Frozen cell thawing device

Abstract

To provide a frozen cell thawing device that can avoid the effects of cryoprotectants as much as possible when thawing frozen cells.SOLUTION: Provided is a frozen cell thawing device 11 including: an introduction pipe 25 for guiding a liquid medium 26; a melting unit 15 for holding a container 13 for introducing the medium 26 from the introduction pipe 25 while accommodating frozen cells 12 containing a freezing treatment liquid including a cryoprotectant, and applying melting heat to the frozen cells 12 from the medium in contact with the frozen cells 12; a liquid flow unit 34 for generating relative movement of the medium with respect to the frozen cells 12; and a discharge pipe 32 connected to the container 13 for guiding the discharge of the medium containing the thawed cells. When the thawed cells are dispersed in the medium, the concentration of the cryoprotectant in the medium is maintained at 3 vol.% or less, preferably 1 vol.% or less.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a frozen cell thawing device that thaws frozen cells and recovers the cells. [Background technology]

[0002] Patent Document 1 discloses a device for thawing frozen cells that includes a heater for heating a vial. A suspension containing cells is frozen in the vial. The heat of thawing the frozen cells is transferred from the heater to the vial. The frozen cells gradually thaw from the periphery toward the center. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 6661616 Summary of the Invention [Problem to be solved by the invention]

[0004] When freezing cells, a cryoprotectant is mixed into the suspension. Cryoprotectants generally contain dimethyl sulfoxide (DMSO). Since DMSO damages cells after thawing, it is desirable to complete thawing as quickly as possible. However, if cells are exposed to temperatures above 42°C, they will die. On the other hand, if the contact time between cells and DMSO is extended due to low-temperature thawing, the cells will die. Therefore, complex and advanced temperature control is required when heating the vial.

[0005] An object of the present invention is to provide a frozen cell thawing device that can avoid the influence of a cryoprotectant as much as possible when thawing frozen cells. [Means for solving the problem]

[0006] The frozen cell thawing device of the present invention comprises an inlet pipe for guiding a liquid culture medium, a melting unit that holds a container for accommodating frozen cells containing a freezing treatment liquid including a cryoprotectant and for introducing the culture medium from the inlet pipe while containing frozen cells, and applies thawing heat to the frozen cells from the culture medium that comes into contact with the frozen cells, a liquid flow unit that generates relative movement of the culture medium with respect to the frozen cells, and a discharge pipe connected to the container for guiding the discharge of the culture medium containing the thawed cells, and when the thawed cells are dispersed in the culture medium, the concentration of the cryoprotectant in the culture medium is maintained at 3 vol% or less. Effect of the Invention

[0007] As described above, the disclosed frozen cell thawing device can avoid the influence of the cryoprotectant as much as possible when thawing frozen cells. [Brief description of the drawings]

[0008] [Figure 1] 1 is an overall view showing, in outline, the configuration of a frozen cell thawing device according to one embodiment of the present invention; [Diagram 2] FIG. 2 is a schematic diagram illustrating the sensible heat change of a frozen cell. [Diagram 3] FIG. 2 is a schematic diagram illustrating the change in latent heat of a frozen cell. [Figure 4] FIG. 1 is a diagram illustrating a container containing frozen cells used in a simulation of the time required for thawing in a frozen cell thawing device. [Diagram 5] FIG. 1 is a diagram showing an example of the time course and thawing state during thawing in a frozen cell thawing device. [Figure 6] FIG. 1 is a diagram showing an example of time course and temperature change during thawing in a frozen cell thawing device. [Figure 7] FIG. 13 is a diagram showing a pattern of a culture medium introduction operation in the frozen cell thawing device of the embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings.

[0010] (Configuration of frozen cell thawing device 11) FIG. 1 shows a schematic configuration of a frozen cell thawing device according to one embodiment of the present invention. The frozen cell thawing device 11 includes a first container 13 that contains frozen cells 12, and a melting unit 15 that applies melting heat from a culture medium 14 in the first container 13 to the frozen cells 12 contained in the first container 13. Here, the first container 13 can be composed of, for example, a glass vial 13a and a cap 13b that closes the opening of the vial 13a. The frozen cells 12 are contained in the vial 13a. When freezing the cells, a suspension is contained in the vial 13a. The suspension is frozen together with the vial 13a. When storing the frozen cells 12, the frozen cells 12 can be cooled with liquid nitrogen, for example. At this time, the temperature of the frozen cells 12 drops to around minus 196°C. An inlet port 16 and an outlet port 17 are formed in the cap 13b.

[0011] The suspension is composed of a freezing solution and cells dispersed in the freezing solution. The freezing solution contains a culture medium, a cell cryopreservation solution, and human serum albumin. The cell cryopreservation solution contains, for example, dimethyl sulfoxide (DMSO) as a cryoprotectant. The concentration of dimethyl sulfoxide is, for example, 5 vol% to 10 vol% of the freezing solution, specifically set to 5 vol%. Dimethyl sulfoxide can prevent the expansion of water that solidifies within the cells when the cells are frozen. Dimethyl sulfoxide can effectively prevent the death of cells. In addition to dimethyl sulfoxide, examples of cryoprotectants include glycerol, ethylene glycol, and propylene glycol. The above cryoprotectants are cell membrane permeable and have the effect of penetrating into cells and suppressing ice crystal formation, but are known to be transformed into harmful substances through metabolism within the cells and to exhibit biological toxicity.

[0012] A sensible heat change unit 23 that heats the vial 13a from the outside is connected to the first container 13. The sensible heat change unit 23 includes a heater 24 that heats the vial 13a to a temperature lower than the melting temperature of the frozen cells 12. The heater 24 can have a shape that receives the vial 13a and supports the vial 13a. Since the temperature of the heater 24 is limited to a temperature lower than the melting temperature of the frozen cells 12, melting of the frozen cells 12 can be avoided by heat transfer from the vial 13a. On the other hand, the temperature of the frozen cells 12 can be brought as close to the melting temperature as possible by sensible heat change. Here, the temperature of the heater 24 is set to, for example, minus 1°C. Alternatively, the temperature of the heater 24 may be set to a high temperature within a range that does not kill the cells, for example, 37°C to 42°C, specifically 42°C, in order to accelerate the sensible heat change.

[0013] Since the temperature of the frozen cells 12 is heated to as high a temperature as possible by sensible heat change prior to thawing by the sensible heat change unit 23, the thermal energy of the culture medium can be efficiently utilized for latent heat change, and thawing of the frozen cells 12 based on the culture medium can be promoted. If the temperature of the frozen cells 12 is lower than the melting temperature of the culture medium, the culture medium in contact with the frozen cells 12 freezes, which hinders thawing of the frozen cells 12. However, if the temperature of the frozen cells 12 is increased, the freezing of the culture medium can be avoided even if the culture medium comes into contact with the frozen cells 12, and the culture medium can efficiently thaw the frozen cells 12.

[0014] A tilt unit 21 is coupled to the first container 13 to rotate the first container 13 around the horizontal axis 18. The tilt unit 21 includes a motor 22, for example, connected to a heater 24, to drive the heater 24 around the horizontal axis 18. The motor 22 includes, for example, a drive shaft that rotates around its axis in response to the supply of power. The rotational force of the drive shaft can be transmitted to the heater 24 via a gear mechanism. The tilt unit 21 can change the attitude of the first container 13 around the horizontal axis 18. The first container 13 can be held at a first position where the central axis of the vial (cylinder) 13a coincides with the vertical direction, and a second position where the cap 13b is positioned below the bottom of the vial 13a. In the second position, the discharge port 17 is positioned below the introduction port 16.

[0015] One end of an inlet pipe 25 that defines one flow path is connected to the inlet port 16 of the cap 13b. The inlet pipe 25 can be composed of a tube made of soft resin, for example. The other end of the inlet pipe 25 is connected to a second container 27 that holds a liquid culture medium 26. A culture medium suitable for the cells contained in the frozen cells 12 is used as the culture medium 26. The culture medium 26 can provide a survival environment for the cells, for example, at room temperature. The liquid culture medium 26 is introduced from the inlet pipe 25 to the first container 13. The introduced culture medium comes into contact with the frozen cells 12. In this way, the heat of fusion is transferred from the culture medium 26 to the frozen cells 12.

[0016] A heating unit 31 is connected to the second container 27 for heating the culture medium 26 contained in the second container 27. The culture medium 26 in the second container 27 can be heated to a temperature above room temperature by the action of the heating unit 31. Thermal energy is supplied to the culture medium 26. The culture medium 26 is heated prior to introduction into the first container 13. However, the temperature of the culture medium 26 is maintained below the cell death temperature. Here, the temperature of the culture medium 26 is limited to 37°C or less.

[0017] One end of a discharge pipe 32 that defines one flow path is connected to the discharge port 17 of the cap 13b. The other end of the discharge pipe 32 is connected to a third container 33 that collects the thawed cells. The discharge pipe 32 guides the discharge of the medium containing the thawed cells from the first container 13 to the third container 33. The suspension that has been thawed and diluted with the medium flows into the third container 33.

[0018] A pump 34 is connected to the inlet pipe 25 to move the culture medium from the second container 27 toward the first container 13. The pump 34 can function as a liquid flow unit that generates a relative movement of the culture medium with respect to the frozen cells 12 in the first container 13. For example, a tube pump can be used for the pump 34. Since the first container 13 is sealed, the pressure of the pump 34 can promote the discharge of the diluted suspension from the first container 13. The flow rate of the culture medium can be adjusted according to the operation of the pump 34. By adjusting the flow rate in this way, the concentration of dimethyl sulfoxide in the culture medium can be maintained at 3 vol% or less when the thawed cells in the first container 13 are dispersed in the culture medium. Since the thawed cells contact the culture medium containing dimethyl sulfoxide at a concentration of 3 vol% or less, the death of the cells due to dimethyl sulfoxide can be reduced regardless of the contact time with the culture medium. The cells can be thawed well.

[0019] In the frozen cell thawing device 11, the concentration of dimethyl sulfoxide in the medium is preferably maintained at 1 vol% or less when the thawed cells are dispersed in the medium, which further reduces cell death and allows the cells to be thawed well.

[0020] A control unit 35 is connected to the motor 22, the heater 24, the heating unit 31, and the pump 34. The control unit 35 supplies control signals to the motor 22, the heater 24, the heating unit 31, and the pump 34, respectively. The motor 22 drives the first container 13 around the horizontal axis 18 between the first position and the second position in response to the supply of the control signal. The heater 24 contacts the first container 13 at a temperature specified by the control signal in response to the supply of the control signal. The control signal can be generated, for example, based on the output of a temperature sensor attached to the vial 13a. The heating unit 31 contacts the second container 27 at a temperature specified by the control signal in response to the supply of the control signal. The control signal can be generated, for example, based on the output of a temperature sensor incorporated in the second container 27 to detect the temperature of the medium 26. The control unit 35 can be composed of, for example, a microprocessor unit (MPU). In this case, the operation of the control unit 35 can be realized based on software stored in a memory in the control unit 35.

[0021] Next, the operation of the frozen cell thawing device 11 will be described. Frozen cells 12 are prepared. The frozen cells 12 correspond to a coagulated body of the suspension. The suspension is contained in a first container 13 and frozen together with the first container 13. When preparing the suspension, the cells are mixed into a freezing treatment liquid. The freezing treatment liquid is formed, for example, of 50 vol% culture medium, 34 vol% cell freezing preservation liquid, and 16 vol% human serum albumin. Here, the freezing treatment liquid contains dimethyl sulfoxide at a concentration of 5 vol%. Dimethyl sulfoxide can effectively prevent the death of cells when they are frozen. When preserving the frozen cells 12, the first container 13 containing the frozen cells 12 is cooled, for example, with liquid nitrogen.

[0022] A first container 13 of frozen cells 12 is set in a melting unit 15. The first container 13 is mounted and supported, for example, on a heater 24. An inlet pipe 25 is connected to an inlet port 16 of the cap 13b. The inlet pipe 25 connects a second container 27 to the first container 13. An outlet pipe 32 is connected to an outlet port 17 of the cap 13b. The outlet pipe 32 connects a third container 33 to the first container 13. A control unit 35 controls the tilt unit 21. Here, the first container 13 is positioned in a first position about a horizontal axis 18.

[0023] The second container 27 holds a liquid culture medium 26. The control unit 35 controls the heating unit 31. A control signal is supplied from the control unit 35 to the heating unit 31. The heating unit 31 heats the culture medium 26 in the second container 27. The culture medium 26 can be heated to a temperature higher than room temperature. Here, the heating unit 31 is maintained at, for example, 37°C.

[0024] The control unit 35 controls the sensible heat change unit 23. The control unit 35 supplies a control signal to the heater 24. The heater 24 heats the frozen cells 12 in the vial 13a. As shown in FIG. 2, thermal energy is transferred from the vial 13a to the frozen cells 12. A sensible heat change is realized in the frozen cells 12. The temperature of the frozen cells 12 increases. The frozen cells 12 are warmed to below the melting temperature. The temperature of the frozen cells 12 is maintained below the melting temperature.

[0025] The control unit 35 controls the pump 34. The liquid medium 26 flows from the second container 27 into the first container 13. As shown in FIG. 3, the medium 38 contacts the surface of the frozen cells 12 in the first container 13. Thermal energy (heat of fusion) 39 is transferred from the medium 38 to the surface of the frozen cells 12. The frozen cells 12 melt. The thawed suspension can be dispersed in the medium 38 immediately. The thawed suspension is diluted with the medium 38. The cells 41 are dispersed in the medium 38. Since the suspension is pushed away from the surface of the frozen cells 12 by the flow of the medium 38, the concentration of the cryoprotectant can be regulated in a good range. In this way, the effect of the cryoprotectant on the cells 41 can be avoided as much as possible. Regardless of the duration of thawing, the death of the cells 41 due to the cryoprotectant can be reduced. The cells 41 can be thawed well. Prior to the introduction of the medium 38, the medium 38 can be warmed to a temperature higher than room temperature. The thawing of the frozen cells 12 can be accelerated.

[0026] Here, the control unit 35 controls the operation of the pump 34 according to the temperature of the frozen cells 12. A control signal is supplied from the control unit 35 to the pump 34. The control signal specifies the operation amount of the pump 34 based on the volume of the suspension melted by convection heat transfer in the medium 38. The operation amount of the pump 34 determines the flow rate of the medium 38. Based on the determined flow rate, the concentration of dimethyl sulfoxide in the medium 38 is maintained at 1 vol% or less when the thawed cells 41 are dispersed in the medium 38. Since the thawed cells 41 contact the medium 38 containing dimethyl sulfoxide at a concentration of 3 vol% or less, preferably 1 vol% or less, death of the cells 41 due to the cryoprotectant can be reduced regardless of the contact time with the medium 38. The cells 41 can be thawed well.

[0027] The medium 38 containing the thawed cells 41 flows from the first container 13 into the third container 33. Thus, the cells 41 are collected in the third container 33. The cells 41 can be separated from the medium 38, for example, by the action of a centrifuge. The control unit 35 may control the tilt unit 21 during thawing of the frozen cells 12. In response to the control, the first container 13 can be positioned at a second position about the horizontal axis 18. In the second position, the discharge port 17 of the cap 13b is located below the frozen cells 12, so that the discharge of the thawed cells 41 can be promoted.

[0028] In this embodiment, the heater 24 of the sensible heat change unit 23 heats the vial 13a at a temperature lower than the melting temperature of the frozen cells 12. Melting of the frozen cells 12 due to heat transfer from the heater 24 can be avoided. Melting of the frozen cells 12 separated from dilution can be prevented. In this way, the cells 41 can be well protected from a high concentration of cryoprotectant. Cell death due to the cryoprotectant can be reduced. On the other hand, since the temperature of the frozen cells 12 is heated to as high a temperature as possible by sensible heat change prior to melting, the thermal energy 39 of the medium 38 can be efficiently used for latent heat change. Thawing of the frozen cells 12 based on the medium 38 can be promoted. Moreover, if the temperature of the frozen cells 12 is raised as high as possible, freezing of the medium 38 can be avoided even if the medium 38 comes into contact with the frozen cells 12. The medium 38 can efficiently thaw the frozen cells 12. If the temperature of the frozen cells 12 is lower than the melting temperature of the medium 38, the medium 38 in contact with the frozen cells 12 will freeze. This will cause problems in thawing the frozen cells 12.

[0029] When thawing the frozen cells 12, the "temperature at which there is sufficient heat to thaw the cells" is expressed by the following formula. i is the mass of the frozen cell 12 [kg], θ i is the initial temperature of the frozen cells [℃], C i is the specific heat of the frozen cell [J / (kg K)], L is the latent heat of the frozen cell [J / kg], M w is the mass of the medium [kg], θ w is the initial temperature of the medium [℃], and C wis the specific heat of the medium [J / (kg K)].

[0030]

number

[0031]

number

[0032] Based on [Math 2], the "temperature with sufficient heat to thaw cells" shown in Table 1 was calculated.

[0033] [Table 1]

[0034] Here, the specific heat C i = 2040 [J / (kg K)] (specific heat of ice at 0°C), specific heat C w = 4180 [J / (kg K)] (specific heat of water at 20°C), and latent heat L = 3.336 x 10 5 [J / kg].

[0035] Next, assuming a situation in which culture medium is supplied to a container containing frozen cells, we simulated the time required for thawing, and obtained the calculation results shown in Table 2.

[0036] [Table 2]

[0037] As shown in Figure 4, frozen cells and culture medium were placed in a cylindrical container. The container was assumed to be insulated in the calculations. Convective heat transfer was not considered. Only temperature changes (sensible heat changes) and phase changes (latent heat changes) due to thermal conduction were considered. The calculations were performed centrally, assuming that there was no temperature gradient in the radial direction of the cylinder. As shown in Figure 5, in Example 1, it was calculated that the amount of melting per unit time was maintained roughly constant. As shown in Figure 6, it was calculated that the temperature was maintained roughly constant on the surface of the frozen cells regardless of the passage of time.

[0038] (Actions and Effects of Frozen Cell Thawing Device 11) In the frozen cell thawing device 11 of this embodiment, the heat of fusion is transferred to the frozen cells 12 from the culture medium in contact with the frozen cells 12. The thawed suspension can be dispersed in the culture medium immediately. The thawed suspension can be diluted with the culture medium. Since the suspension is pushed away from the frozen cells 12 by the flow of the culture medium, the concentration of dimethyl sulfoxide contained in the cell cryopreservation solution can be regulated within a good range. In this way, the influence of the cryoprotectant can be avoided as much as possible when thawing the frozen cells. Regardless of the duration of thawing, cell death due to dimethyl sulfoxide contained in the cryoprotectant is reduced, and the cells can be thawed well.

[0039] (Example) The present inventors prepared a suspension of iris pigmented epithelial cells. The cell concentration in the freezing solution was 1.0 to 2.0x10 6The suspension was set to [live cells / mL]. The freezing treatment solution was composed of 50 vol% medium, 34 vol% cell freezing solution, and 16 vol% human serum albumin. The freezing treatment solution contained dimethyl sulfoxide at a concentration of 5 vol%. The suspension was sealed in a 5 mL storage container. The storage container was slowly cooled in a freezer at minus 80°C. The cells were frozen in the storage container. The total amount of frozen cells and medium was adjusted to 30 mL. As shown in Figure 7, the medium was introduced into the storage container in several patterns. In Example 7, two storage containers were directly connected. In this verification, a heating unit was used instead of the sensible heat change unit 23. The experimental conditions and results are shown in Table 3.

[0040] [Table 3]

[0041] In Table 3, for "cell viability," the total number of viable cells, including remaining frozen cells, was counted, and for "cell yield," the cells contained in the culture medium discharged from the discharge pipe 32 were counted. [Explanation of symbols]

[0042] 11 Frozen cell thawing device 12 Frozen cells 13 Container (First Container) 15 Melting Unit 23 Sensible Heat Change Unit 25 Inlet piping 26 Culture medium (in the second container) 31 Heating unit 32 Discharge piping 34 Liquid flow unit (pump) 38 Culture medium (in the first container)

Claims

1. an introduction pipe for introducing a liquid culture medium; a melting unit that holds a container into which the medium is introduced from the introduction pipe while accommodating frozen cells containing a freezing treatment liquid including a cryoprotectant, and applies melting heat to the frozen cells from the medium that contacts the frozen cells; A liquid flow unit that generates a relative movement of the culture medium with respect to the frozen cells; a discharge pipe connected to the container for guiding the discharge of the medium containing the thawed cells; Equipped with When the thawed cells are dispersed in the medium, the concentration of the cryoprotectant in the medium is maintained at 3 vol% or less. Frozen cell thawing device.

2. The frozen cells contain the cryoprotectant in an amount of 5 vol% to 10 vol%. The apparatus for thawing frozen cells according to claim 1 .

3. When the thawed cells are dispersed in the medium, the concentration of the cryoprotectant in the medium is maintained at 1 vol% or less. The apparatus for thawing frozen cells according to claim 1 .

4. A heating unit is provided for heating the medium prior to introduction into the container. The apparatus for thawing frozen cells according to claim 1 .

5. A sensible heat change unit is connected to the container and heats the container. The apparatus for thawing frozen cells according to claim 1 .

6. The cryoprotectant contains dimethyl sulfoxide. The apparatus for thawing frozen cells according to claim 1 .

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