JPH02300101A - Ice inoculation and ice inoculator in freeze storage apparatus for biological sample - Google Patents

Abstract

PURPOSE:To provide the title ice inoculation method and ice inoculator so designed that within a freeze storage apparatus made up of vapor phase and liquid phase sections, a biological sample-stored vessel is immersed in the liquid phase section with its upper end part exposed to the vapor phase section, and the vapor phase section is cooled to bring a storage liquid projected into the vapor phase section to a supercooling state followed by freezing a storage liquid enveloping a sample. CONSTITUTION:Within a freeze storage apparatus 1 made up of (A) a liquid phase section 12 with brine and (B) a vapor phase section 13, straws 6a and tubes 6b each holding both biological sample A and storage liquid B are placed so as to project their upper end parts into the vapor phase section. The apparatus 1 is additionally equipped with a circulating unit 3 for brine circulation, a refrigeration machine 2 and a ice inoculator 4, and when ice inoculation becomes necessary to prevent the biological samples A in the straws and/or tubes from supercooling, a coolant is made to flow from an ice-inoculating coolant channel 42 via an ice-inoculating chiller 41 into the refrigeration machine. The storage liquid near the upper end part is brought to a supercooling state due to rapid fall in the temperature of the vapor phase section, and with this storage liquid as nucleus, a storage liquid enveloping the samples A is frozen in a state free from supercooling.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an ice planting method and an ice planting apparatus in an apparatus for cryopreserving biological samples such as animal and plant cells, embryos, and fertilized eggs.

[Conventional technology]

There are various types of devices for cryopreserving biological samples, one of which is to store the sample together with a sample preservation solution in a container called a tube, straw, etc. Some products are soaked in a liquid phase and the liquid phase is cooled by a refrigerator.

A problem with cryopreservation of biological samples in such devices is that simply cooling the sample and preservation solution will cause supercooling during the sample freezing process, and the sudden temperature rise due to freezing after supercooling will cause the sample to deteriorate. There is a problem that the healthy survival rate decreases.

In order to prevent such supercooling, ice planting is generally performed, but the ice planting method includes: (1) mechanically vibrating and impacting the container; (2) placing a needle cooled with liquid nitrogen in a storage solution. ■Place tweezers cooled with liquid nitrogen in contact with the outer wall of the container;■Place an ice planting plate with a liquid nitrogen passage in contact with the part of the container that is exposed to the gas phase of the cryopreservation device.
■Place an ice planting plate equipped with a cooling element that utilizes the Peltier effect in contact with the part of the cryopreservation device that is exposed to the gas phase.
Such methods are known.

[Problems to be Solved by the Invention] However, in the method of applying vibration or impact, it is time-consuming to apply the vibration or impact, and there is no guarantee that all containers will have six seeds or uniform ice planting.

In addition, with the method using cooled needles or tweezers, it is time-consuming to apply ice to each container, and there is a time lag between the start and end of the ice application, resulting in the freezing of the sample. There will be variations in the status. Furthermore, when planting ice, it is necessary to open the container storage chamber of the cryopreservation device one by one, which causes the room temperature to rise, making it impossible to quickly freeze the sample.

In the case of an ice-planting plate that flows liquid nitrogen, there is an advantage that the ice-planting operation can be started for all containers at the same time, but liquid nitrogen is expensive and the management of the liquid nitrogen is difficult.

In the case of an ice planting plate that employs a cooling element based on the Peltier effect, the cooling element is expensive.

Therefore, the present invention provides a biological sample cryopreservation apparatus in which a biological sample storage container is immersed in a liquid phase with its upper end exposed to a gas phase, and the liquid phase is cooled with a refrigerator. An ice planting method that can reliably and inexpensively plant ice on all the containers in front without any dispersion without having to open them, and an ice planting device that implements the method.
The purpose is to serve one person.

[Means to solve the problem]

In the present invention, the refrigerator has the ability to cope with the load when the lowest temperature is reached, and this capacity becomes excessive at temperatures around which ice planting is required, so that part of the freezing capacity is used during ice planting. It was completed by focusing on the fact that the above purpose could be achieved by directing the water to the planting of ice.

That is, the present invention provides an ice planting method for a biological sample cryopreservation apparatus in which a biological sample storage container is immersed in a liquid phase with its upper end exposed to a gas phase, and the liquid phase is cooled with a refrigerator. The present invention provides an ice planting method, characterized in that, at a time when the container requires ice planting, ice is planted by applying a part of the refrigerating capacity of the 111th refrigerator to the container in the gas phase section. .

Furthermore, the present invention provides an apparatus for carrying out the above-mentioned method for biological sample cryopreservation in which a biological sample storage container is immersed in a liquid phase with its upper end exposed to a gas phase, and the liquid phase is cooled with a refrigerator. An ice-planting device in the apparatus, comprising an ice-planting cooler provided in the gas phase section, and a part of the refrigerant of the refrigerator flowing through an expansion mechanism to the ice-planting cooler at least during ice planting. An ice planting device comprising: an ice planting refrigerant circuit that connects the cooler and a refrigerator; Pickled in
An ice-planting device for a biological sample cryopreservation device that cools the liquid phase part with a refrigerator, the ice-planting member being provided on a rack that supports the container so as to be in contact with the upper end of the container, and the container rack. When the ice-planting member is placed in a predetermined position, the ice-planting cooler is provided in the gas phase section so that the ice-planting member comes into contact with the ice-planting member, and at least a part of the refrigerant of the refrigerator is supplied to the ice-planting member through an expansion mechanism during ice-planting. The present invention provides an ice-planting device characterized by comprising an ice-planting refrigerant circuit that connects the ice-planting cooler and a refrigerator for flowing ice to the ice-planting cooler.

[Function] According to the method of the present invention, at a time when planting ice is required, a part of the freezing capacity of the refrigerator is allocated to planting ice in the preservation solution in the sample storage container.

According to one ice-planting device according to the present invention, when ice-planting is required, a part of the refrigerant after leaving the condenser of the refrigerator is transferred to the ice-planting refrigerant circuit in the cryopreservation device. The sample is passed through a cooler, whereby the gas phase is cooled to a lower temperature than the liquid phase, and ice is planted on top of the storage solution in the sample storage container.

According to another ice planting device of the present invention, at the time when ice planting is required, part of the refrigerant after leaving the condenser of the refrigerator is transferred to the freezing storage device by the ice planting refrigerant circuit. The ice is poured into an ice cooler, thereby cooling the ice planting member in contact with the ice cooler, and then planting ice on the upper part of the storage solution in the container that is in contact with the ice cooling member.

〔Example] Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 shows a cryopreservation apparatus 1 equipped with an embodiment of the present invention.
It shows.

The cryopreservation apparatus 1 includes a box body 10 that can be airtightly closed by Mll, which contains brine to provide a liquid phase portion 12, and a gas phase portion 13 provided above the liquid phase portion. can be cooled down to a predetermined temperature by a refrigerator 2.

The brine in the liquid phase part 12 is transferred to the box l by the circulation unit 3.
After being pulled out of the box and cooled to a predetermined temperature, it is returned to the inside of the box l.

The circulation unit 3 includes a cooling chamber 31, a pipe 32 connecting the bottom of the box l and the bottom of the cooling chamber 31, a pump unit 33 provided at the top of the cooling chamber 31, and a liquid phase part between the pump unit 33 and the box l. It consists of a pipe 34 that connects the.

The refrigerator 2 is equipped with a compressor 2L condenser 22, a cooler 23 consisting of a cooling pipe installed in the cooling chamber 31 of the circulation unit, and piping 24 connecting these.
An expansion mechanism 25 is connected to the pipe connecting the expansion mechanism 2 and the cooler 23, and an electromagnetic on-off valve SV2 is connected between the expansion mechanism and the condenser. A check valve CV2 is connected to.

Note that the cooling chamber 31 of the circulation unit has a built-in heater 35 for controlling the temperature of the brine.

This heater is operated by a program temperature controller 5 based on a liquid temperature signal from a sensor 36 installed in the liquid phase part inside the box body 1.
It is turned on and off under control from 1. The controller 51 is connected to an operating section 52.

Note that the compressor 21, condenser fan 221, pump unit 33, solenoid valve SV2, and stirring device 14 of the gas phase section 13 are also connected to the operating section 52, and are operated based on instructions from the operating section. .

The ice planting device attached to this cryopreservation device is configured as follows.

That is, the ice-planting device 4 includes an ice-planting cooler 41 consisting of a cooling pipe installed in the gas phase inside the box 1, and an ice-planting refrigerant circuit 42 that connects the cooler 41 and the refrigerator 2. It is equipped with The refrigerant circuit 42 includes a pipe 421 that guides a part of the refrigerant that has exited the condenser 22 of the refrigerator to the cooler 41, and a pipe 422 that returns the refrigerant that has exited the cooler 4I to the cooler 23 side of the refrigerator. In the pipe 421, there is an electromagnetic on-off valve S near the condenser.
(1) An expansion mechanism 423 is connected to the side closer to the cooler, and a check valve CVI is connected to the pipe 422. Valve SVI is turned on and off by operating section 52.

According to this cryopreservation device 1, the brine in the box 1 is contained in the box 1 and W! circulates between the cooling chamber 31 of the ring unit 3,
Meanwhile, in the cooling chamber 31, it is cold 21! It is cooled by the cooler 23 of the machine and adjusted to a predetermined temperature by the heater 35. In this way, the liquid phase inside the box 1 is maintained at a temperature suitable for cryopreserving biological samples.

In the liquid phase part, there is a straw 6 containing biological sample A and storage solution B.
A and tube 6b are supported by a rack R and soaked in brine. The upper ends of the straw 6a and the tube 6b come out into the gas phase, and the storage solution within the straw reaches the upper end of the straw.

Note that while the refrigerator 2 is in operation, the electromagnetic on-off valve SV2 remains open.

The ice planting by the ice planting device 4 is carried out as follows.

In order to prevent overcooling of the biological sample A in the straw or tube, the solenoid valve SVl in the ice-planting refrigerant circuit 42 is opened at or before the time when ice-planting is required, and the refrigerant leaving the condenser 22 is A part of the ice is passed through the expansion mechanism 423 and the ice planting cooler 41 and returned to the refrigerator main circuit.

Therefore, the temperature of the gas phase portion 13 within the box body 1 drops more quickly than that of the liquid phase portion 12 due to the cooling action of the cooler 41. In this way, the storage liquid near the upper end of the straw 6a and the tube 6b protruding into the gas phase part 13 is cooled with a rapid temperature drop gradient, brought to a supercooled state, and then ice is planted. After the ice is planted, using this as a core, the storage solution surrounding the straw and sample A in the tube is frozen in a state where there is no supercooling, or even if there is supercooling, the supercooling state is negligible.

In this way, the sample Δ in the straw 6a and the tube 6b can be frozen and preserved without being damaged by supercooling.

An example of the state of temperature drop in the liquid phase and gas phase during the sample freezing process is shown in FIGS. 2 and 3.

In FIGS. 2 and 3, line L1 indicates the temperature drop state of the preservation solution surrounding sample A in the sample storage container cooled by the liquid phase section 12, and line L2 shown by a broken line indicates the temperature drop of the preservation solution in the sample storage container. It shows the state of temperature drop in the uppermost part of the liquid. Moreover, line L3 shows the state of temperature drop in the gas phase portion.

Point 1-Lla on line L1 is storage solution B in the container.
The freezing start point of the part surrounding biological sample A is shown. Further, a point L2a on the line L2 indicates the freezing point.

The temperature program pattern shown in Figure 2 is based on the fact that the refrigerator has sufficient refrigerating capacity, so a portion of the refrigerator refrigerant continues to flow through the ice-planting refrigerant circuit not only when ice-planting is required, but also thereafter. The temperature program pattern in Figure 3 shows a case where the freezing capacity of the refrigerator does not have much margin when the brine is at a low temperature. The figure shows the case where the ice planting device is stopped by closing.

FIG. 4 shows a modification of the apparatus shown in FIG. 1. In this apparatus, the brine circulation unit 3 in the apparatus shown in FIG. It is located in

FIG. 5 shows a cryopreservation device equipped with an ice planting device 6 according to another embodiment of the present invention.

The basic structure of this cryopreservation apparatus 6 is the same as the apparatus shown in FIG. 1, and parts indicated by the same reference numerals as in FIG. 1 are the same as the apparatus parts in FIG. The ice-planting device 6 attached to this cryopreservation device consists of an ice-planting cooler 61 installed directly above the liquid phase portion 12 in the box 1 of the storage device and along the wall of the box, and It is provided with an ice planting refrigerant circuit 62 connected to a1 and an ice planting plate 63 that comes into contact with and separates from the cooler 61.

The ice-planting refrigerant circuit 62 includes a pipe 622 that guides a part of the refrigerant leaving the condenser 22 of the refrigerator to the cooler 61 via the electromagnetic on-off valve SVI and an expansion mechanism 621, and a pipe 622 that leads the refrigerant leaving the cooler 61 to the refrigerator 61. The piping 623 returns to the cooler 23 side, and a check valve CVI is connected to the piping 623.

The ice planting plate 63 is made of a material with good thermal conductivity (e.g. copper, aluminum, brass, etc.), and is made of a container (in this example, a straw 6) that holds the biological sample and preservation solution.
It is provided at the upper end of a container rack 7 that supports a large number of containers (indicated by a). When the container rack is placed in a predetermined position within the box l so that the straw 6a is immersed in the liquid phase portion 12 with the upper end remaining, the peripheral portion of the ice-planting plate 63 comes into contact with the cooler 61.

As shown in FIGS. 6 and 7, the container rack 7 is made up of three plates 71, 72 and 73 in the upper, middle and lower tiers connected and fixed at predetermined intervals by a plurality of vertical members 74. The plates 71, 72 are provided with a number of holes 70 into which straws are inserted, and the upper plates 1-71
A handle 75 is fixedly attached to the handle. The planting ice plate 63 is
It is provided on the lower surface of the upper plate 71, and the plate also has straw through holes 6 corresponding to the straw through holes 70.
31, and the hole diameter is formed to such an extent that a straw passed through this can come into contact with the ice planting plate.

The ice planting plate 63 has an inner surface of the straw through hole 631 which should be in contact with the straw and a portion 6 which should be in contact with the cooler 61.
All but 32 are surrounded by a heat insulating material 633.

Since the operation of the cryopreservation apparatus shown in FIG. 5 is similar to that of the cryopreservation apparatus 1 shown in FIG. 1, the explanation thereof will be omitted here. The method of planting ice using the ice planting device 6 shown in FIG. 5 is as follows.

That is, at or before the time when ice-planting is required, the solenoid valve SVI in the ice-planting refrigerant circuit 62 is opened and a part of the refrigerant that has exited the condenser of the refrigerator is transferred to the cooler via the expansion mechanism 621. In this way, the ice-planting plate 63 is cooled by the cooling 2S61, and the temperature of the upper end of each straw 6a is lowered more quickly than the liquid phase part by the ice-planting plate 63, whereby each straw Plant ice at the upper end of the storage solution in 6a. The ice planting pattern by this ice planting device can be, for example, the pattern shown in Figures 2 and 3, and the freezing pattern of sample A at the bottom of the straw is also similar to M shown in Figures 2 and 3. It can be done.

Note that the ice planting plate 63 in this ice planting device is taken out together with the straw 6a when the straw 6a is taken out from the box l together with the rack 7.

Note that the present invention is not limited to the above-mentioned embodiments, and it goes without saying that it can be implemented in various other ways.

〔Effect of the invention〕

According to the present invention, in a biological sample cryopreservation apparatus in which a biological sample storage container is immersed in a liquid phase with its upper end exposed to a gas phase, and the liquid phase is cooled with a refrigerator, each container storage chamber is It is possible to provide an ice planting method that can simply, reliably, and inexpensively plant ice in all storage containers at the same time without having to open them, and an ice planting device that implements the method.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an example of an ice planting device according to the present invention and an example of a cryopreservation device equipped with the same, and FIGS. A diagram showing an example of a temperature drop pattern when planting ice and freezing a sample; FIG. 4 is a diagram showing a modification of the apparatus shown in FIG. 1; FIG.
The figure shows an ice planting device according to another embodiment of the present invention and an example of a cryopreservation device attached thereto, FIG. 6 is a plan view of a container rack, and FIG. 7 is a sectional view of a container rack and an ice planting plate. . l... Cryopreservation device 12... Liquid phase section 13... Gas phase section 2... Freezer 4... Ice planting device 41... Ice planting cooler 42... For ice planting Refrigerant circuit 6... Ice planting device 61... Ice planting cooler 62... Ice planting refrigerant circuit 63... Ice planting plate 6a... Straw 6b... Tube 7... Container rack.

Claims (3)

[Claims] (1) In an ice-planting method for a biological sample cryopreservation apparatus in which a biological sample storage container is immersed in a liquid phase with its upper end exposed to a gas phase, and the liquid phase is cooled with a refrigerator, at least the container An ice-planting method, characterized in that, at a time when ice-planting is required, a part of the refrigerating capacity of the refrigerator is applied to the container in the gas phase section to plant ice. (2) An ice-planting device for a biological sample cryopreservation apparatus in which a biological sample storage container is immersed in a liquid phase with its upper end exposed to a gas phase, and the liquid phase is cooled with a refrigerator, comprising: An ice-planting cooler provided in a gas phase part, and a refrigerator and a refrigerator for flowing at least a part of the refrigerant of the refrigerator to the ice-planting cooler through an expansion mechanism during ice planting. An ice planting device characterized by comprising a connected ice planting refrigerant circuit. (3) An ice-planting device for a biological sample cryopreservation apparatus in which a biological sample storage container is immersed in a liquid phase with its upper end exposed to a gas phase, and the liquid phase is cooled with a refrigerator, comprising: An ice planting member is provided on a rack that supports containers so as to be in contact with the upper end of the container, and an ice planting member is provided in the gas phase portion so that the ice planting member is in contact with the container rack when the container rack is placed at a predetermined position. an ice cooler; and an ice-planting refrigerant circuit connecting the ice-planting cooler and the refrigerator for flowing at least a part of the refrigerant of the freezer to the ice-planting cooler via an expansion mechanism during ice planting. An ice planting device characterized by comprising: