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In-vitro expansion method, kit and application of umbilical cord blood NK (nature killer) cells

A technology for NK cells and in vitro expansion, applied in the field of cell engineering, can solve the problems of low yield of NK cells, high cost of separation and expansion, and poor safety, so as to simplify the cell preparation process, avoid safety risks, and reduce preparation costs Effect

Pending Publication Date: 2017-10-20
BEIJING JING MENG STEM CELL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. Cells need to be sorted, and the sorting technology affects the activity of NK cells, and sorting equipment is also needed;
[0007] 2. Trophoblast cells are required, and the introduction of K562 cells greatly affects the safety of expanding NK cells;
[0008] 3. The operation is complicated and the technical versatility is poor;
[0009] 4. The cost of separation and amplification is high;
[0010] 5. The use of animal-derived ingredient additives has poor safety;
[0011] 6. Low production of NK cells

Method used

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  • In-vitro expansion method, kit and application of umbilical cord blood NK (nature killer) cells
  • In-vitro expansion method, kit and application of umbilical cord blood NK (nature killer) cells
  • In-vitro expansion method, kit and application of umbilical cord blood NK (nature killer) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1, in vitro expansion of umbilical cord blood NK cells and detection

[0080] 1. Expansion of umbilical cord blood NK cells in vitro (activation culture protocol 1)

[0081] Such as figure 1 As shown, the in vitro expansion method of umbilical cord blood NK cells of the present invention comprises the following steps:

[0082] 1) Separation of umbilical cord blood mononuclear cells

[0083] 1.1 Take 100mL of fresh anticoagulated cord blood (sample 1) and cryopreserved and resuscitated cord blood (sample 2, the cord blood is provided by the Obstetrics and Gynecology Department of the Armed Police General Hospital and approved by the Hospital Ethics Committee), and add 100mL of PBS (formula: NaCl 8.0g, KCl 0.2g, NaCl 2 HPO 4 1.44g, KH 2 PO 4 0.24g, add distilled water to 1000mL, adjust pH 7.4) to dilute cord blood;

[0084] 1.2 Add 20mL of sample density separation solution (patent number: ZL201110456878.2, medical device record number: Jinghai Machine...

Embodiment 2

[0117] Embodiment 2, in vitro expansion of umbilical cord blood NK cells (activation culture scheme two)

[0118] Such as figure 1 As shown, the in vitro expansion of umbilical cord blood NK cells includes the following processes:

[0119] 1) Isolation of umbilical cord blood mononuclear cells from cryopreserved and resuscitated umbilical cord blood

[0120] Take 10ml of frozen and resuscitated umbilical cord blood (sample 3), the method is the same as in Example 1.

[0121] 2) activating and culturing umbilical cord blood NK cells (the control group is embodiment 1)

[0122] With GMP S&XFM TM - CD lymphocyte culture medium (patent application number: 201310082166.8; medical device record number: Jinghai Equipment No. 20150008) to adjust the mononuclear cell density to 2×10 6 A / mL (0.5~5×10 6 / mL is acceptable, preferably 1-3×10 6 cells / mL), the cell suspension was inoculated into cell culture flasks, and then injected into GMP S&XFM TM -Add 2μg / mL (1-10μg / mL, preferabl...

Embodiment 3

[0126] Example 3, in vitro expansion of umbilical cord blood NK cells and detection (activation culture scheme three)

[0127] Such as figure 1 As shown, the in vitro expansion of umbilical cord blood NK cells includes the following processes:

[0128] 1) Isolation of umbilical cord blood mononuclear cells from cryopreserved and resuscitated umbilical cord blood

[0129] Take 10ml of frozen and resuscitated umbilical cord blood (sample 3), the method is the same as in Example 1.

[0130] 2) Activation and culture of cord blood NK cells

[0131] With GMP S&XFM TM - CD lymphocyte culture medium (patent application number: 201310082166.8; medical device record number: Jinghai Equipment No. 20150008) to adjust the mononuclear cell density to 2×10 6 A / mL (0.5~5×10 6 / mL is acceptable, preferably 1-3×10 6 cells / mL), the cell suspension was inoculated into cell culture flasks, and then injected into GMP S&XFM TM - Add 2μg / mL (1-10μg / mL is acceptable, preferably 1-5μg / mL) zoled...

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Abstract

The invention discloses an in-vitro expansion method, a kit and an application of umbilical cord blood NK (nature killer) cells. The in-vitro expansion method of the umbilical cord blood NK cells comprises the processes of activated culture and enrichment culture of the umbilical cord blood NK cells from isolated cord blood mononuclear cells. Compared with existing methods for isolating and expanding the umbilical cord blood NK cells, the in-vitro expansion method has the advantages that cell sorting is needless, trophoblast cells are needless, operation is simple, universality is good, cost is low, adopted reagents do not contain components of animal origin, safety is good, the yield of the NK cells is high, purity of the obtained cord blood NK cells is up to 90% or higher, the total number of the cells can be up to 10<10-11> / part of cord blood, and the killing capacity for tumor cells is high, thereby playing a role in preparation of tumor immunotherapy drugs and tumor immunotherapy.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to an in vitro expansion method of umbilical cord blood NK cells and a kit thereof and their application in preparing tumor immunotherapy drugs and tumor immunotherapy. Background technique [0002] Natural killer cells (Nature killer cells, NK) are the third type of lymphocytes except T and B cells, and their cell morphology is also different from T and B cells. NK cells are large particles containing azurophilic granules in the cytoplasm Lymphocytes widely exist in lymphoid organs and peripheral tissues, accounting for 5%-10% of the total lymphocytes in normal peripheral blood, 1%-2% in spleen, and NK cells also exist in lymph nodes and bone marrow. As an important part of human natural immunity, NK cells are important immune regulatory cells for the body to resist infection and prevent cell malignant transformation, and play a very important role in immune su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0646C12N2500/42C12N2501/2302C12N5/0635C12N5/0638C12N5/0636A61K39/4644C12N5/0634A61K39/4613C12N2501/2315A61K35/17C07K14/52A61P35/00C12N5/0665C12N2501/2318C12N2501/999
Inventor 武晓云刘学敏康会彦王云虹樊晓翔李志刚丁炜武立华李金艳沈殿富张慧珍
Owner BEIJING JING MENG STEM CELL TECH
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