6 can each obtain 1.52×10 6 and 1.70×10 6 cells / g umbilical cord tissue block, and the cells harvested in Example 5 were the most, which were 1.49 times and 1.33 times that of Example 4 and Example 6, respectively.

[0139] In summary, suspension culture can harvest more primary human umbilical cord mesenchymal stem cells than flat culture of umbilical cord tissue blocks, and the umbilical cord is cut into a volume of 9-25mm when it is separated. 3 The culture period was the shortest when the tissue blocks were isolated, and the maximum number of primary human umbilical cord mesenchymal stem cells were obtained.

[0140] (2) Primary cell culture cycle: the number of days required for umbilical cord tissue fragments to be isolated from the umbilical cord, cultured initially, cultured secondary, and collected from the primary cells.

[0141] The primary cell culture period of Example 2 was 14.33±0.58 days, which was significantly shorter than that of Comparative Example 3 (16.67±1.53 days) and Comparative Example 4 (16.33±1.15 days).

[0142] In Examples 4-6, primary human umbilical cord mesenchymal stem cells were observed crawling out from the periphery of the umbilical cord tissue blocks on day 4 of culture. The primary culture periods of Examples 4-6 were 16 days, 14 days, and 16 days, respectively.

[0143] (III) The number of colonies formed after primary cells crawl out from the edge of the tissue block: The number of colonies formed by primary cells of human umbilical cord mesenchymal stem cells was counted on the 4th day, 6th day and 8th day of the primary culture, and on the 4th day and 6th day of the secondary culture. The specific method is as follows: Divide the T75 culture flask into 9 areas, and then count the number of colonies of primary cells in each area respectively. The sum of the colonies in the 9 areas is the number of colonies in the culture flask. According to the formula: the number of colonies formed after primary cells crawl out from the edge of the tissue block = the sum of the number of colonies in the culture flask / the weight of the umbilical cord tissue block. The test results of Example 2, Comparative Example 3 and Comparative Example 4 are shown in Table 1, the test results of Example 3, Comparative Example 5 and Comparative Example 6 are shown in Table 2, and the test results of Examples 4-6 are shown in Table 3.

[0144] Table 1

[0145]

[0146] Note: *P<0.05; **P<0.01.

[0147] As shown in Table 1, in the primary culture stage, the number of colonies observed in Example 2 on the 4th, 6th and 8th days was significantly greater than that of the other two groups. In particular, on the 8th day, the number of colonies formed per gram of umbilical cord tissue block in Example 2 was 40.66±14.92 per gram of umbilical cord tissue block, which was much greater than that of Comparative Example 3 (21.51±15.11 per gram of umbilical cord tissue block, Comparative Example 3 vs. Example 2 P < 0.05) and Comparative Example 4 (16.08±3.35 per gram of umbilical cord tissue block, Comparative Example 4 vs. Example 2 P < 0.01). In the secondary culture stage, the number of colonies observed in Example 2 on the 4th and 6th days was also greater than that of the other two groups. This indicates that when the umbilical cord is separated, the tissue block of the umbilical cord is cut to a volume of 9-25 mm. 3 It is more suitable for primary cells to crawl out from the edge of the tissue block.

[0148] Table 2

[0149]

[0150] As shown in Table 2, in the primary culture stage, the number of colonies observed in Example 3 and Comparative Example 6 on the 6th and 8th days was significantly more than that in Comparative Example 5, and the colony distribution of Comparative Example 5 was uneven, with large size differences. The number of colonies in Example 3 and Comparative Example 6 was comparable, but the colonies of the former were larger and more evenly distributed. In the secondary culture stage, 55.94 ± 12.91 and 61.43 ± 18.54 colonies were observed in Example 3 and Comparative Example 6 on the 6th day, respectively, with no statistically significant difference between the two. The three groups of colony distributions were all relatively even, with multiple colonies often forming a single piece. The above explanation shows that the amount of liquid added for the first time in the tissue block and the amount of liquid added during the transfer of the secondary culture tissue block are less, which will affect the primary cells from crawling out of the edge of the tissue block. Compared with 10 mL, adding 14 mL and 18 mL of primary complete medium is more conducive to the formation of colonies during primary culture.

[0151] Table 3

[0152]

[0153] As shown in Table 3, the number of colonies observed in Example 5 on days 4, 6, and 8 was significantly greater than that in Examples 4 and 6. On day 8, the number of colonies formed per gram of umbilical cord tissue mass in Example 5 was 43.49 per gram of umbilical cord tissue mass, which was 1.90 times and 1.77 times that of Example 4 (22.90 per gram of umbilical cord tissue mass) and Example 6 (24.63 per gram of umbilical cord tissue mass), respectively. In the secondary culture stage, the number of colonies observed in Example 5 on days 4 and 6 was also greater than that of Examples 4 and 6. On day 6, the number of colonies formed per gram of umbilical cord tissue mass in Example 5 was 58.53 per gram of umbilical cord tissue mass, which was 1.51 times and 1.46 times that of Example 4 (38.74 per gram of umbilical cord tissue mass) and Example 6 (40.04 per gram of umbilical cord tissue mass), respectively. The above results show that compared with the secondary culture by flat-laying method, the secondary culture of umbilical cord tissue blocks can obtain more colonies, and the umbilical cord tissue blocks are cut into a volume of 9-25mm when the umbilical cord is separated. 3 It is more suitable for primary cells to crawl out from the edge of the tissue block.

Claims

1. A method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells, characterized in that: The following steps are involved: Initial cultivation: S1 pre-processed umbilical cord tissue; S2 cuts the umbilical cord tissue into small pieces to obtain umbilical cord tissue blocks; S3 Add primary complete culture medium to the umbilical cord tissue pieces and inoculate them according to weight; S4 was placed in an incubator and cultured for 9-11 days; S5 Cell medium change: Perform the first medium change after culturing for 4 days. Aspirate and discard the supernatant of the primary complete medium, then add the primary complete medium again. Change the medium every other day thereafter. Observe the cell growth status under a microscope before each medium change. When the cell confluence reaches 80-85% and more than 50% of the colonies are formed, collect the primary cells of the initial culture. S6: Transfer the umbilical cord tissue pieces, discard the cell culture supernatant, then wash the primary cultured cells, add dissociation solution respectively, observe the cell digestion under a microscope, and terminate the digestion when the cells become round and fall off in large pieces; S7 collected the cell suspension and calculated the number of cells obtained from the initial culture; Secondary culture: S8: After the initial culture, the umbilical cord tissue pieces are removed and transferred to a new culture bottle, and new primary complete culture medium is added for secondary culture; S9 was placed in an incubator and cultured for 4-6 days; S10 cell culture medium change: Change the medium every other day, and observe the cell growth status under a microscope before each change. When the cell confluence reaches 80-85% and more than 50% of the colonies are formed, collect the primary cells for secondary culture; S11: Aspirate and discard the umbilical cord tissue pieces and cell culture supernatant, then wash the secondary cultured cells, add dissociation solution, and observe the cell digestion under a microscope. When the cells become round and fall off in large pieces, digestion is terminated. S12: Collect the cell suspension of the secondary culture and calculate the number of cells obtained from the secondary culture.

2. The method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The inoculation culture method in step S3 is tile culture or suspension culture.

3. The method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The volume of the umbilical cord tissue block is 9-25 mm 3 .

4. The method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The mass-to-volume ratio of the umbilical cord tissue block to the primary complete culture medium in step S3 and step S8 is 1 g: (14-18) mL.

5. The method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The primary complete culture medium comprises a solute and a solvent, wherein the solute comprises 5-15 vol% fetal bovine serum, 40-60 ng / mL epidermal growth factor EGF, and 5-15 ng / mL basic fibroblast growth factor bFGF, and the solvent is DMEM / F-12 culture medium.

6. The method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells according to claim 5, characterized in that: The DMEM / F-12 culture medium includes 150-155ug / L L-glutamine, 105-115ug / L sodium pyruvate, 3.5-3.6g / L HEPES, 2.3-2.5g / L sodium bicarbonate and DMEM / F-12 basal culture medium.

7. The method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The environmental conditions of the incubator are 37° C. and 1-5 vol% CO 2 .

8. The method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The dissociation solution is recombinant trypsin, and the digestion termination step is to add fetal bovine serum to terminate the digestion.

9. The method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: In the steps S7 and S12, the trypan blue staining counting method is used to detect the cell number.

10. An application of the method for efficiently isolating and culturing primary human umbilical cord mesenchymal stem cells according to any one of claims 1 to 9, characterized in that: Applied in the culture of human umbilical cord mesenchymal stem cells.

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