Separation method and culture method for umbilical cord mesenchymal stem cells

A separation method and stem cell technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of insufficient adherence and growth support ability of primary cells, avoid heterologous contamination, and achieve good results. Supporting role, effect of guaranteeing batch stability

Active Publication Date: 2014-02-19
BEIJING DONGFANG HUAHUI BIOMEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the common problem of the current serum-free medium is that it has ins

Method used

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  • Separation method and culture method for umbilical cord mesenchymal stem cells
  • Separation method and culture method for umbilical cord mesenchymal stem cells
  • Separation method and culture method for umbilical cord mesenchymal stem cells

Examples

Experimental program
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Embodiment 1

[0067] This embodiment provides a method for isolating and culturing umbilical cord mesenchymal stem cells, which includes the following steps:

[0068] 1. Obtain 15cm of healthy newborn umbilical cord tissue, and wash it with PBS buffer solution containing 2wt% penicillin-streptomycin (final concentration of penicillin 100U / mL, final concentration of streptomycin 0.01g / 100mL) to fully remove blood stains;

[0069] 2. Cut the washed umbilical cord evenly into small sections of 3-4cm in length, perform mechanical separation, bluntly peel off Wharton’s jelly, remove the umbilical artery and umbilical vein at the same time, use ophthalmic scissors to cut the stripped Wharton’s jelly into 1mm 3 -3mm 3 small pieces to obtain Wharton's jelly tissue pieces.

[0070] 3. Take a 10cm petri dish, coat it with 0.2wt% gelatin, and place it at 37°C, 5% CO 2 Incubate for 1 hour in an incubator with no Ca 2+ , Mg 2+ Wash with PBS buffer to remove residual gelatin.

[0071] 4. Resuspend ...

Embodiment 2

[0080] This embodiment provides a method for isolating and culturing umbilical cord mesenchymal stem cells, which includes the following steps:

[0081] 1. Obtain 10cm of umbilical cord tissue from caesarean section neonates, and use PBS buffer solution containing 5wt% penicillin-streptomycin (final concentration of penicillin 100U / mL, final concentration of streptomycin 0.01g / 100mL) to fully remove blood stains;

[0082] 2. Cut the washed umbilical cord evenly into 3-4cm long sections, perform mechanical separation, bluntly peel off Wharton's jelly, and remove the umbilical artery and umbilical vein at the same time;

[0083] 3. Cut the peeled Wharton's glue into 1mm 3 -3mm 3 Add 15 mL of a mixture of collagenase A with a concentration of 1 mg / mL and neutral protease with a concentration of 1 mg / mL (the volume ratio of the two is 1:2), and place at 37 ° C, 5% CO 2 Digested in an incubator for 2 hours, during which time it was taken out every 20 minutes to detect the digesti...

Embodiment 3

[0089] This embodiment provides a method for comparing and analyzing the isolation and culture of different umbilical cord mesenchymal stem cells, which includes the following steps:

[0090] 1. Obtain umbilical cord tissue of 15cm caesarean section neonates, and fully remove blood stains with PBS buffer containing 5wt% penicillin-streptomycin (final concentration of penicillin 100U / mL, final concentration of streptomycin 0.01g / 100mL);

[0091] 2. Divide the washed umbilical cord into three sections evenly, perform mechanical separation respectively, bluntly peel off Wharton's jelly, and remove the umbilical artery and umbilical vein at the same time;

[0092] 3. Take a 10cm petri dish, coat the petri dish with gelatin with a concentration of 0.1wt%, overnight at 4°C (8-12 hours), and use Ca-free 2+ , Mg 2+ Wash with PBS buffer to remove residual gelatin;

[0093] 4. Cut the stripped Wharton's glue into 1mm 3 -3mm 3 Among them, the two groups were respectively added with 5...

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Abstract

The invention relates to a separation method and a culture method for umbilical cord mesenchymal stem cells. The separation method comprises the following steps: thoroughly cleaning umbilical cord tissue of a healthy newborn by using a PBS (phosphate buffer solution) containing streptomycin and penicillin, and removing blood; shearing the umbilical cord into small sections uniform in length, and mechanically separating, bluntly stripping Wharton' s jelly, and removing umbilical arteries and umbilical veins; uniformly shearing the Wharton' s jelly; re-suspending the sheared Wharton' s jelly through an MSCs (mesenchymal stem cells) culture medium, inoculating to a culture dish with laid gelatin, and putting in a CO2 culture box for cultivation; conducting centrifugal separation to obtain tissue blocks and a cell resuspension solution. The culture method comprises the following steps: enwrapping the culture dish, discarding the gelatin, and washing with the PBS; inoculating the separated out tissue blocks and the cell resuspension into the culture dish; performing digestive subculture after cell fusion growth rate reaches 80-90%.

Description

technical field [0001] The invention relates to a method for separating and culturing umbilical cord mesenchymal stem cells, belonging to the technical field of bioengineering. Background technique [0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells that widely exist in connective tissue and organ interstitium. They have good proliferation ability and multi-lineage differentiation potential. Differentiation of functional cells such as cardiomyocytes, hematopoietic cells, and liver cells. Because MSCs are convenient to source, easy to isolate, expand and purify in vitro, and can still maintain stemness after passage and expansion, they are ideal seed cell sources for tissue engineering, genetic engineering and cell therapy. In addition, MSCs have a strong immune regulation function and low immunogenicity in transplantation, and have broad application prospects in the treatment of end-stage diseases such as end-stage liver disease and autoimmune diseases such as...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 赵侃王春有赵宇刘湘连李莉莉
Owner BEIJING DONGFANG HUAHUI BIOMEDICAL TECH
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