(19)
(11)EP 3 413 709 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
01.04.2020 Bulletin 2020/14

(21)Application number: 17705340.2

(22)Date of filing:  10.02.2017
(51)International Patent Classification (IPC): 
A01N 1/02(2006.01)
(86)International application number:
PCT/EP2017/052987
(87)International publication number:
WO 2017/137552 (17.08.2017 Gazette  2017/33)

(54)

APPARATUS FOR CRYOPRESERVING A PLURALITY OF CELLULAR SAMPLES AND METHOD FOR CRYOPRESERVING A PLURALITY OF CELLULAR SAMPLES

VORRICHTUNG ZUR KRYOKONSERVIERUNG VON MEHREREN ZELLPROBEN UND VERFAHREN ZUR KRYOKONSERVIERUNG VON MEHREREN ZELLPROBEN

APPAREIL POUR LA CRYOPRÉSERVATION D'UNE PLURALITÉ D'ÉCHANTILLONS CELLULAIRES ET PROCÉDÉ DE CRYOCONSERVATION D'UNE PLURALITÉ D'ÉCHANTILLONS CELLULAIRES


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 12.02.2016 EP 16155424

(43)Date of publication of application:
19.12.2018 Bulletin 2018/51

(73)Proprietor: F. Hoffmann-La Roche AG
4070 Basel (CH)

(72)Inventors:
  • BAUER, Rüdiger
    80689 München (DE)
  • HAMMEL, Markus
    82377 Penzberg (DE)
  • LOEDER, Sandra
    81476 Muenchen (DE)
  • PIENKNY, Robert
    82377 Penzberg (DE)
  • VON LEDEBUR-WICHELN, Clara
    81475 Muenchen (DE)
  • WEHNER, Steffen
    91052 Erlangen (DE)

(74)Representative: Altmann Stößel Dick Patentanwälte PartG mbB 
Dudenstrasse 46
68167 Mannheim
68167 Mannheim (DE)


(56)References cited: : 
WO-A1-2005/036136
US-A- 4 580 409
WO-A2-2008/047154
US-A1- 2013 111 931
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    Field of the invention



    [0001] The present invention relates to an apparatus and a method for cryopreserving a plurality of cellular samples. The terms "cryopreservation" and "cryopreserving", respectively, as used herein refers to a process of preserving cellular samples by cooling to and storing at ultra low temperatures below approximately -130°C. The term "cellular sample" as used herein refers to eukaryotic cells. Usually, the process of cooling and freezing is carried out by means of a refrigerant such as liquid nitrogen.

    Related art



    [0002] The successful cryopreservation of cells is an important prerequisite for the production of biopharmaceuticals (Seth, G.: Freezing mammalian cells for production of biopharmaceuticals; Methods 56, p. 424-431, 2012). However, cryopreservation of cellular samples is still a challenge in order to avoid a deterioration of the quality of the cells.

    [0003] When cell containing suspensions are cooled, the suspension will not necessarily crystallize when the freezing point of the cellular solution is reached. Rather, the suspension will be super-cooled. Super-cooling is defined as a lowering of the temperature of a liquid below its freezing point without the liquid freezing or solidifying (Wilson, P. W.: Ice Nucleation in nature: supercooling point (SCP) measurements and the role of heterogeneous nucleation. Cryobiology 46, p. 88-98, 2003). The formation of the first ice crystal, called "seeding", in the super-cooled solution occurs either spontaneous over a range of temperatures or by means of selective induction, e.g. by a cold pulse or mechanically by rocking at a specific temperature (Mazur, Peter: Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247. C125-C142. 1984). When freezing multiple cellular samples, an induction of seeding is desirable to avoid extensive super-cooling and allow simultaneous crystallization of all samples (Morris, G.J. & Acton, E.: Controlled ice nucleation in cryopreservation--a review. Cryobiology 66, p. 85-92, 2013).

    [0004] Due to the phase change from liquid to solid during seeding, crystallization enthalpy ("latent heat") is set free which causes a temperature rise in the sub-cooled medium up to the freezing point. If ice crystals are formed in concentrated solutions, the solved substances are not incorporated into the ice but accumulate in the fluid between the ice crystals and, thus, may increase the concentration of the solution. The cell components do not freeze and become super-cooled. The subsequent processes depend on the cooling rate. If the cooling rate is sufficiently low, the cells lose water due to exosmosis such that the intracellular substances as saccharides and salts concentrate. Thus, super-cooling within the cells is prevented. Further, the chemical potential of the intracellular water remains in equilibrium with the chemical potential of the extracellular water. As a result, the cells dehydrate without intracellular freezing (Mazur, Peter: Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247. C125-C142. 1984).

    [0005] However, if the cooling rate is too low, solution effects and osmosis can damage cells (Fowler, A. & Toner, M.: Cryo-injury and biopreservation. Ann. N. Y. Acad. Sci. 1066, p. 119-135, 2005). In contrast, if the cooling rate is high, the cells may not dehydrate fast enough in order to achieve the equilibrium. Thus, the intracellular liquid is gradually super-cooled until the phase change from liquid to solid takes place and ice is formed within the cells (Mazur, Peter: Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247. C125-C142. 1984).

    [0006] In summary, viability of cells after re-vitalization is dependent on several factors. Of high importance are the seeding step and the cooling rate. Cells, which are cooled too fast, are destroyed due to the formation of intracellular ice and re-crystallization effects when being re-vitalized. Cells, which are cooled too slowly, are exposed to solution effects and osmotic damage.

    [0007] Accordingly, apparatuses for cryopreserving cellular samples have to be constructed in a way to guarantee exact, simultaneous and reproducible seeding and subsequent cooling to avoid cooling rates that are too low or too high.

    [0008] Conventional apparatuses do not sufficiently fulfil this challenge as these may not cryo-preserve a large number of cellular samples under homogenous conditions. Particularly, the temperature distribution between the single cellular samples stored in vials is inhomogeneous during freezing as the vials are arranged spatially one after another. This is due to the cubic shape of the cooling chamber which causes turbulences, stalls and uneven distribution of the injected refrigerant. These effects cause an inhomogeneous heat removal depending on the vial position. Thus, the temperature within the cellular samples may not be exactly controlled, which is particularly relevant for inducing the ice crystallization. Therefore, there are significant differences in the cooling rates between the respective vials and deviations from the target temperature distribution. Further, in many conventional freezing devices, the seeding function for inducing the ice crystallization is not suitable to synchronously induce the ice crystallization as the refrigerant does not come into contact with each vial. Thus, the formation of ice crystals occurs spontaneously and not controlled. Further, the removal of the latent heat is difficult or problematic as the latent heat may not be removed efficiently. Accordingly, the degree of the sub-cooling process up to the formation of ice crystals within the vials and the further process vary from each other. In consequence, inhomogeneous conditions result in high variance in respect to product quality within a cell bank and between different cell banks.

    [0009] US 2013/0111931 A1 discloses a method and system for controlled rate freezing and nucleation of biological materials contained in vials or other containers. The materials are rapidly cooled within a cooling unit via forced convective cooling using a uniform flow of cryogen in proximity to each of the plurality of vials. In addition, pressure control induced nucleation and temperature quench induced nucleation may be used to provide control over the nucleation temperature of the material in the plurality of vials. US 4 580 409 discloses a freezing device, wherein a crystallization inductor formed by a cooled metal bar can be moved by a motor such as to bear against a plurality of straws that are arranged in parallel, thus providing a thermal shock to initiate crystallization.

    Summary



    [0010] Disclosed herein are an apparatus and a method for cryopreserving a plurality of cellular samples allowing a freezing of vials in a simultaneous and reproducible manner.

    [0011] In accordance with the invention, an apparatus as defined in claim 1 and a method as defined in claim 10 are provided. Preferred embodiments are defined in the dependent claims.

    [0012] As used in the following, the terms "have", "comprise" or "include" or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions "A has B", "A comprises B" and "A includes B" may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements.

    [0013] Further, it shall be noted that the terms "at least one", "one or more" or similar expressions indicating that a feature or element may be present once or more than once typically will be used only once when introducing the respective feature or element. In the following, in most cases, when referring to the respective feature or element, the expressions "at least one" or "one or more" will not be repeated, non-withstanding the fact that the respective feature or element may be present once or more than once.

    [0014] Further, as used in the following, the terms "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting alternative possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way.

    [0015] According to the disclosed apparatus for cryopreserving a plurality of cellular samples, the apparatus comprises a cooling chamber, a cooling device comprising means configured to cool an interior of the cooling chamber, wherein the cooling device further comprises cooling tubes that are separate from the means configured to cool the interior of the cooling chamber and that are arranged within the cooling chamber, wherein the cooling device is configured to provide a flow of refrigerant through the cooling tubes, and a support for supporting a plurality of vials for storing cellular samples, wherein the support is movable relative to the cooling tubes such that the plurality of vials is engageable with the cooling tubes.

    [0016] The term "cooling tubes" as used herein refers to tubes or pipes through which a refrigerant may flow. Particularly, the cooling tubes are part of a so called closed cooling system such that the refrigerant is not supplied into the cooling chamber by means of the cooling tubes, i.e. the cooling tubes do not comprise openings or orifices in communication with the cooling chamber. The cooling chamber and the interior thereof may be separately cooled by means of the cooling device as will explained in more detail below.

    [0017] According to the apparatus, the support may receive a plurality of vials or may be loaded with a plurality of vials. The support may be moved so as to bring the plurality of vials in contact with the cooling tubes when being loaded with vials. Thus, the vials may be engaged by the cooling tubes at a target point of time and/or at a target temperature. With other words, the support is movable relative to the cooling tubes to an extent or in a manner to a position allowing the plurality of vials to be engaged by the cooling tubes. Accordingly, it is possible to initiate or induce a crystallization process within the vials by the engagement of the vials with the cooling tubes. Particularly, crystallization of the cellular samples may be locally induced and not completely across an outer surface of the cellular sample.

    [0018] The support may be movable such that the plurality of vials is engageable with the cooling tubes at a predetermined and adjustable height of the vials. The support may be adjusted to change the height of the engagement point at the vial. The height may correspond to a liquid surface of the cellular samples or any other desired point at the vial. The term "height of a vial" as used herein refers to a certain position at the vial in a longitudinal direction along which a vial usually extends. Particularly, the height is determined as a position in a direction from the bottom to the top of the vial. The position of contact at the predetermined height may thus be determined by the position of the liquid level of the cellular sample within the vial. Thus, too much radiation of the coldness into the vials is prevented. Further, the cooling impulse may be locally limited. Particularly, the heterogeneous temperature distribution resulting from the cooling impulse may only occur in a local limited manner and should extend within the vials as small as technically possible.

    [0019] The cooling tubes may be arranged parallel to one another. Thus, an even distribution of the cooling tubes is provided. This is particularly achieved if the cooling tubes are evenly spaced apart from one another.

    [0020] The support may comprise a plurality of compartments for receiving the plurality of vials. The compartments may be arranged such that the plurality of vials is arrangeable between the cooling tubes. For example, the compartments are arranged as lines, which extend parallel to one another.

    [0021] Advantageously, the cooling tubes and the compartments are arranged parallel to one another. Thus, the distance of the compartments to the cooling tubes and by which the support has to be moved for engaging the vials with the cooling tubes is identical for each vial.

    [0022] The compartments may be movable relative to the support. Thus, a kind of fine tuning regarding the positioning of the vials relative to the cooling tubes is provided which allows each vial to be individually engaged with a cooling tube in an identical manner. This may be realized if the compartments are movable in the support, for example.

    [0023] The compartments may be adapted to fix the plurality of vials. Thus, the vials are securely received by the compartments.

    [0024] The compartments may be formed such that the plurality of vials may be directly engageable with the cooling tubes. Thus, the coldness of the cooling tubes may be directly introduced in the vials without any constructional member inbetween.

    [0025] The support may be movable on rails. Thus, a rather simple construction may be used for moving the support.

    [0026] The cooling device may be adapted to provide a sufficient flow of refrigerant through the cooling tubes such that the cooling tubes reach a cooling tube outer surface seeding temperature within a precooling time. The term "precooling time" as used herein refers to a time which is relatively defined with respect to a time, in which the cooling chamber may be cooled to the seeding temperature. Particularly, the precooling time is shorter than the time within which the cooling chamber may be cooled to the seeding temperature. With other words, the cooling tubes reach the cooling tube outer surface seeding temperature within the precooling time and before the seeding temperature of the cooling chamber is reached. Thus, it is ensured that the seeding process may be initiated within a desired time for maintaining the quality of the cellular samples.

    [0027] The cooling device may comprise a ring-shaped tube portion. The cooling tubes may be connected to the ring-shaped tube portion. Thus, a sufficient supply of the refrigerant into the cooling tubes is provided for reaching the cooling tube outer surface seeding temperature in the precooling time. The term "cooling tube outer surface seeding temperature" as used herein refers to a temperature defined at the outer surface of the cooling tubes which is suitable to induce a seeding process of the cellular samples. With other words, the temperature of the cooling tubes at the outer surface thereof must be sufficient low in order to allow inducing a seeding process of the cellular samples when the vials engage the cooling tubes. The temperature at the outer surface of the cooling tubes may be detected by means of a temperature sensor arranged at the outer surface of the cooling tubes.

    [0028] The ring-shaped tube portion surrounds the cooling tubes. The cooling tubes may comprise ends connected with the ring-shaped tube portion. Thus, each cooling tube may be supplied with refrigerant from the ring-shaped tube portion for reaching the cooling tube outer surface seeding temperature in the precooling time.

    [0029] The ring-shaped tube portion may comprise inlets for supplying refrigerant. Thus, the refrigerant may be supplied at several locations to the ring-shaped tube portion which serves to better distribute the refrigerant to the ring-shaped tube portion.

    [0030] The ring-shaped tube portion may be separated into segments, wherein each segment is associated with at least one of the inlets. Thus, each segment may be supplied with refrigerant.

    [0031] The ring-shaped tube portion may be separated into the segments by means of separating walls. Thus, each segment is independent from another segment and may be individually supplied with refrigerant.

    [0032] The inlets may be evenly spaced apart from one another. Thus, a sufficient supply with refrigerant is provided. This also causes a sufficient flow of the refrigerant through the cooling tubes for reaching the cooling tube outer surface seeding temperature in the precooling time.

    [0033] An inner diameter of the ring-shaped tube portion may be larger than an inner diameter of the cooling tubes. Thus, it is ensured that a sufficient amount of refrigerant may be supplied to the cooling tubes from the ring-shaped tube portion.

    [0034] The cooling device may be adapted to provide refrigerant flow from bottom to top of the cooling tubes until reaching cooling tube outer surface seeding temperature within the precooling time. The terms "bottom" and "top" as used herein refer to orientations relative to the cooling chamber. As the cooling chamber is basically oriented in a vertical direction and the direction of gravity, respectively, the refrigerant is supplied into the cooling tubes at a lower side of the cooling tubes or at a position below the cooling tubes and is discharged from the cooling tubes at a top side of the cooling tubes or at a position above the cooling tubes even though the cooling tubes may be arranged in a horizontal direction and a direction perpendicular to the direction of gravity, respectively. Thus, after the refrigerant has been used and comprises a higher temperature and / or a phase change from liquid to gas, the refrigerant may be withdrawn from the cooling tubes using the conventional convection effects. Thereby, the accumulation of gaseous refrigerant within the cooling tubes is prevented.

    [0035] The cooling chamber may be cylindrical. Thus, a symmetric arrangement is provided which improves temperature laminar, plug-flow of refrigerant within the cooling chamber.

    [0036] The apparatus may further comprise an inner casing, in which the cooling chamber is located, and an outer casing housing the inner casing. Thus, the cooling chamber may be thermally isolated.

    [0037] The means configured to cool an interior of the cooling chamber may be or may comprise nozzles for injecting a refrigerant for cooling the interior of the cooling chamber. Thus, the refrigerant may be distributed within the cooling chamber for homogenously lowering the temperature.

    [0038] The nozzles may be disposed in a space between the inner casing and the outer casing. Thus, the nozzles may be arranged in a compact manner.

    [0039] The inner casing may comprise a lower side and a top side, wherein the lower side and the top side may comprise orifices. For example, the lower side and the top side may comprise perforated plates, wherein the perforated plates comprise the orifices. The perforation plates may comprise a perforation ratio of 5 % to 15 %. The perforation ratio may vary over the plate radius. With other words, the perforation ratio of a radial outer portion of the perforated plates may be different from a radial middle portion and/or a radial inner portion. Further, the perforation ratio of the perforated plate at the lower side may be different from the perforation ratio of the perforated plate at the top side. The term "perforation ratio" as used herein refers to the ratio of the cross-sectional area or opening area of the orifices to or in relation to the surface area of the perforation plate in which the orifices are formed. Thus, the refrigerant may be evenly distributed. Particularly, the perforated plates cause a stow or jam of the refrigerant and a homogenous and laminar flow of the refrigerant in the space between the inner casing and the outer casing.

    [0040] The apparatus may further comprise a fan disposed between the top side of the inner casing and the outer casing. Thus, a refrigerant flow can be generated.

    [0041] The cooling device may comprise an inlet, which is located in the space between the inner casing and the outer casing, and distribution tubes connecting the inlet to the nozzles. Thus, the refrigerant may be distributed throughout the cross section of the cooling chamber. This is particularly achieved by an even distribution of the nozzles in the space between the inner casing and the outer casing.

    [0042] The nozzles may be located within a common plane. Thus, the nozzles comprise a predetermined identical distance to the support.

    [0043] The inlet may be located on a central axis of the cooling chamber. Thus, identical flows of the refrigerant from the inlet to the nozzles are provided.

    [0044] The inlet and the distribution tubes may be located with a minimum of contact area between the distribution tubes and the flow in inner and outer casing. Thus, the distribution tubes of inlet generate a minimum of convective cooling of the flow along the tubes. Only the outcoming refrigerant cools the flow.

    [0045] The apparatus may further comprise a middle casing arranged between the inner casing and the outer casing. The middle casing may comprise an outlet for refrigerant. The outer casing may comprise an outlet for refrigerant. The outlet of the middle casing and the outlet of the outer casing are connected to one another. Thus, a predetermined flow of refrigerant is provided.

    [0046] The middle casing may comprise a lower side and a top side. The outlet of the middle casing may be located in the lower side, wherein the outer casing may comprise a lower side and a top side, wherein the outlet of the outer casing may be located in the top side. Thus, a flow of the used refrigerant from a bottom to a top of the casings is provided.

    [0047] The support may be adapted to support the plurality of vials in a common plane. Thus, the laminar plug-flow of refrigerant removes equal heat equivalents at each vial.

    [0048] The cooling device may be adapted to supply the cooling tubes with refrigerant in a liquid state such that a seeding process of the cellular samples in the vials is initiated by means of an engagement of the vials with the cooling tubes. Thus, the cellular samples may be frozen under defined conditions.

    [0049] According to the disclosed method for cryopreserving a plurality of cellular samples, an apparatus as described above is used. The method comprises the steps:
    • cooling the cooling chamber to a temperature hold point,
    • providing cellular samples in a liquid state in a plurality of vials,
    • loading the support with the plurality of vials,
    • holding the cooling chamber at the temperature hold point for purpose of temperature synchronization in the plurality of vials,
    • cooling the cooling chamber to a seeding temperature in the vials,
    • supplying a refrigerant through the cooling tubes for a precooling time such that the cooling tubes reach a cooling tube outer surface seeding temperature before the seeding temperature is reached,
    • moving the support relative to the cooling tubes when the cooling tube outer surface seeding temperature and the seeding temperature are met such that the plurality of vials is engaged with the cooling tubes for a predetermined time so as to initiate a seeding process in the cellular samples,
    • cooling the cooling chamber to a final temperature, and
    • controlled removal of latent heat until the final temperature is reached.


    [0050] Thus, the cellular samples may be cryopreserved under defined conditions without having negative impact on the viability of the cellular samples. Particularly, by means of the engagement of the vials with the cooling tubes, a local and timely limited sub-cooling of the cellular samples is provided.

    [0051] As the cooling chamber is pre-cooled to a temperature hold point before the support is loaded with a plurality of vials, a first cooling step of the vials when loaded into the cooling chamber is accelerated or takes less time because the cooling chamber and the vials do not have to be pre-cooled together. Further, as the temperature within the cooling chamber is held at the temperature hold point, it is ensured that each of the vials has an identical temperature before being further processed and being cooled to the seeding temperature, respectively. Thus, the vials may be cooled to the seeding temperature under moderate conditions in a short time.

    [0052] The temperature hold point may be from -2 °C to 10 °C. According to another embodiment, the temperature hold point may be and preferably from 0 °C to 5 °C such as 4 °C.

    [0053] The seeding temperature may be from a freezing point of the cellular sample to -15 °C. The temperature hold point and/or the seeding temperature may be defined as temperatures in the cellular samples.

    [0054] The cooling tube outer surface seeding temperature may be defined as a temperature on an outer surface of the cooling tubes which allows to induce the seeding process of the cellular samples. With other words, the temperature on the outer surface of the cooling tubes is determined as a temperature being sufficient low to allow inducing the seeding process of the cellular samples when the vials engage the cooling tubes.

    [0055] The final temperature may be from -120°C to -190°C such as -185°C.

    [0056] The predetermined time may be from 0.5 minutes to 3.0 minutes. According to another embodiment, the predetermined time may be from 1.5 minutes to 2.5 minutes such as 2.0 minutes. Thus, the seeding process may be initiated within a relative short time.

    [0057] The precooling time may be from 0.1 minutes to 5.0 minutes. As the cooling tube outer surface seeding temperature is reached within the precooling time, the cooling tube outer surface seeding temperature is reached within a relative short time.

    [0058] As the plurality of vials may be engaged with the cooling tubes for the predetermined time, seeding occurs in a timely controlled manner.

    [0059] Particularly, the seeding process may be initiated by means of local engagement of the plurality of vials with the cooling tubes such that crystallization of cellular samples is locally induced.

    [0060] The refrigerant may be supplied to the cooling tubes until reaching the cooling tube outer surface seeding temperature such that a local crystallization seed is formed at the cellular sample for the predetermined time when the plurality of vials is engaged with the cooling tubes.

    [0061] With other words, the seeding process may be initiated by means of local engagement of the plurality of vials with the cooling tubes for a predetermined time until reaching the cooling tube outer surface seeding temperature such that crystallization of the cellular samples is enforced by a temporary, local and strong or intensive cold spot in the cellular samples.

    [0062] The refrigerant may be supplied with the cooling tubes with the cooling tube outer surface seeding temperature such that all of the cellular samples crystallize substantially at the same time.

    [0063] The cooling tube outer surface seeding temperature may be from -130 °C to -200 °C. According to another embodiment, the cooling tube outer surface seeding temperature may be from -140 °C to -190 °C such as -180 °C.

    [0064] The plurality of vials may be moved relative to the support so as to be engaged with the cooling tubes.

    [0065] The seeding temperature may be close to a freezing temperature of the cellular sample. The term "close to a freezing temperature" as used herein refers to a temperature having a deviation of not more than 4 K below the freezing temperature.

    [0066] The vials may be disengaged from the cooling tubes when the cooling chamber is cooled to the seeding temperature.

    [0067] When seeding is induced in the plurality of vials, the vials are disengaged from the cooling tubes.

    [0068] Subsequently, the cooling tubes are cooled to the final temperature such that latent heat of the cellular samples may be removed.

    [0069] The latent heat may be removed by means of dissipation. Particularly, the latent heat is removed by controlling velocity and temperature of a gaseous refrigerant flow within the cooling chamber.

    [0070] The final temperature may be from -120 °C to -190 °C. According to another embodiment, the final temperature may be from -125 °C to -190 °C such as -180 °C.

    Short description of the figures



    [0071] Further features and embodiments of the invention will be disclosed in more detail in the subsequent description of embodiments. The embodiments are schematically depicted in the figures. Therein, identical reference numbers in these figures refer to identical or functionally comparable elements. The invention is defined in the appended claims.

    [0072] In the figures:
    Figure 1
    shows an apparatus for cryopreserving a plurality of cellular samples,
    Figure 2
    shows a cross-sectional view of the apparatus,
    Figure 3
    shows a plain view of the cooling tubes,
    Figure 4
    shows an operation of the support, and
    Figure 5
    shows the further operation of the support.

    Detailed description of the embodiments



    [0073] Figure 1 shows an apparatus for cryopreserving a plurality of cellular samples. The apparatus 100 comprises a cooling chamber 102, a cooling device 104 and a support 106. The cooling chamber 102 is cylindrical. Particularly, the cooling chamber 102 is circular cylindrical. The apparatus 100 further comprises an inner casing 108, in which the cooling chamber 102 is located, or which defines the cooling chamber 102, an outer casing 110 housing the inner casing 108, and a middle casing 112 arranged between the inner casing 108 and the outer casing 110. The inner casing 108, the outer casing 110 and the middle casing 112 may be configured to form a modular casing assembly 114 comprising an upper part 116 and a lower part 118. With other words, each of the inner casing 108, the outer casing 110 and the middle casing 112 is separated into two parts forming the upper part 116 and the lower part 118 as will be explained in more detail below. The upper part 116 is arranged on the top of the lower part 118. The outer casing 110 houses the inner casing 108 such that a space 120 is formed between the inner casing 108 and the outer casing 110. More particularly, the space 120 is formed between the inner casing 108 and the middle casing 112.

    [0074] The inner casing 108 comprises a lower side 122 and a top side 124. The lower side 122 and the top side 124 comprise orifices 126. More particularly, the lower side 122 and the top side 124 comprise perforated plates 128. The perforated plates 128 comprise the orifices 126. Needless to say, the lower side 122 and the top side 124 may be designed as perforated plates 128. The perforated plates 128 comprise a perforation ratio of 5 % to 15 % such as 10 %. The perforation ratio may vary over the plate radius. Further, the perforation ratio of the perforated plate 128 at the lower side 122 may be different from the perforation ratio of the perforated plate 128 at the top side 124. The orifices 126 may comprise circular cross-sectional areas. By means of the orifices 126, the space 120 is in fluid communication with the cooling chamber 102. The middle casing 112 comprises a lower side 130 and a top side 132. The middle casing further comprises an outlet 134. The outlet 134 of the middle casing 112 is located in the lower side 130. The outer casing 110 comprises a lower side 136 and a top side 138. The outer casing 110 further comprises an outlet 140. The outlet 140 of the outer casing 110 is located in the top side 138. The outlet 134 of the middle casing 112 and the outlet 140 of the outer casing 110 are connected to one another. For example, the outlets 134, 140 are connected to one another by means of a channel 142, space or the like formed between the outer casing 110 and the middle casing 112. The apparatus 100 further comprises a fan 144. The fan 144 is disposed between the top side 124 of the inner casing 108 and the outer casing 110.

    [0075] Figure 2 shows a cross-sectional view of the apparatus 100. The cooling device 104 is adapted to cool an interior of the cooling chamber 102. For this reason, the cooling device 104 comprises means configured to cool an interior of the cooling chamber 102 as will be explained in further detail below. The cooling device 104 further comprises cooling tubes 146 that are separate from the means configured to cool the interior of the cooling chamber 102. The cooling tubes 146 are arranged within the cooling chamber 102. The cooling device 104 is configured to provide a flow of refrigerant through the cooling tubes 146. The cooling tubes 146 are arranged parallel to one another. Further, the cooling tubes 146 are evenly spaced apart from one another. The cooling device 104 further comprises a ring-shaped tube portion 148. The cooling tubes 146 are connected to the ring-shaped tube portion 148. Particularly, the ring-shaped tube portion 148 surrounds the cooling tubes 146. The cooling device 104 further comprises nozzles 150 for injecting a refrigerant into the cooling chamber so as to cool the interior of the cooling chamber 102. Thus, the nozzles 150 serve as means to cool the interior of the cooling chamber 102. The nozzles 150 are disposed in the space 120 between the inner casing 108 and the outer casing 110. The cooling device 104 further comprises an inlet 152, which is located outside the casing assembly 114, and distribution tubes 154 connecting the inlet 152 to the nozzles 150. As shown in Figure 2, the nozzles 150 are evenly distributed in the space 120 between the inner casing 108 and the outer casing 110. For example, the distribution tubes 154 extend radially outward from the inlet 152 and mainly below the casing assembly 114. For example, the distribution tubes 154 are arranged in a star-shaped manner around the inlet 152. Further, the distribution tubes 154 extend upward laterally outside the casing assembly 114 and extend through the lower part 118 into the space 120 so as to be connected to the nozzles 150. It is to be noted that the lengths of the distribution tubes 154 are identical such that distances from the nozzles 150 to the inlet 152 are identical. Merely as an example, 10 nozzles 150 are shown in Figure 2 arranged at even angular positions around the inlet 152. Needless to say, more or less than 10 nozzles 150 may be provided such as 8, 12, 20 depending on the respective geometry of the apparatus 100. The nozzles 150 are located within a common plane 156. The inlet 152 is located on a central axis 158 of the cooling chamber 102. The central axis 158 corresponds to a cylinder axis of the cylindrically shaped cooling chamber 102.

    [0076] Figure 3 shows a plain view of a part of the cooling device 104. Particularly, Figure 3 shows the cooling tubes 146 and the ring-shaped tube portion 148. As shown in Figure 3, the cooling tubes 146 comprise ends 160 connected to the ring-shaped tube portion 148. Further, the ring-shaped tube portion 148 comprises inlets 162 for supplying a refrigerant into the ring-shaped tube portion 148. The ring-shaped tube portion 148 is separated into segments 164. Each segment 164 is associated with at least one of the inlets 162. Particularly, the ring-shaped tube portion 148 is separated into the segments 164 by means of separating walls 166. The segments 164 are adapted to allow a substantially sufficient flow of the refrigerant through the cooling tubes 146. The inlets 162 are evenly spaced apart from one another. Further, an inner diameter of the ring-shaped tube portion 148 is larger than an inner diameter of the cooling tubes 146. With this construction, the cooling device 104 is adapted to provide a refrigerant flow from a bottom to top as will be explained in further detail below. Particularly, Figure 3 shows 20 cooling tubes 146 and four segments 164 separated from one another by means of three separating walls 166. By means of these segments 164, the refrigerant evenly distributes through the cooling tubes 146. More particularly, the segments 164 shown at the left and right sides with respect to the illustration of Figure 3 supply five cooling tubes 146 with refrigerant while the inner segments 164 supply four cooling tubes with refrigerant.

    [0077] As shown in Figures 1 and 2, the support 106 is adapted to support a plurality of vials 168 for storing cellular samples. The support 106 comprises a plurality of compartments 170 for receiving the plurality of vials 168. For example, the support 106 comprises 100, 200 or even more compartments 170 for receiving 100, 200 or even more vials 168. With other words, each compartment 170 is adapted to receive one vial 168. The compartments 170 are arranged such that the plurality of vials 168 is arrangeable between the cooling tubes 146. As shown in Figures 1 and 2, the compartments 170 are arranged as lines 172, which extend parallel to one another. Further, the cooling tubes 146 and the compartments 170 are arranged parallel to one another. Further, the compartments 170 are movable relative to the support 106. Particularly, the compartments 170 are movable in the support 106 as will be described in further detail below. The compartments 170 are adapted to fix the plurality of vials 168. For example, the compartments 170 comprise elastic clamps or the like which are configured to securely hold the vials 168 in their position.

    [0078] Figure 4 shows the support 106 in a first operation state and Figure 5 shows the support 106 in a second operation state. As shown in Figures 4 and 5, the support 106 is movable relative to the cooling tubes 146 such that the plurality of vials 168 is engageable with the cooling tubes 146 as shown in Figure 5. For example, the support 106 may be movable on rails 174. The compartments 170 are formed such that the plurality of vials 168 is directly engageable with the cooling tubes 146. With other words, the compartments 170 are formed such that the cooling tubes 146 may directly engage the vials 168 without any further constructional member between the cooling tubes 146 and the vials 168 during engagement. Particularly, the support 106 is movable such that the plurality of vials 168 is engageable with the cooling tubes 146 at a predetermined height 176 of the vials 168. The height 176 corresponds to a liquid surface 178 of cellular samples 180 stored in the vials 168 and is defined as a position along a longitudinal direction in which the vials 168 extend. The support 106 can be adjusted to change the engagement point in height 176 of a vial 168. The support 106 is adapted to support the plurality of vials 168 in a common plane 182. The cooling chamber 102 is adapted to be arranged such that the common plane 182 is perpendicular to a direction of gravity. The cooling device 104 is adapted to supply the cooling tubes 146 with refrigerant in a liquid state such that a seeding process of the cellular samples and the vials 168 is initiated by means of engagement of the vials with the cooling tubes 146.

    [0079] Hereinafter, a method for cryopreserving a plurality of cellular samples 180 using the apparatus 100 will be described. Basically, the cellular samples 180 are provided in a liquid state in the plurality of vials 168 at a beginning of the method. The cooling chamber 102 is cooled to a temperature hold point. The temperature hold point is close to, but above the freezing temperature of the cellular samples 180. In order to cool the cooling chamber 102 to the temperature hold point, the cooling device 104 supplies a refrigerant such as liquid nitrogen from the inlet 152 through the distribution pipes 154 to the nozzles 150. The nozzles 150 are configured to spray or inject the refrigerant into the space 120. Further, the fan 144 is operated. The refrigerant discharged from the nozzles 150 flows along the lower side 122 of the inner casing 108 as indicated by arrows 184 in Figure 1 and may enter the interior of the cooling chamber 102 through the orifices 126 of the perforated plate 128 at the lower side 122 of the inner casing 108.

    [0080] Then, the support 106 is loaded with the plurality of vials 168. In the interior of the cooling chamber 102, the refrigerant removes heat from the vials 168 and the cellular sample 180 provided in the support 106. Thereby, the refrigerant is heated and flows from the bottom to the top of the cooling chamber 102 caused by convection effects and by being sucked from the fan 144 as indicated by arrow 186. The heated refrigerant leaves the cooling chamber 102 through the orifices 126 of the perforated plate 128 at the top side 124 of the inner casing 108 and enters the space 120 again. The fan 144 blows the heated refrigerant in lateral and downwards directions towards the outlet 134 of the middle casing 112 as indicated by arrows 188, 190. The refrigerant then flows from the outlet 134 of the middle casing 112 to the outlet 140 of the outer casing 110 within the channel 1 146 as indicated by arrows 192 and is discharged from the outer casing 110 through the outlet 140 thereof. The cooling chamber 102 is held at the temperature hold point so as to allow temperature synchronization in the plurality of vials 168. With other words, the cooling chamber 102 is held at the temperature hold point until all of the vials 168 have the same temperature. The temperature hold point is from 0°C to 5°C such as 2°C. Thereafter, the cooling chamber 102 is cooled to a seeding temperature in the vials 168. The cooling chamber 102 is further cooled to the seeding temperature by means of the cooling device 104 which supplies more refrigerant to the nozzles 150 and the interior of the cooling chamber 102. The seeding temperature is from a freezing point of the cellular samples to -15°C such as -5°C. At the same time, a refrigerant is supplied through the cooling tubes 146 for a precooling time such that the cooling tubes 146 reach a cooling tube outer surface seeding temperature before the seeding temperature is reached. The refrigerant is supplied to the cooling tubes 146 from the ring-shaped tube 148 which in turn is supplied with the refrigerant through the inlets 162. The precooling time is from 0.1 minutes to 5.0 minutes such as 3.0 minutes. The cooling tube outer surface seeding temperature is from -130 °C to -200 °C and particularly from -140 °C to -190 °C such as -150°C. The cooling device provides a refrigerant flow from bottom to top through the cooling tubes 146 until reaching the cooling tube outer surface seeding temperature within the precooling time.

    [0081] When the cooling tube outer surface seeding temperature and the seeding temperature are met, i.e. the cooling tube outer surface seeding temperature corresponds to the seeding temperature, the support 106 is moved relative to the cooling tubes 146 such that the plurality of vials 168 is engaged with the cooling tubes 146 for a predetermined time so as to initiate a seeding process in the cellular samples 180. Particularly, the support 106 is movable such that the plurality of vials 168 is engageable with the cooling tubes 146 at the predetermined height 176 of the vials 168 corresponding to the liquid surface 178 of cellular samples 180. The predetermined time is from 0.5 minutes to 3.0 minutes, particularly from 1.5 minutes to 2.5 minutes such as 2.0 minutes. The plurality of vials 168 is moved relative to the support 106 so as to be engaged with the cooling tubes 146. More particularly, the compartments 170 in which the vials 168 are provided are moved in the support 106 so as to allow a kind of fine-tuning and to individually engage each of the vials 168 with the cooling tubes 146. The seeding process is initiated by means of local engagement of the plurality of vials 168 with the cooling tubes 146 for the predetermined time such that crystallization of the cellular samples 180 is locally induced. With other words, the seeding process is initiated by means of local engagement of the plurality of vials 168 with the cooling tubes 146 for a predetermined time until reaching the cooling tube outer surface seeding temperature such that crystallization of the cellular samples 180 is enforced by a temporary, local and strong or intensive cold spot in the cellular samples. The refrigerant is supplied through the cooling tubes 146 until reaching the cooling tube outer surface seeding temperature such that a local crystallization seed is formed at the cellular samples 180 for the predetermined time when the plurality of vials 168 is engaged with the cooling tubes 146. Particularly, the refrigerant is supplied through the cooling tubes 146 with the cooling tube outer surface seeding temperature such that all of the cellular samples 180 crystallize substantially at the same time.

    [0082] When seeding has been induced in the plurality of vials 168, the vials 168 are disengaged from the cooling tubes 146. Further, the cooling chamber 102 is cooled to a final temperature. The final temperature is from -120°C to -190°C such as -185°C. Until the final temperature is reached, latent heat is controlled removed from the cellular samples 180. The latent heat is removed by controlling velocity and temperature of a gaseous refrigerant flow within the cooling chamber 102. The gaseous flow of the refrigerant is generated by operation of the fan 144. The latent heat is removed by means of dissipation.

    List of reference numbers



    [0083] 
    100
    apparatus
    102
    cooling chamber
    104
    cooling device
    106
    support
    108
    inner casing
    110
    outer casing
    112
    middle casing
    114
    casing assembly
    116
    upper part
    118
    lower part
    120
    space
    122
    lower side
    124
    top side
    126
    orifices
    128
    perforated plates
    130
    lower side
    132
    top side
    134
    outlet
    136
    lower side
    138
    top side
    140
    outlet
    142
    channel
    144
    fan
    146
    cooling tubes
    148
    ring-shaped tube portion
    150
    nozzle
    152
    inlet
    154
    distribution tubes
    156
    common plane
    158
    central axis
    160
    ends
    162
    inlets
    164
    segments
    166
    separating wall
    168
    vial
    170
    compartment
    172
    line
    174
    rails
    176
    height
    178
    liquid surface
    180
    cellular sample
    182
    common plane
    184
    arrow
    186
    arrow
    188
    arrow
    190
    arrow
    192
    arrow



    Claims

    1. Apparatus (100) for cryopreserving a plurality of cellular samples (174), comprising a cooling chamber (102),
    a cooling device (104) comprising means configured to cool an interior of the cooling chamber (102), wherein the cooling device (104) further comprises cooling tubes (146) that are separate from the means configured to cool the interior of the cooling chamber (102) and that are arranged within the cooling chamber (102), wherein the cooling device (104) is configured to provide a flow of refrigerant through the cooling tubes (146), and
    a support (106) for supporting a plurality of vials (168) for storing cellular samples (180), wherein the support (106) is movable relative to the cooling tubes (146) such that the plurality of vials (168) is engageable with the cooling tubes (146).
     
    2. Apparatus (100) according to claim 1, wherein the support (106) is moveable such that the plurality of vials (168) is engageable with the cooling tubes (146) at a predetermined and adjustable height (176) of the vials (168), wherein, in use, the height (176) may correspond to a liquid surface (178) of the cellular samples (180).
     
    3. Apparatus (100) according to any claim 1 or 2, wherein the cooling tubes (146) are arranged parallel to one another.
     
    4. Apparatus (100) according to any one of claims 1 to 3, wherein the support (106) comprises a plurality of compartments (170) for receiving the plurality of vials (168), wherein the cooling tubes (146) and the compartments (170) are arranged parallel to one another, wherein the compartments (170) are arranged such that the plurality of vials (168) is arrangeable between the cooling tubes (146), and wherein the compartments (170) are moveable relative to the support (106) such that each of the plurality of vials (168) is individually engageable with at least one of the cooling tubes (146).
     
    5. Apparatus (100) according to any one of claims 1 to 4 wherein the cooling device (104) is adapted to provide a sufficient flow of refrigerant through the cooling tubes (146) such that the cooling tubes (146) reach a cooling tube outer surface seeding temperature within a precooling time.
     
    6. Apparatus (100) according to claim 5, wherein the cooling device (104) is adapted to provide a refrigerant flow from bottom to top of the cooling tubes (146) until reaching the cooling tube outer surface seeding temperature within the precooling time.
     
    7. Apparatus (100) according to any one of claims 1 to 6, further comprising an inner casing (108), in which the cooling chamber (102) is located, and an outer casing (110) housing the inner casing (108), wherein the inner casing (108) comprises a lower side (122) and a top side (124), wherein the lower side (122) and the top side (124) comprise orifices (126), wherein the lower side (122) and the top side (124) comprise perforated plates (128), and wherein the perforated plates (128) comprise the orifices (126).
     
    8. Apparatus (100) according to claim 7, wherein the perforated plates (128) comprise a perforation ratio of 5 % to 15 %.
     
    9. Apparatus (110) according to any one of claims 1 to 8, wherein the support (106) is adapted to support the plurality of vials (168) in a common plane (178).
     
    10. Method for cryopreserving a plurality of cellular samples (180) using an apparatus (100) according to any one of claims 1 to 9, comprising the following steps:

    - cooling the cooling chamber to a temperature hold point,

    - providing cellular samples (180) in a liquid state in a plurality of vials (168),

    - loading the support (106) with the plurality of vials (168),

    - holding the cooling chamber (102) at the temperature hold point so as to allow temperature synchronization in the plurality of vials (168),

    - cooling the cooling chamber to a seeding temperature in the vials (168),

    - supplying a refrigerant through the cooling tubes (146) for a precooling time such that the cooling tubes (146) reach a cooling tube outer surface seeding temperature before the seeding temperature is reached,

    - moving the support (106) relative to the cooling tubes (146) when the cooling tube outer surface seeding temperature and the seeding temperature are met such that the plurality of vials (168) is engaged with the cooling tubes (146) for a predetermined time so as to initiate a seeding process in the cellular samples (180),

    - cooling the cooling chamber (102) to a final temperature, and

    - controlled removal of latent heat until the final temperature is reached.


     
    11. Method according to claim 10, wherein the seeding process is initiated by means of local engagement of the plurality of vials (168) with the cooling tubes (146) for a predetermined time such that crystallization of the cellular samples (180) is locally induced.
     
    12. Method according to claim 10 or 11, wherein the refrigerant is supplied through the cooling tubes (146) until reaching the cooling tube outer surface seeding temperature such that a local crystallization seed is formed at the cellular sample for the predetermined time when the plurality of vials (168) is engaged with the cooling tubes (146).
     
    13. Method according to any one of claim 10 to 12, wherein the predetermined time is from 0.5 minutes to 3.0 minutes, and wherein the precooling time is from 0.1 minutes to 5.0 minutes.
     
    14. Method according to any one of claim 10 to 13, wherein the refrigerant is supplied through the cooling tubes (146) with the cooling tube outer surface seeding temperature such that all of the cellular samples (180) crystallize substantially at the same time.
     
    15. Method according to any one of claims 10 to 14, wherein the temperature hold point is from 0°C to 5°C, wherein the seeding temperature is from a freezing point of the cellular samples to -15°C, wherein the final temperature is from -120°C to -190°C, and wherein the cooling tube outer surface seeding temperature is from -130 °C to -200 °C.
     
    16. Method according to any one of claims 10 to 15, wherein the support comprises a plurality of compartments for receiving the plurality of vials (168), and the plurality of compartments is moved relative to the support so as to individually engage each vial with a cooling tube in an identical manner.
     
    17. Method according to any one of claims 10 to 16, wherein the vials (168) are disengaged from the cooling tubes (146) when seeding is induced in the plurality of vials (168).
     
    18. Method according to any one of claims 10 to 17, wherein the latent heat is removed by controlling velocity and temperature of a gaseous refrigerant flow within the cooling chamber (102).
     


    Ansprüche

    1. Vorrichtung (100) zur Kryokonservierung einer Vielzahl von Zellproben (174), umfassend eine Kühlkammer (102),
    eine Kühlvorrichtung (104) umfassend Mittel, die dazu eingerichtet sind, ein Inneres der Kühlkammer (102) zu kühlen, wobei die Kühlvorrichtung (104) ferner Kühlrohre (146) umfasst, die von den Mitteln zum Kühlen des Inneren der Kühlkammer (102) getrennt sind und die innerhalb der Kühlkammer (102) angeordnet sind, wobei die Kühlvorrichtung (104) dazu eingerichtet ist, einen Kältemittelfluss durch die Kühlrohre (146) bereitzustellen, und
    eine Halterung (106) zum Halten einer Vielzahl von Röhrchen (168) zum Aufnehmen von Zellproben (180), wobei die Halterung (106) relativ zu den Kühlrohren (146) beweglich ist, sodass die Vielzahl von Röhrchen (168) mit den Kühlrohren (146) in Eingriff gebracht werden kann.
     
    2. Vorrichtung (100) nach Anspruch 1, wobei die Halterung (106) so beweglich ist, dass die Vielzahl von Röhrchen (168) mit den Kühlrohren (146) in einer vorbestimmten und einstellbaren Höhe (176) der Röhrchen (168) in Eingriff bringbar ist, wobei die Höhe (176) im Betrieb einer Flüssigkeitsoberfläche (178) der Zellproben (180) entsprechen kann.
     
    3. Vorrichtung (100) nach Anspruch 1 oder 2, wobei die Kühlrohre (146) parallel zueinander angeordnet sind.
     
    4. Vorrichtung (100) nach einem der Ansprüche 1 bis 3, wobei die Halterung (106) eine Vielzahl von Fächern (170) zum Aufnehmen der Vielzahl von Röhrchen (168) aufweist, wobei die Kühlrohre (146) und die Fächer (170) parallel zueinander angeordnet sind, wobei die Fächer (170) so angeordnet sind, dass die Vielzahl von Röhrchen (168) zwischen den Kühlrohren (146) angeordnet werden kann, und wobei die Fächer (170) relativ zur Halterung (106) so beweglich sind, dass jedes der Vielzahl von Röhrchen (168) einzeln mit mindestens einem der Kühlrohre (146) in Eingriff bringbar ist.
     
    5. Vorrichtung (100) nach einem der Ansprüche 1 bis 4, wobei die Kühlvorrichtung (104) dazu angepasst ist, einen ausreichenden Kältemittelfluss durch die Kühlrohre (146) bereitzustellen, sodass die Kühlrohre (146) innerhalb einer Vorkühlzeit eine Keimtemperatur der Kühlrohraußenfläche erreichen.
     
    6. Vorrichtung (100) nach Anspruch 5, wobei die Kühlvorrichtung (104) dazu angepasst ist, innerhalb der Vorkühlzeit einen Kältemittelfluss von der Unterseite zur Oberseite der Kühlrohre (146) bis zum Erreichen der Keimtemperatur der Kühlrohraußenfläche bereitzustellen.
     
    7. Vorrichtung (100) nach einem der Ansprüche 1 bis 6, ferner umfassend ein Innengehäuse (108), in dem die Kühlkammer (102) angeordnet ist, und ein Außengehäuse (110), das das Innengehäuse (108) aufnimmt, wobei das Innengehäuse (108) eine Unterseite (122) und eine Oberseite (124) umfasst, wobei die Unterseite (122) und die Oberseite (124) Öffnungen (126) umfassen, wobei die Unterseite (122) und die Oberseite (124) Lochplatten (128) umfassen und wobei die Lochplatten (128) die Öffnungen (126) umfassen.
     
    8. Vorrichtung (100) nach Anspruch 7, wobei die Lochplatten (128) ein Lochverhältnis von 5 % bis 15 % aufweisen.
     
    9. Vorrichtung (110) nach einem der Ansprüche 1 bis 8, wobei die Halterung (106) dazu angepasst ist, die Vielzahl von Röhrchen (168) in einer gemeinsamen Ebene (178) zu halten.
     
    10. Verfahren zum Kryokonservieren einer Vielzahl von Zellproben (180) unter Verwendung einer Vorrichtung (100) nach einem der Ansprüche 1 bis 9, umfassend die folgenden Schritte:

    - Kühlen der Kühlkammer auf einen Temperaturhaltepunkt,

    - Bereitstellen von Zellproben (180) in flüssigem Zustand in einer Vielzahl von Röhrchen (168),

    - Beladen der Halterung (106) mit der Vielzahl von Röhrchen (168),

    - Halten der Kühlkammer (102) auf dem Temperaturhaltepunkt, um die Temperatursynchronisation in der Vielzahl von Röhrchen (168) zu ermöglichen,

    - Kühlen der Kühlkammer auf eine Keimtemperatur in den Röhrchen (168),

    - Zuführen eines Kältemittels durch die Kühlrohre (146) während einer Vorkühlzeit, sodass die Kühlrohre (146) eine Keimtemperatur der Kühlrohraußenfläche erreichen, bevor die Keimtemperatur erreicht wird,

    - Bewegen der Halterung (106) relativ zu den Kühlrohren (146), wenn die Keimtemperatur der Kühlrohraußenfläche und die Keimtemperatur erreicht sind, sodass die Vielzahl von Röhrchen (168) für eine vorbestimmte Zeit mit den Kühlrohren (146) in Eingriff gebracht wird, um einen Keimvorgang in den Zellproben (180) einzuleiten,

    - Kühlen der Kühlkammer (102) auf eine Endtemperatur, und

    - kontrolliertes Abführen der latenten Wärme, bis die Endtemperatur erreicht ist.


     
    11. Verfahren nach Anspruch 10, wobei der Keimvorgang durch lokalen Eingriff der Vielzahl von Röhrchen (168) mit den Kühlrohren (146) für eine vorbestimmte Zeit eingeleitet wird, sodass die Kristallisation der Zellproben (180) lokal eingeleitet wird.
     
    12. Verfahren nach Anspruch 10 oder 11, wobei das Kältemittel durch die Kühlrohre (146) zugeführt wird, bis die Keimtemperatur der Kühlrohraußenfläche erreicht ist, sodass für die vorbestimmte Zeit ein lokaler Kristallisationskeim an der Zellprobe gebildet wird, wenn die Vielzahl von Röhrchen (168) mit den Kühlrohren (146) in Eingriff gebracht ist.
     
    13. Verfahren nach einem der Ansprüche 10 bis 12, wobei die vorbestimmte Zeit 0,5 Minuten bis 3,0 Minuten beträgt, und wobei die Vorkühlzeit 0,1 Minuten bis 5,0 Minuten beträgt.
     
    14. Verfahren nach einem der Ansprüche 10 bis 13, wobei das Kältemittel durch die Kühlrohre (146) mit einer Keimtemperatur der Kühlrohraußenfläche so zugeführt wird, dass alle Zellproben (180) im Wesentlichen gleichzeitig kristallisieren.
     
    15. Verfahren nach einem der Ansprüche 10 bis 14, wobei der Temperaturhaltepunkt zwischen 0 °C und 5 °C liegt, wobei die Keimtemperatur zwischen einem Gefrierpunkt der Zellproben und -15 °C liegt, wobei die Endtemperatur zwischen -120 °C und -190 °C liegt, und wobei die Keimtemperatur der Kühlrohraußenfläche zwischen -130 °C und -200 °C liegt.
     
    16. Verfahren nach einem der Ansprüche 10 bis 15, wobei die Halterung eine Vielzahl von Fächern zur Aufnahme der Vielzahl von Röhrchen (168) umfasst und die Vielzahl von Fächern relativ zur Halterung so bewegt wird, dass jedes Röhrchen einzeln in gleicher Weise mit einem Kühlrohr in Eingriff kommt.
     
    17. Verfahren nach einem der Ansprüche 10 bis 16, wobei die Röhrchen (168) von den Kühlrohren (146) gelöst werden, wenn in der Vielzahl der Röhrchen (168) eine Keimung eingeleitet wird.
     
    18. Verfahren nach einem der Ansprüche 10 bis 17, wobei die latente Wärme durch Steuern der Geschwindigkeit und Temperatur eines gasförmigen Kältemittelflusses innerhalb der Kühlkammer (102) abgeführt wird.
     


    Revendications

    1. Appareil (100) pour la cryopréservation d'une pluralité d'échantillons cellulaires (174), comprenant
    une chambre de refroidissement (102),
    un dispositif de refroidissement (104) comprenant un moyen conçu pour refroidir un intérieur de la chambre de refroidissement (102), le dispositif de refroidissement (104) comprenant en outre des tubes de refroidissement (146) qui sont séparés du moyen conçu pour refroidir l'intérieur de la chambre de refroidissement (102) et qui sont disposés à l'intérieur de la chambre de refroidissement (102), le dispositif de refroidissement (104) étant conçu pour fournir un flux de réfrigérant à travers les tubes de refroidissement (146), et
    un support (106) pour supporter une pluralité de flacons (168) pour le stockage d'échantillons cellulaires (180), le support (106) étant mobile par rapport aux tubes de refroidissement (146) de sorte que la pluralité de flacons (168) puisse venir en prise avec les tubes de refroidissement (146).
     
    2. Appareil (100) selon la revendication 1, le support (106) étant mobile de sorte que la pluralité de flacons (168) puisse venir en prise avec les tubes de refroidissement (146) à une hauteur (176) prédéfinie et réglable des flacons (168), lors de l'utilisation, la hauteur (176) pouvant correspondre à une surface liquide (178) des échantillons cellulaires (180).
     
    3. Appareil (100) selon la revendication 1 ou 2, les tubes de refroidissement (146) étant disposés parallèlement les uns aux autres.
     
    4. Appareil (100) selon l'une quelconque des revendications 1 à 3, le support (106) comprenant une pluralité de compartiments (170) pour recevoir la pluralité de flacons (168), les tubes de refroidissement (146) et les compartiments (170) étant disposés parallèlement les uns aux autres, les compartiments (170) étant disposés de sorte que la pluralité de flacons (168) puisse être disposée entre les tubes de refroidissement (146), et les compartiments (170) étant mobiles par rapport au support (106) de sorte que chaque flacon de la pluralité de flacons (168) puisse venir individuellement en prise avec au moins un des tubes de refroidissement (146).
     
    5. Appareil (100) selon l'une quelconque des revendications 1 à 4, le dispositif de refroidissement (104) étant conçu pour fournir un flux suffisant de réfrigérant à travers les tubes de refroidissement (146) de sorte que les tubes de refroidissement (146) atteignent une température d'ensemencement de surface extérieure de tube de refroidissement dans un temps de pré-refroidissement.
     
    6. Appareil (100) selon la revendication 5, le dispositif de refroidissement (104) étant conçu pour fournir un flux de réfrigérant de bas en haut des tubes de refroidissement (146) jusqu'à ce qu'il atteigne la température d'ensemencement de surface extérieure de tube de refroidissement dans le temps de pré-refroidissement.
     
    7. Appareil (100) selon l'une quelconque des revendications 1 à 6, comprenant en outre une enveloppe intérieure (108), dans laquelle est située la chambre de refroidissement (102), et une enveloppe extérieure (110) logeant l'enveloppe intérieure (108), l'enveloppe intérieure (108) comprenant un côté inférieur (122) et un côté supérieur (124), la face inférieure (122) et la face supérieure (124) comprenant des orifices (126), le côté inférieur (122) et le côté supérieur (124) comprenant des plaques perforées (128), et les plaques perforées (128) comprenant les orifices (126).
     
    8. Appareil (100) selon la revendication 7, les plaques perforées (128) comprenant un taux de perforation de 5 % à 15 %.
     
    9. Appareil (110) selon l'une quelconque des revendications 1 à 8, le support (106) étant conçu pour supporter la pluralité de flacons (168) dans un plan commun (178).
     
    10. Procédé de cryopréservation d'une pluralité d'échantillons cellulaires (180) à l'aide d'un appareil (100) selon l'une quelconque des revendications 1 à 9, comprenant les étapes consistant à :

    refroidir la chambre de refroidissement jusqu'à un point de maintien de la température,

    fournir des échantillons cellulaires (180) à l'état liquide dans une pluralité de flacons (168),

    charger le support (106) avec la pluralité de flacons (168),

    maintenir la chambre de refroidissement (102) au point de maintien de la température de sorte à permettre la synchronisation de la température dans la pluralité de flacons (168),

    refroidir la chambre de refroidissement à une température d'ensemencement dans les flacons (168),

    fournir un réfrigérant à travers les tubes de refroidissement (146) pendant un temps de pré-refroidissement tel que les tubes de refroidissement (146) atteignent une température d'ensemencement de surface extérieure de tube de refroidissement avant que la température d'ensemencement ne soit atteinte,

    déplacer le support (106) par rapport aux tubes de refroidissement (146) lorsque la température d'ensemencement de surface extérieure de tube de refroidissement et la température d'ensemencement sont atteintes, de sorte que la pluralité de flacons (168) vienne en prise avec les tubes de refroidissement (146) pendant un temps prédéfini de manière à initier un processus d'ensemencement dans les échantillons cellulaires (180),

    refroidir la chambre de refroidissement (102) à une température finale, et

    éliminer de manière contrôlée la chaleur latente jusqu'à ce que la température finale soit atteinte.


     
    11. Procédé selon la revendication 10, le processus d'ensemencement étant initié au moyen d'une mise en prise locale de la pluralité de flacons (168) avec les tubes de refroidissement (146) pendant un temps prédéfini de sorte que la cristallisation des échantillons cellulaires (180) soit induite localement.
     
    12. Procédé selon la revendication 10 ou 11, le réfrigérant étant fourni à travers les tubes de refroidissement (146) jusqu'à ce qu'il atteigne la température d'ensemencement de surface extérieure de tube de refroidissement de sorte qu'un germe de cristallisation locale soit formé au niveau de l'échantillon cellulaire pendant le temps prédéfini où la pluralité de flacons (168) vient en prise avec les tubes de refroidissement (146).
     
    13. Procédé selon l'une quelconque des revendications 10 à 12, le temps prédéfini étant de 0,5 minute à 3,0 minutes, et le temps de pré-refroidissement étant de 0,1 minute à 5,0 minutes.
     
    14. Procédé selon l'une quelconque des revendications 10 à 13, le réfrigérant étant fourni à travers les tubes de refroidissement (146) avec la température d'ensemencement de surface extérieure de tube de refroidissement de sorte que tous les échantillons cellulaires (180) cristallisent sensiblement en même temps.
     
    15. Procédé selon l'une quelconque des revendications 10 à 14, le point de maintien de la température étant de 0 °C à 5 °C, la température d'ensemencement étant comprise entre un point de congélation des échantillons cellulaires et -15 °C, la température finale étant de - 120 °C à -190 °C, et la température d'ensemencement de surface extérieure de tube de refroidissement étant de - 130 °C à -200 °C.
     
    16. Procédé selon l'une quelconque des revendications 10 à 15, le support comprenant une pluralité de compartiments pour recevoir la pluralité de flacons (168), et la pluralité de compartiments étant déplacée par rapport au support de sorte à mettre individuellement en prise chaque flacon avec un tube de refroidissement de manière identique.
     
    17. Procédé selon l'une quelconque des revendications 10 à 16, les flacons (168) étant séparés des tubes de refroidissement (146) lorsque l'ensemencement est induit dans la pluralité de flacons (168).
     
    18. Procédé selon l'une quelconque des revendications 10 à 17, la chaleur latente étant éliminée en contrôlant la vitesse et la température d'un flux de réfrigérant gazeux dans la chambre de refroidissement (102).
     




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    Cited references

    REFERENCES CITED IN THE DESCRIPTION



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    Patent documents cited in the description




    Non-patent literature cited in the description