CN101443445B - Culture system and method for propagation of human blastocyst-derived stem cells - Google Patents

Abstract

The present invention relates to a culture system for and a method for propagation of human blastocyst-derived stem cells (hBS cells) upon enzymatic dissociation into a single cell suspension. The culture system for propagation of human blastocyst-derived stem (hBS) cells comprises i) human feeder cells at a density of at least 50,000 cells/cm<2>, ii) one or more dissociation agents for dissociation of hBS cell colonies into a single cell suspension, and iii) a supportive culture medium, which culture system makes it possible to propagate hBS cells by dissociation of hBS cell colonies into a single cell suspension at each consecutive passage for an extended time period, while maintaining the significant characteristics of hBS cells.

Description

The culture systems and the method that are used for propagation of human blastocyst-derived stem cells

Invention field

The present invention relates to be used for be dissociated into culture systems and the method that single-cell suspension liquid comes propagation of human blastocyst-derived stem cells (human blastocyst-derived stem cell, hBS cell) based on enzymatic.

Background of invention

Traditionally, human blastocyst-derived stem cells (hBS cell) is maintained on mEF (MEC) feeder layer, and breed people such as [, WO03055992, Bresagen] Heins through manual work cutting and the single small pieces that shift colony.This traditional cultural method is time and effort consuming very, but so far can be with the preferred cultural method of hBS clone long term maintenance in stable standard state, and therefore is the proper method that is suitable for keeping on a small scale cultivation.Yet, also exist many technological deficiencies relevant with this tradition culture systems.For example, how many cells it possibly quantitatively have be transferred in the small pieces hardly, brings negative impact for repeatable and stdn thus.Because with the low consistency of automatic technology (for example robot and bio-reactor), be limited based on the production process that enlarges in proportion of these traditional culture systems, and need for example microscope of a large amount of man-hour, lab space and equipment on the contrary.In addition; Be used for terminal analysis and the cell sorting technology that is used for through the subculture thing of the cell of sorting through what for example density gradient, FACS (fluorescence automated cell separating method) or magnetic bead sorting method were carried out; And the cell transfecting that carries out through for example electroporation or viral reagent technology, dissociated implement on unicellular than go up at cell small pieces (piece) or aggregate (cluster) implement more preferred.

Therefore, with regard to required time and labor, enzymatic dissociates making that the propagation of hBS cell is more effective.In addition, when going down to posterity, the hBS cell dissociation is become unicellular and will make that their ability quilts are quantitative more accurately and experience many schedule of operation that will enlarge the potential use range of application of hBS cell, and this will help robotization propagation program thus.

Having several groups to attempt enzymatic dissociates; But little report is based on and is dissociated into single-cell suspension liquid when going down to posterity and breeds the hBS cell; But depend on the contrary through using collagenase IV to go down to posterity; Obtained a certain size aggregate [people such as Brimble thus; Bresagen,

-people such as Jansson].For example;

-Jansson has reported and when avoiding unicellular going down to posterity in the culture systems at no feeder cell, has adhered to and the survival increase; Simultaneously Brimble and Bresagen reported they utilize the aggregate of about 10～100 cells size but also further clearly advise avoiding in the process of going down to posterity, being dissociated into unicellular because it has negative impact (Human Embryonic Stem Cell Protocols) to viability.The enzymatic that also has other groups to carry out the hBS cell dissociates; And find in order to make the cell adjustment be adapted to said enzyme; Must hBS clone set up in the process or the very early stage process that goes down to posterity in hBS clone in just with cellular exposure in said enzyme people such as [] Cowan; Thus, the hBS clone (traditional method is set up and cultivated) of having only the hBS clone set up according to these comparatively recent enzymatic schemes rather than great majority to exist at present can be applicable to enzymatic and goes down to posterity and possible fairly large propagation of robotization and amplification.In addition, be dissociated into when unicellular, only described the relatively low rate that goes down to posterity and passed than (1: 3) people such as [] Cowan, this means the low possibility that this propagation is expanded in proportion more massive propagation with dividing when being employed in when going down to posterity enzymatic.

Except above-mentioned with enzymatic in going down to posterity is dissociated into unicellular relevant technical difficulty; The problem relevant with quality [people such as Draper, people such as Buzzard, Mitalipova have also been reported with the stability of hBS clone of propagation; People such as Enver, people such as Andrews].For example, Draper, Buzzard and Mitalipova have described the introducing of chromosome aberration, for example the acquisition of karyomit(e) 12 and 17q.Cowan has also clearly indicated genetic instability, and regular karyotyping is carried out in suggestion thus.Enver and Andrews described towards through cultivate to adapt to or through the conversion of the clone of exoadaptation, said clone begins to be similar to people embryo carcinous (EC) cell maybe possibly show the versatility of change and the outer hereditary change aspect caryogram.Viewed these variations maybe be owing to culture systems excessively optionally in the enzymatic breeding of hBS cell, and it helps resisting specifically the cell of population pressure and stress, and these cells begin to occur in the 15th～20 generation usually.

Therefore, need the such culture systems of exploitation, its permissions is unicellular when when going down to posterity, hBS cell enzymatic being dissociated into, in addition with the high score biography than (split ratio), i.e. positive controls for high proliferation rates, and do not produce the problems referred to above.Such culture systems is a target of the present invention.

The invention summary

The invention provides the culture systems that has for the supportive culture environment of height of hBS cell; It allows, and enzymatic is dissociated into unicellular in the process that at every turn goes down to posterity; And in the time period that prolongs, for example surpassed for 20 generations, do not jeopardize not differentiation, multipotency and the normal state of clone.The supportive environment of this of the culture systems that this paper appeared has been offset the selective pressure that pair cell causes in unicellular generation process.

Culture systems provided by the invention is to be used for the culture systems that propagation of human blastocyst-derived is done (hBS) cell, and it comprises:

I) density is at least 50,000 cell/cm ²People's feeder cell,

Ii) one or more be used for the hBS cell colony be dissociated into single-cell suspension liquid dissociate agent and

Iii) supportive substratum,

This culture systems maybe be at the time period internal breeding hBS cell that prolongs through in each continuous passage, the hBS cell colony being dissociated into that single-cell suspension liquid makes, and keeps the notable feature of hBS cell simultaneously.

In addition; When through when going down to posterity, hBS cell colony enzymatic being dissociated into single-cell suspension liquid when breeding; The problem relevant with quality that run into before the invention solves with the low stability of hBS cell that obtained; Surpassed for 20 generations because the hBS cell of in culture systems of the present invention, breeding can be bred, for example surpassed for 30 generations, and keep the notable feature of hBS cell simultaneously.

Invention is described

As stated, a main aspect of the present invention relates to a kind of culture systems, and its permission is bred the hBS cell through hBS cell enzymatic is dissociated into single-cell suspension liquid, and does not lose the notable feature of hBS.The system that is used to breed the hBS cell of the present invention comprises:

I) density is at least 50,000 cell/cm ²People's feeder cell,

Ii) one or more be used for the hBS cell colony be dissociated into single-cell suspension liquid dissociate agent and

Iii) supportive substratum,

This culture systems maybe be at the time period internal breeding hBS cell that prolongs through in each continuous passage, the hBS cell colony being dissociated into that single-cell suspension liquid makes, and keeps the notable feature of hBS cell simultaneously.The hBS cell can be bred in culture systems of the present invention above 20 generations, for example surpassed for 25 generations, surpassed for 30 generations, surpassed for 35 generations, or surpassed for 40 generations, and still keep the notable feature of this type of cell.

Other aspects of the present invention are to be used to breed hBS cell and isolating culture systems subsequently, and said system comprises:

I) density is at least 50,000 cell/cm ²People's feeder cell,

Ii) one or more be used for the hBS cell colony be dissociated into single-cell suspension liquid dissociate agent and

Iii) supportive substratum,

Iv) be used for mixing the magnetic-particle of feeder cell,

This culture systems makes possibly separate feeder cell from the hBS cell.

The separation efficiency of said system can be at least 50%, for example at least 70%, at least 80%, at least 90%, at least 99%, and wherein separation efficiency means the per-cent of the feeder cell overall number that magnetic force attracted that is applied in.

As used herein, term " propagation " means breeding hBS cell, so that enlarge the hBS cell colony, promptly enlarges the quantity of hBS cell.Yet culture systems that is used to breed as herein described and method also can be used for simply keeping the purpose of hBS clone.

As used herein, term " notable feature of hBS cell " means, two, three, four, five, six or seven in the following characteristics:

I) when growing on the feeder cell of inactivation aspect mitotic division, show the multiplication capacity under undifferentiated state, and/or

Ii) in hBS cell proliferation process, show stable karyotype, promptly undistorted, and/or

Iii) maintain the potentiality that develop into the verivate of all types germinal layer in external and the body, and/or

Iv) show at least two kinds in the following molecular marked compound: OCT-4; SEAP, glucide epi-position SSEA-3, SSEA-4; TRA 1-60; The protein core of TRA 1-81 and keratin sulfate/CHS pericellular stromatin glycan of being discerned by monoclonal antibody GCTM-2, and/or

V) do not show molecular marked compound SSEA-1 or other differentiation marker things, and/or

Vi) keep its versatility, and form teratoma in being injected into the mouse of non-responsiveness the time in vivo, and/or

Vii) can break up.

These characteristics can according to described in embodiment 7 and 8 at regular intervals, in for example every 1-20 generation,, in for example every 5-15 generation or per 10 generations, analyzed.

Parent material

Culture systems of the present invention can be used for breeding the hBS cell or be maintained at the clone that this paper is called " chief cell system (master cell line) ".As used herein; " chief cell system " refers to parent's culture of hBS clone; It maintains in traditional culture systems of utilizing mechanical to cut the hBS cell colony when going down to posterity and " chief cell system " also refers to vitrified (vitrified) parent culture.

Feeder cell

In culture systems of the present invention, need intensive feeder layer, so that make said system be enough to support that hBS is unicellular, said hBS is unicellular more responsive to surrounding environment than the hBS cell in the aggregate clearly.Therefore, the people's feeder cell density in the culture systems of the present invention is about 50,000 to about 500,000 cells/cm ², for example, about 50,000 to about 400,000 cells/cm ², about 50,000 to about 300,000 cells/cm ², about 50,000 to about 200,000 cells/cm ², about 60,000 to about 200,000 cells/cm ², about 70,000 to about 200,000 cells/cm ²Usually, inoculating the hBS cell about 1 to about 10 days before, for example, about 1 to about 5 days, feeder cell were inoculated in the culture vessel in about 2 to about 4 days.Alternatively, before inoculation hBS cell, can substratum be changed at least once, for example, at least twice, at least three times, at least four times or at least five times.

The suitable people's feeder cell that are used for culture systems of the present invention can derive from people's tissue, and perhaps they can be external sources.

People's tissue that people's feeder cell can derive from comprises: newborn infant, teenager or adult's tissue, and it further comprises the tissue that derives from skin (comprising foreskin), umbilical cord, muscle, lung, epithelium, placenta, uterine tube, gland (glandula), matrix (stroma) or breast.People's feeder cell can derive from the cell type that belongs to the group of being made up of human fibroblasts, fibrocyte, myocyte, keratinocyte, endotheliocyte and epithelial cell.The example of particular cell types of people's feeder cell of can be used for deriving comprises: the umbilical cord mesenchyma cell; Placenta inoblast and/or fibrocyte; The placenta endotheliocyte; Birth back HFF 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 gland endometrial cell; Adult's matrix endometrial cell; Become the human breast carcinoma parenchyma; Become the HEC; Become human epithelial cell or adult's keratinocyte.

In a particular of the present invention, people's feeder cell are inoblasts, preferably derive from people's neonatal human foreskin fibroblast.HFF's feeder cell systems can be through following manner from setting up from the boy baby's who has implemented posthetomy skin samples: said skin samples places the culture vessel that contains suitable aseptic culture medium; Said substratum for example is a basic medium; For example DMEM (Dulbecco ' s Modified Eagle ' s Medium) or IMDM (Iscove ' s Modified Dulbecco ' s Medium); Wherein be supplemented with mammalian blood serum; For example FBS or human serum perhaps are supplemented with serum substitute and 1%PEST (v/v).Preferably, substratum is the IMDM (Invitrogen) that is supplemented with 10% (v/v) human serum and 1% (v/v) PEST.The cell monolayer that acquisition converges after about 5 days to about 30 days.Now can be with about 2 days to about 10 days, for example about 4 days to about 9 days, about 5 days to about 8 days interval, with one or more agent of dissociating, for example TrypLE ^TMSelect dissociates through enzymatic feeder cell is gone down to posterity.After setting up, can come feeder cell are tested with regard to the suitable selection of human pathogen, to guarantee their health.Particularly, can be according to the clone of following examples 3 said acquisition HFF feeder cell.

In another embodiment, employed feeder cell can be to be purchased the feeder cell that can get in culture systems of the present invention, for example; HFF cell (American Type Culture Collection, CRL-2429 ATCC, Manassas; VA) or human embryonic fibroblast's cell (American Type Culture Collection; CCL-110 ATCC, Manassas, VA).

Employed feeder cell can also be immortalization or genetically modified among the present invention." immortalization " of feeder cell means, the acquisition of the ability of in cultivation, growing through the inferior division of theory unlimited.Have several method, wherein a kind of method is to come transformant and/or the expression through reverse transcriptase of telomere protein (TERT) with for example virus, retrovirus for this reason.TERT is inactivation in most cells, but when expressing hTERT, said cell can be kept is enough to avoid duplicating old and feeble telomere length exogenously.

In addition, two kinds of cell types, i.e. feeder cell or hBS cell, in any can pass through magnetic and modify to allow coming separately blended cell colony through applying magnetic force.In a preferred embodiment of the invention, employed feeder cell are modified through magnetic among the present invention.The magnetic of feeder cell is modified and can be accomplished through following manner, and soon cell colony is exposed to and can passes through several methods, for example mixes the magnetic-particle in the cell through electroporation, endocytosis or phagolysis.

In addition, feeder cell can be through genetic modification, thereby have the specific gene that is integrated in the genome.These gene codified purpose affinity tags or the synthetic known biomolecules useful, for example growth factor, for example bFGF (Prostatropin) to the hBS cell.These genetic modifications also can be induced feeder cell apoptosis and the removal of inducing that makes it possible to obtain feeder cell.

In culture systems of the present invention employed people's feeder cell aspect growth by inactivation, so that the feeder cell of keeping the relative fixed number surpass the hBS cell to avoid the feeder cell growth.The growth inactivation of people's feeder cell can be according to currently known methods or as being implemented in following examples 1 and 4, through with antimitotic agent for example MTC handle cell and carry out.Alternatively, can through giving the radiation of cell doses, for example be enough to cause the gamma-radiation of cell cycle arrest according to currently known methods, make people's feeder cell aspect growth by inactivation.

Before the growth inactivation of feeder cell, can they cultivations be had on the supportive substratum at the feeder cell for particular cell types.When feeder cell are the human fibroblasts; Substratum can be selected from Dulbecco improvement Eagle substratum (DMEM); Iscove improves Dulbecco substratum (IMDM), and it is supplemented with mammalian blood serum for example human serum or FBS or serum substitute and further be supplemented with PEST.Feeder cell can directly be planted in the implantation culture vessel or planted on the matrix components of culture vessel.It is about 1 to about 40%v/v that this substratum can be supplemented with concentration, for example about 5 serum to about 20%v/v or 10%v/v, for example FBS or human serum, and/or be supplemented with one or more microbiotic, for example penicillium mould and Streptomycin sulphate.In a specific embodiment, it is about 0.1% to about 10%v/v that culture medium supplemented has concentration, and about 0.5% to about 2%v/v, or penicillium mould-Streptomycin sulphate of preferred 1%v/v.Feeder cell can be with about 2 to about 21 days in this substratum, and for example about 3 to about 10 days, about 4 to about 8 days interval; For example per 7 days, through using one or more agent of dissociating, with 1: 2 to 1: 20; For example 1: 2 to 1: 10,1: 2 to 1: 8 branch passed than going down to posterity.After about 2 to 10 generations, for example after 3 to 8 generations, feeder cell can be like the above-mentioned inactivation of growing.

Behind the growth inactivation, can substratum with the support hBS of cell inoculation in the culture vessel that is coated with the suitable matrix material in, be described to support that all substratum of hBS cell proliferation all are suitable below promptly.The example of suitable substrate material can comprise recombinant human fibronectin polypeptide, people's placenta cells epimatrix or gelatin, for example, recombinant human gelatin.Suitable gelatin concentration is about 0.1%w/v.With about 50,000 to about 500,000 cells/cm ², for example, about 60,000 to about 200,000 cells/cm ², or about 70,000 to about 100,000 cells/cm ²Density inoculation feeder cell.Ideally, between the 2nd generation and the 10th generation, for example the feeder cell between the 3rd generation and the 8th generation are as feeder cell.Particularly, can be according to following examples 1 and the 3 said feeder cell of cultivating.

In a particular of the present invention, feeder cell are (xeno-free) feeder cell of no allos material, and promptly they never directly or indirectly are exposed to the material in non-human animal source, for example cell, tissue and/or body fluid and verivate thereof.Embodiment 3 and 4 has described the foundation and the cultivation of clone of the no allos material of HFF's feeder cell jointly.

The agent of dissociating

The present invention relates to a kind of culture systems, wherein the hBS cell colony can be dissociated into single-cell suspension liquid when going down to posterity.As used herein, term " single-cell suspension liquid " means and contains single celled hBS cell suspending liquid in fact,, is lower than 10% that is, for example is lower than 8%, is lower than 6%, is lower than 4%, is lower than 2%, or to be lower than 1% cellular entities can be aggregate.The aggregate of any remnants can be alternatively through using cellular filter remove, the function of said cellular filter can be collected aggregate through said hole dimension and let unicellular passing through as the sieve with certain hole dimension.As stated, this is a critical step, because unicellular state is harsh for the hBS cell, Here it is has found before why aspect cell survival and have challenge aspect the stability of survivaling cell and the quality.

When using under the situation in culture systems of the present invention, one or more agent of dissociating can be enzyme, sequestrant or one or more enzymes and/or one or more combination of chelating agents.Correspondingly, said one or more agent of dissociating can be at least two kinds, for example at least three kinds, and at least four kinds, the combination of at least five kinds of agent of dissociating.

The enzyme that is suitable for culture systems of the present invention can comprise proteolytic ferment and/or collagenolysis enzyme; For example, trypsinase, trypsin-like, Dispase II, Dispase II appearance, PRONASE A, PRONASE A appearance, collagenase, collagenase appearance and matrix metalloproteinase.Specially suitable enzyme can be the enzyme of reorganization.

Suitable sequestrant can be a divalent cation chelators, for example, and EDTA, EGTA and HEDTA.

At step I i) in when dissociating the hBS cell colony, can also add the DNA enzyme except that said one or more dissociate the agent, this will prevent the caused caking by the DNA that from the disruptive cell, discharges.

The combination of agent of dissociating is particularly suitable for culture systems of the present invention.Such example has TrypLE ^TMSelect (Invitrogen), Accutase ^TM(Chemicon) and Accumax ^TM(Chemicon).TrypLE ^TMSelect comprises recombinant trypsin appearance enzyme and EDTA, and it is no animal component.Accutase ^TMComprise proteolyze and collagenolysis activity and EDTA, it does not contain the composition of Mammals composition or bacterial origin.Remove Accutase ^TMComposition outside, Accumax ^TMAlso comprise the DNA enzymic activity.

In addition, dissociate agent or its combination of said one or more can be no allos material, and promptly they never directly or indirectly are exposed to the material in non-human animal source, for example cell, tissue, and/or body fluid and verivate thereof.

Substratum

The substratum that is suitable for culture systems of the present invention must be highly to support the growth of hBS cell.Suitable medium can be the definite composition substratum with complete known composition.Suitable supportive substratum comprises through the basic medium that replenishes, for example DMEM or IMDM.Supportive substratum can be supplemented with one or more following components: mammalian blood serum; For example FBS or human serum; serum substitute; Penicillium mould, Streptomycin sulphate, non-essential amino acid; L-glutaminate, beta-mercaptoethanol and hrbFGF (people's recombination basic fibroblast growth factor).

Also can use other substratum in the present invention.Such substratum can contain salt, VITAMINs, energy source (for example glucose), mineral substance and amino acid.To be added to the suitable growth factor in the substratum for example can be, GABA, pipecolic acid, lithium chloride and transforming growth factor-beta (TGF β) and bFGF.In addition, such substratum can chemically confirmed composition.

Substratum also can be a conditioned medium, promptly with before acting on the substratum of hBS cell proliferation with the feeder cell contacted substratum of people's feeder cell for example.Conditioned medium comprises the factor that is discharged by feeder cell, and it supports hBS cell proliferation more than non-conditioned medium thus.In addition, this substratum can be no allos material, and this hBS cell in propagation will be used for the medical use of any kind and thereby be even more important must have clinical criteria the time.

Said supportive substratum can be supplemented with serum; For example FBS or human serum; Perhaps alternatively serum substitute, for example

serum substitute.Said supportive substratum can replenish has an appointment 1% to about 40% serum, and for example, about 5% to about 20% serum, for example 10% serum.

Said supportive substratum can be supplemented with one or more growth factors that is selected from the group that makes it possible to keep significant hBS cell characteristic; Form by following material for said group: EGF (Urogastron), HGF (pHGF), neurotrophin; Fibroblast growth factor; For example acid FGF and/or Prostatropin (bFGF), preferably, people's recombination basic fibroblast growth factor (hrbFGF).In one embodiment, said supportive culture medium supplemented has the hrbFGF of suitable concn.The suitable concn of hrbFGF can be about 0.5 to about 1000ng/ml hrbFGF, and for example, about 1 to about 500ng/ml hrbFGF, and about 2 to about 200ng/ml or about 4 to the scope of about 100ng/ml hrbFGF.

Four kinds of suitable medium are described below.Yet, considered that other substratum also can use, as long as they provide the nutritive ingredient of liquid form for the hBS cell; It is inorganic components; For example trace elements, and organic composition, for example amino acid, salt, mikrobe, energy provider, glucide (comprising carbohydrate) etc.

A kind of suitable medium of using in the present invention is to be supplemented with the VitroHES of 4ng/mlhrbFGF at least ^TMSubstratum.

Another kind can be used for suitable culture medium of the present invention and is made up of DMEM or other basic mediums;

Dulbecco improvement Eagle substratum for example, its be supplemented with 20%

serum substitute and the following composition of final concentration separately that is in: 50 units/ml penicillium mould, 50 μ g/ml Streptomycin sulphates, 0.1mM non-essential amino acid, 2mM L-glutaminate, 100 μ M beta-mercaptoethanols, 4ng/ml hrbFGF at least.

Can be used for another outer a kind of suitable medium (hBS cell culture medium) of the present invention is made up of following:

Dulbecco improvement Eagle substratum, its be supplemented with 20%

serum substitute and the following composition of final concentration separately that is in: 50 units/ml penicillium mould, 50 μ g/ml Streptomycin sulphates, 0.1mM non-essential amino acid, 2mM L-glutaminate, 100 μ M beta-mercaptoethanols and also be supplemented with 4ng/ml hrbFGF at least.

Other a kind of suitable medium of using in the present invention is the substratum of no allos material, and it comprises the substratum that is suitable for breeding the hBS cell.A kind of suitable basic medium is a Dulbecco improvement Eagle substratum (DMEM).Yet other substratum also can work.Except that the basic medium of no allos material, the substratum of said no allos material can also contain human serum, hrbFGF, L-glutaminate or glutamax, non-essential amino acid, beta-mercaptoethanol, penicillium mould and/or Streptomycin sulphate.The concentration of human serum is preferably about 1%v/v to about 30%v/v human serum in the substratum of no allos material, and for example, about 10%v/v is about 30%v/v human serum extremely, and about 15%v/v is about 25%v/v human serum and more preferably 20%v/v human serum extremely.The concentration of hrbFGF is preferably about 2ng/ml to about 100ng/ml hrbFGF in the substratum of no allos material; For example, about 5ng/ml is to about 50ng/ml hrbFGF, and about 5ng/ml is to about 25ng/ml hrbFGF; About 5ng/ml is to about 15ng/ml hrbFGF, for example 4ng/ml hrbFGF at least.The concentration of L-glutaminate or

is preferably about 0.5mM to about 20mM in the substratum of no allos material; For example; About 0.75mM is to about 10mM; About 1mM is to about 5mM, for example 2mM.The concentration of non-essential amino acid is preferably about 0.01mM to about 1mM in the substratum of no allos material, and for example, about 0.03mM is to about 0.8mM, and about 0.05mM is to about 0.6mM, and about 0.07mM is to about 0.4mM, and about 0.09mM is to about 0.2mM, for example 0.1mM.The concentration of beta-mercaptoethanol is preferably about 10 μ M 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.The concentration of penicillium mould is extremely about 200 units/ml of about 5 units/ml in the substratum of no allos material, and for example, about 10 units/ml is to about 150 units/ml, and about 25 units/ml is to about 100 units/ml, and about 25 units/ml is to about 75 units/ml, for example 50 units/ml.The concentration of Streptomycin sulphate is extremely about 200 μ g/ml of about 5 μ g/ml in the substratum of no allos material, and for example, about 10 μ g/ml are to about 150 μ g/ml, and 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.In a specific embodiment, said substratum is the DMEM that is supplemented with 1-30%v/v human serum and 2-100ng/ml hrbFGF.In one embodiment, said basic medium contains the 20%v/v human serum.In another embodiment, said basic medium contains 4ng/ml hrbFGF at least.

Duplication of production has gone out the high-quality human serum of the substratum that is used for above-mentioned no allos material in our laboratory.With regard to many standard infectious agents blood has been carried out testing (hepatitis B, C, HIV, HTLV and syphilis) at hospital's Blood Center.Therefore, the human serum that in the substratum of no allos material, uses preferably prepares through following steps:

1) healthy human blood is collected in the bag that non-heparin encapsulates,

2) shake the bag about 0.5 hour to about 5 hours that said non-heparin encapsulates, for example, about 0.5 hour about 2 hours time period extremely,

3) at least 10 hours the time period of bag that the said non-heparin of incubation encapsulates under the highest 5 ℃ temperature,

4) alternatively, select, for example, do not have the opaqueness of not solidified fibrin, liquid phase according to solidifying quality,

5) from the material that solidifies, isolate serum,

6) the said serum of sterile filtration,

7) from least 15 donors, compile serum,

8) freezing serum before the use.

Preferred culture systems

In a preferred embodiment, culture systems of the present invention comprises

I) density is at least 50,000 cell/cm ²People newborn infant's foreskin feeder cell,

Ii) be used for the hBS cell colony is dissociated into the TrypLE of single-cell suspension liquid ^TMSelect and

Iii) as the VitroHES of 4ng/ml hrbFGF at least that is supplemented with of supportive substratum ^TM,

This culture systems maybe be at the time period internal breeding hBS cell that prolongs through in each continuous passage, the hBS cell colony being dissociated into that single-cell suspension liquid makes, and keeps the notable feature of hBS cell simultaneously.

An improvement provided by the invention is, making maybe be through being dissociated into hBS cell colony enzymatic in the single-cell suspension liquid hBS cell that repeats to go down to posterity, and keeps the notable feature of hBS cell simultaneously.

The program of dissociating

Becoming single-cell suspension liquid to handle the hBS cell colony through the agent of dissociating with one or more hBS cell dissociation accomplishes.After removing employed substratum, can in PBS, wash the hBS cell colony, this can exhaust calcium and magnesium.In colony, add one or more agent of dissociating, and allow its through suitable temperature for example 37 ℃ work one suitable period, promptly the outer regions until the hBS cell colony begins from feeder layer, to gather.When said one or more agent of dissociating contains one or more enzymes, then the appropriate amount of enzymic activity be about 10 units/mL to about 5000 units/mL, for example, about 100 units/mL is to about 500 units/mL.Behind incubation, available volumetric pipette is pulverized repeatedly, so that help said one or more agent of dissociating cell sheet branch to be split unicellular to obtain hBS.Since the existence of said one or more agent of dissociating may in case the ability that the hBS cell forms the clone when being inoculated on people's feeder cell have a negative impact; Therefore; Can be according at least 1: 4; For example at least 1: 5 branch passes and is at least 50,000 cell/cm than said single-cell suspension liquid is dispensed to one or more density that contain ²The culture vessel of people's feeder cell in before reduce the effectiveness of said one or more agent of dissociating.The effectiveness of said one or more agent of dissociating can for example reduce through following manner: physical removal; For example carry out centrifugal, filter or deposition is separated with agent that hBS cell and said one or more are dissociated; Dilute said one or more agent of dissociating; One or more suppressor factor that add said one or more agent of dissociating, or one or more substrates of said one or more agent of dissociating of excessive adding.Alternatively, said one or more agent of dissociating can the oneself suppress, and promptly they have the capability that suppresses himself function in section back sometime, for example Accutase ^TMAnd Accumax ^TMSituation.Dissociate after the agent removing said one or more, be resuspended in the supportive substratum of the present invention the hBS that is obtained is unicellular, to obtain the single-cell suspension liquid of hBS cell.

Divide and pass ratio

Can the hBS cell in the single-cell suspension liquid that is obtained be dispensed in the culture vessel of one or more people's feeder cell that contain as stated preparation, be about to the hBS cell inoculation to feeder cell.The key character of this seeded process is; Culture systems of the present invention allows cell with at least 1: 4; For example at least 1: 5 branch passes than distributing; This means that the hBS cell is assigned to such feeder cell zone, this zone for through use one or more agent of dissociating the hBS cell dissociation is become single-cell suspension liquid before hBS cell oneself five times of zone that dissociate.Depend in degree of converging, the speed of growth and the homogeneity of back of dissociating about the standard of selecting specific branch to pass ratio based on the hBS cell colony in culture vessel of morphological observation and counting.Above-mentioned factor is high more, can adopt high more branch to pass stability and the quality that compares and do not jeopardize cell, and the expansion of the number of the hBS cell that is arrived is big more.Suitable branch passes ratio can be at about 1: 4 or 1: 5 to about 1: 5000, and for example, about 1: 20 to about 1: 1000, branch wherein commonly used passed than being about 1: 20 to about 1: 500 scope in about 1: 50.

Separate

Be used for the appropriate method that feeder cell and hBS cell are separated from each other can be comprised the following steps:

I) before inoculation, feeder cell are exposed to the feeder cell of magnetic-particle to obtain to modify through magnetic;

Ii) after changing substratum alternatively, inoculation was also cultivated the hBS cell at least one day subsequently on the feeder cell of modifying through magnetic;

The population mixture and the culture vessel of feeder cell and hBS cell are broken away from, and through using one or more for example TrypLE of agent that dissociates ^TMSelect with said colony be dissociated into single-cell suspension liquid and

Iv) feeder cell and hBS cell are assigned to through applying magnetic force.

Separation efficiency in the said method can be at least 50%, for example at least 70%, at least 80%, at least 90%, at least 99%, and wherein separation efficiency means the per-cent of the feeder cell overall number that magnetic force attracted that is applied in.

The analysis of notable feature

Whether having kept the notable feature of hBS cell in order to study the hBS cell, is that SA001 and SA121 transfer in the culture systems of the present invention and cultivated for 20～40 continuous generations with Cellartis ' hBSC.In the 20th generation and when above, thoroughly characterize said hBS cell.With the morphology of microscopic evaluation individual cells and the morphology of colony, it has disclosed the normal morphology (Fig. 1) of hBS cell and colony thereof.Use expression (Oct-4, SSEA-3, SSEA-4, Tra1-60, Tra1-81, SSEA-1) (embodiment 7, Fig. 2 and Fig. 5) of immunohistochemistry/histological chemistry's evaluation of markers thing on protein expression level.The heredity sign of implementing through karyotyping and fish analysis discloses, and caryogram has been kept and surpassed 20 generations (embodiment 8, Fig. 3 and Fig. 5).Through in the SCID mouse, forming the versatility of the hBS cell that teratoma assesses according to the present invention to be obtained, it discloses the hBS cell and after surpassing for 20 generations, is polyenergic undifferentiated hBS cell (embodiment 9, Fig. 4 and Fig. 5).

Other examples that hBS cell of cultivating according to the present invention is characterized can be the analyses of clone's survival; For example to different culture thing combination and parameter (for example, feeder cell type and density, dissociate agent and be exposed to time of the agent of dissociating) the unicellular execution CFA method of the similar number inoculated.Two important parameters identifying among the present invention are: the selection of the agent of i) dissociating; It tends to for the number of formed colony, to have importance; The ii) selection of feeder cell type, it tends to have importance for formed being cloned in for the quality that is in the undifferentiated state aspect.Differentiation grade (grade of differentiation) can be analyzed through the morphology in the microscope successively, and possibly be associated with the marker representation analysis about known not differentiation and differentiation marker thing of previous completion.

Can further characterize stem cell and blastocyst-derived stem cells with regard to its telomerase activation, said telomerase activation can be tested with the test kit that for example is called Telomerase PCR ELISA test kit (Roche).This test kit utilizes the intrinsic activity of Telomerase, wherein through (PCR) increases to product and with enzyme-linked immunosorbent assay (ELISA) it detected through the polymerase chain reaction.Telomerase activation also can be measured through QPCR.

Cytodifferentiation state among the present invention can be tested through the QPCR about specific gene in addition.Following Short Description its how to implement.Can adopt mechanical means that the hBS cell colony of undifferentiated or differentiation is broken away from as complete colony and culture plate, and in PBS, wash and be stored in-80 ℃.Further, can be according to manufacturer's specification sheets, for example using, Qiagen RNeasy Mini Kit extracts RNA.Use suitable test kit, for example Bio-Rad iScript First Strand Synthesis Kit (according to manufacturer's specification sheets) carries out rt, and under appropriate condition, carries out QPCR in Rotorgene 3000 (Corbett Research).Can be in identical samsara quantitative all genes, and if possible, can come the differentiation state of more several samples through calculate the mathematical index of sample (mathematical indices) alone based on genetic marker.(more detailed scheme is described among the WO2006094798.)

Employed component alone in the invention that this paper respectfully presented; For example feeder cell, substratum and blastocyst; And the hBS clone of cultivating according to the present invention; Can test with regard to human pathogen before use, for example, mycoplasma, 1 and 2 type human immunodeficiency viruses, B-mode and hepatitis C, cytomegalovirus, 1 and herpes simplex types 2 virus, Epstein-Barr virus and human papillomavirus.Not having human pathogen is important for the cell of hBS clone and differentiation or any clinical application of deriving from the other biological material of this type of clone.

Other aspects of the present invention

The invention enables when going down to posterity, the hBS cell colony to be dissociated into unicellularly, this provides several the improvement that surmount existing culture systems and method.Several the application that are particularly suitable for through the hBS cell that uses culture systems according to the present invention to obtain have below been summarized, further to stress benefit provided by the present invention and improvement.

Compare with existing culture systems and method, the present invention helps the propagation of hBS cell, and this is because the present invention passes than becoming possibility big branch, allows the expansion of hBS cell quantity greatly thereby compare with existing method.This means, the invention provides improved possibility about the expansion scale that enlarges the hBS cell in proportion.Owing to becoming the possible single-cell suspension liquid that when going down to posterity, obtains, the present invention passes (scalable) that can change the in proportion production that makes it possible to realize the hBS cell than this combination with high score; This can also become the object of robotization, and the culture systems that is used to breed the hBS cell that saves time with expense is provided thus.

The agent of dissociating of employed said one or more can comprise TrypLE when using the production that the disclosed culture systems of this paper carries out changing in proportion ^TMSelect, Accutase ^TMAnd Accumax ^TMIn at least a.

Reach as the single celled hBS cell in the single-cell suspension liquid that obtains according to culture systems of the present invention through use; Make it possible to utilize known quant program and device to come the hBS cell is carried out accurate quantification; For example, NucleoCounter, Hemocytometer Manual Count, Flowcytometer Automated Count.Like this quantitatively be important so that can stdn with improve all types program that the hBS cell possibly experience.

In addition; When going down to posterity, make the hBS cell become unicellular these cells that also make and to experience different cells separation as known in the art and cell sorting technology; For example; Density gradient media, based on the chromatography of antibody or the magnetic bead of antibody sandwich, for example for the resistates of hBS cell with feeder cell separated.Alternatively; The single-cell suspension liquid of the hBS cell that obtains can experience different types of sorting technology; For example, FACS (fluorescence automated cell separating method) or magnetic bead sorting method, density gradient centrifugation, (affine) chromatographic separation will be for example in order to separate through the hBS cell of the hBS of transfection cell with untransfected.

HBS cell in the described unicellular solution can be the good starting point that is used for processes, promptly carries out limited dilution cloning (limiting dilution cloning) from hBS clone, to produce the clone with specific characteristic.

In addition, for the utilization of the hBS cell that further promotes to be cultivated, can from feeder cell, isolate the hBS cell.Hereinafter, a kind of such separation method has been described.

Being used for from a kind of possibility method of feeder cell separation hBS cell is to allow little iron particle to mix in one of said cell type.Mixing like this can be passed through the spontaneous completion of cell (for example through endocytosis or phagolysis), perhaps accomplishes through electroporation.Cell can be exposed to the suitable iron particle that is suspended in the substratum before inoculation.Feeder cell can be perhaps to stagnate aspect the mitotic division with alternative mode through what ametycin was handled before being exposed to the iron particle.The iron particle can have few types or label (brand).They can also be Fe2+ ion or Fe3+ ion or its mixture.In one embodiment of the invention, used Endorem ^TMThe iron particle also can have the diameter of 1.0nm to 50nm, for example 2.0 and 40nm between diameter, 4.0 and 30nm between diameter.Iron particulate concentration can change between 0.1ug/ml and 560ug/ml, and for example 0.2 to 300ug/ml, and 0.5 to 100ug/ml, and 0.75 to 10ug/ml, and 1.0 to 3.0ug/ml.Cell can be exposed to ferruginous solution about 1 minute to about 48 hours, and for example about 20 minutes to about 12 hours, for example about 60 minutes to about 5 hours, for example about 2 to 3 hours.After being exposed to magnetic-particle, can feeder cell being inoculated in the culture vessel and change substratum.In case formed the layer and until the inoculation feeder cell after at least one week, just the unicellular solution with the hBS cell is seeded on the feeder cell.Can the hBS cell keep be cultivated on ferruginous feeder cell 1 day for example at least 2, at least 4, at least 7, at least 10, at least 20 days at least.

Further, separation can be carried out through following manner: produce the single-cell suspension liquid of mixed cell population, and subsequently said cell suspending liquid is exposed to magnetic force.This magnetic force can derive from size with wherein keep remaining isolated cells container or manage compatible any suitable magnet.Said isolating a kind of possible summary description is in embodiment 13.

Through using the hBS cell that obtains according to culture systems of the present invention also to be particularly suitable for experiencing the cell transfecting program; This is because the unicellular state of hBS cell has been avoided the cytogamy in the electroporation process, has avoided the blended clone thus and has improved transfection efficiency.

In addition, be suitable in the porous flat plate assay method through using the hBS cell that obtains according to culture systems of the present invention, because the single-cell suspension liquid that is obtained can easily be uniformly distributed in the porous flat plate.Therefore, the single-cell suspension liquid of hBS cell can be used in the porous flat plate assay method, for example so that carry out the toxotest of different compounds or in the drug development program, identify drug candidates.

Inoculation is dissociated into single celled hBS cell to be used for specific end use on low feeder cell density

In some cases, maybe be advantageously with the cell inoculation in the single-cell suspension liquid to feeder cell, wherein feeder cell are for example to be lower than about 50,000 cells/cm ², about 10,000 cells/cm ², about 15,000 cells/cm ², about 20,000 cells/cm ², about 25,000 cells/cm ²Low density exist.This possibly be appropriate, for example as intermediate steps, promptly uses or before the system that is being used for deriving from hBS clone the cell type of differentiation uses in the discriminating that is used for toxotest.In such application, when from feeder cell, isolating the hBS cell, low feeder cell density possibly be favourable.

Description of drawings

Fig. 1:

Utilize hFF and enzyme (TrypLE Select ^TM) as the morphology of the unicellular hBS cell colony of cultivating and going down to posterity.

A) after their the enzymatic first time dissociates, the hBS cell colony is adapted to.

B) observed zone (in new system, going down to posterity for the first time) in the adjustment process in early days with foreign cell colony.

C) size of aggregate, i.e. unicellular in enzymolysis goes down to posterity process.

D) the single-cell suspension liquid of the hBS cell on hFF.

E) dissociated back two days at enzymatic, be adapted to the hBS cell of current system through adjustment.

F) the hBS cell colony that adapts to through adjustment.

G) the hBS cell colony of homogeneous (to upper right).

H) has the culture hole of hBS cell colony, overview.

Scale is 50 μ m (c), and 100 μ m (a, b, g), 200 μ m (d), 250 μ m (e, f), 1.66mm (h).

Fig. 2:

In following description of drawings, " SA001 TrypLe " refers to the hBS cell from Cellartis ' s clone SA001, and it is through using TrypLE Select ^TMBreed and " SA002 TE " refers to the hBS cell from same cell system, it is through using trypsinase-EDTA to breed.Using TrypLE Select ^TMAnd after trypsinase-EDTA surpasses 20 enzymatics and go down to posterity, the immunohistochemical staining of SA001:

Carry out painted SA001 TrypLe:Oct-4 (a), TRA-1-81 (c), SSEA-4 (e) and SEAP (g) with regard to following material.Carry out painted SA001 TE:Oct-4 (b), TRA-1-81 (d), SSEA-4 (f) and SEAP (h) with regard to following material.

Scale is 100 μ m (a-g).Scale is 250 μ m (g).

Fig. 3:

Using TrypLE Select ^TMAfter carrying out going down to posterity for 25 times, the caryogram of SA001 and FISH

(a) karyomit(e) from SA001 TrypLE is that diploid is normal.The figure illustrates representational caryogram.(b) SA001 TE, diploid is normal.(c d) proves that to the selected chromosomal fish analysis from SA001 TrypLE (c) and SA001 TE (d) these cells are that XY and diploid are normal, chromosome x (blueness), Y (gold), 13 (redness), 18 (aquas) and 21 (greens).

Fig. 4:

Versatility test in the body of SA001.Using TrypLE Select ^TMAnd trypsinase-EDTA surpasses the teratoma after going down to posterity for 20 times.

After carrying out respectively 27 times and 22 enzymatics go down to posterity, (a, c is e) with SA001 TE (b, d, teratomatous histologic analysis f) from SA001 TrypLE.(a, b) neuroderm (ectoderm), (c, d) cartilage (mesoderm), (e, f) secretory epithelium (entoderm)

Scale be 25 μ m (a, c, e) with 50 μ m (b, d, f).

Fig. 5:

Using TrypLE Select ^TMAfter carrying out 20 unicellular going down to posterity of enzymatic, the sign of SA121.

(a) Oct-4 immunohistochemical staining, (b) TRA-1-60, (c) SSEA-3, (d) SSEA-4, (e) SEAP.(f) carrying out after 20 unicellular enzymatics go down to posterity the normal caryogram of the diploid of SA121TrypLE.(g-i): 23 teratomas of opisthogenesis that go down to posterity: (g) neuroderm (ectoderm), (h) cartilage (mesoderm), (i) secretory epithelium (entoderm) from SA121.Scale: a-e:100 μ m, g-i:50 μ m.

Fig. 6:

Use TrypLE ^TMSelect carries out unicellular enzymatic and goes down to posterity and cause clone survival to improve.When at TrypLE ^TMAfter Select handles with cell transfer when hFF goes up, cause the number of formed hBS cell colony and trypsinase-EDTA processing Comparatively speaking to be increased to 3 times (p=00.1).If with dissociated hBS cell bed board on hFF, if then with their bed boards on mEF, Comparatively speaking cause the number of good colony significantly to increase (p=0.2).Institute's data presented is MV+standard error (n=3).

Fig. 7:

Cultivate the hFF cell after a week.(A) shown the hFF that is collected on the magnet and (B) shown a small amount of remaining hFF cell that in suspension-s, exists.

Fig. 8:

At human embryonic fibroblast (American Type Culture Collection, CCL-110 ATCC, Manassas, the use TrypLE that carries out on VA) ^TMThe unicellular enzymatic of Select goes down to posterity.

Embodiment

Embodiment 1

The cultivation of HFF's feeder cell and as the purposes of feeder layer

From American type culture collection (American Type Culture Collection) (CRL-2429 ATCC; Manassas; VA) obtain to be purchased the hFF that can get; And cultivation is being supplemented with Iscove ' s DMEM (Gibco Invitrogen Corporation, Paisley, the Scotland of 10%FBS (Invitrogen) and 1% penicillium mould-Streptomycin sulphate; Http:// www.invitrogen.com) in.(weekly) pair cell goes down to posterity to use trypsinase-EDTA (Invitrogen) to compare termly with the branch biography between 1: 2 and 1: 8.The confluent monolayer of hFF is handled (10 μ g/ml, 2.5 hours) with Mitomycin-C (Sigma), and is being supplemented with 4ng/ml people's recombination basic fibroblast growth factor (hrbFGF, VitroHES Invitrogen) ^TMIn the substratum with 70,000-80,000 cell/cm ²Density be plated in the IVF ware that 0.1% gelatin (Sigma) encapsulates.Between the 4th generation and the 12nd generation, the hFF cell is used as feeder cell.

Embodiment 2

Human embryonic fibroblast's cultivation and as the purposes of feeder layer

From American type culture collection (CCL-110 ATCC; Manassas; VA) obtain to be purchased the human embryonic fibroblast that can get; And cultivation is being supplemented with Iscove ' s DMEM (Gibco Invitrogen Corporation, Paisley, the Scotland of 10%FBS (Invitrogen) and 1% penicillium mould-Streptomycin sulphate; Http:// www.invitrogen.com) in.(weekly) pair cell goes down to posterity to use trypsinase-EDTA (Invitrogen) to compare termly with the branch biography between 1: 2 and 1: 8.Human embryonic fibroblast's confluent monolayer is handled (10 μ g/ml, 2.5 hours) with Mitomycin-C (Sigma), and is being supplemented with 4ng/ml people's recombination basic fibroblast growth factor (hrbFGF, VitroHES Invitrogen) ^TMIn the substratum with 70,000-80,000 cell/cm ²Density be plated in the IVF ware that 0.1% gelatin (Sigma) encapsulates.Between the 4th generation and the 12nd generation, the human embryonic fibroblast is used as feeder cell, Fig. 8.

Embodiment 3

The foundation of HFF's feeder cell systems (for example clone hFF003)

Will from implemented posthetomy 8 the week age boy aseptic being collected among the aseptic IMDM (Invitrogen) that contains 2 * qingfengmeisu qiong of people's foreskin sample.The skin explant is placed the 25cm2 tissue culture flasks of former generation (Becton Dickinson) that contains IMDM substratum (Invitrogen), 1% penicillium mould-Streptomycin sulphate (Gibco Invitrogen Corporation) and 10% human serum.After about 10 days, set up confluent monolayer.Use TrypLE ^TMSelect (Invitrogen) pair cell carries out continuous passage.After expansion, to they tests, all results are all negative with regard to lineup's pathogenic agent of standard (mycoplasma, 1 type and 2 type HIV, B-mode and hepatitis C, cytomegalovirus, 1 type and herpes simplex types 2 virus, Epstein-Barr virus, human papillomavirus).

Embodiment 4

The foreskin inoblast clone of (in-house) foundation prepares feeder layer internally

Do not have at bed board before human fibroblasts's feeder cell of allos material, at room temperature use 0.1% recombinant human gelatin (Fibrogen) to encapsulate through hole that tissue culture medium (TCM) is handled at least 1 hour.The confluent monolayer of the hFF003 of the no allos material that will in IMDM, 10% human serum and 1% penicillium mould-Streptomycin sulphate, grow then (8 generations of the 5th generation to the) cell is handled (10 μ g/ml, 2.5 hours) with Mitomycin-C (Sigma).The feeder cell that to handle through Mitomycin-C in substratum with 200,000 cells/2.89cm ²Be plated in the IVF hole (Becton Dickinson), said substratum is based on the DMEM (as above-mentioned) that is supplemented with 10% (v/v) human serum, 1% penicillium mould-Streptomycin sulphate, 1%Glutamax, 0.5mmol/L beta-mercaptoethanol and 1% non-essential amino acid (Gibco Invitrogen Corporation).Before placement has its inner cell mass cell and derives from its blastocyst of cell or hBS cell; Substratum is replaced by DMEM (as above-mentioned), and it is supplemented with 20% (v/v) human serum, 4ng/mLhrbFGF, 1% penicillium mould-Streptomycin sulphate, 1%Glutamax, 0.5mmol/L beta-mercaptoethanol and 1% non-essential amino acid (Gibco Invitrogen Corporation) now.(with identical substratum described in the embodiment 3.)

Embodiment 5

HBSC is transferred to the enzymatic proliferated culture medium

Like previous [Heins; Noakssson] and WO03055992 described in; Set up and characterized hBS clone SA001, SA002, SA002.5, SA121, SA167, SA348, SA461 and SA502 (Cellartis AB;

Sweden, http://www.cellartis.com).Such material can obtain from Cellartis AB, also can pass through NIH stem cell register office Http:// stemcells.nih.gov/ Research/registry/Obtain.Cellartis AB has two kinds of hBS clones (SA001 and SA002) and passes through the subclone (SA002.5) of the obtainable SA002 of NIH.These hBS clones are used for the present invention continually.All employed hBS clones all be given the ratification and register by Britain stem cell bank steering committee (UK Stem Cell Bank Steering Committee), and SA001, SA002 and SA002.5 are ratified by MEXT (Japan).Before test, in the IVF ware, these clones are maintained at the VitroHES that is supplemented with 4ng/ml hrbFGF ^TMIn the substratum (Vitrolife AB, Kungsbacka, Sweden, http://www.vitrolife.com) on the mEF of Mitomycin-C inactivation feeder layer, and through using microscopic capillary to carry out manual cut as cutting and means of transferring.Be transferred to enzymatic dissociates for the hBS cell is cultivated from tradition, remove employed substratum, and wash a petridish with PBS (Invitrogen).In each IVF ware, adding volume then is the TrypLE of 0.5mL ^TMSelect (Invitrogen), Accutase ^TM(Chemicon) or trypsinase/EDTA (Invitrogen).Ware carries out incubation under 37 ℃, begin to gather from feeder layer until the external region of hBS cell colony.Through pulverizing repeatedly the cell sheet is smashed into single-cell suspension liquid then, transfer in the centrifuge tube with volumetric pipette, and with 400g centrifugal 5 minutes.Abandoning supernatant, with the granular pellet resuspended of hBS cell in fresh VitroHES ^TMIn the substratum, and single-cell suspension liquid is inoculated in the IVF ware, said IVF ware contains the intensive feeder layer of the hFF of inactivation.

Embodiment 6

Through adopting as the go down to posterity enzymatic propagation of the hBS cell that carries out of single-cell suspension liquid

In order to carry out enzymatic propagation, the hBS cell is maintained in the culture systems, said culture systems is by highdensity hFF feeder cell and the VitroHES that is supplemented with 4ng/ml hrbFGF ^TMSubstratum is formed.Through dissociating to remove the initial enzymatic of substratum with PBS (Invitrogen) washing petridish.In each ware, adding volume then is the TrypLE of 0.5mL ^TMSelect (Invitrogen), Accutase ^TM(Chemicon) or trypsinase/EDTA (Invitrogen).Ware carries out incubation under 37 ℃, begin to gather from feeder layer until the external region of hBS cell colony.Through pulverizing repeatedly the cell sheet is smashed into single-cell suspension liquid then with volumetric pipette.Subsequently cell suspending liquid is transferred in the centrifuge tube, and with 400g centrifugal 5 minutes.Abandoning supernatant, and with the granular pellet resuspended of hBS cell in fresh VitroHES ^TMIn the substratum.Compare cell inoculation in new IVF ware to pass at the branch between 1: 4 or 1: 5 and 1: 500 then, said IVF ware contains the intensive feeder layer of the hFF of inactivation.In the process that goes down to posterity several times at first in new system, be called in the process of adjustment program, use lower branch to pass ratio at us.Be no more than go down to posterity for 5 times after, pass than cutting apart hBS clone with the branch between 1: 20 and 1: 500.Culture is maintained in the incubator that is under 37 ℃ and 95% humidity.Used fresh VitroHES in per 2～3 days ^TM+ 4ng/ml hrbFGF replacement exhausted substratum.According to the speed of growth of each hBS clone, cell was gone down to posterity in per 6～12 days.When submitting the application to, SA001 and SA121 clone have all been cultivated and have been surpassed 20 generations (wherein having kept normal caryogram), and further in it is supplemented with the normal cultured base of 10%DMSO, have carried out freeze thawing according to the slow freezing method of routine.

Embodiment 7

The immunohistochemistry of hBS cell and tissue chemical analysis

In 4% paraformaldehyde, the hBS cell culture is fixed 15 minutes, infiltrationization is 5 minutes in 0.5%trition solution (Sigma-Aldrich), and the 5%FBS that is used in subsequently among the PBS (Invitrogen) seals.Cell is incubated overnight under 4 ℃ with an anti-solution.Employed one anti-Oct-4, TRA-1-60, TRA-1-81, SSEA-1, SSEA-3 and the SSEA-4 (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.southernbiotech.com) of being specific to.At room temperature with two anti-(Jackson Immunoreserach Laboratories) incubations that are conjugated with FITC or Cy3 60 minutes.Nucleus is redyed with DAPI (Sigma).According to the specification sheets of manufacturer (Sigma-Aldrich), use the alkaline phosphatase activities detection kit to measure alkaline phosphatase activity.Use Nikon Eclipse TE-2000 U fluorescent microscope to assess and write down dyeing.After surpassing for 20 generations, SA001 and SA121 demonstrate all above-mentioned hBS cell characteristics.

Embodiment 8

Heredity characterizes

For karyotyping, incubation hBS cell digests with trypsinase in the presence of Colchiceinamidum, and is fixing, and is placed on the slide glass.Dyeing through DAPI makes karyomit(e) visual, uses to be equipped with appropriate filter mirror and software (CytoVision; Applied Imaging; Santa Clara CA, Http:// www.appliedimagingcorp.com) inverted microscope arrange and record.

Analyze for fluorescence in situ hybridization (FISH), according to manufacturer's specification sheets and do less modification, use the test kit that can get that is purchased that comprises to the probe of karyomit(e) 12,13,17,18,21, X and Y.Under the inverted microscope that is equipped with appropriate filter mirror and software (CytoVision), analyze slide glass.

In system of the present invention, cultivate above after 20 generations, SA001 and SA121 all show normal caryogram.More increase for the time, confirm that FISH is normal.

Embodiment 9

Versatility analysis in the body

As said in early days [people such as Heins], form through the teratoma in immunodeficient mouse (SCID) and to assess versatility.In brief, undifferentiated hBS cell colony cut mechanically is become 200x200-μ m sheet, and be placed on severe associating SCID mouse (C.B-17/lcrCrl-scidBR through surgical operation; Charles River Laboratories) under the scrotum.Mouse are put to death in 8 week backs, cut tumour and in 4% paraformaldehyde, fix.Be evaluated in the painted paraffin section of h and E, whether there is the undifferentiated people's tissue that derives from all three kinds of germinal layers on the histology, for example, neuroderm, cartilage and intestines appearance epithelium.

Even after surpassing for 20 generations, all three kinds of germinal layers have all been confirmed in SA001 that in coming comfortable system of the present invention, cultivates and the teratoma of SA121.

Embodiment 10

The cultivation and the sign of other hBS clone in the enzymatic legacy system

Except that above mentioned hBS clone SA001 and SA121, hBS clone SA167 and SA002 also successfully transfer to and are incubated in the aforesaid enzymatic legacy system.

The characteristic that is incubated at the hBS clone in the said enzymatic legacy system is shown in the following table.

Abbreviation: p, enzymatic passage number; TE, trypsinase-EDTA; TrypLE, TrypLE Select; Undiff, not differentiation; ND, undetermined; Endo, entoderm; Ecto, ectoderm; Meso, mesoderm.

Embodiment 11

The robustness of present method, repeatability and general applicability

The enzymatic of being invented that is used for is cultivated a big advantages of hBSC system and is, confirmed it can stablize, strong and easily breed several clones.Seven kinds of different hBSC systems have been recycled and reused for assessment and have set up said method.In addition, confirm also that it also is stable with fast with the adjustment program of carrying out cultural method subsequently that being used to of being invented set up hBS clone for several different hBS clone.The number of times of every kind of clone foundation is shown in the bracket: SA001 (6), SA002 (5), SA002.5 (＞3), SA167 (3), SA348 (＞4), SA461 (＞10) and SA502 (2).In the industriallization cell cultures is produced, can implement the cultivation that continuous foundation according to the present invention comes to enlarge in proportion hBS clone.

Embodiment 12

The CFA method

Said enzymatic goes down to posterity and the holding capacity of culture systems in order to test, through using TrypLE ^TMSelect, Accutase ^TMOr the hBS cell (on mEF, adopting machinery to go down to posterity) that trypsinase-EDTA cultivates tradition is dissociated into unicellular.Dilute said hBS cell, and inoculate on the hFF or mEF in new IVF ware, produce about 350 and 700 hBS cell/cm with two kinds of density ²

Changed substratum in every 2-3 days.After the about week, remove substratum, and (specification sheets according to manufacturer (Sigma-Aldrich) carries out alkaline phosphatase staining subsequently for Gibco, invitorgen) washed cell with 1 * PBS.Number to the hBS cell colony that from all four test group, obtains is counted.For the sxemiquantitative assessment of the number of undifferentiated hBS cell colony, if the colony above 50% is undifferentiated when visual observation in inverted microscope, then these colonies are designated as the positive.These tests are carried out in duplicate, and triplicate.SA002.5 is used for these tests with hBS clone.

No matter use which kind of enzyme, about 90% colony is cited as undifferentiated (referring to Fig. 6) on hFF.At 200 Accutase that use through assessment ^TMIn the colony of handling, 191 are judged as undifferentiated.As relatively, if with dissociated hBS cell bed board on mEF, then obtain the remarkable reduction of good colony number; 60% the colony of only having an appointment is cited as undifferentiated (Fig. 6).In two kinds of dilutions being tested, quality discrepancy is similar.Therefore, use as the hFF of feeder cell be used to carry out dissociated TrypLE ^TMSelect seemingly is used to promote the most favourable combination of clone's survival of undifferentiated multipotency hBS cell.

Embodiment 13

Utilize magnetic-particle from the hBS cell, to separate hFF

The T-75 bottle that the hFF cell that converges is housed is handled 2-3 hour to suppress cell proliferation with ametycin.After ametycin was handled, hFF with 1 * PBS washing repeatedly passed through to use 1 * trypsinase-EDTA or 1 * TrypLE afterwards ^TMSelect is dissociated into it unicellular.The aliquots containig of cell is counted in hematimeter, and with cell dilution to proper concn.If the magnetic-particle (Endorem of different concns to be tested ^TM), then cell suspending liquid dividing to different pipes, the magnetic-particle with the difference amount is added in the pipe then.In pipe, mix hFF and magnetic-particle then, afterwards with the suspension-s bed board in the IVF ware (Falcon) that encapsulates through gelatin.Each IVF ware is inoculated about 200,000 hFF cells.

The Endorem that is tested ^TMConcentration is in the scope of 5.6ug to 560ug/ hole and 200,000 cells, and it is corresponding to 2.8ug/ml to 280ug/ml.

After inoculation 1-2 days, change 100% substratum, this has removed most of iron particle unconjugated and not endocytosis.Change behind the substratum about 24 hours, the hBS cell is added on the plate with hFF feeder cell.

For hFF is collected on the magnet, in 1 * PBS the rinsing cell once, and with trypsinase-EDTA or 1 * TrypLE ^TMSelect is dissociated into unicellular.Then cell suspending liquid is transferred in the eppendorf pipe and placed with magnet and closely contact.Let cell attachment several minutes to the magnet, from pipe, remove rest solution then.Afterwards, remove pipe from magnet, and Xiang Guanzhong adding suitable medium or 1 * PBS, the solution weight that will contain feeder cell then suspends to carry out cell counting and checking separation efficiency.

On magnet, caught at least 90% hFF (90% separation efficiency) (referring to Fig. 7), no matter the different concns of being tested.When will be through on the hFF of hBS cell inoculation in magnetic that enzymatic goes down to posterity the time, hBS cell attachment, propagation also form cell colony (data not shown).

Reference

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Claims (14)

1. be used for propagation of human blastocyst-derived and do the culture systems of (hBS) cell, it comprises:

I) density is 50,000 cells/cm ²To 500,000 cells/cm ²Derive from newborn infant or the fibroblastic people's feeder cell of teenager,

Ii) be used for when going down to posterity the hBS cell colony is dissociated into the agent combination of dissociating of single-cell suspension liquid at every turn; It comprises one or more enzymes that are selected from trypsinase, Dispase II, PRONASE A, collagenase and matrix metalloproteinase and be selected from EDTA, EGTA and HEDTA one or more sequestrants and

Iii) be used for the supportive substratum of human blastocyst-derived stem cells growth, it is selected from DMEM, IMDM and VitroHES ^TM

2. according to the culture systems of claim 1, wherein said feeder cell derive from people's neonatal human foreskin fibroblast.

3. according to the culture systems of claim 1, wherein said people's feeder cell aspect growth by inactivation.

4. according to the culture systems of claim 3, wherein said people's feeder cell handle through MTC and aspect growth by inactivation.

5. according to the culture systems of claim 3, wherein said people's feeder cell through radiation aspect growth by inactivation.

6. according to the culture systems of claim 1, wherein inoculated said people's feeder cell before in 1 to 10 day at the said hBS cell of inoculation.

7. according to the culture systems of claim 6, wherein before the said hBS cell of inoculation, substratum is changed at least once.

8. according to the culture systems of claim 1, wherein said enzyme is the enzyme of reorganization.

9. according to the culture systems of claim 1, it is no allos material that the wherein said agent of dissociating is made up.

10. according to the culture systems of claim 1, the wherein said agent combination of dissociating is selected from and is purchased the TrypLE that can get ^TMSelect, Accutase ^TMOr Accumax ^TM

11. according to the culture systems of claim 1, wherein said supportive culture medium supplemented has 0.5 to 1000ng/ml hrbFGF.

12. according to the culture systems of claim 1, wherein said supportive culture medium supplemented has 1% to 40%v/v serum.

13. according to the culture systems of claim 12, wherein said serum is FBS or human serum.

14. according to the culture systems of claim 1, said culture systems comprises:

I) density is at least 70,000 cell/cm ²People's neonatal human foreskin fibroblast feeder cell,

Ii) be used for the hBS cell colony is dissociated into the TrypLE of single-cell suspension liquid ^TMSelect and

Iii) be supplemented with the VitroHES of 4ng/ml hrbFGF at least ^TMSupportive substratum,

This culture systems maybe be at the time period internal breeding hBS cell that prolongs through in each continuous passage, the hBS cell colony being dissociated into that single-cell suspension liquid makes, and keeps the notable feature of hBS cell simultaneously.

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