Abstract:
The present application relates to devices for the vitrification of biological samples, including embryos, oocytes and biological tissues. Suitably, the devices are automated devices which require little if any operator intervention or sample handling, thereby reducing contamination, improving success rates and increasing efficiency. Also provided are methods of vitrifying a biological sample, suitably using the disclosed devices.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The present application relates to devices for the vitrification of biological samples, including embryos, oocytes and biological tissues. Suitably, the devices are automated devices which require little if any operator intervention or sample handling, thereby reducing contamination, improving success rates and increasing efficiency. Also provided are methods of vitrifying a biological sample, suitably using the disclosed devices. 
         [0003]    2. Background of the Invention 
         [0004]    Freezing and preservation of biological samples had traditionally been carried out using a slow-freezing process. Those methods were accomplished by tabletop machines that slowly cooled a sample over the period of several hours to cryogenic temperatures. These “slow freezing methods” have recently being supplanted by sample “vitrification,” which allows for rapid freezing and storing of specimens in a matter of minutes instead of hours. 
         [0005]    However, current vitrification devices require significant operation by a skilled lab technician. Success rates of vitrification and warming viable samples vary widely depending on the method used and the skill of the lab technician performing the procedure. Such methods traditionally require a lab technician to prepare individual samples for freezing, handle each separately and carry out specified methods. These methods, however, are not only time-consuming and inefficient, but can result in variations in sample vitrification due to differences in technician training and technique. This makes it extremely difficult to evaluate and compare various protocols. In addition, many manual vitrification methods expose the specimen directly to liquid nitrogen (i.e., open system), which provides rapid cooling (vitrification), but can lead to introduction of contaminants (fungi, pathogens etc.) to the specimen, as well as sample loss and damage. 
         [0006]    The inventors have identified a need for protocols and devices that can be used to repeatedly and efficiently vitrify biological samples, including for use in fertility preservation and tissue banking. 
       SUMMARY OF PREFERRED EMBODIMENTS 
       [0007]    The present application provides automated vitrification devices, as well as methods of vitrification that meet the needs identified above. 
         [0008]    In embodiments, automated vitrification devices are provided. Suitably, such devices comprise a cryo-protectant holder, a cryo-protectant dispenser, a sample holder oriented to allow a sample in the sample holder to be contacted with cryo-protectant from the cryo-protectant dispenser, a sample sealing device, a coolant holder oriented to allow a sealed sample to be placed in a coolant in the coolant holder, and a control module operably connected to the cryo-protectant dispenser, the sample holder and the sample sealing device. 
         [0009]    Suitably, the cryo-protectant dispenser is operably connected to a driving mechanism that controls the movement of the cryo-protectant dispenser. In embodiments, the various components of the devices are housed within a chamber, the chamber comprising at least one opening for introduction of the sample to the sample holder. 
         [0010]    In embodiments, the sample holder is a rotating sample holder that moves the sample from a position that allows introduction of the sample, to a position that allows introduction of the cryo-protectant from the cryo-protectant dispenser. Suitably, the rotating sample holder further moves the sample from the position that allows introduction of the cryo-protectant to a position that allows sealing of the sample by the sample sealing device. 
         [0011]    In embodiments, the devices further comprise a drying mechanism for removing excess cryo-protectant from the sample. Suitably, the sample sealing device comprises a sealing mechanism and a cutting mechanism for first sealing the sample, then cutting the sealed sample, and then releasing the sealed sample from the sample holder into the coolant holder. Suitably, the coolant in the coolant holder is liquid nitrogen. 
         [0012]    In embodiments, the sealing mechanism comprises an adhesive film and the cutting mechanism comprises a first die and a second that align together. 
         [0013]    In further embodiments, automated vitrification devices are provided that comprise a cryo-protectant holder, a cryo-protectant dispenser, a sample holder oriented to allow a sample in the sample holder to be contacted with cryo-protectant from the cryo-protectant dispenser, a drying mechanism for removing excess cryo-protectant from the sample, a sample sealing device comprising a sealing mechanism and a cutting mechanism for first sealing the sample, then cutting the sealed sample. The devices also comprise a coolant holder oriented to allow the sealed sample to be placed in a coolant in the coolant holder and a control module operably connected to the cryo-protectant dispenser, the sample holder and the sample sealing device. Suitably, the various device components are housed within a chamber, the chamber comprising at least one opening for introduction of the sample to the sample holder. 
         [0014]    Also provided are containers for a biological sample, comprising a support member having a top surface and a bottom surface, a sample retention area (e.g., a substantially circular hole) traversing the support member from the top surface to the bottom surface and a porous mesh positioned in the sample retention area. Suitably, the sample retention area allows for introduction of the biological sample onto the porous mesh and removal of a fluid from the porous mesh. Suitably, the sample retention area comprises an extension surrounding said hole, extending at least above said top surface of said support member. In embodiments, the containers further comprise a frame supporting the container. 
         [0015]    Also provided are methods of vitrifying a biological sample. Such methods suitably comprise placing a biological sample in a sample holder, placing a cryo-protectant in a cryo-protectant holder, dispensing the cryo-protectant from the cryo-protectant holder onto the sample, removing excess cryo-protectant from the sample via a drying mechanism, sealing the sample, cutting the sealed sample, and transferring the sealed sample into a coolant in a coolant holder. 
         [0016]    Suitably, the dispensing, removing, sealing, cutting and transferring are automated via a control module. 
         [0017]    Further embodiments, features, and advantages of the embodiments, as well as the structure and operation of the various embodiments, are described in detail below with reference to accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0018]      FIG. 1  shows an exemplary automated vitrification device as described herein. 
           [0019]      FIG. 2  shows a cryo-protectant holder and sample holder of an exemplary automated vitrification device as described herein. 
           [0020]      FIG. 3  shows a cryo-protectant dispenser, a cryo-protectant holder and sample holder of an exemplary automated vitrification device as described herein. 
           [0021]      FIG. 4  shows a drying mechanism of an exemplary automated vitrification device as described herein. 
           [0022]      FIG. 5  shows a cryo-protectant holder, sample holder and sample sealing device of an exemplary automated vitrification device as described herein. 
           [0023]      FIG. 6  shows and alternative view of a cryo-protectant holder, sample holder and sample sealing device of an exemplary automated vitrification device as described herein. 
           [0024]      FIG. 7  shows a sealed sample of an exemplary automated vitrification device as described herein. 
           [0025]      FIG. 8  shows a sealed sample and a coolant holder of an exemplary automated vitrification device as described herein. 
           [0026]      FIG. 9A  shows an alternative view of an exemplary automated vitrification device as described herein. 
           [0027]      FIG. 9B  shows an additional exemplary automated vitrification device as described herein. 
           [0028]      FIG. 9C  shows an alternative view of an exemplary automated vitrification device as described herein. 
           [0029]      FIG. 10  shows an exemplary sample holder/container for a biological sample as described herein. 
           [0030]      FIG. 11A  shows an additional exemplary sample holder/container for a biological sample as described herein. 
           [0031]      FIG. 11B  shows an additional exemplary sample holder/container for a biological sample as described herein. 
           [0032]      FIG. 12A  shows an exemplary control system for use with an automated vitrification device as described herein. 
           [0033]      FIG. 12B  shows an additional exemplary control system for use with an automated vitrification device as described herein. 
       
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0034]    It should be appreciated that the particular implementations shown and described herein are examples and are not intended to otherwise limit the scope of the application in any way. 
         [0035]    The published patents, patent applications, websites, company names, and scientific literature referred to herein are hereby incorporated by reference in their entireties to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter. 
         [0036]    As used in this specification, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%. 
         [0037]    Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present application pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of ordinary skill in the art. 
         [0038]    In embodiments, an automated vitrification device  100 , for example as shown in  FIG. 1 , is provided. 
         [0039]    As used herein “automated” refers to a device that operates, i.e., proceeds step-by-step through various mechanical movements and actions, with little or no intervention or control from a human operator. In general, the automated devices described herein are controlled via computer or other suitable control module. 
         [0040]    The term “vitrification” as used herein refers to the solidification of a liquid by an extreme elevation of viscosity as a result of the rapid cooling of a sample below about −110° C., suitably below about −150° C., to preserve the sample and allow it to be stored for extended periods, e.g., days, weeks, months, years, etc. 
         [0041]    As shown in  FIG. 1 , automated vitrification device  100  suitably comprises cryo-protectant holder  102 . In embodiments, cryo-protectant holder  102  comprises a base  204  that contains one or more reservoirs  206  suitable for receiving and holding a cryo-protectant. Base  204  suitably sits on a rotating platform  202 , which rotates about rotation point  210 . As used herein, “cryo-protectant” or “cryoprotectant” refers to a composition (suitably a fluid solution) that is used to protect biological samples from freezing damage due to ice crystal formation. Exemplary cryo-protectant solutions are known in the art, and include for example, solutions comprising ethylene glycol, dimethyl sulfoxide, propane-1,2-diol (PrOH), etc., and mixtures thereof. Suitably, base  204  comprises multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) reservoirs  206  that can be used to hold different cryo-protectants. As described herein, it is often necessary during a vitrification procedure to contact a sample with more than one cryo-protectant (i.e., cryo-protectants having different compositions). 
         [0042]    Cryo-protectant solutions for use in the devices and methods described herein suitably comprise ethylene glycol (EG), dimethyl sulfoxide (DMSO), sucrose and/or trehalose, and serum substitute supplement (SSS™) in modified human tubal fluid (mHTF) (suitably from Irvine Scientific, Santa Ana, Calif.). An exemplary cryo-protectant solution suitably comprises about 2% to about 30% EG, about 2% to about 20% DMSO, about 0.05 to about 1.0M sucrose and/or about 0.05 to about 1.0M trehalose, and about 5% to about 30% SSS™ in mHTF. More suitably, exemplary cryo-protectants comprise about 5% to about 20% EG, about 5% to about 10% DMSO, about 0.1 to about 0.5M sucrose and/or about 0.1 to about 0.5M trehalose, and about 15% to about 25% SSS in mHTF. 
         [0043]    Exemplary cryo-protectant solutions comprise 1) about 7.5% EG, about 7.5% DMSO, about 0.1M sucrose and about 20% SSS in mHTF; 2) about 10% EG, about 10% DMSO, about 0.3M sucrose and about 20% SSS in mHTF; or 3) about 20% EG, about 10% DMSO, about 0.5M sucrose, about 0.5M trehalose, and about 20% SSS in mHTF. In embodiments, such cryo-protectant solutions are suitably used for vitrification of oocytes. 
         [0044]    Additional exemplary cryo-protectant solutions comprise 1) about 7.5% EG, about 7.5% DMSO, about 0.1M sucrose, and about 20% SSS in mHTF; 2) about 10% EG, about 10% DMSO, about 0.3M sucrose, about 20% SSS in mHTF; or 3) about 20% EG, about 10% DMSO, about 0.5M sucrose, about 0.5M trehalose and about 20% SSS in mHTF. In embodiments, such cryo-protectant solutions are suitably used for vitrification of blastocysts. 
         [0045]    As used herein, “biological material” and “biological sample” are used interchangeably and include material obtained from a human, plant or animal source, and includes for example, tissues, blood, cells, bone, oocytes, embryos, sperm, eggs, ovarian tissue, gamete, gonadal tissue, testicular tissue, etc. 
         [0046]    Vitrification device  100  also suitably comprises cryoprotectant dispenser  104  as shown in  FIG. 1 . Cryo-protectant dispenser  104  is suitably oriented above cryo-protectant holder  102 , as shown in  FIG. 3 , however in other embodiments, cryo-protectant dispenser  104  can be located below cryo-protectant holder  102 , or can be in any other suitable orientation. 
         [0047]    Cryo-protectant dispenser  104  suitably is operably connected to driving mechanism  302  that controls its movement in any suitable direction, (i.e., in any x, y or z direction, or combination of these directions) so as to remove (e.g., suction) cryo-protectant from cryo-protectant holder  102  via nozzles  304  or other suitable mechanism. 
         [0048]    Automated vitrification device  100  also comprises sample holder  208 , shown in  FIG. 2  and  FIG. 3 . Sample holder  208  is suitably oriented so as to allow a sample in the sample holder to be contacted with one or more cryo-protectants from cryo-protectant dispenser  104 . For example, cryo-protectant that is removed from reservoirs  206  via nozzles  304  can be administered/delivered to the sample in sample holder  208 . Cryo-protectant dispenser  104  is able to control the amount of cryo-protectant administered to the sample and can administer multiple cryo-protectants to a single sample in any order, at any time, for any duration and in any amount desired by the operator via a control module as described herein. 
         [0049]    For example, in embodiments, cryo-protectant dispenser administers cryo-protectants to a biological sample in the amounts and for the durations indicated below. Drying mechanism  106  is suitably used to remove excess cryo-protectant at the desired times/intervals prior to application of the next cryo-protectant. 
         [0050]    Described below is an exemplary cryo-protectant protocol for vitrification of oocytes:
       A solution comprising about 7.5% EG+about 7.5% DMSO+about 0.1M sucrose+about 20% SSS in mHTF is applied to the biological sample for 5 minute;   Excess solution is removed;   A solution comprising about 10% EG+about 10% DMSO+about 0.3M sucrose+about 20% SSS in mHTF is applied to the biological sample for about 1 minute;   Excess solution is removed;   A solution comprising about 20% EG+about 10% DMSO+about 0.5M sucrose+about 0.5M trehalose+about 20% SSS in mHTF is applied to the biological sample for about 40 seconds. This 40 second time suitably also includes sealing the sample prior to vitrification, as described herein.       
 
         [0056]    Described below is an exemplary cryo-protectant protocol for vitrification of blastocysts:
       A solution comprising about 7.5% EG+about 7.5% DMSO+about 0.1M sucrose+about 20% SSS in mHTF is applied to the biological sample for about 10 minutes;   Excess solution is removed;   A solution comprising about 10% EG+about 10% DMSO+about 0.3M sucrose+about 20% SSS in mHTF is applied to the biological sample for about 1 minute;   Excess solution is removed;   A solution comprising about 20% EG+about 10% DMSO+about 0.5M sucrose+about 0.5M trehalose+about 20% SSS in mHTF is applied to the biological sample for about 60 seconds. This 60 second time suitably also includes sealing the sample prior to vitrification, as described herein.       
 
         [0062]    As shown in  FIGS. 1 ,  5  and  6 , automated vitrification device  100  suitably further comprises sample sealing device  110 / 108 / 112 , having upper  110  and lower  112  sections. In embodiments, as shown in  FIGS. 5 and 6 , sample sealing device  110 / 108 / 112  suitably comprises a sealing mechanism  108  and a cutting mechanism ( 602  and  604 ). 
         [0063]    Automated vitrification device  100  suitably also comprises coolant holder  114 , as shown in  FIG. 8 . Suitably, coolant holder  114  is oriented to allow a scaled sample  702  to be placed in a coolant in the coolant holder. 
         [0064]    As discussed herein, the automated vitrification devices described throughout are suitably under the control of a control module, suitably a computer or similar device, which is connected to one or more of the various components of the vitrification device so as to allow for automation of a vitrification process carried out by the device. Suitably, a control module (not shown) is operably connected to cryoprotectant dispenser  104 , sample holder  208  and sample sealing device  110 / 108 / 112 . In additional embodiments, a control module can be further connected to drying mechanism  106 , cryo-protectant holder  102 , as well as any other components. 
         [0065]      FIG. 12A  shows an exemplary control system  1200  for use with the automated vitrification devices described herein. In exemplary embodiments, control system  1200  comprises a National Instruments USB D/A Board  1202  running a custom program  1204  written in LabView. As shown in  FIG. 12A , the D/A Board suitably controls three servos, including drying mechanism  106  servo  1206 , cryo-protectant dispenser  104  servo  1208 , and cryo-protectant dispenser suction (not shown) servo  1210  using pulse width modulation. The D/A Board is also connected stepper motor controller  1212  using a serial connection. Stepper motor controller  1212  suitably controls four stepper motors; cryo-protectant holder  102  rotation motor  1214 , Sample Sealing Device  110 / 108 / 112  Motor  1216 , Cryo-Protectant Dispenser  104  Positioning Motor  1218 , and Sealant Tape Advancement (not shown) Motor  1220 .  FIG. 12B  shows an additional exemplary control system  1200 ′ for use with the automated vitrification devices described herein. Exemplary control system  1200 ′ comprises components, including motors/servos, analogous to those disclosed in control system  1200 . 
         [0066]    In exemplary embodiments, the various components of automated vitrification device  100 , as described herein, are housed within chamber  116 , as shown in  FIGS. 1 ,  9 A,  9 B and  9 C. In embodiments, chamber  116  comprises at least one opening (not shown) that allows for introduction of a sample to sample holder  208 . Suitably, chamber  116  is a metal enclosure that contains the various device components described herein. 
         [0067]    As described herein, sample holder  208  is suitably a rotating sample holder attached to base  202  and connected to rotation point  210 , which moves the sample from a position that allows introduction of the sample, to a position that allows introduction of the cryo-protectant from cryo-protectant dispenser  104 . For example, as shown in  FIG. 1 , a sample is introduced into sample holder  208  in a first position. Sample holder  208  is then rotated to a position below cryo-protectant dispenser  104 , in which cryo-protectant dispenser  104  is also able to remove cryo-protectant from reservoirs  206  of cryo-protectant holder  102  (see e.g.,  FIGS. 1 ,  2  and  3 ) and then administer the cryo-protectant to the samples. 
         [0068]    Suitably, cryo-protectant dispenser  104  utilizes a stepper motor to drive two ACME screws that move the main exposure carriage (e.g.,  302 ). The dispenser suitably uses a six-bar linkage driven by a linear solenoid to retrieve cryo-protectant from the reservoirs and place them on the samples. Each nozzle (suitably 2, 3, 4, 5, 6, 7, 8, 9 10, etc. nozzles) is independently controlled by its own solenoid, which draws the cryo-protectant into the nozzles and pushes them out using a suitable device, such as a syringe. 
         [0069]    The rotating sample holder is further able to move the sample from the position where the cryo-protectant is introduced, to a position that allows sealing of the sample by sample sealing device  110 / 108 / 112 , as in  FIG. 5 . As shown in  FIGS. 5 and 6 , sample sealing device suitably comprises sealing mechanism  108 . In exemplary embodiments, sealing mechanism  108  comprises an adhesive or other suitable film  504 , for example an adhesive tape. As shown in  FIG. 5 , in embodiments, sealing mechanism  504  is wound over arms  502  that create an envelope or pocket into which sample holder  208  is inserted, as shown in  FIG. 5 . Sealing mechanism  504  in the form of adhesive tape can suitably be stored on storage roll  506  (or multiple storage rolls as shown in  FIG. 9B ) prior to use in sample sealing device  110 / 108 / 112 . As shown in  FIG. 5 , sample holder  208  is rotated into position with sealing mechanism  504  via a rotation platform about rotation point  210 . 
         [0070]    Sample sealing device  110 / 108 / 112  suitably comprises a cutting mechanism for first sealing the sample ( FIG. 5 ), then cutting the sealed sample ( FIG. 6 ), and then releasing the sealed sample  702  from the sample holder into the coolant holder  114 . 
         [0071]    In exemplary embodiments, cutting mechanism comprises a first die  602  (i.e., an upper die  602  on upper portion  110  of sealing device  110 / 108 / 112 ) and a second die  604  (i.e., a lower die on lower portion  112  of sealing device  110 / 108 / 112 ) that align together, as shown in  FIG. 6 . It should be understood that orientation of upper and lower die are for illustration purposes, and any suitable orientation can be used. In addition, other mechanisms to cut and seal sample  208  can also be used. As shown in  FIG. 6 , first die  602  and second die  604  are brought into contact so as to seal and cut the sample via collapsing arms  512 . A person of ordinary skill in the art will readily be able to envision other suitable mechanisms for sealing and cutting sample  208 . 
         [0072]    Sealing sample utilizing a sealing mechanism such as an adhesive film or tape provides a mechanism by which the sample can be vitrified, but without actually directly contacting the sample with liquid nitrogen, thereby reducing contamination and sample loss or damage. 
         [0073]    Suitably, a stepper motor is used to drive the movement of the sealing device  110 / 108 / 112 , and a slot-and-linkage arrangement is used to control the movement. The assembly has mating upper  602  and lower  604  dies that press the sealing mechanism, e.g., sealant tape, onto containers  208 , cut the tape, punch out the containers from their handling frame and tilt to put the containers into coolant, as described herein. 
         [0074]    The sealing tape is placed in the machine on a roll  506  (or multiple rolls as shown in  FIG. 9B ) and is tensioned by a spring-loaded roller and arm  502  (or multiple arms as shown in  FIG. 9B ). The tape attaches to a roller, which is driven by a stepper motor to pull the sealing tape into an envelope-type arrangement. The specimens are then rotated into the envelope where they are sealed, punched out and vitrified. 
         [0075]      FIG. 7  shows a sealed sample  702 , comprising sealing mechanism  504  covering and securing sample  208 , as well as individual separated support members  209  of the sample holder  208 . The lower portion  112  of sealing device  110 / 108 / 112  suitably comprises raised portions  510  on platform  508  of second die  604 , see  FIGS. 5 and 7 . 
         [0076]    As lower die  604  is lowered, it contacts rod  704 , which causes the die to tilt, thereby releasing the sealed sample  702  into the coolant holder  114 . Suitably, coolant holder  114  comprises individual cryo-vials  802 , each individually containing a coolant, suitable for receiving the sealed samples. In exemplary embodiments, the coolant is liquid nitrogen. Other suitable coolants include, for example, liquid helium, etc. 
         [0077]    As shown in  FIGS. 1 and 4 , the automated vitrification device  100  suitably further comprises a drying mechanism  106 , for removing excess cryo-protectant from the sample. Drying mechanism  106  suitably comprises multiple (i.e., 2 or more, 3, 4, 5, 6, 7, 8, 9, 10 or more) individual drying elements  402  (suitably a cloth or cotton swab or other suitable adsorbent material) positioned in a base  404 . As shown in  FIG. 1 , following the addition of cryo-protectant to the sample  208  with cryo-protectant dispenser  104 , the sample is rotated about rotation point  210 , so that the samples are directly above drying elements  402 . It should be noted that this orientation is for illustrative purposes only, and in other embodiments, the drying mechanism  106  can be positioned above the sample holder, or any other suitable orientation can be used. 
         [0078]    Drying mechanism  106  is used to remove excess cryo-protectant (surplus cryo-protectant that is not necessary for the vitrification as well as cryo-protectant that may interfere with additional cryoprotectants) prior to introduction of a subsequent cryo-protectant to the sample. As described herein, vitrification methods suitably utilize multiple different cryo-protectants, each or which requires contact with a sample for a particular amount of time and in a particular order. 
         [0079]      FIG. 9A  shows an additional view of an automated vitrification device as described herein, showing suitable orientations of the various components described throughout.  FIG. 9B  shows an additional automated vitrification device as described herein, showing suitable orientations of the various components described throughout.  FIG. 9C  shows a photograph of an assembled automated vitrification device as described herein. 
         [0080]    Exemplary materials for use in the various components of the automated vitrification devices described herein are well known in the art and include for example, various metals, plastics, polymers, ceramics and glasses. Methods for mechanically connecting the various components of the devices herein are also well known and readily implemented by one of ordinary skill in the art. 
         [0081]    In additional embodiments, automated vitrification device  100  as shown in  FIGS. 1 ,  FIGS. 9A ,  9 B and  9 C, is provided. The device suitably comprises cryo-protectant holder  102 , cryo-protectant dispenser  104  (suitably also comprising suction assembly  902  to provide suction for cryo-protectant dispenser  104 , though the suction assembly can also be integrated into dispenser  104 ) and sample holder  208  oriented to allow a sample in the sample holder to be contacted with cryo-protectant from the cryo-protectant dispenser  104 . The device suitably further comprises drying mechanism  106  for removing excess cryo-protectant from the sample, sample sealing device  110 / 108 / 112  comprising sealing mechanism  504  and a cutting mechanism  602 / 604  for first sealing the sample, then cutting the sealed sample  702 . The device also comprises coolant holder  114  oriented to allow sealed sample  702  to be placed in a coolant in the coolant holder. Suitably, a control module is operably connected to cryo-protectant dispenser  104 , sample holder  208  and sample sealing device  110 / 108 / 112 . In embodiments, the various components of the device shown in  FIG. 1 ,  FIGS. 9A ,  9 B and  9 C, are housed within chamber  116 , the chamber comprising at least one opening for introduction of the sample to the sample holder  208 . 
         [0082]    As described herein, suitably cryo-protectant dispenser  104  is operably connected to a driving mechanism that controls the movement of cryo-protectant dispenser. Suitably, sample holder  208  is a rotating sample holder that moves about rotation point  210 , so that the sample rotates or moves from a position that allows introduction of the sample, to a position that allows introduction of one or more cryo-protectants from cryo-protectant dispenser  104 . Suitably, the rotating sample holder further moves the sample about rotation point  210  from the position that allows introduction of cryo-protectant to a position that allows sealing of the sample by sample sealing device  110 / 108 / 112 . As described herein, suitably the sealing mechanism  504  comprises an adhesive film, and a cutting mechanism comprising first die  602  and second die  604  that align together so as to seal the sample and then cut the scaled sample. 
         [0083]    Suitably, sealed sample  702  is released from the sample holder into coolant holder  114 , suitably a coolant holder containing liquid nitrogen, for example, in one or more cryo-vials  802 . 
         [0084]    In further embodiments, containers for a biological sample are provided. The terms “containers for a biological sample,” “sample holder” and “sample container” are used interchangeably throughout to refer to a device suitable for receiving a sample and allowing that sample to be manipulated for vitrification, e.g., allows for the introduction and removal of cryo-protectants, allows for sample sealing and vitrification, and allows for ultimate storage. 
         [0085]    As shown in  FIG. 10 , an exemplary container for a biological sample  208  suitably comprises support member  209  having a top surface and a bottom surface. Container for a biological sample  208  also suitably comprises sample retention area  1002 . Sample retention area  1002  suitably traverses support member  209  from the top surface to the bottom surface, thereby forming a hole passing through support member  209 . 
         [0086]    Sample retention area suitably comprises a porous mesh (not shown) positioned in the sample retention area. As used herein, “porous mesh” refers to any suitable material, i.e., cloth, polymer, plastic, metal, organic and synthetic sponges, etc. The porous mesh provides a surface for retention of the biological sample on the mesh, was also allowing for removal of a fluid, e.g., a cryo-protectant, from the mesh, by allowing the cryo-protectant to pass through the mesh. Suitably, sample retention area  1002  allows for introduction of a biological sample onto the porous mesh and removal of a fluid (e.g., cryo-protectant) from the porous mesh. Suitably, sample retention area  1002  is a substantially circular hole traversing support member  209 . 
         [0087]    As shown in  FIG. 11A , in exemplary embodiments, sample retention area  1002 ′ can comprise an extension surrounding the hole, extending at least above the top surface of support member  209 . This extension allows for the sample retention area to accommodate larger sample/cryo-protectant volumes as the increased height of the extension effectively provides a deeper retention area. 
         [0088]      FIG. 11B  shows a further container for a biological sample  208  as described herein. 
         [0089]    As shown in FIGS.  10  and  11 A- 11 B, container for a biological sample  208  suitably optionally comprises frame  1004  supporting container(s)  209 . Use of frame  1004  allows for the preparation of a number of individual containers, each separate, but connected via a single frame surrounding the containers. In this way, multiple containers can be filled with samples, all of which are held together via frame  1004 , for placement in the automated vitrification devices described throughout. 
         [0090]    Exemplary materials for preparing the sample holders/containers described herein are well known in the art, and include for example various metals, plastics, polymers, ceramics, glasses, etc. Suitably, the containers are made from a two-part thermosetting plastic that starts as two liquids and hardens into a hard plastic. This method allows for casting a metal, plastic or cloth mesh in the sample retention area  1002 . Additional methods for preparing the containers, include for example, injection molding. 
         [0091]    In additional embodiments, sample handling apparatuses are provided. As shown in FIGS.  10  and  11 A- 11 B, suitably such apparatuses comprise plurality of containers for a biological sample  208 , each container comprising a support member  209  having a top surface and a bottom surface, a substantially circular hole traversing the support member from the top surface to the bottom surface (i.e., sample retention area  1002 ). Suitably, a porous mesh is positioned in the substantially circular hole. In exemplary embodiments, the substantially circular hole allows for introduction of a biological sample onto the porous mesh and removal of a fluid from the porous mesh. The plurality of containers  208  are suitably supported by frame  1004 . 
         [0092]    In exemplary embodiments the apparatus further comprises an extension surrounding the hole, extending at least above the top surface of support member  209 . 
         [0093]    In still further embodiments, methods of vitrifying a biological sample are provided. Suitably, such methods comprise placing a biological sample in sample holder  208  and placing a cryo-protectant in a cryo-protectant holder  102 , suitably into reservoirs  206  of cryo-protectant holder  102 . In exemplary embodiments, the placing of the biological sample in sample holder  208  comprises placing samples in each of the individual sample retention areas  1002  (e.g., holes) in the support members of a sample holder. Suitably, placing of the biological sample in the sample holder is carried out by a human operator, though it can be an automated process as well. In addition, placing the cryo-protectant (including multiple cryo-protectants) in the cryo-protectant holder is also suitably carried out by a human operator, though in further embodiments, this can also be an automated process. 
         [0094]    The methods suitably further comprise dispensing the cryo-protectant from the cryo-protectant holder onto the sample. Suitably, this dispensing is carried out by cryo-protectant dispenser  104 , for example, via a nozzle  304 . The methods further comprise removing excess cryo-protectant from the sample via drying mechanism  106 . 
         [0095]    The methods suitably further comprise sealing and cutting the sealed sample. As described herein, suitably such sealing is carried out with the use of a sample sealing device  110 / 108 / 112  comprising sealing mechanism  504  and a cutting mechanism  60  for first sealing the sample, then cutting the sealed sample  702 . Suitably, the sealing is carried out with adhesive film. In exemplary embodiments, the cutting comprises cutting the sealed sample with first die  602  and second die  604 , that align together to cut the sealed sample. 
         [0096]    Suitably, sealed sample  702  is then transferred into a coolant in a coolant holder  114 , e.g., in a cryo-vial comprising liquid nitrogen, utilizing the various device components as described throughout. 
         [0097]    As described herein, suitably the dispensing, removing, sealing, cutting and transferring that are carried out in the various methods described herein are automated via a control module, e.g., via a computer control or similar device. 
         [0098]    Suitable computer control modules and programs for controlling and manipulating the various components of the devices as described herein are well known in the art and are readily determined and implemented by one of ordinary skill in the art. 
         [0099]    In still further embodiments, the devices described herein can also be utilized for warming or thawing frozen biological samples (i.e., bringing biological samples to room temperature, or close to room temperature, so that they can be further utilized as desired), including for examples biological samples that have been vitrified utilizing the devices and methods described throughout. Thus, in embodiments the same device can be used to vitrify a biological sample and also warm or thaw the biological sample. 
         [0100]    In embodiments, a sealed biological sample in a container for a biological sample as described herein is unsealed to expose the biological sample. Unsealing can be performed by an operator or can be an automated function of the devices described herein. 
         [0101]    Following unsealing of the biological sample, the container with the biological sample is placed inside of the device, suitably on a sample holder as described herein. One or more warming solutions are then added to the biological sample. In embodiments, the warming solutions are held in the reservoirs of the cryo-protectant holder prior to being removed by cryo-protectant dispenser and then administered to the biological samples. The order, time and duration of administering the warming solutions to the biological samples is suitably controlled via a control module as described herein. 
         [0102]    Suitably, the warming solutions are maintained at room temperature (e.g., about 20-30° C.) prior to administration to the biological samples, though if desired they can be warmed to higher temperatures. The warming solutions are utilized to slowly and safely raise the temperature of the biological samples to about room temperature prior to additional processing or use as desired. 
         [0103]    Warming solutions suitably comprise sucrose, trehalose and SSS in mHTF. Suitable warming solutions comprise about 0.1 to about 1 M sucrose, about 0.05 to about 1 M trehalose and about 10% to about 30% SSS in mHTF. More suitably, warming solutions comprise about 0.2 to about 0.5 M sucrose, about 0.1 to about 0.5 M trehalose and about 15% to about 25% SSS in mHTF. 
         [0104]    Exemplary warming solutions comprise 1) about 0.5M sucrose, about 0.5M trehalose and 20% SSS in mHTF; 2) about 0.3M sucrose, about 0.3M trehalose and about 20% SSS in mHTF; or 3) about 0.2M sucrose, about 0.1M trehalose and about 20% SSS in mHTF. In embodiments, such warming solutions are suitably used for warming of oocytes. 
         [0105]    Additional exemplary warming solutions comprise 1) about 0.5M sucrose, about 0.5M trehalose and about 20% SSS in mHTF; 2) about 0.3M sucrose, about 0.3M trehalose and about 20% SSS in mHTF; or 3) about 0.2M sucrose, about 0.1M trehalose and about 20% SSS in mHTF. In embodiments, such warming solutions are suitably used for warming of blastocysts. 
         [0106]    For example, in embodiments, cryo-protectant dispenser administers warming solutions to a biological sample in the amounts and for the durations indicated below. Drying mechanism  106  is suitably used to remove excess warming solution at the desired times/intervals prior to application of the next warming solution. 
         [0107]    Described below is an exemplary warming solution protocol for warming/thawing of oocytes:
       A solution comprising about 0.5M sucrose+about 0.5M trehalose+about 20% SSS in mHTF is applied to the biological sample for about 1 minute at room temperature;   Excess solution is removed;   A solution comprising about 0.3M sucrose+about 0.3M trehalose+about 20% SSS in mHTF is applied to the biological sample for about 3 minutes at room temperature;   Excess solution is removed   A solution comprising about 0.2M sucrose+about 0.1M trehalose+about 20% SSS in mHTF is applied to the biological sample for about 3 minutes at room temperature;   Excess solution is removed;   A wash solution comprising about 20% SSS in mHTF is applied to the biological sample for about 3 minutes at room temperature;   Excess solution is removed;   The wash solution is applied again for about 3 minutes.       
 
         [0117]    Described below is an exemplary warming solution protocol for warming/thawing of blastocysts:
       A solution comprising about 0.5M sucrose+about 0.5M trehalose+about 20% SSS in mHTF is applied to the biological sample for about 2-3 minutes at room temperature;   Excess solution is removed;   A solution comprising about 0.3M sucrose+about 0.3M trehalose+about 20% SSS in mHTF is applied to the biological sample for about 3 minutes at room temperature;   Excess solution is removed;   A solution comprising about 0.2M sucrose+about 0.1M trehalose+about 20% SSS in mHTF is applied to the biological sample for about 3 minutes at room temperature;   Excess solution is removed;   A wash solution comprising about 20% SSS in mHTF is applied to the biological sample for about 3 minutes at room temperature;   Excess solution is removed;   The wash solution is applied again for about 3 minutes.       
 
         [0127]    It will be readily apparent to one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of any of the embodiments. 
         [0128]    It is to be understood that while certain embodiments have been illustrated and described herein, the claims are not to be limited to the specific forms or arrangement of parts described and shown. In the specification, there have been disclosed illustrative embodiments and, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.