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Method for in vitro preparation of hepatocyte induced by umbilical cord blood hematopoietic stem cell

A technology of hematopoietic stem cells and hepatocyte growth factors, which is applied in the field of in vitro induction of hepatocytes, can solve the problems of stem cells not being suitable for growth and insufficient amount of hepatocyte transplantation cells, and achieve obvious effects and high proportions

Inactive Publication Date: 2006-10-11
EAST CHINA UNIV OF SCI & TECH
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Problems solved by technology

[0008] Cultivating and proliferating hepatocytes in vitro is an idea to solve the problem of insufficient amount of hepatocyte transplantation cells, but stem cells isolated from donors are not suitable for growth in vitro

Method used

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  • Method for in vitro preparation of hepatocyte induced by umbilical cord blood hematopoietic stem cell
  • Method for in vitro preparation of hepatocyte induced by umbilical cord blood hematopoietic stem cell
  • Method for in vitro preparation of hepatocyte induced by umbilical cord blood hematopoietic stem cell

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Embodiment 1

[0050] After umbilical cord blood was subjected to density gradient centrifugation with lymphocyte separation medium (Ficoll), mononuclear cells were collected and washed twice with IMDM medium. The isolated monocytes were divided into 2×10 6 Inoculate cells / mL into IMDM medium containing 20% ​​fetal bovine serum, place at 37°C, 5% CO 2 , in an incubator with 100% humidity, the medium was changed every 7 days, and the cells were collected, and the culture was terminated after 3 weeks.

[0051] Extract the total RNA of the cultured cells, and use RT-PCR technology to detect the expression of human serum albumin gene in the cultured cells. The expression of the specific gene of serum albumin can be detected in the cells after 1 week of in vitro induction (see the arrow indicated ), and the expression level of the gene increases with the culture time, see figure 1 . In the figure, 1: Marker; 2: Initial cells; 3: Cells after 1 week of induction; 4: Cells after 2 weeks of induct...

Embodiment 2

[0054] After the umbilical cord blood was subjected to density gradient centrifugation with lymphocyte separation medium (Ficoll), mononuclear cells were collected and washed twice with IMDM medium, and the cells were suspended in IMDM medium at a concentration of 1×10 8 cells / 300uL, add 50uL magnetic bead-coupled mouse anti-human CD34 antibody, react at 4°C for 30 minutes, wash with PBS containing 2mM EDTA, 0.5% human serum albumin, and remove non-CD34 on the separation column in a magnetic field + After the cells were washed, CD34 cells were separated from the magnetic field with PBS containing 2mM EDTA and 0.5% human serum albumin. + Cells were eluted and resuspended in IMDM medium.

[0055] The isolated CD34 + cells at 1 x 10 5 Inoculate cells / mL into IMDM medium containing 20% ​​fetal bovine serum, place at 37°C, 5% CO 2 , 100% humidity incubator. In the first seven days of culture, only the expanded cytokines human stem cell factor (SCF), human interleukin 3 (IL-3) a...

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Abstract

This invention discloses a method for producing liver cells by inducing haemopoietic stem cells in vitro, comprising: inoculating the haemopoietic stem cells or CD34+ cells into the culture media containing embryonic bovine serum, human stem cell factor, human interleukins 3(IL-3), interleukins 6(IL-6), human liver cell growth factor, human epidermal growth factor(EGF), human acdic fibroblast growth factor, human basic fibroblast growth factor(bFGF), human leukemia inhibitory factor(LIF) and oncostatin M(OSM), and inducing the producing of liver cells in vitro. By applying the method of this invention to induce liver cells from the haemopoietic stem cells, it can acquire a high proportion of liver cells in the culture with good effects.

Description

technical field [0001] The invention relates to a method for inducing the generation of hepatocytes in vitro, in particular to a method for inducing the generation of hepatocytes from umbilical cord blood hematopoietic stem cells in vitro. Background technique [0002] As we all know, a large number of people die every year due to various liver diseases, and the number of patients with liver injury, cirrhosis and liver failure is even greater. [0003] The liver is one of the important organs of the human body. Its functions on the human body are mainly reflected in three aspects: (1) Secretion of bile: liver cells continuously generate bile acids and secrete bile to help the digestion and absorption of fat in the small intestine. (2) Metabolic function: This mainly includes glucose metabolism, protein metabolism, fat metabolism, vitamin metabolism and hormone metabolism. (3) Detoxification function: the liver is the main detoxification organ in the human body, which can pr...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/0789
Inventor 蔡海波谭文松金颖陈祎祺
Owner EAST CHINA UNIV OF SCI & TECH
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