CN101892193A - Method for culturing stem cells in human cumulus cell feeder layer - Google Patents
Method for culturing stem cells in human cumulus cell feeder layer Download PDFInfo
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- CN101892193A CN101892193A CN201010244744XA CN201010244744A CN101892193A CN 101892193 A CN101892193 A CN 101892193A CN 201010244744X A CN201010244744X A CN 201010244744XA CN 201010244744 A CN201010244744 A CN 201010244744A CN 101892193 A CN101892193 A CN 101892193A
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Abstract
The invention discloses a method for culturing stem cells in a human cumulus cell feeder layer, which comprises the following steps: 1, centrifuging waste cumulus cells from a process of treating a person with barrenness by using an in-vitro fertilization embryo transplant technique with a centrifuge; 2, removing supernate obtained by centrifugation to obtain suspension of the cumulus cells, adding a cumulus cell culture medium, mixing the mixture uniformed, filling the mixture into the centrifuge, adding the cumulus cell culture medium into the sediment obtained after the centrifugation to obtain suspension of the cumulus cells; 3, adding gelatin into each hole of a four-hole vessel, placing the vessel at room temperature, and absorbing the gelatin solution out completely for later use; adding the suspension of the cumulus cells at a certain density in the four-hole vessel, and placing the vessel in a CO2 incubator; taking the four-hole vessel out of the CO2 incubator 24 hours later, and changing for stem cell culture solution; and performing the continuous cell culture of the grown stem cell 4 to 7 days later. In the invention, the cells are readily available, the culture method is easy to operate, and the application in clinic is easy to promote.
Description
Technical field
The present invention relates to the method for culturing stem cells, is a kind of method of culturing stem cells in human cumulus cell feeder layer.
Background technology
At present, human embryo stem cell mainly is to cultivate on the mouse embryo fibroblasts feeder layer, though the method for this culturing stem cells stability is better, and, the feeder layer of mouse does not meet the clinical application requirement, has the potentially dangerous of propagating xenogenesis pathogeny body.For this reason, the major subjects that the feeder layer of Humanized cell preparation was studied in recent years for those skilled in the art reports, can there be the inoblast of skin, lung, foreskin and embryonic origin in humanized's feeder layer cells source.Different feeder layers is made effect and is had a long way to go.For example: the foreskin sample carries out feeder layer when making, and effect is than the inoblast difference of embryonic origin etc.And skin, lung, embryo are difficult for obtaining, and also there is ethics problem in the human embryonic fibroblast.Therefore, how can adopt a kind of easy acquisition and not have humanized's feeder layer cells of ethics problem, for clinical application provides reliable feeder layer, be those skilled in the art's major subjects of research at present.
Summary of the invention
The objective of the invention is, a kind of method of culturing stem cells in human cumulus cell feeder layer is provided, its cell is obtained easily, the cultural method easy handling is easy to promote application clinically.
The present invention is achieved through the following technical solutions for achieving the above object: a kind of method of culturing stem cells in human cumulus cell feeder layer comprises the steps:
1. adopt the patient of embryo transfer technology in vitro fertilization treatment Infertility to get behind the ovum 3-4 hour, oocyte corona cumulus complex was inserted the transparent resin acid enzyme of 80units/ml liquid interior 30 seconds, slough ovum cumulus cell on every side with kapillary piping and druming, cumulus cell is collected in the centrifuge tube, add cumulus cell substratum 4-6ml, adopt whizzer under 1000 rotating speeds centrifugal 5 minutes, contain Unidasa 2.45mg in the wherein transparent resin acid enzyme liquid, high sugared DMEM substratum 10ml;
2. the supernatant liquor after centrifugal is discarded, obtain the cumulus cell suspension, in the cumulus cell suspension, add 0.25% trypsinase 1ml, placed 1-2 minute behind the mixing;
3. the substratum 4ml that adds cumulus cell in the cumulus cell suspension of step in 2. again behind the mixing, inserted in 1000 whizzers that change centrifugal 5 minutes;
4. with step in 3. the supernatant liquor after centrifugal discard, adding the cumulus cell substratum in throw out, to be adjusted to density be 5 * 10
4/ ml obtains the cumulus cell suspension;
5. get four hole wares, add 0.1% gelatin 0.5ml in every hole, place at room temperature after 5 minutes standby after the whole sucking-offs of gelatin liquid;
6. after every hole adds the cumulus cell suspension of 1ml step in 4. in the four hole wares of step in 5., be placed on CO
2Interior 24 hours of incubator, CO
2CO in the incubator
2Gas concentration is 5%, CO
2Temperature in the incubator is 37 ℃;
7. after 24 hours at CO
2Take out four hole wares in the incubator, inhale and remove the cumulus cell substratum, change stem cell media, add stem cell nutrient solution 1ml, every hole adds stem cell 6-8 group, and every contains stem cell 90-110;
8. the stem cell media of step in 7. changed 50% every 1-2 days, the embryonic stem cell group that to grow up after 4-7 days chooses every with glass needle is cut into the 4-8 piece, transfer in the new cumulus cell feeder layer with the glass capillary sucking-off and to cultivate, new cumulus cell feeder layer is the cumulus cell feeder layer that 1.-6. obtains according to step;
9. with the stem cell media that more renews in the four old hole wares, when residual stem cell group grew up again, repeating step operation 7. up to the cumulus cell apoptosis, can not be kept stem cell growth.
The method of a kind of culturing stem cells in human cumulus cell feeder layer of the present invention, stem cell media is made by following volume ratio: 80% embryonic stem cell with substratum, 20% human embryo stem cell with blood serum substituting product, 1% nonessential amino acid, 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 4ng/ml Prostatropin, 1000IU/ml leukaemia inhibitory factor.
Cumulus cell substratum of the present invention is made by following volume ratio: high sugared DMEM substratum 90% and inactivated fetal bovine serum 10%.
The invention has the advantages that: the waste cell that adopts the patient of embryo transfer technology treatment Infertility in vitro fertilization, cell obtains easily, cultural method is easy and simple to handle, method is easy to promote, do not need to handle through mitomycin C or gammairradiation, avoided because of necrocytosis, intracellular matter may cause the sudden change of embryonic stem cell and influence the maintenance of normal karyotype, do not have animal derived cell contamination, do not have the potentially dangerous of propagating xenogenesis pathogeny body.For clinical application provides reliable feeder layer.
Embodiment
Embodiment
The method of a kind of culturing stem cells in human cumulus cell feeder layer of the present invention comprises the steps:
1. adopt the patient of embryo transfer technology in vitro fertilization treatment Infertility to get behind the ovum 3-4 hour, oocyte corona cumulus complex was inserted the transparent resin acid enzyme of 80units/ml liquid interior 30 seconds, slough ovum cumulus cell on every side with kapillary piping and druming, cumulus cell is collected in the centrifuge tube, add cumulus cell substratum 4-6ml, adopt whizzer under 1000 rotating speeds centrifugal 5 minutes, contain Unidasa 2.45mg in the wherein transparent resin acid enzyme liquid, high sugared DMEM substratum 10ml;
2. the supernatant liquor after centrifugal is discarded, obtain the cumulus cell suspension, in the cumulus cell suspension, add 0.25% trypsinase 1ml, placed 1-2 minute behind the mixing;
3. the substratum 4ml that adds cumulus cell in the cumulus cell suspension of step in 2. again behind the mixing, inserted in 1000 whizzers that change centrifugal 5 minutes;
4. with step in 3. the supernatant liquor after centrifugal discard, adding the cumulus cell substratum in throw out, to be adjusted to density be 5 * 10
4/ ml obtains the cumulus cell suspension;
5. get four hole wares, add 0.1% gelatin 0.5ml in every hole, place at room temperature after 5 minutes standby after the whole sucking-offs of gelatin liquid;
6. after every hole adds the cumulus cell suspension of 1ml step in 4. in the four hole wares of step in 5., be placed on CO
2Interior 24 hours of incubator, CO
2CO in the incubator
2Gas concentration is 5%, CO
2Temperature in the incubator is 37 ℃;
7. after 24 hours at CO
2Take out four hole wares in the incubator, inhale and remove the cumulus cell substratum, change stem cell media, add stem cell nutrient solution 1ml, every hole adds stem cell 6-8 group, and every contains stem cell 90-110;
8. the stem cell media of step in 7. changed 50% every 1-2 days, the embryonic stem cell group that to grow up after 4-7 days chooses every with glass needle is cut into the 4-8 piece, transfer in the new cumulus cell feeder layer with the glass capillary sucking-off and to cultivate, new cumulus cell feeder layer is the cumulus cell feeder layer that 1.-6. obtains according to step;
9. with the stem cell media that more renews in the four old hole wares, when residual stem cell group grew up again, repeating step operation 7. up to the cumulus cell apoptosis, can not be kept stem cell growth, after 4-7 days to the stem cell that the grows up cultivation of going down to posterity.
Stem cell media of the present invention is made by following volume ratio: 80% human embryo stem cell with substratum, 20% human embryo stem cell with blood serum substituting product, 1% nonessential amino acid, 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 4ng/ml Prostatropin, 1000IU/ml leukaemia inhibitory factor.
Cumulus cell substratum of the present invention is made by following volume ratio: high sugared DMEM substratum 90% and inactivated fetal bovine serum 10%.
Claims (3)
1. the method for a culturing stem cells in human cumulus cell feeder layer is characterized in that: comprise the steps:
1. adopt the patient of embryo transfer technology in vitro fertilization treatment Infertility to get behind the ovum 3-4 hour, oocyte corona cumulus complex was inserted the transparent resin acid enzyme of 80units/ml liquid interior 30 seconds, slough ovum cumulus cell on every side with kapillary piping and druming, cumulus cell is collected in the centrifuge tube, add cumulus cell substratum 4-6ml, adopt whizzer under 1000 rotating speeds centrifugal 5 minutes, contain Unidasa 2.45mg in the wherein transparent resin acid enzyme liquid, high sugared DMEM substratum 10ml;
2. the supernatant liquor after centrifugal is discarded, obtain the cumulus cell suspension, in the cumulus cell suspension, add 0.25% trypsinase 1ml, placed 1-2 minute behind the mixing;
3. the substratum 4ml that adds cumulus cell in the cumulus cell suspension of step in 2. again behind the mixing, inserted in 1000 whizzers that change centrifugal 5 minutes;
4. with step in 3. the supernatant liquor after centrifugal discard, adding the cumulus cell substratum in throw out, to be adjusted to density be 5 * 10
4/ ml obtains the cumulus cell suspension;
5. get four hole wares, add 0.1% gelatin 0.5ml in every hole, place at room temperature after 5 minutes standby after the whole sucking-offs of gelatin liquid;
6. after every hole adds the cumulus cell suspension of 1ml step in 4. in the four hole wares of step in 5., be placed on CO
2Interior 24 hours of incubator, CO
2CO in the incubator
2Gas concentration is 5%, CO
2Temperature in the incubator is 37 ℃;
7. after 24 hours at CO
2Take out four hole wares in the incubator, inhale and remove the cumulus cell substratum, change stem cell media, add stem cell nutrient solution 1ml, every hole adds stem cell 6-8 group, and every contains stem cell 90-110;
8. the stem cell media of step in 7. changed 50% every 1-2 days, the embryonic stem cell group that to grow up after 4-7 days chooses every with glass needle is cut into the 4-8 piece, transfer in the new cumulus cell feeder layer with the glass capillary sucking-off and to cultivate, new cumulus cell feeder layer is the cumulus cell feeder layer that 1.-6. obtains according to step;
9. with the stem cell media that more renews in the four old hole wares, when residual stem cell group grew up again, repeating step operation 7. up to the cumulus cell apoptosis, can not be kept stem cell growth.
2. the method for a kind of culturing stem cells in human cumulus cell feeder layer according to claim 1 is characterized in that: stem cell media is made by following volume ratio: 80% embryonic stem cell with substratum, 20% human embryo stem cell with blood serum substituting product, 1% nonessential amino acid, 2mM L-glutaminate, 0.1mM beta-mercaptoethanol, 4ng/ml Prostatropin, 1000IU/ml leukaemia inhibitory factor.
3. the method for a kind of culturing stem cells in human cumulus cell feeder layer according to claim 1, it is characterized in that: described cumulus cell substratum is made by following volume ratio: high sugared DMEM substratum 90% and inactivated fetal bovine serum 10%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010244744XA CN101892193A (en) | 2010-08-04 | 2010-08-04 | Method for culturing stem cells in human cumulus cell feeder layer |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010244744XA CN101892193A (en) | 2010-08-04 | 2010-08-04 | Method for culturing stem cells in human cumulus cell feeder layer |
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| Publication Number | Publication Date |
|---|---|
| CN101892193A true CN101892193A (en) | 2010-11-24 |
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| CN201010244744XA Pending CN101892193A (en) | 2010-08-04 | 2010-08-04 | Method for culturing stem cells in human cumulus cell feeder layer |
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2010
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Application publication date: 20101124 |