Method for obtaining stem cell by using serum of same cord blood

A technology of umbilical cord blood and stem cells, applied in biochemical equipment and methods, microorganisms, tissue culture, etc., can solve the problem of limited application of the number of cells, and achieve a good effect of attached growth

Inactive Publication Date: 2009-09-09
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Hematopoietic stem cells (HSCs) can be widely used in gene therapy, tumor purification and bone marrow transplantation, etc. It has become a routine method for the reconstruction of hematopoietic function in cancer patients after radiotherapy and chemotherapy, but the limited number of cells greatly limits its clinical application

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  • Method for obtaining stem cell by using serum of same cord blood
  • Method for obtaining stem cell by using serum of same cord blood
  • Method for obtaining stem cell by using serum of same cord blood

Examples

Experimental program
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Embodiment 1

[0025] In this embodiment, serum for culture and mononuclear cells are simultaneously obtained from the same umbilical cord blood.

[0026] Umbilical cord blood comes from healthy puerpera who give birth at term or babies born at term by cesarean section, and the amount of cord blood collected is 120mL. After the umbilical cord blood is collected, mix it evenly with PBS at a volume ratio of 1:1, and use a density of 1.077g.mL -1 The Ficoll cell separation liquid was centrifuged at 2500 rpm for 25 minutes on a density gradient, and the volume ratio of the Ficoll separation liquid to the umbilical cord blood was 1:1-1:2. After centrifugation, as figure 2 As shown, the upper layer is serum, collected and placed in a sterile centrifuge tube for later use; the middle buffy coat layer was taken to obtain mononuclear cells (Mononuclear cells, MNCs), and the separated cells were washed twice with IMDM basal medium (1000rpm, 5 minutes), adjust the cell density for later use.

Embodiment 2

[0028] This example is the co-cultivation of UCB-HSCs and UCB-MSCs under static conditions.

[0029] The freshly isolated UCB-MNCs were divided into 2 × 10 6 cells.mL -1 The density was inoculated into a 6-well plate covered with cytodex-3 microcarriers in advance, and 20% of the same cord blood-derived serum was added, at 37°C, 5% CO 2 , 20%O 2 and under saturated humidity conditions for 12 days. Count every 24 hours and observe with light microscope.

[0030] Experimental results: The same serum derived from umbilical cord blood can well support the in vitro expansion of UCB-HSCs and UCB-MSCs, by figure 2 It can be seen that the attachment and growth of UCB-MSCs on cytodex-3 microcarriers is in good condition.

Embodiment 3

[0032] This example is the separation of UCB-HSCs and UCB-MSCs after co-cultivation under static conditions.

[0033] After the cultivation, use a dropper to blow up the cytodex-3 microcarriers into a suspension, suck out the suspension and place it in a sterile centrifuge tube. After standing for 5 minutes, the cytodex-3 microcarriers settle on the bottom layer, as Figure 4 shown. Aspirate the upper cell suspension, wash the cytodex-3 microcarrier with 5mL IMDM, let it stand again, and repeat three times.

[0034]The separated supernatant cell suspension was centrifuged to obtain UCB-HSCs, and the UCB-MSCs attached to cytodex-3 microcarriers were digested with 0.125% trypsin and 0.02% EDTA to harvest.

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Abstract

The invention discloses a method for obtaining stem cell by using serum of the same cord blood, belonging to the field of biotechnology and tissue engineering. Serum of the same cord blood is used to replace fetal calf serum, no cell factor or stroma cell support is added, and the strategy of combining a microcarrier and a bioreactor is adopted, thus realizing co-culture of cord blood source hemopoietic stem cell and mesenchymal stem cell. The method of the invention can support fine augmentation of UCB-HSCs and UCB-MSCs under three-dimensional conditions, completely replace the role of fetal calf serum and avoid immunological rejection among variants; cytodex-s microcarrier can provide adhesion surface for UCB-MSCs with adherence growth characteristics, so that the UCB-MSCs can realize possible adherence suspension culture in a three-dimensional dynamic environment; the natural settling method is adopted to separate and obtain the suspending UCB-HSCs and the UCB-MSCs which adheres to the microcarrier.

Description

technical field [0001] The invention belongs to the field of biotechnology and tissue engineering, and relates to a method for harvesting stem cells by using the same cord blood serum, in particular to a method for simultaneously obtaining cord blood hematopoietic stem cells and mesenchyme by using the same cord blood serum Stem Cell Approach. technical background [0002] Hematopoietic stem cells (HSCs) can be widely used in gene therapy, tumor purification and bone marrow transplantation, etc. It has become a routine method for the reconstruction of hematopoietic function in cancer patients after radiotherapy and chemotherapy, but the limited number of cells greatly limits its clinical application. An effective way to solve this problem is to implement large-scale expansion of HSCs in vitro. Although hematopoietic stem cell transplantation can significantly improve the quality of life of cancer patients after radiotherapy and chemotherapy, most patients will inevitably s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08
Inventor 刘天庆殷轶群关水鲍春雨葛丹李香琴崔占峰马学虎
Owner DALIAN UNIV OF TECH
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