Abstract:
Successful cryopreservation by the vitrification method depends on high chilling speed. Practitioners of vitrification prefer to use liquid nitrogen as the chilling cryogen due to its inherent safety and low cost. Plunging vitrification cryocontainers in to a quiescent pool of liquid nitrogen invariably results in a chilling rate less than the theoretical potential. The shortfall is attributed to the well-known Leidenfrost effect. The purpose of this invention it to provide improve chilling rates during vitrification using liquid nitrogen. One feature of this invention is a contacting device that invokes convective heat transfer principles to increase chilling speed. In another feature of this invention, cryogen velocity is derived from a self-pressurized dewar containing a saturated cryogen. The self-pressurization is achieved by ambient heating of the dewar&#39;s contents. In another embodiment, a sub-cooled cryogen, such as propane, is used in tandem with a saturated cryogen, such as LN2, in a self-pressurized dewar.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. provisional patent application entitled “Rapid Chilling Device for Vitrification”, Ser. No. 61/021,661 filed on Jan. 17, 2008. Said provisional application is incorporated herein by reference. 
     
    
     TECHNICAL FIELD 
       [0002]    This invention is in the field of devices for the cryopreservation of biological specimens. 
       BACKGROUND 
       [0003]    Cryopreservation is practiced in the life sciences for the purpose of halting biological activity in valuable cell(s) for an extended period of time. One factor in the success of cryopreservation is reducing or eliminating the deleterious effect of ice crystal formation. Sophisticated methods are needed to thwart the natural tendency of water to freeze into ice during cryopreservation. “Vitrification” is such a method. Vitrification can be described as a rapid increase in fluid viscosity upon fast chilling that traps water molecules in a random orientation. Its success is predicated on avoiding the formation of cell-damaging ice altogether. 
       Vitrification 
       [0004]    The initial step in vitrification is to dehydrate a cell or cells with an aqueous solution (“vitrification media”) containing permeating and/or non-permeating cryoprotectants (“CPA”). The cell or cells, together with a small quantity of vitrification media, comprise a “biological specimen.” The biological specimen is then placed in a suitable cryocontainer. A cryocontainer is a container that is suitable for use at cryogenic temperatures. As used herein, “cryogenic temperatures” means temperatures colder than −80° C. 
         [0005]    The biological specimen is then rapidly chilled by immersion in a cryogenic fluid, such as liquid nitrogen (“LN2”). With a proper combination of chilling speed and CPA concentration, intracellular water will attain a solid, innocuous, glassy (vitreous) state rather than an orderly, damaging, crystalline ice state. 
         [0006]    Clinical vitrification has two primary goals. The first is long term storage in a cryogen such as LN2. The second is recovery of a biologically viable cell or cells after warming. Vitrification media, however, are often toxic to cells when the cells are warm. The time exposure of cells to vitrification media during dehydration and warming therefore (not “thawing” since ice is not formed) must be carefully controlled to avoid cellular injury. 
         [0007]    Slow chilling speeds require high, relatively toxic concentrations of vitrification media, such as 60% w/w CPA concentration. At fast chilling speeds, lower, less toxic concentrations can be used. If chilling speeds of 10 6 ° C./minute or greater were attainable, vitrification could be achieved with no cryoprotectants at all. 
         [0008]    It is desirable to chill quickly; the faster the better. Directly plunging a biological specimen into LN2 achieves rapid chilling, but may expose biological specimens to contamination. Commercially available liquid nitrogen may contain bacterial and fungal species which are viable upon warming. Furthermore, it has been reported that vitrified cells can be infected by viral pathogens placed in the LN2. 
         [0009]    The potential of infection has led to the development of closed cryocontainers where the biological specimen is placed in a container and sealed before chilling in LN2. The cryocontainer also serves as a storage device to isolate the biological specimen from pathogen-containing cryogen during long-term storage. 
         [0010]    One of the limitations to achieving the fastest possible chilling speed with either direct plunge open cryocontainers or closed cryocontainers is that contact of the initially warm vitrification device with LN2 will result in the generation of an insulating nitrogen vapor coating around said device. The vapor barrier results from the boiling of the liquid nitrogen when it contacts the warm sample. This is known as the Leidenfrost effect. Nitrogen vapor is a thermal insulator which significantly diminishes chilling speed. If the Leidenfrost effect were reduced or eliminated, chilling speeds would be faster and less toxic vitrification media could be used which would potentially lead to better clinical outcomes. 
         [0011]    There is a need therefore, for a method and apparatus to increase the cooling rate of biological specimens during vitrification by overcoming the Leidenfrost effect. 
       SUMMARY OF THE INVENTION 
       [0012]    The Summary of the Invention is provided as a guide to understanding the invention. It does not necessarily describe the most generic embodiment of the invention or all species of the invention disclosed herein. 
       Improved Vitrification Chilling Apparatus and Method 
       [0013]    The inventions described herein comprise chilling devices and methods that eliminate or reduce the Leidenfrost effect in vitrification. In the case where the chilling cryogen is a saturated liquid (e.g. LN2), high cryogen velocity is used to forcefully displace boiled-off vapors. In the case where the cryogen is a subcooled liquid chilled by a refrigerant, a simple means is described for controlling the temperature of the cryogen without freezing it. 
         [0014]    In one embodiment of this invention, a saturated cryogen is held in a closed dewar whose exterior is exposed to room temperature. Ambient warming pressurizes the dewar and this pressure forces the saturated cryogen out of the dewar as a fluid jet. This jet is directed onto the vitrification device and forcefully displaces the evolved vapors to prevent formation of the Leidenfrost effect. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0015]      FIG. 1  illustrates two prior art vitrification devices. The first is a closed device and the second is an open carrier. 
           [0016]      FIG. 2  illustrates an inventive closed vitrification device. 
           [0017]      FIG. 3  illustrates the Leidenfrost effect on vitrification devices and means to mitigate it. 
           [0018]      FIG. 4  schematically shows how pressure can be converted into cryogen velocity. 
           [0019]      FIG. 5  illustrates two types of open contacting devices. 
           [0020]      FIG. 6  illustrates a vessel-type contacting device. 
           [0021]      FIG. 7  illustrates the features of an inventive dewar containing a saturated cryogen. 
           [0022]      FIG. 8  illustrates the relationship between vapor pressure and temperature for nitrogen. 
           [0023]      FIG. 9A  illustrates the features of a closed contactor. 
           [0024]      FIG. 9B  illustrates a clamp for use with the closed contactor. 
           [0025]      FIG. 10  illustrates how a contactor can be used to vitrify a biological specimen in an open cryocontainer. 
           [0026]      FIG. 11  illustrates how a contactor with a converging/diverging nozzle can be used to vitrify a biological specimen in a closed cryocontainer. 
           [0027]      FIG. 12  illustrates how a contactor that exhibits the vena contracta effect can be used to vitrify a biological specimen in a closed cryocontainer. 
           [0028]      FIG. 13  illustrates a method to vitrify a specimen in a cryocontainer using a contactor and a self-pressurizing dewar of liquid nitrogen. 
           [0029]      FIG. 14  illustrates the features of a dewar having two reservoirs. The first reservoir is for a saturated cryogen that pressurizes the dewar. The second is for a subcooled cryogen that will be used to vitrify a biological specimen. 
       
    
    
     DETAILED DESCRIPTION 
       [0030]    The following detailed description discloses various embodiments and features of the invention. These embodiments and features are meant to be exemplary and not limiting. 
         [0031]    As used herein, except for temperature and unless specifically indicated otherwise, the term “about” means within +/−20% of a given value for a parameter. For temperature, “about” means +/−2° C. of a given value. 
         [0032]    A variety of biological cells can be aseptically cryopreserved (vitrified) using the present invention. One category of cells is mammalian developmental cells such as sperm, oocytes, embryos, morulae, blastocysts, and other early embryonic cells. These cells may be cryopreserved during assisted reproduction procedures. Another category is stem cells that are used in regenerative therapies. The broadest category is any cell or group of cells that can be vitrified with the available chilling speeds of this invention. 
       Vitrification Devices 
       [0033]    Cryocontainers used as vitrification devices span a wide range of designs which can benefit from this invention. Exemplary cryocontainers are described below. 
         [0034]      FIG. 1  shows longitudinal sections of generally tubular elements of an exemplary cryocontainer  100  comprising a cryocontainer tube  102 . This device uses aspiration vacuum to draw a biological specimen into the cryocontainer tube. Both ends of the cryocontainer tube are initially open. A syringe (not shown) is attached to first opening  104  of the cryocontainer tube. The syringe creates a vacuum that draws biological specimen  106  into the second opening  108  of the cryocontainer tube. A biological specimen comprises vitrification media  110  and one or more cells  112 . Referring to item  120 , both ends of the cryocontainer tube are then heated and crimped to create aseptic seals  122  and  124 . The cryocontainer containing biological specimen  126  is now prepared for chilling. 
         [0035]    Item  140  in  FIG. 1  illustrates an open carrier for vitrification. It comprises a handle  142  attached to a shaft  144 . The shaft is attached to a loading cantilever  146 . A biological specimen  148  is placed upon the cantilever. 
         [0036]      FIG. 2  illustrates longitudinal sections of generally tubular elements of an exemplary cryocontainer with deformable walls. Said walls may comprise a shape memory material. This cryocontainer is more fully described in copending US patent application “Shape-Shifting Vitrification Device”, Ser. No. 12/267,708. Said application and all continuations in part thereof, are incorporated herein by reference. 
         [0037]    The cryocontainer comprises a shuttle  200  and sheath  220 . The shuttle comprises a tube  202  with a notch  204  cut in the end to provide a channel  206 . A biological specimen  208  is placed on the channel. 
         [0038]    The sheath comprises a tubular body  222  with a first end  224  heat-sealed. A second end  226  is open. The portion of the sheath corresponding to the position of the biological specimen is deformable. Thus after the shuttle is loaded into the sheath, the deformable section may be crimped such that it touches the biological specimen. This increasing the heat transfer rate to said biological specimen. If the deformable wall is a shape memory material, it may return to its uncrimped shape when the cryocontainer is warmed. This facilitates removal of the biological specimen. 
         [0039]    Item  240  shows the shuttle and sheath assembled and sealed  242 , but prior to crimping. 
         [0040]    Item  260  shows a cross section of the cryocontainer at section A-A immediately after loading and sealing. The biological specimen  262  is surrounded by air  264 . 
         [0041]    Item  280  shows the same cross section after crimping. A significant portion  282  of the deformable wall thus contacts the biological specimen. Thus the rate of heat transfer to the biological specimen will be increased during both cooling and warming. 
       Heat Transfer 
       [0042]      FIG. 3  illustrates how a cryocontainer reacts to being plunged by conventional means into a bath of LN2. 
         [0043]    Item  300  shows the dynamics of the heat transfer in the portion of the cryocontainer  302  that contains the biological specimen  306 . Plunging is performed into a quiescent pool  304  of LN2. The cryocontainer is within 20° C. of room temperature before the plunge. The contact of the LN2 with the relatively very hot cryocontainer surface  308  causes the LN2 to initially vaporize and form a vapor cloud  310  (stagnant gas) surrounding the cryocontainer. Subsequent heat transfer is from the cryocontainer&#39;s vapor coated surface  312  through the stagnant vapor to the opposite surface  314  having contact with the cryogen. The stagnant gas is nitrogen vapor and is a thermal insulator leading to low heat conduction rates. This is the so-called Leidenfrost effect. The chilling rate of the biological specimen is controlled by the rate that heat can be conducted across the stagnant gas layer. 
         [0044]    If the cryocontainer&#39;s outside diameter is D, then we can estimate the heat transfer zone&#39;s length  316  to be wider than the footprint  318  of the biological specimen by about 2D. The rapid chilling methods described herein are preferably applied to the entire heat transfer zone. 
         [0045]    According to the present invention, the Leidenfrost effect can be reduced if the cryogen is caused to flow at a high velocity over the cryocontainer. The cryogen velocity can be oriented in either direction along the principal axis of the cryocontainer,  320  and  322 . Alternatively, the cryogen can be urged to flow transversely  324  across the cryocontainer or a suitable combination of transverse and longitudinal flow. 
         [0046]    Item  340  illustrates the chilling of a vitrification device  342  using flowing cryogen  344  (“inbound stream”). Item  346  depicts a control volume within the flowing cryogen having a length equal to the heat transfer zone  348 . We can assume, due to the jetting characteristics of the cryogen flow, that there is no heat or mass transfer across surface  350  of the control volume. The warm cryocontainer heats the flowing cryogen  352  within the control volume. Therefore, all the heat from the cryocontainer leaves the control volume in cryogen flow  354  (“outbound stream”). The cryogen flow across cryocontainer surface  356  induces convective heat transfer. This mode of heat transfer is superior to conduction through a stagnant gas layer. All the heat released by the cryocontainer leaves the control volume in the outbound stream. 
         [0047]    This invention contemplates inbound streams having velocities that will renew the contents of the control volume hundreds of times during the time it takes to vitrify. Boiled-off vapors will be displaced out of the control volume by the momentum and shear forces arising from this velocity. A Reynolds number of at least 1,000 and more is suitable such that the flow is turbulent. 
       Cryogen Velocity 
       [0048]      FIG. 4  illustrates a method for accelerating a cryogen to the required velocity. A container  402  with a cryogen  404  is at a pressure  406  above the pressure  408  of the surroundings  410 . Leading from this container is a tube  412  with an exit  414  that empties into the surroundings. The pressure in the container urges the cryogen to flow through the tube and exit into the surroundings at a velocity  416 . The fluid velocity at the exit is dependent on the pressure in the container and the frictional losses in the tube. 
         [0049]    If the cryogen is a saturated liquid, such as LN2, some of it may flash into vapor as it passes through the tube. This can be minimized by insulating  418  the tube, keeping its length  424  short, minimizing frictional losses due to fluid motion, and using low thermal conductivity materials, such as plastics. The system is effective nonetheless even if a significant portion of the LN2 is in vapor form as it passes over the cryogenic container containing the biological sample. 
         [0050]    Another method to accelerate a cryogen is to contact a high velocity gaseous non-condensing stream (e.g. helium) with the cryogen. This contact urges the gaseous stream and cryogen to form a moving entrainment stream. 
         [0051]    The container with a saturated cryogen will create a high velocity of cryogen in exchange for the propensity to flash into vapor. The pressure of the container, therefore, can be adjusted through experimentation to give an optimum value for heat transfer for a given geometry of cryocontainer such that the velocity is high without undue flashing. 
       Cryogen Contact Using an Open Contactor Apparatus 
       [0052]      FIG. 5  illustrates open contactor apparati for contacting a high velocity cryogenic fluid with a cryocontainer. 
         [0053]    Item  500  depicts a cryocontainer  502  in proximity to a cryogen velocity source  504 . A cryogen velocity source is an apparatus that can urge a cryogen to flow in a desired direction. An example would be a pressurized container with an outlet pipe. The cryogen stream  506  is in the form of a jet with a preferred diameter of 5-10 times the heat transfer zone. It has a more preferred diameter of 1.5-3 times the heat transfer zone. The cryogen stream is directed onto the heat transfer zone to chill it and achieve vitrification of its contents. The cryogen stream can be a gas, subcooled liquid, saturated liquid or mixtures thereof. There may be a multitude of cryogen streams to insure complete coverage of the cryocontainer&#39;s periphery. An example would be two opposing streams  506  and  508 . After vitrifying, the cryocontainer is placed in a bath of cryogen for long-term storage. 
         [0054]    Item  520  depicts a cryocontainer  522  canted at an angle. Suitable angles are in the range of 2° to 45° from the horizontal. Surrounding the distal tip of the cryocontainer is a three-sided enclosure  524  that forms a channel  526  (View A-A). Suitable widths and heights of the channel are 1.5 to 10 times the diameter of the cryocontainer. Liquid cryogen from velocity source  528  forms a stream of cryogen  530  that impacts the cryocontainer at or near the heat transfer zone. The flow rate of the cryogen stream is sufficient to form a channel flow  532  (View A-A) that completely immerses the cryocontainer, chills it, and vitrifies its contents. After vitrifying, the cryocontainer is placed in a bath of cryogen for long-term storage. 
         [0055]      FIG. 6  depicts an open vessel  600  containing a liquid cryogen  602  that may be either a subcooled or saturated liquid. Attached to the vessel is an agitation system comprising a motor  604  that is connected to a shaft  606  that is connected to an agitator  608 . The spinning agitator urges the contents of the vessel to flow in a manner depicted by arrows  610 . The nature of the flow pattern formed may be different with an alternative agitation system. An external recirculation loop driven by a pump is a suitable alternative. A magnetic stirring system is also suitable. The key element is fluid velocity within the confines of the vessel. Before the plunge, the cryocontainer  612  is oriented with the heat transfer zone close to the cryogen&#39;s surface. The cryocontainer is then plunged into the cryogen to a depth  614  that immerses the heat transfer zone. Exposure to the cryogen vitrifies the contents of the cryocontainer. If the cryogen is a saturated liquid, the cryogen&#39;s velocity in the open vessel minimizes the Leidenfrost effect. 
         [0056]    When vitrifying using an open cryocontainer to hold the biological specimen, the cryogen&#39;s velocity must be limited. This is to prevent displacing the biological specimen from the carrier and into the cryogen. 
       Saturated Cryogen Dewar 
       [0057]      FIG. 7  illustrates the use of a pressurized container to generate a high velocity stream of cryogenic fluid. One way to pressurize a container of a saturated cryogen (such as LN2) is to place the cryogen in a sealed container, such as a closed dewar  700 , and allow it to absorb ambient heat. The ambient heat then evaporates a portion of the cryogenic fluid thus raising the head space pressure. 
         [0058]    The dewar comprises top assembly  702  and bottle  704 . The top assembly comprises a release valve  706 , a bleed valve  708 , an outlet pipe  710 , and a pressure relief valve  712 . The bottle comprises a chamber  714  containing LN2  716  and head space  718  containing nitrogen vapor. The wall  720  of the bottle is a vacuum insulated double wall. The vacuum thermally insulates the contents of the bottle. The exterior of the bottle may be insulated  722  to protect the hands of the user from cold temperatures. Connected to the release valve is a dip tube  724  that extends into the bottle to draw out LN2. The top assembly is joined to the bottle by a threaded connection  726 . 
         [0059]    To use the dewar, the assemblies are first separated. A quantity of LN2 is poured into the bottle to form a level  728 . The top assembly is then screwed onto the bottle. 
         [0060]    The pressure relief valve has two settings: “purge” and “pressure”. In the purge setting, the pressure relief valve is opened and the head space pressure is the same as the ambient pressure  730 . In the pressure setting, the pressure relief is set and the head space then pressurizes as ambient heat is absorbed by the LN2 and the LN2 vaporizes. The head space pressure  732  will rise until the set point of the pressure relief valve is reached. The relief valve will then open to release excess pressure  734  thus maintaining the LN2 at a constant pressure. Well insulated, hand-held dewars can maintain their setpoint pressure for a day by minimizing the amount of LN2 that boils off. 
         [0061]    To dispense  736  LN2 from the dewar, the release valve  706  is opened to allow the head space pressure to urge the LN2  738  up the dip tube and out the outlet pipe. 
         [0062]    An important characteristic of the flowing LN2 stream is the relative proportion of liquid and vapor nitrogen. It is desirable to have a high proportion of liquid nitrogen. To achieve this, the design of the dewar should utilize plastics or similar materials having low thermal conductivity and/or heat capacity for the dip pipe and outlet pipe so that a minimal amount of LN2 is boiled in chilling said pipes. The length  740  of the outlet pipe should also be kept at a minimum to limit this parasitic heating. The entire fluid pathway from the bottle to the surroundings should minimize frictional losses due to fluid motion. The total liquid content of the exit stream may be 80% by volume or greater. 
         [0063]    To reduce parasitic heating, the bleed valve  708  can be left open between vitrification sessions. A small flow of cold gaseous nitrogen will then flow through and chill the outlet pipe. This flow also prevents backwash of ambient air into the outlet pipe. Air may contain moisture that can freeze on cold surfaces and potentially cause a blockage. 
         [0064]    For some embodiments, it may be preferred to dispense gaseous nitrogen through the outlet pipe. This is easily achieved by utilizing a shorter dip pipe that draws from the head space rather than the LN2. 
         [0065]    About 40 grams of LN2 might be consumed for the vitrification of a typical biological specimen held in a typical cryocontainer. Bleed nitrogen consumption to keep the exit pipe chilled and purged of ambient air might be 25 grams per hour. A typical laboratory vitrifying a plurality of specimens might require two devices so that one can be recharged with LN2 while the other is in use. 
         [0066]      FIG. 8  illustrates the relationship between vapor pressure and temperature for LN2. If the pressure relief value of the cryogenic container of  FIG. 7  is set to vent at 1 bar absolute, the corresponding temperature  802  is the normal boiling point of LN2, −196° C. If the pressure relief valve is set to 1.6 bar, the LN2 in the bottle will warm  804  to −192° C. Higher temperatures  806 ,  808  can be achieved with higher pressure settings. Higher temperatures might be required for applications where a second cryogen, such as propane or octafluropropane is held in the same dewar (see below). The higher temperature is necessary so that the second cryogen won&#39;t freeze. 
       Cryogen Contact Using a Closed Contactor Apparatus 
       [0067]      FIG. 9A  illustrates a sectional view of a closed contactor apparatus  900  comprising a cylindrical body  902 , a cryogen entrance  904 , a cryocontainer opening  906  and an exhaust  908 . The entrance is adapted to receive a flow of cryogenic fluid. The opening is adapted to admit said cryocontainer into said contactor. The body is adapted to direct said cryogenic fluid to said cryocontainer when said cryocontainer is loaded through said opening. The exhaust is adapted to direct said cryogenic fluid away from said cryocontainer when said cryocontainer is loaded through said opening. 
         [0068]    The contactor&#39;s overall length  910  is about 6 cm. The length  912  of the exhaust is similarly about 6 cm. The cylindrical body comprises an axis  914 . The ID  916  of the cylindrical body is about 1.7 cm. Suitable IDs of the cylindrical body can range from 1.5 to 10 times the diameter of the cryocontainer. Suitable diameters of cryocontainers can range from 100 microns to 2.5 mm. If a cryocontainer had a 2 mm diameter, for example, then a suitable ID of the cylindrical body would be 3 mm. 
         [0069]    The cylindrical body further comprises a bushing  918  and a sleeve  920 . The axis  922  of the sleeve may be coincident with the axis of the cylindrical body or it may be offset or at an angle thereto. The clearance between the ID of the sleeve and the cryocontainer should be kept to a minimum. However, the clearance should be sufficient to allow easy ingress and egress of the cryocontainer. 
         [0070]    The cross section of the cylindrical body may be circular, square or other appropriate shape. If the cross section of the cylindrical body is different than a circle, then the “ID” of the body is the minimum distance across its cross section. Similarly, if the opening for the cryocontainer is noncircular, then the “ID” of the opening refers to the minimum distance across its cross section. 
         [0071]    Materials to fabricate these parts should be suitable for cryogenic temperatures. Components that are in contact with the cryogen upstream of the heat transfer zone of the cryocontainer may have low thermal conductivity and low heat capacity. Plastics have these attributes. The sleeve needs to be flexible to allow clamping around the vitrification device. A suitable material for the sleeve is polytetrafluroethylene. 
         [0072]    Referring to  FIG. 9B , Clamp  940  comprises a body  942  having a jaw  944  that is held shut by spring  946 . Moving handles  948  and  950  together opens up the jaw. 
         [0073]    Referring back to  FIG. 9A , the body of the contactor is attached to a source of flowing cryogen (not shown) at the cryogen entrance. Arrows  924  indicate the general flow of cryogen through the contactor. The diameter of the cryogen supply tube attached to the cylindrical body can be significantly larger than the diameter of said cylindrical body. It would be attached to the cylindrical body by a reducing union. This would minimize the pressure drop and hence flashing (if the cryogen is saturated) from the cryogenic supply reservoir to the closed contactor apparatus. 
         [0074]    In order to vitrify, the end of a cryogenic container is inserted through the sleeve. The container is inserted until the heat transfer zone is located where it will be immersed within the general flow of the cryogen. Once in place, the open jaw of the clamp is placed over the sleeve at  926 . The handles of the clamp are released which allows the jaw to squeeze the sleeve to engage the cryogenic container. The purpose of clamping the cryogenic container to the sleeve is to keep the cryocontainer from being pushed out of the sleeve when the cryogen flows. The clamped sleeve, however, does not need to be vapor or liquid tight. Other suitable clamps may also be used. 
         [0075]    Once the cryogenic container is in place, the cryogen flow is initiated and the biological specimen is vitrified. The cryogen can be a gas, subcooled liquid, saturated liquid or a combination thereof. After vitrification, the clamp is released and the cryocontainer is placed in long term cryogenic storage. 
         [0076]      FIGS. 10-12  illustrate different embodiments of the closed contactor design. In all cases, the cryogen entrance is attached to a cryogen source that can dispense either a gas, subcooled liquid, or a saturated liquid. Also, different vitrification devices can be engaged as described above in the illustrations of the embodiments. 
         [0077]      FIG. 10  illustrates a contactor  1000  loaded with an open vitrification device  1002 . Due to the delicate nature of an exposed biological specimen  1010 , the contactor may also be comprised of a restricting orifice  1004  at the cryogen entrance. It may also be preferred to have the cryogen source to be a gas. Cryogen dispensed  1006  traverses the contactor as shown by the arrows  1008 . The flow of cryogen contacts the open carrier and vitrifies the biological specimen. 
         [0078]      FIG. 11  illustrates a contactor  1100  loaded with a closed vitrification cryocontainer. A converging/diverging nozzle  1102  is designed to shape  1104  the flow of the incoming cryogen to a very narrow annular stream in the proximity  1106  of the heat transfer zone. Smooth transitions  1108  are needed on both sides of the heat transfer zone to preserve the pressure for conversion to velocity. The flow of cryogen  1110  vitrifies the biological specimen  1112 . The gap between the vitrification cryocontainer and the nozzle may be 0.2 to 1.5 mm. The upstream pressure may be 0.2 to 3 bar above ambient. The pressure  1114  in the exhaust is ambient. Therefore, virtually all of the pressure drop occurs in the nozzle. Cryogen velocity in the heat transfer zone may be 0.5 to 25 meters per second (m/s). This velocity is sufficient to prevent the formation of the Leidenfrost effect. 
         [0079]    The axis of the contactor body may be about coincident with the axis of the opening for the cryocontainer such that when the cryocontainer inserted through said opening, the heat transfer zone can be located in the throat of the converging diverging nozzle. 
         [0080]      FIG. 12  shows a contactor  1200  loaded with a closed vitrification cryocontainer  1202 . A flow orifice  1204  is designed to shape  1206  the flow of the incoming cryogen to a downstream vena contracts  1208  in the proximity of the heat transfer zone. The flow of cryogen  1210  vitrifies the biological specimen  1212 . 
         [0081]      FIG. 13  illustrates a contactor  1302  attached  1306  to a dewar  1304  containing LN2. Prior to vitrification, cold gaseous nitrogen gas from a bleed valve  1308  travels through an outlet pipe  1310 . This chills the contactor to limit parasitic heating. This flow also prevents ambient air that may contain moisture, from entering the contactor at the exhaust  1312  or cryocontainer opening  1314 . 
         [0082]    During vitrification, a cryocontainer is placed through the opening and the LN2 is turned on by opening release valve  1316 . The LN2 velocity  1318  in the outlet tube is relatively slow as compared to its velocity  1320  in the heat transfer zone. This is highly desirable as it reduces the pressure drop from the dewar to the contactor. The pressure in the head space of the dewar is set high enough such that the velocity of the LN2 in the heat transfer zone is at least 0.5 m/s. 
       Two Chamber Dewar 
       [0083]      FIG. 14  illustrates how a saturated cryogen dewar can be used to dispense a subcooled cryogen. An exemplary subcooled cryogen is propane which has a boiling point of −42° C. and a freezing point of −188° C. If propane is cooled to just above its freezing point, it can be used as a flowing cryogen with minimal potential for Leidenfrost effect. 
         [0084]    Item  1400  is a double-chambered dewar wherein the first chamber  1402  serves as a reservoir for LN2. The second chamber  1404  serves as a reservoir for subcooled propane. The headspaces of the two reservoirs are in physical communication with each other such that the pressures therein are about equal. The first and second reservoirs are also in thermal communication with each other such that their temperatures are about equal. 
         [0085]    The pressure relief valve  1406  has a set point that raises the head space pressure such that the LN2 temperature is just above the freezing point of propane. Two bar absolute is a suitable pressure (item  806 ,  FIG. 8 ). 
         [0086]    To load the second chamber, a source of propane gas  1408  is attached at the outlet  1410  of the dewar. A bleed valve  1412  is opened which allows the propane gas to enter the second chamber through a vapor dip pipe  1414 . Heavier gaseous propane will displace the gaseous nitrogen and condense into a liquid  1416  and accumulate in the second chamber. An external scale can be used to control the loading of the propane. When the filling is complete, the propane source is disconnected. 
         [0087]    The vapor dip pipe now communicates with gaseous nitrogen in the head space so the bleed valve reverts its function back to providing bleed nitrogen gas. 
         [0088]    To dispense liquid propane, head space pressure  1418  urges the liquid propane  1420  to move through a dip pipe  1422  and hence though a release valve  1424  and into the outlet tube  1426 . A contactor can be attached to the dewar at the outlet to enable vitrification with the liquid propane. 
         [0089]    Propane is a flammable substance. A suitable non-flammable cryogen is octafluropropane (R218) having a boiling point of −37° C. and a freezing point of −183° C. Octafluropropane requires a head space pressure of 3.5 bar absolute to prevent freezing of the cryogen (item  808 ,  FIG. 8 ). 
       CONCLUSION 
       [0090]    While the disclosure has been described with reference to one or more different exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the disclosure. In addition, many modifications may be made to adapt to a particular situation without departing from the essential scope or teachings thereof. Therefore, it is intended that the disclosure not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention.