CN1483817A - Method for cloning human embryonic stem cell without animal component - Google Patents
Method for cloning human embryonic stem cell without animal component Download PDFInfo
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- CN1483817A CN1483817A CNA021307229A CN02130722A CN1483817A CN 1483817 A CN1483817 A CN 1483817A CN A021307229 A CNA021307229 A CN A021307229A CN 02130722 A CN02130722 A CN 02130722A CN 1483817 A CN1483817 A CN 1483817A
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Abstract
The present invention provides a technology for cloning embryonic stem cell utilizing substitution of human cell nucleus and human egg mother cell nucleus. Said technology includes the following main steps: collecting and preparing human donor cell; accelerating ovulation and taking egg; removing ovarium mound cell around egg mother cell; substitution of human egg mother cell nucleus and human cell nucleus; activating the nucleus-substituted human egg mother cell and external culture and development to blastosphere. The cloned embryonic stem cell has no any animal component, and can be used for creating embryonic stem cell system by means of external culture, and can be used for providing basis of clinical medical research of stem cell therapy, cloning tissue and organ, at the same time for controlling development of human clone technology.
Description
One, background technology situation
First undifferentiated mouse embryo stem cell (Embryonic stem cells, ES cell) is to be 1981, and the Evans of univ cambridge uk and Kaufman will postpone the attached embryo who plants and cultivate, and separates and gets from 4-6 days blastaea inner cell masses.1994, Wheeler separated the ES cell that has obtained pig with Robert etc.Up to the blastaeas of outside human body, being fertilized such as Thomson of Univ Wisconsin-Madison USA in 1998 and Israel Rambam medical center ItskovitxEldor, be separated to the ES cell.The same year, the Shamblott etc. of Johns Hopkins University separated the totipotency stem cell that obtains the people from people's archeocyte.The Britain scientist used nuclear transplantation clone embryos technology in 1997, had cultivated in the world first somatic cell clone sheep-how sharp.The success of this technology has promoted the research of human cloning embryonic stem cell greatly.The ES cell has totipotency, and multiplication capacity is strong, differentiation potential is huge, and directional induction in vivo and in vitro is divided into various cells, tissue and organ, is used to substitute, repair cell, tissue and the organ that disease is decreased.Treat present irremediable numerous disease.Listed the six big hot topic sciemtifec and technical spheres that merited attention in 2002 on the recent U.S. " science " magazine, stem-cell research is positioned at the first place, and what seize the life science commanding elevation future will be the stem cell tissue engineering technique.
Since the nineties in last century, no matter it is the ES cell that comes from animal or human's class that scientist carries out discovering of ES cellular segregation and post-directed training aspect, external can both directed differentiation be various cells, as endotheliocyte, blood cell, neurocyte, chondrocyte, muscle cell, liver and islet cells etc.The ES cell can be induced to differentiate into the stem cell of all tissues of body and organ needs in vivo and in vitro.The formation of stem cell function new ideas and the generation and the development of new technology will need the disease of tissue repair and cellular replacement therapy to bring new treatment to wish for hemopathy, cardiovascular diseases, malignant tumour, nervous system disorders, diabetes and other.Because ES cell source is difficult and wayward in vitro differentiation is induced, thereby influenced the prospect of ES cell that adopt as cell and tissue repair treatment.
Cell, tissue, organ transplantation treatment are the principles of individual character treatment, and xenogenesis, allogeneic stem cell are used for clinical meeting and cause immunological rejection.Its major cause is histocompatibility complex (MHC) difference.Therefore source and the quantity of stem cell are the problems of cell therapy most critical, the development of stem cell tissue engineering technique and perfect, to make people can use the stem cell of own gene or stem cell-derived new organization organ, substitute pathology or old and feeble tissue and organ, treatment is irremediable multiple disease at present.So it is imperative that a kind of technology of the specific human embryo stem cell of homologue that can provide is provided at present.Clone people from the directed purpose differentiation of stem cells of the ES of body gene cell induction transplantation treatment, or human cloning are organized, the organ engineering is solve cell, tissue transplantation treatment basic, are 21 century international research focuses for this reason.
There are at present ox, rabbit, sheep class ES clone to cultivate successful report in the world.Korea S's report adopts the ox ovum as the recipient cell that the human nucleus shifts, and can obtain human embryo stem cell.Priorities such as professor Chen Xigu of Zhongshan Medical Univ. of China use " nuclear transplantation " technology is got home the human skin cell nuclear transplantation in the rabbit oocyte, through 2000 test of many times, successfully clone more than 100 human embryos, wherein part is grown " morula " stage.Adopt ox ovum and rabbit ovum as recipient cell human cloning ES cell,, advantage such as be easy to obtain though exist the ovum source abundant relatively.But the human embryo stem cell of cloning as recipient cell with zooblast contains the animal composition, and the treatment that is used for human diseases has certain danger.
The present invention has set up a kind of human body cell of using, through making up human embryo stem cell clone technology from the body gene with the nucleus substitution of human oocyte.This method is at first set up the human cell line, and body-cell neucleus transplanting in the human oocyte of stoning, is grown to blastaea external embryonic cell is cultivated.These embryonic stem cells and deutero-cell thereof and tissue carry out autotransplantation, can not produce immunological rejection after the transplanting.This invention helps the reconstruction of patient's pathological tissues and function thereof, and the contribution that human treatment's sex clone is studied will be huge.
Two, apply for a patent technology
1, sets up human cloning gordian technique platform.
2, make up the embryonic stem cell clone technology platform of people from the body gene.
Three, set up the purpose and the meaning of this technology
1, provides and make up the human embryonic stem cell methods identical, for cell and gene therapy provide " material " to originate with the individual human genotype.Human embryo stem cell has totipotency, and multiplication capacity is strong, differentiation potential is huge, and directional induction in vivo and in vitro is divided into various cells, tissue and organ, is used to substitute, repair cell, tissue and the organ that disease is decreased.We can say that this technology is a Golden Bridge, have only that other technologies just can be led to desirable perfect other shore by it.This technology is that following clinical treatment Journal of Sex Research has been established solid foundation:
First: human embryo stem cell is directly used in multiple diseases such as clinical transplantation treatment tetter, nervous system disorders, hemopathy after directional induction in vitro is divided into required specific stem cell.
Second: carry therapeutic gene with stem cell,, be used for the treatment of heredopathia through being induced to differentiate into into somatocyte.
The the 3rd: utilize the interdepartmental system differentiation potential of stem cell, be used for the reparation of autologous tissue and organ, can avoid the immunological rejection of allotransplantation.
The 4th: foundation changes the universal mhc gene type embryonic stem cell line of structure or tailors one and the isostructural ES clone of receptor MHC through heredity.Select for use arbitrarily for the patient.
The 5th: the ES cell can be used as the target cell of gene targeting, and by gene targeting, the whole and foreign gene of adjustable point carries out genetic modification to the gene relevant with human diseases, seeks the new way of cure diseases.
The the 6th: set up the embryonic stem cell bank that comprises multiple mhc gene type, to satisfy requirement of different patients.
The 7th: stem cell as seed cell, is united cultivation with the biodegradable stent material, at the lived planting body of external structure, repair tissue damaged or regenerating tissues and organ.
The the 8th: set up stem cell bank, be used for the reparation from sick damage of body or old and feeble histoorgan also can be used for the reparation of allohisto compatibility or organ etc. after joining type.
2, make up the gordian technique platform of human embryo stem cell for human cloning.Can obtain human cloning by the following method: (1) obtains required clone person's somatocyte, with its source as donor nuclei; (2) acquisition aspiration donor's ovocyte; (3) remove the nuclear of ovocyte; (4) mode by microinjection moves to the donorcells consideration convey in the enucleation oocyte; (5) merge, activate fused cell through electricity; (6) the vitro culture fused cell is to morula: (7) are transplanted to 4 embryonic cells, morula or blastaea in volunteer's body, make it to develop into fetus (ethics, law allow and can carry out).
According to above-mentioned, can see obviously that sharpest edges of the present invention are to adopt this technology that human embryo stem cell from the body gene can be provided, for having rejection transplantation treatment technology, human cloning tissue and organ do not lay the foundation.For scientific research and clinical treatment Journal of Sex Research afford ample material.Be induced to differentiate into cell brand-new, no immune different originality, tissue and the organ that needed by human body is wanted in that external ES cell is orientable, be used for replacing cell, tissue, the organ of pathology, can avoid the generation of graft-rejection, treat present irremediable numerous disease, show broad clinical application prospect.
Four, method steps
Human cloning embryonic stem cell technology mainly comprises following steps:
1, preparation people donorcells is set up clone.The somatocyte that is used for nuclear transplantation can be available from various cells of donor and tissue, the donor somatocyte of preparation system comprise human skin cell, inoblast and peripheral blood lymphocyte, etc.Somatocyte system is meant the somatic cell culture back of going down to posterity is used.Peripheral blood lymphocyte is the ideal cell type, and cell is easy to separate and obtains.Can directly be used as donorcells from the isolated mononuclearcell of peripheral blood, also can be in vitro culture after 24 hours, as donorcells
Going down to posterity of somatocyte system cultivated by after the trypsin treatment, with the RPMI1640 cell culture fluid that contains 10%-15% people's bleeding of the umbilicus serum (AB type) and 100U/L penicillin-100 μ g/L Streptomycin sulphate, at 37 ℃, 5%CO
2Environment is cultivated down.
As donorcells is lymphocyte, then prepares donorcells according to the following steps.Extract donor peripheric venous blood 5ml, every milliliter of blood adds the abundant mixing of equivalent physiological saline with heparin (500U/ml) 30 μ l anti-freezings in anticoagulation, then with sharp suction pipe along tube wall amount lymphocyte layering liquid (proportion is 1.077) such as addings grade slowly, centrifugal 2000rpm/min, 20min draws buffy coat with sharp suction pipe, place the test tube of Hank ' s liquid, behind the mixing, 1000-1500rpm/min, centrifugal 10min, abandon supernatant, cell suspension is washed 2 times in 2ml Hank ' s liquid again.With the RPMI1640 cell culture fluid that contains 15% people's bleeding of the umbilicus serum, at 37 ℃, 5%CO
2Cultivated 24 hours under the condition, as the source of nuclear donor.
With epidermic cell during, get donor skin fritter and be positioned over earlier and act on 5 minutes among the 0.02%EDTA at ambient temperature as donorcells; Insert then after cold digestion in 0.25% trypsinase (4 ℃) spends the night, separate skin bit, corium and epidermis separately with tweezers, take out epidermis and be cut into small pieces with scissors, put in the new trypsinase, 37 ℃ digested 30-60 minute, with suction pipe piping and druming gently repeatedly, make into cell suspension, behind the husky net filtration of 80 order stainless steels, low-speed centrifugal, supernatant is abandoned in suction, adds Eagle liquid and 15% Cord blood serum in the precipitation, makes cell suspension, change in the culturing bottle 37 ℃ over to, 5%CO
2Cultivate 48h-72h under the condition, after also can continuing to go down to posterity, as nuclear donor.
2, induced ovulation is gathered human oocyte after technical finesse, obtains non-nucleus egg mother cell.Contribute voluntarily the ovum person from menstrual cycle 21 days start injection Wy-42462s 0.1mg, inject and change half amount after 7 days, up to HCG injection; Menstrual onset 3-5 days (due to illness people's particular case and decide) injection every day fruit receives fragrant 2-3 and props up, up to HCG injection.When follicular development to 16-18mm, when having 1-2, behind HCG injection 1000 units 11-12 hour under the vagina B ultrasonic monitors, with the puncture needle of 15G, from ovary follicle, aspirate liquor folliculi, place the plate that contains the IVF2.0 developing medium, cultivated 4-6 hour, ovocyte is reached maturity.Optimize then and exist polarity corpusculum, kytoplasm the ovocyte that is in II stage metaphase of mound cellular layer of sufficient amount to be arranged evenly, on every side as acceptor ovocyte (because this stages of cell can enough be activated, so can be used for nuclear transplantation).The cell that optimizes is placed in the 35ml plate, washes 3 times with the TCM199 cleaning medium.
Through with Unidasa (1 milligram/ml) handle after, in the TCM199 cleaning medium by being the method that 140-180 micron glass pipette moves liquid with internal diameter repeatedly promptly with physical method, remove ovocyte cumulus cell on every side, clean the ovocyte stripped off with the TCM199 cleaning medium then, again their are moved into and contain in TCM199 developing medium and cytochalasin B (7.5ug/ml) solution after 5 minutes, prepare stoning.Adopt little transfer pipet to hold ovocyte, puncture zona pellucida to obtain a breach with micro-needle in addition, from then on the breach place draws with micro pipette and removes polarity corpusculum and kytoplasm on every side.Promptly draw the kytoplasm 1/5-2/5 of polar body and polar body near region,, clean enucleation oocyte, and in the TCM199 developing medium that contains 15% bleeding of the umbilicus serum, cultivate to obtain non-nucleus egg mother cell.
In addition, use polarizing light source, remove genetic material in polar body and the kytoplasm, can obtain the acceptor ovocyte of stoning at microscopically.
3, donorcells nuclear is moved in the human oocyte of stoning, through activating, at the embryonic stem cell of ectogenesis Cheng Ziti gene.Before donorcells nuclear is injected into the acceptor ovocyte, behind the TCM199 cleaning medium cleaning non-nucleus egg mother cell that contains 15% bleeding of the umbilicus serum, they are moved on in the solution of cytochalasin B, draw somatic cell nuclear with the microinjection pin, from the zona pellucida breach, somatic cell nuclear is injected on the acceptor ovocyte tenuigenin limit of stoning.Merge medium at electricity and (contain N.F,USP MANNITOL 0.3M, 0.1mmol/L CaCL
2, 0.1mmol Mg
2SO
4) in make electricity consumption fusion instrument (BTX ECM2001), with the 120-140 volt of 1 second each 80-120 microsecond at interval/centimetre twice electric pulse make cell realize the electricity fusion, cell after the fusion is by after washing 2 times at 6-DMAP, put into 6-DMAP (TCM199 of 6-DMAP 2.5mmol/L 15% people's bleeding of the umbilicus serum) solution and be activated 37 ℃, 5%CO in the TCM199 medium that contains 15% people's bleeding of the umbilicus serum
2Environment is cultivated the activated cell down.
4, fused cell is cultivated through external continuation and is developed into embryo, morula, blastaea.(TCM-199+15% bleeding of the umbilicus serum, 37 ℃, 5%CO2) cultivation is 2-13 days, makes cell fission become 2 embryonic cells to morula or blastaea in external suitable cell culture environment for fused cell after the activation.
5, set up embryonic stem cell line.The cell of morula inner cell mass was digested 1-6 hour at 4 ℃-37 ℃ with 2.5% trypsinase, obtain single embryonic stem cell, people ES clone is set up in the cultivation that continues to go down to posterity.This stem cell line further directional induction develops into the various tissue stem cells of human body, is used for the cell biological treatment of clinical patient and research and the clinical treatment that clone cell, tissue and organ are cultivated in the inside and outside.
6, from the affirmation of body gene embryonic stem cell: the difference from the embryonic stem cell and the donor gene of body gene of getting that human embryo stem cell adopts that PCR-STR9 site sequencing analysis makes up.
Provide following examples further to specify the present invention now.
Embodiment 1
The preparation donorcells.Extract donor peripheric venous blood 5ml, every milliliter of blood adds the abundant mixing of equivalent physiological saline with heparin (500U/m1) 30 μ l anti-freezings in anticoagulation, then with sharp suction pipe along tube wall amount lymphocyte layering liquid (proportion is 1.077) such as addings grade slowly, centrifugal 2000rpm/min, 20min draws buffy coat with sharp suction pipe, place the test tube of Hank ' s liquid, behind the mixing, 1000rpm/min, centrifugal 10min, abandon supernatant, cell suspension is washed 2 times in 2ml Hank ' s liquid again.With the RPMI1640 cell culture fluid that contains 15% people's bleeding of the umbilicus serum, at 37 ℃, 5%CO
2Cultivate 24h under the condition, as the source of nuclear donor.
Preparation acceptor ovocyte.Contribute voluntarily the ovum person from menstrual cycle 21 days start injection Wy-42462s 0.1mg, inject and change half amount after 7 days, up to HCG injection; Menstrual onset 3-5 days injection fruit receives fragrant 2-3 and props up every day, up to HCG injection.When follicular development to 16-18mm, when having 1-2, in HCG injection 1000 units during 21:00 in evening, the next day at 8 in the morning under the vagina B ultrasonic monitors, with the puncture needle of 15G, draw liquor folliculi from ovary, place the plate that contains the IVF2.0 developing medium, cultivated 4-6 hour.Screening has polar body, uniform kytoplasm and the ovocyte of the mound cellular layer of sufficient amount is arranged on every side then, is placed in the 35ml plate, washes 3 times with the TCM cleaning medium.After handling with Unidasa (1mg/ml), in the TCM199 cleaning medium by promptly move the method for liquid repeatedly with superfine glass pipette with physical method, remove ovocyte cumulus cell on every side, then their immigrations are contained in the solution of TCM199 developing medium and cytochalasin B.Adopt the micrurgy apparatus to hold ovum, with behind the micro-needle puncture zona pellucida, from ovocyte, remove the kytoplasm that part comprises first polar body in addition, promptly draw the kytoplasm 1/5-2/5 of polar body and polar body near region, to obtain non-nucleus egg mother cell, as recipient cell.
Before donorcells nuclear is injected into the acceptor ovocyte, behind TCM199 developing medium cleaning non-nucleus egg mother cell, draw somatic cell nuclear with the microinjection pin, from the zona pellucida breach, somatic cell nuclear is injected on the acceptor ovocyte tenuigenin limit of stoning.In electricity fusion medium, make electricity consumption fusion instrument (BTX ECM2001), twice electric pulse of 140 volts/centimetre with the 1 second each 80-120 microsecond in interval makes cell realize that electricity merges, cell after the fusion cleans 10 times at the TCM199 that contains 10% people's bleeding of the umbilicus serum, after 6-DMAP solution is washed 2 times, put into 6-DMAP (TCM199 of 6-DMAP 2.5mmol/L15% people bleeding of the umbilicus serum) and be activated in solution 1.5-2 hour, in the TCM199 cleaning medium that contains 15% people's bleeding of the umbilicus serum, cultivate the activated cell under 37 ℃, 5%CO2 environment.Fused cell was cultivated 2-3 days through external continuation, made cell development become 2 embryonic cells, morula, blastaea.The foundation of embryonic stem cell line be with the inner cell layer of morula or blastaea with 2.5% trypsinase 4 ℃-37 ℃ digestion 1-6 hour, obtain single embryonic stem cell, cultivation continues to go down to posterity.
Five, the good effect that uses this patented technology to produce
This invention provides ripe body-cell neucleus transplanting to make up embryonic stem cell technology from the body gene. This technology Success, afford ample material for adopting embryonic stem cell from the body gene to carry out the clinical treatment Journal of Sex Research; For Human cloning tissue and organ do not have rejection transplantation treatment technology and lay the foundation; Embryonic stem cell is external orientable Be induced to differentiate into various cells, tissue and organ brand-new, no immune different originality that needed by human body is wanted, be used for Replace cell, tissue, the organ of pathology, can avoid the generation of graft-rejection, treatment can not be treated at present Numerous disease (such as blood disease, AIDS, tumour etc.), show wide potential applicability in clinical practice, with right The development of Medical Biology produces great impetus, and can produce obvious economic results in society.
Claims (13)
1, a kind of method of cloning the people of no animal component from body gene embryonic stem cell, this method may further comprise the steps: a, preparation people donorcells; B, oocyte collection are cultivated; C, remove the cumulus cell around the ovocyte, puncture ovocyte zona pellucida removes polarity corpusculum and kytoplasm on every side to obtain breach from this breach, obtains non-nucleus egg mother cell; D, donorcells nuclear is moved in the non-nucleus egg mother cell; E, fused cell develop into morula, blastaea through activating the back vitro culture; F, blastomere digestion is obtained single embryonic stem cell, set up embryonic stem cell line, it is characterized in that described ovocyte is by to volunteer's induced ovulation, gathers human oocyte.
2, the method for a kind of embryonic stem cell of cloning people according to claim 1 is characterized in that induced ovulation, gathers human oocyte.The donor is from 0.1 milligram of 21 days menstrual cycles start injection Wy-42462, injects to change half amount after 7 days, up to HCG injection, injection 3-5 days every days of menstrual onset fruit receives fragrant 2-3 and props up, up to HCG injection, when follicular development to the 16-18 millimeter, when having 1-2, HCG injection 1000 units.Inject back 11-12 hour under the vagina B ultrasonic monitors, adopt puncture needle, from ovary follicle, aspirate liquor folliculi, place the plate that contains developing medium, cultivated 4-6 hour, ovocyte is reached maturity, preferably exist polarity corpusculum, kytoplasm the ovocyte that is in II stage metaphase of mound cellular layer of sufficient amount to be arranged evenly, on every side then as the acceptor ovocyte, the cell that optimizes is placed in the plate, cleans with TCM199.
3, the method for a kind of embryonic stem cell of cloning people according to claim 1 is characterized in that the human embryo stem cell gene that makes up comes from donorcells fully.
4, the method of a kind of embryonic stem cell of cloning people according to claim 1, the stoning that it is characterized in that described step c ovocyte is that preferred ovocyte is handled through Unidasa, method by moving liquid with internal diameter 140-180 micron glass pipette repeatedly in the TCM199 cleaning medium that contains 10% bleeding of the umbilicus serum, remove ovocyte cumulus cell on every side, clean with the TCM199 cleaning medium then, again they are moved into and contain the TCM199 developing medium, in DMSO and the cytochalasin B solution 5 minutes, adopt little transfer pipet to hold ovocyte, puncture zona pellucida to obtain a breach with micro-needle, draw polarity corpusculum and kytoplasm on every side from this breach with microcapillary, promptly draw the kytoplasm of polar body and polar body near region, to obtain non-nucleus egg mother cell, clean enucleation oocyte, and in the TCM199 developing medium, cultivate.
5, the method for a kind of embryonic stem cell of cloning people according to claim 4, what it is characterized in that the polar body near region kytoplasm drawn is the 20-40% of kytoplasm total amount.
6, the method for a kind of embryonic stem cell of cloning people according to claim 1 is characterized in that the donorcells system for preparing among the step a is mainly the lymphocyte in the peripheral blood.
7, the method for a kind of embryonic stem cell of cloning people according to claim 6 is characterized in that preferred donorcells is the lymphocyte that obtains in in-vitro separation or the lymphocyte cultivated through 24 hours RPMI-1640s.
8,, it is characterized in that somatocyte system stores by the cultivation of going down to posterity, freezing method according to the method for the described a kind of embryonic stem cell of cloning people of claim 1.
9, the method for a kind of embryonic stem cell of cloning people according to claim 8, it is characterized in that in the culturing process that goes down to posterity of described somatocyte system, do not contain animal serum in the nutrient solution, be mainly RPMI1640 and add 10%-20% people's bleeding of the umbilicus serum and 100U penicillin-Streptomycin sulphate, at 37 ℃, the 5%CO2 environment is cultivated down.
10, the method for a kind of embryonic stem cell of cloning people according to claim 1, it is characterized in that activating in the steps d is to make the electricity consumption fusion instrument in merging medium, with the 120-140 volt of 1 second each 80-120 microsecond at interval/centimetre electric pulse make cell realize the electricity fusion, cell after the fusion is by washing at 6-DMAP 2 times, putting into 6-DMAP solution activates, in the TCM199 cleaning medium that contains 10% people's bleeding of the umbilicus serum 37 ℃, 5%CO
2Environment is cultivated down.
11, the method for a kind of embryonic stem cell of cloning people according to claim 10 is characterized in that containing 0.3M/L N.F,USP MANNITOL, 0.01mmol/L CaCl in the described electricity fusion medium
2, 0.1mmol/LMg
2SO
4
12, the method for a kind of embryonic stem cell of cloning people according to claim 10, the vitro culture that it is characterized in that fused cell are in the TCM199 developing medium that contains 15-20% people's bleeding of the umbilicus serum and 100U penicillin-Streptomycin sulphate, put 37 ℃, 5%CO
2Environment was cultivated 2-3 days down, made cell fission become 2 embryonic cells to morula or blastaea.
13, the method for a kind of embryonic stem cell of cloning people according to claim 1, the foundation that it is characterized in that step f embryonic stem cell line be with the inner cell layer of morula or blastaea with 2.5% trypsinase 4 ℃-37 ℃ digestion 1-6 hour, obtain single embryonic stem cell, cultivation continues to go down to posterity.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754654A (en) * | 2016-12-02 | 2017-05-31 | 内蒙古大学 | Mammalian artificial embryo based on stem cell builds method and its model that animal cloning is bred |
| CN101233226B (en) * | 2005-06-22 | 2017-08-11 | 阿斯特利亚斯生物治疗股份公司 | The suspension culture of human embryo stem cell |
| CN109641015A (en) * | 2015-10-09 | 2019-04-16 | 儿童医疗中心有限公司 | By removing tri-methylated increase human somatic cell nuclear transfer (SCNT) efficiency of histone H 3-lysine, and the method and composition of increase mankind NT-ESC derivative |
| CN110426949A (en) * | 2019-06-27 | 2019-11-08 | 南京航空航天大学 | A kind of unicellular operation micro-nano control method can be used for nucleus extraction |
| CN110551682A (en) * | 2018-06-01 | 2019-12-10 | 深圳华大生命科学研究院 | Kits and methods for dissociation of animal embryos |
-
2002
- 2002-09-18 CN CNA021307229A patent/CN1483817A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101233226B (en) * | 2005-06-22 | 2017-08-11 | 阿斯特利亚斯生物治疗股份公司 | The suspension culture of human embryo stem cell |
| CN109641015A (en) * | 2015-10-09 | 2019-04-16 | 儿童医疗中心有限公司 | By removing tri-methylated increase human somatic cell nuclear transfer (SCNT) efficiency of histone H 3-lysine, and the method and composition of increase mankind NT-ESC derivative |
| CN106754654A (en) * | 2016-12-02 | 2017-05-31 | 内蒙古大学 | Mammalian artificial embryo based on stem cell builds method and its model that animal cloning is bred |
| CN110551682A (en) * | 2018-06-01 | 2019-12-10 | 深圳华大生命科学研究院 | Kits and methods for dissociation of animal embryos |
| CN110426949A (en) * | 2019-06-27 | 2019-11-08 | 南京航空航天大学 | A kind of unicellular operation micro-nano control method can be used for nucleus extraction |
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