Abstract:
The invention relates to a cryoapparatus for freezing and/or thawing a sample ( 1 ), especially in the cryopreservation of a biological sample ( 1 ), with a coolable cooling space ( 3 ) and with a sample container ( 10 ) arranged in the cooling space ( 3 ) for temporarily receiving the sample ( 1 ) when freezing or thawing the sample ( 1 ). It is suggested that the sample container ( 10 ) can be tempered separately from the cooling space. Furthermore, the invention includes an associated operating method.

Description:
BACKGROUND OF THE INVENTION 
   The invention relates to a cryoapparatus for freezing and thawing a sample, especially in the cryopreservation of a biological sample, as well as to an associated operating method. 
   In the known cryopreservation biological samples are frozen while maintaining vitality and subsequently thawed out again as needed while maintaining vitality. Examples of usage for such a cryopreservation are the storage of embryos for stem cell research or the preservation of sperm for a subsequent artificial fertilization. It is important for the maintenance of vitality in cryopreservation that predetermined chronological temperature characteristics are maintained during the freezing as well as during the thawing of the biological sample. 
   Therefore, so-called automatic freezers are used for the cryopreservation of biological samples that usually use liquid nitrogen with a boiling point of −196° C. as cooling agent and have a cooling space in which the temperature can be adjusted by controlling the supply of the cooling agent in order to achieve the desired chronological temperature characteristics when freezing or thawing biological samples. 
   During the freezing of biological samples they are inserted into the cooling space of the automatic freezer and subsequently cooled down in accordance with the given chronological temperature characteristics from a starting temperature of a freezing process to a target temperature of the freezing process. After the target temperature of the freezing process has been attained the frozen biological samples are then extracted from the cooling space of the automatic freezer and, e.g., stored in a cryotank. 
   During the thawing of frozen biological samples they are extracted, e.g., from a cryotank and inserted into the cooling space of the automatic freezer. The temperature of the frozen biological samples is subsequently raised in accordance with predetermined chronological temperature characteristics from a certain starting temperature of a thawing process to a target temperature of the thawing process. After the target temperature of the thawing process has been attained, the thawed sample is extracted from the cooling space of the automatic freezer and further used. 
   A disadvantage of the previously described known automatic freezer is that the sample is exposed during the insertion into the cooling space and during the extraction from the cooling temperature to undesirable temperature influences that thermally damage the biological sample and can adversely influence the maintenance of vitality. 
   Furthermore, the previously described known automatic freezer has the disadvantage of an unsatisfactory precision during the adjusting of the given chronological temperature characteristics during freezing and thawing, which is caused by the relatively large volume of the cooling space and the therewith associated problems of regulation technology. 
   The invention therefore has the task of providing an improved cryoapparatus for freezing and/or thawing a sample and of creating a corresponding operating method. 
   SUMMARY OF THE INVENTION 
   The invention comprises the general technical teaching of arranging a sample container in the relatively large cooling space of a cryoapparatus (e.g., of an automatic freezer) in which container the sample is frozen and/or thawed, wherein the sample container can be tempered separately from the cooling space. This means that different temperatures can be adjusted in the sample container and in the cooling space. 
   On the one hand, the separate adjustability of the temperature in the sample container offers the advantage that the chronological temperature characteristics during freezing and thawing that is important for the maintenance of the vitality of the biological sample must be adjusted only for the smaller volume inside the sample container, which is possible in a substantially more precise manner as concerns the regulation technology than the exact tempering of the entire cooling space of the automatic freezer. 
   On the other hand, the separate adjustability of the temperature in the sample container independently of the rest of the cooling space offers the advantage that during the insertion and the extraction of the sample the previously described problematic temperature effects that were damaging to vitality and result from deviations from the given start- and target temperatures can be avoided. 
   The cryoapparatus in accordance with the invention preferably has a lifting apparatus with which the sample container can be lowered and/or raised in the cooling space in a controlled or regulated manner. 
   On the one hand this vertical motion of the sample container in the cooling space makes it possible to take into account the vertical temperature stratification in the cooling space. Thus, the temperature in the cooling space decreases from the top downward so that the sample container has preferably been lowered at the end of a freezing process in order that the frozen sample can be extracted without problematic temperature effects of the surrounding medium in the cooling space. In contrast thereto, at the end of the thawing process the sample container is preferably in a raised position in order that the thawed sample can be extracted avoiding problematic temperature effects of the extremely cold medium present at the bottom of the cooling space. 
   On the other hand the vertical movement of the sample container by the lifting apparatus in a variant of the invention makes possible a tempering of the sample container in that the sample container is raised or lowered in accordance with the desired temperature within the vertical temperature stratification in the cooling space. Therefore, in this variant of the invention the sample container can be tempered separately from the cooling space even without a separate cooling apparatus in that the sample container is appropriately raised or lowered. 
   It is advantageous here if the sample container has a temperature sensor that measures the temperature in the sample container or at the level of the sample container or under it or above it in order that the vertical motion of the sample container in the cooling space can be regulated in a temperature-dependent manner. 
   It is especially advantageous here if several temperature sensors are attached to the sample container at different vertical distances to the sample container, which makes it possible to determine a local vertical temperature gradient. The regulation of the upward or downward motion of the sample container can then be regulated in a very sensitive manner for local temperature variations in the cooling space. 
   However, there is also the alternative possibility that the sample container is arranged stationarily in the cooling space, the sample container being preferably located on the bottom of the cooling space. The temperature change at the opening of the sample container is not achieved in this case by raising or lowering the sample container but rather can be realized, e.g., by a heating that heats the cooling space, the heating apparatus preferably heating only an upper partial area of the cooling space. The heating apparatus is preferably turned on when a sample is to be inserted into the sample container at the beginning of a freezing process or when the sample is extracted from the sample container at the end of a thawing process. The heating of the upper area of the cooling space then prevents a problematic thermal adverse influencing of the sample when the sample is being inserted or extracted. On the other hand, the heating apparatus is preferably turned off when the sample is extracted from the sample container at the end of a freezing process or when the sample is inserted into the sample container at the beginning of a thawing process. The turning off of the heating then also prevents the frozen sample from being thermally damaged when being inserted or extracted. 
   A vertical temperature stratification with a lower cold layer and an upper warm layer is preferably present in the cooling space. This is advantageous because the sample container can be raised or lowered for the extraction or the insertion of the sample into the temperature layer that comes the closest to the actual sample temperature, thus avoiding a thermal adverse influencing of the sample. 
   In such a vertical temperature stratification within the cooling space the warm layer preferably has a temperature that corresponds substantially to a given starting temperature of a freezing process or of a given target temperature of a thawing process whereas the cold layer has a temperature that corresponds substantially to a given target temperature of the freezing process or to a given starting temperature of the thawing process. This is advantageous because the two temperature layers in the cooling space then always have the correct temperature in order to avoid a thermal adverse influencing of the sample during the extraction of the sample and during its insertion into the sample container. 
   In a preferred exemplary embodiment of the invention the sample container is thermally insulated and comprises a cover that can be opened in order to move the sample and to insert the sample. The thermal insulation of the sample container is important if the tempering of the sample container takes place actively by a cooling and/or heating since the temperature adjustment in the sample container is then only minimally influenced by the ambient temperature inside the cooling space. 
   However, it is alternatively also possible that the sample container is not thermally insulated from the cooling space. This is especially meaningful if the tempering of the sample container does not take place actively by a heating or cooling apparatus but rather by a vertical motion of the sample container within the vertical temperature stratification of the cooling space, as already described above. 
   In a preferred exemplary embodiment of the invention at least one substantially vertically running shaft with a wall of a heat-conducting material is arranged in the cooling space wherein the sample container can move vertically in the shaft. The good thermal conductivity of the shaft wall advantageously results in an approximately constant vertical temperature gradient in the shaft so that a certain temperature can be associated with each height in the shaft. This advantageously makes it possible to do without temperature sensors on the sample container. 
   It should furthermore be mentioned that an extractable, thermally insulated transport container is arranged in the cooling space in order to extract the sample from the cooling space after the freezing or to insert it into the cooling space for thawing. At the beginning of a thawing process the sample is then extracted from the transport container and transferred into the sample container, where the sample is then thawed. In the same manner the sample is extracted from the sample container at the end of a freezing process and transferred into the transport container, that can then be extracted from the cooling space and, e.g., inserted in a cryotank. 
   The cooling space itself is preferably designed as a vat in the cryoapparatus in accordance with the invention and covered at its top by an extractable protective hood that is preferably at least partially transparent in order to make a visual monitoring possible. 
   However, there is also the alternative possibility of doing without a protective hood so that the cooling space is open at the top. 
   It should furthermore be mentioned that several storage surfaces can be arranged in the cooling space at different heights that correspond to different temperatures in accordance with the vertical temperature stratification in the cooling space. 
   Finally, it should also be mentioned that the invention comprises not only the previously described cryoapparatus in accordance with the invention but also a corresponding operating method that results from the present description. 

   
     BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS 
     Other advantageous further developments of the invention are characterized in the subclaims or are explained in detail below together with a description of the preferred exemplary embodiments of the invention using the figures. 
       FIG. 1  shows a schematized cross-sectional view of a cryoapparatus in accordance with the invention for freezing and thawing a biological sample in a manner that maintains its vitality, 
       FIG. 2  shows a schematized cross-sectional view of a modified exemplary embodiment of a cryoapparatus in accordance with  FIG. 1 , 
       FIG. 3  shows a schematized cross-sectional view of an alternative exemplary embodiment of a cryoapparatus in accordance with the invention with an additional heating apparatus, 
       FIG. 4  shows a schematized cross-sectional view of a simplified exemplary embodiment of such a cryoapparatus that in contrast to the other cryoapparatuses does not have a protective hood, 
       FIG. 5A  shows a simplified cross-sectional view of another exemplary embodiment of a cryoapparatus in accordance with the invention, 
       FIG. 5B  shows a cross-sectional view of a lifting apparatus of the cryoapparatus in  FIG. 5A  for lifting or lowering the sample, 
       FIG. 6  shows a simplified cross-sectional view of another exemplary embodiment of a cryoapparatus in accordance with the invention with several lifting apparatuses for the simultaneous freezing or thawing of several samples independently of each other, 
       FIG. 7A  shows a freezing process in the exemplary embodiment in accordance with  FIG. 1  in the form of a flowchart, 
       FIG. 7B  shows a thawing process in the exemplary embodiment in accordance with  FIG. 1  in the form of a flowchart, 
       FIG. 8A  shows a freezing process in the exemplary embodiment in accordance with  FIGS. 2 and 5A ,  5 B in the form of a flowchart, and 
       FIG. 8B  shows a thawing process in the exemplary embodiments in accordance with  FIGS. 2 and 5A ,  5 B. 
   

   DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
   The cryoapparatus shown in  FIG. 1  makes it possible to freeze and thaw a biological sample  1  while maintaining vitality in that sample  1  is cooled down or heated during freezing and thawing in accordance with given chronological temperature characteristics. 
   To this end, the cryoapparatus comprises a cryovat  2  that encloses a cooling space  3 , the cryovat  2  comprising a wall  4  of a thermally insulating material. 
   The cooling of the cooling space  3  takes place by liquid nitrogen contained in a cooling agent container  5 , that is represented only schematically here, and inserted via a controllable cooling agent valve  6  into the cooling space  3 . 
   The insertion of the liquid nitrogen into the cooling space  3  takes place here indirectly via a porous buffer material  7  with which the inside of the wall  4  of the cryovat  2  is jacketed, a grating  8  of a material that conducts heat well (e.g., copper) being arranged on the inside of the buffer material  7 . The buffer material  7  prevents the supplied liquid nitrogen from collecting on the bottom of the cooling space  3  in the form of a so-called nitrogen lake. Instead, the supplied liquid nitrogen is released by the buffer material  7  uniformly through the grating  8  into the cooling space  3 . 
   The cryovat  2  is covered on its top by a removable protective bell  9 , the protective bell  9  being transparent in order to make possible a visual monitoring of the cooling space  3 . 
   A sample container  10  is present in the cooling space  3  that can be raised and lowered in the vertical direction by a lifting apparatus  11 , that is represented here only schematically, as will be described in detail later. 
   The temperature sensor  12  is located on the sample container  10 , measures the temperature T ACTUAL1  in the sample container  10  and passes it on to the control apparatus  13 , that also controls the cooling agent valve  6  and adjusts the temperature in the cooling space  3  with it. 
   Furthermore, the sample container  10  has a cooling apparatus  14  that is represented only schematically and cools the inner space of the sample container  10  with a cooling capacity P COOL , given by the control apparatus  13 ,as will also be described in detail later. The cooling apparatus  14  can utilize, e.g., the liquid nitrogen contained in the cooling agent container  5  but other cooling techniques can also be used. 
   Moreover, it should be mentioned that the sample container  10  is thermally insulated and has a sealable cover  15  that is also thermally insulated, the cover  15  being opened in order to insert the sample  1  into the sample container  10  and to extract it from the sample container  10 . 
   Furthermore, another temperature sensor  16  is arranged on the bottom of the cooling space  3  that measures the temperature T ACTUAL2  at the bottom of the cooling space  3  and passes it on to the control apparatus  13 . The control apparatus then regulates the temperature T ACTUAL2  to a given theoretical value by appropriately controlling the cooling agent valve  6 . 
   In addition, a removable transport container  17 , that is thermally insulated and has a removable cover, is located on the bottom of the cooling space  3 . The transport container  17  makes it possible to transfer the cryofrozen sample  1  from the cooling space  3  into a cryotank that is not represented here without the sample  1  being warmed up during this transfer and being thermally damaged as a result. Moreover, the transport container  17  makes it possible to transfer the cryofrozen sample  1  out of a cryotank, that is not represented here, into the cooling space  3  in order that the frozen sample  1  can be subsequently thawed in the sample container  10 , which will be described in detail later. 
   Finally, the control apparatus  13  is connected to a traditional personal computer  18 , wherein any chronological temperature characteristics for the freezing or thawing process can be programmed on the personal computer  18 . 
   A freezing process for the cryoapparatus according to  FIG. 1  is described in the following with reference made to the flowchart in  FIG. 7A . 
   In this process, the control apparatus  13  regulates temperature T ACTUAL2  at the bottom of the cooling space  3  by suitably controlling the cooling agent valve  6  down to the given target temperature of the freezing process. 
   Furthermore, the empty sample container  10  is moved by the lifting apparatus  11  in the cooling space  3  upward into the position shown in dotted lines so that the mouth of the sample container  10  rests at an opening of the cover  5  above the cryovat  2 . 
   The cover  15  of the sample container  10  is then opened, whereupon the sample  1  is inserted into the sample container  10  and the cover  15  of the sample container is closed. During this insertion of the sample  1  into the sample container  10 , the sample  1  is hardly adversely affected thermally since the temperatures in this area are close to the starting temperature of the freezing process. 
   After the sealing of the sample container  10  its inner space is then cooled by the cooling apparatus  14 , the control apparatus  13  regulating predetermined chronological temperature characteristics in that the cooling apparatus  14  regulates the cooling apparatus  14  in accordance with the temperature T ACTUAL  measured by the temperature sensor  12 . 
   After the conclusion of the freezing the sample container  10  is then moved down into the cooling space  3  by the lifting apparatus  11 , a temperature prevailing in the lower area of the cooling space  3  that substantially corresponds to the target temperature of the freezing process. During the subsequent opening of the cover  15  and the extraction of the sample  1  from the sample container  10 , the frozen sample  1  is then hardly adversely affected thermally since the surrounding medium in the cooling space  3  has almost the same temperature. 
   The sample  1  is then transferred after the extraction from the sample container  10  into the transport container  17 , whereupon the transport container  17  with the cryofrozen sample  1  located in it is removed from the cooling space  3  and can be inserted, e.g., in a cryotank. 
   A thawing process of the cryoapparatus according to  FIG. 1  will be described in the following with reference made to the flowchart in  FIG. 7B . 
   In this process, the cooling space  3  is cooled to the extent that the temperature T ACTUAL2  at the bottom of the cooling space  3  substantially corresponds to the starting temperature of the thawing process. 
   The transport container  17  with the cryofrozen sample  1  located in it is then subsequently extracted from a cryotank that is not represented here and placed on the bottom of the cooling space  3 . 
   Then, the cover  15  of the sample container  10  is opened just as the cover of the transport container  17  is also opened. 
   The sample  1  located in the transport container  17  is then extracted from the transport container  17  and transferred into the sample container  10 , whereupon the cover  15  of the sample container  10  is then closed. During the transfer of the sample  1  from the transport container  17  into the sample container  10 , the sample  1  is hardly adversely affected thermally since the temperatures at the bottom of the cooling space  3  are substantially equal to the temperature of the sample  1 . 
   After the sealing of the sample container  10 , the sample container  10  is then moved vertically upward into the position shown in dotted lines by the lifting apparatus  11  in the cooling space  3 . 
   The inner space of the sample container  10  is subsequently heated to predetermined chronological temperature characteristics by reducing the cooling capacity P COOL  of the cooling apparatus  14 , the control apparatus  13  regulating the cooling capacity P COOL  as a function of the measured temperature T ACTUAL1  in order to attain the desired chronological temperature characteristics in the sample container  10  during the thawing of the sample  1 . 
   After the end of the thawing process the cover  15  of the sample container  10  is then opened, whereupon the thawed sample  1  is extracted from the sample container  10 . 
   The sample  1  is hardly adversely affected thermally by the surrounding medium even in this extraction of the thawed sample  1  from the sample container  10  since the temperatures in this area are relatively high. 
   The alternative exemplary embodiment of a similar cryoapparatus shown in  FIG. 2  will now be described in the following, which largely coincides with the previously described exemplary embodiment of a cryoapparatus shown in  FIG. 1 . In order to avoid repetitions the previous description is therefore extensively referred to, wherein the same reference numerals are used for corresponding components. 
   This exemplary embodiment has the particularity that the cryoapparatus does not have a separate, actively operating cooling apparatus  14 . 
   Instead, the tempering of the sample container  10  takes place here by a vertical lowering or raising of the sample container  10  in the cooling space  3 . Due to the vertical temperature stratification in the cooling space  3  the temperature in the sample container  10  also changes so that given chronological temperature characteristics can also be adjusted during the thawing or freezing of the sample. The lowering or raising of the sample container  10  takes place here controlled by the control apparatus  13  as a function of the temperature T ACTUAL1  in the sample container  10  measured by the temperature sensor  12 . 
   In contrast to the exemplary embodiment in accordance with  FIG. 1 , the sample container  10  is not thermally insulated here in order that the inner space of the sample container  10  can assume, as a function of its particular height inside the cooling space  3 , the associated temperature. 
   The characteristics of the freezing process results here from the flowchart according to  FIG. 8A  whereas the characteristics of a thawing process is shown in the flowchart according to  FIG. 8B . 
   The alternative exemplary embodiment of a cryoapparatus in accordance with the invention and shown in  FIG. 3  will now be described in the following, which cryoapparatus coincides partially with the previously described exemplary embodiment so that in order to avoid repetitions the previous description is therefore referred to, wherein the same reference numerals are used for corresponding components. 
   A particularity of this exemplary embodiment is at first that the sample container  10  is stationarily arranged in the cooling space  3  on the bottom of the cooling space  3  so that the lifting apparatus  11  can be dispensed with. 
   Another particularity of this exemplary embodiment is that a heating apparatus  19  is attached to the inner wall of cooling space  3  in its upper area and heats the upper partial area of cooling space  3 . 
   The heating apparatus  19  is turned on by the control apparatus  13  before a sample is inserted into the sample container  10  at the beginning of a freezing process. This heats the upper partial area of the cooling space  3 , which prevents thermal damage to the sample during the insertion into the sample container  10 . 
   Moreover, the heating apparatus  19  is also turned on by the control apparatus  13  before a thawed sample is extracted at the end of a thawing process from the sample container. This also prevents the thawed sample from being thermally damaged during the extraction from the sample container  10 . 
   In contrast thereto, the heating apparatus  19  is turned off when the sample is extracted from the sample container  10  at the end of a freezing process and transferred into the transport container  17 . 
   In addition, the heating apparatus  19  is also turned off when the sample is transferred at the beginning of a thawing process from the transport container  17  into the sample container  10 . 
   This turning off of the heating apparatus  19  prevents for its part an adverse thermal influencing of the cryocooled sample during the transfer between the sample container  10  and the transport container  17 . 
   The alternative exemplary embodiment shown in  FIG. 4  coincides almost completely with the previously described exemplary embodiment shown in  FIG. 3 . Only the protective bell  9  was omitted. 
   The alternative exemplary embodiment of a cryoapparatus in accordance with the invention shown in  FIGS. 5A and 5B  and coinciding in part with the previously described exemplary embodiments will now be described in the following, so that in order to avoid repetitions the previous description is partially referred to, wherein corresponding components are characterized by the same reference numerals. 
   A particularity of this exemplary embodiment is that several stepped storage surfaces  20  are arranged on the bottom of the cooling space  3  on which cryosubstrates can be temporarily stored. Due to the vertical temperature stratification in the cooling space  3 , the individual storage surfaces  20  are located at different temperatures so that a stepped temperature profile can be successively moved through in time in that a cryosample is stored successively on the different storage surfaces  20 . 
   Another particularity of this exemplary embodiment is that several shelf-like storage surfaces  21  are arranged superposed in the upper area of the cooling space  3  that also have different temperatures due to the vertical temperature stratification in the cooling space  3  and make it possible to store a sample. 
   Furthermore, the cryoapparatus in this exemplary embodiment has a lock  22  via which the samples can be brought into the cooling space  3  and extracted from the cooling space  3 . 
   Moreover, the protective bell  9  is provided with intervention zones  23  with which an operator can perform manipulations in the cooling space  3 . 
   The cooling of the cooling space  3  takes place here by a nitrogen lake  24  produced on the bottom of the cooling space  3 . 
   The tempering of the sample  1  in the form of a cryosubstrate in the sample container  10  takes place here, however, in a different manner, as will be described in the following. 
   To this end, a vertically running shaft  25  is arranged in the cooling space  3  which shaft consists of a good heat-conductive material (e.g., copper) and an approximately constant vertical temperature gradient is produced in the shaft  25  due to the good heat conductivity. 
   The sample container  10  can be raised or lowered in a vertical direction by a motor  26  via a rotatable spindle  27  in the shaft  25  in order to temper the sample container  10  in accordance with the vertical temperature stratification in the sample space  3 . 
   It is furthermore apparent from  FIG. 5B  that several temperature sensors  28 - 31  are arranged above and below the sample container  10  at different heights and measure a local vertical temperature gradient, wherein the temperature sensors  28 - 31  are connected to a regulating apparatus that controls the motor  26  in such a manner that the sample container  10  is always located at the right height in the cooling space  3  in order that the desired chronological temperature characteristics are achieved during the freezing and thawing of sample  1 . 
   Furthermore, the enlarged cross-sectional view in  FIG. 5B  shows that the carriage-shaped sample container  10  contains a cryosubstrate  32  and a cover  33  and containers for the samples  1 . 
   The alternative exemplary embodiment shown in  FIG. 6  will now be described that largely coincides with the previously described exemplary embodiment shown in  FIGS. 5A and 5B , so that in order to avoid repetitions the previous description of these figures is extensively referred to, wherein the same reference numerals are used for corresponding components. 
   A particularity of this exemplary embodiment is that several shafts  25  are arranged adjacent to each other in the cooling space  3  in each of which a sample container  10  can be raised and lowered in a vertical direction in order to adjust the desired temperature in accordance with the vertical temperature stratification in the cooling space  3 . This arrangement advantageously makes possible parallel freezing and thawing processes on several samples. 
   The invention is not limited to the previously described preferred exemplary embodiments but rather a plurality of variants and modifications is possible that also makes use of the inventive concept and therefore falls within its scope of protection.