CN1865435A - Human embryo stem cell and its culture method - Google Patents
Human embryo stem cell and its culture method Download PDFInfo
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- CN1865435A CN1865435A CNA2005100260103A CN200510026010A CN1865435A CN 1865435 A CN1865435 A CN 1865435A CN A2005100260103 A CNA2005100260103 A CN A2005100260103A CN 200510026010 A CN200510026010 A CN 200510026010A CN 1865435 A CN1865435 A CN 1865435A
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Abstract
The invention provides a method for obtaining human embryo stem cells in vitro, and the human embryo stem cell lines obtained through the method, especially human embryo stem cell line SH-HES1 having a docket number of CCTCC-C200503.
Description
Technical field
The present invention relates to field of biology.More particularly, the present invention relates to a kind of vitro culture and obtain the method for human embryonic stem cell and the human embryonic stem cell that obtains with this method.
Background technology
Human embryo stem cell is a kind of a kind of cell with unlimited self and multidirectional differentiation potential that derives from human body early embryo inner cell mass.Utilize human embryo stem cell can make people understand basic biological question such as early embryo development rule, mechanism of cell differentiation.Make more that people are interested to be, human embryo stem cell can be divided into into any cell in the human body, therefore in the future may make the special cells that needs clinically in the laboratory, this all may produce revolutionary impact to some disease that is difficult to effect a radical cure such as diabetes, Parkinson's disease, Spinal injury, presenile dementia etc.If human embryo stem cell and organizational project are combined, can in the laboratory, make the needed organ of patient in the future, its meaning is undoubtedly far-reaching.Simultaneously, human embryo stem cell can also be as the experiment material of drug screening, poisonous substance examination, than experimentation on animals more near the truth of human body.In theory, the quantity of human embryonic stem cell is The more the better, can satisfy different patients' needs like this.And the human stem cell cording that different people embryos sets up has different mhc genes, so the immunological characteristic between these clones is different.This also is the first cause that will set up stem cell bank in the world.In addition, finding at present also has certain difference on differentiation potential between the different stem cell lines, that is to say, though the basic characteristics between the stem cell line are consistent, remains differentiated between them.
Therefore, still to press for building of human embryonic stem cell be scheme and new human embryonic stem cell in this area.
Summary of the invention
For achieving the above object, one aspect of the present invention provides a kind of method at external acquisition human embryonic stem cell, and described method comprises:
(1) zona pellucida of blastaea is removed in digestion, then whole blastaea is put on the feeder layer cells of being made by l cell and cultivates, in former generation,, the prescription of nutrient solution was: DMEM, 20% foetal calf serum, 0.06-0.2mmol/L beta-mercaptoethanol, 0.06-0.2mmol/L non-essential amino acid, 1-2mmol/L glutamine, 30-60U/ml penicillin, 30-60 μ g/ml Streptomycin sulphate;
(2) after the cell cultures of step 1 is carried out 8-10 days, cell mass is cut into small pieces, inoculate on the fresh l cell feeder layer cells and cultivate, the nutrient solution prescription is KO-DMEM, 20% serum substitute 0.06-0.2mmol/L beta-mercaptoethanol, 0.06-0.2mmol/L non-essential amino acid, the 1-2mmol/L glutamine, 30-60U/ml penicillin, 30-60 μ g/ml Streptomycin sulphate, 3-5ng/ml bFGF;
(3) when the cell of step 2 grew into for the 4th～8 generation, remove nutrient solution, at saturated humidity, 37 ℃, 5%CO
2Carry out had digestive transfer culture with 0.05% pancreas enzyme-EDTA and cultivate down, separate obtaining human embryonic stem cell.
In a preferable embodiment, human embryo stem cell goes down to posterity with 1: 5～1: 10 ratio in step (3).
In another preferable embodiment, in the step (1) used former generation nutrient solution prescription be: DMEM, 20% foetal calf serum, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 1mmol/L glutamine, 50U/ml penicillin and 50 μ g/ml Streptomycin sulphates.
In another preferable embodiment, nutrient solution prescription used in the step (2) is: KO-DMEM, 20% serum substitute, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 1mmol/L glutamine, 50U/ml penicillin, 50 μ g/ml Streptomycin sulphates and 4ng/ml bFGF.
The present invention also provides a kind of usefulness the above-mentioned human embryonic stem cell of stating the method acquisition on the other hand.Preferably, described human embryonic stem cell is human embryonic stem cell SH-HES1, and its preserving number is CCTCC-C200503.
The accompanying drawing summary
Fig. 1 has shown the 66th generation of the human embryonic stem cell SH-HES1 of the present invention that observes at 4 times of inverted phase contrast microscopes.
Embodiment
Specifically describe the present invention below in conjunction with embodiment.
1, human embryo stem cell former be commissioned to train foster
After obtaining people's blastaea (from clinical IVF is residue blastaea after in vitro fertilization, obtaining) of growth regulation 5 days or the 6th day, with 5mg/ml Protease (PRONASE A, Sigma) digestion zona pellucida.Whole blastaea is put into carry out on the feeder layer cells that is prepared into by l cell former be commissioned to train foster.In former generation,, the prescription of nutrient solution was: DMEM, 20% (W/V) foetal calf serum (Hyclone), 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 1mmol/L glutamine, 50U/ml penicillin, 50 μ g/ml Streptomycin sulphates.The primary cell growth cuts into plurality of small blocks with cell mass with the tip that pasteur pipet pulls into after about 9 days, inoculates on the fresh feeder layer cells.This moment, the nutrient solution prescription changed KO-DMEM into, 20% (W/V) serum substitute (Gibco), 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 1mmol/L glutamine, 50U/ml penicillin, 50 μ g/ml Streptomycin sulphates, 4ng/ml bFGF.Cultivate commitment at human embryo stem cell, all must go down to posterity again after with the cell mass morsel, when in one 4 hole ware, enough undifferentiated cells being arranged, just can use the method for had digestive transfer culture instead with the method for cut mechanically.
2, the had digestive transfer culture of human embryo stem cell is cultivated
(1) when human embryo stem cell grows into the 4th～6 day, inhale and remove nutrient solution, wash one time with PBS (no calcium, magnesium), inhale and remove PBS.
(2) add under 0.05% (W/V) pancreas enzyme-EDTA room temperature that pre-temperature crosses and digested 1～2 minute, represent that digestion is more abundant when big space appears in feeder layer cells, blow and beat cell gently several times with the 1ml suction pipe, the feeder layer cells nutrient solution that adds the 6ml preheating stops digesting.
(3) with cell transfer in the 15ml centrifuge tube, centrifugal 1000rpm, 5min.
(4) abandon supernatant, cell precipitation is assigned on the new feeder layer cells by suitable proportion with the resuspended back of human embryo stem cell nutrient solution, rock culture dish with X and Y direction and make the cell uniform distribution, cell is put in the incubator cultivates at last.The ratio of going down to posterity that human embryo stem cell is general is 1: 5～1: 10.
The human embryo stem cell culture condition is saturated humidity, 37 ℃, 5%CO
2
3, frozen human embryo stem cell
(1) with the frozen storing liquid for preparing (the 90%W/V foetal calf serum, 10%V/VDMSO) place standby on ice.
(2) discard nutrient solution, wash one time, inhale and remove PBS with PBS (no calcium, enzyme).
(3) add under the 0.05%W/V pancreas enzyme-EDTA room temperature that pre-temperature crosses and digested 1～2 minute, represent that digestion is more abundant when big space appears in feeder layer cells, blow and beat cell gently several times with the 1ml suction pipe, the feeder layer cells nutrient solution that adds the 6ml preheating stops digesting.
(4) with cell transfer in the 15ml centrifuge tube, centrifugal 1000rpm, 5min.
(5) abandon supernatant, cell precipitation is resuspended with frozen storing liquid, immediately the cell branch is installed in the frozen pipe after blowing and beating twice.Frozen pipe is transferred in the cell cryopreservation box-70 ℃ of preservations spend the night, transferred to prolonged preservation in the liquid nitrogen in second day.General each 10cm
2Tissue Culture Dish can freeze 8 cells.
This human embryonic stem cell SH-HES1 is preserved in Chinese typical culture collection center (CCTCC) on March 23rd, 2005, and preserving number is CCTCC-C200503.
4, recovery human embryo stem cell
(1) the frozen pipe of human embryo stem cell is taken out from liquid nitrogen, 37 ℃ of water-baths are until almost all dissolvings of ice cube.
(2), be added to lentamente in the human embryo stem cell nutrient solution of preheating after with the cell sucking-off with the 1ml suction pipe with the frozen pipe of 75% alcohol wipe.
(3) cell centrifugation 1000rpm, 5min.
(4) be inoculated in the six hole wares with human embryo stem cell nutrient solution re-suspended cell post precipitation, rock culture dish with X and Y direction and make the cell uniform distribution, cell is put in the incubator cultivates.
5, identifier's embryonic stem cell line
(1) the human embryo stem cell surface marker is identified and genetic expression
Undifferentiated human embryo stem cell express cell surface antigen S SEA3, SSEA4, TRA1-60, TRA1-81, alkaline phosphatase (AKP) etc., not express cell surface antigen S SEA-1.These antigens can detect with the method for immunofluorescence.Method with RT-PCR also can detect undifferentiated human embryo stem cell expression Oct-3, Sox2, genes such as FGF4, Rex1.Human embryonic stem cell SH-HES1 express cell surface antigen S SEA4, TRA1-60, TRA1-81, alkaline phosphatase (AKP), not express cell surface antigen S SEA-1.Represent undifferentiated gene FGF4, SOX2, LeftyA, OCT-4, TDGF1, Thy-1, Rex-1, Nanog in human embryo stem cell SH-HES1, to express.Cell-surface antigens detects with IiT.Identify the not expression of differentiation gene with the method for RT-PCR.
(2) differentiation capability in the human embryo stem cell body
Can produce teratoma after being expelled to human embryo stem cell in immunodeficient mouse (SCID-BEIGE) body, if teratoma contains the cell in three germinal layer sources, reference's embryonic stem cell has the versatility of differentiation in the body substantially.Because teratoma needs the long period to form, therefore should carry out this detection as early as possible.With paraffin embedding, the teratomatous organizational composition of the painted methods analyst of HE.
The result shows that the teratoma that the human embryonic stem cell SH-HES1 that the present invention obtains forms contains epidermis (ectoderm), cartilage (mesoderm), fatty tissue (mesoderm), serosity body of gland (entoderm), simple columnar epithelium tissues such as (entoderms).
(3) human embryo stem cell vitro differentiation ability
Human embryo stem cell can be spontaneous or be induced differentiation under external suitable condition, and the available special antibody test of this differentiation is arrived.If can detect the cell that human embryo stem cell can be divided into three germinal layer sources, provable substantially its has the versatility of differentiation under conditions in vitro.Detect the special cells that cells in vitro is differentiated to form with IiT.
Human embryonic stem cell SH-HES1 of the present invention can form embryoid (EB), the spontaneous differentiation of cell energy after the embryoid adherent culture, method with immunofluorescence detects these cell expressing Actin muscles (mesoderm), cytokeratin 7 (entoderm), neurofilament protein 70 (ectoderm) and neural early protein (nestin, ectoderm) antigen.
SH-HES1 clone of the present invention except in vivo with the external ability that all has to the differentiation of three germinal layers, can also form cardiac-like muscle cell, so the potential of its differentiation should be widely with the ability of independently beating.
(4) human embryo stem cell caryogram
Normal human embryo stem cell should have the normal karyotype of twice body, generally identifies with the method for G band colour developing.Human embryonic stem cell SH-HES1 detected when 15 generations and 23 generations has normal twice body caryogram.
(5) Telomerase is identified
Telomerase is a kind of reversed transcriptive enzyme, can be the length that template prolongs fringes of chromosome with self-contained RNA, therefore plays an important role aspect chromosome length and the decision cell survival keeping.Most of somatocyte are not expressed Telomerase, and the length of telomere shortens along with the increase in years.The embryonic cell high expression level Telomerase of tumour cell and active growth.Through identifying human embryonic stem cell SH-HES1 high expression level Telomerase of the present invention.
Claims (6)
1. the method at external acquisition human embryonic stem cell is characterized in that, described method comprises:
(1) zona pellucida of blastaea is removed in digestion, then whole blastaea is put on the feeder layer cells of being made by l cell and cultivates, in former generation,, the prescription of nutrient solution was: DMEM, 20% foetal calf serum, 0.06-0.2mmol/L beta-mercaptoethanol, 0.06-0.2mmol/L non-essential amino acid, 1-2mmol/L glutamine, 30-60U/ml penicillin, 30-60 μ g/ml Streptomycin sulphate;
(2) after the cell cultures of step 1 is carried out 8-10 days, cell mass is cut into small pieces, inoculate on the fresh l cell feeder layer cells and cultivate, the nutrient solution prescription is KO-DMEM, 20% serum substitute 0.06-0.2mmol/L beta-mercaptoethanol, 0.06-0.2mmol/L non-essential amino acid, the 1-2mmol/L glutamine, 30-60U/ml penicillin, 30-60 μ g/ml Streptomycin sulphate, 3-5ng/ml bFGF;
(3) when the cell of step 2 grew into for the 4th～8 generation, remove nutrient solution, at saturated humidity, 37 ℃, 5% CO
2Carry out had digestive transfer culture with 0.05% pancreas enzyme-EDTA and cultivate down, separate obtaining human embryonic stem cell.
2. method according to claim 1 is characterized in that, wherein human embryo stem cell goes down to posterity with 1: 5～1: 10 ratio in step (3).
3. method according to claim 1, it is characterized in that, in the step (1) used former generation nutrient solution prescription be: DMEM, 20% foetal calf serum, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 1mmoL/L glutamine, 50U/ml penicillin and 50 μ g/ml Streptomycin sulphates.
4. method according to claim 1, it is characterized in that, nutrient solution prescription used in the step (2) is: KO-DMEM, 20% serum substitute, 0.1mmol/L beta-mercaptoethanol, 0.1mmol/L non-essential amino acid, 1mmol/L glutamine, 50U/ml penicillin, 50 μ g/ml Streptomycin sulphates and 4ng/ml bFGF.
5. human embryonic stem cell that obtains with the described method of claim 1.
6. human embryonic stem cell according to claim 5 is characterized in that, described human embryonic stem cell is human embryonic stem cell SH-HES1, and its preserving number is CCTCC-C200503.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338300B (en) * | 2007-07-06 | 2010-06-23 | 李凌松 | Process for culturing human embryo sexual fold stem cell |
| CN104059875A (en) * | 2014-04-28 | 2014-09-24 | 浙江省淡水水产研究所 | Primary culture method of embryonic cell tissue pieces of Chinese softshell turtle |
| CN108707600A (en) * | 2018-05-31 | 2018-10-26 | 东北农业大学 | The one unicellular fusion method of boar mouse cell |
-
2005
- 2005-05-20 CN CNA2005100260103A patent/CN1865435A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338300B (en) * | 2007-07-06 | 2010-06-23 | 李凌松 | Process for culturing human embryo sexual fold stem cell |
| CN104059875A (en) * | 2014-04-28 | 2014-09-24 | 浙江省淡水水产研究所 | Primary culture method of embryonic cell tissue pieces of Chinese softshell turtle |
| CN104059875B (en) * | 2014-04-28 | 2016-05-18 | 浙江省淡水水产研究所 | The primary culture method of Shelled Turtle Trionyx Sinensis embryonic cell tissue block |
| CN108707600A (en) * | 2018-05-31 | 2018-10-26 | 东北农业大学 | The one unicellular fusion method of boar mouse cell |
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