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Method for large scale preparation of liver stem cells, and uses of liver stem cells

A technology for large-scale preparation of liver stem cells, applied in the field of large-scale preparation and application of liver stem cells, can solve problems such as difficult to meet clinical needs, achieve the effect of maintaining pluripotency, improving efficiency, reducing opportunities and workload

Active Publication Date: 2015-05-20
GZ CEDICINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This preparation method can effectively isolate HSCs from fetal liver tissue, but its disadvantage is that it can only isolate up to 10 HSCs from fetal liver tissue at a time. 8 of hepatocytes, the number of HSCs after culture is only 10 7
However, this method uses the pig liver stem cells extracted by the shredding method and enzyme digestion method, and the cell number is only 4.8-6.4×10 7 Pork liver per leaf (about 500g), difficult to meet clinical needs

Method used

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  • Method for large scale preparation of liver stem cells, and uses of liver stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Conventional methods for separation and culture of liver stem cells

[0044] Objective: To isolate hepatic stem cells from rat embryonic liver tissue

[0045] 1. Materials and methods

[0046] A) Material

[0047] 1. Reagents and solvents

[0048] 1) Sterilized water for injection, production unit: self-made

[0049] 2) Liver stem cell culture medium (DMEM / F12 medium, containing B27 (1:50), bFGF (20ng / mL), EGF (20ng / mL), LIF (10ng / mL) and heparin (5μg / mL). );

[0050] 3) Physiological saline and Hank’s buffer;

[0051] 4) Alcohol;

[0052] 5) Collagenase;

[0053] 6) Pancreatin;

[0054] 7) DMEM / F12 medium;

[0055] 8) Surgical instruments

[0056] 2. Experimental animals and breeding conditions

[0057] 2.1 Experimental animals

[0058] 1) Grade and strain: SPF SD rat

[0059] 2) Animal management: animals are kept and managed by personnel who have obtained laboratory animal management qualifications

[0060] 3) Purchase time: November 13, 2010.

[0061] 4) Age at purchase: 6-8 w...

Embodiment 2

[0103] Example 2: The method for preparing stem cells from the large-scale liver of the present invention

[0104] Objective: To adopt a large-scale preparation method to isolate and purify liver stem cells from young animal liver tissues

[0105] 1. Materials and methods

[0106] A) Material

[0107] 1. Newborn suckling pigs are killed around one month, and their livers are taken and weighed;

[0108] 2. Liver stem cell culture medium (DMEM / F12 medium, containing B27 (1:50), bFGF (20ng / mL), EGF (20ng / mL), LIF (10ng / mL) and heparin (5μg / mL). );

[0109] 3. Physiological saline and Hank’s buffer;

[0110] 4. Alcohol;

[0111] 5. Collagenase;

[0112] 6. Pancreatin;

[0113] 7. DMEM / F12 medium;

[0114] 8. Electric homogenization pump, centrifuge and surgical instruments

[0115] Two) Method

[0116] 1. Separate the liver tissue from suckling pigs (100 days after birth) under aseptic conditions, weigh them, and add DMEM medium or DMEM medium containing 0.01% collagenase according to 1:2.

[0117]...

Embodiment 3

[0129] Example 3: Comparison of the effects of two different methods for extracting suckling pig liver stem cells

[0130] The purpose of this example is to explore the difference between the manual method and the electromechanical method in the efficiency of hepatic stem cell preparation.

[0131] 1. Material

[0132] 1. Newborn suckling pigs around March;

[0133] 2. Liver stem cell culture medium (DMEM / F12 medium, containing B27 (1:50), bFGF (20ng / mL), EGF (20ng / mL), LIF (10ng / mL) and heparin (5μg / mL). );

[0134] 3. Physiological saline and Hank’s buffer;

[0135] 4. Alcohol;

[0136] 5. Collagenase;

[0137] 6. Pancreatin;

[0138] 7. DMEM / F12 medium;

[0139] 8. Electric homogenizer, centrifuge and surgical instruments

[0140] 2. Methods and steps

[0141] 3.1 Isolation of liver stem cells

[0142] After the suckling pig is sacrificed under aseptic conditions, the liver tissue is separated, weighed, and DMEM medium is added according to 1:2, and approximately 1 cm3 squares are initially...

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Abstract

The invention relates to a method for large scale preparation of liver stem cells, and uses of the liver stem cells in the fields of cell transplantation, stem cell culture, amplification, directional differentiation and tissue formation. The preparation method of the liver stem cells includes the following steps: extracting the liver stem cells from animal or human liver tissues through using a mechanical method, enzymatic method and mechanical-enzymatic method and density gradient centrifuge method combined technology; and carrying out in-vitro large scale culture, amplifying, carrying out liver stem cell identification, and freezing the liver stem cells for later use. The liver stem cell preparation efficiency of the method disclosed in the invention is about 100-10000 times higher than that of conventional methods, and the method can be used in tissue engineering and the production of liver stem cell preparations or stem cell drugs in order to provide a new method for the treatment of human diseases.

Description

Technical field [0001] The present invention relates to a method for preparing hepatic stem cells (HSCs), in particular to a method for separating, culturing, purifying stem cells, and expanding and culturing HSCs on a large scale, and its application in cell transplantation, stem cell culture, tissue engineering, regeneration Medical and pharmaceutical applications. Background technique [0002] Stem cells are a group of cells that exist in the normal human body and have a high degree of self-replication and differentiation. In recent years, with the deepening of stem cell research, it has been discovered that stem cells can be used to regenerate various tissues and organs, such as liver, pancreas, nerve tissue, bones, and tendons. [0003] Due to the source and isolation reasons, the stem cells currently used in clinical practice mainly include adult bone marrow mesenchymal stem cells (BMSCs) and hematopoietic stem cells (HSCs), human peripheral blood stem cells, and human embry...

Claims

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Application Information

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IPC IPC(8): C12N5/0735C12N5/074A61K35/407A61P1/16
Inventor 邹清雁丁先风刘海霞兰丹唐时幸刘志成杜少茵
Owner GZ CEDICINE BIOTECH CO LTD
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