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Preparation method of nourishing layer for culturing hemopoietic stem cells

A technology for hematopoietic stem cells and cell culture, which is applied in the field of preparation of trophoblasts for hematopoietic stem cell culture, can solve problems affecting hematopoietic stem cell expansion, apoptosis, anoikis and the like, achieve good application prospects and improve expansion efficiency , the effect of alleviating HSPC deficiency

Pending Publication Date: 2017-05-31
SICHUAN NEO LIFE STEM CELL BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when MSCs are used as trophoblasts to cultivate hematopoietic stem cells, MSCs will proliferate in large numbers, and after a period of culture, they will fall off and apoptotic in large numbers, producing a large number of lysosomes and other substances, and changing the pH value of the medium, which has a great impact on Expansion of hematopoietic stem cells, leading to apoptosis
It has been reported that treating MSC cells with mitomycin can prevent their proliferation, but the residual mitomycin will have a very adverse effect on the hematopoietic stem and progenitor cells to be expanded

Method used

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  • Preparation method of nourishing layer for culturing hemopoietic stem cells
  • Preparation method of nourishing layer for culturing hemopoietic stem cells
  • Preparation method of nourishing layer for culturing hemopoietic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of trophoblast for hematopoietic stem cells

[0036] 1. Mesenchymal stem cells were prepared according to the following method

[0037] (1) Take a fresh umbilical cord, place it in a 90mm glass dish, cut off a section of umbilical cord about 10cm long, pour it into normal saline, rinse the umbilical cord, and wash away the blood stains on the surface.

[0038] (2) Use ophthalmic scissors to cut the umbilical cord to a small section of about 1.5 cm and cut it open. Rinse to remove blood stains in the blood vessel, and transfer the umbilical cord to another clean plate.

[0039] (3) Pour in normal saline and rinse the umbilical cord again; repeat the rinse 2-3 times to remove blood stains.

[0040] (4) Prepare 4-6 plastic petri dishes with a diameter of 100mm and mark them. Put a 1.5cm long umbilical cord in each dish (discard the excess umbilical cord tissue) and add 2ml of culture medium (DMEM+F12+10% fetal bovine serum ). Use ophthalmic scissor...

Embodiment 2

[0063] Example 2 Preparation of trophoblast for hematopoietic stem cells

[0064] 1. Mesenchymal stem cells are commercially available products.

[0065] 2. Preparation method of umbilical cord MSC cytotrophoblast

[0066] (1) Prepare mobile concentration of MSC cell suspension:

[0067] With embodiment 1.

[0068] (2) Take a 6-well plate, add 0.8ml rat tail collagen to each well, and place it in a 37°C incubator for use.

[0069] (3) Take out the pretreated 6-well plate from the incubator and wash it twice with sterile PBS.

[0070] (4) The MSC cell suspension was mixed according to 1.5x10 5 Cells / well were seeded into a 6-well plate and shaken gently horizontally to mix.

[0071] (5) Place the 6-well plate in a 37°C incubator, and when the cell confluence reaches 80%, it can be used as a feeder layer to inoculate CD34+ hematopoietic stem cells.

Embodiment 3

[0072] Example 3 Preparation of trophoblast for hematopoietic stem cells

[0073] 1. Mesenchymal stem cells: the preparation method is the same as in Example 1.

[0074] 2. Preparation method of umbilical cord MSC cytotrophoblast

[0075] (1) Prepare mobile concentration of MSC cell suspension:

[0076] With embodiment 1.

[0077] (2) Take a 6-well plate, add 1.2ml rat tail collagen to each well, and place it in a 37°C incubator for use.

[0078] (3) Take out the pretreated 6-well plate from the incubator and wash it twice with sterile PBS.

[0079] (4) The MSC cell suspension was mixed according to 1.5x10 5 Cells / well were seeded into a 6-well plate and shaken gently horizontally to mix.

[0080] (5) Place the 6-well plate in a 37°C incubator, and when the cell confluence reaches 90%, it can be used as a feeder layer to inoculate CD34+ hematopoietic stem cells.

[0081] The beneficial effect of the present invention is illustrated in the mode of experimental example as ...

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Abstract

The invention discloses a novel method for preparing a nourishing layer for culturing hemopoietic stem cells. The method comprises the following steps: (1) taking a cell culture plate and coating collagen to obtain the collagen coated cell culture plate; (2) taking the hemopoietic stem cells and inoculating into the cell culture plate coated with the collagen in the step (1); culturing until the cell fusion degree is 70 percent to 90 percent and obtaining the product. The nourishing layer prepared by the method can provide powerful supporting effect on in-vitro amplification of hemopoietic stem progenitor cells (HSPC); the amplification efficiency of the HSPC is remarkably improved and the problem of the disadvantage of the HSPC can be extremely alleviated; researches and application of HSPC medical values are accelerated.

Description

technical field [0001] The invention relates to a method for preparing a trophoblast for hematopoietic stem cells. Background technique [0002] Umbilical cord blood is considered an important resource in addition to bone marrow transplantation for the treatment of hematological diseases, especially hematological tumors. Since the first successful clinical transplantation of umbilical cord blood in 1988, its application has made great progress along with the development of cell therapy research. Although the use of umbilical cord blood in hematopoietic reconstruction is still at a relatively low level compared to bone marrow and peripheral blood, more than 25,000 successful cord blood transplants to date still raise awareness of this once-discarded valuable resource. Full of confidence. As a source of hematopoietic stem cells (Hematopoietic Stem Cell, HSC), umbilical cord blood has two natural advantages over bone marrow and peripheral blood. First, the risk of graft-versu...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0037C12N5/0647C12N2500/90C12N2502/1352C12N2533/54
Inventor 罗茂文陈强杨本艳姿段海峰张博
Owner SICHUAN NEO LIFE STEM CELL BIOTECH
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