JP5607896B2 - Cryopreservation method, freezing device, cryopreservation system - Google Patents

Description

  The present invention is a device for cryopreserving cells and tissues, in particular, human iPS cells and human ES cells, these cells using a flash freezing method suitably used for cryopreserving animals and human embryos, etc. The present invention relates to a device and a method particularly suitably used for cryopreserving tissue.

  Various techniques relating to devices and methods for storing cells in a frozen state have been reported. For example, the composition of a medium (frozen solution) that is suitably used for rapidly freezing stem cells such as primate ES cells is described in Patent Document 1 (hereinafter referred to as a first conventional example). Specifically, a DAP medium containing DMSO, propylene glycol, a medium, and acetamide at specific concentrations is disclosed. A simple vitrification method, which is a stem cell freezing method using this medium, is also disclosed in the first conventional example. In the simple vitrification method, cells to be frozen, such as stem cells, are enclosed in a container that is not easily contaminated, such as an enclosed tube, using the medium, and the enclosed tube is immersed in liquid nitrogen, followed by rapid freezing. It is a method of thawing. Specifically, human ES cell colonies recovered by the passage operation are centrifuged, and the supernatant is removed to preliminarily pelletize the cells. Add 200 μL of DAP medium to cells, suspend gently, and transfer to a prepared cryopreservation tube (container). Grasp the tube with tweezers (after sealing the tube lid to make it an enclosed tube) and attach it to liquid nitrogen. Freeze in liquid nitrogen for 30-60 seconds, completely freeze to the inside, and then transfer to a liquid nitrogen storage container.

  Here, since the DAP medium has strong cytotoxicity, it is described that the process from adding the medium to the cells and immersing the tube in liquid nitrogen should be performed as quickly as possible, and within 15 seconds as a guideline.

  Also in Non-Patent Document 1 (hereinafter referred to as the second conventional example), aiming for 15 seconds or less as the time from the addition of the medium to the above-mentioned cells until the tube is immersed in liquid nitrogen, this operation is performed as much as possible on-ice. It is described that the cell should not be warmed.

WO2005 / 045007

Human pluripotent stem cell maintenance culture practice protocol (2008), RIKEN Center for Developmental and Regenerative Sciences, Human Stem Cell Research Support Office, 2008 edition, page 12

  The above conventional examples include (i) addition of a medium to cells, (ii) suspension of cells, (iii) transfer to a tube, (iv) sealing of the tube, (v) holding and transporting the tube, (vi It is necessary to perform a series of steps consisting of a number of steps of immersing the tube in liquid nitrogen in a short time. Therefore, there is a problem that the operator needs to fully master the operation of a series of these critical processes in advance, and it is necessary to pay close attention to the operation, which places a heavy burden on the operator. Here, since cells must be handled in a safety cabinet to prevent contamination before being sealed in a sealed container, operations from the steps (i) to (v) are performed in the safety cabinet. There are many restrictions. Moreover, since the container containing liquid nitrogen is difficult to decontaminate and is generally installed outside the safety cabinet, there is a restriction that the transport distance in the latter half of (v) becomes long. Under such restrictions, if it takes time to operate and it takes time, conversely, if you make a mistake such as operating procedure wrong or incomplete operation due to too much time constraint, adverse effects on cells will be caused. There is a problem that there is a risk of deteriorating or losing (losing) valuable stem cells.

  In addition, since the critical process has a time constraint, only one sample can be processed at a time, and when many samples are frozen, there is a problem that work efficiency is low. According to the second conventional example, when the number of passages of human pluripotent stem cells such as human iPS cells and ES cells increases, the frequency of abnormalities in proliferation, differentiation potential, undifferentiation, karyotype, etc. gradually increases. It is said that it is better to conduct experiments with relatively young passage numbers, and it is recommended to make as many frozen stocks of cells as possible. Despite the demand for freezing a large number of cells, there is a problem that the work efficiency is low and the burden on the operator is extremely large.

  Therefore, the present invention pays attention to the above-mentioned problems, and when cells are frozen and stored in a freezing solution, the burden on the operator is reduced, damage to the cells is suppressed, and the cells are frozen in a short time. An object is to provide a cryopreservation method, a cryodevice, and a cryopreservation system that perform preservation.

In order to achieve the above object, a cryopreservation method according to the present invention is a cryopreservation method in which a suspension of a sample and a freezing solution for the sample is formed and the suspension is cryopreserved. Forming a freezing device having a first section for storing the second section and a second section for storing the frozen liquid, and a hollow tube for storing the frozen liquid at a boundary between the first section and the second section ; A nozzle formed at a tip of the tube and capable of holding the frozen liquid by surface tension, and the first operation is performed by a non-contact operation that is an operation of applying an acceleration output from the nozzle to the freezing device . The freezing liquid stored in the two compartments is formed by a supply means capable of supplying the sample stored in the first compartment, the non-contact operation is performed on the freezing device, and the freezing liquid is supplied to the sample via the supply means. It is characterized by doing.

  Further, the supply means includes a hollow tube for storing the frozen liquid, and a nozzle formed at a distal end of the tube and capable of holding the frozen liquid by surface tension. It is an operation of applying an acceleration output from the nozzle to the freezing device.

Furthermore, the cryopreservation method according to the present invention is a cryopreservation method in which a suspension of a sample and a frozen solution for the sample is formed and the suspension is cryopreserved. A freezing device having a compartment and a second compartment for containing the frozen liquid is formed, and a boundary between the first compartment and the second compartment is formed at a hollow tube for containing the frozen liquid and a tip of the tube. A second nozzle formed of a ferromagnetic material; and a magnet that is adsorbed by the ferromagnetic material and seals the second nozzle; and a magnetic field at which the magnet leaves the second nozzle. The frozen liquid stored in the second section is stored in the first section by a non-contact operation, which is an operation of outputting the frozen liquid from the second nozzle by applying and releasing the sealing of the second nozzle. The freezing device formed by a supply means capable of supplying to the prepared sample Wherein performs non-contact operation, and supplying the frozen liquid to the sample through the feed means.

A freezing device according to the present invention that embodies the cryopreservation method is a freezing device that firstly forms a suspension of a sample and a freezing solution for the sample and cryopreserves the suspension. The freezing device includes a container having an opening, a lid capable of sealing the opening, a first compartment for containing the sample, and a second compartment for containing the frozen liquid . And a supply means for supplying the frozen liquid to the sample by a non-contact operation. The supply means is formed integrally with the lid and can be inserted from the opening. A hollow tube that stores the frozen solution, and a nozzle that is formed at a tip of the tube and can hold the frozen solution by surface tension, and the non-contact operation is configured to increase an acceleration that the frozen solution outputs from the nozzle. It is an operation to apply .

  Second, the freezing device includes a container having an opening and a lid capable of sealing the opening, and the supply means is formed integrally with the lid and can be inserted from the opening. A hollow tube that stores the frozen solution and a nozzle that is formed at the tip of the tube and that can hold the frozen solution by surface tension, and the non-contact operation generates an acceleration that the frozen solution outputs from the nozzle. It is an operation to apply.

  Third, the hollow tube or the nozzle is filled with a mesh-like filler.

Fourth, a freezing device that forms a suspension of a sample and a freezing solution for the sample and cryopreserves the suspension, wherein the freezing device includes a container having an opening, the opening Forming a boundary that divides the inside of the container into a first compartment that contains the sample and a second compartment that contains the frozen solution, and the frozen solution is removed by a non-contact operation. Supply means for supplying to the sample, wherein the supply means is opened on the lid side and inserted into the container, is held in contact with the inner wall of the container, and is held by the container from the opening side. An intermediate unit capable of injecting the frozen liquid, an output hole formed on the insertion side of the intermediate unit, and formed to seal the output hole, holding the frozen liquid and freezing by the touched operation Having a second filler for outputting liquid to the container Together with the non-contact operation, characterized in that the freezing liquid is operated to apply an acceleration output from the output port through the second filling material.

Fifth, a freezing device that forms a suspension of a sample and a freezing solution for the sample and cryopreserves the suspension, the freezing device including a container having an opening, the opening Forming a boundary that divides the inside of the container into a first compartment that contains the sample and a second compartment that contains the frozen solution, and the frozen solution is removed by a non-contact operation. Supply means for supplying to the sample, the supply means comprising: a hollow tube for storing the frozen liquid; a second nozzle formed of a ferromagnetic material at a tip of the tube; and the ferromagnetic material. A magnet that is attracted by a body and seals the second nozzle, and the non-contact operation applies a magnetic field by which the magnet separates from the second nozzle to seal the second nozzle. The operation of outputting the frozen liquid from the second nozzle by releasing the stop And wherein the door.

  The specimen cryopreservation system according to the present invention includes a container holder that holds the freezing device, and a drive device that causes the container holder to rotate and eccentrically move, and the container holder includes the container The freezing device is held with the output side of the supply means facing the outer peripheral side of the container holder at the periphery of the holder, and the driving device rotates the container holder to generate centrifugal force generated in the freezing device. To output the frozen liquid from the supply means and supply the frozen liquid to the sample, and eccentrically move the container holder, stir the sample and the frozen liquid, suspend the sample in the frozen liquid, and The holder is detachable from the driving device and can be immersed in a freezing refrigerant.

The sample cryopreservation system of the present invention further includes a container holder that holds the freezing device, a second magnet that is mounted on the container holder and generates the magnetic field, and a second drive that moves the container holder eccentrically. And the second magnet attracts the magnet to the second magnet side by the magnetic field, releases the seal of the second nozzle, and discharges the frozen liquid to the second nozzle. And the second driving device causes the container holder to move eccentrically, stirs the sample and the frozen liquid, and suspends the sample in the frozen liquid. In addition, the container holder is detachable from the second driving device and can be immersed in a freezing refrigerant.
The container holder can hold a plurality of the freezing devices.

  According to the cryopreservation method and the freezing device according to the present invention, first, the sample and the frozen liquid are separated in the freezing device and can be stored independently. That is, the step of injecting the freezing liquid into the freezing device and the step of supplying the freezing liquid to the sample stored in the freezing device are separated. And since a frozen liquid is supplied to a sample by non-contact operation, the above-mentioned process (i) and (ii) can be processed automatically and continuously. In the present invention, since only the freezing device is used to mix, disperse, and freeze the cells and the frozen solution consistently, the transfer step corresponding to step (iii) can be omitted in the present invention. Therefore, there is no need for the operator to perform operations such as supplying the frozen solution to the sample with a pipette, etc., and not only the time to freeze storage of the suspension can be shortened, but also the unevenness of the time can be suppressed. The damage to the sample can be stably suppressed.

  Second, since the supply means is integrally formed with the lid of the freezing device, the frozen liquid can be stored in a state where the supply means is separated from the container. Since the container sealing process is completed before the cells and the frozen liquid come into contact with each other, the container sealing process corresponding to the above step (iv) is excluded from the critical process time count, Work efficiency can be improved. The supply means consists of a hollow tube and a nozzle formed at the tip of the tube. Since the frozen liquid can be supplied to the sample by applying acceleration, specifically centrifugal force, to the supply means. It can be mounted on a simple device such as a separator, and the frozen liquid can be easily supplied to the sample without human work.

  Thirdly, by filling the hollow tube or nozzle with a mesh-like filler, the resistance of the freezing liquid can be increased and the freezing liquid can be prevented from leaking from the nozzle before the non-contact operation. it can.

  Fourthly, by inserting the intermediate unit, which is separated from the lid and the container and opened on the lid side, into the container, the injection of the frozen liquid into the supply means is facilitated and the working efficiency is improved.

  Fifth, the supply means has a hollow tube for storing the frozen liquid, a second nozzle formed of a ferromagnetic material at the tip of the tube, and is sucked by the ferromagnetic material to seal the second nozzle. The non-contact operation is performed by applying a magnetic field that causes the magnet to leave the second nozzle and outputting the frozen liquid from the second nozzle, thereby freezing the second nozzle. It is not necessary to use the surface tension of the liquid, and the inner diameter of the second nozzle can be increased, so that the supply of the frozen liquid to the sample can be performed in a short time, and the time to cryopreservation is further shortened. be able to.

  Further, according to the cryopreservation system for a sample according to the present invention, a step (corresponding to the above (i)) of adding a frozen solution to a cell or the like by a non-contact operation represented by centrifugal motion is realized, and an eccentric circle A step (corresponding to (ii)) of suspending cells or the like in a frozen solution can be realized by a non-contact operation typified by exercise. Here, the meaning of non-contact means that in order to perform these steps, it is not necessary to open the lid of the container, and it is not necessary to insert and operate a pipette inside the container.

  Furthermore, according to the cryopreservation system for a sample according to the present invention, since the sealing of the second nozzle can be released by magnetic force, the inner diameter of the second nozzle can be increased, and the supply of the frozen liquid to the sample can be performed. A cryopreservation system that can be performed in a short time and has a shorter time to cryopreservation. Since the container holder is detachable from the driving device or the second driving device, the freezing device can be immersed in the freezing refrigerant together with the container holder immediately after suspension, and the working efficiency can be improved.

  In addition, since the container holder can hold a plurality of freezing devices, the working efficiency is improved, and the suspension of the sample and the freezing liquid is simultaneously formed in each freezing device, so that work unevenness can be suppressed.

It is the schematic which shows typically the cross section of the freezing device by the 1st Example of this invention. It is the schematic which shows typically the structure of the freezing device by the 1st Example of this invention. It is the schematic which shows typically the structure of the one-touch desorption mechanism of the container holder of the stirring centrifuge by the 1st Example of this invention. It is the schematic which shows typically the shape of the container holder by the 1st Example of this invention. It is a flowchart which shows typically operation | movement of the freezing device by the 1st Example of this invention. It is an example of the result of having evaluated the viable cell rate after thawing | decompression of the cell frozen according to the 1st Example of this invention and the prior art example. It is the schematic which shows typically the cross section of the container holder by the 1st modification of the 1st Example of this invention. It is the schematic which shows typically the cross section of the container holder by the 2nd modification of the 1st Example of this invention. It is the schematic which shows typically the cross section of the freezing device by the 3rd Example of this invention. It is the schematic which shows typically the cross section of the holder by the 3rd Example of this invention, and its usage. It is the schematic which shows typically the structure of the freezing device by the 4th Example of this invention. It is the schematic which shows typically the usage method of the freezing device by the 4th Example of this invention. It is the schematic which shows typically the structure and preparation procedure before use of the freezing device by the 4th Example of this invention.

  Hereinafter, the present invention will be described in detail with reference to embodiments shown in the drawings. However, the components, types, combinations, shapes, relative arrangements, and the like described in this embodiment are merely illustrative examples and not intended to limit the scope of the present invention only unless otherwise specified. .

  FIG. 1 shows a schematic diagram of a freezing device. The cryopreservation method according to the present invention is a cryopreservation method in which a suspension of a sample and a freezing solution for the sample is formed and the suspension is cryopreserved, and the first compartment 2 for storing the sample is stored. And a freezing device 1 having a second section 3 for storing the frozen liquid, and a freezing operation in which the boundary between the first section 2 and the second section 3 is stored in the second section 3 by a non-contact operation. A freezing means that is formed by supply means capable of supplying the liquid to the sample stored in the first compartment 2, performs the non-contact operation on the freezing device 1, and supplies the frozen liquid to the sample. The device 1 is a freezing device 1 that forms a suspension of a sample and a freezing solution for the sample and cryopreserves the suspension. The freezing device 1 includes a first compartment 2 that houses the sample. A second compartment 3 for storing the frozen liquid, and the first compartment 2 The second to form a boundary between the compartment 3, and has a supply means for supplying to the sample by a non-contact manipulating the freezing liquid.

  In the freezing device 1 according to the present embodiment, the supply means includes a liquid storage unit 13 that is a hollow tube that stores the frozen solution, and the frozen solution that is formed at the tip of the tube by the surface tension of the frozen solution. While having the nozzle 13a which can be hold | maintained, the said non-contact operation is operation which applies the acceleration (centrifugal force) which the said frozen liquid outputs from the said nozzle 13a.

  FIG. 1 is a schematic cross-sectional view of a freezing device 1 according to this embodiment. FIG. 2 is a schematic cross-sectional view of the upper unit 10 and the lower unit 20 which are main components of the freezing device 1 according to this embodiment. A boundary between the first compartment 2 and the second compartment 3 is formed in the freezing device 1 by the liquid storage portion 13 and the nozzle 13a constituting the supply means. The freezing device 1 according to this embodiment includes an upper unit 10 and a lower unit 20.

  A schematic configuration of the upper unit 10 is as follows. 11 is a cap, 12 is a packing, 13 is a liquid storage section, 14 is an opening, and 15 is a filler. The liquid storage unit 13 is basically a hollow tube, and has a capillary nozzle 13a having an inner diameter of about 0.3 mm at its tip (downward in the figure), and is formed integrally with the cap 11. And the nozzle 13a can be inserted from the opening part 20a of the lower unit 20 (the container main body 21 mentioned later). The opening 14 is a through hole provided in the side wall of the liquid storage unit 13 and communicates the inside and outside of the hollow tube. The filler 15 is filled inside the tip of the liquid storage unit 13. As the filler 15, super fine grade quartz wool manufactured by Nippon Quartz Glass (currently Tosoh) was used. The packing 12 is made of flat donut-shaped silicon rubber and is shown independently of the cap 11 in the figure. However, since both are used integrally, the following description will be made assuming that the cap 11 includes the packing 12.

  A schematic configuration of the lower unit 20 is as follows. 21 is a container main body, 22 is a container bottom part, 23 is a screw part. The container bottom portion 22 is a part of the inner surface of the container body 21, and the screw portion 23 is a part of the outer surface of the container body 21. Therefore, in this embodiment, the lower unit 20 is synonymous with the container body 21. A female screw corresponding to the screw portion 23 (male screw) is formed on the inner side of the cap 11, but the figure number is omitted.

  Incidentally, the cap unit obtained by removing the liquid storage unit 13 from the upper unit 10 of the freezing device 1 and the lower unit 20 (that is, the container main body 21) respectively correspond to the cap of the cryotube and the container main body according to the prior art. A conventional cryotube cap is hereinafter referred to as a conventional cap 11 '(not shown). Therefore, it can be said that the freezing device 1 according to the present embodiment employs a configuration in which the liquid storage portion 13 is newly provided in the cap portion based on the conventional cryotube.

  A stirring centrifuge suitably used in the present embodiment will be described. In this example, a stirring centrifuge NSD-12J manufactured by Nisshin Rika Co., Ltd. was used as the stirring centrifuge. This apparatus has a container holder capable of holding 12 (plural) containers, and has both functions of centrifugal operation and stirring operation. The standard container holder corresponds to a 2 mL centrifuge tube, but by using a custom-made container holder having a container holder hole that fits the outer diameter of the lower unit of the freezing device 1, the freezing device 1 is accommodated in the container holder hole. used. In this stirring centrifuge, the container holder is fixed to the apparatus main body with a fixing screw. The container holder can be removed by removing the fixing screw, but it takes time and effort to remove the fixing screw. Therefore, in this embodiment, the container holder fixing mechanism in this product has been improved, and a one-touch detachment mechanism capable of instantaneously detaching the container holder has been newly developed and adopted.

  FIG. 3 is a schematic cross-sectional view of the one-touch desorption mechanism for the container holder of the stirring centrifuge in the present embodiment. 100 is a container holder, 110 is a fixture, 120 is a C-ring, and 130 and 140 are nuts. FIG. 4 is a top view schematically showing the shape of the container holder 100 of the stirring centrifuge employed in this embodiment. 101 is a container hole, 102 is a center hole, and 103 is a container holder hole.

  Therefore, the cryopreservation system of the present embodiment includes a container holder 100 that holds the freezing device 1 and a stirring centrifuge that serves as a driving device that causes the container holder 100 to rotate and eccentrically move. The freezing device 1 is held at the periphery of the container holder 100 with the output side of the supply means (the liquid storage unit 13 and the nozzle 13a) facing the outer peripheral side of the container holder 100, and the driving device holds the container holder 100 in place. The rotating liquid is rotated and the freezing liquid is output from the supplying means by the centrifugal force generated in the freezing device 1 and supplied to the sample, and the container holder 100 is eccentrically moved to stir the sample and the freezing liquid. The sample is suspended in the freezing liquid, and the container holder 100 is configured to be detachable from the driving device and immersed in a freezing refrigerant.

  Next, an outline of the operation of the cryopreservation system using the freezing device according to the first embodiment will be described. FIG. 5 is a flowchart schematically showing the operation of this embodiment. Step 1001 is a preparation process, Step 1002 is a centrifugation process, Step 1003 is an agitation process, and Step 1004 is an immersion process in a freezing refrigerant such as liquid nitrogen. The outline of the operation of the freezing device according to the present invention is as follows. In the preparation step (step 1001), pellets such as cells are stored in the container bottom 22 of the lower unit 20 of the freezing device 1, and the frozen liquid is stored in the liquid storage unit 13. The freezing device 1 was put in a sealed state by fitting the unit 10 into the lower unit 20 and installed in the container holder 100 of the stirring centrifuge. In the centrifugation step (step 1002), centrifugal acceleration of the agitating centrifuge is used to apply centrifugal acceleration to the frozen liquid, so that the frozen liquid is liquid-free without contact (in other words, without directly operating the frozen liquid or cells). The container was dropped from the storage unit 13 to the container bottom 22. In the stirring step (step 1003), the pellets of cells and the like were mixed with the frozen solution and suspended by the stirring operation of the stirring centrifuge. In the immersion step in liquid nitrogen (step 1004), the freezing device 1 together with the container holder was detached from the stirring centrifuge with one touch, and immersed in a liquid nitrogen bath to freeze cells and the like.

Next, the cryopreservation system using the freezing device of Example 1 will be described in detail.
As a preliminary preparation, first, the centrifugal adjustment knob (Spindown) of the stirring centrifuge NSD-12J is set to 5.3 memory, the stirring adjustment knob (Speed) is set to 7.5 memory, and the container holder 100 is installed in the stirring centrifuge. A balance container (such as a new freezing device 1 that does not contain cells or a frozen solution) was appropriately installed in the container holder 100 according to a standard method. A liquid nitrogen bath was prepared near the stirring centrifuge. The centrifugal adjustment knob 5.3 memory corresponds to a condition of about 2400 rpm in terms of rotation speed and about 400 × g in terms of centrifugal force. The stirring control knob (Vortex) corresponds to about 1200 rpm in terms of the rotational speed of the eccentric circular motion.

  As a first procedure of the preparation process (step 1001), the upper unit 10 was removed from the freezing device 1, and a conventional cap 11 'was used instead to obtain a pellet such as cells. Specifically, a suspension of cells and the like is stored in the lower unit 20 of the freezing device 1 and sealed with a conventional cap 11 ′, and placed in a centrifuge and centrifuged for 5 minutes with a centrifugal force of 170 × g. The cap 11 'was removed and the supernatant was largely removed. The cap 11 'was sealed, centrifuged again for 1 minute, the cap 11' was removed, and the supernatant was completely removed to obtain a pellet of cells or the like at the container bottom 22 of the lower unit 20. The lower unit 20 was cooled to 4 ° C. before the start of use, and the cooling centrifuge was set to 4 ° C. for use in the centrifugation step, and the supernatant removal operation and storage after the operation was completed were set to 4 ° C. It is preferable to operate on an electronic cooling plate or the like while keeping cells or the like cold.

  As a second procedure of the preparation step (step 1001), the frozen liquid was stored in the liquid storage unit 13. The second procedure can be performed during the centrifugation of the first procedure. Specifically, 200 microliters of freezing solution (freezing solution for primate ES cells manufactured by Reprocell) is used in the liquid storage unit 13 of the upper unit 10 removed in the first step, and a micro-dispensing pipetter is used through the opening 14. And injected. During the injection, the central axis of the upper unit 10 was held horizontally so that the opening 14 was vertically upward. In addition, bubbles and frozen liquid were alternately injected into the capillary nozzle portion 13a at the tip of the liquid storage portion 13 using a pipettor.

  Next, a liquid leakage confirmation test was performed on the liquid storage unit 13 storing the frozen liquid. The liquid leakage means that the frozen liquid falls from the liquid storage unit 13 before the centrifugation process. In the liquid leakage confirmation test, the cap 11 is placed vertically on the upper unit 10 in which the frozen liquid is stored in the liquid storage unit 13 on the (empty) lower unit 20 different from that in which pellets such as cells are stored. Even if the nozzle 13a is fitted so that the nozzle 13a faces vertically downward, and the sealing operation is repeated, light vibrations are applied, or left, the frozen liquid is transferred from the nozzle 13a at the tip of the liquid storage unit 13 to the lower unit 20 Confirmed that it would not fall into

  When the liquid storage part 13 storing the frozen liquid was prepared by the above method, most of the individuals did not leak. This is because the nozzle 13a is a capillary and can hold a frozen liquid by surface tension, the situation where gas and liquid are alternately present inside the capillary, the filler 15 filled inside the tip, etc. It is considered that the situation where the surface tension and the liquid flow resistance are high was achieved by the synergistic effect, and the frozen liquid could be prevented from dropping due to gravity or the like. The nozzle 13a has a narrower capillary diameter, and a fine particle filler having a surface area larger than that of quartz wool is used as the filler 15 or a platter-like filler prepared by sintering fine particles is used to obtain a liquid. By increasing the flow resistance, the fall of the frozen liquid can be prevented without alternately forming gas and liquid inside the capillary tube.

  Although there were individuals that leaked liquid with a very low probability, these defective products were excluded in this confirmation process, and only non-defective products that passed the confirmation test were used in the next process. Of course, the confirmation test in the preparation process can be omitted by performing the confirmation test and the selection of non-defective products during the manufacturing process. In the present embodiment, the method of injecting the frozen liquid through the opening 14 when stored in the liquid storage unit 13 has been described as an example. However, the frozen liquid is discharged from the nozzle portion at the tip of the liquid storage unit 13 with a microdispensing pipettor (first tip). Can also be injected (using a thick pipette tip). In this case, the position of the opening 14 can be provided at an upper position in FIG. 2, that is, a position closer to the connection portion of the liquid storage portion 13 with the cap 11. In this case, the length of the liquid storage unit 13 can be shortened. As an extreme example, the opening 14 can be formed not only as a through-hole on the side surface of the liquid storage part 13 but also as a notch of the liquid storage part 13 (at the connection part with the cap 11).

  As a third procedure of the preparation process (step 1001), the upper unit 10 prepared in the second procedure is fitted on the lower unit 20 containing the cell pellet prepared in the first procedure, and the freezing device 1 is sealed. Was placed in the container holder 100 of the stirring centrifuge and the lid of the stirring centrifuge was closed. Inside the freezing device 1, pellets of cells and the like are stored in the container bottom portion 22, and a frozen solution is stored in the liquid storage portion 13, both of which exist in independent compartments. In other words, the frozen solution and the cells are not in contact with each other or mixed in the preparation steps so far. Therefore, since it is not counted in the critical process time so far, the operation can be performed without being limited in time.

  Next, in the centrifugation step (step 1002), the centrifugal switch (Spindown) of the stirring centrifuge was turned on for 1 second, and then turned off and left for 3 seconds. At this time, the output side (nozzle 13a side) of the supply means (liquid storage unit 13, nozzle 13a) is arranged to face the outer peripheral side of the container holder 100. By the centrifugal operation at 400 × g for 1 second, the frozen liquid stored in the liquid storage unit 13 in the freezing device 1 was output from the nozzle 13b by centrifugal force and dropped to the container bottom 22 to come into contact with pellets such as cells. Therefore, in this embodiment, the critical process time count starts from this point. Here, the memory of the centrifugal adjustment knob can be appropriately selected according to the number of containers and the like so as to obtain a predetermined centrifugal force. Further, in this embodiment, 3 seconds is adopted as a waiting time until the rotation is almost stopped after the end of the centrifugal operation. Therefore, this process time is 4 seconds.

  Next, in the stirring step (step 1003), the stirring switch (Vortex) of the stirring centrifuge was turned on for 2 seconds, and then the switch was turned off. During the stirring operation, the lid of the stirring centrifuge was opened. By a stirring operation for 2 seconds, the frozen solution was mixed with pellets of cells and the like to obtain a suspension of cells and the like. This process time is 2 seconds.

  Next, in the immersion process (step 1004) in liquid nitrogen, the freezing device 1 was removed from the stirring centrifuge together with the container holder 100, moved to the liquid nitrogen bath, and immersed in the liquid nitrogen bath. Thereby, the cells and the like were instantaneously frozen. As described above, in the present embodiment, since a mechanism capable of detaching the container holder 100 with one touch is used, the container holder 100 can be instantaneously removed from the stirring centrifuge. The time for this step (until immersion in liquid nitrogen) is about 2 seconds. The detailed structure and operation of this one-touch desorption mechanism will be described below.

  The cross section of the fixture 110 is convex, the horizontally long lower part and the vertically long upper part are integrally formed, and a male screw is formed on the upper side surface. The fixture 110 is connected to the centrifugal mechanism of the stirring centrifuge, and rotates together with the fixture 110 about the vertical center line in FIG. The centrifugal mechanism of the stirring centrifuge is connected to the stirring mechanism and causes the fixture 110 to move eccentrically. A container holder boss (not shown) provided on the fixture 110 engages with the container holder hole 103. Further, the C-ring 120 is fixed around the central axis of the upper portion of the fixture 110 by nuts 130 and 140. Since the lower slope of the C ring 120 is in pressure contact with the upper edge of the center hole 102 of the container holder 100, the container holder 100 is fixed to the fixture 110. Accordingly, the rotation and eccentric circular motion of the fixture 110 are transmitted to the container holder 100, and act on the container accommodated in the container hole 101 of the container holder 100, such as centrifugation or stirring.

  Incidentally, when the upper part of the stirring centrifuge or the fixture 110 is fixed in the state of FIG. 3 and a force for pulling up the container holder 100 is applied, the C-ring 120 contracts, and the center hole 102 of the container holder 100 becomes the C-ring. The container holder 100 is disengaged from the fixture 110 by sliding around 120. That is, the container holder 100 can be detached from the stirring centrifuge with one touch. On the contrary, by pushing the container holder 100 between the fixture 110 of the stirring centrifuge and the C ring 120, the container holder 100 can be attached to the stirring centrifuge with one touch.

  In the present embodiment, the C-ring 120 is used as a mechanical component for enabling the container holder 100 to be detached from the stirring centrifuge with one touch. However, the mechanism that can be used for this purpose is not limited to the C-ring system. Examples of other adoptable mechanisms include various methods used for fixing an optical disk to a shaft, such as a method using a claw formed of an elastic body, an electromagnetic chuck method, and an air balloon chuck method. There are a method of detachably fixing the container holder to the fixture by a magnetic force such as an electromagnet or a permanent magnet, an O-ring method, and the like. By adopting these various methods, the container holder 100 can be detached from the stirring centrifuge with one touch.

  The container holder 100 employed in this embodiment includes twelve container holes 101 as shown in FIG. Accordingly, in the preparation step (step 1001), independent freezing devices 1 can be prepared for a plurality of (maximum 12) samples, and each can be installed in one container holder. In addition, since the centrifugation step (step 1002), the stirring step (step 1003), and the immersion step in liquid nitrogen (step 1004) are all performed with the container holder 100 as a unit, a plurality of samples can be collectively operated. That is, in this embodiment, a plurality of samples can be collectively processed for critical processes. By adopting the container holder 100 having more container holes 101, the number of samples that can be collectively processed can be increased to 12 or more. By adopting a larger stirring centrifuge, it is possible to further increase the number of samples that can be processed simultaneously.

  In this embodiment, the critical process time, that is, the time from when the frozen solution and the cells contact each other until they are immersed in liquid nitrogen, is the total of the time from step 1002 to step 1004, that is, about 8 seconds. . In other words, since it is about half of 15 seconds, which is an indication of the critical process time described in the prior art, there is little adverse effect on the cells. In addition, the operation of the critical process in this embodiment is only a simple operation of operating two switches, opening the lid, one-touch removal of the container holder, and putting it in the liquid nitrogen bath, and skill and attention are unnecessary. Since there is almost no risk of failure, the burden on the operator is small and the risk of adverse effects on cells is also low.

  In this embodiment, the frozen liquid is held by the surface tension of the capillary nozzle 13a and the filler 15. However, it is possible to hold the frozen liquid using other principles. For example, a valve (not shown) supported by an elastic body is provided in the nozzle 13a. Normally, the valve (not shown) is in a closed state, and the valve (not shown) opens only when strong centrifugal acceleration is applied, and the frozen liquid is generated. It is also possible to adopt a falling configuration.

  The effect of this example was evaluated using cells. Adherent human cell lines (HT-29) were cultured as cells according to the ATCC protocol, and 2 million cells detached and recovered 4 days after seeding were used as experimental materials. It was frozen by the method of either this example or the second conventional example. These frozen cells were thawed by the method described on page 10 of the second conventional example, and the viability of the cells was evaluated. An example of the result is shown in FIG. By the way, the cells used in the experiment were peeled and collected and then kept in the medium at ice temperature for about 6 hours, and the viable cell ratio just before the experiment was 98.3%. The freezing experiment by the method of the present invention was performed three times, and the latter two performed freezing treatment on a plurality of samples simultaneously. As is clear from FIG. 6, the viable cell ratio after thawing was equal to or higher than that of the conventional method for any sample. The same experiment was repeated a plurality of times, but similar results were obtained in all cases (not shown). Since the cells frozen by the method of the present invention have a high viable cell ratio after thawing, it was confirmed that the method of the present invention has little adverse effect on the cells and is highly reliable. The reason for this is considered that the critical process time of the present invention is about 8 seconds, which is about half of the conventional method, and is not easily affected by a highly toxic freezing solution at room temperature.

Therefore, the present embodiment has a specific effect that a plurality of samples can be frozen at the same time with high reliability in a shorter time and with a simple operation.
In the present embodiment, the container holder 100 attached as a standard to the stirring centrifuge is used, but a container holder having a slightly different shape can be manufactured and used for the purpose of the present invention. FIG. 7 shows a schematic cross-sectional view of the container holder 100 ′ according to the first modification. As shown in the figure, the container holder 100 ′ is similar to the container holder 100 of the first embodiment, except that the portion holding the container is steeply inclined and the container is held at an angle close to the horizontal. Moreover, the point which added the mark which shows the position of the opening part 14 to the cap 11 of the freezing device 1 also differs.

  The operation in the case of using this modification is substantially the same as that of the first embodiment, but differs in the following points. First, in the second procedure of the preparation process (step 1001), when storing the frozen liquid in the liquid storage section 13 of the freezing device 1, the bubbles and the frozen liquid are alternately placed inside the capillary nozzle at the tip of the liquid storage section 13. The step of injecting was omitted. In addition, after the frozen liquid was stored in the liquid storage unit 13, the opening 14 was vertically upward, in other words, the central axis of the upper unit 10 was kept horizontal (no liquid leakage confirmation test was performed). Next, in the third procedure of the preparation step (step 1001), the upper unit 10 (containing the frozen solution) prepared in the second procedure is inserted into the lower unit 20 (containing the cell pellet) prepared in the first procedure. The freezing device 1 was obtained by sealing and installed in the container holder 100 ′ of the stirring centrifuge. During this time, the posture was maintained so that the opening 14 was always vertically upward. That is, after putting the frozen liquid into the freezing device 1, the tip nozzle portion is kept horizontal and does not face vertically downward, so there is no possibility that the frozen liquid leaks.

  The effect peculiar to this modification is that there is no possibility that the frozen liquid leaks, so that the likelihood of the shape of the liquid storage portion 13 of the freezing device 1, particularly the nozzle 13a at the tip thereof is high, and the creation is easy. When storing the frozen liquid, it is not necessary to alternately inject the bubbles and the frozen liquid into the nozzle 13a, so that the operation is simple.

  As a second modification, it is also possible to use a swing rotor type container holder as the container holder. In particular, as the swing rotor, it is preferable to employ a swing rotor that can be set in an initial state in which the container can be held substantially horizontally as conceptually shown in FIG. When the swing rotor rotates, as conceptually shown in FIG. 8B, the swing rotor changes to a normal swing rotor state by centrifugal force. That is, the part holding the container takes a free angle in consideration of the centrifugal force and the acceleration of gravity. Similar to the first modification, the second modification can be combined with the freezing device 1 having the liquid storage part 13 with a small surface tension and liquid flow resistance of the tip nozzle part. Furthermore, in the state of FIG. 8B, gravity acceleration can be used for the fall of the frozen liquid, and therefore, it is only necessary to perform centrifugation for a very short time (from FIG. 8A to the state of FIG. 8B). This is a unique effect.

  Therefore, according to the cryopreservation method and the freezing device according to the present embodiment, first, the sample and the frozen liquid are separated in the freezing device 1 and can be stored independently. That is, the step of injecting the freezing liquid into the freezing device and the step of supplying the freezing liquid to the sample stored in the freezing device are separated. And since a frozen liquid is supplied to a sample by non-contact operation, the process of (i) and (ii) described in the prior art can be processed automatically and continuously. In this embodiment, since only the freezing device 1 is used to mix, disperse, and freeze cells and the like in a consistent manner, the transfer step corresponding to step (iii) described in the prior art is omitted in the present invention. can do. Therefore, there is no need for the operator to perform operations such as supplying the frozen solution to the sample with a pipette, etc., and not only the time to freeze storage of the suspension can be shortened, but also the unevenness of the time can be suppressed. The damage to the sample can be stably suppressed.

  Secondly, since the supply means (the liquid storage unit 13 and the nozzle 13a) are integrally formed with the upper unit 10 which is a lid of the freezing device 1, the supply means can be stored in a state where the supply means is separated from the container. it can. And since the sealing process of the lower unit 20 as the container is completed before the cells and the frozen liquid come into contact with each other, the sealing process of the container corresponding to the process (iv) described in the prior art is critical. Therefore, it is possible to improve the work efficiency by excluding the time count of a simple process. The supply means includes a liquid storage portion 13 which is a hollow tube and a nozzle 13a formed at the tip of the liquid storage section 13, and supplies the frozen liquid to the sample by applying acceleration, specifically centrifugal force, to the supply means. Therefore, it can be mounted on a simple device such as a centrifuge, and the frozen solution can be easily supplied to the sample without human work.

  Third, by filling the liquid supply unit 13 or the nozzle 13a, which is a hollow tube, with the mesh-like filler 15, the liquid flow resistance of the frozen liquid is increased, and the frozen liquid is discharged from the nozzle before the non-contact operation. Can be prevented from leaking.

  In addition, according to the cryopreservation system using the freezing device of this example, a step (corresponding to (i) of the prior art) of adding a frozen solution to cells or the like by a non-contact operation represented by centrifugation is realized. In addition, it is possible to realize a step (corresponding to (ii)) of suspending cells or the like in a frozen solution by a non-contact operation represented by an eccentric circular motion. In addition, since the container holder 100 constituting the cryopreservation system can hold a plurality of freezing devices 1, the working efficiency is improved and a suspension of the sample and the freezing solution is simultaneously formed in each freezing device 1, Work unevenness can also be suppressed. Since the container holder 100 is detachable from the drive device, the working efficiency can be improved because the freezing device can be immersed in the freezing refrigerant together with the container holder 100 immediately after suspension.

  The configuration of the freezing device and cryopreservation system according to Example 2 is similar to that of Example 1, except that a stirring centrifuge having a mechanism for mechanically braking after the end of the centrifugal operation is employed, and the waiting time after centrifugation. This is different from the above in that the agitation time is shortened by increasing the agitation intensity setting in the agitation process, the operation of the agitation centrifuge is controlled by a PLC (logic controller), and the operation is automated.

  Specifically, the cover portion of the stirring centrifuge NSD-12J was modified to provide an electric brake mechanism that can press an elastic body such as a sponge against the container holder. Conventionally, a waiting time of about 3 seconds was required until the container holder substantially stopped after centrifugation, but the rotation of the container holder was stopped within 1 second by operating the electric brake after the centrifugal operation was completed. Also, the centrifugal switch (stirring centrifuge), stirrer switch, and electric brake switch are wired to the PLC, and the centrifuge process (brake operation) can be performed with a single start switch by creating a sequence that automatically controls the operation of the agitator centrifuge with the PLC And the stirring step can be performed fully automatically.

In the preparatory stage, the stirring control knob (Speed) was set to MAX memory and used.
In the centrifugation step (step 1002), by applying an electric brake after the end of the centrifugal operation, the waiting time until the container holder almost stopped was shortened to about 1 second, and the process time was set to 2 seconds.
In the stirring process (step 1003), stirring was performed for 1 second.
Otherwise, the same configuration and procedure as in Example 1 were adopted.

  In this embodiment, the critical process time is a total of 5 seconds. This is 1/3 of the conventional example, and since the critical process time is extremely shorter than before, there is very little adverse effect on the cells, and there is a time margin, and the process up to stirring with only one start switch operation. Therefore, there is a specific effect that the burden on the operator is small.

  In this embodiment, an example in which NSD-12J is modified and used as a stirring centrifuge has been described. However, another stirring centrifuge, for example, Biosan MSC-3000 can also be used. Because the brake that MSC-3000 is equipped with is powerful, the time until the container holder is almost stopped after centrifugation is short. Moreover, since the operation of centrifugation and stirring can be programmed, automation is possible without externally attaching a PLC. The stirring centrifuge that can be used in the present invention is not limited to NSD-12J, and various commercially available devices can be selected or modified according to the purpose.

  The configuration of the freezing device and cryopreservation system according to the third embodiment is similar to that of the first embodiment, but the structure of the liquid storage unit 13 is different, and the mechanism for dropping the frozen liquid onto the cells is different.

  In the third embodiment, the supply means is adsorbed to the liquid storage unit 13 which is a hollow tube for storing the frozen liquid, the second nozzle 13b formed of a ferromagnetic material at the tip of the tube, and the second nozzle 13b. And a small ball 16 of the magnet that seals the second nozzle 13b. In the non-contact operation, a magnetic field that causes the small ball 16 of the magnet to separate from the second nozzle 13b is applied to the second nozzle 13b. This is an operation for outputting the frozen liquid from the second nozzle 13b by releasing the sealing of 13b.

  Specifically, in the freezing device 1b employed in this example, as schematically shown in FIG. 9, the capillary nozzle 13b at the tip of the liquid storage unit 13 which is a component of the upper unit 10b is replaced with Teflon (registered trademark). As a new component, a small ball 16 of a magnet covered with Teflon (registered trademark) is provided at the tip of the nozzle. Moreover, the container holder 30 was provided as typically shown in FIG. The container holder 30 is comprised from the storage part 32 which has the hollow which can accommodate the freezing device 1b, and the side wall part 33 which has the 2nd magnet 31 formed with the strong alnico magnet. As schematically shown in the lower part of FIG. 10 ((a), (b)), the storage part 32 and the side wall part 33 are detachable from each other, and the second magnet 31 built in the side wall part 33 is small in the freezing device 1b. It is installed at a position facing the sphere 16. In this embodiment, not the stirring centrifuge but a simple stirring device having no centrifugal mechanism is used as the second driving device.

  The outline of the operation of the third embodiment is the same as that of the third embodiment except for the following points. Since the magnet sphere 16 is attracted to the nozzle 13b (nickel) by magnetic force and seals the tip of the nozzle 13b, no leakage of liquid occurs when the frozen liquid is stored in the liquid storage portion 13 of the freezing device 1b. . In this embodiment, in the freezing liquid addition step (step 1002 ′), which is an alternative to the centrifugation step (step 1002), the freezing device 1b is previously installed in the storage portion 32 of the container holder 30 (with the side wall portion 33 removed) (FIG. 10 (a)). Next, the side wall portion 33 of the container holder 30 was fitted into the storage portion 32.

  Then, due to the action of the second magnet 31, the magnet sphere 16 is detached from the nozzle at the tip of the liquid storage portion 13 and is attracted to the wall surface of the container body 21 in the vicinity of the second magnet 31 of the container holder 30 (FIG. 10). (B)). Since the small ball 16 of the magnet that seals the tip of the nozzle 13b disappears, the frozen liquid falls from the liquid storage unit 13 to the cells. In the stirring step (step 1003 ′), the freezing device 1 b was stirred together with the container holder 30 by the stirring device. Finally, in the immersion step in liquid nitrogen (step 1004 '), the freezing device 1b was moved to the liquid nitrogen bath together with the container holder 30 and immersed in the liquid nitrogen bath. FIG. 9 illustrates the container holder 30 that houses one freezing device 1b. However, the container holder 30 includes a housing portion having a plurality of depressions capable of housing a plurality of freezing devices 1b and a plurality of second magnets 31. It is also possible to use in combination with the side wall. With this configuration, a large number of freezing devices 1b can be simultaneously processed in parallel.

  In this embodiment, the holding and dropping of the frozen liquid are controlled in a non-contact manner using magnetic force as an opportunity. Therefore, there is a unique effect that a centrifugal mechanism is not necessary and a problem of liquid leakage hardly occurs.

  Further, according to the freezing device and the cryopreservation system according to the present embodiment, the sealing of the second nozzle 13b can be released by magnetic force, so that the inner diameter of the second nozzle 13b can be increased, and the frozen liquid The sample can be supplied to the sample in a short time, and the time until cryopreservation can be further shortened. Since the container holder 100 is detachable from the second drive device as in the first embodiment, the freezing device can be immersed in the freezing refrigerant together with the container holder 100 immediately after suspension, thereby improving work efficiency. Can be made. In addition, since the container holder 100 can hold a plurality of freezing devices 1, work efficiency is improved and a suspension of the sample and the frozen liquid is simultaneously formed in each freezing device 1, thereby suppressing work unevenness. Can do.

  The configuration of the freezing device according to the fourth embodiment is similar to that of the first embodiment, but the structure of the liquid storage unit 13 is different. In Example 4, the freezing device 1c includes a lower unit 20c serving as a container having an opening, and an upper unit 10c serving as a lid capable of sealing the opening. An intermediate unit 40 that is inserted into the unit 20c, is held in contact with the inner wall of the lower unit 20c and is held by the lower unit 20c, and can be injected with a frozen liquid from the opening side, and an output hole formed on the insertion side of the intermediate unit 40 The second nozzle 13d and the second nozzle 13d are formed to seal the second nozzle 13d, hold the frozen liquid, and output the frozen liquid to the lower unit 20c by a contact operation. At the same time, the non-contact operation is an operation in which the frozen liquid applies an acceleration output from the nozzle 13d via the second filler 15c. In this way, the frozen liquid is separated from the upper unit 10c and the lower unit 20c, and the intermediate unit 40 opened on the upper unit 10c side is inserted into the lower unit 20c, so that the frozen liquid can be easily injected into the supply means. Work efficiency is improved.

  FIG. 11 is a schematic cross-sectional view of the upper unit 10c, the intermediate unit 40, and the lower unit 20c constituting the freezing device 1c according to the present embodiment. The upper unit 10c is equivalent to the cap 11 (including the packing 12) in the first embodiment. The intermediate unit 40 corresponds to the liquid storage portion 13c in the first embodiment, but has a nozzle 13d having a large diameter instead of a capillary-shaped nozzle portion at the lower end of the hollow tube, and the second filler 15c. As an alternative to quartz wool, it has an eye plate, does not have an opening 14 (see FIG. 2), the intermediate unit 40 is configured independently of the cap (upper unit 10c), and is detachably attached to the lower unit 20c. The point which has the knob 17 formed in the wing shape on the inner wall of a pipe, etc. differ. The lower unit 20 c is the same as that of the first embodiment except that the lower unit 20 c includes the protrusions 24.

  The outline of the operation of the fourth embodiment is similar to that of the first embodiment. In the preparation step (step 1001), pellets such as cells were stored in the container bottom 22 of the lower unit 20c of the freezing device 1c, and then the intermediate unit 40 was stored in the lower unit 20c (lower in FIG. 12). Here, since the intermediate unit 40 is supported by the protrusion 24, it does not fall on the container bottom 22. The freezing device 1c is put into a sealed state by injecting the frozen liquid into the inside (on the eye plate) from the upper opening of the liquid storage unit 13c and fitting the upper unit 10 into the lower unit 20c (which stores the intermediate unit 40). It was placed in the container holder of the stirring centrifuge. The operation after the centrifugation step is the same as that in the first embodiment. At the time of thawing, the upper unit 10c is loosened and removed, and then the knob 17 is held and pulled up with (sterilized) tweezers to pull out the intermediate unit 40 from the lower unit 20c and store the frozen cells in the container bottom 22. A thawing operation was performed after obtaining the lower unit 20c.

  The upper unit 10c and the lower unit 20c are combined in advance (FIG. 13 (a)) to form a pair of packages, and the intermediate unit 40 is combined with a cylinder 41 and a piston 42 (FIG. 13 (b)) that circumscribe it, and both are packed. Both packages can be sterilized with gamma rays and provided to the user. In this case, details of the preparation process are as follows. First, in the first procedure of the preparation step, a cell pellet is formed in the lower unit 20c using a combination of the upper and lower units. In the second procedure of the preparation step, the intermediate unit 40 is aseptically press-fitted from the cylinder 41 into the lower unit 20c using the piston 42 (FIG. 13 (c)). Hereinafter, the procedure after the storage of the frozen liquid is as described above.

  For the eye plate used as the filler 15c, a flat plate formed by sintering polystyrene fine particles was used. The material, porosity, cross-sectional area, thickness and the like of the eye plate can be arbitrarily selected. By appropriate selection, it is possible to satisfy the two conditions that the frozen liquid does not leak in the preparation process and that the frozen liquid falls quickly in the centrifugation process.

  In this embodiment, the liquid storage portion 13 is formed as the intermediate unit 40 independently of the upper unit 10c, but the liquid storage portion having a shape similar to that of the intermediate unit 40 in this embodiment is formed integrally with the upper unit. It is also possible to do.

  This embodiment has a unique effect that the structure is simple, the manufacturing is easy, and the cost is low, because a capillary-shaped nozzle portion, a through hole, and quartz wool are not used for the liquid storage portion 13c. Since the frozen liquid may be injected from the upper opening of the liquid container 13c having a large diameter, there is no need to inject through the opening 14 (see FIG. 2), and bubbles and frozen liquid are alternately injected into the capillary. Since it is not necessary, there is a specific effect of high operability.

  By applying the present invention, when rapidly freezing cells or tissues, the critical process time can be shortened, the operation can be simplified and automated, and the burden on the operator can be reduced. The risk of adverse effects can be reduced, and multiple samples can be processed simultaneously, improving efficiency. Therefore, by using the cell freezing device and method according to the present invention, there is an industrial applicability that cells and tissues can be maintained at low cost and high quality.

1 ......... Freezing device, 1b ......... Freezing device, 1c ......... Freezing device, 10 ......... Upper unit, 10b ......... Upper unit, 10c ......... Upper unit, 11 ......... Cap, 11 ' ......... Conventional cap, 12 ......... Packing, 13 ......... Liquid container, 13a ......... Nozzle, 13b ......... Nozzle, 13c ......... Liquid container, 13d ......... Second nozzle, 14 ......... Opening part, 15 ......... Filler, 15c ......... Second filler, 16 ......... Magnet ball, 17 ......... Knob, 20 ......... Lower unit, 20a ......... Opening portion, 20c ............ Lower unit, 21 ......... Container main body, 22 ......... Container bottom, 23 ......... Threaded portion, 24 ......... Protrusion, 30 ......... Container holder, 31 ......... Second Magnets, 32... Storage section, 33... Side wall section, 40. 41 ......... Cylinder, 42 ......... Piston, 100 ......... Container holder, 101 ......... Container hole, 102 ......... Center hole, 103 ......... Container hole, 110 ......... Fixing tool 120 ......... C-ring, 130 ......... Nut, 140 ......... Nut, Step 1001 ......... Preparation step, Step 1002 ......... Centrifuge step, Step 1003 ......... Stirring step, Step 1004 ......... Liquid Immersion process in nitrogen.

Claims (9)

A cryopreservation method for forming a suspension of a sample and a frozen solution for the sample and cryopreserving the suspension,
Forming a freezing device having a first compartment containing the sample and a second compartment containing the frozen liquid;
A boundary between the first section and the second section, a hollow tube that stores the frozen solution, and a nozzle that is formed at a tip of the tube and can hold the frozen solution by surface tension, Formed by a supply means capable of supplying the frozen liquid stored in the second section to the sample stored in the first section by a non-contact operation that is an operation of applying an acceleration output from the nozzle to the freezing device by the frozen liquid,
A cryopreservation method comprising performing the non-contact operation on the freezing device, and supplying the frozen liquid to the sample via the supply unit. A cryopreservation method for forming a suspension of a sample and a frozen solution for the sample and cryopreserving the suspension,
Forming a freezing device having a first compartment containing the sample and a second compartment containing the frozen liquid;
The boundary between the first compartment and the second compartment is adsorbed by the ferromagnetic material, a hollow tube that stores the frozen liquid, a second nozzle formed of a ferromagnetic material at the tip of the tube, and the ferromagnetic material. A magnet that seals the second nozzle, and the second nozzle is released by applying a magnetic field that separates the second nozzle from the second nozzle to release the sealing of the second nozzle. Formed by a supply means capable of supplying the frozen liquid stored in the second compartment to the sample stored in the first compartment by a non-contact operation that is an operation of outputting the frozen liquid from the nozzle,
A cryopreservation method comprising performing the non-contact operation on the freezing device, and supplying the frozen liquid to the sample via the supply unit. A freezing device that forms a suspension of a sample and a freezing solution for the sample and cryopreserves the suspension,
The freezing device is
A container having an opening;
A lid capable of sealing the opening;
The container, a first compartment for containing said sample, said forming a second compartment for containing the frozen liquid, the boundary that divides the a supply means for supplying to the sample by a non-contact operating the freezing liquid Have
The supply means is formed integrally with the lid and can be inserted through the opening to store the frozen liquid, and a nozzle formed at the tip of the pipe and capable of holding the frozen liquid by surface tension. Have
The non-contact operation is an operation of applying an acceleration output from the nozzle by the frozen liquid . The freezing device according to claim 3 , wherein the hollow tube or the nozzle is filled with a mesh-like filler. A freezing device that forms a suspension of a sample and a freezing solution for the sample and cryopreserves the suspension,
The freezing device is
A container having an opening;
A lid capable of sealing the opening;
A supply means for forming a boundary dividing the container into a first compartment for containing the sample and a second compartment for containing the frozen solution, and supplying the frozen solution to the sample by a non-contact operation; Have
The supply means includes
An intermediate unit capable of injecting the frozen liquid from the opening side, the lid side being opened and inserted into the container, abutting against the inner wall of the container and held by the container;
An output hole formed on the insertion side of the intermediate unit;
The second hole is formed so as to seal the output hole, holds the frozen liquid, and outputs the frozen liquid to the container by the contacted operation.
The non-contact operation, freezing device, characterized in that the freezing liquid is operated to apply an acceleration output from the output port through the second filling material. A freezing device that forms a suspension of a sample and a freezing solution for the sample and cryopreserves the suspension,
The freezing device is
A container having an opening;
A lid capable of sealing the opening;
A supply means for forming a boundary dividing the container into a first compartment for containing the sample and a second compartment for containing the frozen solution, and supplying the frozen solution to the sample by a non-contact operation; Have
The supply means includes a hollow tube for storing the frozen liquid, a second nozzle formed of a ferromagnetic material at a tip of the tube, and the suction of the ferromagnetic material to seal the second nozzle. And a magnet to
The non-contact operation is an operation of outputting the frozen liquid from the second nozzle by applying a magnetic field in which the magnet is detached from the second nozzle to release the sealing of the second nozzle. freezing device, characterized in that. A cryopreservation system using the freezing device according to any one of claims 3 to 5 ,
The cryopreservation system comprises:
A container holder for holding the freezing device;
A drive device for rotating and eccentrically moving the container holder;
The container holder is
Holding the freezing device in a state where the output side of the supply means is directed to the outer peripheral side of the container holder at the periphery of the container holder;
The drive device
The container holder is rotated, the frozen liquid is output from the supply means by centrifugal force generated in the freezing device and supplied to the sample, and the container holder is eccentrically moved, whereby the sample and the frozen liquid are To suspend the sample in the frozen solution,
The container holder is
A cryopreservation system, wherein the cryopreservation system is detachable from the driving device and can be immersed in a freezing refrigerant. A cryopreservation system using the freezing device according to claim 6 ,
The cryopreservation system comprises:
A container holder for holding the freezing device;
A second magnet mounted on the container holder for generating the magnetic field;
A second drive device for moving the container holder in an eccentric circle,
The second magnet is
The magnet is attracted to the second magnet side by the magnetic field, the sealing of the second nozzle is released, the frozen liquid is output from the second nozzle, and the frozen liquid is supplied to the sample,
The second driving device includes:
Eccentric circular movement in the container holder, stirring the sample and the frozen liquid to suspend the sample in the frozen liquid,
The container holder is
A cryopreservation system, wherein the cryopreservation system is detachable from the second drive device and can be immersed in a freezing refrigerant. The container holder is cryopreservation system of a sample according to claim 7 or 8, characterized in that the freezing device is more maintainable.

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