CN101384703A

CN101384703A - A method for obtaining a xeno-free hBS cell line

Info

Publication number: CN101384703A
Application number: CNA2006800460047A
Authority: CN
Inventors: H·塞姆; R·斯特雷赫尔; S·J·许尔纳; C·埃勒斯特罗姆; K·弗雷吉; K·莫亚; E·K·克尔玛丽
Original assignee: Cellartis AB
Current assignee: Takara Bio Europe AB
Priority date: 2005-10-07
Filing date: 2006-10-06
Publication date: 2009-03-11

Abstract

A method for obtaining a stable xeno-free hBS cell line, xeno-free hBS cell lines obtained according to said method and use thereof. The method comprises the steps of: i) removing the zona pellucida from a blastocyst to obtain trophectodermenclosed inner cell mass by a xeno-free procedure, ii) at least partly removing the trophectoderm to obtain isolated inner cell mass cells by a xeno-free procedure, iii) placing the inner cell mass cells on a layer of human feeder cells in a xeno-free medium, iv) co-culturing of the inner cell mass cells with human feeder cells for a time period of from about 5 days to about 50 days in a xeno-free medium, v) releasing the inner cell mass cells or cells derived thereof from trophectoderm overgrowth, if any, by a xeno-free procedure, vi) selectively,; transferring the inner cell mass cells or cells derived thereof to a fresh layer of human feeder cells in a xeno-free medium to obtain xeno-free hBS cells, vii) propagating the xeno-free hBS cells by co-culturing with human feeder cells in a xeno-free medium to obtain a xeno-free hBS cell line. The xeno-free hBS cell line is suitable for use in medicine and in in vitro testing.

Description

Be used to obtain do not have the method for the hBS clone of allos material

Invention field

HBS cell and its purposes of the no allos material that the present invention relates to obtain the method for (xeno-free) hBS cell of stable no allos material, obtains according to described method.

Background of invention

Stem cell is the unique ability that has self and produce the cell of specialization or differentiation.Although the most cells of health for example heart cell and skin cells is responsible for carrying out the function of specialization, thereby stem cell forms not responsible function of carrying out specialization before the cell type of specialization at its received signal.What make the stem cell uniqueness is the ability of its multiplication capacity and its specialization that becomes.For many years, the investigator concentrates on always and finds to use stem cell to replace the method for cell that lose, ruined or sick and tissue.Up to the present, great majority research concentrates on two types stem cell, embryonic stem cell and adult stem cell.The zygote of (pre-implanted) before embryonic stem cell derives from and implants, promptly blastocyst forms the soma cell and is present in the adult organism body, for example is present in marrow, epidermis and the intestines.Multipotency detects (Pluripotency test) can produce all cells although shown the stem cell (after this being called blastocyst derived stem cell (blastocyst-derivedstem cell) or BS cell) that derives from embryo or blastocyst in organism, comprise sexual cell, but adult stem cell has more limited cell bank in the progeny cell type.

Perhaps the most far-reaching potential application of hBS impact cell is to produce cell and the tissue that can be used for so-called cell therapy.Numerous disease or illness are to be caused by the interruption of cell function or systemic destruction.Today, organ and the tissue with donations is used for replace diseased or ruined tissue usually.Unfortunately, the number of the illness of these methods treatment that up to the present is adapted to pass through has surpassed and can obtain to be used for the transplanted organ number.The availability of hBS cell is keeping the lead further investigation of effective ways of different cell fates (for example producing the neurone of β cell, myocardial cell and the generation Dopamine HCL of Regular Insulin) of these cells in the future in for example diabetes, myocardial infarction and the parkinsonian hope that increases day by day of using based on the treatment of cell of degenerative disease (degenerative diseases) with development is used for.

Yet, all present obtainable people's blastocyst derived stem cells (hBS) tie up to its derive and/or keep during some the point on directly or indirectly be exposed to animal material.A potential consequence using the hBS cell that is polluted by xeno in the patient is the possibility that increased transplant rejection people such as [, 2005] Martin JM.In addition, xenobiotic exposes (xeno-exposure) has increased the risk that shifts inhuman pathogenic agent in any clinical application.Therefore, the harm relevant with use the hBS cell be exposed to xeno in the patient of expection makes this type of cell not be suitable for clinical application.In order to try hard to overcome these problems, several research groups use no raiser's matrix (feeder-free matrices) [people such as xu C, 2001] or the feeder cell in people source carry out the deriving of hBS clone [people such as Richards M, 2002, people such as Inzunza J] and cultivate people such as [, 2003] Richards M.Yet, up to the present still can not in the system that does not have the allos material fully, derive and cultured continuously hBS clone.In order to obtain and to cultivate and do not have the hBS of allos material cell fully, must overcome 3 major obstacles.The first, must develop the scheme of the hBS clone that is used under the condition of no allos material, deriving new.Carry out inner cell mass (inner cell mass) separation (ICM) by the immunosurgery method, described immunosurgery method is to comprise the method for using guinea pig serum classically.We report [people such as Heins, 2004] in early days: can get rid of the immunosurgery method, thereby can carry out the separation of ICM cell and be not exposed to the component of animal.The second and the 3rd, it is essential that raiser's culture systems of no allos material combines with the use of the substratum of no allos material.Because what cause that xeno pollutes is not minimum medium, but foetal calf serum (FBS) that generally uses or the serum that comprises various animal proteinums dives for thing, and whether we determine to check the hBS cell can cultivate for a long time in human serum.Be used for about human serum the hBS cell cultures purposes before report the cultivation stability that considerably less and this cultivation only shows short-term, promptly be less than the stability in 10 generations people such as [, 2002 and 2004] Richards.The difficulty that the substratum of the additional human serum of the use that other people report makes the hBS cell maintain undifferentiated state encourages the present inventor to seek the completely specified culture condition of development at the very start promptly not to be had raiser's culture and has the substratum (definite substratum) of complete known composition and concentration rather than the hBS cell culture system of development end user and synthetic component.Yet the present inventor can have been developed based on human fibroblasts and the proliferating system that is used for the hBS cell that comprises the substratum of serum described herein.HBS clone SA121 (setting up according to the method for WO2003055992) now successfully cultivates generation more than 50 in based on the substratum of this serum.

In addition, the present inventor has been developed and is used to derive and cultivate the system that does not have the people of allos material feeder cell fully.Reported the people's feeder cell human foreskin fibroblast for example that has no allos material before, although in many cases in the actual foundation of feeder cell and culturing process feeder cell contact with many animal materials.

Reported in the present invention only use satisfy regulate the component that requires for example people or synthetic source component be used for deriving and keeping the successful exploitation of the system that does not have the allos material fully of cultivation of hBS cell.Method described herein has been avoided the direct or indirect exposure to animal ingredient fully, the hBS cell that can obtain not have fully the allos material thus with further with the unrestricted source that acts on the developing cells therapy and other purposes (for example being used for the discovery of medicine and exploitation, toxicity test), be used for for example manufacturing of antibody and vaccine of medicine.

Detailed Description Of The Invention

The present invention relates to be used to obtain do not have the method for the hBS clone of allos material, the method comprising the steps of:

I) remove the inner cell mass that zona pellucida (zona pellucida) surrounds with the acquisition trophectoderm by the operation of no allos material from blastocyst,

Ii) remove trophectoderm at least in part obtaining isolating inner cell mass cell by the operation of no allos material,

Iii) the inner cell mass cell is placed on the people's feeder layer in the substratum of no allos material,

Iv) inner cell mass cell and people's feeder cell are cultivated about 5 days altogether to about 10 days time bar in the substratum of no allos material,

V) if any, the operation by no allos material discharges the inner cell mass cell or derives from its cell from the trophectoderm of hypertrophy,

Vi) optionally, by with inner cell mass cell or the cell transfer that derives from it to the Freshman feeder layer in the substratum of no allos material, with the hBS cell of the no allos material of acquisition,

Vii) breed the hBS cell of no allos material to obtain the hBS clone of no allos material by in the substratum of no allos material, being total to cultivation with people's feeder cell.

In specific embodiment, the invention provides the method for the hBS clone that is used to obtain above-mentioned no allos material, but the starting point in this method can be the arbitrary step in the above-mentioned steps, that is, method of the present invention can comprise step I i)-step vii), step I ii)-step vii), step I v)-step vii), step v)-step vii), step vi)-step vii) or step vii) (or the step of mentioning below viii)).

As used herein, term " no allos material " wish material that expression never directly or indirectly is exposed to the non-human animal source for example cell, tissue and/or body fluid with and derivative.

In order to keep the hBS clone of the no allos material that obtains according to the present invention, method can further comprise step

Viii) by in the substratum of no allos material, cultivating altogether and with about 2 days to about 20 days with people's feeder cell, made passage to suitable timed interval of about 12 in for example about 4 days, thus the clone of the no allos material of propagation.

The viii) middle timed interval of selecting of step depends on the cell growth.

Above-mentioned steps viii) also is a special aspects of the present invention.

May want in no feeder layer culture systems, to keep the hBS clone of the no allos material of acquisition, thereby method of the present invention can further comprise step

Ix) hBS clone that will not have an allos material is transferred to no allos material and no feeder layer culture systems.

Can carry out step I x according to WO2004099394), this paper is integrated with in described application by reference.

Step I)

Zona pellucida is the thick extracellular matrix around Mammals ovum (ovum) that is rich in glycoprotein.Can be by using a) acidic solution, b) recombinase for example Unidasa or PRONASE A (pronase) or c) mechanical means remove zona pellucida with in step I from zygote) obtain the inner cell mass of trophectoderm encirclement.Can observe the degradation process of zona pellucida by the visual inspection of microscopically.

As used herein, term " inner cell mass that trophectoderm surrounds " is intended to be illustrated in zygote is experienced step I) the back material that obtains, wherein remove zona pellucida by acidic hydrolysis, enzymic digestion or mechanical means.

The recombinase of Shi Yonging has the aminoacid sequence corresponding to the human amino acid sequence of these enzymes herein, and promptly the recombinase that herein uses has the human amino acid sequence but produces by reorganization.

When removing zona pellucida by the use acidic solution, blastocyst was experienced acidic solution about 5 seconds to about 180 seconds, for example about 10 seconds to about 120 seconds, about 15 seconds to about 90 seconds, about 20 seconds to about 60 seconds, about 30 seconds to about 50 seconds.

Importantly, the pH of acidic solution is enough low for the carbohydrate structure of hydrolysis zona pellucida, that is, the pH of acidic solution is about 2 to about 3, for example about 2.5 to about 2.8, and for example 2.5.Can use any suitable acid.In a preferred embodiment of the invention, acidic solution be have pH 2.5 ± 0.3 acid tyrode's solution (Acid Tyrodes solution) (Sigma).

If, then blastocyst is accepted the solution of one or more recombinases by using one or more recombinases to remove zona pellucida.One or more recombinases have the aminoacid sequence corresponding to the human amino acid sequence of these enzymes.

The example of suitable enzyme is for example recombinate PRONASE A, reorganization Unidasa and recombinant trypsin.The other example of suitable cellular enzymes Digestive system is reorganization or that do not have the allos material and the enzyme of combined protein hydrolysis and CA, for example Accutase potentially in the step (i) ^TM(Chemicon) and be the enzymic digestion liquid of reorganization or that do not have the allos material, combined protein hydrolysis potentially, procollagen and DNA enzymic activity Accumax for example ^TM(InnovativeCell Technologies).

Provide suitable concentration and treatment time below:

PRONASE A: 10U/ml carried out about 2 to about 20 minutes, and for example about 2 to about 10 minutes, the time bar of about 2 to about 5 minutes or about 3 to 4 minutes (the best).

Unidasa: 70.000U/ml carries out about 2 to about 240 minutes time bar.

People's recombinant trypsin: 5-10.000U carries out about 0.5 to about 30 minutes time bar.

Also can remove zona pellucida, wherein for example under by means of microscopical visual inspection, use glass capillary by mechanical means.

Step I i)

After removing zona pellucida, be the inner cell mass experience step I i that trophectoderm surrounds with the remainder of blastocyst) to remove trophectoderm at least in part.Selectively, spontaneous blastocyst of hatching directly can be experienced step I i), thereby skip over step I).

Can be by using a) acidic solution, b) one or more recombinases or c) mechanical means carries out step I i).

When carrying out step I i by the use acidic solution) time, will be in step I) in the inner cell mass experience acidic solution that surrounds of the trophectoderm that obtains, carried out about 5 seconds to about 180 seconds, for example about 10 seconds to about 120 seconds, about 15 seconds to about 90 seconds, about 20 seconds to about 60 seconds, about 30 seconds to about 50 seconds.

Importantly, the pH of acidic solution is enough low for the protein properties structure of hydrolysis trophectoderm, and promptly the pH of acidic solution is about 2 to about 3, for example about 2.5 to about 2.8, and for example 2.5.Can use any suitable acid.In a preferred embodiment of the invention, acidic solution is the acid tyrode's solution (Sigma) of pH 2.5 ± 0.3.

If, the inner cell mass of trophectoderm encirclement is experienced the solution of one or more recombinases by using one or more recombinases to remove trophectoderm at least in part.One or more recombinases are the recombinases that have corresponding to the aminoacid sequence of the human amino acid sequence of these enzymes.

Suitable enzyme is the recombinant protein lytic enzyme, and for example serine protease comprises recombinant trypsin and TrypLE ^TMSelect.If use TrypLE ^TMSelect is usually by with step I) in the inner cell mass that surrounds of the trophectoderm that obtains experience the undiluted TrypLE that promptly uses concentration ^TMSelect carried out about 0.5 to 10 minute, for example about 0.5 to about 8 minutes, about 0.5 to about 5 minutes, about 1 to about 3 minutes, for example carried out step I i in 1.5 minutes).If the use recombinant trypsin is usually by with step I) in the inner cell mass that surrounds of the trophectoderm that obtains experience the recombinant trypsin of about 5.000U to about 10.000U, carry out carrying out in about 0.5 minute to about 30 minutes step I i).

The other example of step suitable cellular enzymes Digestive system in (ii) is reorganization or that do not have the allos material, the enzyme of combined protein hydrolysis and CA, for example Accutase potentially ^TM(Chemicon) and be enzymic digestion liquid reorganization or that do not have the allos material, combined protein lytic enzyme, procollagen enzyme and DNA enzymic activity potentially Accumax for example ^TM(Innovative CellTechnologies).

Also can remove trophectoderm at least in part, wherein can carry out described method as cutting tool by using glass capillary by mechanical means.Use microscope visually easily to carry out the detection of inner cell mass cell.

Step I ii)

Step I ii) in, usually by using glass capillary with step I i at microscopically) afterwards the material of gained place on people's trophoderm.In addition, if desired, for example PRIMARIA of suitable culture dish can pack people's trophoderm into

In the plastic culture dish.If desired, available suitable substrate material is coated with the culture surface of shop culture dish, as long as this substrate material is no allos material.The material that is suitable for this background comprises recombinant human gelatin.

Step I v)

The step I of method of the present invention v) in, with inner cell mass co-culture of cells about 5 days to about 50 days, for example about 5 days to about 30 days, about 5 days to about 20 days or about 5 days were to about 15 days time bar, thus amplifying cells colony.In one embodiment of the invention, the inner cell mass cell is cultivated 10 days time bar in step I in v) altogether.

Can carry out once or the replacing of more times substratum to about substratum of 80%, about 40% to about 60% by changing about 20% to about 100% for example about 30% between common incubation period in v) in step I at the inner cell mass cell.In one embodiment of the invention, about 50% substratum carries out once or the replacing of more times substratum by changing between common incubation period at the inner cell mass cell in v) in step I.Can be with about 2 days to about 14 days, carried out once or the replacing of more times substratum to about 7 days timed interval in for example about 4 days.

Because when when step I is placed the inner cell mass cell in ii), may residual trophectoderm cell being placed on people's feeder layer, therefore can regularly use microscopical visual inspection, whether disturb the growth of the cell in inner cell mass cell or inner cell mass source to see trophectoderm.Bringing cell into step proper time point v) is inspection by microscopically, in the time of a couple of days, observed raised growth in.

In the process that step I is bred in v), some in these cells may begin their and do (BS) transformation to the blastocyst source at the inner cell mass cell.Therefore, the cell colony that obtains in v) of step I can comprise the inner cell mass cell with and the deutero-cell be the BS cell.

Step v) and vi)

As mentioned above, the inner cell mass cell can be polluted by residual trophectoderm with the cell that derives from it.If situation is like this, trophectoderm tends to surround the cell in inner cell mass somatocyte and its source so.Can be by using a) mechanical means or b) one or more recombinases discharge the inner cell mass cell or derive from its cell from the trophectoderm of this hypertrophy.

Suitable mechanical means is to use glass capillary as cutting tool.Optionally cut out the cell mass cell or derive from its cell based on the visual inspection of microscopically, then it is transferred on the Freshman feeder layer in the substratum of no allos material that (step vi) with the hBS cell that obtains no allos material.

Selectively, can (for example, one or more recombinant protein lytic enzymes comprise recombinant trypsin and TrypLE by using one or more recombinases in v) in step ^TMSelect) from the trophectoderm (if any) of hypertrophy, discharge inner cell mass cell or derive from its cell.If use recombinant trypsin to discharge the inner cell mass cell in v) or derive from its cell, usually with the inner cell mass cell or derive from its cell and about 5.000U to the trypsinase incubation of about 10.000U about 0.5 minute to about 30 minutes in step.As use TrypLE ^TMSelect discharges the inner cell mass cell in v) or derives from its cell in step, usually with the inner cell mass cell or derive from its cell and the undiluted TrypLE that promptly uses concentration ^TMSelect together incubation about 0.5 to about 15 minutes.

Step (v) and (the other example of suitable cellular enzymes Digestive system (cell enzymaticsolution) is for example Accutase of enzyme reorganization or that do not have the allos material, combined protein hydrolysis and CA potentially vi) ^TM(Chemicon) and be enzymic digestion liquid reorganization or the hydrolysis of combined protein potentially, procollagen and the DNA enzymic activity of not having the allos material Accumax for example ^TM(Innovative Cell Technologies).

After from trophectoderm (if any), discharging the inner cell mass cell or deriving from its cell, select the inner cell mass cell or derive from its cell based on the visual inspection of microscopically, (step vi) with the hBS cell of the no allos material of acquisition then it to be transferred to Freshman feeder layer in the cultivation of no allos material.

Step vii)

Step vii) in, by cultivating the hBS cell of breeding no allos material altogether, thereby obtain the hBS clone of no allos material with people's feeder cell.Can carry out a generation during the propagation at the hBS of no allos material in vii) or more generations go down to posterity in step, wherein according to the visual inspection selectivity of the microscopically hBS cell that goes down to posterity.Can use glass capillary to carry out these as cutting tool goes down to posterity.Selectively, can use for example TrypLE of one or more recombinases ^TMSelect, recombinant trypsin and/or recombinant collagen enzyme carry out a generation in vii) or more generations go down to posterity in step.

If use TrypLE ^TMSelect is usually by using the undiluted TrypLE that promptly uses concentration ^TMSelect carries out carrying out step vii) in about 0.5 minute to about 15 minutes.

If the use recombinant trypsin is undertaken carrying out step vii) in about 0.5 minute to about 30 minutes by the recombinant trypsin of using the extremely about 10.000U of about 5.000U usually.

If use the recombinant collagen enzyme, undertaken carrying out step vii) in about 1 minute to about 40 minutes by the recombinant collagen enzyme that uses about 200U/ml usually.

(the other example of suitable cellular enzymes Digestive system is for example Accutase of enzyme reorganization or that do not have the allos material and combined protein hydrolysis and CA potentially to step vii) ^TM(Chemicon) and be enzymic digestion liquid reorganization or that do not have the allos material and combined protein hydrolytic activity, CA and DNA enzymic activity potentially Accumax for example ^TM(InnovativeCell Technologies).

Being used for substratum that individuality goes down to posterity can be identical or can be different.

Step viii)

The propagation of the hBS clone of no allos material is consistent with the vii) middle propagation of describing of step.

In one embodiment of the invention, can cut apart by machinery and for example use glass capillary to carry out the passage of step in viii) as cutting tool.Selectively, can be by using for example TrypLE of one or more recombinases ^TMSelect, recombinant trypsin and/or recombinant collagen enzyme carry out the passage of step in viii).Use TrypLE ^TMThe concentration of Select, recombinant trypsin and recombinant collagen enzyme and incubation time are respectively with vii) described the same for step.

(the other example of the suitable cellular enzymes Digestive system viii) is for example Accutase of enzyme reorganization or that do not have the allos material and combined protein hydrolytic activity and CA potentially to step ^TM(Chemicon) and be enzymic digestion liquid reorganization or that do not have the allos material and combined protein hydrolytic activity, CA and DNA enzymic activity potentially Accumax for example ^TM(Innovative Cell Technologies).

Step I x)

In particular of the present invention, at step I X) in the hBS clone that obtains is transferred to no allos material, in no raiser's the culture systems, the supported matrix that described culture systems comprises suitable no allos material is recombinant human gelatin for example, recombinant human fibronectin polypeptide, the substratum of people's placenta cells epimatrix and suitable no allos material, the substratum of described no allos material can (step I be ii) with the foundation that is used for hBS clone, iv), vi), vii)) or to be used for the substratum of no allos material of keeping on people's feeder cell (step viii)) identical or different.In order to keep the undifferentiated growth of hBS clone, for example the recombinant bfgf of high density and/or the activator of WNT approach replenish this substratum but the conditioning of end user's feeder cell does not have the substratum of allos material or the available suitable factor.

People raiser's preparation

Under the condition of no allos material, obtain step I ii), vi), vii) and the people's feeder cell that use in the arbitrary step viii).People's feeder cell derive from healthy people's tissue and can obtain by biopsy.

The tissue that can comprise embryo, fetus, newborn infant, teenager or adult from its people tissue that produces people's feeder cell, it also comprises and derives from the tissue that skin comprises foreskin, umbilical cord, muscle, lung, epithelium, placenta, uterine tube, gland (glandula), matrix or mammary gland.People's feeder cell can derive from and human fibroblasts, fibrocyte (fibrocyte), myocyte, keratinocyte, cell type that endotheliocyte is relevant with epithelial cell.The example that can be used for producing the particular cell types of people's feeder cell comprises embryo fibroblast, the extraembryonic endoderm cell, the extraembryonic mesoderm cell, fetal fibroblast and/or fibrocyte, the fetus myocyte, fetal skin cell, the fetus pneumonocyte, the fetus endotheliocyte, the human fetal epithelium cell, umbilical cord mesenchyma cell (umbilical chord mesenchymal cell), placenta inoblast and/or fibrocyte, the placenta endotheliocyte, birth back human foreskin fibroblast and/or fibrocyte, birth back myocyte, birth back skin cells, birth back endotheliocyte, adult skin inoblast and/or fibrocyte, become human muscle cell, adult's uterine tube endotheliocyte, adult's endometrial glandular cell (adult glandular endometrial cell), adult's endometrial stromal cell (adult stromal endometrial cells), become the human breast carcinoma parenchyma, become human endothelial cell, become the human epithelial cell or become human keratinized cell.

But immortalization or genetic modification are used for feeder cell of the present invention.The immortalization of feeder cell means in cultivation the acquisition of growth by the ability of the division number of times of theory unlimited.Have the several method be used to carry out immortalization, method is to realize immortalization with virus for example, retrovirus transformant and/or the expression by reverse transcriptase of telomere albumen (TERT).TERT is non-activity in most cells, but when exogenous expression hTERT, cell can keep being enough to avoid the telomere length of replicability aging.

In addition, can carry out genetic modification, special genes is integrated into genome feeder cell.These gene codified purpose marks or be used for known to the useful biomolecules of hBS cell for example somatomedin (for example bFGF's (basic fibroblast growth factor) is synthetic.

When people's feeder cell derived from the hBS cell, the cell that derives from the hBS cell can be an inoblast.

In one embodiment of the invention, people's feeder cell derive from newborn infant people's foreskin.

In particular of the present invention, people's feeder cell are inoblasts, preferably derive from people's neonatal human foreskin fibroblast.

Can get people's foreskin sample from the boy baby of ring cutting.Can in aseptic suitable medium, for example comprise in the aseptic IMDM substratum of 2X gentamicin (Invitrogen) and sterilely select sample.The skin explant is placed for example 25cm of tissue culture flasks ²Pericardium (primaria) tissue culture flasks (Becton Dickinson) in, this culturing bottle is equipped with the human serum of IMDM substratum (Invitrogen), 1% penicillin-Streptomycin sulphate (Gibco Invitrogen Corporation) and 10% (people such as Tallheden, 2005)After about 10 days, the individual layer that converges is set up.Use TrypLE ^TMSelect (Invitrogen) continuous passage cell.It has been observed by the present inventors that each suitable time bar between going down to posterity is about 2 to about 20 days, going down to posterity at least 15 times usually is suitable for example going down to posterity for maximum 20 times.After amplification, they are detected with regard to the list (comprising mycoplasma, 1 type and 2 type HIV, hepatitis B and hepatitis C, cytomegalovirus, 1 type and herpes simplex types 2 virus, Epstein-Barr virus and human papillomavirus) of human pathogen.

The substratum of no allos material

Substratum can be any minimum medium that is suitable for the cell proliferation of people's inner cell mass.A kind of suitable medium is Dulbecco ' s Modified Eagle ' the s substratum (DMEM) that is supplemented with the recombinant bfgf of the human serum of 1-30%v/v and 2-100ng/ml.In one embodiment, minimum medium comprises the human serum of 20% v/v.In another embodiment, minimum medium comprises the recombinant bfgf of 10ng/ml.Can use other minimum mediums, if they with the liquid form for the inner cell mass cell matrix provide nutritive ingredient (be inorganic components for example the trace element and organic composition for example amino acid, salt, VITAMIN, energy supplier, carbohydrate comprise sugar etc.).Importantly, substratum is no allos material.

Be used for step I ii), iv), vi), vii) and/or viii) the substratum of the no allos material of arbitrary step comprise the minimum medium of the propagation that is suitable for people's inner cell mass cell.A kind of suitable minimum medium is a Dulbecco ' s Modified Eagle ' s substratum (DMEM).Yet other minimum mediums can be worked too.Except the minimum medium of no allos material, the substratum of no allos material can further comprise human serum, recombinant bfgf, L-glutaminate or glutamax, non-essential amino acid, beta-mercaptoethanol, penicillin and/or Streptomycin sulphate.

Preferably about 1% v/v of the concentration of human serum is to the human serum of about 30% v/v in the substratum of no allos material, for example about 10% v/v is to the human serum of about 30% v/v, about 15% v/v is to the human serum of about 25%v/v and the more preferably human serum of 20% v/v.

The preferably about 2ng/ml of the concentration of recombinant bfgf is to the recombinant bfgf of about 100ng/ml in the substratum of no allos material, for example about 5ng/ml is to the recombinant bfgf of about 50ng/ml, approximately 5ng/ml is to the recombinant bfgf of about 25ng/ml, approximately 5ng/ml is to the recombinant bfgf of about 15ng/ml, for example recombinant bfgf of 10ng/ml.

L-glutaminate or Glutamax in the substratum of no allos material

Concentration preferably approximately 0.5mM is to about 20mM, for example approximately 0.75mM is to about 10mM, approximately 1mM is to about 5mM 2mM for example.

Preferably approximately 0.01mM is to about 1mM for the concentration of non-essential amino acid in the substratum of no allos material, and for example approximately 0.03mM is to about 0.8mM, and approximately 0.05mM is to about 0.6mM, and approximately 0.07mM is to about 0.4mM, and approximately 0.09mM is to about 0.2mM, for example 0.1mM.

The preferably about 10 μ M of the concentration of beta-mercaptoethanol are to about 200 μ M in the substratum of no allos material, and for example about 25 μ M are to about 175 μ M, and about 50 μ M are to about 150 μ M, and about 75 μ M are to about 125 μ M, for example 100 μ M.

Preferably about 5 the unit/ml of the concentration of penicillin are to about 200 unit/ml in the substratum of no allos material, for example about 10 unit/ml are to about 150 unit/ml, about 25 unit/ml are to about 100 unit/ml, for example about 25 unit/ml are to about 75 unit/ml, for example 50 unit/ml.

The preferably about 5 μ g/ml of the concentration of Streptomycin sulphate are to about 200 μ g/ml in the substratum of no allos material, for example about 10 μ g/ml are to about 150 μ g/ml, about 25 μ g/ml are to about 100 μ g/ml, and about 25 μ g/ml are to about 75 μ g/ml, for example 50 μ g/ml.

The substratum of same other no allos materials also can be used for one or more single step of the present invention, for example be used to breed step (vii) with (viii).This substratum can comprise salt, VITAMIN, energy derive (for example glucose), mineral substance (mineral) and amino acid.The suitable somatomedin that adds in substratum can be for example GABA, nipecotic acid, lithium chloride and transforming growth factor-beta (TGF β) and bFGF.In addition, can chemically defined this substratum.Therefore, can in the substratum except the substratum that comprises serum, go down to posterity or breed the hBS clone that deutero-among the present invention does not have the allos material.

The preparation of human serum

The good human serum of the quality of production repeatedly in our laboratory (people such as Tallheden, 2005)Detect blood at the Blood Center of hospital with regard to the pathogenic agent (hepatitis B, hepatitis C, HIV, HTLV and syphilis) of many standards.

Therefore, preparation process is iii), iv), vi), vii) and the human serum that uses in the arbitrary step viii) through the following steps

A) in the sack that is not coated with the shop heparin, collect the healthy human blood,

B) shake the described sack that is not coated with the shop heparin, carried out about 0.5 hour to about 5 hours, for example about 0.5 hour about 2 hours time bar extremely,

C) incubation is not coated with the sack of shop heparin under 5 ℃ temperature at the most, carries out at least 10 hours time bar

D) randomly, for example do not solidify the selection of the opaqueness of not the existing of fibrin (non-clottedfibrin), liquid phase based on solidifying quality

E) with serum and the material separation of solidifying

F) serum that obtains in the sterile filtration step d)

G) compile serum from least 15 donors

H) use preceding freezing serum.

In specific embodiment of the present invention, the method that is used to obtain not have the hBS clone of allos material comprises step:

1) by at room temperature using acid tyrode's solution incubation blastocyst about 10 seconds to about 10 minutes, preferably approximately 30 seconds is removed zona pellucida to about 60 seconds time bar from blastocyst and to the small part trophectoderm, thereby obtains isolating inner cell mass cell,

2) the inner cell mass cell is placed on the human foreskin fibroblast feeder layer in the substratum of the no allos material that comprises DMEM, human serum, recombinant bfgf, L-glutaminate or glutamax, non-essential amino acid, beta-mercaptoethanol, penicillin and Streptomycin sulphate

3) in the substratum of no allos material, inner cell mass cell and human foreskin fibroblast feeder cell are cultivated about 5 days altogether to about 15 days time bar, the substratum of the no allos material of replacing at least 50% in wherein per 3 to 5 days.

4) if any, by using TrypLE ^TMSelect (Invitrogen) handles the cell that discharges the inner cell mass cell or derive from it from the trophectoderm of hypertrophy as enzyme,

5) optionally, by with inner cell mass cell or the cell transfer that derives from it to the hBS cell of the fresh human foreskin fibroblast feeder layer in the substratum of no allos material with the no allos material of acquisition,

6), thereby obtain the hBS clone of no allos material by in the substratum of no allos material, cultivating the hBS cell of breeding no allos material altogether with the human foreskin fibroblast feeder cell.

In specific embodiment, the invention provides the method for the hBS clone that obtains above-mentioned no allos material, but the starting point in the method can be the arbitrary step in the above-mentioned steps, that is, method of the present invention can comprise step 2) to step 6), step 3) to step 6), step 4) to step 6), step 5) to step 6) or step 6) (or step 7 (mentioning)) as following.

Can carry out the keeping of hBS clone of the no allos material that obtains in the step 6) by following further step, promptly

7) by in the substratum of no allos material, cultivating altogether with the human foreskin fibroblast feeder cell, changed the substratum of at least 50% no allos material and at interval in per 3 to 5 days, the hBS clone that for example per 3 to 8 days pair cells go down to posterity and breed no allos material with reasonable time.

Can in step 1), carry out the removal of zona pellucida by the visual inspection of microscopically.

About the details and the details of the preferred concentration of single component in the substratum of above-mentioned no allos material, with reference to step 2), 3), 5), 6) and 7) in the substratum of the no allos material that uses.

In step 3), can regularly carry out visual inspection to see trophectoderm and whether disturb the inner cell mass cell or to derive from the growth of its cell.

In one embodiment of the invention, by using glass capillary to shift the inner cell mass cell according to the visual inspection of microscopically or deriving from its cell as cutting tool selectivity in step 5).

The going down to posterity of propagation that is suitable for the hBS clone of the no allos material in the step 7) at interval depends on the growth of cell, and goes down to posterity and undertaken by using glass capillary manually to shift.

Other aspects

The other aspects of the present invention that are described below should be with reference to using details and the details of discussing under the superincumbent main aspect.

Another aspect of the present invention relates to the method that is used to obtain do not have the hBS clone of allos material, and it comprises step:

I) blastocyst that never has zona pellucida at least in part of the operation by no allos material is removed trophectoderm obtaining isolating inner cell mass cell,

Ii) the inner cell mass cell is placed on the people's feeder layer in the substratum of no allos material,

Iii) in the substratum of no allos material, inner cell mass cell and people's feeder cell are cultivated altogether, are carried out about 5 days to about 50 days time bar,

Iv) if any, the operation by no allos material discharges the inner cell mass cell or derives from its cell from the trophectoderm of hypertrophy,

V) optionally, by with inner cell mass cell or the cell transfer that derives from it to the Freshman feeder layer in the substratum of no allos material, with the hBS cell of the no allos material of acquisition,

Vi) by in the substratum of no allos material, cultivating the hBS cell of breeding no allos material altogether to obtain the hBS clone of no allos material with people's feeder cell.

Another aspect of the present invention relates to the method that is used to obtain do not have the hBS cell of allos material, and it comprises step:

I) the near small part inner cell mass cell that do not contain trophectoderm is placed on people's feeder layer in the substratum of no allos material,

Ii) inner cell confluent monolayer cells and people's feeder cell are cultivated in the substratum of no allos material altogether, are carried out about 5 days to about 50 days time bar,

Iii) if any, the operation by no allos material discharges the inner cell mass cell or derives from its cell from the trophectoderm of hypertrophy,

Iv) optionally, by with inner cell mass cell or the cell transfer that derives from it hBS cell to the Freshman feeder cell in the substratum of no allos material with the no allos material of acquisition,

V) by in the substratum of no allos material, cultivating the hBS that breeds no allos material altogether, to obtain the hBS clone of no allos material with people's feeder cell.

I) near small part does not contain the inner cell mass cell and the cultivation altogether in no allos material of people's feeder cell of trophectoderm, carries out about 5 days to about 50 days time bar,

Ii) if any, the operation by no allos material discharges the inner cell mass cell or derives from its cell from the trophectoderm of hypertrophy,

Iii) optionally, by with inner cell mass cell or the cell transfer that derives from it to the Freshman feeder layer in the substratum of no allos material, with the hBS cell of the no allos material of acquisition,

Iv) by in the substratum of no allos material, cultivating the hBS cell of breeding no allos material altogether to obtain the hBS clone of no allos material with people's feeder cell.

The invention still further relates to the method that does not rely on the hBS clone that is used to obtain described no allos material no allos material hBS clone propagation and keep cultivation.Substratum and people's feeder cell described herein based on human serum can be done necessary change to adapt to enrichment procedure.More detailed content vii) and viii) is presented in this specification sheets down in step.

The sign of the hBS clone of no allos material

The invention still further relates to the hBS clone of the no allos material that obtains by method of the present invention.The hBS cell that this type of cell is not had the allos material directly or indirectly is exposed to any non-human animal's material, mean method all components in steps be not exposed to any non-human animal's material, for example any material that derives from non-human mammal.Therefore, at people's feeder cell of deriving used according to the invention under without any situation about exposing to any non-human animal's material.The foundation of the hBS clone of no allos material and keep during any organic materials of using be that the people originates or the material of synthetic, semisynthetic or reorganization.

The hBS clone of no allos material of the present invention keeps self and versatility at reasonable time in the time limit, therefore it is stable in the time limit at reasonable time.In this manual, term " stable " is intended to expression when growing on people's feeder layer of the present invention, surpassed for 50 weeks, for example surpasses for 40 weeks, surpasses for 30 weeks, surpasses for 20 weeks, surpasses the ability of breeding in undifferentiated state in 15 weeks.Therefore the hBS clone of no allos material of the present invention is immortal in theory.

The hBS clone of the no allos material that obtains by the method according to this invention can be used for the preparation of noble cells.Therefore the invention still further relates to the cell of this type of differentiation.

In addition, the hBS cell of no allos material of the present invention or clone can experience freezing and melt.In specific embodiment, can be freezing and the hBS clone of melting the no allos material that obtains among the present invention according to the method for vitrification that provides at WO2004098285 (it integrates with this paper by reference) by Cellartis before.

In order to increase the homogeneity of hBS cell culture, the clone who describes among the cell experience WO2005059116 (it integrates with this paper by reference) that obtains among the present invention can be derived.

The hBS clone of the no allos material that obtains by the present invention has satisfied general requirement.Clone has the one or more features in the following feature, has at least 4,5,6,7 or 8 features in the following feature especially.Therefore, this clone

I) when when the feeder cell (mitotically inactivatedfeeder cell) of mitotic division deactivation are gone up growth, show the ability of in undifferentiated state, breeding that surpassed for 15 weeks, and/or

Ii) show normal euploid karyotype, and/or

Iii) show in the training period stable karyotype and/or

Iv) maintenance develops into the potential of the derivative of all types of germinal layers in vitro and in vivo, and/or

V) show following molecule marker OCT-4, alkaline phosphatase, carbohydrate epitope (carbohydrate epitope) SSEA-3, SSEA-4, TRA 1-60, TRA 1-81 and by at least two molecule markers in the protein core of the keratan sulfate/chondroitin sulfate pericellular stromatin glycan (proteinglycan) of monoclonal antibody GCTM-2 identification, and/or

Vi) not display molecule mark SSEA-1 or other differentiation markers, and/or

Vii) keep its versatility and when being injected into the mouse of immunocompromised host, form teratoma in vivo, and/or

Viii) can break up.

The hBS clone of no allos material of the present invention is showed following standard: for the positive reaction of Oct-4, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 with at least 1 in the negative reaction of SSEA-1, and for example at least 2, at least 3, at least 4, at least 5 or at least 6 standards.

Below, the method that is used for characterizing with regard to differential period, versatility and caryogram the hBS of no allos material has been described.These class methods can be used for investigating the hBS cell that obtains according to the present invention and whether satisfy above-mentioned standard.

Immunohistochemistry

Can analyze the hBS clone of no allos material with regard to immunohistochemistry mark Oct-4, TRA-1-60, TRA-1-81, SSEA-1, SSEA-3 and the SSEA-4 of undifferentiated cell to monitor its differentiation state.

Alkaline phosphatase

Alkaline phosphatase and telomerase activation are taken as the mark that does not break up the hBS cell usually.Can measure active by existing any test kit that is purchased.

External versatility

Can detect the versatility of the hBS clone of no allos material by the cultivation that allows colony spontaneous differentiation in culture dish carry out about 3 to 4 weeks, per 2 to 7 days replacing one subcultures.In order to identify cell, use the immunohistochemical analysis colony from 3 different germinal layers.Suitable antibody can be at ectodermic beta tubulin, at mesoblastic ASMA (α smooth muscle actin) with at endoblastic HNF3 β (liver nucleofactor 3 β).

Versatility-teratoma in the body

Analyst BS clone whether keep a method of versatility be with cell xenotransplantation to immunodeficient mouse to obtain tumour, teratoma.The dissimilar tissue of finding in the tumour should be represented all 3 germinal layers.

Severe associativity immune deficiency (SCID)-mouse (lacking bone-marrow-derived lymphocyte and the lymphocytic strain of T) can be used for the neoplastic analysis of monster.Can people BS cell be placed testis or place under the scrotum by operation.In testis or kidney, can in the scope of 000 cell of 10 000-100, transplant the BS cell.After about 1 month, tangible usually to tumour.Killed mouse then after 1 to 4 months, the cutting tumour is fixed to be used for paraffin or freezing microtome section analysis.Analyze tumor tissues by immunohistochemical method then.Can use specific marker at all 3 germinal layers.

Gene characterizes: karyotyping and original position fluorescent hybridization (FISH) and telomerase activation

Use trypsinase-Jim Sa or DAPI dyeing to manifest to be tried the karyomit(e) of the hBS clone of not having the allos material.Analyze for fluorescence in situ hybridization (FISH), can use the obtainable test kit that is purchased of the probe that comprises karyomit(e) 12,13,17,18,20,21 and sex chromosome (X and Y) according to having improved slightly manufacturers instruction.In the inverted microscope that is equipped with suitable spectral filter and software, further analyze slide glass.

Can further characterize stem cell and blastocyst derived stem cell with regard to its telomerase activation, wherein can use the test kit that for example is called Telomerase PCR ELISA test kit (Roche) to detect the activity of described Telomerase.Test kit carries out the amplification of product and uses enzyme-linked immunosorbent assay (ELISA) to detect the inside activity that it utilizes Telomerase by using polymerase chain reaction (PCR).Equally can be by the activity of QPCR measuring junction granzyme.

Gene characterizes: QPCR

Can be by further detect the differentiation state of the cell among the present invention at the QPCR of specific gene.Below, describe how to carry out this detection briefly: can mechanically separate hBS cell colony undifferentiated or differentiation with the form of complete colony,, store down at-80 ℃ then with the PBS washing from culture dish.Can for example using according to manufacturers instruction, Qiagen RNeasyMini test kit further extracts RAN.Thereby use suitable test kit for example the Bio-Rad iScript First StrandSynthesis test kit (according to manufacturers instruction) among the Rotorgene3000 (Corbett Research) carry out reverse transcription and under appropriate condition, carry out QPCR.If possible, can be in identical reaction quantitative all genes and can be by calculating differentiation state based on the more several samples of quantification index (mathematical indices) of the individual sample of genetic marker.(more detailed scheme is described in WO2006094798).

Detection at the human pathogen list

Be used for individual components of the present invention herein, for example feeder cell, serum and blastocyst can be before use, and the hBS of no allos material clone after setting up immediately human pathogen it is detected, described human pathogen is for example mycoplasma, 1 type and 2 type human immunodeficiency viruses, hepatitis B and hepatitis C, cytomegalovirus, 1 type and herpes simplex types 2 virus, Epstein-Barr virus and human papillomavirus.

Not the existing of human pathogen learned any clinical application of material yes very important for the hBS clone and the cell of no allos material or the other biological that derives from clone.

Sialic acid Neu5Gc detects

Can further detect the hBS cell of the no allos material that produces according to the present invention with regard to sialic acid Neu5Gc (it is that film is in conjunction with glycan molecule).The negative findings of this detection can be considered the direct or indirect exposure that does not take place non-human animal's material.

The hBS cell of no allos material of the present invention or the purposes of clone

The hBS clone of no allos material of the present invention can be used for for example progenitor cell and the further preparation of the cell of differentiation of all 3 germinal layers of derivative of its differentiation, the feature of the cell type of the differentiation that the cell display of described further differentiation is different, for example liver cell sample feature, Cardiomyocyte-like feature, neuron feature.

GMP (good Production Flow Chart (Good Manufacturing Procedure)) produces

The hBS clone of no allos material of the present invention can be further used for GMP production, and for example the clinical GMP of the cell of hBS cell and/or its differentiation produces.In addition, can under GMP and/or cGMP condition, carry out the deutero-method that is used to not have the allos material described herein, but clone and derivative (people Nat.Med 2005 such as Martin) so that clinical application to be provided.

Medical usage

The hBS cell of no allos material of the present invention or the derivative of its differentiation can be used in the medical science.The derivative that does not for example have the hBS clone of allos material or its differentiation can be used for the manufacturing of pharmaceutical prod, and described pharmaceutical prod is used to prevent and/or treat pathological conditions and/or the disease that is caused by tissue degeneratiaon (tissuedegeneration).In addition, the hBS cell of no allos material or the derivative of its differentiation can be used for the manufacturing of pharmaceutical prod, and described pharmaceutical prod is used for the treatment of and/or prevents metabolism pathological conditions and/or disease.

The hBS cell that obtains by method of the present invention can be used for the manufacturing of medicine, and described medicine is used for the hBS Transplanted cells of no allos material is gone into Mammals with prevention or treat disease.Specific aspect is to use the hBS cell of no allos material in autotransplantation, promptly only is used to not have the hBS cell of allos material or the preparation of clone from people's material of described particular patient.

Can imagine many different types of diseases, the cell of no allos material wherein of the present invention or clone can be suitable for comprising the disease of hepatopathy, cardiovascular disorder (comprising myocardial infarction), degenerative disease (for example neurodegenerative disease comprises Parkinson's disease and Alzheimer), diabetes.

Such medicine can comprise and is dispersed in for example hBS cell of the no allos material of the hBS cell of the undifferentiated no allos material in the aqueous vehicles or differentiation of pharmaceutically acceptable vehicle.Vehicle can comprise one or more additives that is selected from pH regulator agent, stablizer, sanitas, osmotic pressure regulator and physiologically acceptable salt; And/or one or more are selected from the reagent of therapeutic active substance, preventative active substance, graft immigration promotor (engraftment improvingagent), vigor promotor (viability improving agent), differentiation accelerator (differentiation improving agent) and immunosuppressor.

Regenerative medicine and cell therapy (Regenerative medicine and celltherapy)

Be divided into the multipotency and the ability (carrying out the proliferating cells type to particular tissue type) of complete differentiated tissues cell type and/or progenitor cell type owing to it, the cell that the method by no allos material provided herein produces is provable to be exceedingly useful in regenerative medicine.Along certain bar after for example the biological pathway of multipotent cardiac progenitor cell makes cytodifferentiation, can imagine the treatment of cardiac-related diseases, or for example make cell after hepatic progenitor cells differentiation (as for example proposing among the WO2006034873), can imagine the treatment of liver relative disease, or, can imagine for example multiple sclerosis of sacred disease making cell after the neural progenitor cell differentiation, hypoxia injury (hypoxic injury), the local asphyxia damage, traumatic injury (traumatic injury), the treatment of Parkinson's disease and myelinoclasis disease (demyelition disorder).

The other progenitor cell type that derives from the hBS clone of the no allos material that obtains as described here can be mesoblastic cell (having generation also produces bone and cartilage except the heart cell type potential), maybe can be endoblastic cell (have and also produce for example potential of β cell of pancreas cell type).

For restore funcitons in the heart that suffers cardiac infarction (cardiac infarction), essential myocardial cell and the neovascularity replaced.In the time can obtaining clinical compliance hBS clone (for example hBS clone that produces according to method provided herein), may use this type of cell-derived subsequently can be transplanted and the progenitor cell assessed of the potential use in the people just.This type of progenitor cell can have further original position and be divided into the sex change tissue, for example the potential of the cell type at the position of cardiac infarction.

Can be used for the treatment of the relevant illness of cardiac muscle with for example necrosis, apoptosis, damage, functional disorder (dysfunctional) or morphological abnormalities in addition from the cell of the hBS clone of no allos material differentiation.This type of illness comprises, but be not limited to ischemic heart disease, cardiac infarction, rheumatic heart disease, endocarditis, autoimmunization heart trouble, valvular heart disease, congenital heart disease, heart rhythm disorders and cardiac insufficiency (cardiac insufficiency).Thereby this type of can be bred and cell with differentiation of the potential that is divided into heart cell type (comprising myocardial cell, endotheliocyte and smooth muscle cell) can be suitable for treating most of cardiac conditions and disease by the cardiac damage that the local asphyxia that reverses, suppresses or prevention is caused by myocardial infarction causes.

In other respects, cytodifferentiation is from the cytodifferentiation of the no allos material that provides herein and can be used for treating the cardiac conditions that is characterised in that abnormal heart rhythm, for example irregular pulse.

Preferably by the heart to the experimenter use (preferably by being injected into heart) treatment effective dose cell treat.The treatment effective dose is the amount that is enough to produce useful or the clinical effectiveness wanted, can use this dosage with 1 time or more times.Depend on the heart tissue type that needs reparation, can use injection the different zones of heart.Can use and to use opening the thoracic cavity or enter the back by any suitable blood vessel based on the method for conduit.The effective dose of cell can be based on following factor, the lapse of time after for example body weight, age, physiological situation, medical history, infarct size and local asphyxia take place.The using of cell comprises preferably treated the experimenter with immunosuppressant scheme before this is used, to suppress this repulsion.

Other purposes

The hBS clone of the no allos material that obtains by method of the present invention or the derivative of its differentiation also can be used for medical research, because they are suitable for studying for example external model of people's degenerative disease of human disease very much.

In drug discovery in pharmaceutical industry for example and the drug development and in the toxotest of the chemical pharmaceutical prod of all kinds, find the hBS cell of no allos material itself and from the potential application of its deutero-clone and cell colony.Today, the extensive and high flux screening of drug candidates depends on usually and provides about compound binding affinity and specific information but biochemical measurement method about the information of function seldom or not is provided.Functional screening depends on based on the screening of cell and uses the more weak biology of clinical cognation usually, for example can be with heavy body economical and that produce apace bacterium or yeast.Continuously the bigger model species of clinical cognation are used in the screening of wheel, but these species are more expensive and screening process is non-when expending.Existence is based on the screening implement of the cell type of people's primary cell or immortalization, but these cells owing to the reason of losing vital functions because of vitro culture and conversion supply with or availability on be restricted.The acquisition of the hBS cell of the no allos material that breaks up under the condition of design and hBS cell is provided new and ability uniqueness that has heavy body but do not weaken clinical cognation based on the mensuration of people's cell.

By heavy body being combined with improved clinical meaning the hBS cell of no allos material can being used for high flux screening.In the hBS cell, use gene targeting accurately the ability of modifying gene group (cytodifferentiation of genetic modification become or be not divided into various cell types) allow to use this technology by therapeutic active substance preliminary and that the postsearch screening evaluation is new.

Therefore, the present invention relates in other respects be used for by determining of obtaining of method of the present invention

I) production of monoclonal antibody

Ii) in vitro toxicity examination,

The iii) in-vitro screening of potential drug material, or

The iv) user of the hBS cell of the evaluation of potential drug material.

Description of drawings

Fig. 1 shows handling with acid tyrode's solution with the blastocyst before removing zona pellucida and trophectoderm part under 40 x magnifications.

Fig. 2 shows handling with acid tyrode's solution with the blastocyst after removing zona pellucida and trophectoderm part under 40 x magnifications.

Fig. 3 shows the A under 10 x magnifications) hBS clone SA611 in the 7th generation, the 4th day.

Fig. 4 shows for not breaking up hBS cell marking A) positive reaction of ALP..

Fig. 5 shows for the positive reaction of not breaking up hBS cell marking SSEA-4 with for the negative reaction of differentiation hBS cell marking SSEA-1, and the both was in for the 6th generation and under 20 x magnifications.

Fig. 6 shows for the positive reaction of not breaking up hBS cell marking Tra 1-60 and Tra 1-81, and the both was in for the 6th generation and under 20 x magnifications

Fig. 7 show under 20 x magnifications after 21 to 28 days spontaneous differentiation of course in the 6th generation for the positive reaction of entoderm mark HNF-3 β and neuroderm marks beta tubulin.

Fig. 8 shows extra morphology and the immunohistology feature of the hBS clone SA611 of no allos material.(A) show blastocyst (scale bar (scale-bar) 25um).(B) morphology of the SA611 after being presented at that the condition of no allos material is following and going down to posterity for 12 times (scale bar=100um).(C-H) being presented at 12 backs of going down to posterity uses Oct-4 (C), SSEA-1 (D), Tra1-60 (E), Tra1-81 (F), SSEA-3 (G), SSEA-4 (H) to carry out the immunofluorescence of undifferentiated SA611.(please note that (C) and the image in (E) are to use the amphophilic images of different two anti-generations.) the scale bar is 50um at (C), (E) and (G), at (F) be 100um (H).

Fig. 9 shows the hereditary feature of the hBS clone SA611 of the no allos material in the 9th generation.(A) show that karyomit(e) is diploid and normal.(B) and (C) show that the chromosomal in situ hybridization fluorescence of selecting from SA611, this in situ hybridization fluorescence proof cell are that XY and normal dyeing body 12 and 17 are diploid.Shown further that (C) X chromosome shows blue, Y chromosome shows golden yellow, and karyomit(e) 13 shows red, and karyomit(e) 18 shows that light green and karyomit(e) 21 show green (this manifests with black and white almost is impossible).

Figure 10 show the hBSC of no allos material be SA611 in vivo) (at (A), (C) with (E)) and in the affirmation of the multipotency of external (at (B), (D) with (F)): under the condition of no allos material at the teratomatous histologic analysis that goes down to posterity for 11 times afterwards from SA611: (A) neuroderm (ectoderm), (C) cartilage (mesoderm), (E) secretory epithelium (entoderm).Use the SA611:(B of immunofluorescence analysis vitro differentiation in 2 to 4 weeks of back of going down to posterity) β-III-tubulin positive neuron (ectoderm), (D) the positive smooth muscle actin (mesoderm) of ASMA and (F) HNF3 β (Foxa2) positive cell (entoderm).Scale bar 50 μ m (A, B, D, E, F), scale bar 100 μ m (C).

Embodiment

Embodiment 1-obtains and cultivates the hBS cell of no allos material

Obtaining The residue people embryo who obtains donating after the local Ethics Committee agreement of University and the approval from clinical (IVF) in vitro fertilization processing.The embryo of donations is cultivated into blastocyst in 5 day age at the substratum that routine is used for the IVF processing.According to Gardner with blastocyst be classified as 4AA be classified as according to WO2003055992 quality be A (have many different tight fillings the ICM cell expansion blastocyst and have the trophectoderm of the adhesion of many cells).The serum that the inner cell mass cell is placed no allos material by the human foreskin fibroblast feeder cell of ametycin inactivation on before, at room temperature use acid tyrode's solution (Medicult) (promptly using concentration) to handle blastocyst 15 to 30 seconds removing the part of zona pellucida and trophectoderm, the serum of described no allos material comprises and has about 270 osmotic pressure, be supplemented with 20% (v/v) human serum, 4ng/mL people's recombinant bfgf, 1% penicillin-Streptomycin sulphate, 1% Glutamax, 0.5mmol/l the DMEM of beta-mercaptoethanol and 1% non-essential amino acid (GibcoInvitrogen Corporation).(referring to Fig. 1 and 2.) under 37 ℃, in air, contain 5%CO then ₂Condition under the incubation blastocyst.Changed 50% substratum in per 2 to 3 days, after 10 days, mechanically with passage to fresh hFF raiser.Since the 2nd generation, use glass capillary as cutting and the means of transferring hBS cell (clone SA611) that mechanically goes down to posterity.Approximately once in a week they are gone down to posterity, and cultivated above 11 generations (October, 2005) when priority application.(referring to Fig. 3).The SA611 hBS clone of (October, 2006) no allos material is cultivated altogether and was surpassed for 30 generations when PCT enters.

The foundation of embodiment 2-human foreskin fibroblast feeder cell systems (for example clone hFF003)

To under aseptic condition, be collected among the aseptic IMDM (Invitrogen) that contains the 2X gentamicin from people's foreskin sample of big boy of 8 weeks of ring cutting.The skin explant is placed 25Gm ²The former generation tissue culture flasks (primariatissue culture flask) (Becton Dickinson) that IMDM substratum (Invitrogen), 1% penicillin-Streptomycin sulphate (GibcoInvitrogen Corporation) and 10% human serum are housed in.After about 10 days, set up the individual layer that converges.Use TrypLE ^TMSelect (Invitrogen) is with the cell continuous passage.After amplification, it is detected with regard to the list (mycoplasma, 1 type and 2 type HIV, hepatitis B and hepatitis C, cytomegalovirus, 1 type and herpes simplex types 2 virus, Epstein-Barr virus and human papillomavirus) of human pathogen.

The preparation of embodiment 3-feeder layer

Before the human fibroblasts that coated plate does not have an allos material raises, at room temperature use 0.1% recombinant human gelatin (Fibrogen) to be coated with tissue culture hole, shop, carry out 1 hour shortest time.Use ametycin (Sigma) to handle the confluent monolayer of hFF003 (the 5th to the 8 generation) cell that is grown in the no allos material in IMDM, 10% human serum and the 1% penicillin-Streptomycin sulphate then, carried out 2.5 hours.In substratum (described substratum is based on the DMEM (as above) that is supplemented with 10% (v/v) human serum, 1% penicillin-Streptomycin sulphate, 1% Glutamax, 0.5mmol/l beta-mercaptoethanol and 1% non-essential amino acid (Gibco Invitrogen Corporation)), the raiser that ametycin is handled is with 200000 cell/2.89cm ²Be coated with and be layered on the IVF aperture (Becton Dickinson).Have its inner cell mass cell and before the blastocyst of the cell of its generation or hBS cell in placement, substratum is replaced by now changes the DMEM (as above) that is supplemented with 20% (v/v) human serum, 10ng/mL people's recombinant bfgf, 1% penicillin-Streptomycin sulphate, 1% Glutamax, 0.5mmol/l beta-mercaptoethanol and 1% non-essential amino acid (Gibco InvitrogenCorporation) into.(same medium of describing among the embodiment 1).

Embodiment 4-is based on the preparation of the substratum of serum

By in the plastics bag that under about 8 ℃ blood collecting be not coated with the shop heparin after yesterday from least 15 healthy individual (Blodcentralen, Sahlgrenska UniversityHospital) blood sample obtains human serum, wherein serum and coagulated substance can be separated.With the further sterile filtration of serum, compile in the proper ratio with freezing.(at Blodcentralen, Sahlgrenska University_Hospital comprises that with regard to pathogenic agent the standard combination of hepatitis B, hepatitis C, HIV, HTLV and syphilis detects the blood before using.)

Other components of describing among serum that melts by adding from 20% (v/v) to DMEM (as above) and the embodiment 1 further prepare substratum.

Embodiment 5-is by carry out freezing of the vitrifying of the hBS cell of no allos material and melt

Is cell SA611 counting generation for example freezing in the 25th generation with melting hBS according to the method for describing among the WO2004098285.Prepare two kinds of solution A and B (solution A: the DMSO for preparing among 10% ethylene glycol of sterile filtration, 10% the Cryo-PBS; Solution B: the DMSO for preparing among the 0.3M trehalose of sterile filtration, 20% ethylene glycol, 10% the Cryo-PBS).So that (identical mode was cut the colony of the hBS clone SA611 of selection when Sweden) the cutting cell went down to posterity to carry out routine for Swemed Labs International, Billdal with working as use stem cell cutting tool.At first, be transferred to then in the 25ml solution B cell fragment incubation 1 minute in 500ml preheating (37 ℃) solution A, incubation 30 seconds, and then be transferred in the drop of the solution B of newly joining incubation 20 to 30 seconds.Volume is approximately 40-50 μ l.The cell fragment is pumped into preparation is used for vitrified suction pipe, use tackiness agent (bond) sealing suction pipe then.Again suction pipe is immersed in the liquid nitrogen.

After several days, the suction pipe that refrigerated SA611 is housed that melts as described below:

Prepare two kinds of solution C and D (solution C: the trehalose of in Cryo-PBS, preparing of sterile filtration 0.2M; Solution D: the trehalose of in Cryo-PBS, preparing of sterile filtration 0.1M).With solution C and D and hBS-substratum 37 ℃ of following preheatings.Take out the suction pipe of the sealing that vitrified SA611 is housed in people's liquid nitrogen container.Suction pipe was at room temperature kept 10 seconds, in 40 ℃ of water-baths, melt (in the several seconds) fast then.The scissors that autoclaving is crossed in use one is cut off suction pipe at the obstruction end, uses syringe that content is released from suction pipe and enters solution C.With hBS cell incubation 1 minute in 500 μ l solution C, be transferred in the 500 μ l solution D incubation 5 minutes then.Under stereoscopic microscope, rinsing hBS cell fragment fast in based on the substratum of the serum of no allos material is seeded in it then based on human fibroblasts's feeder cell of the no allos material in the substratum of serum in culture dish.Culturing cell (at 37 ℃ of following incubations) is counted the new colony number of setting up then, and goes down to posterity with the viablity of hBS cell after the checking vitrifying.

The sign of the hBS clone of embodiment 6 no allos materials

Immunohistochemistry and tissue chemical analysis

HBS cell colony culture is fixing in 4% Paraformaldehyde 96, change thoroughly then.After successive washing and sealing step, cell resisted under 4 ℃ with one be incubated overnight.One of use resists for Oct-4, TRA-1-60, TRA-1-81, SSEA-1, SSEA-3 and SSEA-4 (Santa Cruz Biotechnology; SantaCruz, CA; Http:// www.southernbiotech.com) be specific.Two anti-being used for that FITC-or Cy3-put together are detected.With DAPI (Vectashield; Vector Laboratories, Burlingame, CA; Http:// www.vectorlabs.com) redye nucleus.Scheme (Sigma-Aldrich Stockholm, Sweden according to manufacturer; Http:// www.sigmaaldrich.com) determine the activity of alkaline phosphatase (ALP).

In order to identify cell, use following antibody to carry out immunohistochemical analysis as mentioned above: to use HNF3 β (Santa CruzBiotechnology, Santa Cruz for the endoderm cell from 3 kinds of different germinal layers; Http:// www.southernbiotech.com).Use ASMA (company) to detect mesoblastema and use 'beta '-tubulin-III mAb (Sigma-Aldrich) evaluation neuroderm cell.

Use is purchased obtainable test kit and reacts according to scheme (Sigma-Aldrich) detection of alkaline phosphatase (ALP) of manufacturer.

Detecting at ALP (referring to Fig. 4), Oct-4, Tra1-60, Tra1-81, SSEA-3 and SSEA-4 in undifferentiated colony is the male reaction, and to detect at SSEA-1 (referring to Fig. 5,6,8) be negative reaction.(referring to Fig. 4-6).

Chromosome karyotype analysis and FISH

The hBS cell that is designed for the gene sign is transferred to mouse embryo fibroblasts, carry out going down to posterity or being transferred to for twice matrix offset plate (Matrigel plate) (BectonDickinson), and then further cultivated about 10 days.Lure incubation cell under the situation that the plain A of cancer (Calyculin A) exists at the calyx cavernous body then,,, use ethanol then and freeze acetic acid and fix by hypotonic processing lysing cell by centrifugal collecting cell.Use trypsinase-Jim Sa or DAPI dyeing to manifest karyomit(e).Analyze for fluorescence in situ hybridization (FISH), use to comprise and be purchased the availability test kit at the probe of karyomit(e) 12,13,17,18,20,21 and sex chromosome (X and Y) according to having improved slightly manufacturers instruction.Be equipped with suitable spectral filter and software (CytoVision; Applied Imaging; Santa Clara CA; Http:// www.appliedimagingcorp.com) inverted microscope analyze down slide glass.In the 9th to 10 generation hBS clone SA611 being carried out gene characterizes.HBS clone SA611 has the diploid normal karyotype by the karyotyping proof.Caryogram is 46 XY.The chromosomal fish analysis of selecting has been verified this discovery.

The caryogram of having confirmed SA611 is normal.(referring to Fig. 9).

External versatility

At first by making the spontaneous differentiation or by undifferentiated hBS cell colony is transferred to the Matrigel that allows their spontaneous differentiation thereon on the raiser of hBS cell colony ^TMDetect the versatility of SA611 on the coated plate (Becton Dickinson).In both cases, when differentiation when being induced, with substratum be converted to VitroHESTM (Vitrolife, Kungsbacka, Sweden).After the cultivation in 3 to 4 weeks, by the immunohistochemical analysis colony to identify cell from 3 different germinal layers.

Identified the positive reaction of just early stage entoderm mark HN3 β (being also referred to as Foxal sometimes), ectoderm mark 'beta '-tubulin and ASMA (α-smooth muscle actin).(about the reaction of HNF3 β and 'beta '-tubulin referring to Fig. 7, about all 3 marks referring to Figure 10 (B, D, F)).

Versatility in the body

For the versatility character of the hBS clone SA611 that surveys no allos material in vivo, undifferentiated hBS cell cluster is implanted in below the scrotum of SCID mouse.The appearance proof SA611 of ectoderm (neuroderm, Figure 10 A), mesoderm (cartilage, Figure 10 C) and entoderm (intestines sample epithelium, Figure 10 E) tissue in the abnormal embryoma has showed the interior differentiation capability of feature gonosome of multipotency hBS cell.

In a word, the hBS clone SA611 of no allos material stably expresses the heredity and the phenotypic characteristic of undifferentiated pluripotent human BS cell.

Reference

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Claims

1. obtain the method for the hBS clone of no allos material, described method comprises step:

I) remove the inner cell mass that zona pellucida surrounds with the acquisition trophectoderm by the operation of no allos material from blastocyst,

Iv) inner cell mass cell and people's feeder cell are cultivated about 5 days altogether to about 50 days time bar in the substratum of no allos material,

Vi) optionally, with inner cell mass cell or the cell transfer that derives from it to the Freshman feeder layer in the substratum of no allos material, thereby the hBS cell of the no allos material of acquisition,

Vii) by in the substratum of no allos material, cultivating the hBS cell of breeding no allos material altogether, thereby obtain the hBS clone of no allos material with people's feeder cell.

2. the method for claim 1, it also comprises step

Viii) breed the clone of no allos material: in the substratum of no allos material, cultivate altogether and with about 2 days to about 20 days, cell was gone down to posterity to about 12 days suitable timed interval in for example about 4 days with people's feeder cell by following manner.

3. claim 1 or 2 method, it also comprises step

Ix) hBS clone that will not have an allos material is transferred to no allos material and no raiser's culture systems of the substratum of the supported matrix that comprises suitable no allos material and no allos material.

4. each method in the aforementioned claim, wherein by using a) acidic solution, b) for example Unidasa or PRONASE A of one or more recombinases, or c) mechanical means carries out step I).

5. the method for claim 4 is wherein carried out step I by the use acidic solution).

6. the method for claim 5 wherein by blastocyst being experienced acidic solution about 5 seconds to about 180 seconds, for example about 10 seconds to about 120 seconds, about 15 seconds to about 90 seconds, about 20 seconds to about 60 seconds, was carried out step I in about 30 seconds to about 50 seconds).

7. claim 5 or 6 method, wherein the pH of acidic solution is about 2 to about 3, for example about 2.5 to about 2.8, for example 2.5.

8. each method in the claim 5 to 7, wherein acidic solution is acid tyrode's solution.

9. each method in the aforementioned claim is wherein by using a) acidic solution, b) one or more recombinases, or c) mechanical means carries out step I i).

10. the method for claim 9 is wherein carried out step I i by the use acidic solution).

11. the method for claim 10, wherein by with step I) in the inner cell mass experience acidic solution that surrounds of the trophectoderm that obtains carry out step I i), the time of wherein experiencing acidic solution is about 5 seconds to about 180 seconds, for example about 10 seconds to about 120 seconds, about 15 seconds to about 90 seconds, about 20 seconds to about 60 seconds, about 30 seconds to about 50 seconds.

12. the method for claim 10 or 11, wherein the pH of acidic solution is about 2 to about 3, for example about 2.5 to about 2.8, for example 2.5.

13. each method in the claim 10 to 12, wherein acidic solution is acid tyrode's solution.

14. the method for claim 9 is wherein by using one or more recombinases to carry out step I i).

15. the method for claim 14, wherein said one or more recombinases are recombinant protein lytic enzymes.

16. the method for claim 15, wherein said one or more recombinant protein lytic enzymes are selected from recombinant trypsin and TrypLE ^TMSelect.

17. each method among the claim 14-16 is wherein by with step I) in the inner cell mass that surrounds of the trophectoderm that obtains experience about 5.000U and carried out step I i in about 0.5 minute to about 30 minutes to the about recombinant trypsin of 10.000U).

18. each method among the claim 14-17 is wherein by with step I) in the undiluted TrypLE that promptly uses concentration of inner cell mass experience that surrounds of the trophectoderm that obtains ^TMSelect about 0.5 for example about 0.5 to about 8 minutes, about 0.5 to about 5 minutes, about 1 to about 3 minutes, for example carried out step I i in 1.5 minutes to about 10 minutes).

19. each method in the aforementioned claim, wherein the time bar of step I in v) be step I v) in about 5 days to about 30 days, for example about 5 days to about 20 days or about 5 days to about 15 days.

20. each method in the aforementioned claim, wherein between the common incubation period of the inner cell mass cell of step I in v) by changing about 20% to about 100%, for example about 30% carries out once or the replacing of more times substratum to about substratum of 80%, about 40% to about 60%.

21. each method in the aforementioned claim, wherein about 50% substratum carries out once or the replacing of more times substratum by changing between the common incubation period of the inner cell mass cell of step I in v).

22. the method for claim 20 or 21 wherein with about 2 days to about 14 days, was carried out once or the replacing of more times substratum to about 7 days timed interval in for example about 4 days.

23. each method in the aforementioned claim, wherein, if any, step v) in by using mechanical means from the trophectoderm of hypertrophy, to discharge the inner cell mass cell or deriving from its cell.

24. the method for claim 23, wherein mechanical means comprises that the use glass capillary is as cutting tool.

25. each method in the aforementioned claim, wherein, if any, step v) in by using one or more recombinases from the trophectoderm of hypertrophy, to discharge the inner cell mass cell or deriving from its cell.

26. the method for claim 25, wherein said one or more recombinases are recombinant protein lytic enzymes.

27. the method for claim 26, wherein said one or more recombinant protein lytic enzymes are selected from recombinant trypsin and TrypLE ^TMSelect.

28. each method in the claim 25 to 27 is wherein by carrying out step v) in about 0.5 minute to about 30 minutes with inner cell mass cell or the cell that derives from it and about 5.000U to the about recombinant trypsin incubation of 10.000U.

29. each method in the claim 25 to 28 is wherein by with the inner cell mass cell or derive from its cell and the undiluted TrypLE that promptly uses concentration ^TMSelect incubation together carried out step v) in about 0.5 minute to about 15 minutes.

30. each method in the aforementioned claim is wherein carried out once during the propagation of the hBS cell of the no allos material of step in vii) or more times goes down to posterity.

31. the method for claim 30 is wherein carried out selectivity to the hBS cell and is gone down to posterity.

32. the method for claim 30 or 31 is wherein by using mechanical means to go down to posterity.

33. the method for claim 30 or 31 is wherein by using one or more recombinases to go down to posterity.

34. the method for claim 33, wherein said one or more recombinases are selected from TrypLE ^TMSelect, recombinant trypsin and recombinant collagen enzyme.

35. each method in the claim 33 to 34 was wherein carried out step vii) in about 0.5 minute to about 30 minutes by the recombinant trypsin incubation that uses the extremely about 10.000U of about 5.000U.

36. each method in the claim 33 to 34 is wherein by using the undiluted TrypLE that promptly uses concentration ^TMSelect incubation about 0.5 carried out step vii) to about 15 minutes.

37. each method in the claim 33 to 34 is wherein by using about 200U/ml recombinant collagen enzyme incubation to carry out step vii) in about 1 minute to about 40 minutes.

38. each method in the aforementioned claim is wherein cut apart by machinery and is carried out going down to posterity of the cell of step in viii).

39. the method for claim 38 is wherein cut apart by using glass capillary to carry out machinery as cutting tool.

40. each method in the aforementioned claim is wherein by using one or more recombinant proteins to carry out the passage of step in viii).

41. the method for claim 40, wherein said one or more recombinases are selected from TrypLE ^TMSelect, recombinant trypsin and recombinant collagen enzyme.

42. each method in the claim 40 to 41 was wherein carried out step viii) in about 0.5 minute to about 30 minutes by the recombinant trypsin incubation that uses the extremely about 10.000U of about 5.000U.

43. each method in the claim 40 to 41 is wherein by using the undiluted TrypLE that promptly uses concentration ^TMSelect incubation about 0.5 carried out step viii) to about 15 minutes.

44. each method in the claim 40 to 41 is wherein by using about 200U/ml recombinant collagen enzyme incubation to carry out step viii) in about 1 minute to about 40 minutes.

45. each method in the aforementioned claim is wherein at step I x) in the supported matrix of the no allos material that uses optional from recombinant human gelatin, recombinant human fibronectin polypeptide, people's placenta cells epimatrix.

46. each method in the aforementioned claim, wherein step I x) in the no allos material that uses substratum can with step I ii), iv), vi), vii) and the substratum of the no allos material that uses in the arbitrary step viii) identical or different.

47. each method in the aforementioned claim is wherein at step I x) in the substratum of the no allos material that the uses feeder cell of can choosing carry out conditioning, or it can replenish the suitable factor to keep undifferentiated growth.

48. the method for claim 47, the optional activator of the wherein suitable factor from recombinate bFGF and WNT approach.

49. each method in the aforementioned claim wherein obtains under the condition of no allos material in step I ii), iv), vi), vii) and the people's feeder cell that use in the arbitrary step viii).

50. the method for claim 49, wherein people's feeder cell derive from healthy people's tissue.

51. the method for claim 49 or 50, wherein people's feeder cell are the skin flbroblast that derive from newborn infant people's foreskin.

52. each method in the aforementioned claim, wherein step I ii), iv), vi), vii) and/or the substratum of the no allos material that uses in the arbitrary step viii) comprise the minimum medium that is suitable for the cell proliferation of people's inner cell mass.

53. the method for claim 52, wherein minimum medium is DMEM.

54. the method for claim 52 or 53, the substratum that does not wherein have the allos material also comprises about 2ng/ml to about 100ng/ml recombinant bfgf, for example about 5ng/ml is to about 50ng/ml recombinant bfgf, approximately 5ng/ml is to about 25ng/ml recombinant bfgf, and about 5ng/ml is the 15ng/ml recombinant bfgf extremely approximately.

55. each method in the claim 52 to 54, the substratum that does not wherein have the allos material also comprises the 10ng/ml recombinant bfgf.

56. each method in the claim 52 to 55, the substratum that does not wherein have the allos material also comprises about 1%v/v to about 30%v/v human serum, and for example about 10%v/v is the 30%v/v human serum extremely approximately, approximately the extremely about 25%v/v human serum of 15%v/v.

57. each method in the claim 52 to 56, the substratum that does not wherein have the allos material also comprises the 20%v/v human serum.

58. each method in the aforementioned claim wherein prepares in step I ii), iv), vi), vii) and the human serum that uses in the arbitrary step viii) through the following steps

C) the described sack that is not coated with the shop heparin of incubation under 5 ℃ temperature at the most carries out at least 10 hours time bar,

D) randomly, for example do not exist the opaqueness of not solidifying fibrin, liquid phase to select based on solidifying quality,

E) with serum and the material separation of solidifying,

F) serum that obtains in the sterile filtration step d),

G) compile serum from least 15 donors,

H) use preceding freezing serum.

59. obtain the method for the hBS clone of no allos material, the method comprising the steps of:

1) by at room temperature using acid tyrode's solution incubation blastocyst about 10 seconds to about 10 minutes, preferably approximately 30 seconds to about 60 seconds time bar removes zona pellucida and to the small part trophectoderm from blastocyst, thereby obtains isolating inner cell mass cell,

3) in the substratum of no allos material, inner cell mass cell and human foreskin fibroblast feeder cell are cultivated altogether, carried out about 5 days to about 15 days time bar, the substratum of the no allos material of replacing at least 50% in per 3 to 5 days,

5) optionally, on inner cell mass cell or the cell transfer that derives from it the fresh human foreskin fibroblast feeder layer to the substratum that does not have the allos material, thereby obtain the hBS cell of no allos material,

60. the method for claim 59, it further comprises step

7) breed the hBS clone of no allos material by following manner: in the substratum of no allos material, cultivate altogether with the human foreskin fibroblast feeder cell, the substratum of the no allos material of replacing at least 50% in per 3 to 5 days, with with reasonable time at interval, pair cell went down to posterity in for example per 3 to 8 days.

61. hBS clone by the no allos material of each method acquisition in the aforementioned claim.

62. the hBS clone of the no allos material of claim 61, it is not exposed to any non-human animal's material directly or indirectly.

63. the hBS clone of the no allos material of claim 61 or 62, use therein any organic materials is that the people originates or synthetic, semisynthetic or the reorganization material.

64. the hBS clone of each no allos material in the claim 61 to 63, wherein animal is a Mammals.

65. the hBS clone of each no allos material in the claim 61 to 64, it shows in the following standard at least 1, for example at least 2, at least 3, at least 4, at least 5 or at least 6 standards: for the positive reaction of 0ct-4, TRA-1-60, TRA-1-81, SSEA-3 and/or SSEA-4 with for the negative reaction of SSEA-1.

66. the hBS clone of each no allos material is used to prepare the purposes of the derivative of its differentiation in the claim 61 to 65.

67. by the hBS clone of the no allos material of each method acquisition or the purposes of derivative in medical science of its differentiation in the claim 1 to 60.

68. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used for the purposes of the manufacturing of pharmaceutical prod, described pharmaceutical prod is used to prevent and/or treat pathological conditions and/or the disease that is caused by tissue degeneratiaon.

69. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used for the purposes of the manufacturing of pharmaceutical prod, described pharmaceutical prod is used for the treatment of and/or prevents metabolic pathological conditions and/or disease.

70. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used for the purposes of medical research.

71. be used for studying for example purposes of the external model of people's degenerative disease of human disease by the hBS clone of the no allos material of each method acquisition or the derivative of its differentiation in the claim 1 to 60.

72. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used for the purposes of drug discovery method.

73. be used for the purposes that in vitro toxicity is tested by the hBS clone of the no allos material of each method acquisition or the derivative of its differentiation in the claim 1 to 60.

74. the hBS clone of the no allos material that obtains by each method in the claim 1 to 60 or the derivative of its differentiation are used to screen the purposes of purpose.