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Stem cell in-vitro multiplication culture system and method

A culture system and in vitro amplification technology, applied in the field of biomedicine, can solve the problems of donor injury, failure to find a single umbilical cord blood, and limited number of stem cells, so as to save storage space, reduce physical storage capacity, and reduce possible damage Effect

Inactive Publication Date: 2019-02-22
章毅 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bone marrow or peripheral blood HSCs transplantation has extremely high requirements on the type of donor and recipient. Patients often have to wait for a long time to find a suitable donor. When the number of stem cells obtained in a single collection is insufficient, multiple collections are required. Donors cause great harm; for cord blood HSCs transplantation, the number of stem cells in a single cord blood is limited, which will lead to some adult patients unable to find a single cord blood that meets clinical transplantation conditions

Method used

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  • Stem cell in-vitro multiplication culture system and method
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  • Stem cell in-vitro multiplication culture system and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation and Purification of Umbilical Cord Blood CD34+ Cells

[0033] After obtaining the informed consent of the puerpera, obstetricians and medical staff aseptically collected cord blood from healthy full-term newborns, transported the blood samples to a 10,000-level aseptic laboratory, and performed preparation operations in a 100-level aseptic biological safety cabinet. The operator draws about 10 mL of umbilical cord blood from the umbilical cord blood sample, measures the number of white blood cells in the whole blood, and adds 60 g / L of hydroxyethyl starch to the remaining blood sample, umbilical cord blood: hydroxyethyl starch = 1:4 (v / v), Gently mix to promote erythrocyte sedimentation, then place the blood sample in a large-capacity low-temperature centrifuge and centrifuge at 630rpm for 6.5min at 8-12°C, then manually press the slurry to separate the upper layer of plasma from the centrifuged blood sample , white blood cells (including hematopoi...

Embodiment 2

[0034] Example 2 In vitro expansion and culture of hematopoietic stem cells

[0035] The CD34+ cells prepared and purified according to the method described in Example 1 were inoculated on a 12-well plate (Corning Costar Company, the United States), and a control group and an expansion group were set up, and the cell inoculation density was 1 × 10 4 / mL.

[0036] The expansion group cells were added with expansion medium, 2mL / well, and the expansion medium contained: serum-free medium (StemCellTechnologies, Vancouver, BC) as the base medium, 10% (v / v) fetal bovine serum (Gibco, U.S. ), 4.5mM Nicotinamide, 20ng / mL TPO, 50ng / mL IL-6, 60ng / mL FLT-3 Ligand, 40ng / mL SCF, 20ng / mL IL-3, 30ng / mL G-CSF, and 15ng / mL EPO. The culture medium of the control group was the expansion medium without nicotinamide, and the dosage was 2 mL / well. Place cells at 37°C, 5% CO 2 , and a sterile incubator with saturated humidity, and the cells were fully replaced every 5 days. When the cell densit...

Embodiment 3

[0038] Example 3 Determination of hematopoietic stem cell colony-forming ability

[0039] The hematopoietic stem cells prepared and purified according to the method described in Example 1 were diluted with 5% glacial acetic acid, and the cells were counted by trypan blue staining. The culture medium adjusted the cell suspension concentration to 1×10 4 / mL, mix thoroughly, then, draw the cell suspension with a sterile syringe, add 2mL / well into a six-well culture plate (Corning Costar, USA), repeat three holes; gently shake the culture plate to make the cells evenly fill the whole At the bottom of the culture plate, mark the sample number and inoculation time with a marker pen above the culture well of the culture plate, draw a "cross" on the bottom of the culture well to divide the bottom into 4 areas, so that the colonies can be counted later, and finally, culture Plates were placed at 37°C, 5% CO 2 , saturated humidity in a sterile cell incubator, and then observe and coun...

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Abstract

The invention relates to a stem cell in-vitro multiplication method. A multiplication culture system containing niacinamide substances is adopted, a serum-free culture medium is used as a basic culture medium, and 8%-12% of fetal bovine serum, a cytokine composition and niacinamide substances are added. The cytokine composition includes a stem cell factor, thrombopoietin, an FLt3 ligand, G-CSF, IL-3 and IL-6. Compared with conventional culture systems, the culture system can effectively increase the number of hematopoietic stem cells and the cell proportion of CD34+ / Lin- and CD34+ / CD38- when culturing CD34+ cells for 1-12 weeks and is helpful to solving of the problem of insufficient cells in hematopoietic stem cell transplantation.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a composition for in vitro expansion of stem cells, in particular to a culture system for in vitro expansion of hematopoietic stem cells, and an in vitro rapid expansion and culture method for hematopoietic stem cells. Background technique [0002] Hematopoietic stem cells (HSCs) are a type of adult stem cells present in hematopoietic tissues and blood, which have the ability of self-renewal and the potential to differentiate into all mature blood cells. HSCs are the earliest stem cells discovered and studied by people. They appear in the yolk sac at the second week of human embryonic development, transfer to the embryonic liver at about the fourth week, and enter the bone marrow for hematopoiesis at the fifth month of pregnancy. [0003] It has been found clinically that the occurrence and progression of most malignant hematological diseases, such as acute myeloid leukemia...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2500/30C12N2501/11C12N2501/12C12N2501/125C12N2501/13C12N2501/14C12N2501/145C12N2501/165C12N2501/22C12N2501/2301C12N2501/2302C12N2501/2303C12N2501/2306C12N2501/231C12N2501/2312C12N2501/235C12N2501/25C12N2501/26C12N2501/727
Inventor 章毅伍婷陈侃俊李冉陈亮陆薇
Owner 章毅
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