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Non-animal-source serum-free culture medium for umbilical cord blood stem cells

A technology of serum-free medium and mixture, which is applied in the field of cord blood stem cell serum-free medium, can solve the problems of high price, uncertain chemical composition of serum-free medium, difficulty in ensuring the consistency of medium batches, etc., and achieve clear properties , avoid instability, and ensure consistency

Active Publication Date: 2015-03-11
内蒙古干细胞医学工程技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] These serum-free media are chemically indeterminate and expensive, making it difficult to ensure batch-to-batch consistency

Method used

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  • Non-animal-source serum-free culture medium for umbilical cord blood stem cells
  • Non-animal-source serum-free culture medium for umbilical cord blood stem cells
  • Non-animal-source serum-free culture medium for umbilical cord blood stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: the preparation of culture medium

[0021] Preparation of recombinant human insulin storage solution: Weigh 100mg of recombinant human insulin and dissolve it in 40ml of water for injection, add dropwise 2M HCl until the recombinant human insulin is completely dissolved, and make the volume to 50ml, prepare a 20mg / ml storage solution, and store at 4°C.

[0022] Preparation of human transferrin stock solution: Weigh 100mg of human transferrin and dissolve it in 40ml of water for injection, until it is completely dissolved, dilute to 50ml, prepare a 20mg / ml stock solution, and store at 4°C.

[0023] The medium SFM composition of the present invention is as follows:

[0024]

[0025] More preferably, the medium SFM composition of the present invention is as follows:

[0026]

[0027]

[0028] The amino acids mentioned in the table above are L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine , L-Phenylalanine, L-Thre...

Embodiment 2

[0033] Example 2: Umbilical cord blood separation

[0034] Source of umbilical cord blood: The umbilical cord blood comes from the obstetrics department of Beijing Armed Police Hospital. , the average volume of 80 ~ 130ml. The separation process is as follows:

[0035] 1: After diluting the fresh cord blood and the buffer solution PBS at a volume ratio of 1:1, slowly add to the upper layer of the separation solution Ficoll along the wall of the test tube. Pay attention to keep the two interfaces clear and prevent blood from mixing into the separation solution.

[0036] 2: Centrifuge at 1800rpm for 15min, gently insert the white mist layer (the second layer from top to bottom) with a capillary pipette, gently suck out the mononuclear cells in this layer along the tube wall, and put it into another centrifuge tube.

[0037] 3: The resulting mononuclear cell suspension was washed with one volume of PBS, centrifuged at 1500 rpm for 10 min, and washed 3 times.

[0038] 4: Count...

Embodiment 3

[0039] Example 3: Cell Culture

[0040] The medium SFM prepared in Example 1 of the present invention and the serum-free medium X-VIVO developed by Lonza were used to culture cord blood stem cells respectively, and the cell morphology was observed under a microscope, see figure 1 , the results showed that at 3 days, the suspension cells of umbilical cord blood stem cells cultured in X-VIVO serum-free medium were round, and many adherent cells were spindle-shaped, and the cell density was higher than that of the serum-free medium (SFM) provided by the present invention. However, at 12 days and 18 days, the umbilical cord blood stem cells in the two serum-free media were round in shape with high uniformity.

[0041] Centrifuge at 1000rpm for 10min every three days after culturing, change the medium in half, and count the number of cells by trypan blue staining. The results are shown in figure 2 , showing that at 3 days, the total number of umbilical cord blood stem cells culture...

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Abstract

The invention relates to the field of biology, and discloses a non-animal-source serum-free culture medium which essentially comprises an IMDM (Iscove Modified Dulbecco Medium), L-glutamine, sodium bicarbonate, recombinant human insulin, recombinant human transferrin, recombinant human albumin, 2-mercaptoethanol, phytohaemagglutinin (PHA), lipid, amino acid, vitamins, trace elements, interleukin-3(IL-3), stem cell factor, (SCF), Fit3-L, IL-6 and granulocyte colony-stimulating factor (G-CSF). The non-animal source serum-free culture medium is clear in chemical components, free from animal sources and serum and safe and ideal in cell cultivation, avoids the doped animal components and unstability of batches, and the results of cultured umbilical cord blood stem cells show that the total cellular score, the cell phenotype and the secretory cell factors are normal, so that the non-animal-source serum-free culture medium has good industrial application prospect.

Description

technical field [0001] The invention relates to the field of biology, in particular to a serum-free medium for umbilical cord blood stem cells with clear chemical components and no animal source. Background technique [0002] As an alternative source of stem cells, cord blood is rich in hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells. Umbilical cord blood stem cells are a kind of primitive progenitor cells discovered in recent years that have the same multipotential differentiation potential as bone marrow stem cells and peripheral blood stem cells. Umbilical cord blood stem cells have the ability to self-renew and proliferate, and can differentiate into various cells or tissues under the influence or induction of specific factors. As a substitute for bone marrow and peripheral blood stem cells, umbilical cord blood stem cells can rebuild bone marrow hematopoiesis and immunity, and are used to treat bone marrow failure, malignant and non-m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789
Inventor 武晓云康会彦吕岩刘学敏王云虹王黎明高锦
Owner 内蒙古干细胞医学工程技术研究中心
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