Stem cell separating liquid and its separating method

A technology for separating liquid and stem cells, applied in the field of stem cell separation, can solve the problem of inability to separate liver stem cells, and achieve the effect of improving the separation effect

Active Publication Date: 2007-12-19
GZ CEDICINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] The above methods are mostly used to isolate bone marrow mesenchymal stem cells, but the current experiments show that hepatic stem cells cannot be effectively isolated from human and mouse fetal liver by the above methods

Method used

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  • Stem cell separating liquid and its separating method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment one: the preparation of stem cell separation liquid

[0054] 1. Materials

[0055] 1. Diatrizoate meglumine (Sigma), hydroxymethylcellulose (Sigma), cesium chloride (Sigma), polysucrose (Ficoll400, phamacia)

[0056] 2. Potassium dihydrogen phosphate, sodium hydroxide (Guangzhou Chemical Reagent Factory)

[0057] 3. Hydrometer (1.000-1.100, 1.100-1.200)

[0058] 2. Method

[0059] Weigh 3 g of diatrizoate, 2 g of hydroxymethylcellulose, 0.2 g of cesium chloride, and 3 g of polysucrose. Add the above components into a 100ml graduated cylinder in turn, add 0.01mol / L phosphate buffer to 100ml, and obtain the concentrated solution of the stem cell separation solution. Measure its specific gravity with a hydrometer to be 1.123g / ml.

[0060] When in use, dilute the concentrated solution of the above-mentioned stem cell separation solution with 0.01mol / L phosphate buffer solution to the required specific gravity, which is the working solution.

[0061] The abo...

Embodiment 2

[0063] Example 2: Isolation of Rat Hepatic Stem Cells

[0064] This example uses the stem cell separation liquid obtained in Example 1.

[0065] 1. Materials

[0066] 1. BALB / C mice at 12-14 days pregnant.

[0067] 2. Imported premium fetal bovine serum (fetal bovine serum, FBS, Gibco), imported FBS and domestic newborn calf serum (newborn calf serum, NCS; provided by Hangzhou Sijiqing Institute of Bioengineering Materials). DM EM / F12 (sigma), non-essential amino acids (×100, Gibco), human basic fibroblast growth factor (basic fibroblast growth factor, bFGF, 10ug, Gibco), β-mercaptoethanol (β-Mercaptoethanol, β- ME; Nacalai Tesoue INC Kyoto, Japan), bovine serum albumin (BSA), conalbumin (Conalbumin, Sigma). Carbon tetrachloride (carbontetrachloride, CCl4), D-galactosamine (D-galactosamine, D-Galn).

[0068] 3. Liver stem cell medium: 5% FBS (domestic or imported), 10% domestic NCS, 0.14mM β-ME, 40ng.ml-1 conalbumin, 10ng.ml-1 human bFGF, 1% non-essential Amino acids in D...

Embodiment 3

[0091] Example Three: Isolation of Porcine Bone Marrow Stem Cells

[0092] This example uses the stem cell separation liquid obtained in Example 1.

[0093] 1. Materials

[0094] 1. Equipment: ultra-clean bench, horizontal centrifuge, inverted microscope and ordinary microscope. Centrifuge tubes and balances, bone marrow puncture needles and blood collection bags. Flow cytometer, drying box, gloves and surgical gown.

[0095] 2. Reagents: Meglumine Diatrizoate (Sigma Company), Hydroxymethyl Cellulose (Sigma Company), Cesium Chloride (Sigma Company), Polysucrose (Ficoll-400, Phamacia Company), Potassium Dihydrogen Phosphate, Sodium Hydroxide (both Guangzhou Chemical Reagent Factory), Rhodamine 123 (Sigma Company).

[0096] 3. BD Procount progenitor Cell Enumeration Kit (BD company, stem cell enumeration kit, batch number: NO.340498).

[0097]4. Lymphocyte Separation Solution (Shanghai Reagent No. 2 Factory), the specific gravity is 1.076g / ml.

[0098] 5. Cell culture medi...

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Abstract

The present invention relates to stem cell separating and purifying technology, and is especially one kind of stem cell separating liquid and its usage in separating stem cell. The stem cell separating liquid consists of meglucamine diatrizoate 0-10 weight portions, methylol cellulose 0-10 weight portions, cesium chloride 0-1.0 weight portions, and sucrose polymer 0-10 weight portions. It is prepared through mixing the said ingredients and adding 0.01 mol / l concentration phosphate buffering liquid. When it is applied, it is diluted with 0.01 mol / l concentration phosphate buffering liquid to density of 1.080-1.120 g / ml. The present invention has high stem cell separating effect.

Description

technical field [0001] The invention relates to the field of stem cell separation and purification, in particular to a stem cell separation liquid and a method for stem cell separation. Background technique [0002] In 1998, Thomson and Gearhart laboratories in the United States reported the successful establishment of human embryonic stem cells (embryonic stem cells, ES cells) and embryonic germ cells (Embryonic germ cells, EG cells), which inspired and aroused people's interest in ES cells and their orientation. A passion for differentiated research. This scientific and technological achievement will become the latest research field with the most development and application prospects. In recent years, research on stem cells has made people see new hope. Stem cells are cell populations existing in the normal human body, with a high degree of self-replication and differentiation capabilities. With the deepening of research, it has been discovered that stem cells can be us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/08C12N5/071C12N5/074C12N5/0789
Inventor 邹清雁孔祥平郑业华杨联萍刘惠平
Owner GZ CEDICINE BIOTECH CO LTD
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