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Serum-free culture medium for hematopoietic stem cell in vitro expansion culture and application thereof

A serum-free culture medium and culture medium technology, applied in the fields of molecular biology, medicine, and cell biology, can solve the problems of no HSC and limited knowledge, and achieve the goal of maintaining quality, overcoming insufficient amplification and no tumorigenic risk. Effect

Inactive Publication Date: 2016-06-29
GUANGDONG XTEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, when HSCs are used for treatment, they also face a serious problem: there are not enough HSCs for transplantation
[0003] The in vitro expansion and culture of HSC is a good solution, but also a very challenging technology, based on the following reasons: ①The in vitro expansion of HSC requires continuous self-renewal ability and is also expanding. prevent their differentiation; however, there is limited understanding of the physiological mechanisms that regulate HSC self-renewal and prevent HSC expansion; ②The number of expanded stem cells is closely related to the success rate of transplantation

Method used

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  • Serum-free culture medium for hematopoietic stem cell in vitro expansion culture and application thereof
  • Serum-free culture medium for hematopoietic stem cell in vitro expansion culture and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, preparation of hematopoietic stem cell serum-free medium

[0063] 1000ml of IMDM basal culture medium (Gibco31980030), recombinant human insulin (Escherichia coli recombinant expression, LIFE company), recombinant human transferrin (yeast recombinant expression, Invitria company), recombinant human serum albumin (Invitria company, Cellastim), β- Mercaptoethanol (Invitrogen, 21985-023), ethanolamine (Sigma, E0135), cholesterol (Sigma, C1231), linoleic acid (Sigma, L1012) and oleic acid (Sigma, O1383) were mixed to obtain hematopoietic stem cell IMDM culture system 1 .

[0064] Add L-glutamine to the above-mentioned hematopoietic stem cell IMDM culture system before the cells are cultured to obtain the hematopoietic stem cell IMDM culture system 1; add stem cell factor (SCF) to the above-mentioned hematopoietic stem cell IMDM culture system 2 in the process of cell culture , interleukin 3 (IL-3), interleukin 6 (IL-6), thrombopoietin (TPO), erythropoietin (E...

Embodiment 2

[0066] Example 2, application of hematopoietic stem cell serum-free medium in culturing hematopoietic stem cells

[0067] 1. Collection of umbilical cord blood and isolation of mononuclear cells

[0068] The umbilical cord blood from the placenta was collected, and each umbilical cord blood was taken from a maternal and child health hospital (pregnant women are required to have no obstetrics, medical complications, and other blood diseases). Store at ℃. All samples were separated within 12 hours after collection, and sibling cord blood was centrifuged using Ficoll lymphocyte separation medium density gradient. Specific steps are as follows:

[0069] 1. Centrifuge the collected cord blood at 2000r / min for 30min. Carefully aspirate the middle buffy coat layer, add more than 5 times the volume of IMDM culture solution to fully aspirate, wash, centrifuge at 1500r / min for 10min, and discard the supernatant. Repeat the above operation and wash twice in total. After the final cen...

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Abstract

The invention discloses a serum-free culture medium for hematopoietic stem cell in vitro expansion culture and a preparation method thereof. The culture medium for in vitro expansion of the hematopoietic stem cell is free from introducing exogenous gene expression, and the genome stability of the original stem cell is unchanged, the tumorigenic risk is avoided, and the stem cell transplantation safety problem caused by feeder layer cell and other exogenous cell mixing is not involved; through the combined application of various cytokines, a proper culture condition and properexpansion time are selected to overcome the defect that the expansion number is insufficient, the number is effectively expanded, and the quality is maintained at the same time, namely, the effect of maintaining hematopoietic reconstitution capacity through the expansion of the early hematopoietic progenitor cell and reservation of the HSC is achieved.

Description

technical field [0001] The invention belongs to the research fields of cell biology, molecular biology and medicine, and relates to a serum-free medium for in vitro expansion and culture of hematopoietic stem cells, more specifically, to a serum-free culture for in vitro expansion and culture of hematopoietic stem cells base and its applications. Background technique [0002] Hematopoietic stem cells (HSCs) refer to the most primitive cell population that has not matured in the body and is responsible for generating all blood cells. HSC is a pluripotent stem cell, which is called "universal cell" in medicine. Hematopoietic stem cells have high self-renewal ability and multiple differentiation potentials, and can produce all mature blood cells, such as red blood cells, white blood cells, platelets and lymphocytes. HSC is a kind of heterogeneous and immature hematopoietic precursor cells, which can rebuild the whole hematopoietic system during life, differentiate into B cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2500/24C12N2500/32C12N2500/36C12N2500/44C12N2500/46C12N2500/90C12N2501/125C12N2501/14C12N2501/145C12N2501/22C12N2501/2303C12N2501/2306C12N2501/33C12N2501/998
Inventor 乔志平单磊
Owner GUANGDONG XTEM BIOTECH CO LTD
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