CN102160546A - Systems and methods for cryopreservation of cells - Google Patents
Systems and methods for cryopreservation of cells Download PDFInfo
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- CN102160546A CN102160546A CN2010105209439A CN201010520943A CN102160546A CN 102160546 A CN102160546 A CN 102160546A CN 2010105209439 A CN2010105209439 A CN 2010105209439A CN 201010520943 A CN201010520943 A CN 201010520943A CN 102160546 A CN102160546 A CN 102160546A
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Abstract
A fluid sample vessel includes inlet and vent tube fittings formed at one end of a container with an opposite open end closed by a needle septum. A support cap is removably engaged to the container to support the container and protect terminal ends of inlet and vent tubular branches coupled to the fittings. The support cap includes a pair of opposite legs with outwardly directed tabs for mounting within a centrifuge while supporting the cryopreservation container.
Description
The cross reference of related application
The application is that the application number of submitting on December 17th, 2008 is 12/337, the part continuation application of 237 unsettled U.S. Patent applications also requires its priority, this U.S. Patent application is common unsettled U.S. Patent application 11/765,000 and common unsettled International Patent Application PCT/US2007/071,545 part continuation application also requires the priority of these two applications, U.S. Patent application 11/765,000 and International Patent Application PCT/US2007/071, the 545th, submit to the name that also all requires on June 20th, 2006 to submit to be called the provisional application 60/814 of " system and method for freezing preservation cell " on June 19th, 2007,982 priority, whole disclosures of this application merge to this paper as a reference.
Technical field
The application relates to storage method and the relevant apparatus that is used for freezing preservation cell (for example mammiferous cell) and tissue samples/sample.
Background technology
Cell often is frozen preservation temporarily to prolong survival ability and the validity on its biomedicine with organizing.The procedure division ground of freezing preservation comprises cell put into and comprises electrolyte and during refrigerating process in the aqueous solution of chemical compound (antifreezing agent) of protection cell.This antifreezing agent is the molecule of small-molecular weight normally, for example glycerine, propane diols, ethylene glycol or methyl-sulfoxide (DMSO).
When these solution were cooled to the temperature that is lower than its freezing point slightly, solution remained in liquid condition.It is this that it keeps liquid situation to be called surfusion under the phase transition temperature of solution.In the time of under the aqueous solution is further cooled to its freezing point, cold excessively degree aggravation.When nonintervention, be not more than 15 ℃ some place under the freezing point usually, the hydrone in the solution is with spontaneous nucleation, and pure water will be condensed into ice.
Changing into from liquid state the solid-state transition process, solution moves on to low free energy state from high free energy state, thereby causes exothermic reaction.The heat that produces during this phase transformation makes the sample instantaneous heating, and sample temperature raises in this process.Surrounding environment (for example device of just freezing preservation sample) remains on temperature constant state or continues cooling (cooling means that depends on use) simultaneously.Then, along with the heat in the sample dissipates, make sample experience quick cooldown rate to rebulid heat balance in the imbalance of the heat between sample and cooling device during this incident.This in many cases quick cooldown rate causes the formation of ice in the cell, and this causes cell death usually.The quality of sample, the heat-transfer character of sample container, the cooling scheme of use and the basic low-temperature biological characteristic of cell are depended in the formation of ice usually in this cell.
Relation between frozen state and the biosystem has attracted human a lot years.As far back as 1683, Boyle, Robert (Robert Boyle) found that some fishes and frog can survive the of short duration time period in subfreezing temperature, if a maintenance of moisture is not frozen in its body.The freezing preservation of artificial induction at first 1948 by Polge, Smith and Parkes by chancing on glycerine to poultry and Niu Jingzi and be found for erythrocytic anti-freezing characteristic subsequently.In nearer period, to begun one's study the basic process of control relation of the interesting scientist of the natural phenomena relevant and biomedical applications with freezing biosystem.At first, be known that the temperature of decline causes metabolic activity to be suppressed, and therefore cause reducing of speed, the degeneration of apotrophic biosystem will take place under this speed that reduces.But, the beginning that refrigerating process can not be contemplated as the people; It causes the chemistry that can expect, heat and extreme variation electrical characteristics usually, to change the iuntercellular interaction system of intracellular organelle, cell membrane and the precision relevant with tissue and organ.Certainly, even therefore the extreme complexity of known the simplest biological cell it should be noted that the seemingly-dead reversible state by freezing gained is fully possible.
Because the at first discovery of the anti-freezing effect of glycerine and the discovery subsequently of widely used infiltration antifreezing agent methyl-sulfoxide (DMSO), many researchers have been devoted to mainly to pass through the preservation of cell or tissue of the method for experience.The freezing preservation scheme of most cells suspension has been set up the anti-freezing additive of the infiltration of using molar concentration can realize freezing survival rate.By using these artificial antifreezing agents, many flexibilities are added to process of cryopreservation.For example, in order to be issued to best survival rate in the situation of not adding antifreezing agent (CPA), human erythrocyte need be cooled with the speed about 1000 ℃/min.But under the situation of the glycerine that has 3.3M (30%), the survival rate of this cell type goes up in cooldown rate 2-3log scope (2-3log range) and keeps about 90%.As predictable, CPA concentration is high more, and is big more in the possibility of seepage failure between interpolation/removal matter era, and therefore need more concern in these processes.
In arbitrary process of cryopreservation, the solution that is comprised will be cold excessively below freezing point at it, find any nucleating point of crystallization up to it.When by the freezing preservation of freeze-thaw method, freezing in the extracellular medium intentionally causes by the seeding that was in cold minuent place.If freezing is not to be caused by seeding, when solution was cooled under its equilibrium freezing point by enough lowlands, ice will form naturally so.Because this process is actually arbitrarily, will at random take place under the uncertain temperature so freeze; Thereby, will be alterable height utilizing the sample survival rate between the repeated in experiments of same freezing scheme.And the extreme crystallization meeting fast of height over-cooled solution causes the damage of pair cell and tissue when causing forming ice.Further, show, when cold excessively height, cause have the possibility of freezing in the cell of damageability significantly to increase so if the extracellular freezes.This phenomenon is because the freezing cell dehydration that causes postpones beginning, the bigger possibility that this causes ICW to keep increase and therefore cause freezing in cell.
As mentioned above, from liquid state between solid-state tour, solution moves on to low free energy state from high free energy state, the thermal unbalance between this sample that causes continuing heating and the cooling device that continues to cool off.This imbalance causes the serious deviation with the cooldown rate of the regulation of particular cell types at last, and causes the possibility of cell damage in this process.
In order to prevent that these possible damaged conditions from taking place, the step in process of cryopreservation often comprises intervention, to introduce ice crystal near the solution solidifies point in the solution of extracellular.This process that is called " seeding " is carried out near sample being cooled to solution solidifies point, is used in the outside of metal device (for example tweezers or metal bar) the contact sample container of precooling in the cryogen (for example liquid nitrogen) then.This seeding step produces ice crystal in the solution of extracellular, and " model " is provided, and by model, the subcooled water molecule in the solution organises and produces more ice.But the seeding sample in this mode is time-consuming, and is frozen in the cell and make sample be in risk under the situation that the cooling device that is used for this program shifts out and because this seeding method can inadvertently cause by temporary transient at it.
Need avoid the freezing saved system of the above-mentioned problem relevant with unbalance condition.Also need to need not to be performed at present such system of the auxiliary seeding step that forms with the ice crystal that causes control.Also need to be convenient to the freezing storage device of the solution of the problems referred to above.Needed freezing storage device also can provide to be simplified it and is used to obtain and stores the cell of wanting freezing preservation and the method for tissue.
Summary of the invention
Need and to satisfy aspect several by of the present invention in these and other of freezing preservation field.In one aspect of the invention, be provided with automatic one-tenth nuclear device, be used for before the liquid freezing that freezing preservation holder comprises, being incorporated in the freezing preservation holder.Described device comprises the synthetic that size is suitable for being incorporated into the elongated hollow tube in the freezing preservation holder and is arranged on the formation ice-nucleus in the hollow tube.The two ends of pipe are sealed, and at least one end is to use diaphragm seal, and this film is impermeable and be permeable for the liquid that is included in the freezing preservation holder for the synthetic that forms ice-nucleus.Preferably, two ends comprise that this film is to allow the sample liquid inflow device and to pass through device.
In a preferred embodiment, the synthetic that forms ice-nucleus is a sterol, and more preferably is cholesterol.Cholesterol can be the coating on the hollow tube inside, the solid matrix in perhaps can being set to manage.
In another aspect of this invention, provide freezing preservation holder, it can use with automatic one-tenth nuclear device.In one embodiment, freezing preservation holder comprises the elastic tubular main body, and it has and is beginning the open closed port that liquid sample is incorporated into the end in the main body and limits at the end opposite place of main body of being used for.This port is suitable for being pierced through with the Extract sample body by entry needle.Rear open end is heat sealed in liquid sample is introduced into holder.Automatically become nuclear device and the inside that is fixed to tubular body, so that its entry needle that can not be pierced the port of sealing touches with entering the mouth biasing.
In another embodiment, freezing storage device comprises the container that is used to receive with the storaging liquid sample, this container have opening in the container inlet fitting and be installed to the adapter of accessory.Adapter has the first tubulose branch road and the second tubulose branch road, and the second tubulose branch road terminates in the pipe strips for joint parts.The diaphragm seals first tubulose branch road, its septation is suitable for being pierced through by entry needle.Freezing storage device also is provided with the pipe that at one end joins the pipe strips for joint parts on the second tubulose branch road to, and the closure member that is in the end opposite place of pipe.
The closure member that is used for second branch road at first is a barrier film, and this barrier film can be pierced through so that sample liquid is introduced in the holder by entry needle.Container can be below being initially located in atmospheric pressure pressure with promote sample liquid from the injector delivery to the holder in.In case sample liquid is transferred, then the pipe on second branch road is heat sealed and just is cut off to form the final closure member of second branch road on the pipe strips for joint parts.The device of sealing experiences freezing reconciliation ice scheme then.After thawing, the interior barrier film of first branch road that can use syringe to pass through adapter is extracted sample liquid out.
Can predict, the present invention will provide a kind of simple reproducible system, be used for using introducing ice and reducing cold at many different cell freezings.The present invention has conceived method and apparatus, is used for the inducing of extracellular ice crystal by control during freezing preservation cell of being based upon of solid matrix device and the tissue, and ice-nucleus will be formed naturally on the solid matrix device.
The present invention surpasses existing system and method has multiple advantages.Now, the most methods that causes controlled ice-nucleus is trouble, be difficult to regeneration, though and when operation technique, point to the very big font of freeze-thaw survival rate of the increase of many cells and tissue, still to repeatedly check.So far, most of normally used method scopes from the metal object of freezing (being refrigerated to-196 ℃ usually) or cotton wipe away contact pipe-type bottles or tubule simply the side to being designed for the refining plant that sprays liquid nitrogen in the zonule of sample.But, even under optimum condition, carry out, mechanically introducing ice crystal and can cause to form enough big ice crystal by this way to allow breeding fully in the solution of whole extracellular, or owing to approach observed great freezing rate in the sample portion of the position that metal object or liquid nitrogen spray point to most on container, thereby cause cell damage and loss.
In a structure, the liquid sample device comprises the container that is used to receive with the storaging liquid sample, and container is formed by the elongate body of one.Main body is sealed by inlet tube accessory and the ventilation duct accessory of opening to hollow inside from openend qualification hollow inside and in end opposite.On the one hand, a phosphor bodies also limit extend in the hollow inside and be arranged on the inlet tube accessory and the ventilation duct accessory between wall.The openend of container is by the entry needle diaphragm seals.Device comprises that also the adapter main body that is installed to each pipe fitting, adapter main body have inlet tubulose branch road and ventilation tubulose branch road, and the tubular branch road that ventilates comprises the filter cell that is arranged on wherein.
On the other hand, be provided with supporting cover, it has the bottom that removably joins container to, and the top that extends beyond inlet tube accessory and ventilation duct accessory when supporting cover joins container at the end opposite place of container.Container can limit at least two recesses at its outer surface, but the bottom of while supporting cover comprises the elongated protrusion of at least two elastic deflections, and each elongated protrusion has the protuberance of the inside sensing in corresponding that is configured to receive in the recess extensiblely.
On the one hand, the top of supporting cover comprises the cylindrical wall that surrounds inlet tube accessory and ventilation duct accessory, but also comprises the inwall of the side of the opposite side that is positioned at inlet tube accessory and ventilation duct accessory.In another embodiment, the bottom of supporting cover comprises two relative slender leg, and each supporting leg is included in the protuberance that its free end outwards points to.Slender leg can be longer than elongated protrusion.
Use described device to obtain to be used to analyze or be used for the method for the liquid sample of freezing preservation, be contemplated that and comprise following beginning step: adapter main body is installed on each pipe fitting, adapter main body has corresponding to the tubular branch road of inlet of inlet tube accessory and ventilation duct accessory and the tubular branch road that ventilates, and the tubular branch road that will enter the mouth is connected to fluid supply.Method is proceeded with following step: insert the liquid in the container by the inlet tube accessory, by the ventilation duct accessory and the tubular branch road that ventilates the air in the container is discharged simultaneously, the position in the top of support cap cuts off and the tubular branch road of sealed entry and the tubular branch road that ventilates then.
When the container complete closed, support cap is installed on the container.This assembly is installed in the centrifuge, and this centrifuge is operated with thing and fluid separation applications on will floating on the surface with centrifugal force, and the thing on floating on the surface directly is exposed to the entry needle barrier film.Use extends through the entry needle of barrier film, and the thing on will floating on the surface is extracted out in container.Then, remaining liquid can be frozen preservation, and wherein support cap is mounted or is not installed on the container.
Description of drawings
Fig. 1 is the view of automatic one-tenth nuclear device according to an embodiment of the invention;
Fig. 2 is the view in conjunction with the known freezing preservation holder of automatic one-tenth nuclear device shown in Figure 1;
Fig. 3 a is the view of system's pipe-type bottles of the enclosed elastic that is used for freezing preservation liquid sample according to another embodiment of the present invention, and pipe-type bottles is expressed as the original state that is used to carry sample;
Fig. 3 b is the view of the pipe-type bottles shown in Fig. 3 a, and it is sealed to be expressed as the pipe-type bottles end;
Fig. 4 is the perspective view of cell freezing save set according to another embodiment of the invention;
Fig. 5 is the perspective view that is used for the adapter of device shown in Figure 4;
Fig. 6 is the exploded view of device shown in Figure 4;
Fig. 7 is the perspective view according to the cell freezing save set of another embodiment;
Fig. 8 is the side sectional view at the top of device shown in Figure 7;
Fig. 9 is the perspective cut-away schematic view of the bottom of device shown in Figure 7;
Figure 10 is the perspective view according to the freezing storage device of another embodiment;
Figure 11 is the perspective view of the freezing storage device of Figure 10, is installed on this device according to the supporting cover that is disclosed in another feature wherein;
Figure 12 is the device shown in Figure 11 and the view sub-anatomy of supporting cover;
Figure 13 is the enlarged perspective at the top of device shown in Figure 11-12 and supporting cover;
Figure 14 is the perspective view according to the supporting cover of optional embodiment;
Figure 15 a and 15b are the schematic diagrames according to the freezing storage device of Figure 10 double-duty;
Figure 16 is according to the freezing storage device of Figure 14 of a kind of purposes wherein and the schematic diagram of supporting cover.
Embodiment
In order to promote understanding to principle of the present invention, referring now to shown in the accompanying drawing and subsequently the embodiment described in the explanation of writing.Be understandable that and be not intended to thus limit the scope of the invention.Can further be understood that any change and the correction that the present invention includes described embodiment, and comprise the application of the principle of the present invention that can expect usually as technical staff for the field that the present invention relates to.
In one embodiment of the invention, provide automatic one-tenth nuclear device 10, as shown in Figure 1, it relates to the use of the synthetic that can form ice-nucleus.According to the present invention, the synthetic 20 that forms ice-nucleus is incorporated into the inner surface 14 of opening hollow tube 12.In a preferred embodiment, pipe is made of plastics.The nucleation synthetic of q.s is introduced in the pipe to form solid matrix in pipe, allows liquid flow to pass through pipe simultaneously.
In a preferred embodiment, the nucleation synthetic is the crystal cholesterol.The use of sterol synthetic, particularly cholesterol are known in other field, for example at the cooling water system as shown in United States Patent (USP) 4928493.In using these other, pulverous synthetic is placed in the container, is used to be exposed to the formation of water to help to ice.As described below, what can determine after the experiment is that the crystal cholesterol is nontoxic for sample cell and the liquid (for example blood, stem cell solution and sperm) that preparation is used for freezing preservation.
The end 16 of pipe is by film 18 sealings of permeable solution.Especially, film is permeable for freezing preservation liquid, and is impermeable for the cell or tissue that will preserve.Keep to separate and prevent cell/tissue and the direct contact that forms between the synthetic of ice-nucleus is important.Film at each place, end also comprises any cholesterol crystal, and it can remove and prevent that crystal from polluting liquid on every side from pipe.It also is important that film allows the free-flow of freezing preservation liquid to enter in the pipe 12.Space in pipe and the solid matrix nucleation synthetic also can be full of at first isoosmotic pressure buffer solution is arranged.
Should become the size design of nuclear device 10 to become to be placed in the freezing preservation holder automatically, for example pipe-type bottles 30 as shown in Figure 2 or blood bag 40.Device can be unfixed in holder or be fixed to inner surface.Because the device intention is as the nucleation site that is used to ice formation, so it needn't be big especially.In a particular embodiment, pipe 12 is that 0.25 inch long, diameter are 0.0625 inch.Freezing preservation holder can be full of specific sample or sample and as at suitable Cryosreservation solution known in the art, install 10 simultaneously and remain in the container.Container 30 or 40 is performed freezing preservation scheme then.Because freezing preservation liquid contact with the synthetic 20 that forms ice-nucleus in device, so ice from installing 10 pipe 12 interior a little or do not have spontaneously formation under the cold situation.In the solution around then ice continues to be formed into away from pipe, thereby cause seldom or do not have frozen cell suspension under ice forms in the cold and minimal cell the situation.
In a certain embodiments, first experiment is designed to definite cholesterol that physically is attached to the inside of freezing preservation holder will bring out ice-nucleus.In this embodiment, the sterol solution that works adds in the 3ml methyl alcohol by the dried cholesterol with 0.025g and prepares.The suspension that obtains is placed in 70 ℃ the dry slot subsequently and shakes off and on, dissolves up to all solid-state sterol.The coated 100 μ l sterol solution of commercial available pipe-type bottles also are placed in 75 ℃ the dry slot so that the methyl alcohol evaporation, thereby realize the crystallization again and the adhesion of cholesterol.Pipe-type bottles washes 2-3 time to remove any crystal that is scattered with 1 milliliter of PBS then.
Then, 6% glycerite (to duplicate the freezing preservation medium of typical sperm bank) and 10% DMSO solution (is refrigeration system to duplicate ordinary cells) prepare in PBS, and assess by cooling off with the speed of-5 ℃/min in the doleiform pipe that applies at sterol and uncoated (control) doleiform pipe.In order to reach the statistics rate, 20 comprise DMSO doleiform pipe and 12 doleiform pipes that comprise glycerine are evaluated.Monitor temperature in each doleiform pipe with thermocouple with 1 second interval, so that the release of the latent heat of the decomposition of freezing point solution and thawing.
This result of experiment shows that in DMSO and glycerine freezing point is higher and reduces for the doleiform pipe variations in temperature that is applied by sterol during melting heating (Δ T).These results are summarised in the following table:
In this experiment, some that can also observe crystal come off or cracked (and in the DMSO sample to a certain degree decomposition), thereby cause contaminated aqueous solution (perhaps partly because the inevitable physical operations of container and solution).In order to address this problem, an embodiment of automatic one-tenth nuclear device 10 is provided, wherein 0.25 inch hollow tube is coated with 100 μ l sterol solution and was dried 48 hours in inside.One end of pipe seals with permeable tampon, and the other end of pipe utilizes epoxy resin to be attached to the inside of doleiform pipe lid and was dried 14-24 hour.Support is designed to keep the cholesterol of combination in the environment of isolating, also allow solution (but not allowing cell) to enter to form contact simultaneously.
In second experiment, the human sperm utilizes this automatic one-tenth nuclear device 10 freezing preservations and is analyzed with the freezing sperm of the sample of determining the freezing preservation according to the present invention and the pipe-type bottles that uses standard construction to compare whether have higher post-thaw survival power particularly.In this experiment, obtain the human sperm's sample (from 20 samples of four donors) abandon, and be placed to and continue 30-60 in moist 37 ℃ of couveuses (5% carbonic acid gas, 95% air) until being liquefied.In case be liquefied, utilize isobaric PBS (keeping 37 ℃) that sample is adjusted to 5ml and utilize computer assisted sperm analytical equipment to assess to measure and to write down total initial total amount and activity.Sample is balanced to 6% glycerine by the step-by-step system program of adding up in TEST yolk buffer solution then.After the balance, each sample is divided into three 1.5ml sample sizes and is deposited in the pipe-type bottles that (1) comprise device 10; (2) manually in the standard tubular bottle of seeding (control) energetically; (3) will not accept in seeding (passive control) the standard tubular bottle.
All samples are placed in the refrigerator of control speed and with the speed of-5 ℃/min and are cooled to-8 ℃ from 22 ℃.Under-8 ℃, the cotton swab that has been soaked in the liquid nitrogen is used to begin seeding in the pipe-type bottles of artificial seeding after 3 minutes.Under-8 ℃, sample is cooled off with the speed of-10 ℃/min again to descend-40 ℃ after in addition 7 minutes.Under-40 ℃, speed is enhanced-20 ℃/min, is dropped into liquid nitrogen (LN at-80 ℃ of samples
2) in.
After freezing, by being placed on the sample that thaws on the testing stand (corresponding to the speed of thawing of-300 ℃/min).In case only Sheng ice melts, then glycerine is diluted in 10 minutes period by adding PBS in the mode that splashes into; Sample is rinsed and is suspended in not having the PBS of glycerine then.At last, before back total amount and the activity assessment of thawing, sample is cultivated (37 ℃, moist environment, 5% carbonic acid gas, 95% air) at least one hour.
Second result of experiment shows the activity of utilizing the freezing sample of automatic one-tenth nuclear device of the present invention those samples freezing with utilizing artificial seeding (56.0 ± 3.8%) to compare to keep obviously (p＜0.05) higher (standard error of 66.1 ± 4.7% means (mean ± SEM)).As utilize the variance analysis of technology determined, two seeding schemes obviously high (p＜0.05) are in the sample (43.1 ± 3.7%) of the passiveness control of seeding not.
In the 3rd experiment, what determine is that the cholesterol of combination in long time period can not produce cytotoxinic effect for cultivating sperm.In this experiment, the sperm sample of liquefaction is exposed to the culture plate that is coated with 100 μ l sterol solution.There is not tangible toxic action cultivating between the culture period that 1,2, the 4 activity evaluation forms that carry out during with 8 hours are shown in 8 hours directly to contact for the human sperm with the cholesterol of combination.
Therefore, in sperm freezing preservation process, and use artificial seeding or do not have seeding to compare, automatic one-tenth nuclear device 10 of the present invention is proved to be and can produces the activity afterwards of better thawing.These experimental results show that the ice-nucleus that sterol causes be stablize, method reliably, it can reduce cold and therefore have the quick rising that reduces " flashing " the relevant temperature afterwards that forms at the typical ice crystal of the freezing incident of over-cooled solution under better result's the situation.Apparatus and method of the present invention allow sample not remain in the cooling chamber in during whole freezing with being upset, so do not need the artificial seeding of prior art.
What can be sure of is the sperm stock method of the apparatus and method of the present invention commerce that is particularly suitable for standard.In the sperm bank dis environment of the commerce of standard, handle many samples, and time/manpower limits and can not always allow the meticulous processing controlling the refrigeration of speed or can reach in the laboratory.What can be sure of is that the present invention allows to have the freezing preservation of repeatably sample better than the result of prior art.In addition, the present invention can also realize successful freezing and recovery with sample of low activity, and the sample with low activity is excluded from contributor colony usually.
Similarly, device 10 of the present invention and method have remarkable influence for the candidate stem cell of storing frozen preservation by this way and/or the ability of hemopoietic progenitor cell (PCB HPC): this mode allows stock and time enough to be used for enough the infectious disease screening and the HLA somatotype that will carry out.Freezingly save as preservation from benefiting from gene therapy or because disease or provide chance by the PCB source HPC that is in the newborn patient in the normal hematopoiesis function risk of forfeiture that medical treatment causes by radiotherapy or chemotherapy.Recently, more make great efforts to be devoted to purify the CFU-GM system of selection.The ability of preserving the progenitor cell (cell of for example representing the CD34 surface glycoprotein) of these relative " pure " may make the cumulative volume of the cell suspending liquid of transplanting minimize.But,, therefore can obtain every kilogram of limited quantity that is subjected to the HPC of body weight because the volume ratio sample of bone marrow of the common PCB that obtains is much smaller.This makes compares much important for the effective and best freezing and storing method of PCB source HPC with the situation of the HPC in other source (for example marrow, peripheral blood).Automatic one-tenth nuclear device of the present invention therewith the time before available device the more effective and preferred mode that is used for freezing preservation and recovers these rapid wear samples that provides is provided.What can be sure of is will cause unique scheme in the research and development of the device 10 cord blood stem cell freezing and storing method that is attached to ongoing improvement, to preserve this cell type under the situation of the higher recovery rate of still less manpower.Develop experimental program and be used for verifying the viability of preserving apparatus and method of the present invention at PCB and Cord blood and the ox sperm freezing that is used for commercial device for artificial insemination.In the provisional application 60/814982 of these schemes reference in the above, this description merges to herein by reference.
Another aspect of the present invention proposes the various Cord bloods of freezing preservation source ancestral cells and requires diverse program, and therefore requires to be different from the reservoir vessel that exists under the known program having now.For stock and storage source various kinds of cell type, preferably use the very different freezing scheme that comprises different freezing/heating rates from Cord blood.Prior art is used stored frozen bag (some bags have a plurality of chambers) or pipe-type bottles.But these two systems have substantial limitations.Multi-cavity chamber bag does not allow different freezing rates maybe will be used in CPA in the different chamber.This can not be considered to " closed " system pipe-type bottles under freezing storage temperature, unless use thermosealed external packing, the application of external packing can damage responsive sample.
In order to overcome this limitation, another embodiment of the present invention is that form is the freezing preservation holder of resilient sealing system pipe-type bottles 50, shown in Fig. 3 a and 3b, thereby it makes sample use different schemes to be frozen in the system of sealing separated between the separative element.Pipe-type bottles 50 comprises the elastic tubular main body 52 that at one end has port 54.Port preferably by identical with elastic body but be adapted at thawing after pierced through sterilely to extract the material seal of sample out by entry needle.
Shown in Fig. 3 a, be open during 56 beginnings of the end opposite of pipe-type bottles to allow to introduce liquid sample.In case pipe-type bottles 50 has been filled, end 56 for example is closed by heat seal bar 58, shown in Fig. 3 b, has so just obtained closed system pipe-type bottles 50 and has been used for the freezing of individual unit and storage.Randomly, but preferably, each pipe-type bottles comprises above-mentioned automatic one-tenth nuclear device 10.Shown in Fig. 3 a, device 10 preferably is fixed to the inwall of main body 52, so that its entry needle that can not passed port 52 enters.
Foreseeable is that the pipe-type bottles 50 of present embodiment can be used for a plurality of freeze/thaw schemes at the refrigerated container that separates.Therefore, pipe-type bottles 50 in a row can be supported in the fixture, can use openend 56 to be used to introduce a plurality of liquid sample sample sizes.When each pipe-type bottles was filled, respective end was sealed to be provided for the closed system pipe-type bottles of freezing preservation.
In another embodiment of the present invention, the freezing storage device 60 shown in Fig. 4-6 is provided, it has further been simplified the acquisition sample and it has been ready for use on freezing process.Device comprises that size is used to receive the container 62 of liquid sample.Container 62 at one end comprises inlet fitting 64.As shown in Figure 6, become nuclear device 10 to be introduced in the container automatically by inlet fitting 64.
Inlet fitting holds adapter 65, as being shown specifically in Fig. 5.Adapter comprises tube 66 down, and its size is suitable for closely being assemblied in the inlet fitting 64.Bottom 66 can use the suitable mode of epoxy resin or heat seal or other to be sealed to inlet fitting to provide airtight and liquid-tight seal between container 62 and adapter 65.
Adapter comprises two tubular branch roads 67 and 69.Branch road 67 terminates in the end 68, and end 68 is configured to engage entry needle barrier film 72 (Fig. 6).Second branch road 69 terminates in the accessory 70 jaggy.This accessory 70 jaggy engages hermetically with the end 74a of pipeline 74.The free end 74b of pipeline 74 receives the entry needle barrier film 75 of itself.The place, ends that two entry needle barrier films 72 and 75 are formed at two branch roads 67 and 69 provides airtight and liquid-tight seal.And in a single day barrier film 72,75 is configured to be pierced through in known manner and to be removed entry needle by entry needle will self sealss.
In a specific embodiment, provide pipeline folder 80 when pipeline 74 joins adapter 65 to, to stablize pipeline 74.Folder 80 comprises the part 80 that is configured to slide with attached part 84 on the branch road 67 of adapter, attached part is configured to slide on pipeline 74, as shown in Figure 4.
In case sample has been introduced in the container 62, the branch road 69 of adapter just is permanently sealed predictably.In preferential embodiment, sealing is undertaken by seal elastic pipeline just above accessory 70 jaggy.In case sealed, owing to no longer need, the remainder of pipeline just can be removed.In a particular embodiment, known constriction sealing bar can be used for making simultaneously the part that pipeline flattens, heat seal flattens and cut off unnecessary part.Sealing and cutting are positioned as close to preferably that accessory 70 jaggy carries out so that elastic pipeline 74 does not have remainder to exceed the vertical shell of container 62.
It is desirable to seal the aseptic integrality or the close encapsulation aspect that can not endanger freezing storage device 60 with cutting step.When by barrier film 75 injection samples, branch road 69 keeps sealing in whole process, even after removing entry needle.In case branch road 69 is sealed, the device 60 that comprises liquid sample is just prepared to be used for freezing and to be stored in same egg carton formula container, and this egg carton formula container holds this device during filling step.When sample was fetched in hope, device 60 can take out from egg carton formula container and be used for separating with other device that remains on egg carton formula container and thawing individually.The entry needle barrier film 72 of branch road 67 provides the approach that is used for aseptic sample drawn.Therefore can use syringe needle and syringe to pierce through barrier film and liquid sample is drawn in the syringe.Can abandon empty device 60 then.
In optional embodiment, provide the freezing storage device 100 shown in Fig. 7-9.Device comprises container 102, and it is similar with above-mentioned container 62 aspect the ability that is used for freezing preservation cell suspending liquid, has or do not have the automatic one-tenth nuclear device 10 that is arranged at wherein.In certain embodiments, container 102 has the storage volume of 2-5 milliliter.Container 102 has the top 103 of opening and limits the bottom 104 of opening 105.Top 103 is by covering 110 sealings airtightly, shown in the cross section view of Fig. 8.The top limits annular lip 107, and the following skirt 116 of lid 110 is installed on the annular lip 107.This time skirt preferably is heat sealed on the annular lip in a conventional manner, to form the liquid seal nipple that can resist freezing storage temperature.
In the embodiment of Fig. 7-9, branch road 114 is as the ventilation branch road, rather than conduct is as the outlet branch road in the device 60 of front.Filter cell 125 can be arranged in the passage 121 of accessory 120.In a preferred embodiment, this filter parts is the aseptic micro-filter of 3 μ m.Micro-filter 125 is gas-permeables, but is impermeable basically for the liquid sample or the cell suspending liquid that are stored in the container.
In this embodiment, device 100 is arranged to remove the suspension that stores by the bottom 104 of container 102, rather than removes by lid as the embodiment of front.Therefore, shown in the profile of Fig. 9, the opening 105 in the container is by 130 sealings of entry needle barrier film.Go out diaphragma of mouth 130 and suitably be sealed to container 102 airtightly, for example pass through heat seal.In order to protect barrier film, dismountable cover 132 is provided, it is fixed in container around barrier film.Cover 132 can limit the tearing part 133 of thinner or the area format that dies down, and it can make the central part 135 of cover be removed.Can provide lappet 135 as shown in Figure 7, so that the removal of central part 135.Cover can be the paper tinsel film, and it is fixed in the bottom 104 of container 102 around it.
The occupation mode of freezing storage device 100 is similar to said apparatus 60.Particularly, cell suspending liquid is introduced in the container 102 by inlet branch road 112, and introduces by above-mentioned entry needle barrier film in particular.Alternatively, the inlet branch road can be provided with the entry needle port or the needleless port of standard.In the time of in cell suspending liquid is injected into container, air wherein is discharged from by the filter in the ventilation branch road 114 125.In certain embodiments, micro-filter 125 is configured to for cell suspending liquid porous not basically.Therefore, in case the level of the suspension in the container arrives filter 125, suspension can not pass through filter so, thereby the rising of pressure will stop in the liquid inlet container.
In case container 102 is filled, two branch roads will use for example above-mentioned constriction fusion splicer to carry out heat seal so that container by completely, airtightly the sealing.Certainly, be understandable that entry needle barrier film 130 and cover 132 still are kept perfectly when container 102 is filled.The device 100 that is full of and seals can be frozen storage then.
When cell suspending liquid was extracted in hope, device and content were at first thawed in known manner.In case thaw, cover 132 is removed to expose barrier film 130.Ventilation branch road 113 is cut off to open vent passages 121.Can pierce through barrier film 130 by syringe or similar device then and extract cell suspending liquid.A series of protuberances 106 can be formed on the bottom 104 of container, and it is fit to engage draw-out device or syringe.In certain embodiments, protuberance 106 is configured to dock draw-out device, and in other embodiments, protuberance can be provided with gas thread (LUER threads), is used to engage the pipe joint (LUER fitting) that extracts on the syringe.
According to another embodiment, freezing preservation holder 200 comprises can be by the container of calibrate or pipe-type bottles 201, as shown in figure 10.Holder 200 has the lower end 302 of opening and the upper end 205 of sealing.As seen from Figure 12, container 201 is the main bodys that form the one basically of hollow inside 202 from the lower end 203 of opening.At 205 places, upper end of sealing, the main body of one is configured to form the pair of pipes adapter 207,208 that reaches in the hollow inner 202.Two pipe adapters reach inner interior opening and are separated by wall 210.As in the previous embodiments, adapter 207 can be connected to exhaust outlet, and adapter 208 can be connected to and will be frozen the fluid supply that is kept in the holder 200.Wall 210 prevents that the liquid that enters by adapter 208 is drawn out of from adapter 207 immediately, particularly when as described here when adapter 207 applies negative pressure.
The openend 203 of container 201 is by 215 sealings of entry needle barrier film, and entry needle barrier film 215 can be configured to be similar to above-mentioned entry needle barrier film 130 and play effect similar with it.Entry needle barrier film 215 is kept by cover 217, and cover 217 is sealed to the openend 203 of container by conventional methods, so that the anti-sealing of revealing to be provided.
As further describe, holder 200 is similar to holder 100.But container 201 is modified to hold supporting cover 240, shown in Figure 11-13.Supporting cover 240 comprises bottom 242 and top 244, and the upper and lower all is roughly cylindrical structure and size is fit to sliding fit to container 201.Bottom 242 comprises at least two protuberances 246 of the protuberance 247 with inside sensing.Protuberance 247 sizes are made and are arranged to be locked in and are formed on the container 201 in the corresponding recess 250 (Figure 10).Protuberance 247 inwardly points to so that must be by deflection protuberance 246 outside mobile protuberances when lid 240 slides on container.Protuberance comprises the upper surface 247a of inclination, and when the hope removal was covered, the upper surface 247a of inclination can be pulled out protuberance smoothly from recess 250.Protuberance 247 is set at the middle part of protuberance 246, so that bottom 246a extension below protuberance of protuberance is used for manual engagement to discharge protuberance from recess.Bottom 246a provides leverage to help discharging protuberance, also prevents protuberance simultaneously because accident contacts shifting out of causing.
The top 244 of lid 240 is configured to protect the pipe 221 and 223 that extends from container top.Therefore, top 244 comprises annular wall 252, and it has the inwall 254 of being arranged to be positioned at the side of managing the side, as shown in figure 13.Therefore, when pipe combined with the adapter 207 and 208 of holder 200, wall prevented canal curvature, and this has been avoided destroying fluid-tight sealing and has polluted content.
The lid of revising 260 as shown in figure 14.This lid is configured to support freezing preservation holder 200 particularly in centrifuge or other utensil.In some instances, wish to separate the content of holder 200, for example the thing isolated cell grain on float on the surface with centrifuge.Container 201 may be difficult for being installed in the centrifuge, so the lid of revising 260 provides extra support for container.Particularly, lid 260 comprises bottom 262 and top 264.Top 264 can be similar to be covered 240 top 244 and is configured and comprises that inwall 265 is with the protection pipeline.
In addition, be similar to and cover 240, the bottom 262 of lid 260 comprises at least two protuberances 266, and protuberance 266 has the protuberance 268 that is configured to engage the recess 250 in the container 201.But bottom 262 comprises supporting leg 270, and supporting leg 270 extends beyond the end of protuberance 266.And these supporting legs 270 are terminated at outwards outstanding protuberance 272.Therefore the major part of supporting leg 270 length of 201 from recess 250 along container is extended.Outside protuberance 272 is configured to engage the recess or the installing component of utensil (for example above-mentioned centrifuge).Longer supporting leg 270 has been not only for container provides support, supporting leg and specifically protuberance 272 a kind of mechanism also is provided, be used for engaging releasedly utensil, thereby in utensil, support freezing preservation holder 200 fully.
The use of holder 200 has been described in Figure 15 a and 15b.In two kinds of uses, container is connected to fluid supply, and for example the blood bag 280.The blood bag comprises pipe 282, and pipe 282 is connected to the inlet branch road 223 of adapter main body 220.In one embodiment, adapter 284 can be engaged with between the expansion end 226 and blood bag pipe 282 of inlet branch road.In a kind of scheme shown in Figure 15 a, from being input in the container 201, air overflows by ventilation branch road 221 blood by gravity.In another kind of scheme, shown in Figure 15 b, whole system is sealed, and syringe 290 is connected to ventilation branch road 221.Syringe is drawn back applying negative pressure to container 201, thereby blood is extracted in the container from bag 280.
In case want blood or other liquid of freezing preservation to be pumped in the container, pipeline just is cut off and seals, as shown in figure 16.The end 295 of pipe of sealing is cut off and is present in when Yi Danggai is engaged on the container 201 below the end of covering 260 top 264.Therefore, as shown in figure 16, the end of pipe is fully surrounded and is protected.Can utilize and cover 260 protuberance 272 holder 200 is installed in centrifuge or other storage device.Can further be understood that, lid 260 as support with support have upwards towards the holder of entry needle barrier film 215.When the content of container separates with centrifuge, the thing on floating on the surface will move up naturally, and it can also be extracted out by easily approaching by the entry needle that passes barrier film in this position.In case the thing on floating on the surface is removed, just can remove and cover 260 from container 201, content just can be frozen preservation then.
Will be further appreciated that the described process of Figure 15 a-15b can not use under subsequently the situation of freezing preservation.For example, require the destruction of having stipulated detected blood unit according to the blood stock's of U.S. blood bank association (AABB) standard current blood testing and since current have no idea effectively the blood sample amount to be moved into detect in the container and do not pollute the possibility of whole unit.This requirement causes the destruction by the about centesimal blood unit of all blood bank's processing, and this is being an obvious results aspect cost and the leiphemia.Detect the centrifugal action that container must allow to be used for the cell granulation, the system that keeps sealing simultaneously is to avoid pollution.
Can predict, in certain embodiments, freezing preservation holder 60,100 and 200 container 62,102 and 201 can be provided with initial vacuum.This vacuum helps the extraction of liquid sample in filling the reservoir receptacle step.Owing to lead to each branch road 67/69,112/114 and 221/223 sealed (by corresponding barrier film 72,75 or pass through heat seal), so vacuum can be held a very long time.Lid can be set on each branch road to determine gas-tight seal.In a particular embodiment, container 62,100 and 201 interior initial vacuum can be the pressure under the atmospheric pressure that is between 100mmHg (definitely) and the 160mmHg (definitely).
Freezing preservation holder 60,100 and 200 can be made by the standard material that uses in blood stock and long preservation field under standard freezing conditions (promptly hanging down the temperature to-196 ℃).It is suitable plastic components to be reached meet pathogens standard or other requirement that specific blood carries that current O.S.H.A. recommends.Therefore, the container in the various embodiments described above, accessory and pipeline can be made by the plastics that are fit to, for example polystyrene or polypropylene.In the egg carton formula container for the standard of being installed to, holder (after the sealing elastic pipeline) should meet within 10mm diameter and the 90mm height.Elastic pipeline 74 also must can be resisted freezing storage temperature, and heated sealant and cut off the ability of pipeline can not damage branch road 69 when adapter seal 65 time.In a particular embodiment, elastic pipeline by
Or similar material is made.
Though illustrate and describe the present invention in the description of accompanying drawing and front, what consider is that it is schematically on feature rather than limits.Be understandable that only to have proposed preferred embodiment, wish to protect the institute that falls into purport of the present invention to change, revise and further use.
Claims (18)
1. be used to receive device, comprise from the liquid sample of independent container acquisition:
Be used to receive the container of liquid sample, described container is formed by the elongate body of one, described main body limits hollow inside from openend, and at the end opposite place by opening to the inlet tube accessory of described hollow inside and the sealing of ventilation duct accessory, the main body of described one also limit extend in the described hollow inside and be arranged on described inlet tube accessory and described ventilation duct accessory between wall; With
Seal the entry needle barrier film of described openend.
2. the device of claim 1 also comprises:
Be installed to the adapter main body of described pipe fitting, described adapter main body has the tubular branch road of inlet and the tubular branch road that ventilates, and the tubular branch road of described ventilation comprises the filter cell that is arranged at wherein.
3. the device of claim 2, the tubular branch road of wherein said inlet comprises the expansion end that is suitable for receiving adapter, described branch road is connected to the single pipe that is associated with liquid container.
4. the device of claim 1, wherein said container is made by the plastics that are suitable for the storing blood sample.
5. the device of claim 1, wherein said container is made by the plastics that are suitable for storaging liquid sample under freezing storage temperature.
6. be used to store device, comprise from the liquid sample of independent container acquisition:
Be used to receive the container of liquid sample, described main body has the hollow inside that begins from openend, and is sealed by inlet tube accessory and the ventilation duct accessory of opening to described hollow inside at the end opposite place;
Seal the entry needle barrier film of described openend; With
Supporting cover, it has bottom that removably joins described container to and the top that extends beyond described inlet tube accessory and described ventilation duct accessory when supporting cover joins container at the end opposite place of described container.
7. the device of claim 6, wherein:
Described container limits at least two recesses in its outer surface; And
The described bottom of described supporting cover comprises at least two deflectable elongated protrusion of elasticity, and each elongated protrusion all has the protuberance of inside sensing, and inwardly the protuberance that points to is configured to be received in the described recess in corresponding one extensiblely.
8. the device of claim 7, wherein said protuberance is positioned at along the center of the length direction of described protuberance.
9. the device of claim 6, wherein said top comprise around the cylindrical wall of described inlet tube accessory and described ventilation duct accessory, also comprise the inwall of the side of the opposition side that is positioned at described inlet tube accessory and described ventilation duct accessory.
10. the device of claim 6, the described bottom of wherein said supporting cover comprises two relative slender leg, each of described supporting leg all comprises the protuberance of the outside sensing that is positioned at its free end.
11. the device of claim 10, wherein, described slender leg is longer than described elongated protrusion.
12. the device of claim 6, wherein, described container is made by the plastics that are suitable for the storing blood sample.
13. the device of claim 6, wherein said container is made by the plastics that are suitable for storaging liquid sample under freezing storage temperature.
14. use the method for the device of claim 6, comprising:
Described inlet tube accessory is connected to fluid supply;
Insert the liquid in the described container by described inlet tube accessory, discharge air in the described container by described ventilation duct accessory simultaneously;
Seal described inlet tube accessory and described ventilation duct accessory;
Described supporting cover is installed on the described container;
The described supporting cover of the described container of carrying is placed in the centrifuge;
Thing on floating on the surface from fluid separation applications with centrifuge, the thing on floating on the surface directly is exposed to described entry needle barrier film; And
Use entry needle to extend through described barrier film, extract the thing on floating on the surface in the container.
15. the method for claim 14, wherein liquid is introduced in the container from input by gravity.
16. the method for claim 14 also comprises:
Syringe is connected to described ventilation duct accessory; And
Use syringe in described container, producing negative pressure, thus with liquid in fluid supply suction container.
17. the method for claim 14 also comprises:
Adapter main body is installed to beginning step on the described pipe fitting, and described adapter main body has corresponding to the tubular branch road of inlet of described inlet tube accessory and described ventilation duct accessory and the tubular branch road that ventilates;
Connection Step comprises the tubular branch road of inlet is connected to fluid supply; And
The sealing step, the position that is included in the top that is in supporting cover cuts off and the tubular branch road of sealed entry and the tubular branch road that ventilates.
18. the method for claim 14, wherein said liquid is blood, and described fluid supply is a blood unit bag.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US12/711,929 US8709797B2 (en) | 2006-06-20 | 2010-02-24 | Systems and methods for cryopreservation of cells |
| US12/711929 | 2010-02-24 |
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| CN111448304A (en) * | 2017-12-12 | 2020-07-24 | 国立研究开发法人农业·食品产业技术综合研究机构 | Cell sealing device with suction tube and use thereof |
| US11603518B2 (en) | 2019-01-23 | 2023-03-14 | Asterias Biotherapeutics, Inc. | Dorsally-derived oligodendrocyte progenitor cells from human pluripotent stem cells |
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