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Method for co-culturing natural killer cells by umbilical cord mesenchymal stem cells

A technology of natural killer cells and stem cells, applied in the field of umbilical cord mesenchymal stem cells co-cultivation of natural killer cells, can solve the problems of low cell biological activity, inconsistent cell sources, and high cost of quality control

Pending Publication Date: 2021-04-16
广东壹加再生医学研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in tumor treatment, the recipient is generally a tumor patient, and there is a risk of low cell biological activity and low cell number when using it as a cell source
Although the use of NK cells is not limited by MHC, there is still a certain degree of immunogenicity, which may cause immune rejection in the use of allogeneic donors. Whether the source of NK cells is from autologous or allogeneic adult donors, there is a certain risk of infectious diseases. In the production process, a complete isolation operation is required to prevent cross-contamination. Therefore, adult peripheral blood is used as the source of NK cells. Although the technical requirements are lower and the production is more flexible, the quality of the final product is not stable due to the inconsistent source of cells. The cost of control is high, and the cells of each donor need to be produced and isolated as an independent product batch, which virtually increases the cost of production, but the biological source of umbilical cord blood is the newborn himself, and its biological characteristics are inconsistent
Second, each unit of cord blood is limited by the volume of blood collected, and the total number of cord blood cells per donor is limited

Method used

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  • Method for co-culturing natural killer cells by umbilical cord mesenchymal stem cells
  • Method for co-culturing natural killer cells by umbilical cord mesenchymal stem cells
  • Method for co-culturing natural killer cells by umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: The co-culture cell expansion curve of umbilical cord blood nucleated cells and umbilical cord mesenchymal stem cells. Stem cells to the P5 generation were temporarily frozen and stored in a liquid nitrogen tank after the cells were prepared. According to the technical scheme of the present invention, 1.2*10^7 cells of umbilical cord blood nucleated cells and 1.2*10^8 cells of P5 generation umbilical cord mesenchymal stem cells were resuscitated in a 37°C water bath for each batch. According to the cell counting results, inoculate in 100ml complete co-culture medium at a density of 1.0*10^6cells / ml for umbilical cord blood nucleated cells and 1.0*10^7cells / ml for P5 generation umbilical cord mesenchymal stem cells. Subsequent cultures were cultured in 2L cell culture bags under the conditions of 37°C, saturated humidity, and 5% CO2, and samples were taken and counted every 2 days during the culture process. After sampling and counting, add fresh co-culture c...

Embodiment 2

[0072] Example 2: Colony differentiation statistics after co-culture of umbilical cord blood nucleated cells and umbilical cord mesenchymal stem cells, recovery and washing of umbilical cord blood nucleated cells obtained from five groups of isolated nucleated cells and co-cultured and expanded nucleated cells in Example 1 After washing with DMEM to adjust the cell density to 1*10^6cells / ml, take 100ul and 900ul Stemcell MethoCult human hematopoietic stem cell methylcellulose medium and mix them evenly, and inoculate them in a 24-well plate, with 3 replicate wells for each group. The 24-well plates were cultured at 37°C, saturated humidity, and 5% CO2 for 14 to 16 days, and colonies were counted.

[0073] Results: After 16 days of culture, count the number of hematopoietic cell colonies in the 24-well plate under a microscope as follows:

[0074]

[0075]

[0076] Table 1 Colony differentiation statistics of umbilical cord blood nucleated cells and umbilical cord mesench...

Embodiment 3

[0078] Example 3: Flow cytometric phenotype statistics of umbilical cord blood nucleated cells CD3-CD56+ after sorting, the nucleated cells obtained according to Example 1 of the present invention were sorted by CD3-CD56+ MACS magnetic beads according to the technical scheme of the present invention, using After flow cytometry detection, the cell purity of the NK cell phenotype CD3-CD16+CD56+ before sorting and after sorting was counted respectively.

[0079] Before sorting After sorting Group 1 20.45％ 98.77％ Group 2 19.56％ 97.53％ Group 3 15.34％ 94.35％ Group 4 12.56％ 97.12％ Group 5 14.37％ 96.51％

[0080] Table: 2 is the comparison of NK cell purity before and after MACS CD3-CD56+ sorting

[0081] Conclusion: MACS magnetic bead sorting can effectively purify NK cells and their precursor cells.

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Abstract

The invention provides a method for co-culturing natural killer cells by using umbilical cord mesenchymal stem cells. According to the method, umbilical cord mesenchymal stem cells with the same source as an umbilical cord blood donor and nucleated cells in umbilical cord blood are co-cultured to provide a microenvironment for promoting proliferation and differentiation of the nucleated cells in the umbilical cord blood, in-vitro large-scale amplification of nucleated cells in umbilical cord blood to NK cells and precursor cells of the NK cells is achieved, the requirement for umbilical cord blood raw materials in subsequent large-scale production of the NK cells is lowered, application to industrial production of subsequent NK cell therapy products is achieved, and the umbilical cord blood contains hematopoietic stem cells capable of reconstructing a hematopoietic and immune system of a human body and can be used in hematopoietics stem cell graft. Therefore, the umbilical cord blood becomes an important source of hematopoietic stem cells, particularly a source of hematopoietic stem cells without blood relationship, and is also a very important human biological resource.

Description

technical field [0001] The invention relates to a method for co-cultivating natural killer cells with umbilical cord mesenchymal stem cells, and belongs to the field of cell technology. Background technique [0002] For a long time, traditional treatment methods represented by surgery, radiotherapy and chemotherapy have achieved good results in the treatment of malignant tumors. However, these treatments have limitations for different tumors and are accompanied by obvious side effects. Therefore, it is an urgent need for clinical tumor treatment to find a treatment method that can effectively control tumor growth and metastasis with less damage to patients. With the rapid development and cross-infiltration of tumor biology, immunology, molecular biology and other related disciplines, the research on tumor immunotherapy has advanced by leaps and bounds. Cell therapy has gradually transitioned from laboratory research to effective and safe clinical application. [0003] Nat...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/0775
Inventor 王东福陈智聪卢丽红黄里
Owner 广东壹加再生医学研究院有限公司
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