CN102160546B - The system and method for Cell Cryopreservation - Google Patents

Abstract

Disclose the system and method for Cell Cryopreservation.Liquid sample storage device comprises inlet tube accessory and the ventilation duct accessory at one end place being formed in container, and opposing opened ends is by entry needle diaphragm seals.Supporting cover removably joins container to and also protects the tubular branch road of entrance being connected to accessory and the terminal of ventilating tubular branch road with support vessels.Supporting cover comprises a pair relative supporting leg, and it has protuberance outwardly for being arranged in centrifuge, supports freeze storage container simultaneously.

Description

The system and method for Cell Cryopreservation

The cross reference of related application

The application is the application number submitted 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 the U.S. Patent application 11/765 of CO-PENDING, 000 and the International Patent Application PCT/US2007/071 of CO-PENDING, the part continuation application of 545 also requires these two priority of applying for, U.S. Patent application 11/765, 000 and International Patent Application PCT/US2007/071, 545 is submit to the name also all requiring on June 20th, 2006 to submit to be called the provisional application 60/814 of " system and method for Cell Cryopreservation " on June 19th, 2007, the priority of 982, whole disclosures of this application are merged into herein as a reference.

Technical field

The application relates to storage method for Cell Cryopreservation (such as mammiferous cell) and tissue samples/sample and relevant apparatus.

Background technology

Biological cells and tissues is often frozen preserves temporarily extend survival ability in its biomedicine and validity.The procedure division ground of freezen protective comprise cell put into comprise electrolyte and during refrigerating process the chemical compound (antifreezing agent) of Cell protection the aqueous solution in.The molecule of this antifreezing agent normally small-molecular-weight, such as glycerine, propane diols, ethylene glycol or methyl-sulfoxide (DMSO).

When these solution are cooled to the temperature slightly lower than its freezing point, solution remains in liquid condition.It is this that under the phase transition temperature of solution, it keeps liquid situation to be called surfusion.When the aqueous solution is further cooled under its freezing point, excessively cold degree aggravation.When nonintervention, under usual freezing point, be not more than the some place of 15 DEG C, the hydrone in solution is by spontaneous nucleation, and pure water will be condensed into ice.

Be in solid-state transition process from liquid state, solution moves on to low free energy state from high free energy state, thus causes exothermic reaction.The heat produced during this phase transformation makes sample instantaneous heating, and sample temperature raises in this process.Surrounding environment (such as the device of positive freezen protective sample) remains on temperature constant state or continues cooling (depending on the cooling means of use) simultaneously.Then, along with the dissipate heat in sample, the imbalance of the heat during this event between sample and cooling device makes sample experience quick cooldown rate to re-establish heat balance.This quick cooldown rate causes the formation of thin intracellular ice in many cases, and this causes cell death usually.The basic low-temperature biological characteristic of the quality of sample, the heat-transfer character of sample container, the cooling scheme of use and cell is depended in the formation of this thin intracellular ice usually.

Relation between frozen state and biosystem has attracted the mankind's a lot of year.As far back as 1683, Boyle, Robert (RobertBoyle) 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.Artificial induction's freezen protective first 1948 by Polge, Smith and Parkes by chancing on glycerine to poultry and Niu Jingzi and be found for erythrocytic anti-frozen characters subsequently.In nearer period, the scientist be interested in the natural phenomena relevant to freezing biosystem and biomedical applications has begun one's study the basic process of control planning.First, it is known that the temperature declined causes metabolic activity suppressed, and therefore cause the reduction of speed, under the speed of this reduction, the degeneration of apotrophic biosystem will occur.But refrigerating process can not beginning contemplated by people; Its usually cause the chemistry that can expect, heat with the extreme variation of electrical characteristics, to change the iuntercellular interaction system of intracellular organelle, cell membrane and the precision relevant with organ to tissue.Certainly, even if the extreme complexity of the simplest known biological cell, therefore it should be noted that by the seemingly-dead reversible state of freezing gained be completely possible.

Due to the first discovery of the anti-refrigeration of glycerine and the discovery subsequently of widely used infiltration antifreezing agent methyl-sulfoxide (DMSO), many researchers have been devoted to the preservation of the cell or tissue of the method mainly through experience.Most cells suspension freezen protective scheme has established and has used the anti-freezing additive of infiltration of molar concentration can realize freezing survival rate.By using these artificial antifreezing agents, many flexibilities are added to process of cryopreservation.Such as, in order to be issued to best survival rate in the situation of not adding antifreezing agent (CPA), Human erythrocytes needs to be cooled with the speed of 1000 DEG C/about min.But under the sweet oil condition that there is 3.3M (30%), the survival rate of this cell type is in the upper maintenance about 90% of cooldown rate 2-3log scope (2-3logrange).As predictable, CPA concentration is higher, larger in the possibility of adding/removing seepage failure between matter era, and therefore in these processes, needs more concern.

In arbitrary process of cryopreservation, the solution comprised will be excessively cold below its freezing point, until it finds any nucleating point of crystallization.When by freeze-thaw method freezen protective, freezing in extracellular medium is intentionally caused by the seeding being in cold minuent place.If freezing is not caused by seeding, so when solution is by enough low being cooled under its equilibrium freezing point, ice will self-assembling formation.Because this process is actually arbitrary, will occur at random uncertain temperature so freeze; Thus the sample survival rate between the test of repetition utilizing same freezing scheme will be alterable height.And the extreme crystallization fast of height over-cooled solution can cause the damage to biological cells and tissues when causing forming ice.Further, show, if extracellular freezes and to cause when excessively cold height, so have the possibility of the intracellular ice of damageability significantly to increase.This phenomenon is because the freezing cell dehydration caused postpones to start, and this causes ICW to retain increases and the larger possibility therefore caused at intracellular ice.

As mentioned above, between from liquid state to solid-state tour, solution moves on to low free energy state from high free energy state, the thermal unbalance between this cooling device causing the sample of continuation heating and continuation to cool.This imbalance finally causes the severe deviations with the cooldown rate of the regulation of particular cell types, and causes the possibility of cell damage in this process.

In order to the damaged condition preventing these possible occurs, the step in process of cryopreservation often comprises intervention, to introduce ice crystal near solution freezing point in Extracellular solution.Be called that this process of " seeding " is undertaken by being cooled to by sample near solution freezing point, the metal device (such as tweezers or metal bar) being then used in precooling in cryogen (such as liquid nitrogen) contacts the outside of sample container.This seeding step produces ice crystal in Extracellular solution, and provides " model ", and by model, the subcooled water molecule in solution organises and produces more ice.But seeding sample is in the manner in which time-consuming, and when its by temporary transient shift out from the cooling device for this program and inadvertently can cause intracellular ice and sample is in risk because of seeding methods for this reason.

Need the freezen protective system avoiding the above-mentioned problem relevant to unbalance condition.Such system of the auxiliary seeding step also needing the ice crystal without the need to being performed to cause control at present to be formed.Also need the freezing storage device of the solution being convenient to the problems referred to above.Required freezing storage device also can provide and simplify it for obtaining and store the method for the biological cells and tissues wanting freezen protective.

Summary of the invention

These and other needs in freezen protective field can by of the present invention several in meet.In one aspect of the invention, be provided with and automatically become nuclear device, be incorporated in freezen protective holder for before the liquid freezing that comprises in freezen protective holder.Described device comprises the elongated hollow tube that size is suitable for being incorporated in freezen protective holder and the synthetic being arranged on the formation ice-nucleus in hollow tube.The two ends of pipe are sealed, and at least one end is with diaphragm seal, and this film is impermeable and be permeable for the liquid be included in freezen protective holder for the synthetic forming ice-nucleus.Preferably, two ends comprise this film to allow sample liquid inflow device and to pass through device.

In a preferred embodiment, forming the synthetic of ice-nucleus is sterol, and more preferably cholesterol.Cholesterol can be the coating on hollow tube, or can be set to the solid matrix in pipe.

In another aspect of this invention, provide freezen protective holder, its can with automatically to become together with nuclear device to use.In one embodiment, freezen protective holder comprises elastic tubular main body, its have start open for one end liquid sample is incorporated in main body and the closed port limited at the end opposite place of main body.This port is suitable for being pierced through by entry needle with extracted liquid sample.Be introduced into rear open end in holder at liquid sample to be heat sealed.Automatic one-tenth nuclear device and entrance are offsettingly fixed to the inside of tubular body, touch to make its entry needle that can not be pierced closed port.

In another embodiment, freezing storage device comprises for receiving the container with storage liquid sample body, and this container has opening to the inlet fitting in container and the adapter being installed to accessory.Adapter has the first tubular branches and the second tubular branches, and the second tubular branches terminates in pipe strips for joint parts.Diaphragm seals first tubular branches, its septation is suitable for being injected needle-penetration.Freezing storage device is also provided with the pipe of the pipe strips for joint parts at one end joined on the second tubular branches, and is in the closure member at end opposite place of pipe.

First closure member for the second branch road is barrier film, and this barrier film can be injected needle-penetration to be introduced in holder by sample liquid.Container can the pressure being initially located in below atmospheric pressure with promote sample liquid from injector delivery to holder in.Once sample liquid is transferred, then the pipe on the second branch road is heat sealed and is just cut off with the final closure member forming the second branch road on pipe strips for joint parts.Then the device closed experiences freezing reconciliation ice scheme.After thawing, sample liquid extracted out by the barrier film that syringe can be used to pass through in the first branch road of adapter.

Can predict, the present invention will provide a kind of simple reproducible system, for introducing ice and reduce over cold in many different cell freezings application.This invention contemplates method and apparatus, for the induction of the extracellular ice crystal being based upon Cell Cryopreservation and controlling during organizing by solid matrix device, on solid matrix device, ice-nucleus will be formed naturally.

The present invention surpasses existing system and method has multiple advantages.Now, the most methods causing controlled ice-nucleus be troublesome, be difficult to regeneration, although and point to the very large font of the freeze-thaw survival rate of the increase of many biological cells and tissues when operation technique, still to repeatedly check.So far, most of normally used method scope from wipe with the metal object of freezing (being usually refrigerated to-196 DEG C) or cotton contact pipe-type bottles or tubule simply side to being designed for the refining plant spraying liquid nitrogen in the zonule of sample.But, even if perform at optimum conditions, mechanically introducing ice crystal by this way can cause can not forming enough large ice crystal to allow fully to breed in whole Extracellular solution, or the great freezing rate owing to observing in the sample portion of position pointed on container closest to metal object or liquid nitrogen spray, thus cause cell damage and loss.

In one structure, liquid sample device comprises for receiving the container with storage liquid sample body, and container is formed by the elongate body of one.Main body is limited empty internal from openend and is closed to the inlet tube accessory of empty internal and ventilation duct accessory by opening in end opposite.On the one hand, a phosphor bodies also limits and extends in empty internal and the wall be arranged between inlet tube accessory and ventilation duct accessory.The openend of container is by entry needle diaphragm seals.Device also comprises the adapter main body being installed to each pipe fitting, and adapter main body has entrance tubular branches and ventilation tubular branches, and the tubular branch road that ventilates comprises the filter cell be arranged on wherein.

On the other hand, be provided with supporting cover, it has the bottom removably joining container to, and extends beyond the top of inlet tube accessory and ventilation duct accessory at the end opposite place of container when supporting cover joins container to.Container can limit at least two recesses at its outer surface, simultaneously the bottom of supporting cover comprise at least two can the elongated protrusion of elastic deflection, each elongated protrusion has the protuberance being configured to the inside sensing received in corresponding in recess extensiblely.

On the one hand, the top of supporting cover comprises the cylindrical wall surrounding inlet tube accessory and ventilation duct accessory, but also comprises the inwall of the side of the opposite side being 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 its free end protuberance outwardly.Slender leg can be longer than elongated protrusion.

Described device is used to obtain the method for liquid sample for analyzing or for freezen protective, be contemplated that and comprise following beginning step: adapter main body is installed on each pipe fitting, adapter main body has the tubular branch road of entrance corresponding to inlet tube accessory and ventilation duct accessory and the tubular branch road that ventilates, and tubular for entrance branch road is connected to fluid supply.Method proceeds with step below: insert the liquid in container by inlet tube accessory, discharged by air in container by ventilation duct accessory and the tubular branch road that ventilates, the position then in the top supporting cap cuts off and the tubular branch road of sealed entry and the tubular branch road that ventilates simultaneously.

When container is closed completely, support cap and be installed on container.This assembly is installed in centrifuge, this centrifuge operated with centrifugal force by the thing on floating on the surface and fluid separation applications, the thing on floating on the surface directly is exposed to entry needle barrier film.Use the entry needle extending through barrier film, the thing on floating on the surface is extracted out in container.Then, remaining liquid can be frozen preservation, wherein supports cap and is mounted or is not installed on container.

Accompanying drawing explanation

Fig. 1 is the view of automatic one-tenth nuclear device according to an embodiment of the invention;

Fig. 2 is the view of the known freezen protective holder of the automatic one-tenth nuclear device shown in composition graphs 1;

Fig. 3 a is the view of the system pipe-type bottles of the enclosed elastic for freezen protective liquid sample according to another embodiment of the present invention, and pipe-type bottles is expressed as the original state for carrying sample;

Fig. 3 b is the view of the pipe-type bottles shown in Fig. 3 a, is expressed as pipe-type bottles end and is sealed;

Fig. 4 is the perspective view of cell freezing save set according to another embodiment of the invention;

Fig. 5 is the perspective view for the adapter in the device shown in Fig. 4;

Fig. 6 is the exploded view of the device shown in Fig. 4;

Fig. 7 is the perspective view of the cell freezing save set according to another embodiment;

Fig. 8 is the side sectional view at the top of the device shown in Fig. 7;

Fig. 9 is the perspective cut-away schematic view of the bottom of the device shown in Fig. 7;

Figure 10 is the perspective view of the freezing storage device according to 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 of the another feature be disclosed in wherein;

Figure 12 is the view sub-anatomy of the device shown in Figure 11 and supporting cover;

Figure 13 is the enlarged perspective at the top of the device shown in Figure 11-12 and supporting cover;

Figure 14 is the perspective view of the supporting cover according to embodiment;

Figure 15 a and 15b is the schematic diagram of the freezing storage device according to Figure 10 double-duty;

Figure 16 is the basis wherein freezing storage device of Figure 14 of a kind of purposes and the schematic diagram of supporting cover.

Embodiment

In order to promote the understanding to principle of the present invention, referring now to shown in accompanying drawing and subsequently write embodiment described in explanation.Be understandable that intention does not limit the scope of the invention thus.It is further understood that any change that the present invention includes described embodiment and correction, and comprise the application of the principle of the present invention usually can expected as the technical staff for the field that the present invention relates to.

In one embodiment of the invention, provide and automatically become 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 forming ice-nucleus is incorporated into the inner surface 14 of open hollow pipe 12.In a preferred embodiment, pipe is made of plastics.The nucleation synthetic of q.s is introduced into form solid matrix in pipe in pipe, allows liquid flow by pipe simultaneously.

In a preferred embodiment, nucleation synthetic is crystal cholesterol.The use of sterol synthetic, particularly cholesterol are known in other field, such as, at the cooling water system as shown in United States Patent (USP) 4928493.In using these other, pulverous synthetic is placed in a reservoir, for being exposed to water to contribute to the formation of ice.As described below, what can determine after experiment be crystal cholesterol for preparing for the sample cell of freezen protective and liquid (such as blood, stem cell solution and sperm) is nontoxic.

The end 16 of pipe is sealed by the film 18 of permeable solution.Especially, film is permeable for freezen protective liquid, and is impermeable for the cell or tissue that will preserve.Keep being separated and prevent the direct contact between cell/tissue and the synthetic forming ice-nucleus from being important.Film in each end also comprises any cholesterol crystal, and it can be removed from pipe and prevent the liquid around crystal contamination.It is also important in pipe 12 that film allows freezen protective free flow to enter.Pipe and solid matrix nucleation synthetic in space also can be filled with isoosmotic pressure buffer solution at first.

This automatically becomes the size of nuclear device 10 to be designed to be placed in freezen protective holder, such as pipe-type bottles 30 as shown in Figure 2 or blood bag 40.Device can be unfixed or be fixed to inner surface in holder.Because device intention is as the nucleation site being used for ice formation, therefore it need not be large especially.In a particular embodiment, pipe 12 is 0.25 inch long, diameter is 0.0625 inch.Freezen protective holder can be full of specific sample or sample and as at suitable Cryosreservation solution known in the art, keep in a reservoir with timer 10.Then container 30 or 40 is performed freezen protective scheme.Due in device freezen protective liquid with form the synthetic 20 of ice-nucleus and contact, so ice in the pipe 12 of device 10 when a little or spontaneously formed not having cold.Then ice continues to be formed in the solution of surrounding away from pipe, thus cause when seldom or do not have cold and minimal thin intracellular ice to be formed frozen cell suspension.

In a specific embodiment, the first experiment is designed to determine that the cholesterol of the inside being physically attached to freezen protective holder will bring out ice-nucleus.In this embodiment, the sterol solution worked is prepared by being added in 3ml methyl alcohol by the dry cholesterol of 0.025g.The suspension obtained is placed to subsequently in the dry slot of 70 DEG C and also shakes off and on, until all solid-state sterol dissolve.The coated 100 μ l sterol solution of commercially available pipe-type bottles are also placed on to make methyl alcohol evaporate in the dry slot of 75 DEG C, thus realize recrystallization and the adhesion of cholesterol.Then pipe-type bottles rinses 2-3 time to remove any crystal be scattered with 1 milliliter of PBS.

Then, 6% glycerite (to copy typical sperm bank freezen protective medium) and 10% DMSO solution (to copy ordinary cells system refrigeration system) prepare in PBS, and by carrying out cooling assessing with the speed of-5 DEG C/min in the doleiform pipe that applies at sterol and uncoated (control) doleiform pipe.In order to reach statistics rate, 20 comprise DMSO doleiform pipe and 12 doleiform pipes comprising glycerine are evaluated.The temperature in each doleiform pipe is monitored with the interval of 1 second, with the release of the latent heat of the decomposition and thawing that make freezing point solution with thermocouple.

Result of this experiment shows that in DMSO and glycerine freezing point is higher and reduces for the doleiform pipe variations in temperature applied by sterol melting heating (Δ T) period.These results are summed up in the following table:

In this experiment, some can also observing crystal come off or cracked (and in DMSO sample decomposition) to a certain degree, thus cause contaminated aqueous solution (or partly due to the inevitable physical operations of container and solution).In order to address this problem, provide the embodiment automatically becoming nuclear device 10, wherein 0.25 inch of hollow tube is coated with 100 μ l sterol solution and 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 dried 14-24 hour.Support is designed to the cholesterol keeping combining in the environment of isolation, also allows solution (but not allowing cell) to enter to form contact simultaneously.

In the second experiment, human sperm utilizes this automatic one-tenth nuclear device 10 to carry out freezen protective and is analyzed to determine whether have higher post-thaw survival power particularly compared with the sperm that the sample of freezen protective of the present invention is freezing with using the pipe-type bottles of standard construction.In this experiment, obtain human sperm's sample (20 samples from four donors) of abandoning, and be placed in moist 37 DEG C of couveuses (carbonic acid gas of 5%, the air of 95%) and continue 30-60 until be liquefied.Once be liquefied, isobaric PBS (keeping 37 DEG C) is utilized sample to be adjusted to 5ml and utilizes computer assisted sperm analysis device to carry out assessing to measure and record total initial total amount and activity.Then sample is balanced to 6% glycerine by the step-by-step system program of adding up in TEST yolk buffer solution.After balance, each sample is divided into three 1.5ml sample sizes and is deposited into (1) and comprises in the pipe-type bottles of device 10; (2) will in the standard tubular bottle of cloud seeding (energetically control); (3) will not accept in seeding (passive control) standard tubular bottle.

All samples to be placed in the refrigerator of speed control and to be cooled to-8 DEG C with the speed of-5 DEG C/min from 22 DEG C.After 3 minutes at-8 DEG C, the cotton swab be soaked in liquid nitrogen is used to start seeding in the pipe-type bottles of cloud seeding.After in addition 7 minutes at-8 DEG C, sample is cooled with the speed of-10 DEG C/min again to decline-40 DEG C.At-40 DEG C, speed is raised to-20 DEG C/min, is put into liquid nitrogen (LN at-80 DEG C of samples ₂) in.

After freezing, by being placed on sample that testing stand thaws (corresponding to the Thawing Rate of-300 DEG C/min).Once only surplus ice melts, then glycerine is diluted by adding PBS in the mode of instillation within the period of 10 minutes; Then sample is rinsed and is suspended in the PBS not having glycerine.Finally, before total amount and activity are assessed after thawing, sample is cultivated (37 DEG C, moist environment, 5% carbonic acid gas, 95% air) at least one hour.

The result of the second experiment shows to utilize compared with the freezing sample of automatic one-tenth nuclear device of the present invention those samples freezing with utilizing cloud seeding (56.0 ± 3.8%) and keeps the activity (standard errors (mean ± SEM) of 66.1 ± 4.7% means) that obvious (p < 0.05) is higher.As utilized the analysis of variance technique determined, two seeding schemes are high (p < 0.05) sample (43.1 ± 3.7%) of controlling in the passiveness of non-seeding obviously.

In the 3rd experiment, what determine is that the cholesterol combined in long time period can not produce cytotoxinic effect for cultivation sperm.In this experiment, the sperm sample of liquefaction is exposed to the culture plate being coated with 100 μ l sterol solution.Be shown between the culture period of 8 hours at cultivation 1,2,4 and the 8 little activity evaluation forms carried out constantly and directly contact with the cholesterol combined obvious toxic action is not had for human sperm.

Therefore, in sperm cryopreservation process, with use cloud seeding or do not have compared with seeding, automatic one-tenth nuclear device 10 of the present invention be proved to be can produce better thaw after activity.These experiments prove ice-nucleus that sterol cause be stablize, reliable method, it can reduce over the cold and quick rising of relevant temperature after therefore reduce " the flashing " formed at the typical ice crystal of the freezing event of over-cooled solution when having better result.Apparatus and method of the present invention allow sample can not remain in cooling chamber with being upset in whole pool period, therefore do not need the cloud seeding technology of prior art.

Can it is believed that apparatus and method of the present invention be particularly suitable for the sperm stock method of the business of standard.In the sperm bank dis environment of the business of standard, many samples be processed, and time/manpower limits the meticulous process that always can not allow the refrigeration of speed control or can reach in the lab.It is believed that the present invention allows to have the repeatably sample freezen protective of the result surpassing prior art.In addition, the present invention can also realize successfully freezing and have the recovery of sample of low activity, and the sample with low activity is excluded from contributor colony usually.

Similarly, the ability of the candidate stem cell preserved for storing frozen by this way of device 10 of the present invention and method and/or hemopoietic progenitor cell (PCBHPC) has significant impact: enough infectious disease screenings which allows stock and time enough to be used for carrying out and HLA somatotype.Freezen protective is preserve from benefiting from gene therapy or provide chance due to disease or by the PCB source HPC being in the newborn patient lost in normal hematopoiesis function risk that radiotherapy or chemotherapy are caused by medical treatment.Recently, more make great efforts to be devoted to the system of selection of purification CFU-GM.The ability of preserving the progenitor cell (such as representing the cell of CD34 surface glycoprotein) of these relative " pure " may make the cumulative volume of the cell suspending liquid of transplanting minimize.But, because the volume ratio sample of bone marrow of usual obtained PCB is much smaller, every kilogram of limited quantity by the HPC of body weight therefore can be obtained.This make for the effective of PCB source HPC and best freezing and storing method much important compared with the situation of the HPC (such as marrow, peripheral blood) in other source.Automatic one-tenth nuclear device of the present invention therewith time before available device compare and to provide more effectively and preferred for freezen protective and the mode recovering these rapid wear samples.It is believed that device 10 is attached in the research and development of the cord blood stem cell freezing and storing method of ongoing improvement will cause unique scheme, preserve this cell type with when the recovery rate higher at less manpower.Develop sequence scheme for verify PCB and Cord blood and for the ox sperm cryopreservation in business device for artificial insemination in the viability of apparatus and method of the present invention.These schemes are in above referenced provisional application 60/814982, and this is described through with reference to being merged into herein.

Another aspect of the present invention proposes freezen protective various Cord blood source ancestral cells and requires diverse program, and therefore requires to be different from the reservoir vessel existed under existing known program.In order to stock and storage source are from the various kinds of cell type of Cord blood, preferably use comprise different freezing/the very different freezing scheme of heating rate.Prior art uses stored frozen bag (some bags have multiple chamber) or pipe-type bottles.But these two systems have substantial limitations.Multi-chamber bag does not allow different freezing rates maybe will be used in CPA in different chamber.Pipe-type bottles originally can not be considered to " closed " system at freezen protective temperature, and except the thermosealed external packing of non-usage, 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 freezen protective holder of resilient sealing system pipe-type bottles 50, as best shown in figures 3 a and 3b, its make sample between separative element separated thus close system in use different schemes to be frozen.Pipe-type bottles 50 is included in the elastic tubular main body 52 that one end has port 54.Port is preferably by identical with elastic body but be injected needle-penetration sterilely to extract the material seal of sample out after being adapted at thawing.

As shown in Figure 3 a, be open to allow introducing liquid sample when the end opposite 56 of pipe-type bottles starts.Once pipe-type bottles 50 is filled, end 56 is such as closed by heat seal bar 58, as shown in Figure 3 b, so just obtains closed system pipe-type bottles 50 for the freezing of individual unit and storage.Optionally, but preferably, each pipe-type bottles comprises above-mentioned automatic one-tenth nuclear device 10.As shown in Figure 3 a, device 10 is preferably fixed to the inwall of main body 52, enters to make its entry needle that can not be through port 52.

The foreseeable multiple freeze/thaw schemes being the pipe-type bottles 50 of the present embodiment and may be used in the refrigerated container be separated.Therefore, pipe-type bottles 50 in a row can be supported in fixture, and openend 56 can be used for introducing multiple liquid sample sample size.When each pipe-type bottles is filled, respective end is sealed to be provided for the closed system pipe-type bottles of freezen protective.

In another embodiment of the invention, provide freezing storage device 60 as Figure 4-Figure 6, which further simplifies and obtain sample and be ready for use on freezing process.Device comprises the container 62 of size for receiving liquid sample body.Container 62 at one end comprises inlet fitting 64.As shown in Figure 6, nuclear device 10 is automatically become can be introduced in container by inlet fitting 64.

Inlet fitting holds adapter 65, as being shown specifically in Figure 5.Adapter comprises lower tube 66, and its size is suitable for closely being assemblied in inlet fitting 64.Bottom 66 can use the applicable 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 end is connected in end 68, and end 68 is configured to engage entry needle barrier film 72 (Fig. 6).Second branch road 69 end is connected in 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 end 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, barrier film 72,75 be configured to be pierced through in known manner by entry needle and once remove entry needle will self sealss.

In a specific embodiment, piping clip 80 is provided to stablize pipeline 74 when pipeline 74 joins adapter 65 to.Folder 80 comprises the part 80 that is configured to slide on the branch road 67 of adapter with the part 84 of attachment, and the part of attachment is configured to slide on pipeline 74, as shown in Figure 4.

Container 62 size of freezing storage device 60 is set to be accommodated in " egg carton " formula separator of standard, and this separator is for carrying and storing for cell sample that is freezing and that finally thaw.Several this freezing storage devices 60 loaded predictably from the cell sample of common source can be installed in shared egg carton formula separator.During use, device 60 starts to be stored in the structure shown in Fig. 4, and namely pipeline 74 projects upwards from cell container itself.The size of adapter 65 is arranged so that its vertical shell not extending beyond container and therefore can not interferes with other similar device 60 stored.Pipeline 74 is depicted as the turn of bilge having and extend to vertical enclosure.If the device in egg carton formula container is properly arranged, so pipeline 74 can not be interfered with other cell container.But according to preferred embodiment, it can be flexible can be configured to as required avoid other container 62 with same egg carton formula separator to interfere for can predicting pipeline 74.

Pipeline 74 is also because other reason is preferably flexible.Particularly, branch road 69 and the container 62 of fixing pipeline 74 for filling device 60.Therefore, according to the present invention, elastic pipeline 74 can be operated to allow the new cell sample extracted to be incorporated in container.This introducing is pierced through barrier film 75 by the syringe needle of the liquid sample comprising extraction on the one hand and is carried out.Alternatively, barrier film 75 can being removed from the end 74b of elastic pipeline, being injected directly in pipeline without the need to piercing through film to make sample.In both cases, because pipeline can carry out operating as required, container remains in egg carton formula container, so elastic pipeline 74 is conducive to the step of filling containers 62.

Once sample has been introduced in container 62, the branch road 69 of adapter has just been permanently sealed predictably.In preferred embodiments, sealing is by just carrying out at accessory 70 upper seal elastic pipeline jaggy.Once be sealed, owing to no longer needing, the remainder of pipeline just can be removed.In a particular embodiment, the part that known constriction sealing bar may be used for making pipeline flatten simultaneously, heat seal flattens also cuts off unnecessary part.Sealing and cutting are preferably positioned as close to accessory 70 jaggy and carry out making elastic pipeline 74 not have remainder to exceed the vertical shell of container 62.

It is desirable to seal and cutting step can not endanger sterile integrity or the close encapsulation aspect of freezing storage device 60.When injecting sample by barrier film 75, branch road 69 keeps sealing in whole process, even if after removing entry needle.Once branch road 69 is sealed, the device 60 comprising liquid sample just prepares for freezing and be stored in same egg carton formula container, and this egg carton formula container holds this device during filling step.When sample is fetched in hope, device 60 can take out for separating with other device remained on egg carton formula container from egg carton formula container and thaw individually.The entry needle barrier film 72 of branch road 67 provides the approach for aseptic aspiration sample.Therefore syringe needle and syringe can be used to pierce through barrier film and are drawn in syringe by liquid sample.Then empty device 60 can be abandoned.

In an alternate embodiment of the invention, freezing storage device 100 is as Figure 7-9 provided.Device comprises container 102, and it is similar with above-mentioned container 62 in the ability for Cell Cryopreservation suspension, has or do not have automatic one-tenth nuclear device 10 disposed therein.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 seals airtightly by covering 110, as shown in the cross section view of Fig. 8.Top limits annular flange flange 107, and the lower skirt 116 of lid 110 is arranged in annular flange flange 107.This lower skirt is preferably heat sealed in annular flange flange in a conventional manner, to form the hydraulic seal joint resisting freezen protective temperature.

Lid 110 supports entrance branch roads 112 and ventilation branch roads 114, and each in these two branch roads can be the form of sterile pipes.Entrance branch road 112 can be provided with the entry needle barrier film of standard, such as above-mentioned barrier film 75.Two branch roads 112,114 are arranged on corresponding accessory 118,120, and each accessory limits the passage 119,121 be communicated with container 112.Each branch road is sealed on corresponding accessory airtightly to form the joint of the energy freezen protective of hydraulic seal again.

In the embodiment of Fig. 7-9, branch road 114 as ventilation branch road, instead of as the outlet branch road in such as device 60 above.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 μm.Micro-filter 125 is gas-permeables, but is impermeable substantially for the liquid sample be stored in container or cell suspending liquid.

In this embodiment, the suspension that bottom 104 removal that device 100 is arranged through container 102 stores, instead of if embodiment is above by lid removal.Therefore, as shown in the profile of Fig. 9, the opening 105 in container is sealed by entry needle barrier film 130.Go out diaphragma of mouth 130 and be suitably sealed to container 102 airtightly, such as, pass through heat seal.In order to protect barrier film, provide dismountable cover 132, it is fixed on container around barrier film.Cover 132 can limit the tearing part 133 of area format that is thinner or that die down, and it can make the central part 135 of cover be removed.Lappet 135 as shown in Figure 7 can be provided, so that the removal of central part 135.Cover can be paper tinsel film, and it is fixed on 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 container 102 by entrance branch road 112, and is introduced by above-mentioned entry needle barrier film in particular.Alternatively, entrance branch road can be provided with entry needle port or the needleless port of standard.When cell suspending liquid is injected in container, air is wherein discharged by the filter 125 in ventilation branch road 114.In certain embodiments, micro-filter 125 is configured to substantially can not permeate for cell suspending liquid.Therefore, once the level of suspension in container arrives filter 125, so suspension can not pass through filter, thus the rising of pressure will stop in liquid inlet container.

Once container 102 is filled, use such as above-mentioned constriction fusion splicer is carried out heat seal by two branch roads, is sealed completely, airtightly to make container.Certainly, be understandable that, when container 102 is filled, entry needle barrier film 130 and cover 132 still keep complete.To be full of and then the device 100 sealed can be frozen storage.

When cell suspending liquid is extracted in hope, first device and content are thawed in known manner.Once thaw, cover 132 is removed to expose barrier film 130.Ventilation branch road 113 is cut-off to open vent passages 121.Then can pierce through barrier film 130 by syringe or similar device and extract cell suspending liquid.A series of protuberance 106 can be formed on the bottom 104 of container, and it is applicable to engaging 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 (LUERthreads), for engaging the pipe joint (LUERfitting) extracted on syringe.

According to another embodiment, freezen protective holder 200 comprises the container or pipe-type bottles 201 that can be marked with scale, as shown in Figure 10.The lower end 302 that holder 200 has opening and the upper end 205 closed.As seen from Figure 12, container 201 is the main bodys of the one substantially forming empty internal 202 from the lower end 203 of opening.Upper end 205 place closed, the main body of one is configured to be formed the pair of pipes adapter 207,208 reached in empty internal 202.The opening that two pipe adapters reach in inside is separated by wall 210.As in the previous embodiments, adapter 207 can be connected to exhaust outlet, and adapter 208 can be connected to the fluid supply that will be frozen and be kept in holder 200.Wall 210 prevents the liquid entered by adapter 208 to be drawn out of from adapter 207 immediately, particularly when applying negative pressure to adapter 207 as described here.

The openend 203 of container 201 is closed by entry needle barrier film 215, and entry needle barrier film 215 can be configured to be similar to above-mentioned entry needle barrier film 130 and play similar effect.Entry needle barrier film 215 is kept by cover 217, and cover 217 is sealed to the openend 203 of container by conventional methods, seals to provide anti-dew.

Holder 200 comprises the adapter main body 220 of ventilation duct 221 and inlet tube 223 further.The end of pipe is installed on corresponding accessory 207,208 in a suitable manner to provide the joint of permanent anti-dew sealing.Ventilation duct 221 can preferably include the filter cell 225 be contained in pipe, and it is similar to above-mentioned filter 125.Inlet tube 223 can comprise expansion end 226, for engaging with pipe adapter, as described here.

Further describe as such, holder 200 is similar to holder 100.But container 201 is modified to hold supporting cover 240, as figs 11-13.Supporting cover 240 comprises bottom 242 and top 244, and upper and lower is all roughly cylindrical structure and size is applicable to sliding fit on container 201.Bottom 242 comprises at least two protuberances 246 with the protuberance 247 inwardly pointed to.Protuberance 247 size is made and is arranged to be locked in and is formed at corresponding recess 250 interior (Figure 10) on container 201.Protuberance 247 inwardly points to so that make must by deflecting the outside mobile protuberance of protuberance 246 when lid 240 slides on container.Protuberance comprises the upper surface 247a of inclination, and when lid is removed in hope, the upper surface 247a of inclination makes protuberance be pulled out from recess 250 smoothly.Protuberance 247 is arranged on the middle part of protuberance 246, extends for manual engagement to discharge protuberance from recess below protuberance to make the bottom 246a of protuberance.Bottom 246a provides leverage to be conducive to discharging protuberance, and what also prevent protuberance from causing due to unexpected contact shifts out simultaneously.

The top 244 of lid 240 is configured to protect the pipe 221 and 223 extended from container top.Therefore, top 244 comprises annular wall 252, and it has the inwall 254 being arranged to the side being positioned at pipe side, as shown in figure 13.Therefore, manage with the adapter 207 and 208 of holder 200 in conjunction with time, wall prevents pipe bending, which avoid and destroys fluid-tight sealing and pollution content.

The lid 260 of amendment as shown in figure 14.This lid is configured to support freezen protective holder 200 particularly in centrifuge or other utensil.In some instances, wish the content with centrifuge separated storage device 200, such as, from the thing isolated cell grain floating on the surface.Container 201 may not easily be installed in centrifuge, so the support that the lid 260 of amendment is extra for container provides.Particularly, cover 260 and comprise bottom 262 and top 264.The top 244 that top 264 can be similar to lid 240 is constructed and comprises inwall 265 with protective conduit.

In addition, be similar to lid 240, the bottom 262 of lid 260 comprises at least two protuberances 266, and protuberance 266 has the protuberance 268 being configured to the recess 250 engaged in container 201.But bottom 262 comprises supporting leg 270, supporting leg 270 extends beyond the end of protuberance 266.And these supporting leg 270 ends are connected to protuberance 272 outwardly.Therefore supporting leg 270 extends from recess 250 along the major part of the length of container 201.Outside protuberance 272 is configured to the recess or the installing component that engage utensil (such as above-mentioned centrifuge).Longer supporting leg 270 is not only container and provides support, supporting leg and specifically protuberance 272 a kind of mechanism is also provided, for engaging utensil releasedly, thus support freezen protective holder 200 fully in utensil.

The use of holder 200 is described in Figure 15 a and 15b.In using at two kinds, container is connected to fluid supply, such as blood bag 280.Blood bag comprises pipe 282, and pipe 282 is connected to the entrance branch road 223 of adapter main body 220.In one embodiment, adapter 284 can be engaged with between the expansion end 226 of entrance branch road and blood bag pipe 282.In a kind of scheme shown in Figure 15 a, blood is by gravity from being input in container 201, and air is overflowed by ventilation branch road 221.In another kind of scheme, as illustrated in fig. 15b, whole system is closed, and syringe 290 is connected to ventilation branch road 221.Syringe is opened to apply negative pressure to container 201, thus is extracted in container from bag 280 by blood.

Once want the blood of freezen protective or other liquid to be pumped in container, pipeline is just cut off and seals, as shown in figure 16.The end 295 of the pipe of sealing is present in when being cut off to join on container 201 when cover below the end on top 264 of lid 260.Therefore, as shown in figure 16, the end of pipe is fully surrounded and is protected.The protuberance 272 of lid 260 can be utilized to be arranged in centrifuge or other storage device by holder 200.Can understand further, lid 260 as support with support tool have upwards towards the holder of entry needle barrier film 215.When the content centrifuge of container is separated, the thing on floating on the surface will move up naturally, and in this position, it is readily accessible by the entry needle through barrier film and extracts out.Once the thing on floating on the surface is removed, just can remove lid 260 from container 201, then content just can be frozen preservation.

Will be further appreciated that the process described in Figure 15 a-15b can use when not having freezen protective subsequently.Such as, current blood testing according to the blood stockpile quantity of association of blood bank of the U.S. (AABB) standard requires the destruction defining detected blood unit, because blood sample amount is moved into the possibility not polluting whole unit in inspection instrument by current having no idea effectively.This requirement causes the destruction of the about centesimal blood unit processed by all blood banks, and this is Be very effective in cost and leiphemia.Inspection instrument must be allowed for the centrifugal action of cell granulation, keeps closed system to avoid polluting simultaneously.

Holder 200 meets AABB standard, with the remainder making blood bank or doctor directly can extract blood sample and not entail dangers to blood unit from blood unit.Once blood sample is by from blood bag 280 suction container 201, blood bag pipe 282 can be sealed and be departed from from adapter 284, protects the blood unit in blood bag 280 thus.Then the blood sample in container 201 can be installed in lid 260, to be transported to laboratory and to be separated with centrifuge.Entry needle barrier film 215 allows sterilely to extract the thing on floating on the surface for further analysis, such as, determine cell number, or detects the existence of haemoglobin or microorganism in blood sample.

Can predict, in certain embodiments, the container 62,102 and 201 of freezen protective holder 60,100 and 200 can be provided with initial vacuum.This vacuum contributes in the extraction of filling liquid sample in reservoir receptacle step.Sealed (by corresponding barrier film 72,75 or pass through heat seal) owing to leading to each branch road 67/69,112/114 and 221/223, therefore vacuum can be kept a very long time.Lid can be arranged on each branch road gas-tight seal to determine.In a particular embodiment, in container 62,100 and 201, initial vacuum can be in the pressure under the atmospheric pressure between 100mmHg (definitely) and 160mmHg (definitely).

Freezen protective holder 60,100 and 200 can be made up of the standard material using in blood stockpile quantity and long-term preservation field under standard freezing conditions (i.e. the low temperature to-196 DEG C).Current O.S.H.A. recommends to be that the plastic components making to be applicable to can reach the pathogens standard or other requirement that meet the carrying of specific blood.Therefore, the container in the various embodiments described above, accessory and pipeline can be made up of the plastics being applicable to, such as polystyrene or polypropylene.In order in the egg carton formula container of the standard of being installed to, holder (after sealed elastic pipeline) should meet within 10mm diameter and 90mm height.Elastic pipeline 74 also must can resist freezen protective temperature, and heated sealant the ability of cutting out pipeline when can not damage the branch road 69 when adapter seal 65.In a particular embodiment, elastic pipeline by or similar material is made.

Although illustrate and describe the present invention in accompanying drawing and description above, it is considered that it is characteristically schematic instead of restriction.Be understandable that and only propose preferred embodiment, it is desirable to protect the institute falling into purport of the present invention to change, revise and further apply.

Claims (26)

1., for receiving the device of the liquid sample obtained from independent container, comprising:

For the container of receiving liquid sample body, described container is formed by the elongate body of one, described main body limits empty internal from openend, and closed by inlet tube accessory and ventilation duct accessory at end opposite place, described inlet tube accessory limits ingress path, ingress path has the inlet opens to described empty internal, described ventilation duct accessory defines ventilating path, ventilating path has the ventilation orifice to described empty internal, the main body of described one also limits to extend in described empty internal and exceedes described inlet opens and described ventilation orifice and the wall be arranged between described inlet tube accessory and described ventilation duct accessory, with

Close 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 entrance and the tubular branch road that ventilates, and the tubular branch road of described ventilation comprises filter cell disposed therein.

3. the device of claim 2, the tubular branch road of wherein said entrance comprises the expansion end being suitable for receiving adapter, described branch road to be connected to the independent pipe be associated with liquid container.

4. the device of claim 1, wherein said container is made up of the plastics being suitable for storing blood sample.

5. the device of claim 1, wherein said container is made up of the plastics being suitable for storage liquid sample body at freezen protective temperature.

6., for storing the device of the liquid sample obtained from independent container, comprising:

For the container of receiving liquid sample body, described container has the empty internal from openend, and is closed to the inlet tube accessory of described empty internal and ventilation duct accessory by opening at end opposite place;

Close the entry needle barrier film of described openend; With

Supporting cover, it has the bottom removably joining described container to, and extends beyond the top of described inlet tube accessory and described ventilation duct accessory at the end opposite place of described container when supporting cover joins container to.

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 has the protuberance inwardly pointed to, and the protuberance inwardly pointed to be configured to be received in described recess in corresponding one extensiblely.

8. the device of claim 7, wherein said protuberance is positioned at the center of the length direction along described protuberance.

9. the device of claim 6, wherein said top comprises the cylindrical wall around described inlet tube accessory and described ventilation duct accessory, also comprises the inwall of the side of the opposition side being 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, and each of described supporting leg comprises the protuberance being outwardly positioned at its free end.

The device of 11. claims 10, wherein, described slender leg is longer than described elongated protrusion.

The device of 12. claims 6, wherein, described container is made up of the plastics being suitable for storing blood sample.

The device of 13. claims 6, wherein said container is made up of the plastics being suitable for storage liquid sample body at freezen protective temperature.

The method of the device of 14. use claims 6, comprising:

Described inlet tube accessory is connected to fluid supply;

Insert the liquid in described container by described inlet tube accessory, discharged the air in described container simultaneously by described ventilation duct accessory;

Seal described inlet tube accessory and described ventilation duct accessory;

Described supporting cover is installed on described container;

The described supporting cover of the described container of carrying is placed in centrifuge;

With centrifuge from the thing fluid separation applications is floated on the surface, 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, in container, extract the thing floating on the surface.

The method of 15. claims 14, wherein liquid is introduced in container from input by gravity.

The method of 16. claims 14, also comprises:

Syringe is connected to described ventilation duct accessory; And

Use syringe to produce negative pressure in described container, thus by liquid from fluid supply suction container.

The method of 17. claims 14, also comprises:

Adapter main body is installed to the beginning step on described pipe fitting, described adapter main body has the tubular branch road of entrance corresponding to described inlet tube accessory and described ventilation duct accessory and the tubular branch road that ventilates;

Connection Step, comprises and tubular for entrance branch road is connected to fluid supply; And

Sealing step, is included in and is in position in the top of supporting cover and cuts off and the tubular branch road of sealed entry and the tubular branch road of ventilation.

The method of 18. claims 14, wherein said liquid is blood, and described fluid supply is blood unit bag.

19. 1 kinds of fluid sample devices, comprising:

Container, there is the empty internal from openend, and closed to the inlet tube accessory of described empty internal and ventilation duct accessory by opening at end opposite place, wherein said ventilation duct accessory has at the first opening of described empty internal and second opening contrary with described first opening;

Adapter main body, comprise the ventilation duct being installed to described ventilation duct accessory, the inlet tube being installed to described inlet tube accessory, and gas by aseptic micro-filter, described gas by aseptic micro-filter be received in described ventilation duct instead of in described ventilation duct accessory, described micro-filter is received in described ventilation duct at the second overthe openings of described ventilation duct accessory, and described inlet tube is configured to receive fluid sample; With

Close the entry needle barrier film of described openend.

20. devices as claimed in claim 19, also comprise the cell suspending liquid be contained in the empty internal of container.

21. devices as claimed in claim 20, wherein cell suspending liquid comprises freezen protective medium.

22. devices as claimed in claim 21, wherein cell suspending liquid is in frozen state.

23. devices according to any one of claim 19-22, also comprise the removable lid of protection entry needle barrier film.

24. devices according to any one of claim 19-22, wherein ventilation duct is made up of heat-sealable pipeline.

25. devices according to any one of claim 19-22, wherein inlet tube is made up of heat-sealable pipeline.

26. devices according to any one of claim 19-22, it can resist the low temperature of-196 DEG C low.