Human umbilical cord mesenchymal stem cell serum-free medium and preparation method and method for obtaining human umbilical cord mesenchymal stem cell serum-free culture medium

A serum-free medium and serum-free culture technology, applied in the field of stem cell culture, can solve problems such as poor stability and slow proliferation, and achieve the effects of promoting cell division, increasing proliferation activity, and improving adherent growth and expansion capabilities

Active Publication Date: 2021-05-04
西安艾尔菲生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 cells, which solves the problems of adherent growth speed, slow proliferation and poor stability of human umbilical cord mesenchymal stem cells in the prior art

Method used

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  • Human umbilical cord mesenchymal stem cell serum-free medium and preparation method and method for obtaining human umbilical cord mesenchymal stem cell serum-free culture medium
  • Human umbilical cord mesenchymal stem cell serum-free medium and preparation method and method for obtaining human umbilical cord mesenchymal stem cell serum-free culture medium
  • Human umbilical cord mesenchymal stem cell serum-free medium and preparation method and method for obtaining human umbilical cord mesenchymal stem cell serum-free culture medium

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preparation example Construction

[0054]Method for preparing a human umbilical-tape male mixed stem cell-free medium, including the following specific steps:

[0055]Step 1: Preparation of serum-free cultured concentrate

[0056]Human umbilical-binding mesenchymal stem cells were cultured in a serum-free medium. When the cell fusion was 80-90%, the human umbilical cord serum-mixed stem cell supernatant was collected; filtration, ultrafiltration concentration, and the total protein concentration was determined. 10 mg / ml, resulting in serum-free culture.

[0057]Step 1 People Umbilical Belt Menque Stem Cells in 5 × 103~ 8 × 103 / cm2Turn in the commercially available serum-free medium.

[0058]Step 1 was filtered twice, one of which was used in a 0.8 μm microfiltration membrane, and 0.45 μm microfiltration membrane was used.

[0059]The ultrafiltration concentration in step 1 is ultrafiltrated with a superfiltration system with a cross-filtration system of 3 kD to concentrate.

[0060]The ultrafiltration in step 1 is concentrated to 1...

Embodiment 1

[0079]A human umbilical cord serum-free stem cell serum-free medium preparation, including the following steps:

[0080]Step 1: Preparation of serum-free cultured concentrate

[0081]Human umbilical cord mesenchymal stem cells with 5 × 103 / cm2The culture supernatant was collected in the serum-free medium, and the culture supernatant was collected by 80%, and the supernatant having a volume of 5 L was obtained, and 5 L of the supernatant was filtered through a flat filter, and the filtration filter aperture is respectively. 0.8 μm, 0.45 μm; ultrafiltration concentration with a cellulose ultrafiltration membrane with a 3 kDa of 3 kDa, concentrated to 1 / 15 of the liquid volume of the liquid volume to stop concentration, 305 ml of liquid after concentration, using a BCA protein reagent The cassette was detected by 11.2 mg / ml of the concentrated liquid, and 37 ml of injection was supplemented with water after concentrated liquid, and finally formed a concentrated serum-free culture supernat...

Embodiment 2

[0089]Preparation of a human umbilical cord serum-free cell-free medium, which is as follows:

[0090]Step 1: Preparation of serum-free cultured concentrate

[0091]Human umbilical-tape mesenchymal stem cells at 6 × 103 / cm2The culture supernatant was collected in the serum-free medium, and the culture supernatant was collected at a cell fusion, and the volume of 30 L was collected, and 30 L of the supernatant was sequentially filtered through the flat filter, and the filtered filtration film aperture is respectively. It is 0.8 μm, 0.45 μm; ultrafiltration concentration with a cellulose ultrafiltration membrane having a 3 kDa of 3 kDa, a monoclonate film cover area of ​​0.5 m.2A total of 6 membrane packs, which are concentrated by tangential flow processes, transmitting end liquid discarding treatment, concentration to 1 / 18 of the original microfiltration volume, and the liquid in the ultrafiltration system is vacuum, and the supernatant is concentrated. Liquid 1678 mL; then detecting the...

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Abstract

The human umbilical cord mesenchymal stem cell serum-free medium, preparation method and human umbilical cord mesenchymal stem cell serum-free culture solution disclosed in the present invention include a basal medium and a serum-free culture supernatant concentrate, and also include the following components And its concentration: vitamin composition 5-100ng / ml, grape polyphenols 50-200μg / ml, recombinant human epidermal growth factor 1-50ng / ml, recombinant human basic fibroblast growth factor 1-50ng / ml, recombinant Platelet-derived growth factor-C 1-10ng / ml, recombinant human insulin-like growth factor-1 1-10ng / ml, β-mercaptoethanol 50-200μM, human albumin 1-100μg / ml, glutathione 1- 100μg / ml, sodium bicarbonate 1.2‑3.7g / L. Use human umbilical cord mesenchymal stem cell serum-free medium instead of traditional medium containing animal serum to cultivate human umbilical cord mesenchymal stem cells, avoiding unsafe factors such as stem cells being contaminated by pathogens or animal-derived components and foreign proteins causing human immune reactions .

Description

Technical field[0001]The present invention belongs to the field of stem cell culture, involving human umbilical-tape-serum-free stem cell-free medium, human umbilical-tape serum-free cell-free medium preparation method, and culture methods of human umbilical-cord serum-free stem cell serum culture solution.Background technique[0002]Mequted stem cells are an important member of a stem cell family, from the early mid-staple embryo and octodiliary. The mesenchymal stem cells were earlier in the bone marrow, which was increasingly concerned about its characteristics of multiple differentiation potential, hematopoietic support, promoting stem cell implantation, immunomodulation and self-replication. Bone marrow mesenchymal stem cells have been widely used in clinical practice. At present, there will be a greater application potential in mesenchymal stem cells in the umbilical cord source.[0003]HUC-MSCS (Human Umbilical Cord Mesenchymal Stem Cells, Human Umbilical Covered Stem Cell), is u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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