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Method for sequential culture of human umbilical cord blood mesenchymal stem cells by using two culture media

A technology of mesenchymal stem cells and human umbilical cord blood, applied in the field of cell engineering and biomedicine, can solve the problems of high economic cost, reduced success rate of hUCB-MSCs culture, differential adherence, etc.

Active Publication Date: 2012-07-11
AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the prominent problem of these culture methods is that the success rate of hUCB-MSCs is low, especially after 3-5 generations of subculture in single DMEM / F12, DMEM / MEM and other media, obvious hUCB-MSCs biological morphological characteristics and Function changes, not suitable for research and application with large demand for cells
In addition, in addition to the high economic cost of many commercially available special media, the growth of osteoclast-like cells is often relatively vigorous during the primary culture process, which is obviously not conducive to differential attachment and purification of hUCB-MSCs after subculture, and even greatly reduces the Reduced culture success rate of hUCB-MSCs

Method used

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  • Method for sequential culture of human umbilical cord blood mesenchymal stem cells by using two culture media
  • Method for sequential culture of human umbilical cord blood mesenchymal stem cells by using two culture media
  • Method for sequential culture of human umbilical cord blood mesenchymal stem cells by using two culture media

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Embodiment 1

[0050] Embodiment 1 of the present invention: a method for sequentially culturing human umbilical cord blood mesenchymal stem cells in two culture media, comprising the following two steps:

[0051] (1) Isolation of mononuclear cells: the aseptically collected heparin anticoagulated umbilical cord blood was mixed with D-PBS buffer (take KCl 0.20g, KH 2 PO 4 0.20g, NaCl 8.00g, NaCl 2 HPO 4 ·7H 2 (O2.16g, add ultrapure water to 1000ml, adjust pH to 7.2-7.4, autoclave, cool and store in 4°C refrigerator) After dilution and mixing, add diluted blood to lymph On the cell separation medium (density 1.077g / ml) (purchased from Pharmacia, USA), after centrifugation, absorb the middle buffy coat, wash with D-PBS buffer containing 2mmol / L EDTA, and add 0.83% ammonium chloride to lyse For red blood cells, wash the cells with PBS buffer containing 2% FBS, centrifuge and suspend the pellet in DMEM / F12 medium, stain with 0.4% trypan blue, and observe the cell viability.

[0052] (2) Cul...

Embodiment 2

[0053] Embodiment 2 of the present invention: a method for sequentially culturing human umbilical cord blood mesenchymal stem cells in two culture media, comprising the following two steps:

[0054] (1) Separation of mononuclear cells: Dilute the aseptically collected heparin-anticoagulated umbilical cord blood with D-PBS buffer, mix well, and add the diluted blood to the lymphocyte separation medium (density 1.077 g / ml), after centrifugation, absorb the middle buffy coat layer, wash with D-PBS buffer containing 2mmol / L EDTA, add 0.83% ammonium chloride to lyse the red blood cells, wash the cells with PBS buffer containing 2% FBS, After centrifugation, the precipitate was suspended in DMEM / F12 medium, stained with 0.4% trypan blue, and the cell viability was observed.

[0055] (2) Culture: when the cell viability is >95%, the cell density is 5×10 6 cells / ml, inoculate isolated mononuclear cells in T25 culture flasks, inoculate 2-3 T25 culture flasks with cells obtained from 1...

Embodiment 3

[0056] Embodiment 3: A method for sequentially culturing human umbilical cord blood mesenchymal stem cells in two media, comprising the following steps:

[0057] (1) Separation of mononuclear cells: Dilute the aseptically collected heparin-anticoagulated umbilical cord blood with D-PBS buffer, mix well, add the diluted blood to the lymphocyte separation medium at a volume ratio of 1:1, and centrifuge Aspirate the middle buffy coat, wash with D-PBS buffer containing 2mmol / L EDTA, then add 0.83% ammonium chloride to lyse the red blood cells, wash the cells with PBS buffer containing 2% FBS, centrifuge, and culture the pellet with DMEM / F12 base suspension, stained with 0.4% trypan blue, and observed cell viability.

[0058] (2) Culture: when the cell viability is >95%, use DMEM / F12 with pH 6.5-6.8 containing 10% FBS to culture at 37°C, 5% CO 2 The primary culture was carried out under saturated humidity, half of the medium was changed for the first time after 5 days for the P0 g...

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Abstract

The invention discloses a method for sequential culture of human umbilical cord blood mesenchymal stem cells by using two culture media. A lymphocyte separation medium density gradient centrifugation method is used for separating umbilical cord blood mononuclear cells, a dulbecco modified eagle medium (DMEM) / F12 is used for primary culture of the mesenchymal stem cells of the human umbilical cord blood, and the method increases adherence of the cells, reduces growth of osteoclast like cells remarkably, facilitates formation of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) colonies, and greatly improves successful rate of culture of the hUCB-MSCs. The DMEM / F12 is used continuously for subculturing to a P2 generation, the method is combined by a using enzymatic digestion and differential velocity adherent method simultaneously, and the method facilitates purification of P1-P2 generation cells remarkably. A P3 generation and the following generations are cultured by using an Oricell human umbilical cord mesenchymal stem cell culture medium, marker proteins and good morphological characteristics and growth characteristics of the hUCB-MSCs are maintained, and multilineage differentiation potential of the hUCB-MSCs is maintained. In addition, costs of the culture media adopted by the method are reduced remarkably.

Description

technical field [0001] The invention relates to a method for sequentially culturing human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) with two culture media, which belongs to the technical fields of cell engineering and biomedicine. Background technique [0002] Human umbilical cord blood (hUCB) is the blood in the umbilical cord and the blood vessels near the fetus in the placenta when the fetus is born. mesenchymal stem cells, MSCs). Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) are fibroblast-like stem cells with unlimited proliferation and multilineage differentiation potential, expressing SH2, SH3, SH4, ASMA, MAB1470, CD13, CD29, CD44, CD166 and Cell surface antigens such as CD73, but do not express surface markers such as CD34, CD45, and CD14; they have similar biological characteristics to bone marrow-derived MSCs, but their culture success rate is relatively low. According to statistics, only 25-30% of cord blood samples are cultured The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 余丽梅
Owner AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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