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

An in vitro expansion and stem cell technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the adverse reactions of patients, the roller bottle operation is not very convenient, and is not suitable for large-scale expansion, etc. problems, achieve good proliferation potential, simple operation, and low cost

Active Publication Date: 2012-11-21
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, which contains complement components, and the remaining fetal bovine serum may cause adverse reactions in patients
However, roller bottles that are too large are not very convenient to handle, and microbial safety is a key aspect of cell culture

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0030] With the informed consent of the parturient, the umbilical cord or placenta of healthy fetuses delivered by cesarean section was collected. Before collecting the umbilical cord or placenta, women need to be tested for HIV antibody, hepatitis B virus antibody, hepatitis C virus antibody, Treponema pallidum antibody, alanine aminotransferase, and mycoplasma, etc., and they can only be collected after all qualified to ensure safety.

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

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

[0033] 3. Cut the umbilical cord or placenta into 1mm 3 Put the large and small tissue block into a 200mL blue cap reagent bottle, add 30mL of type II collagenase with a mass / volume ra...

Embodiment 2

[0037] MesenPRO RS TM Optimization of Medium culture system

[0038] MesenPRO RS as the best culture system for in vitro culture of human umbilical cord placental mesenchymal stem cells TM Medium, considering the following factors, we supplement 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 The necessary nutrients. Its functions after cell seeding are roughly as follows. ①Protect cells from damage; ②Promote cell adhesion; ③Promote cell proliferation: A variety of growth factors, especially platelet growth-promoting factors, can promote cell division and are the main factors that promote cell proliferation. In addition, insulin in serum can promote the uptake of glucose and amino acids by cells, which is related to cell division; insulin-like growth factor, which can bind to the insulin receptor expressed by cells, and thus has the same effect as insulin; growth-promoting horm...

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 cell density of the mesenchymal stem cells cultured in the Medium culture system reaches 80%-90%, trypsin digestion to prepare a single cell suspension;

[0047] 2. Inoculate the suspension on a surface area of ​​850cm 2 In the roller bottle, the total number of cells is 1×10 6 (1400 cells / cm 2 ), join 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 evenly distribute the cells in the roller bottle, and then place the roller bottle in INCUDRIVE D-I CO 2 In the incubator rotating system, in order to stabilize the optimal temperature, the roller bottle should be located behind the incubator as much as possible and driven at 4 positions with a wide axial distance;

[0048] 3. At 37℃, rotate for 2 hours a...

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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 high-efficiency 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, are widely present in a variety of tissues throughout the body. They can be cultured and expanded in large quantities in vitro, and are derived from the three germ layers of the inner, middle and outer germ layers under specific conditions. 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 system diseases in the 21st century. It is used in hematopoietic cell transplantation, organ transplantation, bone and cartilage tissue repair, myocardial infarction and liver. The injury has potential application value, and certa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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