CN101497873A - 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 [mu]l 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

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

The application is dividing an application of application number is 2004800124278, denomination of invention is on May 10th, 2004 patent application for " by using the stem cell of closed straw vitrification method cryopreservation of human blastocyst-derived ", the applying date.

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).Described method to still keep after thawing active cell be non-normal temperature and.

Background of invention

Stem cell is the cell type that has self and produce the unique ability of specialization or noble cells.Although most somatocyte, for example heart cell or skin cells are responsible for exercising specific function, and stem cell function before it receives the signal that develops into particular cell types is uncertain.Form described stem cell uniqueness be its multiplication capacity with and the ability of differentiation takes place.For many years, the researchist 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 described 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, zygote is not to be considered to the embryo in after fertilization 10-14 days before implanting the uterus, and therefore when using as the present invention, this class cell is called as blastocyst-derived stem cell or hBS cell herein.Although the versatility check has shown that described embryo or blastocyst-derived stem cell can produce biological intravital all cells, comprise sexual cell, somatic stem cell has more limited derived cell set of types storehouse.

1998, the researchist can isolate embryonic stem cell for the first time and it is cultivated in substratum, referring to for example United States Patent (USP) 5 843 780 and United States Patent (USP) 6,200 806 from people's fertilized oocyte.

Increasing research and progress need to obtain to be suitable for preserving the method for described cell and clone in the stem cells technology field.Cell can be by vitrifying or chilled storage.Because the formation of extracellular ice has destruction, therefore use the low temperature storage of ordinary method to be difficult to be applied to complicated and responsive biologic material.Through the vitrifying process, the sample that contains described 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 quick freezing liquid medium under the situation that no ice crystal forms.The quick freezing process that amorphous glass is put into liquid nitrogen at the suction pipe that directly will for example contain cell forms.Described glass keeps the proper distribution of liquid but preserves with overcooled form.Described glass has avoided ice crystal and described cell to avoid forming relevant primary cellular defect with ice crystal.Therefore, vitrifying is defined as the curing of the amorphous glass sample 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 by using opening drawing and pulling type straw vitrification method to carry out the method for these cells of low temperature storage.The shortcoming of described method is that its opening end that relates to suction pipe contacts with liquid nitrogen, and this may become will be by the source of pollution 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) described in the application to oocyte of mouse.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 clone.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 described cytodifferentiation or death.Usually, the cell with this slow freezing method vitrifying comparatively large vol can cause lower recovery rate (people such as Reubinoff).

Therefore, still with regard to needs development easy handling, relate to the least possible step and in described process, avoid simultaneously or reduce effective method for vitrification at least the risk of the undesired pollution of described cell.Especially, need development to be used for the vitrified effective ways of comparatively large vol of cell or clone (for example hBS or hBS clone).

Invention is described

As mentioned above, effectively the low temperature storage method is blastocyst-derived stem cell line generation and widespread use, and to set up people's blastocyst stem cell bank hereunder necessary.Effectively freezing and deforst technique can be preserved cell and clone effectively.For some purposes, wish in each root suction pipe, to carry out the large 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 by 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) described 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) described cell of incubation in second kind of solution randomly,

V) the cell transfer that will obtain ii) or iv) from step I has a closed straw that permission can comprise at least 20 μ l volume sizes therein to one or more,

Vi) seal described one or more closed straw and

Vii) one or more closed straws of vitrifying.

A very important feature of aforesaid method is can be by vitrified large volume in each suction pipe.The present invention relates to be used for the method for the cell of vitrifying closed straw, described 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 using in embodiment provided by the invention is about 13cm, and diameter is about 2mm and is about the thick extremely thin plastic tube wall of 0.1mm (closed straw, 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, use longer suction pipe just successfully vitrifying even bigger volume so.

Very important step is to use so-called closed straw in another aforesaid method.In the present context, term " closed straw " is used to refer to has the suction pipe that can make the opening end that the biological agents in suitable culture medium (for example cell or clone) 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 resistance to air loss 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 described suction pipe and at the other 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 kapillary at this end.Also can use other appropriate method of this end of sealing.Use sealer (welding, bonding etc.) the sealing the 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 sl. preferably, to be fit to the standard carriage in the liquid nitrogen filling.Described suction pipe is made by plastics, also can comprise that glass (although its may easier fracture) makes by any suitable material.Importantly described 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 the context of the invention " directly contact " or " directly being exposed to " are meant if the surface of biologic material or the existing substratum of described material, solution or material when contact with frozen material, claims biologic material " directly contact " or " direct exposure " in for example frozen material.

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

" vitality " as used herein be meant in for some time biologic material can normal existence, growth and propagation.

According to the present invention, the biologic material (for example hBS cell or hBS clone) of at least 20 μ l volumes is placed closed straw.Described closed straw is exposed in the frozen material (liquid nitrogen suitably) then.In case be exposed to frozen material, described cell begins vitrifying, can store subsequently to thaw in for some time and period afterwards.The biologic material that thaws still keeps vitality.As shown in top, cell does not directly contact with frozen 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 viable cell or clone, particularly the BS cell, from cell-derived BS cell of BS or cell.Described cell can be in any etap.Preferably, described cell derives from animal-origin, comprises Mammals source, including but not limited to people, non-human primates monkey, laboratory animal for example pig, sheep, cow, goat and horse of rat, mouse and hamster, agriculture livestock for example for example.Have in the embodiment of specific purpose in the present invention, described 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 clone, particularly hBS cell or hBS clone.By using method described herein can obtain described cell or clone.

One of them plants vitrification solution (first kind and second kind of solution) can contain one or more cryoprotectants or cryoprotection agent composition.Certain non-toxicity cryoprotectant is preferred.Cryoprotectant is retained in the non-molar ratio that freezes the solute in the water and assists contraction is reduced to minimum level by reducing other.It suppresses the formation of ice crystal, and therefore reduces the zero pour of water.It also can combine with irreducible water by hydrogen and prevent protein denaturation.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 nonelectrolyte can destroy cell.When handling with cryoprotectant, cell can not reach the salt concn of non-processing cell before it reaches much lower temperature.Chemical reaction carries out very slowly and therefore makes the destruction of pair cell drop to minimum level under so low temperature.Usually preferably use the cryoprotectant combination because may have difference between the different sorts.Cryoprotectant also can serve as the osmotic pressure promoting agent.Suitable cryoprotectant can be selected from 1,2 ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), glycerine, propylene glycol, 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 is 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 concn of cryoprotectant (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 cryoprotectants.The concentration of one or more cryoprotectants in first kind and the second kind of solution can be identical or different, and usually in second kind of solution the total concn of cryoprotectant be higher than first kind of concentration in the solution.

In the specific embodiment of the present invention, described cryoprotectant 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, sees in hundreds of plant and animals.Trehalose is an important energy derive and to be presented between pool period be the primary factor of stabilate body.Proved trehalose can reduce film transformation temperature so that itself in addition under the exsiccant condition, 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 by water being evicted from the arrestin qualitative change from described protein surface.

In another embodiment of the invention, described cryoprotectant 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 cryoprotectant 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, white protein, 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, described 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, 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 aforesaid method).

Owing to this step is intended to promote solution equilibria and guarantees that cryoprotectant 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 virose 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 or about 95% or more cell after going vitrifying and cultivate and after on the suitable medium, vitality is arranged.The suitable method for vitrification that goes has been described herein.

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) one or more vitrified closed straws are put into have from about room temperature to about 40 ℃ environment for some 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 cell and

Xiv) randomly further cultivate described 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 visual inspection 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 described cell can be brought out the heat shock effect.

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

Go vitrification solution can comprise cryoprotectant, for example have the active cryoprotectant of osmotic pressure (for example having hypotoxic osmotic pressure promoting agent), 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.Can prevent in the existence of the described disaccharides in extracellular that natural osmotic pressure from working and dump for the cell time enough and be present in intracellular DMSO (or analogue), and replace it by water lentamente.

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

In specific embodiment, one or more cryoprotectants are selected from glycerine, trehalose, sucrose, 1,2 ethylene glycol, DMSO, propylene glycol 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 cryoprotectant 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 cryoprotectant 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 (0.1-0.4mm x0.1-0.4mm).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 the other end opening.Described 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 enclosed appts (Demtek A/S) seals.Before freezing relatively large cell, carry out taking turns freezing and the check of thawing.After thawing, described cell small pieces are seeded on the culture dish with mice embryonic feeder cell (MEF).People BS cell cultures is assessed after once going down to posterity.

All per-cents of mentioning all are v/v.

Following description has provided explanation to suitable method, solution, time period etc.But based on describe, in general terms and guide herein, 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 sterile 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 sterile filtration.

Add aseptic DMSO to final concentration be 20%.

4. 1ml solution A and solution B are added respectively four orifice plates (Nunclon, VWRInternational) in two apertures that separate.Plate is placed under 37 ℃.

5. cut with the glass capillary (World PrecisionInstruments) of the drawing of using sterilization (or from Swemed stem cell parting 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 parting 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 glass capillary 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, must number and as seen manage (visiotubes) SvenskMjolk) with freezing numbering (being clone/date/signature) mark.

Freezing flow process:

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

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

3. then with in described 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 described 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 described 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 hot-sealing device.

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

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

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

Serum substitute and following ingredients

Dulbecco ' s Modified Eagle ' s substratum, described composition final concentration separately is: penicillin 100 units/ml, non-essential amino acid 0.1mM, L-glutaminate 2mM, β-thioglycol 100 μ M, people's recombinant bfgf 4ng/ml (Prostatropin)].

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

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

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 described suction pipe to be connected on the syringe as the blocking thing.Then suction pipe is located to cut off in bonding (welding).With the air in the syringe hBS cell is pushed in the solution C.The cell concentration that will wash in identical aperture should be no more than the contained amount of single suction pipe.

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

5. under stereoscopic microscope, 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 uses glass fiber cell pipe (World PrecisionInstruments) under stereoscopic microscope or draw is transferred to the hBS cell colony on the hBS substratum.

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

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

Chart

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

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

Fig. 3: the recovery people BS clone 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 clone SA001 on the mice embryonic feeder cell.

Fig. 5 (A)-(C): the representative configuration of cultivating the people BS clone SA001 on the mice embryonic feeder cell 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 clone SA001 on the mice embryonic feeder cell 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] and, (E) TRA-1-60[p31], (F) TRA-1-81[p31] and, (G) Oct-4[p31], (H) ALP[p31].

Fig. 8: the caryogram of clone 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 the refrigerated closed straw.

The present invention will be further specified in the following embodiments, and described 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 sterile filtration, 10% DMSO; Solution B: the Cryo-PBS solution of the trehalose of the 0.3M of sterile 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 parting tool.Described cell block sizes should be about 0.1 to 0.4mmX0.1-0.4mm.At first with described 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.Described volume is 40-50 μ l approximately.About 10 cell fritters are sucked into preparation are used for vitrified suction pipe, then with the suction pipe 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 sterile filtration; Solution D: the Cryo-PBS solution of the 0.1M trehalose of sterile 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.Described hBS cell incubation in 500 μ l solution C changed in the 500 μ l solution D in 1 minute then and incubation 5 minutes.Under stereoscopic microscope,, be seeded in then in the culture dish above the mice embryonic feeder cell on the hBS substratum the washing fast in the hBS substratum of hBS cell fritter.Then described 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 the closed straw vitrification and the people BS cell that thaws

People BS cell (clone 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 cell 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 morphocytology) number and the initial vitrified hBS cell fritter number.Prepare 3 suction pipes and assess each clone, and with below the results are shown in of each single suction pipe, the result shows that recovery rate is between 40% and 100%.

Closed straw people BS clone
	SA002 SA181
Thaw and recover 3/7 9/9 5,/10 6/8 10,/10 2/5

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 describing to people BS cell (clone AS 034) and thaw in embodiment 1 and 2.Clearly, only having an appointment 4 BS cell fritters in each opening drawing and pulling type suction pipe can be by vitrifying.Be seeded in the culture dish above the mice embryonic feeder cell back 48 hours the hBS cell colony is assessed and counted.Calculate the recovery rate of thawing with the ratio between (the showing suitable hBS morphocytology) number of the colony after viable the thawing and the initial vitrified hBS cell fritter number.Each clone 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 how viable (absolute number) keeps the cell of acceptable recovery rate simultaneously from each closed straw.

People BS clone AS034
	Closed straw opening drawing and pulling type suction pipe
Thaw and recover 6/9 4/4 6/9 2/4 6,/10 3/4

Embodiment 5

In the vitrifying substratum, 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 substratum 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 (clone SA121) that thaws.The mice embryonic feeder cell were assessed and are counted the hBS cell colony after last 48 hour in being inoculated into culture dish.Calculate the recovery rate of thawing with the ratio between (the showing suitable hBS morphocytology) number of the colony after viable the thawing and the initial vitrified hBS cell fritter number.As if each clone 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.

People BS clone SA121
	Trehalose sucrose
Thaw and recover 5/9 8,/10 8,/10 5,/10 6,/10 9/10

Embodiment 6

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

Except in the vitrifying substratum, 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 (clone SA121) that thaws described in embodiment 1 and 2.The mice embryonic feeder cell were assessed and are counted the hBS cell colony after last 48 hour in being inoculated into culture dish.Calculate the recovery rate of thawing with the ratio between (the showing suitable hBS morphocytology) number of the colony after viable the thawing and the initial vitrified hBS cell fritter number.Each clone has all been prepared three suction pipes and assessed, and with below the results are shown in of each single suction pipe.

Closed straw people BS clone SA121
	But phenanthrene concentration 0mg/ml 10mg/ml 100mg/ml
Thaw and recover 5/5 7,/10 6,/10 9/9 6,/10 3,/10 6,/10 3/10-

Embodiment 7

In the vitrifying substratum, use the comparison between the different concns trehalose

Except using in second kind of vitrification solution (solution B) two kinds of different concns (0.3M and 0.5M) trehalose, all the other are all according to the method vitrifying and the people BS cell (clone 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 cell 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 morphocytology) 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 clones.Each clone has all been prepared three suction pipes and assessed, and the following demonstration of the result of each single suction pipe.Although described 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.

Closed straw people BS clone SA121 and SA002
	Trehalose 0.3M 0.5M
The recovery (SA121) 6,/10 8,/10 5,/10 9,/10 6,/10 8/8 of thawing
	The recovery (SA002) 5/8 7/8 6/8 8/8 7/7 5/7 of thawing

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 clones (SA001, SA002 and AS034) that a large amount of people BS cells are carried out vitrifying (described in above-mentioned embodiment 1) three kinds of different occasions.Each clone has under each occasion〉100 branched pipettes are by vitrifying.From these suction pipes in batches, select 8 to 10 to go vitrifying (described in above-mentioned embodiment 2) and be seeded in above the mice embryonic feeder cell in the culture dish separately randomly.In each culture dish, determine the hBS cell mass of inoculation and the number that adheres to, breeds and show suitable morphologic hBS cell mass.Among described Fig. 1 of the results are shown in, 2 and 3 and show that every branched pipette produces viable hBS cell colony, described hBS cell colony is gone down to posterity and describe its feature 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 (clone SA001).After going vitrifying and inoculation, 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 clone SA002 and AS034, obtained similar result (data not shown).

Embodiment 10

The follow-up feature 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 feature description widely.This comprises surface antigen expression analysis, karyotyping and the check of external and intravital multipotency.Obtain following result by end user BS clone SA001, and also can obtain similar result (data not shown) by end user BS clone SA002 and AS034.

The immunohistochemical staining of undifferentiated hBS cell

The vitrified people BS cell (clone SA001) that goes that to cultivate on mice embryonic feeder cell (MEF) is fixed in PFA, then bores a hole with Triton X-100.After successive washing and sealing step, described cell is carried out incubation with one anti-(by explanation).Subsequently with two anti-detections of puting together.Show nuclear by DAPI dyeing.(SigmaDiagnostics, Stockholm, the specification sheets that Sweden) provides use the commercial test kit of buying to determine the activity of alkaline phosphatase (ALP) according to manufacturer.Going down to posterity during each of being carried out analyzed numbered in the marginal data bracket that is shown among Fig. 7.As indicated in Fig. 7, described 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.By centrifugal collecting cell and use ethanol and Glacial acetic acid is fixed.Use trypsinase-Ji's nurse Sa dyeing to show karyomit(e).As shown in Figure 8, described result does not detect chromosome abnormalty after showing the process vitrifying and going the vitrifying process in described cell.

Telomerase activation

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

The inside activity of described mensuration use side granzyme also detects it with enzyme linked immunological absorption measurement (ELISA) by the described product of pcr amplification.After the vitrifying people BS clone SA001 is being analyzed and cultivating on the mouse feeder cell, 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 clone SA001 in the suspension culture to allow blastocyst body (Eb) to form with the stem cell parting 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 myocardial 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.The described Fig. 9 that the results are shown in.

Differentiation in the body

In order to investigate the versatility of vitrifying people BS cell, undifferentiated cell (clone SA001) is placed under the scrotum with serious immune deficiency (SCID) mouse by operation.Kill the mouse tumor resection after 8 weeks and fixing in PFA.In order to determine existence, the paraffin section of phenodin-Yi red colouring is carried out the histology estimation from the tissue of all three germinal layers.As indicated among Figure 10, keeping its potential that is divided into the cell of described 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 general 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, obtain employed cell (being incorporated herein by reference) by method claimed 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 clone and comprise step:

I) ovocyte (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 cell and blastocyst,

Iii) by mechanical anatomical isolation inner cell mass cell,

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

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

Use is as the fertilized oocyte of the parent material of this method.The quality of fertilized oocyte is extremely important to the quality of the blastocyst that obtained.The step I of described method) the people's blastocyst cell in can derive from refrigerated or fresh human oocyte in vitro fertilization.The method of the suitable ovocyte 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 ovocyte.Therefore, if only use 3 grades of ovocytes, the probability of hBS clone that obtains to satisfy general requirement (below being described in) is just very low.

The fresh fertilized oocyte of donations: at the 0th day described ovocyte is sucked Asp-100 (Vitrolife), and fertilization in IVF-50 (Vitrolife) in the 1st day.Assess described fertilized oocyte according to the 3rd day morphology and cell fission.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).Described fertilized oocyte is further cultivated 3-5 days (being after fertilization 5-7 days).According to the described 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 parent cell of donations: carry out freezing at four cell stage described zygote with Freeze-test kit (Vitrolife) at the 2nd day (after fertilization).The refrigerated fertilized oocyte is stored in the liquid nitrogen.Before 5 term gauges are full, obtain informed consent from the donor.Use Thaw test kit (Vitrolife) the described fertilized oocyte that thaws, and carried out aforesaid method from the 2nd day.

As described above, fresh fertilized oocyte is from 3 grades of quality, and the freezed fertilized egg parent cell is from 1 grade and 2 grades.According to the data that obtain by the method for having set up, the per-cent that develops into the fresh fertilized oocyte of blastocyst is 19%, and 50% freezed fertilized egg parent cell develops into blastocyst.This means may be because the fertilized oocyte quality be higher, and described freezed fertilized egg parent 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 parent 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 parent 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 cell that will have A or B level are cultivated altogether to set up one or more inner cell mass cell colonies.After being coated on the feeder cell, monitor its growth and when described colony when manually going down to posterity (approximately apply back 1-2 week), described cell can be cut and places new feeder cell to cultivate from other cell type with flattening.By using glass capillary, the inner cell mass cell is carried out cut mechanically separate as parting tool.Can easily detect by microscope, and therefore needn't carry out any processing to damage or to remove trophectoderm to ovocyte 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.By relatively using immune excision method contrast to keep the success ratio of the complete Ben Fafa of trophectoderm, found to avoid simpler, the quicker and AT method of immune excision method more effective than immune excision method.These methods make preparation stem cell and these clones of differentiation in viable commercial.From having set up 19 clones (15.5%) 122 blastocysts altogether.By immunity excision method handled 42 blastocysts and wherein 6 successfully set up clone (14%).Handle 80 blastocysts and set up 13 clones (16%) by the inventive method.

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

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

People BS clone separable from natural hatching blastocyst or from the blastocyst of expansion with complete zona pellucida.Step I in aforesaid method) blastocyst is the blastocyst of natural hatching.The blastocyst trophectoderm of hatching can be kept perfectly.Can be with the blastocyst of hatching or have and remove or blastocyst that part is removed zona pellucida places on the feeder cell of deactivation.

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

Blastocyst with complete zona pellucida is carried out of short duration PRONASE A (Sigma) handle the elimination that causes described band.Also can use other type proteolytic enzyme that has with the same or similar protease activity of PRONASE A.After handling, strepto-albumen blastocyst can be coated on the feeder cell of described deactivation.

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

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

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

The major criterion that the hBS clone of general requirement is satisfied in another acquisition is to carry out the blastocyst culture condition.Therefore can be lower than every approximately cm by use ²60,000 cells for example are lower than every approximately cm ²55,000 cells or be lower than every approximately cm ²The feeder cell 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 cell of the density of 45,000 cells are cultivated described stem cell line.These values are applied under the situation of using the mouse feeder cell and are expected in the feeder cell 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 mitotic division deactivation to described feeder cell for fear of undesired feeder cell growth.

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

The stem cell line that obtains by above-mentioned establishment method satisfies general requirement.Therefore, described clone

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

Ii) show normal euploid karyotype and

Iii) keep developing in vitro and in vivo all types germinal layer derived cell potential and

Iv) at least two kinds of following molecule marker: 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 monoclonal antibody GCTM-2 identification and

V) do not show molecule marker 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.

Determine the not differentiation hBS cell that obtains by aforesaid method by following standard: it separates, and fertilized oocyte is a blastocyst before people's implantation, and shows the multiplication capacity under undifferentiated state when cultivating on the feeder cell of mitotic division deactivation; 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 monoclonal antibody GCTM-2 identification, and show expression without any carbohydrate epitope SSEA-1 or other differentiation marker.In addition, interior (teratoma) versatility check of external and body shows the derived cell that can be divided into all germinal layers.

As mentioned above, described method provides pure basically versatility people BS cellular preparations, described people BS cellular preparations i) show on embryo's feeder cell of mitotic division deactivation under undifferentiated state, to have when cultivating and surpass 21 months multiplication capacity; Ii) show normal euploid karyotype; Iii) keep all developing in vitro and in vivo the potential of the derived cell of all germinal layers; Iv) at least two kinds of following molecule marker: 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 monoclonal antibody GCTM-2 identification; V) do not show molecule marker 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, use the antibody at described cell marking in described immunologic assay.The method, hybridizing 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 described establishment method 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 described cell with a temperature resistance with 10% dried milk sealing.Educate described cell with two temperature resistances and examine in washing back roughly with DAPI dyeing showed cell.

Alkaline phosphatase

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

Oct-4RT-PCR

By 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.

Fluorescence in situ hybridization (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 test kit that this analysis CTS used the commerce that contains the probe that is useful on karyomit(e) 13,18,21 and sex chromosome (X and Y) to buy (Vysis.Inc, Downers Grove, IL, USA).At least analyze 200 nucleus for each clone.With described cell suspension in Carnoy ' s fixing agent and drip on the positively charged slide glass.Probe LSI13/21 mixed and be added on the described slide glass and with cover glass with the LSI hybridization buffer cover.Probe CEP X/Y/18 is added on another slide glass 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 described slide glass 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 in direct mode to all karyomit(e)s, can detect a large amount of and the more distortion on the macrostructure.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 by synthetic analogues and microtubule depolymerization reagent in order to improve the condition that is used to measure, but still (the 6X10 that provides of a large amount of cells is provided with the Colchiceinamidum that causes the cell prevention in mid-term, colchicine ⁶Cell/analysis).With cell under the situation that 0.1 μ g/ml Colchiceinamidum exists incubation 1-2 hour, then with the PBS washing and use tryptic digestion.By collecting described cell in centrifugal 10 minutes with 1500rpm.With the fixing described cell of ethanol and Glacial acetic acid and with improved Wrights to the karyomit(e) observation of dyeing.

Comparative genome hybridization

Comparative genome hybridization (CGH) is replenishing karyotyping.CGH produces higher karyomit(e) resolving power 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 solution that comprises methane amide, 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 DAPI II) and described slide glass is being covered with cover glass.At microscopically and with ias slide glass is estimated then.

Telomerase activation

Because high telomerase activation has been confirmed as the standard of hBS cell 6, therefore in described hBS clone, measure telomerase activation.The known lasting reduction of telomerase activation when cell reaches more differentiation state.So to the described active algebraic sum control sample that quantitatively must relate to more early, and can be used as the instrument that detects differentiation.Described method, Telomerase PCR

The inscribe activity of ELISA test kit (Roche) use side granzyme also detects it with enzyme linked immunological absorption measurement method (ELISA) by the described product of polymerase chain reaction (PCR) amplification.Carry out described mensuration according to manufacturers instruction.Result from this mensuration shows that usually the hBS cell has high telomerase activation (〉 1).

Described clone keeps its versatility and form teratoma in vivo when being injected into the mouse of non-responsiveness.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

Analyst BS clone whether kept a method of versatility be with described cell xenotransplantation to immunodeficient mouse to obtain tumour, teratoma.All kinds of tissues of finding in described tumour should be represented all three germinal layers.Existing report shows the various tissues of the tumour that derives from the xenotransplantation immunodeficient mouse, for example voluntary 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 people BS cell in the testis or under the scrotum by operation.In testis or kidney, transplant the hBS cell with the scope of 10 000 to 100 000 cells.Ideally, each clone 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 xenotransplantation in that to produce aspect the tumour effect identical.Therefore, female SCID mouse teratoma model is preferred.Described 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.By immunohistochemical method described tumor tissues is analyzed then.Use is at the specific markers of all three kinds of germinal layers.The described mark that uses is at present: 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 phenodin-Yi red colouring for the gross morphology observation.

In following " setting up embodiment ", described establishment method is described.The embodiment that herein comprises only also limits the scope of the invention never in any form as illustration purpose.Well known to those skilled in the art and all reactants of general method described herein and damping fluid 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 suitable medium is called " BS cell culture medium " or " BS substratum " and can comprises: replenish with 20%

Serum substitute and following ingredients

Dulbecco ' s Modified Eagle ' s substratum, described composition final concentration separately is: penicillin 100 units/ml, Streptomycin sulphate 50 μ g/ml, non-essential amino acid 0.1mM, L glutamine 2mM, β-thioglycol 100 μ M, people's recombinant bfgf (Prostatropin) 4ng/ml.

Another kind of suitable medium is " a BS cell paste substratum ", and it is composed as follows: replenish with 20%

Serum substitute and following ingredients

Dulbecco ' s Modified Eagle ' s substratum, the final concentration separately of described composition is: penicillin 50 units/ml, Streptomycin sulphate 50 μ g/ml, non-essential amino acid 0.1mM, L glutamine 2mM, β-thioglycol 100 μ M.

Term " is stablized " and is meant to have under undifferentiated state when cultivating on the embryo's feeder cell in the mitotic division deactivation and surpasses 21 months multiplication capacity in the present context.

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 surrogate and following ingredients

Dulbecco ' s Modified Eagle ' s substratum, and described composition final concentration separately is: penicillin 50 units/ml, Streptomycin sulphate 50 μ g/ml, non-essential amino acid 0.1mM, L glutamine 2mM, β-thioglycol 100 μ M, people's recombinant bfgf (Prostatropin) 4ng/ml) on the feeder cell (EF) in, replenishes with the 0.125mg/ml hyaluronic acid.After being coated in described blastocyst on the EF cell, monitor its growth and apply about 1-2 after week colony described inner cell mass cell is cut from other cell type when manually going down to posterity and increases at new EF cell by 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 zona pellucida

For blastocyst with complete zona pellucida, after on the EF cellular layer that described blastocyst is directly placed the hBS substratum that is supplemented with hyaluronic acid (0.125mg/ml), (Vitrolife in rS2 (ICM-2) substratum, Gothenburg, Sweden) (10U/ml Sigma) carries out of short duration incubation with the degraded zona pellucida to use PRONASE A.

Set up embodiment 3

Alkaline phosphatase is carried out histochemical stain

Gather in the crops described cell to carry out RT-PCR and histology (alkaline phosphatase) and immunocytochemical assay (referring to following).Isolation of RNA and carry out RT-PCR.Use Rneasy Mini test 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 test kit (Roche) that use is used for RT-PCR carries out cDNA.Use the commercial test 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 clone

On the EMFI substratum of Mouse Embryo Fibroblasts Culture in Vitro in the tissue culture ware: DMEM (Dulbecco ' s Modified Eagle ' s substratum), replenish with 10%FCS (foetal calf serum), 0.1 μ M β-thioglycol, 50 units/ml penicillin, 50 μ g/ml Streptomycin sulphates and 2mM L-glutaminate (GibcoBRL).Use ametycin (10 μ g/ml, 3 hours) that described feeder cell are carried out the mitotic division deactivation.By artificial cutting people BS cell colony is placed on the mouse embryo fibroblast feeder cell of deactivation and increase.

On the mouse embryo fibroblast feeder cell with the mitotic division deactivation of people BS cell cultures in having the tissue culture ware of hBS cell culture medium, described substratum is: replenish with 20%

Serum substitute and following ingredients

Dulbecco ' s Modified Eagle ' s substratum, described composition final concentration separately is: penicillin 50 units/ml, Streptomycin sulphate 50 μ g/ml, non-essential amino acid 0.1mM, L glutamine 2mM, β-thioglycol 100 μ M, people's recombinant bfgf (Prostatropin) 4 nanograms/ml.Described 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 substratum with glass fiber cell pipe, described substratum comprises: replenish with 20%

Serum substitute and following ingredients

Dulbecco ' s Modified Eagle ' s substratum, described composition final concentration separately is: penicillin 50 units/ml, Streptomycin sulphate 50 μ g/ml, non-essential amino acid 0.1mM, L glutamine 2mM and β-thioglycol 100 μ M.Described 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 described hBS cell is taken pictures.Every 4-5 days colony is gone down to posterity.It is just even as big as going down to posterity when described colony can be cut into fritter (0.1-0.3X0.1-0.3mm).When described cell went down to posterity for the first time, it had been cultivated 1-2 week and can be cut into about 4.

Described colony is one by one being focused under the stereoscopic microscope 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 kapillary and place on the new feeder cell (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 described cell can stick on the new feeder cell, and then put into incubator.Change the hBS substratum weekly three times.If colony is gone down to posterity, at these specific Zhou Genghuan two subcultures.Usually carry out " half change ", its be meant a sucking-off half substratum and substitute with fresh, the gentle substratum of equivalent.The substratum of whole volume can be changed if desired.

Set up embodiment 6

Vitrifying hBS cell

Cutting has suitable does not break up morphologic colony from described clone to go down to posterity.With the sterile filtration of 100-200ml liquid nitrogen to the freeze pipe 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, pure and mild 200 μ l DMSO of 2-ethylene) place A to carry out 1 minute and with described colony and place B to carry out 25 seconds.Use closed straw to store described freezing colony.After being transferred to described colony in the suction pipe, put it into rapidly in the freeze pipe that the sterile filtration liquid nitrogen is housed.

Set up embodiment 7

The inoculation fetal mice is raised (EMFi) cell

By down described cell being carried out deactivation with the EMFi substratum incubation that contains ametycin 3 hours at 37 ℃.Cover the IVF culture dish with gelatin.Suck described substratum and wash described cell with PBS.Substitute PBS to separate described cell with trypsinase.Behind the incubation, stop tryptic activity with the EMFi substratum.By the described cell of centrifugal collection, in EM Fi substratum, press the 1:5 dilution, and in the Burker cell, count then.With described cell dilution to final concentration is 170K cell/ml EMFi substratum.Substitute the gelatin in the IVF culture dish and put into incubator with the 1ml cell suspending liquid.Changed the EMFi substratum in postvaccinal second day.

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

Can be in no feeder cell culture system with hBS cell cultures used in the present invention, compare described method advantage with currently known methods 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 the hBS clone that studies show that can not (comprise Matrigel at acellular matrix under undifferentiated state ^TM) in breeding surpass 6 times and go down to posterity.Yet described 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 speed of growth 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 described hBS cell transfer to the initial step of not having the feeder cell culture system.Therefore, describe below and be used for the hBS cell transfer wherein described hBS cell mechanically being cut out from feeder cell to the method for not having the feeder cell culture system.Among the no feeder cell embodiment herein, only use the middle portion of each colony, be carried out separation although the risk that whole colony is emitting the substratum that had feeder cell to pollute in the previous work of people such as Xu is handled by enzyme.In addition, to the lip-deep step of not having feeder cell, 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 cell are cultivated.Matrigel ^TMIn main component be extracellular matrix protein, for example IV class collagen protein and ln.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, whole connection egg α is in vivo and have unique effect aspect the cell proliferation in the external adjusting collagen stroma in the collagen protein acceptor.The sticking limit of the layer protein-specific acceptor that may be formed by the beta 2 integrin alpha 6 and the β 1 of hBS cell great expression also can play main effect at the hBS cell adhesion to stromal surface.Therefore, having a kind of may be that described initial intensive Collagenase IV handles and to have influenced some unfriendly and play and adhere to or the significant surfaces acceptor of survival effect before described cell has adapted to new surface.

Separate the hBS cell transfer to the different technologies that does not have in the feeder cell environment having investigated by 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 scale operation.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 cell system

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

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

As mentioned herein before and proved that in no feeder cell embodiment the key factor of successfully carrying out the hBS cell proliferation is that described hBS cell is transferred to method the no feeder cell culture system from the feeder cell culture system.Therefore, must be by cut mechanically with described hBS cell transfer to there not being the feeder cell culture system, can carry out described cut mechanically as parting tool by using glass capillary.As shown among the embodiment herein, to compare with the culture that enzyme is handled, mechanical separation makes cell stick to Matrigel more extremely effectively ^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 among the embodiment herein see, under no feeder cell condition, cultivate with the cell of breeding and have and the similar mitotic index of cultured cells under the feeder cell condition.

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

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

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

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

C) randomly, described blastocyst-derived stem cell was gone down to posterity by enzymatic and/or mechanical treatment every 3-10 days.

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

The hBS cell is transferred to no feeder cell culture system from the feeder cell culture system

Found that as mentioned above transfer step is a committed step.Therefore, should by the method for mechanical separation or in the feeder cell culture system pair cell carry out mechanical separation and shift.Can carry out this mechanical treatment by the instrument that any suitable parting tool for example has sharp-pointed end and the size that is suitable for cutting.Described instrument can be made by any suitable materials such as plastics or glass, and to have the aseptic sharp-pointed glass capillary parting 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 suitable medium with cell mass from the central authorities of described colony.One or many mechanically separates described cell mass for example to have until described 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 described size is defined as the diameter of described cell mass or colony respectively.

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

No feeder cell embodiment 1

For no feeder cell culture prepares adaptability (conditioned) VitroHES ^TM-substratum (k-VitroHES-substratum)

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 ametycin ²Concentration be seeded in Dulbecco ' s Modified Eagle substratum (D-MEM) is housed be coated with gelatin (0.1%; Sigma) in the flat bottle of cultivation, described culture medium supplemented has 1% penicillin/streptomycin (PEST; 10000 units/ml), 10% foetal calf serum (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 described substratum and use VitroHES with PBS (GibcoBRL/Invitrogen) washing once ^TM-substratum (0.28ml/cm ²) the alternative adaptive phase of carrying out 24 hours.Carry out up to the described VitroHES of three times collection every day from identical mEF culture (during second pass generation) ^TM-acclimatizing culture medium and use the low protein binding strainer of 0.2 μ m (Sarstedt, Landskrona Sweden) carry out sterile filtration.Use fresh or-20 ℃ down freezing mistakes the k-VitroHES-substratum and replenish bFGF (GibcoBRL/Invitrogen) before use with 4ng/ml.If be stored in+can use in 4 ℃ of degree k-VitroHES-substratum to reach a week.When being stored in-20 ℃, be two months, do not detect the sign that biological activity goes down in use.

No feeder cell embodiment 2

HBS clone is transferred on the no feeder cell culture condition

On 10-50 time the mouse feeder cell of going down to posterity that initial hBS clone is remained on that ametycin handled and cultivate at the VitroHES that is supplemented with the rh-bFGF of 4ng/ml (bFGF) ^TMOn-the substratum.

To being used for the hBS cell is transferred to Matrigel from the feeder cell culture ^TMTwo kinds of different technologies in the culture dish that covers are assessed, a kind of with mechanical separation another kind handle with collagenase.(Sweden) with hBS cell dice, described square is represented the middle part of colony for Swemed Lab AB, Bilidal, and described cell separated carefully and is transferred in the HBSS solution by using the stem cell parting tool.Described stem cell parting tool is to have the aseptic sharp-pointed glass capillary 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 enzymically treat (being used for comparison) with collagenase

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

Carry out mechanical separation according to the present invention

In HBSS, after the washing, use the 1ml autospencer mechanically described cell mass to be separated carefully.Show corresponding to the size of handling the cell aggregation that produces by collagenase IV-when being about the 1/10-1/20 of original colony (average 20000 cells/original colony), finish described sepn process when the size of described cell mass as mentioned above.In HBSS, after the washing, described colony is transferred to collagenase IV solution (200 units/ml) separate to begin carrying out enzyme.For two kinds of different technology, each with in described cell inoculation to 4 aperture and under 37 ℃ in 5% CO ₂Middle incubation.Each experiment repeats 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 cell embodiment 1 and 2 result

For optimization hBS cell from feeder cell to the transfer of not having the feeder cell condition, assess two kinds of different technology, 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 by two kinds of different technologies respectively at Matrigel ^TMThe total area of all colonies of last generation compares (P＜0.001).In addition, compare with the isolating culture of enzymatic after applying 6 days that total colony area significantly increases (P=0.036) in the culture of mechanical separation.

No feeder cell embodiment 3

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

In all experiments, use 4 kinds of different clone SA002, AS038, SA121 and SA167.With described clone at Matrigel ^TMOn be cultured to for 35 generations and even remain unchanged in the described morphology appearance of freeze/thaw week after date and other hBS feature.All cultures are made of the hBS cell colony of fully determining of the morphology mark that does not have differentiation.After about 3-6 days by taking that substratum goes down to posterity to described cell away and in each aperture, adding 1ml collagenase 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 described cell then, be suspended in k-VitroHES again ^TMBe seeded in Matrigel in the substratum and with the separation ratio of 1:2 to 1:6 ^TMOn.Second to the 3rd day replacing substratum that described hBS culture is gone down to posterity and going down to posterity at every turn every 5 to 6 days.

The result of no feeder cell embodiment 3

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

No feeder cell embodiment 4

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

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

Thawing of HBS cell

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

No feeder cell embodiment 5

The sign of the hBS cell that no feeder cell 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 mentioned above described 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 described culture with PBS, (HistoLab, Gothenburg Sweden) fix 15 minutes, and wash 3 times in PBS at room temperature to use 4% formaldehyde.It is anti-SSEA-1 ,-3 and-4 (1:200 that employed mono-clonal one resists; Developmental Studies Hybridoma Bank, University ofIowa, IowaCity, 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 the two anti-(1:300 that use appropriate C y3-or FITC to put together; Jackson immunoResearch laboratories, West Grove PA) resists in 4 ℃ of following overnight incubation described one before showing.Equally at room temperature be 4 '-6 ' diamino-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 painted culture of rinsing and use DAKO fluorescence sealing medium (Dakopatts AB,

, Sweden) carry out sealing and under inverted fluorescence microscope (Nikon Eclipse TE2000-U), observing.Use the commercial test 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 by 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 karyomit(e) on slide glass and by using improved Wrights dyeing in sealing.The plate (plates) for preparing the phase in mid-term as previously described.For carrying out fluorescence in situ hybridization (FISH) analysis, use the commercial test kit (MultiVysion that contains the probe that is useful on karyomit(e) 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).By using collagenase 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 injection liquid or from handling control animal with the nascent brain cell of the mouse under viviparous.Injection is after kill described animal eight weeks and tumour is fixed in 4% the paraformaldehyde solution rapidly and uses paraffin embedding.Described teratoma is 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 test kit (Qiagen) to separate from all 4 kinds of Matrigel according to manufacturers instruction ^TMTotal RNA of the hBS clone of cultivating.Use AMV First Strand cDNA Synthesis test 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.Described PCR reaction comprises the decline circulation that four steps are initial, and described 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 muscle primer as internal reference (justice, 5 '-TGGCACCACACCTTCTACAATGAGC-3 ', antisense, 5 '-GCACAGCTTCTCCTTAATGTC-ACGC-3 '; The 400bp product).The sepharose of use 1.5% separates described PCR product by size by gel electrophoresis.End user's liver contrasts over against shining and the water conduct being born as the PCR reaction.

No feeder cell embodiment 4 and 5 result

Whether freezing preservation technology is freezing can find any changing features with thaw clone SA002, AS038, SA121 and SA167 with observation by using.All the four kinds of clones of back of thawing are all survived and are begun to be grown in similar pattern and are coated with Matrigel ^TMCulture dish on.

In the versatility of four kinds under the no feeder cell condition different hBS clones with maintenance is verified and compare with the result of each clone under feeder cell are cultivated of front.By to morphology, do not break up in expression, telomerase activation, caryogram and the body of differentiation marker and investigate to describe these features.

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 clone of having or not feeder cell cultivation.In addition, all four kinds of Matrigel ^TMCell in the clone of breeding all represents high-caliber AP reactivity.

Telomerase activation: at three kinds of Matrigel ^TMAnalyze in the cultured cells system (AS038, SA121 and SA167).Cultivation is at Matrigel ^TMIn the hBS cell be found and have high-caliber telomerase activation.

Karyotyping and FISH: two kinds of Matrigel therein ^TMCultured cells is to carry out karyotyping among AS038 and the SA121.From 3 in 3 cells of clone AS038 with have normal people 46 from 10 discoveries in 12 cells of clone SA121, XY caryogram (figure .10).2 cells of residue from SA121 clone show 45, XY and 42, the abnormal karyotype of XY.However, for the hBS cell of feeder cell and the cultivation of no feeder cell, the seemingly normal event of the variation of caryogram after long-term cultivation.The karyotyping and the Matrigel of the hBS cell that feeder cell 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 cell.Two Matrigel therein ^TMCarry out fish analysis in the clone (SA121 (XY) and SA167 (XX)) of breeding.Chromosome x, Y, 18,13 and 21 are analyzed.For two clones that accept inspection at least 93% is normal.Result from fish analysis is similar to the result of the hBS clone of cultivating from feeder cell.

Teratomatous formation: use two Matrigel ^TMHBS clone SA167 that cultivates and SA002 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 the clone camber express.

No feeder cell embodiment 6

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

Clone SA121 is cultivated Matrigel under no feeder cell condition simultaneously ^TMOn the fetal mice feeder cell, carried out 3 days with cultivating in the plate that applies.The nuclear immunoreactivity of the histone H 3 by phosphorylation is carried out quantitatively mitotic cell number then.Mitotic index in two kinds of cultures is calculated with the speed of growth between the hBS cell that does not relatively have the cultivation of feeder cell and feeder cell.

The result of embodiment 6

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

Claims (35)

1. 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) described cell of incubation in first kind of solution randomly,

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

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

V) the described cell transfer that will obtain ii) or iv) from step I has a closed straw that permission can comprise at least 20 μ l volume sizes therein to one or more,

Vi) seal described one or more closed straw and

Vii) one or more closed straws of vitrifying.

2. the method for claim 1, the size of wherein said closed straw allows the volume that has 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, from about 40 μ l to about 50 μ l.

3. each method in the aforementioned claim, wherein said cell are BS cell or BS clone.

4. each method in the aforementioned claim, wherein said cell are hBS cell or hBS clone.

5. each method in the aforementioned claim, at least a one or more cryoprotectants that comprises in wherein said first and second kinds of solution.

6. the method for claim 5, wherein one or more cryoprotectants are selected from glycerine, trehalose, sucrose, 1,2 ethylene glycol, DMSO, propylene glycol and/or its mixture.

7. each method in the claim 5 or 6, wherein said first and second kinds of solution comprise one or more identical or different cryoprotectants.

8. each method among the claim 5-7, wherein the concentration of one or more cryoprotectants in described first and second kinds of solution is identical or different.

9. each method among the claim 5-8, wherein in described second kind of solution the total concn of cryoprotectant (pressing %v/v, %w/v or M calculates) greater than the concentration in described first kind of solution.

10. each method among the claim 5-9, wherein said cryoprotectant is a trehalose.

11. the method for claim 10, the concentration of wherein said trehalose are from about 0.02M to about 1M, for example from about 0.05M to about 0.9M, from about 0.1M to about 0.8M, from about 0.2M 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.

12. each method among the claim 5-9, wherein said cryoprotectant is a sucrose.

13. the method for claim 12, wherein said concentration of sucrose from about 0.02M to about 1M, for example from about 0.05M about 0.9M extremely, from about 0.1M to about 0.8M, from about 0.2M 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.

14. each method in the aforementioned claim, the wherein at least a viscosity modifier that comprises in described first and second kinds of solution.

15. the method for claim 14, wherein said viscosity modifier be selected from phenanthrene can, Percoll, hyaluronic acid, white protein, polyvinylpyrrolidone, alginic acid, gelatin and glycerine.

16. the method for claim 14 or 15, wherein said viscosity modifier are that phenanthrene can.

17. the method for claim 16, its China and Philippines can concentration be about 150mg/ml at the most, for example be about 100mg/ml at the most, be about 50mg/ml at the most, be about 25mg/ml at the most, be about 15mg/ml at the most or be about 10mg/ml at the most.

18. each method among the claim 14-17, wherein said first and second kinds of solution comprise one or more identical or different viscosity modifiers.

19. each method among the claim 14-18, wherein the concentration of one or more viscosity modifiers in described first and second kinds of solution is identical or different.

20. each method in the aforementioned claim, having at least a kind of in wherein said first and second kinds of solution is aqueous solution.

21. each method in the aforementioned claim is comprising step I i).

22. the method for claim 21, wherein the incubation between carrying out from 5 seconds to about 20 minutes under about 37 ℃ 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 about 1 minute.

23. each method in the aforementioned claim, comprising step I v).

24. the method for claim 23, the incubation between carrying out under about 37 ℃ wherein from 5 seconds to about 10 minutes, 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.

25. the method for claim 23 or 24 was wherein carried out incubation about 30 seconds or shorter time under about 37 ℃ of the place.

26. each method in the aforementioned claim, wherein 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 or about 95% or more cell after going vitrifying and cultivate and after on the suitable medium, vitality is arranged.

27. a cell, by among the claim 1-26 in each defined method described cell is carried out vitrifying.

28. further comprising by the method that comprises the following step, each method among the claim 1-26, described method go vitrifying:

Viii) one or more vitrified closed straws are placed to have temperature and carry out for some time so that the content of closed straw thaws from about room temperature to about 40 ℃ environment,

Ix) open one or many closed straws,

X) use described the third solution (solution C) to carry out washing flow to being included in one or many cell in the closed straw of opening,

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

Xii) randomly with described cell incubation in the 4th kind of solution,

Xiii) randomly from described the 4th kind of solution transfer in-migration from xii) cell and with described cell inoculation on feeder cell and

Xiv) randomly further cultivate described cell.

29. the method for claim 28 comprises step xi), xii) and xiv).

30. the method for claim 29 further comprises step xii).

31. each method among the claim 28-30, the wherein said the 3rd and/or the 4th (if relevant) solution comprises one or more cryoprotectants.

32. the method for claim 31, wherein said one or more cryoprotectants are selected from glycerine, trehalose, sucrose, 1,2 ethylene glycol, DMSO, propylene glycol and or its mixture.

33. the method for claim 32, wherein said one or more cryoprotectants are glycerine, trehalose, sucrose or its mixture.

34. the method for claim 33, the concentration of wherein said cryoprotectant are 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.

35. each method among the claim 28-34, if wherein relevant, the concentration of cryoprotectant is higher than the concentration of cryoprotectant in described the 4th kind of solution in described the third solution.

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