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Cell culture method for promoting rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using three-dimensional cell culture system

A hematopoietic stem cell, three-dimensional cell technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of cell activity limitation, cells cannot rebuild hematopoietic for a long time, and achieve the effect of promoting proliferation

Inactive Publication Date: 2017-06-06
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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AI Technical Summary

Problems solved by technology

③ Limitation of nutrient intake and changes in cell metabolism lead to limitation of cell activity, etc.
Therefore, cells expanded in such culture systems are unable to reconstitute hematopoiesis in the long term

Method used

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  • Cell culture method for promoting rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using three-dimensional cell culture system
  • Cell culture method for promoting rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using three-dimensional cell culture system
  • Cell culture method for promoting rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using three-dimensional cell culture system

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Embodiment Construction

[0029] The technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0030] A cell culture method for promoting rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using a three-dimensional cell culture system, comprising the following steps:

[0031] 1. All materials, reagents, etc. in the examples can be obtained from commercial sources unless otherwise specified.

[0032] 2. Extraction, culture, expansion and identification of stromal cells umbilical cord mesenchymal stem cells (the umbilical cords of newborns were obtained with the knowledge of the puerpera, and the puerpera was in good health.)

[0033] 1) Take out the umbilical cord, soak it quickly in a small beaker pre-filled with 75% alcohol for 30 seconds to 1 minute, take it out and place it in a 150mm plate, wash it twice with PBS, and wash away the alcohol. ...

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Abstract

The present invention provides a cell culture method for promoting the rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using a three-dimensional cell culture system. The cell culture method comprises: 1) extracting and preparing cells adopted as matrix cells during co-culture, and identifying with flow cytometry; 2) treating a micro-carrier or other carriers and other materials used during a three-dimensional culture process so as to be spare; 3) inoculating the matrix cells onto the micro-carrier, and culturing in a three-dimensional culture system; 4) after the matrix cells proliferate to cover more than 80% of the micro-carrier surface, treating the cells to damage mitosis; 5) preparing a homogenized hematopoietic stem cell culture medium; 6) separating PBMC from umbilical cord blood, sorting CD34+ cells after obtaining the PBMC, and identifying with flow cytometry; and 7) culturing the sorted CD34+ cells and the treated matrix cells in the three-dimensional culture system by adding the homogenized hematopoietic stem cell culture medium. According to the present invention, the culture system for promoting the rapid and stable proliferation of the hematopoietic stem cells within a certain time by using the three-dimensional cell culture system and by combining the matrix cells and a variety of cytokines is established, and the theoretical basis and the technical platform are provided for the clinical obtaining of the high-quality hematopoietic stem cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a cell culture method for promoting rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using a three-dimensional cell culture system Background technique [0002] Hematopoietic stem cell transplantation is an effective method for the clinical treatment of severe trauma, malignant blood diseases, severe aplastic anemia, immunodeficiency syndrome, thalassemia and other diseases. Since the end of the 1980s, the world's first umbilical cord blood hematopoietic stem cell transplantation successfully treated thalassemia patients and the gradual establishment and improvement of cord blood banks around the world. It is a good supplement to bone marrow transplantation and has been increasingly used in the treatment of various blood system diseases. At present, more than tens of thousands of children and adults have received UCBT worldwide. However, in clinical ...

Claims

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Application Information

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IPC IPC(8): C12N5/0789C12N11/02
Inventor 郭希民刘东华钱智勇周勇毛宁
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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