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Method for separating hematopoietic stem cells from umbilical cord blood and amplifying CD34 positive cells

A technology of hematopoietic stem cells and positive cells, applied in the field of CD34+ cell preparation, can solve the problems of limited supply of human umbilical cord blood, limited number of CD34+ cells, etc.

Inactive Publication Date: 2017-05-24
BOYALIFE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Isolation and purification of primary CD34 directly from human umbilical cord blood by the above method + cells, in view of the limited supply of human cord blood, the CD34 + The number of cells is also extremely limited

Method used

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  • Method for separating hematopoietic stem cells from umbilical cord blood and amplifying CD34 positive cells
  • Method for separating hematopoietic stem cells from umbilical cord blood and amplifying CD34 positive cells
  • Method for separating hematopoietic stem cells from umbilical cord blood and amplifying CD34 positive cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1. Preparation of CD34 positive cells from umbilical cord blood hematopoietic stem cells

[0121] The formula of the hematopoietic stem cell culture system used in this example is: human low-density lipoprotein (Human LDL) 75 μg / ml, erythropoietin (EPO) 5ng / ml, interleukin 3 (IL-3) 1500IU / ml, stem cell factor (SCF) 25ng / ml, an appropriate amount of animal component-free medium (StemSpan-ACF) was added to the full volume of the dosing solution.

[0122] In this example, after the hematopoietic stem cells were isolated from the collected umbilical cord blood, they were first cryopreserved and resuscitated, and then CD34 + Expansion of cells so that cryopreservation-resuscitation processing does not lose / damage cord blood hematopoietic stem cells and in some cases large cord blood samples can be collected for CD34 as needed + Expansion of cells.

[0123] 1. Cryopreservation of umbilical cord blood hematopoietic stem cells

[0124] After the fresh umbilical cor...

Embodiment 2

[0142] Example 2. Preparation of CD34 positive cells from umbilical cord blood hematopoietic stem cells

[0143] The formula of the hematopoietic stem cell culture system used in this example is: human low-density lipoprotein (Human LDL) 25 μg / ml, erythropoietin (EPO) 15ng / ml, interleukin 3 (IL-3) 500IU / ml, stem cell factor (SCF) 75ng / ml, and an appropriate amount of animal component-free medium (StemSpan-ACF) was added to the full volume of the dosing solution.

[0144] In this example, cryopreservation and resuscitation operations are not performed, but the collected cord blood is directly subjected to CD34 + Expansion of cells.

[0145] (1). Separation of umbilical cord blood hematopoietic stem cells: after the fresh umbilical cord blood is collected, separate umbilical cord blood hematopoietic stem cells to obtain nucleated cells;

[0146] (2). Use a disposable sterile syringe to transfer 5ml of cord blood hematopoietic stem cells obtained in step (1) to a 50ml sterile...

Embodiment 3

[0158] Example 3, Preparation of CD34 positive cells from umbilical cord blood hematopoietic stem cells

[0159] The formula of the hematopoietic stem cell culture system used in this example is: human low-density lipoprotein (Human LDL) 50 μg / ml, erythropoietin (EPO) 10 ng / ml, interleukin 3 (IL-3) 1000IU / ml, stem cell factor (SCF) 50ng / ml, and an appropriate amount of animal component-free medium (StemSpan-ACF) was added to the full volume of the dosing solution.

[0160] In this example, after the hematopoietic stem cells were isolated from the collected umbilical cord blood, they were first cryopreserved and resuscitated, and then CD34 + Expansion of cells.

[0161] 1. Cryopreservation of umbilical cord blood hematopoietic stem cells

[0162] After the fresh umbilical cord blood is collected, it passes through the AXP Automatic Separation System (AXP Auto Xpress TM Platform) to obtain nucleated cells, and then add cell freezing solution (65 parts of DMEM-F12, 10 parts ...

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Abstract

The invention relates to a method for separating hematopoietic stem cells from umbilical cord blood and amplifying CD34 positive cells. The method comprises the following specific steps: separating hematopoietic stem cells from umbilical cord blood; purifying; separating by using a Ficoll separating solution; counting the CD34 positive cells by using a flow cytometer, diluting, inoculating and culturing; changing the solution and culturing; transferring the cells to new plates and culturing; evaluating the cell amplifying effect. The invention further relates to cell products containing the CD34 positive cells prepared by the method and application of the cell products. The method has excellent technical effects as described in the specification.

Description

technical field [0001] The present invention belongs to the field of treating clinical diseases with cell biology techniques. Specifically, the present invention relates to a method for preparing CD34 from umbilical cord blood hematopoietic stem cells + cell method. Background technique [0002] Stem cells mediate regeneration and the transmission of genetic information to subsequent cell generations. They can self-renew and produce differentiated progeny. In recent years, advances have been made in our understanding of the molecular mechanisms underlying the interactions between stem cells and their tissue microecological environment. This has led to a better understanding of the molecular regulatory mechanisms at work in stem cells. [0003] While gene therapy is still experimental, the technology holds the promise of having a major impact on human health. The scope and definition of gene therapy has changed and expanded over the past few years. In addition to correc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789A61K35/51A61P9/14A61P9/10
CPCC12N5/0647A61K35/51C12N2500/36C12N2500/92C12N2501/125C12N2501/14C12N2501/2303
Inventor 许晓椿朱业峰肖海蓉
Owner BOYALIFE
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