Human umbilical cord mesenchymal stem cell serum-free culture medium and preparation method thereof, and acquisition method of human umbilical cord mesenchymal stem cell serum-free culture solution

A serum-free medium and serum-free culture technology, applied in the field of stem cell culture, can solve the problems of poor stability and slow proliferation, and achieve the effect of improving adherent growth and expansion ability

Active Publication Date: 2019-12-13
西安艾尔菲生物科技有限公司
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Problems solved by technology

[0009] The second object of the present invention is to provide a preparation method of serum-free medium for human umbilical cord mesenchymal stem cel...

Method used

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  • Human umbilical cord mesenchymal stem cell serum-free culture medium and preparation method thereof, and acquisition method of human umbilical cord mesenchymal stem cell serum-free culture solution
  • Human umbilical cord mesenchymal stem cell serum-free culture medium and preparation method thereof, and acquisition method of human umbilical cord mesenchymal stem cell serum-free culture solution
  • Human umbilical cord mesenchymal stem cell serum-free culture medium and preparation method thereof, and acquisition method of human umbilical cord mesenchymal stem cell serum-free culture solution

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preparation example Construction

[0054] A preparation method for human umbilical cord mesenchymal stem cell serum-free medium, comprising the following specific steps:

[0055] Step 1: Prepare serum-free culture supernatant concentrate

[0056] Human umbilical cord mesenchymal stem cells were inoculated in serum-free medium for culture. When the cell confluence was 80-90%, the supernatant of human umbilical cord mesenchymal stem cells was collected; filtered, concentrated by ultrafiltration, and added with water to determine the total protein concentration. 10mg / mL to obtain a serum-free culture supernatant concentrate.

[0057] Human umbilical cord mesenchymal stem cells were used in step 1 at 5×10 3 ~8×10 3 / cm 2 Inoculate in commercially available serum-free medium.

[0058] In step 1, filter twice, one of which uses a 0.8 μm microfiltration membrane, and the other uses a 0.45 μm microfiltration membrane.

[0059] In step 1, the ultrafiltration concentration adopts an ultrafiltration system with a mol...

Embodiment 1

[0079] A method for preparing human umbilical cord mesenchymal stem cell serum-free medium, specifically comprising the following steps:

[0080] Step 1: Prepare serum-free culture supernatant concentrate

[0081] Human umbilical cord mesenchymal stem cells in 5×10 3 / cm 2 Inoculate in serum-free medium for culture, collect the culture supernatant when the cell fusion degree is 80%, and obtain a supernatant with a volume of 5 L, filter the 5 L supernatant through a plate filter, and filter membrane pore diameters are respectively 0.8μm, 0.45μm; then use cellulose acetate ultrafiltration membrane with a molecular weight cut-off of 3KDa for ultrafiltration and concentration, stop concentration when the concentration reaches 1 / 15 of the original filtered liquid volume, and obtain 305ml of concentrated liquid, use BCA protein reagent The box detects that the protein content of the concentrated solution is 11.2 mg / ml, add 37 ml of water for injection to the concentrated liquid, a...

Embodiment 2

[0089] Prepare a serum-free medium for human umbilical cord mesenchymal stem cells, the method is as follows:

[0090] Step 1: Prepare serum-free culture supernatant concentrate

[0091] Human umbilical cord mesenchymal stem cells in 6×10 3 / cm 2 Inoculate in serum-free medium for culture, collect the culture supernatant when the cell confluence is 85%, obtain a supernatant with a volume of 30L, filter the 30L supernatant through a plate filter in turn, and filter the filter membranes with different pore sizes 0.8μm, 0.45μm; then use cellulose acetate ultrafiltration membrane with a molecular weight cut-off of 3KDa for ultrafiltration and concentration, and the area of ​​a single membrane is 0.5m 2, a total of 6 membrane packs, using the tangential flow process for concentration, the liquid at the permeate end is discarded, and the concentration is stopped when it is concentrated to 1 / 18 of the volume of the original microfiltrate, and the liquid in the ultrafiltration syste...

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Abstract

The invention discloses a human umbilical cord mesenchymal stem cell serum-free culture medium and a preparation method thereof, and an acquisition method of a human umbilical cord mesenchymal stem cell serum-free culture solution. The culture medium comprises a basic culture medium and a serum-free culture supernatant concentrated solution, and also comprises the following components and concentrations: 5-100 ng/ml of a vitamin composition, 50-200 [mu]g/ml of grape polyphenol, 1-50 ng/ml of a recombinant human epidermal growth factor, 1-50 ng/ml of a recombinant human basic fibroblast growthfactor, 1-10 ng/ml of a recombinant platelet-derived growth factor-C, 1-10 ng/ml of a recombinant human insulin-like growth factor-1, 50-200 [mu]M of beta-mercaptoethanol, 1-100 [mu]g/ml of human serum albumin, 1-100 [mu]g/ml of glutathione, and 1.2-3.7 g/L of sodium bicarbonate. The human umbilical cord mesenchymal stem cell serum-free culture medium is used for replacing a traditional culture medium containing animal serum to culture human umbilical cord mesenchymal stem cells, and unsafe factors such as human immune response caused by stem cell pollution by pathogens or animal-derived components and heterologous protein are avoided.

Description

technical field [0001] The invention belongs to the technical field of stem cell culture, and relates to a serum-free medium for human umbilical cord mesenchymal stem cells, a preparation method for a serum-free medium for human umbilical cord mesenchymal stem cells, and a method for culturing a serum-free culture medium for human umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells are important members of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development. Mesenchymal stem cells were first discovered in the bone marrow, and have attracted increasing attention due to their multiple differentiation potentials, hematopoietic support, promotion of stem cell implantation, immune regulation, and self-replication. Bone marrow mesenchymal stem cells have been widely used clinically, and current research shows that umbilical cord-derived mesenchymal stem cells will also have greater application pote...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2500/12C12N2500/30C12N2500/38C12N2500/44C12N2500/90C12N2501/105C12N2501/11C12N2501/115C12N2501/135C12N2501/998
Inventor 王静王海江肖萍胡春梅
Owner 西安艾尔菲生物科技有限公司
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