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Human NK cell culture system and preparation method thereof

A culture system, NK cell technology, applied in cell culture active agents, cell culture supports/coatings, biochemical equipment and methods, etc., can solve the risk of increasing clinical application, low amplification efficiency and purity, and poor repeatability To achieve the effect of promoting progenitor cell proliferation, improving cell purity, and reducing the probability of contamination

Pending Publication Date: 2021-07-23
苏州科贝生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, NK cells only account for 5-10% of peripheral blood lymphocytes, and the current NK cell expansion method, whether it is cytokine stimulation expansion or trophoblast cell expansion, has low expansion efficiency and purity. There is a disadvantage of poor repeatability
For example, patent CN108300693A discloses a method for in vitro expansion of NK cells. The new artificial antigen-presenting cells CD86-4-1BBL-aAPC are used as feeder cells to expand NK cells. The introduction of exogenous cells increases clinical application risk
Patent CN109628397A also discloses a method for in vitro expansion of NK cells, but the culture period is 14-18 days, which takes a long time

Method used

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  • Human NK cell culture system and preparation method thereof
  • Human NK cell culture system and preparation method thereof
  • Human NK cell culture system and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 In vitro expansion of human umbilical cord blood NK cells

[0055] 1. Coated culture flask: 24-48 hours before inoculation, mix human hepatitis B immunoglobulin (50μg / mL) and human serum albumin (200μg / mL) according to the ratio of 1:1, take 10mL and add it to the 75cm 2 In a culture bottle, mix well, let it stand at room temperature for 1 hour, and then put it in a refrigerator at 4°C for later use.

[0056] Before use, discard the supernatant in the coated culture flask and wash it again with PBS.

[0057] 2. Gradient centrifugation of umbilical cord blood and separation of umbilical cord blood mononuclear cells and plasma:

[0058] (1) Blood collection: Umbilical cord blood was taken from the umbilical cord of full-term healthy newborns, anticoagulated with sodium citrate, and separated within 24 hours after collection.

[0059] (2) Separation: Cord blood was diluted according to the ratio of cord blood: PBS = 1:1, slowly added to an equal volume of human...

Embodiment 2

[0073] Example 2 In vitro expansion of human umbilical cord blood NK cells

[0074] 1. Coated culture flask: 24-48 hours before inoculation, mix human hepatitis B immunoglobulin (100μg / mL) and human serum albumin (500μg / mL) according to the ratio of 1:1, take 10mL and add 75cm 2 In a culture bottle, mix well, let it stand at room temperature for 1 hour, and then put it in a refrigerator at 4°C for later use.

[0075] Before use, discard the supernatant in the coated culture flask and wash it again with PBS.

[0076] 2. Gradient centrifugation of umbilical cord blood and separation of umbilical cord blood mononuclear cells and plasma:

[0077] (1) Blood collection: Umbilical cord blood was taken from the umbilical cord of full-term healthy newborns, anticoagulated with sodium citrate, and separated within 24 hours after collection.

[0078] (2) Separation: Cord blood was diluted according to the ratio of cord blood: PBS = 1:1, slowly added to an equal volume of human lymphocy...

experiment example 1

[0098] Experimental example 1 cell count

[0099] On the 0th and 12th day of cell culture, samples were taken and counted respectively, and the test results are shown in Table 1 below.

[0100] Table 1 Total cell count results

[0101] cell number Day 0(unit) Day 12 (month) Example 1 2×10 7

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Abstract

The invention provides a human umbilical cord blood NK cell culture system and a preparation method, and belongs to the technical field of immune cell therapy. The human umbilical cord blood NK cell culture system in the invention comprises human hepatitis B immune globulin, human serum albumin, a serum-free medium, an activating factor and autologous plasma. Human umbilical cord blood NK cells are obtained through the steps of culture bottle coating, umbilical cord blood gradient centrifugation preparation of umbilical cord blood mononuclear cells and autologous plasma, inoculation, liquid changing, bagging, cell harvesting and the like. According to the invention, the human hepatitis B immune globulin and the human serum albumin are creatively combined for coating a culture bottle; pure factors IL2 and IL15 are combined for use; the clinical use risk is reduced; furthermore, the culture period of the preparation method in the invention is 12 days; the cost is reduced; and the prepared NK cells are good in amplification efficiency, high in purity, large in quantity and capable of meeting clinical requirements.

Description

technical field [0001] The invention relates to the technical field of immune cell therapy, in particular to a human NK cell culture system and a preparation method. Background technique [0002] Surgery, chemotherapy and radiotherapy are the three standard treatment methods for tumors, which play an important role in improving the survival rate and prolonging the survival time of cancer patients. However, there is still a long way to go to cure tumors, and there is an urgent need to develop new treatments. In recent years, immunotherapy, as the fourth treatment method for tumors, is developing rapidly and has received great attention. Immunotherapy is different from the traditional standard treatment to directly kill cancer cells, but to drive out and destroy cancer cells by activating the body's immune system. [0003] Natural killer cells (NK) are large granular lymphocytes derived from hematopoietic cells and are one of the important components of the body's innate imm...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/2315C12N2501/91C12N2500/90C12N2501/998C12N2533/50
Inventor 王珏胡向兵姬云
Owner 苏州科贝生物技术有限公司
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