CN100469870C - Method for separating and purifying nerve stem of human amnion tissue - Google Patents
Method for separating and purifying nerve stem of human amnion tissue Download PDFInfo
- Publication number
- CN100469870C CN100469870C CNB2006100900265A CN200610090026A CN100469870C CN 100469870 C CN100469870 C CN 100469870C CN B2006100900265 A CNB2006100900265 A CN B2006100900265A CN 200610090026 A CN200610090026 A CN 200610090026A CN 100469870 C CN100469870 C CN 100469870C
- Authority
- CN
- China
- Prior art keywords
- amnion tissue
- human amnion
- cell
- separating
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
This invention relates to a method for separating and purifying neural stem cells of human amnion tissue. The method comprises: (1) washing human amnion tissue; (2) separating epithelial cells from human amnion tissue; (3) separating mesenchymal cells from human amnion tissue; (4) separating neural stem cells from mesenchymal cells. The method separates CD133 positive neural stem cells from mesenchymal cells of human amnion tissue by using immunomagenetic method, and cultures neural stem cells by using neural stem cell culture medium. The method can largely reduce the cost for separation and purification of neural stem cells of human amnion tissue, and increase the survival rate of neural stem cells.
Description
Technical field
The present invention relates to a kind of method of separating and purifying nerve stem, particularly related to a kind of from human amnion tissue, the separation and the method for purifying nerve stem cell.
Background technology
At present, utilize Culture of neural stem cells liquid and cell clone technology, the method of separation and purification of nerve thousand cells is mature on the whole from embryo or tire brain, owing to have only stem cell to have the multiple fission activity in the cerebral tissue, so method can effectively be applied to the separation and purification of brain source property neural stem cell, and also there is not at present the associate method of separation and purification of nerve stem cell in the human amnion tissue of documents and materials report.In addition, human amnion tissue is interior except that having the active neural stem cell of multiple fission, also have epithelial stem cell and mescenchymal stem cell, and neural stem cell quantity is far fewer than the tire brain, utilize the neural stem cell in the above-mentioned known method separation and purification human amnion tissue, to raise the cost greatly, reduce the surviving rate of neural stem cell.
Summary of the invention
The object of the invention provide a kind of from human amnion tissue the method for separation and purification of nerve stem cell.
In order to realize this goal of the invention, the invention provides a kind of method of separating and purifying nerve stem of human amnion tissue, it may further comprise the steps:
(a) clean human amnion tissue;
(b) from above-mentioned human amnion tissue, isolate epithelial cell;
(c) from human amnion tissue, isolate the human amnion tissue mesenchymal cell;
(d) from the human amnion tissue mesenchymal cell, isolate neural stem cell;
The invention provides a kind of method of separating and purifying nerve stem of human amnion tissue, wherein: described step (a) may further comprise the steps:
(1) with human amnion tissue rinsing, immersion and sterilization;
(2) human amnion tissue is cut into is about 1-2mm
3Fragment;
(3) the human amnion tissue fragment is sent in the centrifuge tube, added and contain 0.125% tryptic D-hank ' s damping fluid;
(4) mix above-mentioned solution, after mixing above-mentioned solution is placed in the biological oscillator, in temperature be about 37 ℃, the rotating speed of biological oscillator is under the condition of 80-120rpm, jolts 10-20 minute;
(5) remove the supernatant liquor that obtains, finished the cleaning of human amnion tissue;
The invention provides a kind of method of separating and purifying nerve stem of human amnion tissue, wherein: described step (b) may further comprise the steps:
(1) cleaned human amniotic tissues is joined contains in 0.125% tryptic D-hank ' the s damping fluid;
(2) mix above-mentioned solution, after mixing above-mentioned solution is placed in the biological oscillator, in temperature be about 37 ℃, the rotating speed of biological oscillator is under the condition of 400-600rpm, jolts 20-40 minute;
(3) filter suspension with the cell sieve, collect the epithelial cell of from human amnion tissue, separating;
(4) repeat above-mentioned (1) to (3) step 2-3 time, collect all epithelial cells and the counting from human amnion tissue, separated at every turn;
(5) above-mentioned epithelial cell is seeded in the culturing bottle;
The invention provides a kind of method of separating and purifying nerve stem of human amnion tissue, wherein: described step (c) may further comprise the steps:
(1) will isolate the rinsing of epithelial human amnion tissue fragment D-hank ' s damping fluid;
(2) above-mentioned fragment of tissue is joined contain in 0.1% the collagen type v enzyme solution and mix;
(3) mix after, above-mentioned solution is placed in the biological oscillator, in temperature be about 37 ℃, the rotating speed of biological oscillator is under the condition of 400-600rpm, jolts 20-40 minute;
(4) filter suspension with the cell sieve, centrifugal collector's amnion tissue mesenchymal cell and counting;
The invention provides a kind of method of separating and purifying nerve stem of human amnion tissue, wherein: described step (d) may further comprise the steps:
(1) the human amnion tissue mesenchymal cell with above-mentioned collection is inoculated in the Culture of neural stem cells liquid, is cultured to 1 * 10
6-1 * 10
7Number of cells;
(2) method of employing immunomagnetic beads cellular segregation is isolated neural stem cell from the human amnion tissue mesenchymal cell;
The invention provides a kind of method of separating and purifying nerve stem of human amnion tissue, wherein: described Culture of neural stem cells liquid comprises: contain the Prostatropin (bFGF) of 20ng/ml, the epithelical cell growth factor (EGF) that contains 20ng/ml and the DMEM/F12 serum-free medium of B27;
The invention provides a kind of method of separating and purifying nerve stem of human amnion tissue, wherein: the characteristics of expressing CD133 antibody according to neural stem cell, with the CD133 antibody sandwich on immunomagnetic beads, utilize the method for immunomagnetic beads cellular segregation, from the human amnion tissue mesenchymal cell, isolate CD133 positive neurons stem cell.
The present invention compares with the method for separation and purification of nerve stem cell from embryo or tire brain, the present invention is by separating epithelial cell, collect mesenchymal cell and expressing the characteristics of CD133 antibody according to neural stem cell from human amnion tissue, adopt the immunomagnetic beads cytopheresis, from the human amnion tissue mesenchymal cell, isolate CD133 positive neurons stem cell, adopt Culture of neural stem cells liquid to continue culture of neural stem cells neural again, this method greatly reduces the cost of separation and purification of nerve stem cell from human amnion tissue, has improved the surviving rate of neural stem cell.
Embodiment
The invention discloses a kind of method of separating and purifying nerve stem of human amnion tissue, it comprises four big steps, and each big step also comprises several little steps, is four big steps below wherein:
(a) clean human amnion tissue;
(b) from above-mentioned human amnion tissue, isolate epithelial cell;
(c) from human amnion tissue, isolate the human amnion tissue mesenchymal cell;
(d) from the human amnion tissue mesenchymal cell, isolate neural stem cell.
Wherein step (a) may further comprise the steps:
(1) with human amnion tissue rinsing, immersion and sterilization;
(2) human amnion tissue is cut into is about 1-2mm
3Fragment;
(3) the human amnion tissue fragment is sent in the 50ml centrifuge tube, added and contain 0.125% tryptic D-hank ' s damping fluid;
(4) mix above-mentioned solution, after mixing above-mentioned solution is placed in the biological oscillator, in temperature be about 37 ℃, the rotating speed of biological oscillator is under the condition of 100rpm, jolts 15 minutes;
(5) remove the supernatant liquor that obtains, finished the cleaning of human amnion tissue.
Wherein step (b) may further comprise the steps:
(1) cleaned human amniotic tissues is joined contains in 0.125% tryptic D-hank ' the s damping fluid;
(2) mix above-mentioned solution, after mixing above-mentioned solution is placed in the biological oscillator, in temperature be about 37 ℃, the rotating speed of biological oscillator is under the condition of 500rpm, jolts 30 minutes;
(3) filter suspension with the cell sieve, collect the epithelial cell of from human amnion tissue, separating;
(4) repeat above-mentioned (1) to (3) step 3 time, collect all epithelial cells and the counting from human amnion tissue, separated at every turn;
(5) above-mentioned epithelial cell is seeded in the culturing bottle, with as other purposes.
Wherein said step (c) may further comprise the steps:
(1) will isolate the rinsing of epithelial human amnion tissue fragment D-hank ' s damping fluid;
(2) above-mentioned fragment of tissue is joined contain in 0.1% the collagen type v enzyme solution and mix;
(3) mix after, above-mentioned solution is placed in the biological oscillator, in temperature be about 37 ℃, the rotating speed of biological oscillator is under the condition of 500rpm, jolts 30 minutes;
(4) filter suspension with the cell sieve, centrifugal collector's amnion tissue mesenchymal cell and counting are in order to follow-up use.
Wherein step (d) may further comprise the steps:
(1) the human amnion tissue mesenchymal cell with above-mentioned collection is inoculated in the Culture of neural stem cells liquid, is cultured to 1 * 10
6-1 * 10
7Number of cells, Culture of neural stem cells liquid comprises: contain 20ng/ml Prostatropin (bFGF), contain the epithelical cell growth factor (EGF) of 20ng/ml and the DMEM/F12 serum-free medium of B27 (trade name); The consumption of B27 (trade name) can add according to the introduction of product description.
(2) according to the characteristics of neural stem cell expression CD133 antibody, the CD133 antibody sandwich on immunomagnetic beads, is utilized the method for immunomagnetic beads cellular segregation, from the human amnion tissue mesenchymal cell, isolate CD133 positive neurons stem cell.The neural stem cell of separating continues to cultivate through nutrient solution.
More than describing is explanation of the invention, is not that institute of the present invention restricted portion is referring to claim to the qualification of invention, and under the situation of spirit of the present invention, the present invention can do any type of modification.
Claims (7)
1. the method for a separating and purifying nerve stem of human amnion tissue, it is characterized in that: it may further comprise the steps:
(a) clean human amnion tissue;
(b) from above-mentioned human amnion tissue, isolate epithelial cell;
(c) from human amnion tissue, isolate the human amnion tissue mesenchymal cell;
(d) from the human amnion tissue mesenchymal cell, isolate neural stem cell.
2. the method for separating and purifying nerve stem of human amnion tissue as claimed in claim 1, it is characterized in that: described step (a) may further comprise the steps:
(1) with human amnion tissue rinsing, immersion and sterilization;
(2) human amnion tissue is cut into is about 1-2mm
3Fragment;
(3) the human amnion tissue fragment is sent in the centrifuge tube, adds to contain 0.125% tryptic D-hank ' s damping fluid;
(4) mix above-mentioned solution, after mixing above-mentioned solution is placed in the biological oscillator, in temperature be about 37 ℃, the rotating speed of biological oscillator is under the condition of 80-120rpm, jolts 10-20 minute;
(5) remove the supernatant liquor that obtains, finished the cleaning of human amnion tissue.
3. the method for the described separating and purifying nerve stem of human amnion tissue of claim 2, it is characterized in that: described step (b) may further comprise the steps:
(1) cleaned human amniotic tissues is joined contains in 0.125% tryptic D-hank ' the s damping fluid;
(2) mix above-mentioned solution, after mixing above-mentioned solution is placed in the biological oscillator, in temperature be about 37 ℃, the rotating speed of biological oscillator is under the condition of 400-600rpm, jolts 20-40 minute;
(3) filter suspension with the cell sieve, collect the epithelial cell of from human amnion tissue, separating;
(4) repeat above-mentioned (1) to (3) step 2-3 time, collect all epithelial cells and the counting from human amnion tissue, separated at every turn;
(5) above-mentioned epithelial cell is seeded in the culturing bottle.
4. the method for separating and purifying nerve stem of human amnion tissue as claimed in claim 3, it is characterized in that: described step (c) may further comprise the steps:
(1) will isolate the rinsing of epithelial human amnion tissue fragment D-hank ' s damping fluid;
(2) above-mentioned fragment of tissue is joined contain in 0.1% the collagen type v enzyme solution and mix;
(3) mix after, above-mentioned solution is placed in the biological oscillator, in temperature be about 37 ℃, the rotating speed of biological oscillator is under the condition of 400-600rpm, jolts 20-40 minute;
(4) filter suspension with the cell sieve, centrifugal collector's amnion tissue mesenchymal cell and counting.
5. the method for separating and purifying nerve stem of human amnion tissue as claimed in claim 4, it is characterized in that: described step (d) may further comprise the steps:
(1) the human amnion tissue mesenchymal cell with above-mentioned collection is inoculated in the Culture of neural stem cells liquid, is cultured to 1 * 10
6-1 * 10
7Number of cells;
(2) method of employing immunomagnetic beads cellular segregation is isolated neural stem cell from the human amnion tissue mesenchymal cell.
6. the method for separating and purifying nerve stem of human amnion tissue as claimed in claim 5, it is characterized in that: described Culture of neural stem cells liquid comprises: the DMEM/F12 serum-free medium that contains the Prostatropin of 20ng/ml, the epithelical cell growth factor that contains 20ng/ml and B27.
7. the method for separating and purifying nerve stem of human amnion tissue as claimed in claim 6, it is characterized in that: the characteristics of expressing CD133 antibody according to neural stem cell, with the CD133 antibody sandwich on immunomagnetic beads, utilize the method for immunomagnetic beads cellular segregation, from the human amnion tissue mesenchymal cell, isolate CD133 positive neurons stem cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100900265A CN100469870C (en) | 2006-06-23 | 2006-06-23 | Method for separating and purifying nerve stem of human amnion tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100900265A CN100469870C (en) | 2006-06-23 | 2006-06-23 | Method for separating and purifying nerve stem of human amnion tissue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101092608A CN101092608A (en) | 2007-12-26 |
| CN100469870C true CN100469870C (en) | 2009-03-18 |
Family
ID=38991050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100900265A Expired - Fee Related CN100469870C (en) | 2006-06-23 | 2006-06-23 | Method for separating and purifying nerve stem of human amnion tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100469870C (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591643B (en) * | 2008-05-30 | 2011-12-21 | 李荣旗 | Human amniotic membrane tissue-derived mesenchymal cells extract method and subcultring method |
| CN101824398B (en) * | 2009-03-05 | 2012-08-08 | 中日友好医院 | Method for co-culturing, inducing and differentiating dopaminergic neuron by human amniotic epithelial cells and neural stem cells |
| CN101824399B (en) * | 2009-03-05 | 2012-05-30 | 中日友好医院 | Method for inducing and differentiating dopaminergic neuron from human amniotic epithelial cell and application of separated dopaminergic neuron |
| CN111088219B (en) * | 2020-01-16 | 2021-05-04 | 南京鼓楼医院 | Method for separating and culturing vaginal epithelial cells |
| CN117070443B (en) * | 2023-10-18 | 2024-02-06 | 广州正源生物技术有限公司 | Separation method of human amniotic epithelial cells |
-
2006
- 2006-06-23 CN CNB2006100900265A patent/CN100469870C/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| 人类羊膜细胞表达神经干细胞特异性蛋白研究. 蔡哲等.中国康复理论与实践，第11卷第12期. 2005 |
| 人类羊膜细胞表达神经干细胞特异性蛋白研究. 蔡哲等.中国康复理论与实践，第11卷第12期. 2005 * |
| 免疫磁珠体外纯化人胎脑中CD133阳性干细胞的实验研究. 余爽等.神经解剖学杂志，第20卷第3期. 2004 |
| 免疫磁珠体外纯化人胎脑中CD133阳性干细胞的实验研究. 余爽等.神经解剖学杂志，第20卷第3期. 2004 * |
| 羊膜上皮细胞促进共培养神经干细胞存活及分化. 孟晓婷等.吉林大学学报（医学版），第30卷第2期. 2004 |
| 羊膜上皮细胞促进共培养神经干细胞存活及分化. 孟晓婷等.吉林大学学报（医学版），第30卷第2期. 2004 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101092608A (en) | 2007-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105969720B (en) | A kind of Human vascular endothelial's cell culture fluid and its cultural method | |
| CN100469870C (en) | Method for separating and purifying nerve stem of human amnion tissue | |
| CN106715685B (en) | Method for producing cardiomyocyte population derived from pluripotent stem cells | |
| WO2010040262A1 (en) | Methods for isolating animal embryonic mesenchymal stem cells and extracting secretion substance thereof | |
| CN101886060A (en) | Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells | |
| CN104450611A (en) | Primary separation and culture method of human amniotic mesenchymal stem cells | |
| CN103966159B (en) | Human plactnta Subaerial blue green algae and stem cell bank construction process thereof | |
| CN101735983B (en) | Method for separating and purifying oligodendrocyte precursor cells | |
| CN107164326A (en) | A kind of method of the neural precursor in 3D culture autologous fats MSCs sources | |
| CN110438067A (en) | Human skin fibroblasts and preparation method thereof | |
| CN118755667A (en) | Culture medium of menstrual mesenchymal stem cells and extraction culture method thereof | |
| CN110964693A (en) | Separation method of umbilical cord mesenchymal stem cells | |
| CN105695409B (en) | A kind of naked mole oligodendrocyte precursor cells cultural method | |
| CN102191220B (en) | Method for isolating and purifying schwann cells from adipose tissue | |
| CN116836934B (en) | Osteosarcoma organoid culture solution, culture reagent combination and culture method | |
| CN106497863B (en) | A kind of separation, purifying and the cultural method of cornea of rats endothelial cell | |
| CN112159796A (en) | Primary isolation method and application of human umbilical cord-derived mesenchymal stem cells | |
| CN107513519A (en) | A kind of cultural method of schwann cell | |
| CN113736737B (en) | Primary glioma-related fibroblast culture method | |
| CN109666642B (en) | Method for in vitro separation and purification of oligodendrocyte precursor cells of tree shrew cerebral cortex | |
| CN104560875A (en) | Method for separating and culturing trigeminal root ganglion neurons of mammal in vitro and application of method | |
| CN115181722A (en) | In-vitro separation and culture method of goose skeletal muscle satellite cells | |
| CN109439614B (en) | Exosome preparation for maintaining and restoring hair papilla cell stem property | |
| US20150147409A1 (en) | Adipose stromal vascular fraction-conditioned medium | |
| CN107365743B (en) | Culture method of oligodendrocyte precursor cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090318 Termination date: 20120623 |