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In vitro efficient amplification method for human umbilical cord blood hematopoietic stem cells

A technology for hematopoietic stem cells and umbilical cord blood, applied in the biological field, can solve the problems of trauma, high viral infection rate, limited sources, etc., and achieve the effects of stable biological properties, stable product quality, and easy control.

Inactive Publication Date: 2016-08-17
ANHUI HUIEN BIOTECH
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  • Summary
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, MSCs are mainly derived from adult bone marrow, but the number and proliferation and differentiation potential of adult bone marrow MSCs decrease with the age of the donor, and the virus infection rate is high, and the collection is traumatic, so its source is limited

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Collection of umbilical cord blood

[0025] Under aseptic conditions, the umbilical cord blood of newborns who were born with full-term natural delivery or caesarean section was extracted by needle aspiration, put into a centrifuge tube, anticoagulated with heparin, and stored in a cryopreservation;

Embodiment 2

[0026] Example 2: In vitro culture and expansion of umbilical cord mesenchymal stem cells

[0027] ① Isolation of mononuclear cells: Within 12 hours after the neonatal umbilical cord blood was drawn, mononuclear cells (MNC) were separated with Fi-coll-Hypaque separation medium, and washed 3 times with MDM medium;

[0028] ②Put the above isolated mononuclear cells into 1 g / L type I collagenase and digest in a shaker at 37°C for 1 hour, centrifuge at 1 000 rr / mjn (centrifugal radius 16 cm) for 15 minutes after digestion, and suck out the upper layer to suspend The components were filtered through a 100-mesh sieve, and the filtered liquid was centrifuged at 1 000 r / mjn (centrifugal radius 16 cm) for 5 mjn, and the supernatant was discarded;

[0029] ③ Inoculate the digested solution obtained in the above step ② into complete MDM medium (containing 20% ​​fetal bovine serum (FBS), 50 μmol / L 2-mercaptoethanol, 10% fetal bovine serum, 50 μg / mL ascorbic acid, 0 .5 mmol / L glycerol pho...

Embodiment 3

[0036] Embodiment 3: the preparation of bioactive factor

[0037] Melting, mixing, low-temperature separation, ultrafiltration and concentration of the waste liquid collected during step (2) umbilical cord stem cell culture and expansion. 10. Retain biologically active substances, and then perform ultrafiltration through a hollow fiber ultrafiltration column with a molecular weight cut-off of 50KD, and concentrate the ultrafiltered liquid to obtain a composition rich in stem cell bioactive factors;

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PUM

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Abstract

The invention discloses an in vitro efficient amplification method for human umbilical cord blood hematopoietic stem cells. The method comprises the following steps: (1) collecting umbilical cord blood, namely under aseptic conditions, drawing neonatal cord blood during full-term normal delivery or caesarean operation in a needle aspiration manner, putting the neonatal cord blood into a centrifugal tube, carrying out anticoagulation with heparin, and carrying out cryopreservation; (2) carrying out in vitro culture and amplification on umbilical cord mesenchymal stem cells; (3) preparing a bioactive factor; and (4) preparing lysate. The method disclosed by the invention has the advantages that the bioactive factor or substance does not contain any animal derived ingredient, a product with a cytobiology active function can be developed by utilizing the bioactive factor or substance, after the product acts on a human body, organism stem cells can be activated, damaged, diseased and senescent cells can be repaired or replaced, and the bioactive factor or substance has the functions of regulating cellular metabolism of an organism, enhancing immunity of the organism and delaying senescence.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a high-efficiency expansion method for human umbilical cord blood hematopoietic stem cells in vitro. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of tissue stem cells with multi-directional differentiation potential, which can be obtained from various tissues such as bone marrow, peripheral blood, fat, and skin. As a type of pluripotent stem cells, they can Proliferate and differentiate in the direction of osteogenesis, cartilage, muscle, nerve and islet-like cells. At present, MSCs are mainly derived from adult bone marrow, but the number and proliferation and differentiation potential of adult bone marrow MSCs decrease with the age of the donor, and the virus infection rate is high, and the collection is traumatic, which limits its source. Recently, MSCs extracted from umbilical cord or placenta tissue have been developed. It is also a kind of ste...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2500/38C12N2500/42C12N2500/44
Inventor 张正亮刘大海
Owner ANHUI HUIEN BIOTECH
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