Amplification process for umbilical cord mesenchymal stem cells

Abstract

The invention discloses an amplification process for umbilical cord mesenchymal stem cells in the field of stem cell amplification, which comprises the following steps: S101, taking an umbilical cord meeting the requirements, flushing the umbilical cord with flushing fluid, removing blood vessels (artery blood vessels and vein blood vessels) with scissors (tweezers) and the like, stripping out Wharton's jelly tissues, fully cutting the obtained tissues into pieces, and culturing the tissues to obtain the umbilical cord mesenchymal stem cells; forming cell colonies with different sizes, namely P0 generation cells; s102, digesting and collecting the P0-generation cells in the step S101, washing, filtering, purifying, changing liquid and other technical operations to effectively remove various heterogeneous cells and impurities, and performing in-vitro amplification culture to obtain first-generation umbilical cord mesenchymal stem cells, namely P1-generation cells. According to the first, second and third wheel umbilical cord mesenchymal stem cell amplification technology, the technological parameters and the set parameter value range are stable and safe, production technology amplification can be conducted on the basis, and rapid amplification can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of stem cell expansion, in particular to an expansion process for umbilical cord mesenchymal stem cells. Background Art

[0002] Umbilical cord mesenchymal stem cells are primitive cells with the potential for self-replication and differentiation, and are active special biological products. However, the process parameters and parameter value ranges of the existing expansion process for umbilical cord mesenchymal stem cells are relatively unstable and unsafe. Therefore, those skilled in the art provide an expansion process for umbilical cord mesenchymal stem cells to solve the problems raised in the above background technology. Summary of the invention

[0003] The object of the present invention is to provide an expansion process for umbilical cord mesenchymal stem cells to solve the problems raised in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The expansion process for umbilical cord mesenchymal stem cells comprises the following steps:

[0006] S101. Take an umbilical cord that meets the requirements, rinse it with a flushing solution, remove the blood vessels (arterial and venous) with scissors (tweezers), peel off the Wharton's jelly tissue, cut the obtained tissue into pieces, and form cell colonies of varying sizes after tissue culture, i.e., P0 generation cells;

[0007] S102, the P0 generation cells in step S101 are digested and collected, washed, filtered, purified, and the fluid is replaced to effectively remove various foreign cells and impurities, and the first generation of umbilical cord mesenchymal stem cells, i.e., P1 generation cells, are obtained by in vitro amplification and culture;

[0008] S103, the P1 generation cells in step S102 are digested, collected, washed, purified and other technical operations to effectively improve the purity of the cells, and the second generation of umbilical cord mesenchymal stem cells, i.e., P2 generation cells, are obtained by in vitro amplification and culture for about 72 hours;

[0009] S104. P2 cells that meet the quality control standards are expanded and cultured in vitro to P3 to P5 cells;

[0010] S105. Establish a tertiary cell bank of umbilical cord mesenchymal stem cells.

[0011] As a further solution of the present invention: the umbilical cord mesenchymal stem cells at the P1 generation cell culture stage in step S102 are cryopreserved in liquid nitrogen using an appropriate cryoprotectant and stored in a primary cell bank. About 9 to 12 tubes of three umbilical cords are cryopreserved in the same batch, and the number of cells in each tube is 2 to 3*106 cells.

[0012] As a further solution of the present invention: in the step S103, the P2 generation cells need to be tested for cell viability, sterility, mycoplasma, virus, cell phenotype, etc., and after passing the test, they need to be cryopreserved in liquid nitrogen using an appropriate cryoprotectant and stored in a master cell bank of umbilical cord mesenchymal stem cells. About 12-16 tubes of three umbilical cords are cryopreserved in the same batch, and the number of cells in each tube is 2 to 3*106 cells.

[0013] As a further solution of the present invention: in the step S104, the fifth P5 generation cells need to be tested for sterility, mycoplasma, cell viability, cell phenotype, adipogenic, osteogenic and chondrogenic differentiation, etc. After passing the test, appropriate cryoprotectants are used for cryopreservation in liquid nitrogen and the P5 generation cells are stored. About 95-120 tubes of three umbilical cords are frozen in the same batch, and the number of cells in each tube is 2 to 3*106 cells.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention has a first, second and third round of umbilical cord mesenchymal stem cell expansion process, and the process parameters and the set parameter value range are stable and safe, and the production process can be amplified on this basis, and can be rapidly expanded. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a flow chart of the present invention;

[0017] Figure 2 This is the morphology of the first batch of P5 umbilical cord mesenchymal stem cells (10×) in the present invention;

[0018] Figure 3 This is a morphological diagram of the second batch of P5 umbilical cord mesenchymal stem cells in the present invention;

[0019] Figure 4 This is a morphological diagram of the second batch of P5 umbilical cord mesenchymal stem cells in the present invention;

[0020] Figure 5 This is a graph showing the growth of three batches of umbilical cord mesenchymal stem cells in the present invention;

[0021] Figure 6 This is an activity test table of three batches of P5 umbilical cord mesenchymal stem cells in the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] See also Figures 1 to 6 In an embodiment of the present invention, the expansion process for umbilical cord mesenchymal stem cells comprises the following steps:

[0024] S101. Take an umbilical cord that meets the requirements, rinse it with a flushing solution, remove the blood vessels (arterial and venous) with scissors (tweezers), peel off the Wharton's jelly tissue, cut the obtained tissue into pieces, and form cell colonies of varying sizes after tissue culture, i.e., P0 generation cells;

[0025] S102, the P0 generation cells in step S101 are digested and collected, washed, filtered, purified, and the fluid is replaced to effectively remove various foreign cells and impurities, and the first generation of umbilical cord mesenchymal stem cells, i.e., P1 generation cells, are obtained by in vitro amplification and culture;

[0026] S103, the P1 generation cells in step S102 are digested, collected, washed, purified and other technical operations to effectively improve the purity of the cells, and the second generation of umbilical cord mesenchymal stem cells, i.e., P2 generation cells, are obtained by in vitro amplification and culture for about 72 hours;

[0027] S104. P2 cells that meet the quality control standards are expanded and cultured in vitro to P3 to P5 cells;

[0028] S105. Establish a tertiary cell bank of umbilical cord mesenchymal stem cells.

[0029] The umbilical cord mesenchymal stem cells at the P1 generation cell culture stage in step S102 are cryopreserved in liquid nitrogen using an appropriate cryoprotectant and stored in a primary cell bank. About 9 to 12 tubes of three umbilical cords are cryopreserved in the same batch, with each tube containing 2 to 3*106 cells.

[0030] Among them, in step S103, the P2 generation cells need to be tested for cell viability, sterility, mycoplasma, virus, cell phenotype, etc., and after passing the test, they need to be cryopreserved in liquid nitrogen using an appropriate cryoprotectant and stored in the master cell bank of umbilical cord mesenchymal stem cells. Three umbilical cords are cryopreserved in the same batch for about 12-16 tubes, with 2 to 3*106 cells in each tube.

[0031] Among them, in step S104, the fifth P5 generation cells need to be tested for sterility, mycoplasma, cell viability, cell phenotype, adipogenic, osteogenic and chondrogenic differentiation, etc. After passing the test, appropriate cryoprotectants are used for cryopreservation in liquid nitrogen and P5 generation cells are stored. About 95-120 tubes of three umbilical cords are frozen in the same batch, and the number of cells in each tube is 2 to 3*106 cells.

[0032] In this example, the morphological detection of umbilical cord mesenchymal stem cells

[0033] Under the same production process, three batches of umbilical cord mesenchymal stem cells (generation 5) from different umbilical cords were subjected to parallel microscopic morphological tests. Figure 2-4 It can be seen that the three batches of umbilical cord mesenchymal stem cells all showed adherent growth in a spindle shape. When the growth density reached more than 90%, they covered the entire bottom of the dish, and the entire texture was arranged in a spiral shape. There was no significant difference between the batches.

[0034] Cell growth of umbilical cord mesenchymal stem cells:

[0035] Under the same production process conditions, three batches of umbilical cord mesenchymal stem cells from different umbilical cords were cultured in parallel, and cell growth was tested at the 5th generation. As shown in the table, the three batches of umbilical cord mesenchymal stem cells were passaged to the same number of 75cm2 culture flasks at the same passage density, and one 75cm2 culture flask was randomly selected from each batch for cell counting and cell activity testing. The test results showed that the first day of the culture of the three batches of the 5th generation umbilical cord mesenchymal stem cells was the cell growth adaptation period, the 3rd day reached the multiple growth period, the 5th day the cell number reached a peak and the activity was the strongest, and there was no significant difference among the three batches; see Figure 5-6 .

[0036] Detection of surface markers of umbilical cord mesenchymal stem cells:

[0037] Under the same production process, three batches of umbilical cord mesenchymal stem cells from different umbilical cords were cultured in parallel, and cell surface markers were detected at the 5th generation. The three batches of umbilical cord mesenchymal stem cells at the 5th generation all expressed CD105, CD90, and CD73, with expression rates above 95%, while CD34, CD45, and HLA-DR were not expressed or weakly expressed, with expression rates below 2%; there was no statistically significant difference in the expression of each marker among the three batches (P>0.05), as shown in the following table:

[0038] Detection of surface markers of three batches of P5 umbilical cord mesenchymal stem cells

[0039]

[0040] In summary, after the first, second and third rounds of umbilical cord mesenchymal stem cell expansion process, the process parameters and the set parameter value range are stable and safe, and the production process can be scaled up on this basis.

[0041] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A process for the expansion of umbilical cord mesenchymal stem cells, characterized in that: The steps include: S101, take an umbilical cord that meets the requirements, rinse it with a flushing solution, remove the blood vessels with scissors, peel off the Wharton's jelly tissue, cut the obtained tissue into pieces, and form cell colonies of varying sizes after tissue culture, i.e., P0 generation cells; S102, the P0 generation cells in step S101 are digested and collected, washed, filtered, purified, and the fluid is replaced to effectively remove various foreign cells and impurities, and the first generation of umbilical cord mesenchymal stem cells, i.e., P1 generation cells, are obtained by in vitro amplification and culture; S103, the P1 generation cells in step S102 are digested, collected, washed, purified and other technical operations to effectively improve the purity of the cells, and the second generation of umbilical cord mesenchymal stem cells, i.e., P2 generation cells, are obtained by in vitro amplification and culture for about 72 hours; S104. P2 cells that meet the quality control standards are expanded and cultured in vitro to P3 to P5 cells; S105. Establish a tertiary cell bank of umbilical cord mesenchymal stem cells.

2. The process for expanding umbilical cord mesenchymal stem cells according to claim 1, characterized in that: In the step S102, the umbilical cord mesenchymal stem cells at the P1 generation cell culture stage are cryopreserved in liquid nitrogen using an appropriate cryoprotectant and stored in a primary cell bank. About 9 to 12 tubes of three umbilical cords are cryopreserved in the same batch, and the number of cells in each tube is 2 to 3*106 cells.

3. The process for expanding umbilical cord mesenchymal stem cells according to claim 1, characterized in that: In step S103, the P2 generation cells need to be tested for cell viability, sterility, mycoplasma, virus, cell phenotype, etc. After passing the test, they need to be cryopreserved in liquid nitrogen using an appropriate cryoprotectant and stored in the master cell bank of umbilical cord mesenchymal stem cells. Three umbilical cords are cryopreserved in the same batch for about 12-16 tubes, with 2 to 3*106 cells in each tube.

4. The process for expanding umbilical cord mesenchymal stem cells according to claim 1, characterized in that: In step S104, the fifth generation P5 cells need to be tested for sterility, mycoplasma, cell viability, cell phenotype, adipogenic, osteogenic, and chondrogenic differentiation, etc. After passing the test, appropriate cryoprotectants are used for cryopreservation in liquid nitrogen and P5 cells are stored. About 95-120 tubes of three umbilical cords are frozen in the same batch, and the number of cells in each tube is 2 to 3*106 cells.

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