CN1784143B - Cryopreservation of human blastocyst-derived stem cells by use of a closed straw vitrification method - Google Patents

Abstract

An improved method for vitrification of biological cells, especially blastocyst-derived stem cells (BS cells). The method is very mild for the cells that remain viable after they have been thawed. The method comprises, i) transfer of the cells to a first solution (solution A), ii) optionally incubation of the cells in the first solution, iii) transfer the cells obtained in step i) or ii) to a second solution (solution B), iv) optionally incubation of the cells in the second solution, v) transfer of the cells obtained from step iii) or iv) into one or more closed straws with dimensions that allow a volume of at least 20 mul to be contained in them vi) sealing the one or more closed straws, and vii) vitrification of the one or more closed straws. An important feature of the present invention is the use of closed straw and that relatively large volumes can be efficiently vitrified and subsequently thawed.

Description

Through using the stem cell of closed straw vitrification method cryopreservation of human blastocyst-derived

Invention field

The present invention relates to be used for the vitrifying biological cell, the improving one's methods of particularly blastocyst-derived stem cell (BS cell).Said method to still keep after thawing active cell be non-normal temperature and.

Background of invention

Stem cell is the cell type with self and the unique ability that produces specialization or noble cells.Although most somatic cell, for example heart cell or Skin Cell are responsible for exercising specific function, and stem cell function before it receives the signal that develops into particular cell types is uncertain.Form said stem cell unique be its multiplication capacity with and the ability of differentiation takes place.For many years, the researcher is devoted to seek stem cell alternative impaired or the cell of pathology and the method for tissue used.At present, most of research concentrates on two types stem cell, embryonic stem cell and somatic stem cell.The fertilized oocyte that embryonic stem cell derives from the embryo before implanting, i.e. blastocyst, and said somatic stem cell is present in the ripe organism, for example in marrow, epidermis and the intestines.According to many Europe and other national state's laws, fertilized egg is not to be considered to the embryo in after fertilization 10-14 days before implanting the uterus, and therefore when using like the present invention, this type cell is called as blastocyst-derived stem cell or hBS cell herein.Although the versatility check has shown that said embryo or blastocyst-derived stem cell can produce all cells in the organism, comprise reproductive cell, somatic stem cell has more limited derived cell set of types storehouse.

1998, the researcher can isolate embryonic stem cell for the first time and it is cultivated in medium from people's fertilized oocyte, referring to for example United States Patent (USP) 5843780 and United States Patent (USP) 6,200 806.

Increasing research and progress need to obtain to be suitable for preserving the method for said cell and cell-line in the stem cells technology field.Cell can be through vitrifying or chilled storage.Because the formation of extracellular ice has destruction, therefore use the low temperature storage of conventional method to be difficult to be applied to complicated with responsive biologic material.Through the vitrifying process, the sample that contains said cell is quickly cooled to extremely low temperature, the non-crystallizable and hyaloid attitude that forms of water capacity thing.Therefore, vitrifying is the method for snap frozen liquid medium under the situation that no ice crystal forms.The snap frozen process that amorphous glass is put into liquid nitrogen at the suction pipe that directly will for example contain cell forms.Said glass keeps the normal distribution of liquid but preserves with overcooled form.Said glass has avoided ice crystal and said cell to avoid forming relevant primary cellular defect with ice crystal.Therefore, vitrifying is defined as the curing of the amorphous glass appearance attitude of avoiding nucleus to form and growing.

Once the human embryo stem cell low temperature storage was studied and people such as Reubinoff (Human Reproduction, 2001,16,2187-2194) described through using opening drawing and pulling type straw vitrification method to carry out the method for these cells of low temperature storage.The shortcoming of said method is that its openend that relates to suction pipe contacts with liquid nitrogen, and this may become will be by the pollution source of vitrified biomaterial.In addition, because the size of pull suction pipe, people such as Reubinoff carry out vitrified volume and are about 1 μ l.

The method for vitrification that is used to avoid directly contacting with nitrogen is at people such as Lopez-Bejar (Theriogenology; 2002; 58:1541-52) to people such as rabbit embryonic and Chen (HumanReproduction, 2001,16 (11): 2350-56) be able in the application to oocyte of mouse describe.Therefore these methods have all been used the pull mode identical with known opening pull suction pipe and have been had the sealing pull suction pipe that the pull suction pipe that uses with people such as Reubinoff has identical size.Therefore, in these methods, vitrified volume all is 1-2 μ l approximately in each suction pipe.

Slow freezing and method for rapidly thawing once were used for low temperature storage cell-line.Although these methods are fit to be used for for example mouse embryo stem cell of low temperature storage, as if very low for the survival rate of undifferentiated human embryo stem cell, and most said cell differentiation or death.Usually, the cell with this slow freezing method vitrifying larger volume can cause lower recovery rate (people such as Reubinoff).

Therefore, still with regard to needs development easy operating, relate to the least possible step and in said process, avoid simultaneously or reduce effective method for vitrification at least the risk of the undesired pollution of said cell.Especially, need development to be used for the vitrified effective ways of larger volume of cell or cell-line (for example hBS or hBS cell-line).

Invention is described

As stated, effectively the low temperature storage method is blastocyst-derived stem cell line generation with extensive use, also to set up people's blastocyst stem cell bank hereunder necessary.Effectively freezing and deforst technique can be preserved cell and cell-line effectively.For some purposes, hope in each root suction pipe, to carry out big volume vitrifying.For example when in given step (or application), needing a large amount of cells or when cell will be sent with charge free through mailing with its vitrifying state, being exactly this situation.

The present invention relates to be used for the vitrified method of cell, comprise

I) with in cell transfer to the first kind of the solution (solution A),

The ii) said cell of incubation in first kind of solution randomly,

Iii) with step I) or ii) in cell transfer to the second kind of the solution (solution B) that obtains,

The iv) said cell of incubation in second kind of solution randomly,

The cell transfer that v) will obtain ii) or iv) from step I has a closed straw that permission can comprise at least 20 μ l volumes size therein to one or more,

Vi) seal said one or more closed straw and

Vii) one or more closed straws of vitrifying.

A very important characteristic of said method is can be by vitrified big volume in each suction pipe.The present invention relates to be used for the method for the cell of vitrifying closed straw, said closed straw has and allows to hold therein from about 20 μ l to about 250 μ l, for example from about 20 μ l to about 225 μ l; From about 25 μ l to about 200 μ l, from about 25 μ l to about 175 μ l, from about 25 μ l to about 150 μ l; From about 30 μ l to about 125 μ l; From about 30 μ l to about 100 μ l, from about 35 μ l to about 75 μ l, the size from about 40 μ l to about 50 μ l volumes.The suction pipe length of in embodiment provided by the invention, using is about 13cm; Diameter is about 2mm and is about the thick extremely thin plastic tube wall (closed straw of 0.1mm; The mini suction pipe of French; 250 μ l, L ' Aigle, 475 ° of IMV ZA; 133mm, Svensk ).Yet, can imagine the length that the size of the container of hypothesis dress liquid nitrogen allows liquid nitrogen to flood whole suction pipe, as long as the diameter of suction pipe is almost the same with suction pipe as used herein with thickness, just successfully vitrifying even bigger volume of the longer suction pipe of use so.

Very important step is to use so-called closed straw in another said method.In this context; Term " closed straw " is used to refer to has the suction pipe that can make the openend that the biological agents in suitable culture medium (for example cell or cell-line) for example packs in the loading position, but after packing into deadend immediately should end to avoid from undesired cell contamination on every side and also to avoid the undesired risk of polluting from the periphery of cell.The air-tightness sealing at suction pipe two ends is extremely important to the pollution that prevents sample and environment.The hand closed unit that is called CBSSYMS from Cryo Bio System is a kind of suitable system.

In the openings at one side of said suction pipe and at another side stopper to be housed be very important.This stopper allows to suck air so that suction pipe charges into liquid with syringe, in case but its directly contact then polymerization with liquid, seal capillary at this end.Also can use other appropriate method of this end of sealing.Use sealer (welding, bonding etc.) the sealing other end then.Importantly wall to approach and diameter little so that the inclusion in the suction pipe is cooled off fast.Suction pipe length is not too crucial, but is actual needs, and its length is full-length preferably, to be fit to the standard carriage in the liquid nitrogen filling.Said suction pipe is made by plastics, also can comprise that glass (although it possibly rupture more easily) makes by any suitable material.Importantly said material is wanted safety and is not absorbed or h substance, and it is atresia, nontoxic and biocompatibility.

In the present invention used, term " low temperature storage " is meant under extremely low temperature preserved biologic material.

Employed term in context of the present invention " directly contact " or " directly being exposed to " are meant if the surface of biologic material or the existing medium of said material, solution or material when contact with refrigeration material, claims that biologic material " directly contact " or " direct exposure " are in for example refrigeration material.

Employed term " refrigeration material " in the context of the present invention is meant and anyly can causes the vitrified material of biologic material.Because sample does not directly contact with refrigeration material, can use any enough cold refrigerant in theory.Suitable material includes but not limited to liquid gas for example liquid nitrogen, LPG, liquid helium, ethane etc.

" vitality " as used herein be meant in a period of time biologic material can normal existence, growth and propagation.

According to the present invention, the biologic material (for example hBS cell or hBS cell-line) of at least 20 μ l volumes is placed closed straw.Then said closed straw is exposed in the refrigeration material (liquid nitrogen suitably).In case be exposed to refrigeration material, said cell begins vitrifying, can store subsequently to thaw in a period of time and period afterwards.The biologic material that thaws still keeps vitality.Like top demonstration, cell does not directly contact with refrigeration material or directly exposes.Therefore, avoided cell (from external source) pollution and cell risk to the pollution of environment.

Biologic material of the present invention is living cells or cell-line, particularly the BS cell, from cell-derived BS cell of BS or cell.Said cell can be in any developmental stage.Preferably, said cell derives from animal origin, comprises mammal source, comprises but is not limited to for example monkey, laboratory animal for example pig, sheep, cow, goat and horse of rat, mouse and hamster, agriculture livestock for example of people, non-human primates.Have in the embodiment of specific purpose in the present invention, said cell is the human stem cell that comprises people BS cell.

The suitable cell that is used for according to the inventive method is BS cell or BS cell-line, particularly hBS cell or hBS cell-line.Through using method described herein can obtain said cell or cell-line.

One of them plants vitrification solution (first kind and second kind of solution) can contain one or more cryoprotectors or cryoprotection agent composition.Certain non-toxicity cryoprotector is preferred.Cryoprotector is retained in the non-molar ratio that freezes the solute in the water and assists contraction is reduced to minimum level through reducing other.It suppresses the formation of ice crystal, and therefore reduces the freezing point of water.It also can combine to prevent albuminous degeneration through hydrogen with irreducible water.When cell cooled off, aqueous solvent was transformed into extracellular ice, and the non-capillary electrolysis matter in the ever-increasing extracellular of concentration or non electrolyte can destroy cell.When handling with cryoprotector, cell can not reach the salinity that much lower temperature reaches non-processing cell before at it.Chemical reaction carries out to such an extent that very slowly and therefore make the destruction of pair cell drop to minimum level under so low temperature.Usually preferably use the cryoprotector combination because possibly have difference between the variety classes.Cryoprotector also can serve as the osmotic pressure activating agent.Suitable cryoprotector can be selected from 1,2 ethylene glycol, 1,2-trimethylene glycol, dimethyl sulfoxide (DMSO), glycerine, propane diols, sugar (comprising sucrose, trehalose, maltose, lactose) and methyl pentanediol.The concentration that is contained in the single reagent in first kind and/or the second kind of solution usually in the 5-50%v/v scope, for example, from about 5% to about 40%v/v, for example from about 5% to about 25%v/v (first kind of solution) with from about 5% to about 30%v/v (second kind of solution).Usually, the total concentration of cryoprotector (promptly pressing v/v, w/v or M calculates) ratio is high in first kind of solution in second kind of solution.First kind and second kind of solution can comprise one or more identical or different cryoprotectors.The concentration of one or more cryoprotectors in first kind and the second kind of solution can be identical or different, and usually in second kind of solution the total concentration of cryoprotector be higher than first kind of concentration in the solution.

In the specific embodiment of the present invention, said cryoprotector is a trehalose.Be contained in first kind and/or the second kind of solution trehalose concentration usually at about 0.02M to the scope of about 1M, for example from about 0.05M about 0.9M extremely, from about 0.1M about 0.8M extremely; About 0.2M is to about 0.7M; From about 0.3M to about 0.65M, from about 0.4M to about 0.6M, from about 0.45M to about 0.55M.

Usually, sucrose is used for similarly using.Trehalose is the disaccharides of kind of a uniqueness, natural generation, is shown in hundreds of plant and animals.Trehalose is an important energy source and to be presented between pool period be the primary factor of stabilate body.Proved trehalose can reduce film phase transition temperature so that itself in addition under the condition of drying, keep mesomorphic state.Be not subject to any theory, supposed to prevent like this seepage of film in the rehydration process, thereby preserved cell viability.About protein, shown trehalose when cell is in hydration status through water being evicted from CKIs qualitative change property from said protein surface.

In another embodiment of the invention, said cryoprotector is a sucrose.The contained sucrose concentration of first kind and/or second kind of solution is usually in from about 0.02M to the scope of about 1M, for example from about 0.05M about 0.9M extremely, from about 0.1M about 0.8M extremely; About 0.2M is to about 0.7M; From about 0.3M to about 0.65M, from about 0.4M to about 0.6M, from about 0.45M to about 0.55M.

And in another embodiment of the invention, at least a cryoprotector that contains in first and second kinds of solution.

At least one can comprise viscosity modifier in first and second kinds of solution.The suitable viscosity modifier that is used for this paper can be selected from phenanthrene can (Ficoll), Percoll (Percoll), hyaluronic acid, albumin, polyvinylpyrrolidone, alginic acid, gelatin and glycerine.First and second kinds of solution can comprise one or more identical or different viscosity modifiers.The concentration of one or more viscosity modifiers in first and second kinds of solution can be identical or different.

In the specific embodiment of the present invention, said viscosity modifier is that phenanthrene can.But first and/or the highest 150mg/ml of being about of phenanthrene concentration that second kind of solution comprised, the for example the highest 100mg/ml that is about, the highest 50mg/ml that is about, the highest 25mg/ml that is about, the highest 15mg/ml of being about or the highest 10mg/ml that is about.

In one embodiment of the invention, at least a in first and second kinds of solution is aqueous solution.

In the specific embodiment of the present invention, comprise the step I i in the said method).

Owing to this step is intended to promote solution equilibria and guarantees that cryoprotector fully spreads, so possible time span is 10 seconds to 20 minutes, if but have DMSO, should be with cellular exposure long time in slightly toxic DMSO.

Common incubation between carrying out under about 37 ℃ from 5 seconds to about 20 minutes, for example, from about 10 seconds to about 15 minutes; From about 15 seconds to about 10 minutes, from about 20 seconds to about 7.5 minutes, from about 30 seconds to about 5 minutes; From about 40 seconds to about 4 minutes; From about 50 seconds to about 3 minutes, from about 30 seconds to about 2 minutes, from about 45 seconds to about 1.5 minutes or 1 minute.

In further embodiment, also comprised step I v), and common incubation between carrying out from 5 seconds to about 10 minutes under about 37 ℃; For example, from about 10 seconds to about 7.5 minutes, from about 10 seconds to about 5 minutes; From about 15 seconds to about 4 minutes, from about 15 seconds to about 3 minutes, from about 15 seconds to about 2 minutes; From about 20 seconds to about 1 minute, from about 5 seconds to about 1 minute, from about 5 seconds to about 30 seconds or from about 10 seconds to about 30 seconds.

In specific embodiment, comprise step I v), and under 37 ℃, carried out incubation about 30 seconds or shorter time.

The method for vitrification pair cell is very effective and gentle.Usually, about 50% or more many cases as, about 55% or more; About 60% or more, about 65% or more, about 70% or more; About 75% or more; About 80% or more, about 85% or more, about 90% or more about 95% or more cell after going vitrifying, after on the proper culture medium, vitality is arranged with cultivating.The suitable method for vitrification that goes has been described here.

On the other hand, the present invention also relates to vitrified cell of the method for almanac invention.

In further embodiment of the present invention or on the other hand, relate to vitrified method.

Go vitrifying to comprise:

Viii) with one or more vitrified closed straws put into have from about room temperature to about 40 ℃ environment a period of time so that the content of closed straw thaw,

Ix) open one or more closed straws,

X) use the third solution (solution C) to make to be contained in the cell in one or more closed straws of opening to accept washing,

Xi) randomly will be available from step x) in four kinds of solution of cell transfer to the (solution D) of washing and

Xii) randomly cell is carried out incubation in the 4th kind of solution,

Xiii) randomly will be from xii) in the 4th kind of solution cell transfer and be seeded on the feeder cells and

Xiv) randomly further cultivate said cell.

Through step x) after the cell that obtains promptly can be used for any purpose of wanting, and can use any step further to investigate for example vitality with pair cell.

Step viii) relates to thawing of cell.This step only be thaw in the suction pipe content and should in to the perusal of suction pipe, carry out, so the time seems unimportant.Temperature should not surpass 40 ℃ but can be between room temperature to 40 ℃.If cell does not take out from water-bath after thawing apace, temperature higher when thawing said cell can be brought out the heat shock effect.

Therefore, as forcing step, said method also comprises step xi),, xiii) and xiv); Further comprise step xii).

Go vitrification solution can comprise cryoprotector, for example have the active cryoprotector (for example having hypotoxic osmotic pressure activating agent) of osmotic pressure, avoid for example DMSO usually, and preferably use trehalose and sucrose individually or in combination.Disaccharides at this solution middle and high concentration prevents cell rupture, otherwise cell is because of contacting and can break with the solution that does not contain DMSO suddenly.The existence of said disaccharides can prevent that natural osmotic pressure from working and dump for the cell time enough to be present in intracellular DMSO (or analog) in the extracellular, and lentamente through the water displacement.

Therefore, the 3rd and/or the 4th kind of (if relevant) solution comprise one or more cryoprotectors usually.

In specific embodiment, one or more cryoprotectors are selected from glycerine, trehalose, sucrose, 1,2 ethylene glycol, DMSO, propane diols and/or its mixture, particularly glycerine, trehalose, sucrose or its mixture and are fit to use.

The 3rd and/or the 4th kind of solution in the cryoprotection agent concentration usually from about 0.02M to 1M for example from about 0.05M to about 0.9M; From about 0.1M to about 0.8M, from about 0.1M to about 0.7M, from about 0.1M to about 0.6M; From about 0.15M to about 0.5M; From about 0.2M to about 0.4M, and if relevant, the concentration of cryoprotector is higher than the concentration of osmotically active reagent in the 4th kind of solution in the third solution.If relevant usually, the concentration of cryoprotector is higher than the 4th kind of cryoprotection agent concentration in the solution in the third solution.

Provided the summation of the inventive method below.

With the people blastocyst-derived do (hBS) cell colony be cut into small pieces (0.1-0.4mmx 0.1-0.4mm).In 40-50 μ l volume, can be chilled in the closed straw up to the individual cell fritter of 20 (preferably about 10).Closed straw at one end has stopper and at other end opening.Said cell fritter be inhaled in the suction pipe with carry out freezing after, with the cryo-PBS sealing have stopper (valve) an end and at an end of opening with binding agent (welding) with heat locking device (Demtek A/S) seals.Before freezing more a large amount of cell, carry out taking turns freezing and the check of thawing.After thawing, said cell small pieces are seeded on the culture dish with mice embryonic feeder cells (MEF).People BS cell culture is assessed after once going down to posterity.

All percentages of mentioning all are v/v.

Following description has provided explanation to suitable method, solution, time period etc.But based on here describe, in general terms and guide, those skilled in the art can change different factors within the scope of the present invention.

Method for vitrification-summation:

Preparation:

1. preparation is dissolved in the mother liquor of the trehalose formation of Cryo-PBS (available from VitrolifeAB (Gothenburg, Sweden)) by 0.6M.

2. solution A: the 1,2 ethylene glycol of preparation 10% and carry out aseptic filtration in Cryo-PBS.

Add aseptic DMSO to final concentration be 10%.

3. solution B: preparation contains 0.3M trehalose and 20%1 in Cryo-PBS, and the A solution of 2-ethylidene glycol also carries out aseptic filtration.

Add aseptic DMSO to final concentration be 20%.

With 1ml solution A and solution B add respectively four orifice plates (Nunclon, VWRInternational) in two apertures that separate.Plate is placed under 37 ℃.

5. cut with the capillary glass tube (World PrecisionInstruments) of the drawing of using sterilization (or from Swemed stem cell cutting tool) pair cell, method is to cut the blastocyst-derived stem cell colonies of the appropriate morphologic people of selected displaying with the same procedure that goes down to posterity.From the cutting tool of Swemed is the chamber with 25 degree inclination angles and 200 or 300 microns, and design is used for cutting, operating and shift the aseptic sharp capillary glass tube of hBS colony or part hBS colony.It is by Swemed Lab International AB, Bilidal, and Sweden produces.

With hBS number the mark suction pipe (closed straw, French mini-straws, working volume are 250ul; L ' Aigle; IMV ZA475,133mm, SvenskMjolk) must number and with the visible pipe of freezing numbering (being cell-line/date/signature) mark (visiotubes).

Freezing flow process:

1. the glass pipet (Pasteur, VWR International) that uses capillary glass tube (World Precision Instruments) or draw will be chilled in cell transfer in the suction pipe to solution A.

2. with said cell incubation 1 minute in solution A.

3. then with in said cell transfer to (25 μ l) solution B, and in 25 seconds with cell transfer in another fresh solution B (25 μ l).

4. with said cell incubation 25 seconds (the longest) in solution B.The preferred time that is used for the 3rd to the 4th step should be short as far as possible.

5. the syringe (disposable tuberculin syringe, CodanTriplus AB) with 1ml is connected on the 1-1.5cm siloxanes pipe (sterilized), then silicone tube is connected to an end (blocking end) that has continuous plug on the suction pipe.The siloxanes pipe plays sealing function between suction pipe and syringe.At first, the cryo-PBS with about 2-3cm volume is sucked in the suction pipe.Suck the air (referring to Fig. 1) of about 1-2cm then.

6. the cell in the 2cm post is sucked in the suction pipe from solution B at the stereopsis microscopically.

7. suck the 1-2cm air, then suck 0.5-1cm cryo-PBS (serving as extra stopper) at this end.

8. thereby the content of drawing in the suction pipe with syringe expands said stopper so that cryo-PBS contacts with continuous plug.

9. use pair of forceps that syringe is taken away from suction pipe, and then suction pipe is carried out welded closure with apparatus for heat sealing.

10. suction pipe is put into visual pipe (visiotube), and then put into the jar that liquid nitrogen is housed and carry out long term storage.

Remove vitrifying flow process-overall procedure

Preparation:

1. preparation is dissolved in the mother liquor that the trehalose of cryo-PBS constitutes by 0.2M.

2. solution C: the trehalose that 0.2M is dissolved in cryo-PBS carries out aseptic filtration.

3. solution D: preparation 0.1M is dissolved in the trehalose of cryo-PBS and carries out aseptic filtration.

4. 1ml solution C, solution D and hBS medium (referring to following) are put into respectively in the single aperture of 4 orifice plates (Nunclon, VWR International) and with plate and placed 37 ℃ of following incubations.[said hBS medium comprises and replenishes with 20%

serum substitute and

Dulbecco ' s Modified Eagle ' s medium of following ingredients; Said composition final concentration separately is: penicillin 100 units/ml; Dispensable amino acid 0.1mM; L-glutaminate 2mM; β-thioglycol 100 μ M, people's recombinant bfgf 4ng/ml (basic fibroblast growth factor)].

The cell that washing is thawed from DMSO fast is very important, and said DMSO is used for vitrification solution.Having of the trehalose of less toxicity helps progressively changing relatively slowly from vitrification solution to the medium that is used for inoculating cell.Said concentration also can change (5-50%v/v, w/w or w/v) by different efficacies.Also might use other to have hypotoxic cryoprotector.

Thaw

1. from the storage that contains liquid nitrogen is irritated, take out and contain the closed straw of vitrified people BS cell and place the bottle that contains liquid nitrogen.

2. the suction pipe of said sealing was at room temperature placed 10 seconds in air, put into 40 ℃ about 2 seconds of water-bath then." general rag " with the bacterium of going out (allduk) dried suction pipe.

3. use the scissors of the bacterium of going out will have only the blocking end of the suction pipe of blocking thing sealing to cut off.Use a silicone tube said suction pipe to be connected on the syringe as the blocking thing.Then suction pipe is located to cut off in bonding (welding).Air with in the syringe pushes the hBS cell in the solution C.The cell concentration that will in identical aperture, wash should be no more than the contained amount of single suction pipe.

4. with said hBS cell colony incubation 1 minute in solution C.Usually from about 1 minute to about 20 minutes.

5. under stereoscope, use the glass pipet (Pasteur, VWR International) of glass fiber cell pipe (World PrecisionInstruments) or drawing that the hBS cell colony is transferred in the solution D.

6. with hBS cell colony incubation 5 minutes in solution D.Usually from about 5 minutes to about 30 minutes.

7. the glass pipet (Pasteur, VWR International) that under stereoscope, uses glass fiber cell pipe (World PrecisionInstruments) or draw is transferred to the hBS cell colony on the hBS medium.

8. the glass pipet (Pasteur, VWR International) that uses glass fiber cell pipe (World Precision Instruments) or draw during several seconds (＞5 seconds) with said colony from the transfer of hBS medium and be seeded in the culture dish above the mice embryonic feeder cells (MEF).

9. said culture dish being put into incubator further cultivates.

Chart

Fig. 1: the recovery people BS cell-line of thawing in vitrifying with after going vitrifying SA001.

Fig. 2: the recovery people BS cell-line of thawing in vitrifying with after going vitrifying SA002.

Fig. 3: the recovery people BS cell-line of thawing in vitrifying with after going vitrifying SA034.

Fig. 4 (A)-(C): the representative configuration of before being about to vitrifying, cultivating the people BS cell-line SA001 on the mice embryonic feeder cells.

Fig. 5 (A)-(C): the representative configuration of cultivating the people BS cell-line SA001 on the mice embryonic feeder cells when after going vitrifying, going down to posterity for the first time.

Fig. 6: the representative configuration of after going vitrifying, cultivating the people BS cell-line SA001 on the mice embryonic feeder cells when the 18th generation (A), the 23rd generation (B), the 29th generation (C) and the 35th generation (D).

Fig. 7: (A) people BS cell colony [p19], (B) SSEA-1 [p31], (C) SSEA-3 [p31], (D) SSEA-4 [p31], (E) TRA-1-60 [p31], (F) TRA-1-81 [p31], (G) Oct-4 [p31], (H) ALP [p31].

Fig. 8: the caryogram of cell-line SA001 after the vitrifying.

Fig. 9: go vitrified people BS cell (SA001) in external differentiation, the 29th generation.(A) β-III-tubulin, (B) desmin, (C) α-Jia Taidanbai, (D) HNF-3 β.

Figure 10: the 19th generation people BS cell (SA001) differentiation in vivo.(A) entoderm (secretory epithelium), (B) mesoderm (cartilage), (C) ectoderm (neuroderm).

Figure 11: have the syringe that preparation is used for freezing closed straw.

To be further specified among the present invention embodiment below, said embodiment does not also limit the present invention in any way.

Embodiment 1:

Vitrifying hBS cell

Preparation solution A and B (solution A: the cryo-PBS solution of 10% 1,2 ethylene glycol of aseptic filtration, 10% DMSO; Solution B: the Cryo-PBS solution of the trehalose of the 0.3M of aseptic filtration, 10% 1,2 ethylene glycol, 10% DMSO).So that (Sweden) the blastocyst-derived stem cell of cutting people is cut the blastocyst-derived stem cell colonies of the appropriate morphologic people of selected displaying with the same procedure of carrying out routine and going down to posterity for Swemed Labs International, Billdal with using the stem cell cutting tool.Said cell block sizes should be about 0.1 to 0.4mmX0.1-0.4mm.At first with said cell fritter incubation 1 minute in 500 μ l solution A of preheating (37 ℃), be transferred in the 25 μ l solution B then and incubation 30 seconds, and then be transferred in the fresh solution B drop and incubation 30 seconds.Said volume is 40-50 μ l approximately.About 10 cell fritters are sucked into preparation are used for vitrified suction pipe, then suction pipe is used adhesive closure.Suction pipe is inserted in the liquid nitrogen.

Embodiment 2

Vitrified people BS cell thaws

Prepare two kinds of solution C and D (solution C: the Cryo-PBS solution of the 0.2M trehalose of aseptic filtration; Solution D: the Cryo-PBS solution of the 0.1M trehalose of aseptic filtration).Solution C and D and hBS are cultivated based on 37 ℃ of preheatings.From liquid nitrogen container, take out the closed straw that contains vitrified hBS cell (about 10 cell fritters).Suction pipe was at room temperature kept 10 seconds, then promptly in 40 ℃ of water-baths (in the several seconds) thaw.Use the scissors of the bacterium of going out to cut off and use syringe that content is pushed into the solution C from suction pipe at the blocking end of suction pipe.Said hBS cell incubation in 500 μ l solution C was changed in the 500 μ l solution D and incubation 5 minutes in 1 minute then.Under stereoscope,, be seeded in then in the culture dish above the mice embryonic feeder cells on the hBS medium the washing fast in the hBS medium of hBS cell fritter.Then said cell is cultivated (in 37 ℃ of incubations), the number of the new colony set up is counted and gone down to posterity with the vitality of hBS cell after the check vitrifying.Experience the viable cell that recovers after this vitrifying and the step of thawing usually in the scope of 70-100%.

Embodiment 3

Use closed straw vitrification and thaw people BS cell

People BS cell (cell-line SA002, SA121 and SA181) is carried out vitrifying and thaw according to the method for describing in embodiment 1 and 2.The mice embryonic feeder cells were assessed and are counted the hBS cell colony after last 48 hour in being seeded in culture dish.Because each sheet in initial vitrified hBS cell fritter all can produce a colony, so calculate the recovery rate of thawing with the ratio between the colony after viable the thawing (showing suitable hBS cytomorphology) number and the initial vitrified hBS cell fritter number.Prepare 3 suction pipes and assess each cell-line, and below the result of each single suction pipe was shown in, the result showed that recovery rate is between 40% and 100%.

Embodiment 4

Direct comparison between closed straw and the opening drawing and pulling type suction pipe

Except using opening drawing and pulling type suction pipe as the closed straw contrast simultaneously, all the other all carry out vitrifying according to the method for in embodiment 1 and 2, describing to people BS cell (cell-line AS 034) and thaw.Clearly, in each opening drawing and pulling type suction pipe, only having an appointment 4 BS cell fritters can be by vitrifying.Be seeded in the culture dish above the mice embryonic feeder cells back 48 hours the hBS cell colony is assessed and counted.Ratio with between (the showing suitable hBS cytomorphology) number of the colony after viable the thawing and the initial vitrified hBS cell fritter number is calculated the recovery rate of thawing.Each cell-line has all been prepared three suction pipes and assessed, shown the result of each single suction pipe below and show to obtain the achievement that more how viable (absolute number) keeps the cell of acceptable recovery rate simultaneously from each closed straw.

Embodiment 5

In the vitrifying medium, use the comparison between trehalose and the sucrose

According to the method for describing in embodiment 1 and 2; Perhaps except vitrifying and the trehalose in removing the vitrifying medium are used the cane sugar substitution identical with used trehalose concentration; All the other are all according to embodiment 1 and 2 described methods, the vitrifying and the people BS cell (cell-line SA121) that thaws.The mice embryonic feeder cells were assessed and are counted the hBS cell colony after last 48 hour in being inoculated into culture dish.Ratio with between (the showing suitable hBS cytomorphology) number of the colony after viable the thawing and the initial vitrified hBS cell fritter number is calculated the recovery rate of thawing.As if each cell-line has all been prepared three suction pipes and assesses, and having shown the result of each single suction pipe below and being presented in this case using between trehalose and the sucrose does not have significant difference.

Embodiment 6

In the vitrifying medium, use phenanthrene can carry out vitrifying and the people BS cell that thaws

Except in the vitrifying medium, use phenanthrene can (0mg/ml, 10mg/ml and 100mg/ml), all the other are all according to the method vitrifying and the people BS cell (cell-line SA121) that thaws of description in embodiment 1 and 2.The mice embryonic feeder cells were assessed and are counted the hBS cell colony after last 48 hour in being inoculated into culture dish.Ratio with between (the showing suitable hBS cytomorphology) number of the colony after viable the thawing and the initial vitrified hBS cell fritter number is calculated the recovery rate of thawing.Each cell-line has all been prepared three suction pipes and assessed, and below the result of each single suction pipe is shown in.

Embodiment 7

In the vitrifying medium, use the comparison between the variable concentrations trehalose

Except in second kind of vitrification solution (solution B), using two kinds of variable concentrations (0.3M and 0.5M) trehalose, all the other are all according to the method vitrifying and the people BS cell (cell-line SA121) that thaws described in embodiment 1 and 2.When using the 0.3M trehalose in the solution B, solution C contains the 0.2M trehalose and solution D contains the 0.1M trehalose.When using the 0.5M trehalose in the solution B, solution C contains the 0.4M trehalose and solution D contains the 0.2M trehalose.The mice embryonic feeder cells were assessed and are counted the hBS cell colony after last 48 hour in being inoculated into culture dish.With can survive thaw after colony (showing suitable hBS cytomorphology) number and the ratio between initial vitrified hBS cell fritter number calculate the recovery rate of thawing.Carry out two branches and else use the experiment of two kinds of different people BS cell-lines.Each cell-line has all been prepared three suction pipes and assesses, and the result of each single suction pipe shows as follows.Although said result shows that observed two kinds of trehalose situations performance is all good, as if the trehalose of 0.5M is better than the performance of the trehalose of 0.3M in the solution B in the solution B.

Embodiment 8

Use closed straw to assess vitrifying more widely and remove method for vitrification

In order to assess the quality of method for vitrification, use three kinds of different people BS cell-lines (SA001, SA002 and AS034) that a large amount of people BS cells are carried out vitrifying (as described in the above-mentioned embodiment 1) three kinds of different occasions.Under each occasion each cell-line have＞100 branched pipettes are by vitrifying.From these suction pipes in batches, select 8 to 10 to go vitrifying (as described in the above-mentioned embodiment 2) and be seeded in above the mice embryonic feeder cells in the culture dish separately randomly.In each culture dish, confirm the hBS cell mass of inoculation and the number that adheres to, breeds and show suitable morphologic hBS cell mass.Said result is shown among Fig. 1,2 and 3 and shows that every branched pipette produces viable hBS cell colony, said hBS cell colony is gone down to posterity and describes its characteristic according to standard method.

Embodiment 9

The representative configuration of vitrifying and people from front and back BS cell that thaws

Fig. 4 shows the representative configuration of vitrifying forefathers BS colony (cell-line SA001).After going vitrifying and inoculating, the morphological feature (Fig. 5) of undifferentiated people BS cell is bred and showed to viable colony.

Subsequently, according to standard method these cells are bred and go down to posterity and the representative illustration of people BS cell colony is shown in Fig. 6.In people BS cell-line SA002 and AS034, obtained similar result (data not shown).

Embodiment 10

The follow-up characteristic of in closed straw, accepting vitrifying and removing vitrified people BS cell

For identifier BS cell recovers fully in vitrifying with after going the vitrifying process and represents suitable feature, the hBS cell is carried out widely characteristic describe.This comprises the multipotency check in surface antigen expression analysis, karyotyping and external and the body.Obtain following result through end user BS cell-line SA001, and also can obtain similar result (data not shown) through end user BS cell-line SA002 and AS034.

The immunohistochemical staining of undifferentiated hBS cell

The vitrified people BS cell (cell-line SA001) that goes that to cultivate on mice embryonic feeder cells (MEF) is fixed in PFA, then bores a hole with Triton X-100.After continuous washing and sealing step, said cell is carried out incubation with one anti-(by explanation).Subsequently with two anti-detections of puting together.Show nuclear through DAPI dyeing.(SigmaDiagnostics, Stockholm, the specification that Sweden) provides use the commercial kit of buying to confirm the activity of alkaline phosphatase (ALP) according to manufacturer.The numbering that goes down to posterity during each of being carried out analyzed is shown in the marginal data bracket among Fig. 7.Like what Fig. 7 showed, said result shows that people BS cell conforms to SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Oct-4 and ALP demonstration positive staining and with undifferentiated people BS cell, and it is negative to SSEA-1.

Karyotyping

To carry out the cultivation of karyotyping and under the situation that calyculin A exists, carry out incubation, wash with cell culture medium then at the vitrified people BS cell (SA001 system) that goes on the MEF.Through centrifugal collecting cell and use ethanol and glacial acetic acid is fixed.Use trypsase-Ji's nurse Sa dyeing to show chromosome.As shown in Figure 8, said result does not detect chromosome abnormality after showing the process vitrifying and going the vitrifying process in said cell.

Telomerase activation

In order to analyze telomerase activation, according to the specification use Telo TAGGG Telomerase PCR ELISA of manufacturer ^PLUSKit (Roche, Basel, Switzerland).

The interior active of said mensuration use side granzyme also detects it with enzyme linked immunological absorption measurement (ELISA) through the said product of pcr amplification.After the vitrifying people BS cell-line SA001 is being analyzed and cultivating on the mouse feeder cells, and finding that it shows high telomerase activation.High telomerase activation in the HBS cell is relevant with its unlimited splitting ability in cultivation.

Vitro differentiation

Be the versatility that vitrified people BS cell is removed in investigation, (Swemed Lab, Gteborg Sweden) will be transferred to from the not differentiation colony of cell-line SA001 in the suspension culture to allow blastocyst body (Eb) to form with the stem cell cutting tool.Then, Eb is coated in the tissue culture ware, and uses anti-beta tubulin (ectoderm), desmin (mesoderm) simultaneously, the antibody of α alpha-fetoprotein and HNF-3 β (entoderm) carries out the immunohistochemistry estimation to the cell of differentiation.Also observe simultaneously the contractive cell (not shown) that is assembled into the cardiac muscle cell.All things considered, these results show that standing vitrifying is keeping it to become to represent the potential (being that it keeps versatility) of the cell of three kinds of different germinal layers in vitro differentiation with the people BS cell that goes the vitrifying process.Said result is shown in Fig. 9.

Differentiation in the body

In order to investigate the versatility of vitrifying people BS cell, undifferentiated cell (cell-line SA001) is placed under the scrotum with serious immune deficiency (SCID) mouse through operation.Kill the mouse tumor resection after 8 weeks and fixing in PFA.In order to confirm existence, the paraffin section of haematine-Yi red colouring is carried out the histology estimation from the tissue of all three germinal layers.Like what shown among Figure 10, keeping its potential that is divided into the cell of said three the different germinal layers of representative (that is, it keeps versatility) in vivo through vitrifying and the people BS cell that goes the vitrifying process.

Be used to set up the conventional method of the cell that can be used for method for vitrification

Be used to set up the method for the people hBS cell that is suitable for being used for the inventive method.In the disclosed PCT application, the appropriate method that is used to set up the hBS cell has been described with WO 03/055992 (identical applicant) on July 10th, 2003.In one aspect of the invention, through requiring the method for protection to obtain employed cell (being incorporated herein by reference) among the WO 03/055992.

Be used to set up from the blastocyst-derived stem cell of the versatility people of fertilized oocyte or the method for cell-line and comprise step:

I) egg mother cell (having 1 or 2 grade) that randomly uses fertilization to be obtaining blastocyst (randomly having A or B level),

Ii) cultivate altogether setting up one or more inner cell mass cell colonies with feeder cells and blastocyst,

Iii) through mechanical anatomical isolation inner cell mass cell,

Iv) inner cell mass cell and feeder cells are cultivated to obtain blastocyst-derived stem cell line altogether.

V) randomly, breed said blastocyst-derived stem cell line.

Use is as the fertilized oocyte of the starting material of this method.The quality of fertilized oocyte is extremely important to the quality of the blastocyst that obtained.The step I of said method) the people's blastocyst cell in can derive from freezing or fresh human oocyte in vitro fertilization.The method of the suitable egg mother cell of the method that is used to select to be used for WO 03/055992 is described below.Found that important successful standard of the present invention is the appropriate selection to egg mother cell.Therefore, if only use 3 grades of egg mother cells, the probability that obtains to satisfy generally the hBS cell-line that requires (below being described in) is just very low.

The fresh fertilized oocyte of donations: at the 0th day said egg mother cell is sucked Asp-100 (Vitrolife), and fertilization in IVF-50 (Vitrolife) in the 1st day.Assess said fertilized oocyte according to the 3rd day morphology and cell division.Following grade is used for the assessment of fertilized oocyte:

1 grade of fertilized oocyte: level and smooth blastomere, no fragment

2 grades of fertilized oocytes:＜20% fragment

3 grades of fertilized oocytes:＞20% fragment

After assessment in the 3rd day, the fertilized oocyte of 1 grade and 2 grades is transplanted or chilled storage.3 grades of fertilized oocytes are transferred to ICM-2 (Vitrolife).Said fertilized oocyte is further cultivated 3-5 days (being after fertilization 5-7 days).According to the said blastocyst of following level evaluation:

A level blastocyst: had tangible inner cell mass (ICM) amplification at the 6th day

B level blastocyst: do not have amplification but similar A level

C level blastocyst: do not have visible ICM

The freezed fertilized egg mother cell of donations: carry out freezing at four cell stage said fertilized egg with Freeze-kit (Vitrolife) at the 2nd day (after fertilization).Freezing fertilized oocyte is stored in the liquid nitrogen.Obtain informed consent from the donor completely before in 5 term gauges.Use Thaw kit (Vitrolife) the said fertilized oocyte that thaws, and carried out said method from the 2nd day.

As described above, fresh fertilized oocyte is from 3 grades of quality, and the freezed fertilized egg mother cell is from 1 grade and 2 grades.According to the data that obtain through the method for having set up, the percentage that develops into the fresh fertilized oocyte of blastocyst is 19%, and 50% freezed fertilized egg mother cell develops into blastocyst.This means maybe be because the fertilized oocyte quality be higher, and said freezed fertilized egg mother cell can obtain blastocyst better.11% the blastocyst that derives from fresh fertilized oocyte develops into stem cell line, and 15% the blastocyst that derives from the freezed fertilized egg mother cell develops into stem cell line.In a word, 2% fresh fertilized oocyte develops into stem cell line in the fertilized oocyte of cultivating, and has 7% to develop into stem cell line in the freezed fertilized egg mother cell of cultivating.

After carrying out method well known in the art, fertilized oocyte is cultured to the blastocyst stage.At people's such as Gardner Embryo culture systems, Trounson, A.O. and Gardner, the Handbook of in vitro fertilization of D.K. (writing), second edition, CRC Press, Boca Raton, 205-264 page or leaf; People's such as Gardner Fertil Steril, 74, supplementary issue 3,0-086; People's such as Gardner HumReprod, 13,3434,3440; People's such as Gardner J Reprod Immunol (being about to deliver); With people's such as Hooper Biol Reprod, 62, can find the method that is used to prepare blastocyst in the supplementary issue 1,249.

In step I) in randomly derive from after the blastocyst with 1 or 2 grade of fertilized oocyte sets up, the blastocyst and the feeder cells that will have A or B level are cultivated to set up one or more inner cell mass cell colonies altogether.After being coated on the feeder cells, monitor its growth and when said colony goes down to posterity (approximately apply back 1-2 week) even as big as carrying out manual work, can said cell be cut from other cell type and places new feeder cells to cultivate with flattening.Through using capillary glass tube, the inner cell mass cell is carried out machine cuts separate as cutting tool.Can easily detect through microscope, and therefore needn't carry out any processing to damage or to remove trophectoderm to egg mother cell with enzyme and/or antibody to the inner cell mass cell.

Therefore, the method for WO 03/055992 reduces the needs to immunity excision method.Through relatively using the contrast of immune excision method to keep the success rate of the complete Ben Fafa of trophectoderm, found to avoid immune excision method more simply, quicker and AT method is more effective than immune excision method.These methods make preparation stem cell and these cell-lines of differentiation in viable commercial.From having set up 19 cell-lines (15.5%) 122 blastocysts altogether.Through immunity excision method handled 42 blastocysts and wherein 6 successfully set up cell-line (14%).Handle 80 blastocysts and set up 13 cell-lines (16%) through the inventive method.

Cut apart inner cell mass subsequently, said inner cell mass and feeder cells are cultivated to obtain blastocyst-derived dried (BS) cell-line altogether.After obtaining hBS cell-line, randomly breed said cell-line with the amplifying cells amount.Therefore, the stem cell line in blast source can be bred through for example every said stem cell line being gone down to posterity at a distance from 4-5 days.Surpass 4-5 days if said stem cell is cultivated before going down to posterity, the possibility of undesired cell differentiation will increase.

The specific process that pair cell goes down to posterity in raising culture systems is provided among the embodiment 5 that has set up herein.

People BS cell-line separable from natural hatching blastocyst or from the blastocyst of expansion with complete oolemma.Step I in said method) blastocyst is the blastocyst of natural hatching.The blastocyst trophectoderm of hatching can be kept perfectly.Can or have the blastocyst of removing or partly removing oolemma with the blastocyst of hatching places on the feeder cells of deactivation.

At step I i) before through using for example ZD of one or more acid reagents ^TM-10 (Vitrolife, Gothenburg, Sweden), the mixture of one or more enzymes or enzyme for example pronase handle and can carry out the part degraded at least to the oolemma of blastocyst or chemistry plays pleat.

Blastocyst with complete oolemma is carried out of short duration pronase (Sigma) handle the elimination that causes said band.Also can use other type protease that has with the same or similar proteinase activity of pronase., strepto-albumen can blastocyst be coated on the feeder cells of described deactivation after handling.

In embodiments of the invention, can in improving blastocyst and/or inner cell mass cell (if relevant) the reagent that adheres to, carry out step I i to feeder cells) and/or step I is v).The suitable substance that is used for this purpose is a hyaluronic acid.

As if being used for blastocyst is coated in suitable culture medium on the feeder cells can be to be supplemented with hyaluronic hBS medium, and said hyaluronic acid can promote said blastocyst on feeder cells, to adhere to the growth with inner cell mass.Hyaluronic acid (HA) is the important mucopolysaccharides composition of extracellular matrix in the joint.As if it through bringing into play its biological effect with the acceptor of at least two kinds of cell surface receptors: CD44 and the motility (RHAMM) that is used for the HA mediation and with the binding interactions of the albumen of extracellular matrix.Set up the interaction of the surface-active polar head that the positive-effect of HA in the process can be through phosphatide in itself and the cell membrane brings into play at the hBS cell; Thereby stablize the surface tension that therefore said surfactant layer also reduces inner cell mass or blastocyst, said capillary reduction can cause the increase with the feeder cells joint efficiency.Selectively, HA can with its on inner cell mass or blastocyst receptors bind and/or combine and bring into play its forward to influence the biological effect that inner cell mass adheres to and grows with feeder cells.Therefore, other reagent or other that can change surface tension of liquid influences that interactional mode also can be used to substitute hyaluronic acid between blastocyst and the feeder cells.

Cultivation at feeder cells described in the said method is extremely important to setting up of hBS cell-line.The breeding of blastocyst-derived stem cell line can comprise that 3 feeder cells go down to posterity at the most, for example at the most 2 times.

The feeder cells that are suitable for the inventive method are the fibroblasts in embryo or ripe body source for example.Be applied to step I i in the method for the invention) or feeder cells iv) can be identical also can be different and derive from the mammals of the for example any people of comprising of animal origin, mouse, rat, monkey, hamster, frog, rabbit etc.Feeder cells from people or mouse are preferred.

Another obtains to satisfy generally, and the major criterion of the hBS cell-line of requirement is to carry out the blastocyst culture condition.Therefore can be lower than every approximately cm through use ²60,000 cells for example are lower than every approximately cm ²55,000 cells or be lower than every approximately cm ²The feeder cells of the density of 50,000 cells come culturing stem cells so that blastocyst-derived stem cell line is bred.In specific embodiment, the propagation of blastocyst-derived stem cell line comprises with every approximately cm ²The feeder cells of the density of 45,000 cells are cultivated said stem cell line.These values are applied under the situation of using the mouse feeder cells and are expected in the feeder cells of other type also can find proper density.Based on discovery of the present invention, those skilled in the art can find out such proper density.Can carry out the mitosis deactivation to said feeder cells for fear of undesired feeder cells growth.

The blastocyst-derived stem cell line that obtains through above-mentioned method for building up keeps self and versatility in the suitable time period, and in the suitable time period, is stable therefore.Term in this context " is stablized " and is meant on embryo's feeder cells of mitosis deactivation under undifferentiated state, to have when cultivating and surpasses 21 months multiplication capacity.

The stem cell line that obtains through above-mentioned method for building up satisfies general requirement.Therefore, said cell-line

I) show on embryo's feeder cells of mitosis deactivation under undifferentiated state, have when cultivating the multiplication capacity that surpasses 21 months and

Ii) show normal euploid karyotype and

Iii) remain on the derived cell that develops into all types germinal layer in external and the body potential and

Iv) at least two kinds of following molecular labeling: OCT-4 of performance, alkaline phosphatase, carbohydrate epitope SSEA-3, SSEA-4, TRA1-60, TRA1-81 and by the protein core of the keratan sulfate/chondroitin sulfate pericellular stromatin glycan of monoclone antibody GCTM-2 identification and

V) do not show molecular labeling SSEA-1 or other differentiation marker and

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

Vii) can break up.

Confirm the not differentiation hBS cell through the said method acquisition through standards: it separates, and fertilized oocyte is a blastocyst before people's implantation, and shows the multiplication capacity under undifferentiated state when on the feeder cells of mitosis deactivation, cultivating; The karyotype that it is acted normally; It expresses the typical marks of not breaking up the hBS cell; For example OCT-4, alkaline phosphatase, carbohydrate epitope SSEA-3, SSEA-4, TRA1-60, TRA1-81 and by the protein core of the keratan sulfate/chondroitin sulfate pericellular stromatin glycan of monoclone antibody GCTM-2 identification, and show the expression that has no carbohydrate epitope SSEA-1 or other differentiation marker.In addition, interior (teratoma) versatility inspection show of external and body can be divided into the derived cell of all germinal layers.

As stated, said method provides pure basically versatility people BS cellular preparations, said people BS cellular preparations i) show on embryo's feeder cells of mitosis deactivation under undifferentiated state, to have when cultivating and surpass 21 months multiplication capacity; Ii) show normal euploid karyotype; Iii) remain on the potential that all develops into the derived cell of all germinal layers in external and the body; Iv) at least two kinds of following molecular labeling: OCT-4 of performance, alkaline phosphatase, carbohydrate epitope SSEA-3, SSEA-4, TRA1-60, TRA1-81 and by the protein core of the keratan sulfate/chondroitin sulfate pericellular stromatin glycan of monoclone antibody GCTM-2 identification; V) do not show molecular labeling SSEA-1 or other differentiation marker and vi) keep its versatility and when being injected into the mouse of non-responsiveness, form teratoma in vivo and vii) can break up.

At Gage, the Science of F.H., 287:1433-1438 can find the method that is used to detect cell marking in (2000).These methods are well known to those skilled in the art and comprise method for example RT-PCR or immunologic assay, in said immunologic assay, use the antibody to said cell marking.The method, cross method, the karyotyping that are used to detect cell marking is described below, is used to measure telomerase activation and the neoplastic method of monster.These methods can be used for investigating the hBS cell that obtains according to said method for building up and whether satisfy above-mentioned standard.

Immunohistochemistry

Differentiation state to hBS stem cell that continue to cultivate carries out routine monitoring.Being used to monitor the cell surface marker that does not break up the hBS cell is SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81.Fixing people BS stem cell in 4%PFA is then bored a hole with 0.5% Triton X-100.In washing with after, educate said cell with a temperature resistance with 10% dried milk sealing.Educate said cell with two temperature resistances and examine in washing back roughly with DAPI dyeing showed cell.

Alkaline phosphatase

Specification according to manufacturer (Sigma Diagnostics) uses the commercial kit of buying to confirm alkaline phosphatase activity.

Oct-4?RT-PCR

Through use RT-PCR and gene-specific primer to (5 '-CGTGAAGCTGGAGAAGGAGAAGCTG; 5 '-CAAGGGCCGCAGCTTACACATGTTC) and as the GAPDH of house-keeping gene (5 '-ACCACAGTCCATGCCATCAC, 5 '-TCCACCACCCTGTTGCTGTA) measure the mRNA level of transcription factor Oct-4.

FISH (FISH)

Take turns among the FISH with chromosome-specific probe screening one or polysomy one.This technology can detect a large amount of heredity distortion (if existence).For carry out the kit that this analysis CTS used the commerce that contains the probe that is useful on chromosome 13,18,21 and sex chromosome (X and Y) to buy (Vysis.Inc, Downers Grove, IL, USA).At least analyze 200 cell nucleus for each cell-line.With said cell suspension in Carnoy ' s fixative and drip on the positively charged slide.Probe LSI 13/21 mixed and be added on the said slide and with cover glass with the LSI hybridization buffer cover.Probe CEP X/Y/18 is added on another slide with the mixing of CEP hybridization buffer and with identical method.In 70 ℃ of following sex change 5 minutes, then in wet box, hybridized 14-20 hour in 37 ℃.Through dyeing with DAPI II pair cell nuclear behind three washing steps and said slide being analyzed under the inverted microscope that is equipped with suitable filter and software (CytoVision, Applied Imaging).

Karyotyping

Karyotyping allows to study and to provide profuse information with direct mode to all chromosomes, can detect the distortion on a large amount of and the macrostructure more.In order to detect mosaicism, need 30 caryogram at least.Yet, the very time-consuming and technical sophistication of this technology.Can improve mitotic index through synthetic analogues and microtubule depolymerization reagent in order to improve the condition that is used to measure, but still need (the 6X10 that provides of a large amount of cells with the colchicinamide that causes the cell prevention in mid-term, colchicine ⁶Cell/analysis).With cell under the situation that 0.1 μ g/ml colchicinamide exists incubation 1-2 hour, then with the PBS washing and use trypsinization.Through collecting said cell in centrifugal 10 minutes with 1500rpm.With the fixing said cell of ethanol and glacial acetic acid and with improved Wrights to the chromosome observation of dyeing.

Comparative genome hybridization

Comparative genome hybridization (CGH) is replenishing karyotyping.CGH produces higher chromosome resolution and has less challenge technically.In the mixture of DNA, A4, the red dUTP/FITC 12-dUTP of Texas and archaeal dna polymerase 1, separated DNA is carried out nick translation.Carry out the dna fragmentation size (600-2000bp) of agarose gel electrophoresis with the control gained.Precipitate and be suspended in again in the hybridization mixed liquor that comprises formamide, dextran sulfate and SSC with check with reference to DNA.In wet box, on the sex change glass slide that has the phase in mid-term under 37 ℃, hybridize.Roughly washing a back adding antidamping sealing mixture (vectashield, 0.1 μ g/ml DAPIII) and said slide is being covered with cover glass.At microscopically and with ias slide is estimated then.

Telomerase activation

Because high telomerase activation has been confirmed as the standard of hBS cell 6, therefore in said hBS cell-line, measure telomerase activation.The known lasting reduction of telomerase activation when cell reaches more differentiation state.Therefore to said activity quantitatively must relate to algebraical sum control sample more early, and can be used as the instrument that detects differentiation.Said method, the inscribe of Telomerase PCRELISA kit (Roche) use side granzyme is active, also with enzyme linked immunological absorption measurement method (ELISA) it is detected through the said product of PCR (PCR) amplification.Carry out said mensuration according to manufacturers instruction.Result from this mensuration shows that usually the hBS cell has high telomerase activation (＞1).

Said cell-line keeps its versatility and when being injected into the mouse of non-responsiveness, forms teratoma in vivo.In addition, can form the corpusculum in hBS cell source at external these cells.In these two models, can find the cell characteristic of all germinal layers.

Teratomatous formation in the immunodeficient mouse

Whether analyst BS cell-line has kept a method of versatility is to obtain tumour, teratoma with said cell heterograft to immunodeficient mouse.All kinds of tissues of in said tumour, finding should be represented all three germinal layers.Existing report shows the various tissues of the tumour that derives from the heterograft immunodeficient mouse, for example striated muscle, cartilage and bone (mesoderm), intestines (entoderm) and neural lotus throne (ectoderm).Also have, most of tumour is by the organizational composition of spuious (disorganized).

Serious combination immune deficiency (SCID) mouse (lacking the lymphocytic strain of B and T system) is used to the neoplastic analysis of monster.Insert in the testis people BS cell or under the scrotum through operation.In testis or kidney, transplant the hBS cell with the scope of 10000 to 100000 cells.Ideally, each cell-line is used 5-6 mouse at every turn.PRELIMINARY RESULTS show female mice after surgery than male mice stable and in kidney with in testis, carry out heterograft in that to produce aspect the tumour effect identical.Therefore, female SCID mouse teratoma model is preferred.Said tumour showed obviously after about 1 month usually.After 1-4 month, kill mouse and dissect tumour, and be fixed for paraffin or freezing microtome section.Through immunohistochemical method said tumor tissues is analyzed then.Use specific markers to all three kinds of germinal layers.The said mark that uses at present is: E-cadherin, α-smooth muscle actin (mesoderm), α-Jia Taidanbai (entoderm) and the β-III-tubulin (ectoderm) that is used to distinguish mouse tissue and people's tumor tissues.In addition, carry out haematine-Yi red colouring for the gross morphology observation.

In following " setting up embodiment ", said method for building up is described.The embodiment that here comprises only also limits the scope of the invention as illustration purpose never in any form.Well known to those skilled in the art and all reactants of conventional method described herein and buffer solution are easy to obtain, and the experimental program of having set up the preparation that can commercial buy or easily grasp according to those skilled in the art comes.All incubations are all under 37 ℃, at CO ₂Carry out in the gas.

Employed a kind of proper culture medium is called " BS cell culture medium " or " BS medium " and can comprises: replenish with 20%

serum substitute and Dulbecco ' s Modified Eagle ' s medium of following ingredients; Said composition final concentration separately is: penicillin 100 units/ml; Streptomycin 50 μ g/ml; Dispensable amino acid 0.1mM; L glutamine 2mM; β-thioglycol 100 μ M, people's recombinant bfgf (basic fibroblast growth factor) 4ng/ml.

Another kind of proper culture medium is " a BS cyton medium "; It is formed as follows: additional with 20%

serum substitute and

Dulbecco ' s Modified Eagle ' s medium of following ingredients; The final concentration separately of said composition is: penicillin 50 units/ml; Streptomycin 50 μ g/ml; Dispensable amino acid 0.1mM; L glutamine 2mM, β-thioglycol 100 μ M.

Term in this context " is stablized " and is meant when cultivating on the embryo's feeder cells in the mitosis deactivation, under undifferentiated state, to have and surpasses 21 months multiplication capacity.

Set up embodiment

Set up embodiment 1

Foundation is from the undifferentiated stem cell prepared product of the substantially pure of natural blast of hatching

The embryo of the blastocyst-derived fertilization outside freezing or fresh human body of people.Directly place the hBS cell culture medium (to replenish natural blastocyst of hatching with 20%

Serum substitute and

Dulbecco ' s Modified Eagle ' s medium of following ingredients; And said composition final concentration separately is: penicillin 50 units/ml; Streptomycin 50 μ g/ml; Dispensable amino acid 0.1mM; L glutamine 2mM; β-thioglycol 100 μ M; People's recombinant bfgf (basic fibroblast growth factor) 4ng/ml) on the feeder cells (EF) in, replenishes with the 0.125mg/ml hyaluronic acid.After being coated in said blastocyst on the EF cell, monitoring its growth and when applying about 1-2 colony goes down to posterity even as big as manual work after week, said inner cell mass cell is cut from other cell type and increase at new EF cell through cultivation.

Set up embodiment 2

Foundation is from the prepared product of the basic purifying of the undifferentiated stem cell of the blastocyst with complete oolemma

For blastocyst with complete oolemma; After on the EF cell tier that said blastocyst is directly placed the hBS medium that is supplemented with hyaluronic acid (0.125mg/ml); (Vitrolife in rS2 (ICM-2) medium; Gothenburg, (10U/ml Sigma) carries out of short duration incubation with the degraded oolemma Sweden) to use pronase.

Set up embodiment 3

Alkaline phosphatase is carried out histochemical stain

Gather in the crops said cell to carry out RT-PCR and histology (alkaline phosphatase) and immunocytochemical assay (referring to following).Isolation of RNA with carry out RT-PCR.Use Rneasy Mini kit (Qiagen) to prepare total cell RNA according to the recommendation of manufacturer.It is synthetic and use Platinum Taq archaeal dna polymerase (Invitrogen) to carry out PCR that the AMV First Strand cDNA Synthesis kit (Roche) that use is used for RT-PCR carries out cDNA.Use the commercial kit of buying (Sigma) that alkaline phosphatase is carried out histochemical stain according to the recommendation of manufacturer.

Set up embodiment 4

Preparation and cultivation hBS cell-line

On the EMFI medium of Mouse Embryo Fibroblasts Culture in Vitro in the tissue culture ware: DMEM (Dulbecco ' s Modified Eagle ' s medium), replenish with 10%FCS (hyclone), 0.1 μ M β-thioglycol, 50 units/ml penicillin, 50 μ g/ml streptomycins and 2mM L-glutaminate (GibcoBRL).Use mitomycin C (10 μ g/ml, 3 hours) that said feeder cells are carried out the mitosis deactivation.Through manual work cutting people BS cell colony is placed on the mouse embryo fibroblast feeder cells of deactivation and increase.

On the mouse embryo fibroblast feeder cells with the mitosis deactivation of people BS cell culture in having the tissue culture ware of hBS cell culture medium; Said medium is: replenish with 20% Dulbecco ' s Modified Eagle ' s medium of serum substitute and following ingredients; Said composition final concentration separately is: penicillin 50 units/ml; Streptomycin 50 μ g/ml; Dispensable amino acid 0.1mM; L glutamine 2mM; β-thioglycol 100 μ M, people's recombinant bfgf (basic fibroblast growth factor) 4 nanograms/ml.Said colony is even as big as producing BS cell corpusculum after going down to posterity 7 days.

The BS cell colony is cut into the fritter of 0.4X0.4mm and is coated in the NAC culture dish that contains BS cell corpusculum medium with glass fiber cell pipe; Said medium comprises: replenish with 20% serum substitute and Dulbecco ' s Modified Eagle ' s medium of following ingredients; Said composition final concentration separately is: penicillin 50 units/ml; Streptomycin 50 μ g/ml; Dispensable amino acid 0.1mM, L glutamine 2mM and β-thioglycol 100 μ M.Said BS cell corpusculum (comprising cryptomere hBS cell corpusculum) formed in 7-9 days.

Set up embodiment 5

The hBS passage

Before going down to posterity, use the digital camera of Nikon Eclipse TE2000-U inverted microscope (10X object lens) and DXM 1200 that said hBS cell is taken pictures.Colony was gone down to posterity in every separated 4-5 days.It is just even as big as going down to posterity when said colony can be cut into fritter (0.1-0.3X0.1-0.3mm).When said cell went down to posterity for the first time, it had been cultivated 1-2 week and can be cut into about 4.

Said colony is one by one being focused under the stereoscope and cutting on the lattice pattern according to above-mentioned size.Only the structure to inner homogeneous goes down to posterity.Move each colony square with cutting, it is sucked capillary and places on the new feeder cells (maximum 4 days sizes).10-16 square is placed in each new IVF culture dish equably.Culture dish was placed 5 to 10 minutes so that said cell can stick on the new feeder cells, and then put into incubator.Change the hBS medium weekly three times.If colony is gone down to posterity, at these specific Zhou Genghuan two subcultures.Usually carry out " half changes ", it is meant that the half the medium of a sucking-off also substitutes with fresh, the gentle medium of equivalent.Can the medium of whole volume be changed if desired.

Set up embodiment 6

Vitrifying hBS cell

Cutting has suitable does not break up morphologic colony from said cell-line to go down to posterity.With the aseptic filtration of 100-200ml liquid nitrogen to the cryovial of q.s.(A:800 μ l has the Cryo PBS of 1M trehalose to prepare two kinds of solution A and B; 100 μ l 1; The pure and mild 100 μ l DMSO of 2-ethylene; B:600 μ l has the PBS of 1M trehalose, 200 μ l1, the pure and mild 200 μ l DMSO of 2-ethylene) and with said colony place A carry out 1 minute with place B to carry out 25 seconds.Use closed straw to store said freezing colony.After being transferred to said colony in the suction pipe, put it into rapidly in the cryovial that the aseptic filtration liquid nitrogen is housed.

Set up embodiment 7

The inoculation fetal mice is raised (EMFi) cell

Through down said cell being carried out deactivation with the EMFi medium incubation that contains mitomycin C 3 hours at 37 ℃.Cover the IVF culture dish with gelatin.Suck said medium and wash said cell with PBS.Substitute PBS to separate said cell with trypsase.Behind the incubation, stop tryptic activity with the EMFi medium.Through the said cell of centrifugal collection, in the EMFi medium, diluted, and in the Burker cell, count then by 1: 5.With said cell dilution to final concentration is 170K cell/ml EMFi medium.Substitute the gelatin in the IVF culture dish and put into incubator with the 1ml cell suspending liquid.Changed the EMFi medium in postvaccinal second day.

Being used for effectively will be by the hBS cell transfer of feeder cells support to not having the feeder cells cultivating system and the method for long-term breeding hBS cell under no feeder cells condition with the hBS cell

Can be in no feeder cells cultivating system with hBS cell culture used in the present invention, compare said method advantage with known method and be to be transferred cell and keeping stable in the going down to posterity up to 10 times at least.People's such as Richards research shows that hBS cell-line can not (comprise Matrigel at acellular matrix under undifferentiated state ^TM) in breeding surpass 6 times and go down to posterity.Yet said hBS cell can be at Matrigel ^TMIn stable go down to posterity up to 35 times, even freeze/thaw week after date still express the mark and the growth rate of not breaking up the hBS cell and keep roughly suitable.In addition, when mechanical separation is separated with enzyme when comparing, after applying 2 days, observe the significantly survival colony of higher number.Critical step seemingly with said hBS cell transfer to the initial step of not having the feeder cells cultivating system.Therefore, describe below and be used for the hBS cell transfer wherein said hBS cell mechanically being cut out from feeder cells to the method for not having the feeder cells cultivating system.Among the no feeder cells embodiment herein, only use the mid portion of each colony, separated although the risk that whole colony is emitting the medium that had feeder cells to pollute in the previous work of people such as Xu is handled through enzyme.In addition, to the lip-deep step of not having feeder cells, the use of enzyme causes the inactivation of the significant surfaces molecule that relates to cell adhesion and growth in the very fine hBS cell transfer that feeder cells are cultivated.Matrigel ^TMIn main component be extracellular matrix protein, for example IV class collagen and laminin.The activity that it is believed that the integrin of cell surface when combining with extracellular matrix protein becomes the committed step of regulating cell adhesion, survival and breeding.For example, wholely in the collagen acceptor join egg α in vivo and all have unique effect aspect the cell proliferation in the external adjusting collagen stroma.Possibly also can in hBS cell adhesion to stromal surface, play main effect with the sticking limit of the layer protein-specific acceptor that β 1 forms by the beta 2 integrin alpha 6 of hBS cell great expression.Therefore, having a kind of possibly be to have adapted to said initial strong Collagenase IV before the new surface at said cell to handle and influenced some unfriendly and play and adhere to or the significant surfaces acceptor of survival effect.

Separate the hBS cell transfer to the different technologies that does not have in the feeder cells environment having investigated through machinery or enzymatic among the embodiment herein about cell adhesion, survival rate and proliferation.In addition, developed the method for using the pure lines colony that helps long-term breeding and the undifferentiated hBS cell of large-scale production.Equally also investigated the purposes of the conventional freezing preservation technology that is used for freeze/thaw hBS cell.

The hBS cell transfer is not bred to there being the feeder cells system

After cutting inner cell mass, said inner cell mass and feeder cells are cultivated to obtain blastocyst-derived dried (BS) cell-line altogether.After obtaining hBS cell-line, randomly breed said cell-line to increase the amount of cell.

Before in no feeder cells system, the hBS cell being bred, can be with said hBS cell transfer to there not being the feeder cells system.

As before mentioned herein and proved that in no feeder cells embodiment the key factor of successfully carrying out the hBS cell proliferation is that said hBS cell is transferred to the method the no feeder cells cultivating system from the feeder cells cultivating system.Therefore, must be through machine cuts with said hBS cell transfer to there not being the feeder cells cultivating system, can carry out said machine cuts as cutting tool through using capillary glass tube.As shown among the embodiment, to compare with the culture that enzyme is handled, mechanical separation makes cell stick to Matrigel more extremely effectively. here ^TMGo up, breed more apace, and much stable more at cell described in the process of going down to posterity.Therefore, the method that is used to shift the HS cell according to the present invention is handled without any need for enzyme.As seen among the embodiment, under no feeder cells condition, cultivate with the cell of breeding and to have and the similar mitotic index of cultured cells under the feeder cells condition. here

The breeding of blastocyst-derived stem cell line is included in and cultivates said stem cell under the no feeder cells condition of culture; Since under the situation of no feeder cells cultivation hBS cell have many favourable aspect; Therefore for example need not carry out the production of feeder cells, can be easily the hBS cell be enlarged in proportion and produce and do not have that DNA from feeder cells shifts or the risk of other infection according to commercial output.

Therefore, transfer and propagation steps can comprise the steps: under no feeder cells condition

A) through mechanical treatment blastocyst-derived stem cell is transferred on the no feeder cells culture from feeder cells.

B) randomly, cultivate in proper culture medium and/or under the condition of culture at feeder cells on the suitable support substrate said blastocyst-derived stem cell and

C) randomly, whenever through enzymatic and/or mechanical treatment said blastocyst-derived stem cell was gone down to posterity at a distance from 3-10 days.

Usually, comprise i)-iii) the institute in steps.

The hBS cell is transferred to no feeder cells cultivating system from the feeder cells cultivating system

Found that as stated transfer step is a committed step.Therefore, should through the method for mechanical separation or in the feeder cells cultivating system pair cell carry out mechanical separation and shift.Can carry out this mechanical treatment through the instrument that any suitable cutting tool for example has sharp distal and the size that is suitable for cutting.Said instrument can be processed by any suitable materials such as plastics or glass, and to have the aseptic sharp-pointed capillary glass tube cutting tool that 25 degree angles and 200 or 300 microns tube chambers, design be used for cutting, handle and shift hBS colony or part hBS colony be the example of a proper tools.It is by Swemed Lab International AB, Billdal, and Sweden produces.

The hBS cell that shifts is the hBS cell colony and cuts out fritter and be suspended in the proper culture medium with cell mass from the central authorities of said colony.One or many mechanically separates said cell mass for example to have until said cell mass and is at most 50% of original colony, for example about at the most 40%, about 30%, about 20%, about 10%, about 5% size at the most at the most at the most at the most.For example said size is confirmed as the diameter of said cell mass or colony respectively.

Provided the suitable condition that is used for transfer step among the no feeder cells embodiment herein.Certainly these conditions can change in suitable restriction, in the said ken that is limited in those skilled in the art.

No feeder cells embodiment 1

For no feeder cells culture prepares adaptability (conditioned) VitroHES ^TM-medium (k-VitroHES-medium)

For adapting to VitroHES ^TM-medium preparation mEF cell is handled the individual layer mEF cell (second pass generation) converge and with 59000 cells/cm with mitomycin C ²Concentration be seeded in Dulbecco ' s Modified Eagle medium (D-MEM) is housed be coated with gelatin (0.1%; Sigma) in the flat bottle of cultivation, said culture media supplemented has 1% penicillin/streptomycin (PEST; 10000 units/ml), 10% hyclone (FBS) and 2mMGLUTAMAX ^TM-1 fill-in (200mM); All things are all from GibcoBRL/Invitrogen, Carlsbad, CA, USA.Through 24 hours incubation period and after, abandon said medium and use VitroHES with PBS (GibcoBRL/Invitrogen) washing once ^TM-medium (0.28ml/cm ²) the alternative laundering period of carrying out 24 hours.Carry out up to the said VitroHES of three times collection every day from identical mEF culture (during second pass generation) ^TM-acclimatizing culture medium and use the low protein combination filter of 0.2 μ m (Sarstedt, Landskrona Sweden) carry out aseptic filtration.Use fresh or-20 ℃ down freezing mistakes the k-VitroHES-medium and replenish bFGF (GibcoBRL/Invitrogen) before use with 4ng/ml.If be stored in+can use in 4 ℃ of degree k-VitroHES-medium to reach a week.When being stored in-20 ℃, be two months, do not detect the sign that biologically active goes down in use.

No feeder cells embodiment 2

HBS cell-line is transferred on the no feeder cells condition of culture

On 10-50 time the mouse feeder cells of going down to posterity that initial hBS cell-line is remained on that mitomycin C handled and cultivate at the VitroHES that is supplemented with the human alkaline fibroblast growth factor of 4ng/ml (bFGF) ^TMOn-the medium.

To being used for the hBS cell is transferred to Matrigel from the feeder cells culture ^TMTwo kinds of different technologies in the culture dish that covers are assessed, a kind of with mechanical separation another kind use collagenase treated.(Sweden) with hBS cell dice, said square is represented the middle part of colony for Swemed Lab AB, Bilidal, and said cell is separated carefully and is transferred in the HBSS solution through using the stem cell cutting tool.Said stem cell cutting tool is to have the aseptic sharp-pointed capillary glass tube that 25 degree angles and 200 or 300 microns tube chambers, design are used for cutting, handling and shift hBS colony or part hBS colony.It is by Swemed Lab InternationalAB, Billdal, and Sweden produces.

Carry out enzymatic treatment (being used for comparison) with clostridiopetidase A

(200 units/ml Sigma) separate to carry out enzymatic in HBSS, after the washing said cell mass to be transferred to clostridiopetidase A IV solution.With said cell 37 ℃ down with 5% CO ₂Middle incubation 30 minutes.Between incubation period, carry out the mechanical separation of repetition and monitoring separation process under inverted microscope with pipettor.Use KnockOut at incubation deposition (400G carried out 5 minutes) said cell suspension thing later and before in being suspended in the k-VitroHES medium again ^TMD-MEM (GibcoBRL/Invitrogen) washing once.

Carry out mechanical separation according to the present invention

In HBSS, after the washing, use the 1ml automatic pipettor mechanically said cell mass to be separated carefully.Show corresponding to the size of handling the cell aggregation that produces through clostridiopetidase A IV-when being about the 1/10-1/20 of original colony (average 20000 cells/original colony), accomplish said separation process when the size of said cell mass as stated.In HBSS, after the washing, said colony is transferred to clostridiopetidase A IV solution (200 units/ml) separate to begin carrying out enzyme.For two kinds of different techniques, each with in said cell inoculation to 4 aperture and under 37 ℃ in 5% CO ₂Middle incubation.Each tests repetition 4 times, inoculates the cell of same amount at every turn.Calculate the size and the quantity of colony after 2 and 6 days.

No feeder cells embodiment 1 and 2 result

For optimization hBS cell from feeder cells to the transfer of not having the feeder cells condition, assess two kinds of different techniques, a kind of a kind ofly with mechanical separation separate with enzymatic.With respect to the culture that enzyme is handled, mechanical separation makes cell to Matrigel ^TMProduce more effective adhesion and faster propagation.When mechanical separation is separated (Fig. 5) when comparing with enzymatic, after applying 2 days, observe the number of significantly higher survival colony.To after separating through two kinds of different technologies respectively at Matrigel ^TMThe gross area of all colonies of last generation compares (P＜0.001).In addition, the culture that after applying 6 days, separates with enzymatic compares that total colony area significantly increases (P=0.036) in the culture of mechanical separation.

No feeder cells embodiment 3

To cultivating at Matrigel ^TMOn the hBS cell cultivate and go down to posterity

In all experiments, using 4 kinds of different cells is SA002, AS038, SA121 and SA167.With said cell-line at Matrigel ^TMOn be cultured to for 35 generations and even remain unchanged with other hBS characteristic in the said morphology appearance of freeze/thaw week after date.All cultures are made up of the hBS cell colony of fully confirming of the morphology mark that does not have differentiation.After about 3-6 days through taking that medium goes down to posterity to said cell away and in each aperture, adding 1ml clostridiopetidase A IV (solution of 200 units/ml) and incubation 15-20 minute.In order to help cell to come out from surface isolation, carry out mechanical separation, then carry out other 15 minutes incubation.Wash said cell then, be suspended in k-VitroHES again ^TMBe seeded in Matrigel in the medium and with 1: 2 to 1: 6 separation ratio ^TMOn.Whenever second to the 3rd day replacing medium that at a distance from 5 to 6 days said hBS culture is gone down to posterity and going down to posterity at every turn.

The result of no feeder cells embodiment 3

Observe and be based upon Matrigel ^TMOn hBS passage process in, the method for enzymatically treating that need to use clostridiopetidase A IV with colony from surface isolation.With respect to mechanical separation, find that after inoculation enzymatic treatment also makes reproduction speed increase in the process that goes down to posterity.

No feeder cells embodiment 4

Cultivation is at Matrigel ^TMOn the hBS cell freezing preservation and thaw

Before freezing, be that SA002, AS038, SA121 and SA167 handle 20-30 minute so that said cell is separated from each other to 4 kinds of different cells with clostridiopetidase A IV.After centrifugal said cell is transferred in the freezing medium with the concentration that the freezing medium of every ml contains 1,000,000 cells, said medium contains and comprises 10%DMSO, the VitroHES of the bFgF of 30% serum substitute and 4ng/ml ^TM-medium.The final cell suspending liquid that obtains is the mixture of individual cells and cell mass.For a long time in liquid nitrogen before the storage with cryovial (0.5-1.0ml cell suspension thing) be transferred to fast in the Nalgene refrigerated container in-80 ℃ down storage spend the night or stored at least 2 hours.

Thawing of HBS cell

, all cells suspension must prepare k-VitroHES through cryovial being put into 37 ℃ of water-baths before thawing ^TM-medium also carries out preheating.Centrifugal (400G5 minute) preceding to be transferred to said cell suspension thing in the medium of preheating and to carry out 5 minutes.Through adding 1ml k-VitroHES ^TMIn-medium to the aperture to Matrigel ^TMThe aperture that thin layer covers (BD) carries out rehydration and in 37 ℃ of following incubations 30 minutes.With said cell precipitation at k-VitroHES ^TMAgain suspend in-the medium and be transferred to 24-or 6-hole Matrigel ^TMIn the plate.

No feeder cells embodiment 5

The sign of the hBS cell that no feeder cells are cultivated

Be based upon Matrigel ^TMLast back and process freeze/thaw are carried out all and are characterized experiment after the cycle.

Immunocytochemistry: as stated said culture is gone down to posterity, be seeded to 6-or 24-hole Matrigel ^TMCultivated 6 days on the plate and before carrying out immunostaining.Wash said culture with PBS, (HistoLab, Gothenburg Sweden) fix 15 minutes, and in PBS, wash 3 times at room temperature to use 4% formaldehyde.Employed monoclonal one is anti-to be anti-SSEA-1 ,-3 and-4 (1: 200; Developmental Studies Hybridoma Bank, University ofIowa, Iowa City, IA), Tra-1-60, Tra-1-81 (1: 200; Santa CruzBiotechnology, Santa Cruz, the anti-phosphoric acid histone H 3 of antibody CA) and multi-clone rabbit (1: 150; KeLab, Upstate).At two anti-(1: 300 that uses that appropriate C y3-or FITC put together; Jackson immunoResearch laboratories, West Grove PA) resists in 4 ℃ of following incubated overnight said one before showing.Equally at room temperature be 4 '-6 ' diaminourea-2-phenylindone (DAPA of 0.5 μ g/ml with final concentration; Sigma-Aldrich SwedenAB, Stockholm, Sweden) to culture incubation 5 minutes to manifest all cells nuclear.The culture of rinsing dyeing also uses DAKO fluorescence sealing medium (Dakopatts AB,

Sweden) to carry out sealing and under inverted fluorescence microscope (Nikon Eclipse TE2000-U), observes.Use the commercial kit of buying (Sigma-Aldrich) to Matrigel according to manufacturers instruction ^TMThe hBS cell of cultivating carries out alkaline phosphatase (AP) dyeing.

Telomerase activation: results, cracking Matrigel ^TMThe hBS cell of cultivating and according to the ELISA method of manufacturers instruction through PCR-based (Roche Diagnostics GmbH, Mannheim Germany) analyze telomerase activation.

Karyotyping and FISH: the Matrigel that will be used for karyotyping ^TMThe hBS cell of breeding colchicine (0.1 μ g/ml, Invitrogen, Carisbad, CA, USA) in incubation 1 to 3 hour, separate, fixing, (#WS-32 Sigma) shows chromosome on slide and through using improved Wrights dyeing in sealing.Plate (plates) like preparation noted earlier phase in mid-term.For carrying out FISH (FISH) analysis, use the commercial kit (MultiVysion that contains the probe that is useful on chromosome 13,18,21 and sex chromosome (X and Y) that purchases according to manufacturers instruction ^TMPB Multicolour Probe Panel; Vysis, Inc., Downers Grove, IL).Use is equipped with suitable filter and software (CytoVision, Applied Imaging, Santa Clara, inverted microscope analysis slide glass CA).

Teratoma: form experiment for carrying out teratoma, use immune deficiency SCID mouse (C.B-17/lcrCro-scidBR, Charles River Laboratories, Germany).Through using clostridiopetidase A IV (200 units/ml) with Matrigel ^TMThe hBS cell colony enzymatic ground of breeding is opened from surface isolation, mechanically is divided into little cell aggregation and is expelled under the scrotum with the amount of about 50000 to 100000 cells of each organ.With the cryo-PBS parenteral solution or from handling control-animal with the nascent brain cell of the mouse under viviparous.Injection is after kill said animal eight weeks and tumour is fixed in 4% the paraformaldehyde solution rapidly and uses FFPE.With said teratoma be cut into the section of 8 μ m and with Alcian Blue/Van Giesson dyeing to carry out histologic analysis.

The RT-PCR that Oct-4 expresses analyzes: use Rneasy Mini kit (Qiagen) to separate from all 4 kinds of Matrigel according to manufacturers instruction ^TMTotal RNA of the hBS cell-line of cultivating.Use AMV First Strand cDNA Synthesis kit (Roche) synthesizes cDNA and uses Platinum Taq archaeal dna polymerase (Invitrogen) to carry out the PCR reaction from the total RNA of 1 μ g.Said PCR reaction comprises the decline circulation that four steps are initial, and said decline circulates in and repeats twice circulation on each annealing temperature, 94 ℃ of sex change 15 seconds, extends 30 seconds 66 ℃ to 60 ℃ annealing 15 seconds and at 72 ℃.Ensuing circulation comprises that 35 annealing temperatures are 58 ℃ repetition.The forward and the reverse primer sequence that are used for Oct-4 have been described in front.B actin primer as internal reference (justice, 5 '-TGGCACCACACCTTCTACAATGAGC-3 ', antisense, 5 '-GCACAGCTTCTCCTTAATGTC-ACGC-3 '; The 400bp product).The Ago-Gel of use 1.5% separates said PCR product through gel electrophoresis by size.End user's liver contrasts over against shining and the water conduct being born as the PCR reaction.

No feeder cells embodiment 4 and 5 result

Whether freezing preservation technology is freezing can find any changing features with thaw cell-line SA 002, AS 038, SA121 and SA167 with observation through using.All the four kinds of cell-lines of back of thawing are all survived and are begun to be grown in similar pattern and are coated with Matrigel ^TMCulture dish on.

Be able to verify and compare at the versatility of four kinds under the no feeder cells condition different hBS cell-lines and maintenance with the result of each cell-line under feeder cells are cultivated of front.Through to morphology, do not break up in expression, telomerase activation, caryogram and the body of differentiation marker and investigate to describe these characteristics.

Immunocytochemistry: with opposite with anti-SSEA-3, SSEA-4, TRA-1-6-and TRA-1-80 antibody staining (demonstrating the positive clearly immune response that conforms to versatility hBS cell), SSEA-1 expresses and is negative in the hBS cell-line of having or not feeder cells cultivation.In addition, all four kinds of Matrigel ^TMIt is reactive that cell in the cell-line of breeding all represents high-caliber AP.

Telomerase activation: at three kinds of Matrigel ^TMAnalyze in the cultured cells system (AS 038, SA 121 and SA 167).Cultivation is at Matrigel ^TMIn the hBS cell come to light and have high-caliber telomerase activation.

Karyotyping and FISH: two kinds of Matrigel therein ^TMCultured cells is to carry out karyotyping among AS 038 and the SA 121.From 3 in 3 cells of cell-line AS 038 with have normal people 46 from 10 discoveries in 12 cells of cell-line SA 121, XY caryogram (figure .10).2 cells of residue from SA 121 cell-lines show 45, XY and 42, the abnormal karyotype of XY.However, for the hBS cell that feeder cells and no feeder cells are cultivated, the seemingly normal event of the variation of caryogram after long-term cultivation.The karyotyping and the Matrigel of the hBS cell that feeder cells are cultivated in this research ^TMResult after the breeding is similar, is hinting that hBS cell caryogram keeps normal and stable under the condition of these no feeder cells.Two Matrigel therein ^TMCarry out fish analysis in the cell-line (SA 121 (XY) and SA 167 (XX)) of breeding.Chromosome x, Y, 18,13 and 21 are analyzed.For two cell-lines that accept inspection at least 93% is normal.Result from fish analysis is similar with the result of the hBS cell-line of cultivating from feeder cells.

Teratomatous formation: use two Matrigel ^TMHBS cell-line SA 167 that cultivates and SA 002 form teratoma, and the teratoma that the result shows formation provides Matrigel by the cell and the organizational composition of representative from the differentiation of all three germinal layers (entoderm, mesoderm and entoderm) ^TMThe hBS culture of breeding has the evidence that keeps its versatility.

The expression of Oct-4: Oct-4 in all four kinds of cultivations at Matrigel ^TMOn cell-line in highly express.

No feeder cells embodiment 6

At Matrigel ^TMCultivate the comparison of mitotic index with the mitotic index of cultivating the hBS cell on the fetal mice feeder cells of the hBS cell under no feeder cells condition in the plate that applies

Cell-line SA 121 is cultivated the Matrigel under no feeder cells condition simultaneously ^TMOn the fetal mice feeder cells, carried out 3 days with cultivating in the plate that applies.The nuclear immunoreactivity of the histone H 3 through phosphorylation is carried out quantitatively mitotic cell number then.Mitotic index in two kinds of cultures is calculated with the growth rate between the hBS cell that does not relatively have the cultivation of feeder cells and feeder cells.

The result of embodiment 6

Compare with the feeder layer condition, cultivate at no feeder cells (Matrigel ^TM) under culture have similar mitotic index.No feeder cells culture increases roughly the same (about 35 hours) of the hBS cell of time doubly and feeder cells breeding.

Claims (30)

1. a vitrified method that is used for the hBS cell comprises

I) with cell transfer to the first kind of solution (solution A) that contains one or more cryoprotectors,

Ii) with step I) the said cell transfer that obtains to the second kind of solution (solution B) that contains one or more cryoprotectors,

Iii) will be from step I i) the said cell transfer of 20 μ l to 250 μ l that obtains have a closed straw that permission can comprise 20 μ l to 250 μ l volumes size therein to one or more,

Iv) seal said one or more closed straw and

V) said one or more closed straws of vitrifying.

2. the process of claim 1 wherein that said hBS cell is a hBS cell-line.

3. the process of claim 1 wherein that said one or more cryoprotectors are selected from glycerine, trehalose, sucrose, 1,2 ethylene glycol, DMSO, propane diols and/or its mixture.

4. the process of claim 1 wherein that said first and second kinds of solution comprise one or more identical or different cryoprotectors.

5. the process of claim 1 wherein that the concentration of one or more cryoprotectors in said first and second kinds of solution is identical or different.

6. the process of claim 1 wherein that the total concentration of in said second kind of solution cryoprotector is greater than the concentration in said first kind of solution.

7. the process of claim 1 wherein that said cryoprotector is a trehalose.

8. the method for claim 7, the concentration of wherein said trehalose is from 0.02M to 1M.

9. the process of claim 1 wherein that said cryoprotector is a sucrose.

10. the method for claim 9, wherein said concentration of sucrose is from 0.02M to 1M.

11. the process of claim 1 wherein at least a viscosity modifier that comprises in said first and second kinds of solution.

12. the method for claim 11, wherein said viscosity modifier be selected from phenanthrene can, Percoll, hyaluronic acid, albumin, polyvinylpyrrolidone, alginic acid, gelatin and glycerine.

13. the method for claim 11, wherein said viscosity modifier are that phenanthrene can.

14. the method for claim 13, its China and Philippines can concentration be to be 150mg/ml at the most.

15. the method for claim 11, wherein said first and second kinds of solution comprise one or more identical or different viscosity modifiers.

16. the method for claim 11, the concentration of one or more viscosity modifiers in wherein said first and second kinds of solution is identical or different.

17. the process of claim 1 wherein that at least a in said first and second kinds of solution is aqueous solution.

18. the process of claim 1 wherein in step I) in first kind of solution the said cell of incubation.

19. the method for claim 18, the wherein incubation between carrying out under 37 ℃ from 5 seconds to 20 minutes.

20. the process of claim 1 wherein at step I i) in second kind of solution the said cell of incubation.

21. the method for claim 20, the wherein incubation between carrying out under 37 ℃ from 5 seconds to 10 minutes.

22. the method for claim 20 was wherein carried out incubation 30 seconds or shorter time under 37 ℃.

23. the process of claim 1 wherein 50% or more cell going vitrifying and cultivating after on the proper culture medium, vitality is arranged.

24. the method for claim 1, said method further comprise the vitrifying of going through the method that comprises the following step:

Vi) one or many vitrified closed straws are placed to have temperature environment a period of time from room temperature to 40 ℃, so that the content in the closed straw thaws,

Vii) open said one or many closed straws and

Viii) use the third solution (solution C) that contains one or more cryoprotectors that the cell that is included in said one or many closed straw of opening is washed.

25. the method for claim 24 comprises:

Ix) will available from step viii) through the washing cell transfer to the 4th kind of solution (solution D) that contains one or more cryoprotectors,

X) from said the 4th kind of solution transfer in-migration from ix) cell and with said cell inoculation on feeder cells and

Xi) further cultivate said cell.

26. the method for claim 25 is wherein at step I x) in said cell incubation in the 4th kind of solution.

27. the method for claim 24 or 25, wherein said one or more cryoprotectors are selected from glycerine, trehalose, sucrose, 1,2 ethylene glycol, DMSO, propane diols or its mixture.

28. the method for claim 24 or 25, wherein said one or more cryoprotectors are glycerine, trehalose, sucrose or its mixture.

29. the method for claim 28, the concentration of wherein said cryoprotector is from 0.02M to 1M.

30. the method for claim 25, if wherein relevant, the concentration of cryoprotector is higher than the concentration of cryoprotector in the 4th kind of solution in the third solution.

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