Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro

A technology of mesenchymal stem cells and in vitro expansion, applied in the field of in vitro expansion of human umbilical cord placental mesenchymal stem cells, can solve the problems of inconvenient roller bottle operation, unsuitable for large-scale expansion, adverse reactions of patients, etc., and achieve Good proliferation potential, simple operation, low cost effect

Active Publication Date: 2010-09-15
UNION STEMCELL & GENE ENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, DMEM/F12 medium can be used when harvesting umbilical cord mesenchymal stem cells, but it is not suitable for its large-scale expansion
(3) The human MSC culture system that is widely used at present mostly uses fetal bovine serum, w

Method used

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  • Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro
  • Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro
  • Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro

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Experimental program
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Embodiment 1

[0029] Preparation of human umbilical cord placental mesenchymal stem cells:

[0030] With the informed consent of the puerpera, the umbilical cord or placenta of healthy fetuses delivered by cesarean section at term were collected. Before the umbilical cord or placenta is collected, the puerpera needs to be tested for HIV antibody, hepatitis B virus antibody, hepatitis C virus antibody, treponemal pallidum antibody, alanine aminotransferase, mycoplasma, etc., all of which are qualified to ensure safety before collection.

[0031] 1. After the umbilical cord or placenta is removed from the operating table, immerse in 0.9% saline containing antibiotics and store at 4°C;

[0032] 2. Take out the umbilical cord or placenta in the clean bench, and rinse the residual blood on the surface with D-PBS;

[0033] 3. Cut the umbilical cord or placenta into 1mm 3 Put the tissue pieces of different sizes into a 200mL blue cap reagent bottle, add 30mL type II collagenase with a mass / volum...

Embodiment 2

[0037] MesenPRO RS TM Optimization of Medium culture system

[0038] As the best culture system for human umbilical cord placental mesenchymal stem cells cultured in vitro, MesenPRO RS TM Medium, considering the following factors, we supplemented the original medium with a final concentration of 10%-15% FBS, 2mM L-glutamine and gentamicin sulfate: (1) Fetal bovine serum is rich in cell growth necessary nutrients. Its role after cell inoculation generally has the following points. ①Protect cells from damage; ②Promote cell adhesion; ③Promote cell proliferation: A variety of growth factors, especially platelet growth-promoting factor, can promote cell division and are the main factors that promote cell proliferation. In addition, insulin in the serum can promote the uptake of glucose and amino acids by cells, which is related to promoting cell division; insulin-like growth factors can bind to insulin receptors expressed by cells, thus having the same effect as insulin; prol...

Embodiment 3

[0045] Large scale expansion of human umbilical cord placental mesenchymal stem cells under optimal roller bottle culture conditions

[0046] 1. Use MesenPRO RS TM When the mesenchymal stem cells cultured in the Medium culture system reach a cell density of 80%-90%, they are digested with trypsin to prepare a single cell suspension;

[0047] 2. Inoculate the suspension on a surface area of ​​850cm 2 In a roller bottle, the total number of cells is 1 x 10 6 (1400 cells / cm 2 ), joined MesenPRO RS TM Medium culture medium to 100ml, shake the suspension vertically and then slowly rotate the roller bottle horizontally for 1 week, the purpose is to make the cells evenly distributed in the roller bottle, and then place the roller bottle in the INCUDRIVE D-I CO 2 The incubator rotation system, in order to stabilize the optimum temperature, the roller bottle is located at the rear of the incubator as far as possible, and is driven at 4 positions with a wide axial distance;

[00...

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Abstract

The invention discloses a method for amplifying mesenchymal stem cells of a human umbilical cord and a human placenta in vitro. The method comprises the following steps of: using human umbilical cord and placenta as a cell source; digesting tissues by using II type collagenase, centrifuging the product the digestion; removing supernate in the product of the centrifugation; washing the remaining product of the centrifugation by using D-Hank's liquid for three times to obtain single cell suspension; inoculating the single cell suspension in a culture plate or a culture flask; fully replacing the liquid when cell (P0) are adhered after 24 to 72 hours; when 80 to 90 percent of cells are mixed, performing subculture once (P1); inoculating the cells in a roller bottle according to a minimum density of 1,400 cells per cubic centimeter; comparing the growing status, cell amplification time and colony-forming capability of the mesenchymal stem cells between different culture systems to obtain an efficient and stable amplification culture system; and amplifying the mesenchymal stem cells in a large scale in the roller bottle by using the optimal amplification system.

Description

technical field [0001] The invention relates to a method for efficiently and large-scale expansion of mesenchymal stem cells, in particular to a method for in vitro expansion of human umbilical cord placental mesenchymal stem cells. technical background [0002] Mesenchymal stem cells (Mysenchymal Stem Cell, MSC), as a kind of adult stem cells, widely exist in various tissues throughout the body. differentiation. MSC is another research hotspot after embryonic stem cells and hematopoietic stem cells. It has become a practical stem cell for the treatment of various systemic diseases in the 21st century. It has potential application value in injury, and some treatment measures have entered the stage of clinical trials. In recent years, it has been gradually applied to the treatment of cardiovascular and cerebrovascular diseases, liver diseases, bone and muscle degenerative diseases, brain and spinal cord nerve injuries, senile dementia, systemic lupus erythematosus and scler...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 洪敬欣韩俊领钱其军李林芳刘剑
Owner UNION STEMCELL & GENE ENG
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