JP2020115813A - Cell thawing apparatus - Google Patents

Abstract

To provide a cell thawing apparatus by which anyone can easily thaw a cell such as a cultured epidermal cell cryopreserved in a cell freezing container.SOLUTION: A cell thawing apparatus 100 comprises a thawing part 200 and a base part 300. The thawing part 200 is provided with an upper housing 210, a lower housing 220, a pair of thawing surfaces 250 capable of sandwiching the cell freezing container 1, and a planar heater. The base part 300 is provided with a top plate 340 capable of mounting the thawing part 200, and oscillation means capable of oscillating the top plate 340. The thawing surface 250 is capable of wrapping a plurality of the cell freezing containers 1, and capable of thawing the containers even in the case where container shapes or sizes are different. Laminate members including at least a thermal conduction rubber layer or a heat storage gel layer, and the planar heater are severally housed in the upper housing 210 and the lower housing 220, and it is preferable that a frame body capable of holding the laminate members is attached freely detachably.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cell thawing device capable of thawing cells (for example, cultured epidermal cells and cultured fibroblasts) that have been cryopreserved in a cell freezing container.

(Refractory skin ulcer)
The refractory skin ulcer refers to a condition of the skin which is widespread and sometimes infected, which is frequently caused in a bedridden patient or a patient with diabetes mellitus or arteriosclerosis obliterans whose peripheral circulation is deteriorated. It is said that the number of patients who have to undergo amputation of the lower limbs due to intractable skin ulcer is about 10,000 per year, and the survival rate after 5 years is about 30% even if the surgery is performed, and even if they survive, the medical cost is high. Will be required.

(Trial of regenerative medicine for intractable skin ulcer)
There is also an attempt to perform regenerative medicine to transplant self-renewal by transplanting cultured epidermal cells to the affected part of a refractory skin ulcer, instead of the amputation of the lower limbs having the above-mentioned drawbacks. Cultured epidermal cells are stored in a cell freezing container such as a cryotube and are usually frozen and stored at -80°C to -196°C. Since the frozen cells are transported and stored in the cell freezing container at the terminal hospital, it is necessary to thaw the frozen cells contained in the cell freezing container during the operation.

(Conventional thawing technology for cryopreserved cultured epidermal cells: water bath)
As a conventional technique for thawing the frozen epidermal cells, there is a method using a water bath. This is a method in which the water in the bath is heated to about 37° C. lukewarm water, the tube containing the cells is warmed in this lukewarm water, and the cells in the tube are thawed while being rapidly stirred. However, the above-mentioned stirring and thawing operation in the water bath requires not only a high degree of skill, but also the risk of water entering the tube.

(Conventional frozen cell defroster)
In addition to the water bath described above, a frozen cell thawing device as shown in Non-Patent Document 1 is already available. A plurality of open-ended holes are provided on the upper surface of the thawing device, and cell containers are placed in these holes to heat them. However, since the above-mentioned opening hole is made in a predetermined size, it cannot be applied to a container other than a dedicated cell container adapted to this size. There are many hospitals nationwide, and many vendors supply medical equipment to these hospitals. The shape, structure, size, etc. of the cell container are varied depending on this fact, and thus there is a demand for a cell thawing device that can handle all of these cell containers.

JP 58-225019 A JP, 2007-061245, A JP, 2013-252204, A

Catalog No: YS-1 Frozen Cell Thaw Device, BM Equipment Co., Ltd. website, [January 13, 2019 search], Internet <https://www.bmbio.com/product/tabid73.html?pdid1=YS -1>

(Thawing equipment in other fields)
Turning to other fields other than frozen cells, devices for thawing frozen plasma bags have been proposed to date (see Patent Documents 1 to 3).

(Difference between frozen container for frozen plasma and frozen container for frozen cells)
In the case of frozen plasma bags, it is limited to bag-shaped containers, but there are various shapes such as plate-shaped and tube-shaped other than bag-shaped containers for frozen cells. There is a demand for a decompression device that can handle the above.

(Difference between storage temperature of frozen plasma and storage temperature of frozen cells)
Frozen plasma is usually stored at -20°C, but cells are stored at -80°C to -196°C. Therefore, a large amount of heat is required for thawing frozen cells as compared with the case of frozen plasma. In addition, since it contains cells, it is necessary to perform thawing within a short time of 3 minutes or less.

(Patent Document 1 defrosting device)
As described above, all of Patent Documents 1 to 3 are intended to thaw "frozen plasma", but the configuration thereof will be briefly described. The thawing device of Patent Document 1 heats the frozen blood by pressing a bag-shaped container containing frozen blood so that a pair of movable heater plates approach each other, and at the same time, by a drive mechanism including a motor, The heater plate is swiveled to promote convection in frozen blood.

(Pincher of Patent Document 1)
The defrosting target of Patent Document 1 is a bag-shaped container (specifically, a plastic bag) that stores frozen blood and the like. That is, the thawing target is flexible, and the thawing target itself can be deformed when sandwiched by a pair of heaters. Therefore, the holding portion on the heater side may be a simple rigid body (plate-shaped copper sheet), and in Patent Document 1, when the plate-shaped copper sheet comes into contact with the bag-shaped container and presses it, the bag-shaped container is made of copper. It will be deformed according to the sheet shape.

(Problems in Applying the Device of Patent Document 1 to Applications of the Present Invention)
However, in the case of frozen cells, the cell freezing container has a wide variety of shapes. For example, in the case of a tube-shaped container, the container itself does not deform. Therefore, it is necessary to deform the holding part on the heater side to hold the container. is there. Therefore, even if the device of Patent Document 1 is simply applied to the field of frozen cells as it is, the container in which these cells are stored (for example, the hard cylindrical container) cannot be thawed properly.

(Patent Document 2 defrosting device)
Similarly, Patent Document 2 discloses a thawing device in which a blood bag containing frozen plasma is rocked by a rocking means while being sandwiched and heated by a pair of heaters. In Patent Document 2, it is the first and second heat medium bags in which the flowable heat medium is sealed that directly contacts the thawing target.

(Problems in Applying the Device of Patent Document 2 to Applications of the Present Invention)
Although the first and second heat medium bags have flexibility, there is no problem as long as the thawing target (holding target) is a deformable blood bag, but the thawing target is a hard cell freezing container containing frozen cells. In the case of a flat cell freezing container having a sharp edge, there is a concern that the heating medium bag may be ruptured during use or the high temperature heating medium in the bag may be scattered.

Further, the swinging mechanism disclosed in Patent Document 2 moves one end of the sandwiching portion up and down, that is, swings the one end and the other end of the sandwiching portion like a seesaw. If it is necessary to thaw, the present inventors' experience shows that this method of rocking is inadequate when the thaw of frozen cells that requires the application of a high amount of heat in a very short time is assumed, and the rocking mechanism is also improved. There is a need. In other words, the device of Patent Document 2 cannot be applied to the thawing of frozen cells simply as it is.

(Decompression device of Patent Document 3)
Patent Document 3 is also a device for thawing frozen plasma contained in a deformable bag-shaped container, and a pair of melting plates sandwiching this is a hard plate-shaped body made of an aluminum plate or a copper plate. It is the same as the reference 1. Further, the swinging mechanism is similar to that of Patent Document 2 in that the sandwiching portion swings up and down like a seesaw. Therefore, even when the device of Patent Document 3 is applied to the thawing of frozen cells, the above-described problems described in the columns of Patent Documents 1 and 2 are concerned.

(Problems common to Patent Documents 1 to 3)
In addition, as can be said for any of the thawing devices of Patent Documents 1 to 3, as the number of times the freeze container is clamped and heated, the clamp part for clamping and heating becomes unclean due to dirt and the like. In particular, it is necessary not only to replace the contact part) but also to inspect and replace the heater and the like in the holding part. However, the devices of Patent Documents 1 to 3 cannot be said to have a structure that allows easy maintenance thereof.

Further, since any of the thawing devices of Patent Documents 1 to 3 is intended for a relatively large blood bag containing frozen plasma, it is usually intended to pinch and thaw one blood bag with one use. However, the cell freezing container containing frozen cells, which is the object of the present invention, is usually much smaller than a blood bag, and it is desired that a large number of cell freezing containers can be thawed in one use. When sandwiching and thawing the large number of containers at one time, it is desirable that the thermal environment such as heating conditions can be individually controlled for each cell freezing container. There is room for further improvement in these demands and applications.

(Purpose of the present invention)
The present invention has been made in view of such circumstances, and even in a terminal hospital that is not a specialized hospital, anyone can easily store cells (more specifically, cultured epidermal cells) cryopreserved in a container. It is an object of the present invention to provide a cell thawing device capable of thawing.

Another object of the present invention is to provide a cell thawing device which can be applied to cell freezing containers of various shapes and whose maintenance of the thawing part that frequently contacts the container is easy.

Another object of the present invention is to provide a cell thawing device capable of sandwiching a large number of cell freezing containers at one time and individually managing the thermal environment of each cell freezing container.

After intensive studies, the present inventors have found that the above problems can be satisfactorily solved by using a cell thawing device having the following structure, and completed the present invention.

That is, the present invention has the following configurations and features, for example.
(Aspect 1)
A cell thawing device for thawing frozen cells stored in a cell freezing container,
An upper casing, a lower casing, a hinge portion that connects the upper casing and the lower casing in an openable and closable manner, and the cell freezing provided inside the upper casing and the lower casing A pair of defrosting surfaces capable of holding the container, and a defrosting section provided with a planar heater capable of heating the defrosting surface,
A base part provided with a top plate on which the defrosting part can be placed, and a swinging means capable of swinging the top plate,
And
The cell thawing device, wherein the thawing surface can wrap a plurality of the cell freezing containers while being deformed, and can be thawed even when the shape or the size of the cell freezing container is different.
(Aspect 2)
Each of the upper housing and the lower housing contains a laminated member including at least a heat-conducting rubber layer or a heat storage gel layer and the planar heater in an order close to the thawing surface,
The heat conductive rubber layer or the heat storage gel layer has deformability,
The planar heater is a flexible film heater or a flexible rubber heater,
The cell thawing device according to aspect 1, wherein a frame capable of holding the laminated member is detachably attached to the upper casing and the lower casing.
(Aspect 3)
The cell thawing device according to aspect 1 or 2, wherein the thawing surface is covered with a plastic film.
(Aspect 4)
The cell thawing device according to any one of aspects 1 to 3, wherein the thawing unit is detachably attached to the top plate.
(Aspect 5)
The cell thawing device according to any one of aspects 1 to 4, wherein a plurality of the planar heaters are juxtaposed so as to divide the thawing surface.
(Aspect 6)
The cell thawing device according to any one of aspects 1 to 5, wherein the swinging means swings the top plate so as to draw a circle in a plan view of the top plate.
(Aspect 7)
Aspect 1 further characterized in that the base unit further comprises a memory in which a plurality of thaw modes suitable for frozen cells of different types are stored in advance, and a control means capable of selecting one of the thaw modes. 6. The cell thawing device according to any of 6.
(Aspect 8)
The cell thawing device according to any one of aspects 1 to 7, wherein cultured epidermal cells or cultured fibroblasts are used as the frozen cells.
(Aspect 9)
The cell thawing device according to any one of aspects 1 to 8, wherein the cell freezing container is a rigid container having no deformability.

(1. Capable of supporting cell freezing containers of various shapes and sizes)
According to the thawing device of the present invention, unlike the conventional thawing device, since the soft heating surface (and the heat conductive rubber layer having a deformability, the heat storage gel, the film heater, etc. close to the soft heating surface) is formed, It can be heated in such a way that it encloses cell freezing vessels of various sizes. This heating surface can be adapted to a wide variety of cell freezing containers by changing the casing size of the thawing unit and the type, size, number of layers, and combinations of the heat conductive rubber layer and heater.

The defrosting unit is configured to be attachable to and detachable from the base unit, and the upper casing and the lower casing of the defrosting unit are configured to include a frame body that detachably holds the laminated members in these casings. This not only facilitates maintenance of the thawing unit, but also makes it possible to replace the thawing unit with a different thawing unit having various thawing surfaces.

In addition, by applying shaking and vibration suitable for the shape and size of the cell freezing container during heating, cells (cells that progress from the frozen state to the solid-liquid state and then to the liquid state) can be used regardless of the shape of the cell freezing container. Can be uniformly stirred and heat can be evenly transferred to the cells.

(2. Thawing of various cells is possible)
Further, since the thawing apparatus of the present invention has the control means for controlling the heating condition and the swinging condition, the operator can freely change the setting of the thawing condition each time to suit the thawing target.

(3. Multiple decompressions are possible at one time)
Since the heating surface provided in the thawing section is a wide and flexible flat surface due to the presence of a heat conductive rubber layer or a film heater which contains or is close to this surface, a plurality of cell freezing containers can be used as desired heating surfaces. Can be placed in place. As a result, the device of the present invention can uniformly thaw a large number of frozen cells at once, and can also be applied to transplant surgery that requires a large amount of cells over a wide range.

(4. Anyone can easily decompress)
The thawing method using the thawing apparatus of the present invention does not require the skilled operation skill required for the conventional method using the water bath. The operator may operate the device by simply selecting or inputting the defrosting conditions suitable for the defrosting target using the operation panel or the like.

(5. Prevention of foreign matter)
The thawing method using the thawing apparatus of the present invention does not require a warm bath of the cell freezing container, which is essential for the water bath method, and the possibility of foreign matter being mixed in is significantly reduced. In addition, all the parts that come into contact with the cell freezing container are exchangeable (replaceable), and the cells can be thawed in a clean environment at all times.

It is the perspective view and exploded perspective view showing the cell defrosting device of the present invention. It is the front view and side view which showed the cell defrosting apparatus (separated state) of this invention. It is a disassembled perspective view of the defrosting part which showed the main components of the defrosting part. It is the figure which showed the temperature sensor attached to one or more heaters and heat insulating material layers. It is a figure showing the basic composition of the device of the base part. It is the top view and bottom view which showed the principal part of a base part. It is the side view which showed the principal part of a base part. It is the figure which showed the modification of the cell freezing container and frame which are the objects of thawing. It is an image showing the appearance of the decompression unit of the operation panel of the decompression device.

Hereinafter, the present invention will be described based on the following specific embodiments with reference to the accompanying drawings, but the present invention is not limited to these embodiments.

(Outline of device)
The cell thawing apparatus (hereinafter, also simply referred to as “thawing apparatus” or “apparatus”) 100 of the present embodiment mainly includes a thawing section (holding section) 200 and a base section 300. The defrosting unit 200 can be detachably mounted on the upper portion of the base unit 300, as will be described later. Note that FIG. 1B shows a state in which the defrosting unit 200 and the base unit 300 are separated from each other, and FIG. 1A shows the defrosting unit 200 above the base unit 300 (specifically, as described below. It shows a state in which a plurality of cell freezing containers 1 (1A) are placed on a plate) and one of the thawing surfaces 250 of the thawing unit 200 is arranged.

(Decompression target)
The object to be thawed by the device 100 of the present invention is the container 1 (hereinafter, also referred to as “cell freezing container” or “container”) for storing, transporting or storing frozen cells. The frozen cells stored in the container 1 are assumed to be cultured skin cells such as fibroblasts and epidermal cells in the present example, but are not necessarily limited to these cells.

(Specific example of cell freezing container)
There are various types of frozen cell containers 1 having various shapes and sizes. Specific examples of the cell freezing container 1 include, for example, a hard hollow cylindrical container 1B such as a cryotube as shown in FIG. 3 and FIG. 8 and a cell freezing container in which the edge portion 1Ae other than the cell containing area 1Ac is flat. There is 1A. As will be described later, the thawing apparatus 100 of the present invention is capable of sandwiching and heating a single container 1 in accordance with a wide variety of shapes and capacities of the container 1 as described below. It can be said that it is suitable for thawing. Further, in the thawing device 100, since the thawing surface 250 and the laminated members in the vicinity thereof are deformable, as will be described later, even a solid container 1 whose thawing target is not deformable can be wrapped flexibly and comfortably. Is possible.

(Outline of defrosting section)
As shown in FIGS. 1 to 3, the defrosting unit 200 has a pair of an upper housing 210 and a lower housing 220, and a hinge portion 230 that connects these housings so that they can be opened and closed. Further, the defrosting unit 200 is provided with a lock mechanism that holds the closed state of the upper casing 210 and the lower casing 220 in contact with each other. In the embodiment, the two first locking members 241 are juxtaposed to the front edge of the upper casing 210, and correspondingly, the two second locking members 242 are juxtaposed to the front edge of the lower casing 220. ing. The first locking member 241 is provided with a wedge-shaped convex portion 243 extending downward, the second locking member 242 is provided with a locking hole 244 facing upward, and the upper housing 210 and the lower housing 220 are separated from each other. When the wedge-shaped convex portions 243 enter the locking holes 244 when they come into contact with each other, the wedge-shaped convex portions 243 are temporarily held and fixed by engaging the wedge-shaped convex portions 243 with an engaging member (not shown).

(Upper case)
In the upper housing 210, layered members having substantially the same dimensions (approximately A5 size in the embodiment) are housed inside in a stacked state. In the example shown in FIG. 3, a heat insulating material layer 211, a heat conductive rubber layer 212, a planar heater (preferably a film heater as shown or a rubber heater not shown (for example, silicon rubber) in order from the upper casing 210). (Heater) 213 and a heat conductive rubber layer 214 are laminated, and these members 211 to 214 are integrally held inside the upper housing 210 by a frame body 215 having an opening 216. Therefore, most of the lower surface of the lowermost heat-conducting rubber layer 214 (that is, the upper thawing surface 250) is exposed from the opening 216 and comes into contact with and presses the cell freezing container 1 described below from above.

The heat conductive rubber layer 212 is not essential and may be omitted. That is, only the heat conductive rubber layer 214 may be installed on the upper housing 210. Note that, in FIG. 3, for convenience of description, the lock mechanism and the hinge portion 230 described above are omitted.

(Lower case)
On the other hand, also in the lower housing 220, layered members having substantially the same dimensions are housed inside in a laminated state. In this embodiment, the heat insulating material layer 221, the film heater 222, the heat conductive rubber layer 223, the film heater 224, and the heat conductive rubber layer 225 are laminated in this order from the lower housing 220, and the opening 227 is provided. It is integrally held inside the lower housing 220 by the frame 226. Therefore, most of the upper surface of the uppermost heat-conducting rubber layer 225 (that is, the lower thawing surface 250) is exposed from the opening 227 and comes into contact with and presses the cell freezing container 1 described below from below.

In this example, a plurality of film heaters 222 and 224 (two) were prepared in order to increase the amount of heat applied to the cell freezing container 1 per unit time, but the present invention is not limited to this example, and the target of defrosting is not limited. The number of heaters can be increased or decreased depending on the cell freezing container 1. For example, similarly to the upper housing 210, only one heater 224 may be configured.

(Frame that can be attached to and detached from the case)
The frame body 215 and the frame body 226 are preferably detachably attached to the upper housing 210 and the lower housing 220 by known connecting means such as screws S (see FIG. 2A). .. This makes it possible to easily remove a part or all of the laminated members such as the heaters 213, 222, 224 and the heat conductive rubber layers 212, 214, 223, 225, and replace or clean them.

(Modification of frame)
The opening 216 (227) of the frame body 215 (226) shown in FIG. 3 is configured to expose a large part (that is, the defrosting surface 250) of the area inside the housing except the frame body 215 (226). , But is not limited to this. For example, as shown in FIGS. 8C and 8D, the frame 215A (226A) provided with a plurality of openings 216A (227A) corresponding to the outer shape of the cell freezing container 1A and the outer shape of the cell freezing container 1B. A frame body 215B (226B) provided with a plurality of corresponding openings 216B (227B) may be prepared and used.

In this way, by using the openings 216A (227A), 216B (227B) and the like that match the shape and size of the object to be thawed (cell freezing container 1), the cell freezing containers 1A and 1B are framed by 215A (226A). , 215B (226B), it can be more surely held at an appropriate place, and an unexpected situation in which the containers 1A and 1B are accidentally dropped from the thawing unit 200 during the opening/closing operation of the thawing unit 200 or the thawing process. It can be reduced significantly. Further, there is no risk of the containers 1A (1B) contacting each other during thawing. Further, since the cell freezing container 1A (1B) does not move when closing the thawing unit 200, each container 1A (1B) and a film heater (for example, each opening 216A (227A) of the above divided heaters). , 216B (227B) on the projected area) is more reliable, and more appropriate temperature monitoring and heating on the thawing surface 250 are possible.

Even if the shape and dimensions of the container 1 are new, the frame bodies 215A (226A) and 215B (226B) of the modified example may be manufactured by a 3D printer or the like to quickly meet the customer's request. it can.

(Heat conductive rubber layer)
The heat-conducting rubber layers 212, 214, 223, and 225 provided on the upper housing 210 and the lower housing 220 are characterized by having deformability, and preferably have a slight shape memory property. As a result, even if any cell freezing container 1 is placed, these layers are more flexibly deformed in conformity with the container shape, wrap them, and the temperature in the layers can be kept more stable and constant. Like Note that part or all of the heat conductive rubber layers 212, 214, 223, 225 may be replaced with a heat storage gel layer.

(Thawed surface)
The upper and lower thawing surfaces 250 with which the cell freezing container 1 comes in contact are the inner surfaces of the heat conductive rubber layers 214 and 225 in the example shown in FIG. 1 and the like, but these surfaces are made of a plastic film 251 such as a vinyl sheet. It may be covered with, and this may be used as the thawing surface 250 (see FIG. 9C). As a result, if the thawing surface 250 becomes dirty after the device 1 has been used several times, the thawing surface 250 only needs to be replaced with a new one, so that it is possible to reduce the cost and time required for cleaning or replacing the thawing surface 250. ..

(Film heater)
As the film heaters 213, 222, and 224 described above, for example, an etched metal foil (for example, stainless steel foil) sandwiched between polyimide or polyester films from both sides can be used. Since the film-shaped heater is extremely thin (thickness is usually about 0.1 mm to 0.3 mm), it can be deformed according to the deformation of the adjacent heat conductive rubber layer. A rubber heater (not shown) having flexibility may be used.

(Temperature sensor)
As shown in FIG. 4A, a thermistor 241 (15 in the illustrated example) and a temperature sensor of a thermocouple 242 are provided on the upper and lower surfaces of the heaters 213 and 224. Such temperature sensors 241 and 242 send signals to the control means 320 in the base unit 300 through electric wires. For more accurate temperature monitoring, the temperature of each film heater is measured by a plurality of temperature sensors (for example, 8 to 20), the average temperature is calculated by the control means 320, and the average temperature is calculated. It may be used as a representative temperature of the heater.

In addition to the temperature sensor 241, a temperature sensor for preventing overheating and a safety device may be separately provided. For example, as shown in FIG. 4A, thermocouples 242 (two in the illustrated example) and temperature fuses 243 are provided in the heaters 213 and 224 as temperature sensors and safety devices for preventing overheating. On the other hand, as shown in FIG. 4B, a thermocouple 242 is provided on the heat insulating material layer 211 (221).

(Differential heating of the defrosting surface by attaching multiple heaters)
Further, the film heaters 213, 222, and 224 may be attached so that all of the defrosting surfaces 250 are covered with one heater, but as shown in FIG. So as to be divided into sections (blocks), a plurality of (16 in the illustrated example) relatively small heaters (that is, divided heaters) 260 and temperature sensors (in the illustrated example, thermoelectric generators are provided at the centers of the divided heaters 260. Pairs 242) may be juxtaposed. It is not necessary to intentionally limit the number of divisions of the divided heater 260, but if it is the A5 size defrosting surface 250 as described above, about 16 divisions (16 heaters) would be realistic.

Also in this case, a thermocouple 242 may be provided on the heat insulating material layer 211 (221) as a temperature sensor for preventing overheating. A thermal fuse 243, which is difficult to mount on the surface including the split heater 260, may be provided on the heat insulating material layer 211 (221).

(Merit 1 of split heating)
By using a plurality of small film-shaped heaters (hereinafter also referred to as “divided heaters”) 260 and temperature sensors 242 that are attached so as to divide the thawing surface 250, the plurality of cell freezing containers 1 are thawed. When sandwiching and thawing at the same time in the apparatus 100, the amount of heat can be applied to each container 1 while measuring the temperature at pinpoint, so that the temperature of each container 1 can be controlled.

(Advantage 2 of split heating)
Further, when the plurality of split heaters 260 and the temperature sensor 242 are installed, it becomes possible to heat the thawing surface 250 uniformly over the entire surface, as compared with the case where one heater is installed. No matter where the thawing target (cell freezing container 1) is placed, thawing can be performed under substantially the same temperature conditions.

(Advantage 3 of split heating)
In addition, when there are few cell freezing containers 1 to be thawed at one time in the thaw device 100, a region that does not require heating may occur on any of the thaw surfaces 250. For example, the left and right sides of the thawing surface 250 may not be needed and only the central region may need to be heated. In this embodiment of divided heating, if power supply is suppressed only to the divided heaters 260 placed in areas where heating is not required (for example, areas on the left and right sides), power consumption and energy can be saved.

(Merit 4 of split heating)
In addition, the surface including the heater is divided as shown in FIG. 4(c) (scales of fish), so that the flexibility and local movability of the portion are increased, and the shape and size of the container 1 are adapted. Since it becomes easier, the wrapping of these containers 1 becomes easier.

For example, the heat conductive rubber layers 212, 214, 223, 225 and the film heaters 213, 222, 224 having the above-described deformability include the thawing surface 250 or are provided in the vicinity of the thawing surface 250, and thus are different from each other, for example. Even when a plurality of shaped cell freezing containers 1 are sandwiched at the same time, it is possible to sandwich them and heat (melt) them.

(Base part)
Next, the base part 300 will be described in detail. As shown in FIG. 1 and the like, the base portion 300 includes a base portion housing 301, leg portions 302 that support the base portion housing 301, and a top plate 340. In addition, it is preferable that the leg portion 302 is provided with a vibration-proof pad, so that the vibration generated in the base portion 300 is suppressed from being transmitted to an external structure (not shown). In addition, FIG. 1B shows the internal structure of the base portion 300 by a broken line for reference.

(Basic configuration of base part)
Further, FIG. 5 is a diagram conceptually explaining the basic configuration of the base unit 300. The swinging means 310 capable of driving the defrosting unit 200, the swinging operation is controlled, the temperature signal is acquired/processed from the temperature sensor, and the defrosting condition is set/changed/stored in the base unit casing 301. There is provided a control means 320 for this purpose, and a power supply means 330 capable of supplying power to these means 310, 320.

As shown in FIG. 1, the main power switch 350 and the operation panel 360 are provided on the upper surface of the outer surface of the base casing 301, and the heating condition and the swing condition of the heater can be displayed and input. Further, a buzzer 380 is also attached, and it is possible to emit a warning sound, a start-up sound, an end sound, and the like.

Further, although not shown, instead of the function of the buzzer 380 or together with the function of the buzzer 380, a signal regarding the end of thawing or abnormal stop is sent to the control means 320 to the portable terminal of the operator (medical worker such as nurse). You may add a function.

(Connection between base and defroster)
As shown in FIGS. 1B and 2, the upper surface 341 of the top plate 340 is provided with a connection hole 342 capable of accommodating the convex portion 220p provided on the lower surface 220b of the lower housing 220. Accordingly, the defrosting unit 200 including the lower housing 220 can be attached to and detached from the top plate 340 located above the base unit 300. In addition, if a thawing unit having a size different from that of the thawing unit (not shown, for example, a thawing unit having a thawing surface of A4 or B5 size) is prepared, an appropriate size and number of the containers 1 to be thawed can be obtained. The defrosting unit having a size can be appropriately replaced and placed on the base unit 300. In addition, in the drawings such as FIG. 1B, the components (the temperature sensors 241, 242 and the planar heater 213) in the defrosting unit 200 that require power and communication from the power supply unit 330 and the control unit 320 of the base unit 300. 224, etc.) is omitted.

The structure inside the base casing 301 will be described with reference to FIGS. 6 and 7. For convenience of description, FIG. 7 shows the side surface of the base unit 300 after removing the base unit housing 301 and the top plate 340.

(Power supply means)
A power supply inlet 331 and a control/motor power supply 332 are provided in the base casing 301 as constituent members of the power supply means 330. The control/motor power supply 332 converts the voltage of AC100V obtained at the power supply inlet 331 into the voltage of DC24V and supplies electric power to the motor 311.

(Specific configuration of swinging means)
A motor 311, a speed increasing gear group 312, an eccentric shaft 313, a top plate 340, and the like are provided as constituent members of the swinging means 310. In addition to the motor driver 321, the operation monitor board 322 and the control board 323 are provided as constituent members of the control means 320. The operation monitor board 322 and the control board 323 also play a role of setting/monitoring/controlling the thawing temperature.

In addition to the above-mentioned components, a control board relay 324, a heater relay 325, a power fuse 326, etc. are also installed in order to configure an electric circuit.

(Rotation of the defrosting section by the rocking means)
The motor 311 is operated by being supplied with power by the control/motor power supply 332, and the motor driver 321 and the control board 323 control the rotation operation such as the number of rotations. The motor 311 meshes with the gear (312a) of the speed increasing gear group 312 provided on the lower side of the base housing 301, and the power of the motor 311 is generated by the gears 312a, 312b, 312c, The data is sequentially transmitted in the 312d. The gear 312d farthest from the motor 311 in terms of gear transmission is connected to the eccentric shaft 313. The upper portion of the eccentric shaft 313 is connected to the top plate 340.

With the above configuration, when the motor 311 rotates, the gears 312a to 312d of the speed increasing gear group 312 also rotate while increasing the rotation speed. Since the rotation center of the final gear 312d of the speed increasing gear group 312 and the eccentric shaft 313 are deviated from each other by several millimeters (1 to 10 mm, for example, 5 mm), when the speed increasing gear group 312 rotates, the eccentric shaft The 313 and the top plate 340 are driven, that is, rocked while being eccentric by 5 mm. That is, the top plate 340 moves in a plan view so as to draw a circle whose radius of rotation is the distance (deviation) caused by the eccentricity. Further, as shown in FIG. 6A, the eccentric shaft 313 may be connected to the top plate 340 via the connecting plate 343.

As shown in FIGS. 6 and 7, the swinging means 310 may be provided with a plurality of eccentric shafts 313, 314. Specifically, a second eccentric shaft (following side) 314 that is separated from the first eccentric shaft (driving side) 313 in parallel is also provided, and the first eccentric shaft 313 and the second eccentric shaft 314 are provided. Are connected by a transmission belt and gears (not shown). By providing the plurality of eccentric shafts 313 and 314 in this way, even if the weight of the thawing unit 200 increases, the thawing unit 200 can be reliably rocked without any trouble.

In addition to the swinging means 310 described above, a second swinging means (for example, a vibration motor (not shown)) may be installed on the top plate 340 or the like. As a result, not only the above-described rotation operation in the plane direction by the swinging means 310 (swinging at 100 to 1,000 rpm), but also minute vertical vibration by the second swinging means (for example, about 2,000 to 3,000 rpm). Vibration) can be applied to the top plate 340 and the defrosting unit 200.

The control means 320 can set the optimum parameters for realizing the swinging motion according to the shape and the number (capacity) of the cell freezing container 1. Further, in the control unit 320, in addition to the swing frequency, the heating temperature (for example, room temperature to 70° C.), the standby temperature (for example, room temperature to 70° C.), and the temperature increase rate from the lowest temperature (for example, 0 to 100). %), temperature upper limit from the minimum temperature (eg room temperature to 70° C.), thawing time (eg 1 to 10 minutes), and other parameters related to temperature raising programs, and other safety related parameters (eg, overheat prevention temperature) it can.

In addition, the control unit 320 can store a plurality (for example, three types) of thawing conditions in which the above parameters are combined according to the shape and type of the cell freezing container 1 in the memory 327 (see FIG. 5) in advance. Further, it is possible to display the temperature information sequentially acquired from the temperature sensor on a display unit such as the display display 366 (see FIG. 9A), compare it with the safety parameter, or store it in the memory 327. ..

(control panel)
The operation panel 360 shown in FIG. 9A is provided with switches such as a mode switch 361, a start switch 362, a set switch 363, an enter switch 364, and an up/down switch 365, a display display 366, a mode display lamp 367, and the like. An operating status indicator lamp 368 (standby indicator lamp, run indicator lamp, error indicator lamp, etc.) is provided.

(Various switches on the operation panel)
The mode switch 361 can select a desired mode from a plurality of preset defrosting conditions (defrosting modes). The finally determined decompression mode can be confirmed by the lighting state of any mode indicator lamp 367. The start switch 362 can select, for example, start or stop of operation. The set switch 363 can display and switch parameters, for example, in order to change the settings of various parameters constituting the defrosting condition. The enter switch 364 confirms the setting change of the above-mentioned parameter, for example. The up/down switch 365 inputs/changes a parameter setting value, for example.

(Selection and setting of decompression mode on operation panel)
In the defrosting apparatus 100 of the embodiment, up to three types (three modes) of thawing conditions suitable for the cell freezing container 1 or the type of cells can be stored in the memory 327 in advance, but the present invention is not limited to this example, and the number of modes can be increased or decreased. May be. Since the operator only has to select the decompression mode suitable for the decompression target from the plurality of preset decompression modes, the setting change for each decompression target can be done with little trouble. When thawing an object to be thawed that is not suitable for the preset mode (for example, a new type of cell or a cell freezing container of a new shape), use the set switch 363, the enter switch 364, the up/down switch 365, etc. The setting may be changed to the decompression mode.

(How to use the defroster)
Next, an example of a method of using the above-mentioned decompression device 100 will be described.
(1) Connect the power inlet 331 and a power outlet (100 VAC, not shown), and turn on the main power switch 350.
(2) By depressing the mode switch 361 on the operation panel 360, the defrosting condition according to the defrosting target is selected from three types of defrosting modes.
(3) When preheating is started and the set temperature is reached, the buzzer 380 and the operation state indicator lamp 368 notify the defrostable state.
(4) The thawing surface 250 is cleaned with ethanol or the like, and the cell freezing container 1 is set on the thawing surface 250.
(5) The upper casing 210 of the defrosting unit 200 is brought closer to the lower casing 220 to close and lock the defrosting space.
(6) Push down the start switch 362.
(7) When the thawing is completed, the buzzer 380 and the operation status display lamp 368 notify that the thawing is completed.
(8) Unlock the thawing unit 200, lift the upper housing 210 to gently open the thawing space, and take out the cell freezing container 1.
(9) After the cell freezing container 1 is taken out, if the operation status display lamp 368 indicating the defrostable state is on, the above (4) to (8) are performed for another (remaining) cell freezing container 1. Repeat the process. If the light is off, the process starts from the step (3).
(10) When the thawing work is completed for all the cell freezing containers 1, be sure to turn off the main power switch 350 and disconnect the power outlet.

(Maintenance after using the defroster)
After the use of the defrosting device 100, if the defrosting surface 250 is dirty, clean it with disinfecting ethanol or the like. If the defrosting surface 250 is heavily contaminated, the vinyl sheet 251 and the heat conductive rubber layers 212, 214, 223, 225 and the upper and lower frames 215, 226 forming the defrosting surface 250 are replaced with new ones. When performing this maintenance, be sure to turn off the main power switch 350 and disconnect the power outlet for safety.

(Verification test)
An experiment (demonstration test) of actually thawing a frozen cell and measuring the survival rate of the cell was performed using the thawing apparatus 100 of the present invention. In addition, as a comparative example, the cells were thawed in a warm bath using a water bath, and the survival rate of the cells was also measured.

(Culture of cells and measurement of cell viability during culture)
The test cells of this example were cultured according to the following method. That is, 3T3-J2 cells were cultured according to a standard method until they became 80% confluent, and after recovering these cells, they were suspended in a cell preservation solution (10% BS, 10% DMSO, 80% DME medium) and the viability was determined by automatic fluorescence. It measured with the cell counter LUNA-FL (Logos Bio Inc.).

(Cryopreservation of cells)
2 ml each of the cells suspended in the cell preservation solution, a cell freezing container (flat container) 1A (manufactured by JMS) or a cell freezing container (cylindrical container, cryotube) 1B (manufactured by Biologics #88) -3202) and stored frozen in liquid nitrogen according to a standard method.

(Thawing of cells and measurement of viable cell rate)
Cells stored in liquid nitrogen for a certain period of time were thawed using a water bath at 37° C. in a warm bath (comparative example) or the thaw device 100 of the present invention, and the cell viability was measured by the above-mentioned automatic fluorescence cell counter. ..

(Measurement result)
Table 1 shows the measurement results of cell viability under each test condition. In the thawing apparatus of the present invention, an optimal temperature rising program can be set for the frozen cells to be thawed, so that the cells can be thawed in an optimal state. Therefore, the survival rate of the cells was significantly higher than that of the conventional hot bath method (comparative example). Moreover, although the containers 1A and 1B having different shapes and dimensions were used as the cell freezing containers, the defrosting device of the present invention has a better cell viability after thawing, regardless of which shape of the containers 1A and 1B is used. It has been found that can be maintained high.

(Evaluation of cell culture and cell proliferation ability after thawing)
Next, the cells cryopreserved in the cylindrical cell freezing container 1B as described above are thawed by using the thaw device 100 of the present invention, and 1/5 of the amount is thawed in a petri dish (10 cm diameter). The cells were cultured for a week, and the number of cell divisions at the end of the culture was counted to evaluate the proliferation ability.

(Evaluation results)
Table 1 shows the observation results of the number of cell divisions under each test condition. In the thawing apparatus of the present invention, an optimal temperature rising program can be set for the frozen cells to be thawed, so that the cells can be thawed in an optimal state. For this reason, it was revealed that the cells after thawing survived in a state in which the higher cell proliferation ability was retained, as compared with the conventional warm bath method (comparative example).

As described above, by using the cell thawing device of the present invention, even in a terminal hospital that is not a specialized hospital, cells cryopreserved in a cell freezing container (more specifically, cultured fibroblasts or cultured epidermal cells) ) Can be easily unzipped by anyone.

Further, the cell thawing device of the present invention can be applied to cell freezing containers of various shapes, and the maintenance of the constituent members (for example, the thawing surface) of the thawing section that frequently contacts the cell freezing container is easy.

Further, the cell thawing apparatus of the present invention can provide a cell thawing apparatus capable of sandwiching a large number of cell freezing containers at one time and setting and managing the thermal environment for each cell freezing container.

As described above, the present invention not only supports the inter-hospital cooperation using frozen cells in the field of regenerative medicine, but also contributes to the establishment and popularization of cultured cell transplantation as a therapeutic method in place of conventional lower limb amputation. Without a doubt, its industrial utility value is very high.

1, 1A, 1B Cell freezing container 100 Cell thawing device 200 Thawing section 210 Upper casing 211 Heat insulating material layer 212, 214 Thermal conductive rubber layer 213 Sheet heater (film heater)
215 frame body 220 lower housing 221 heat insulating material layer 222, 224 planar heater (film heater)
223, 225 Thermal conductive rubber layer 226 Frame 230 Hinge part 241, 242, 243 Temperature sensor (thermistor, thermocouple, thermal fuse)
250 Defrosting surface 251 Plastic film (vinyl sheet)
260 split heater 300 base portion 301 base portion housing 302 base portion leg 310 swinging means 320 control means 330 power source means 340 top plate 350 main power switch 360 operation panel

Claims (9)

A cell thawing device for thawing frozen cells stored in a cell freezing container,
An upper casing, a lower casing, a hinge portion that connects the upper casing and the lower casing in an openable and closable manner, and the cell freezing provided inside the upper casing and the lower casing A pair of defrosting surfaces capable of holding the container, and a defrosting section provided with a planar heater capable of heating the defrosting surface,
A base part provided with a top plate on which the defrosting part can be placed, and a swinging means capable of swinging the top plate,
And
The cell thawing device, wherein the thawing surface can wrap a plurality of the cell freezing containers while being deformed, and can be thawed even when the shape or the size of the cell freezing container is different. Each of the upper housing and the lower housing contains a laminated member including at least a heat-conducting rubber layer or a heat storage gel layer and the planar heater in an order close to the thawing surface,
The heat conductive rubber layer or the heat storage gel layer has deformability,
The planar heater is a flexible film heater or a flexible rubber heater,
The cell thawing device according to claim 1, wherein a frame body capable of holding the laminated member is detachably attached to the upper casing and the lower casing. The cell thawing device according to claim 1 or 2, wherein the thawing surface is covered with a plastic film. The cell thawing device according to any one of claims 1 to 3, wherein the thawing section is detachably attached to the top plate. The cell thawing device according to any one of claims 1 to 4, wherein a plurality of the sheet heaters are juxtaposed so as to divide the thawing surface. The cell thawing device according to any one of claims 1 to 5, wherein the swinging unit swings the top plate so as to draw a circle in a plan view of the top plate. The base unit further comprises a memory in which a plurality of thawing modes suitable for frozen cells of different types are stored in advance, and a control unit capable of selecting one of the thawing modes. 7. The cell thawing device according to any one of to 6. The cell thawing device according to any one of claims 1 to 7, wherein cultured epidermal cells or cultured fibroblasts are used as the frozen cells. The cell thawing device according to any one of claims 1 to 8, wherein the cell freezing container is a rigid container having no deformability.