Cell freezing device

Abstract

To provide a cell freezer that has improved cooling performance, can cool a substrate to a liquid nitrogen temperature efficiently and for a long time, and stably and instantly freezes the cell-containing droplets dropped onto the substrate.SOLUTION: A cell freezer includes: a substrate 16 onto which cell-containing droplets are dropped; and a cooler that comes into contact with an object for cooling, including the substrate, to cool the object. The cooler is a liquid nitrogen absorber 13 having absorbed liquid nitrogen 11. The liquid nitrogen absorber 13 is fibrous material or sponge foam having wettability to liquid nitrogen.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an apparatus for depositing droplets containing cells (hereinafter referred to as "cell-encapsulating droplets") on a cooled substrate and instantly freezing them. [Background technology]

[0002] In various fields that handle cells, such as regenerative medicine, cell therapy, and clinical fields, cell freezing is an essential technology that allows stable long-term storage of cells. Existing cell freezing methods include slow freezing and rapid freezing. However, these require the addition of cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol to freeze cells (vitrified state) while suppressing the formation of ice crystals within the cells. As a result, problems have been raised regarding the cytotoxicity of cryoprotectants and the dehydration effect caused by osmotic pressure differences inside the cells during the freezing process.

[0003] In response to this, the present inventors have developed an ultra-instantaneous cell freezing method using inkjet technology that does not require any cryoprotectants at all (see Non-Patent Document 1). In this method, cells are encapsulated in microdroplets and ejected from an inkjet head, which are then deposited on a substrate cooled with liquid nitrogen. This allows the inside of the cells to be vitrified before ice crystals form, thereby achieving freezing of the cells. The substrate was cooled by placing the cooled object, including the substrate, on a metal (aluminum) base cooled with liquid nitrogen (see Non-Patent Document 2).

[0004] However, the adhesion between the metal base and the object to be cooled was insufficient, resulting in poor cooling performance, and problems occurred with the temperature rise and cooling rate of the substrate during the freezing and thawing process. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Y. Akiyama et al., “Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing,” Proc. Natl. Acad. Sci. USA, Vol.116(16), pp.7738-7743, 2019. [Non-Patent Document 2] H. watanabe, Y. Akiyama, “Improved and reproducible cell viability in the superflash freezing method using an automatic thawing apparatus,” Cryobiology, 96, pp.12-18, 2020. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above circumstances, the present invention provides a cell freezing device that has improved cooling performance, efficiently cools a substrate to liquid nitrogen temperature over a long period of time, and stably and instantly freezes cell-encapsulating droplets that have landed on the substrate. [Means for solving the problem]

[0007] According to one embodiment of the cell freezing device of the present invention, the device includes a substrate on which droplets containing cells are deposited, and a cooling member that comes into contact with an object to be cooled including the substrate and cools the object to be cooled, the cooling member being a liquid nitrogen absorbent that has absorbed liquid nitrogen. The liquid nitrogen absorbent is a fiber material or sponge foam that has elasticity and wettability with liquid nitrogen, and deforms to fit closely to the object to be cooled. The object to be cooled is placed on the top of the liquid nitrogen absorbent in contact with the object to be cooled so that heat can be transferred thereto. It is characterized by the above.

[0008] In addition, according to one embodiment of the cell freezing device of the present invention, the liquid nitrogen absorbent is It is a rectangular parallelepiped sponge foam, and the object to be cooled is placed on the flat top of the sponge foam. It is characterized by the above. Furthermore, according to one example of the cell freezing device of the present invention, the liquid nitrogen absorbent is a nonwoven fabric sheet wound in a spiral shape, the nonwoven fabric sheet is arranged so that the spiral surfaces form the upper and lower surfaces, and the object to be cooled is placed on the upper part of the spiral surface. Effect of the Invention

[0009] According to the cell freezing device of the present invention, the substrate on which the cell-encapsulated droplets are deposited can be cooled efficiently for a long period of time, thereby achieving the effect of stably and instantly freezing the cells. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram illustrating a cell freezing device and a cryopreservation process according to an embodiment of the present invention. [Diagram 2] 1A and 1B are diagrams illustrating a cell freezing device and a thawing process according to an embodiment of the present invention. [Diagram 3] FIG. 13 is a diagram illustrating a cell freezing device according to another embodiment of the present invention. [Figure 4] 1 is a photograph of a liquid nitrogen adsorbent and a cooled object placed on the liquid nitrogen adsorbent of a cell freezing device according to an embodiment of the present invention. [Diagram 5] 1 is a photograph of an aluminum base of a cell freezing device according to a comparative example and an object to be cooled placed on the aluminum base. [Figure 6] 13 shows the results of measuring the cooling preparation time in an embodiment of the present invention and a comparative example. [Figure 7] 1 shows the results of evaluation of low temperature maintaining performance of an embodiment of the present invention and a comparative example. [Figure 8] 1 is a fluorescent image of an embodiment of the present invention. [Figure 9] 13 is a fluorescence image of a comparative example. [Figure 10] This is an example of using a nonwoven fabric sheet as a liquid nitrogen absorbent. [Figure 11] This is an example of using polypropylene film as a liquid nitrogen absorbent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, an embodiment of the cell freezing device according to the present invention will be described with reference to the drawings, however, the present invention is not limited to the embodiment described here.

[0012] <One embodiment> 1 is a diagram illustrating a cell freezing device and a cryopreservation process according to one embodiment of the present invention. In this embodiment, the cell freezing device is used for instant cell freezing using inkjet technology.

[0013] (Cooling material) Liquid nitrogen 11 is stored in a polystyrene foam container 12. A liquid nitrogen absorbent 13, which is a cooling member, is immersed in liquid nitrogen 11, absorbs the liquid nitrogen 11, and is cooled to the liquid nitrogen temperature (-196°C). The liquid nitrogen absorbent means an absorbent that absorbs liquid nitrogen. For the liquid nitrogen absorbent 13, a fiber material or sponge foam having wettability with respect to the liquid nitrogen 11 can be used. For example, a nonwoven fabric such as polypropylene resin or nylon resin, a fiber such as glass wool, and a sponge foam having open cells made of melamine, urethane, polyvinyl alcohol, etc. can be used. This is because the liquid nitrogen absorbent 13, which is a cooling member, sufficiently contains the liquid nitrogen 11 and is cooled. The wettability can be evaluated by the magnitude of the contact angle θ. The smaller the contact angle θ, the higher the affinity with the liquid nitrogen 11 and the better the wettability. It is desirable for the contact angle θ to be 90° or less. At this time, the capillary phenomenon becomes prominent in the liquid nitrogen absorbent 13, so that the liquid nitrogen 11 permeates the liquid nitrogen absorbent 13 and the liquid nitrogen 11 can be sufficiently contained. In addition, the liquid nitrogen absorbent 13 is preferably made of a fibrous material such as a nonwoven fabric, since the material does not harden even after absorbing the liquid nitrogen.

[0014] When sponge foam is used for the liquid nitrogen absorbent 13, it can be cut into, for example, a rectangular parallelepiped shape. The rectangular parallelepiped shape has a flat top, so the object to be cooled can be stably placed on top. When a nonwoven fabric sheet is used, it can be rolled up like a roll cake and placed so that the spiral surface is at the top and bottom, as shown in Figure 10. The liquid nitrogen 11 soaks into the nonwoven fabric sheet and rises along it.

[0015] In addition to fiber materials and sponge foam, the liquid nitrogen absorbent 13 may also be made of multiple layers of film. For example, polypropylene film may be used as the film material. Examples of the film-layered structure are shown in Figs. 11(a) and 11(b). Fig. 11(a) shows a case where multiple layers of film are stacked and integrated like a bundle, and the stacked cross sections are placed on the upper and lower surfaces, while Fig. 11(b) shows a case where multiple layers of film are rolled and placed on the upper and lower surfaces. In either case, there is a small gap between the overlapping films, and when the film is immersed in liquid nitrogen 11, the liquid nitrogen seeps in through the gap by capillary action, and the liquid nitrogen can be sucked up to the top of the liquid nitrogen absorbent 13.

[0016] (Cooled object) Furthermore, the object to be cooled includes a glass substrate 16, and is composed of an aluminum transporter 14, an aluminum carrier 15, and a glass substrate 16. These are stacked and installed in the order of the transporter 14, the carrier 15, and the substrate 16 from the bottom. The transporter 14 contacts the upper part of the liquid nitrogen absorbent 13 of the cooling member in a heat-transferable manner, and the entire object to be cooled is cooled. The liquid nitrogen absorbent 13 has elasticity and deforms to contact the transporter 14 of the object to be cooled. Therefore, the transporter, carrier 15, and substrate 16 of the object to be cooled can be efficiently cooled to the liquid nitrogen temperature. As a result, the cell encapsulation droplets 18 discharged from the nozzle of the inkjet head 17 (made of glass) can be dropped on the surface of the cooled substrate 16 and instantly frozen.

[0017] In addition, in one embodiment of the cell freezing device of the present invention, although not shown, the container 12 is placed on a two-axis automatic stage, and the automatic stage and inkjet head 17 can be controlled by a computer so that they move in sync, thereby ejecting droplets of cell-encapsulated liquid at any position on the substrate 16.

[0018] Furthermore, the substrate 16 with the frozen cells on its surface can be transferred together with the carrier 15 to a cryovial 19 and stored for a long period of time in an LN (liquid nitrogen) tank 20 at -180°C or below.

[0019] FIG. 2 is a diagram illustrating a cell freezing device and a thawing process according to an embodiment of the present invention. The cell freezing device according to the present invention has excellent low-temperature maintenance performance, and can also be used in the thawing process. In the thawing process, first, the substrate 16 carrying frozen cells is taken out together with the carrier 15 from the cryovial 19 stored in the LN (liquid nitrogen) tank 20, and the substrate 16 and carrier 15 are set on the transporter 14. The transporter 14 is in close contact with the liquid nitrogen absorbent 13 that has absorbed the liquid nitrogen 11 and is cooled to the liquid nitrogen temperature, and cools the carrier 15 and substrate 16 set on the transporter 14. The transporter 14 is provided with a spring hinge (not shown) at the base as a rotation drive source, and a rotation torque is applied to the transporter 14. However, the transporter 14 is locked by an aluminum lock plate (not shown), and the rotation of the transporter 14 is suppressed. Then, when the lock plate is pulled forcefully, the transporter 14 rotates at high speed. The substrate 16 carrying the frozen cells is transported to just above the thawing medium 21 warmed to 37°C, and as shown in Figure 2, the substrate 16 drops and is immersed in the thawing medium 21 due to the inertia of rotation. This prevents the cells from heating up and allows the cells to thaw instantly. The time required for thawing, including the time it takes to drop, is approximately 21 ms.

[0020] <Other embodiments> 3 is a diagram illustrating a cell freezing device according to another embodiment of the present invention, in which the cell freezing device is used particularly for flash freezing of embryos (fertilized eggs).

[0021] Liquid nitrogen 11 is stored in a polystyrene foam container 12. The liquid nitrogen absorbent 13, which is a cooling member immersed in the liquid nitrogen 11, absorbs the liquid nitrogen 11 and is cooled to the liquid nitrogen temperature (-196°C). As in the above-mentioned embodiment, the liquid nitrogen absorbent 13 can be made of a fiber material or sponge foam that is wettable by the liquid nitrogen 11, and can be made of nonwoven fabric such as polypropylene resin or nylon resin, fiber such as glass wool, and sponge foam having open cells made of melamine, urethane, polyvinyl alcohol, etc. If the contact angle θ with the liquid nitrogen 11 is 90° or less, the liquid nitrogen absorbent 13 has high affinity and excellent wettability, and can sufficiently contain the liquid nitrogen 11.

[0022] Furthermore, the object to be cooled is composed of only the substrate 16. The material of the substrate 16 may be metal such as aluminum, titanium, titanium alloy, or ceramic such as alumina. The substrate 16 is in contact with the liquid nitrogen absorbent 13 of the cooling member in a heat-transferable manner, and the entire object to be cooled is cooled. The liquid nitrogen absorbent 13 has elasticity and deforms to come into close contact with the substrate 16, and can efficiently cool the substrate 16. Then, the liquid containing the embryo is sucked with a tapered pipette, and for example, 1 to 3 μL of minute droplets are dropped and deposited on the substrate 16. Since the substrate 16 is cooled to the liquid nitrogen temperature, the droplets 24 containing the embryo are instantly frozen and vitrified. Then, although not shown, the droplets can be placed together with the substrate in a cryotube that has been cooled in advance, and stored in liquid nitrogen. Also, after placing the substrate in a melting liquid and melting it, and confirming the embryo, it can be cultured. EXAMPLES

[0023] The low temperature performance and cell survival rate of a cell freezing device (Example) using the liquid nitrogen adsorbent according to the present invention as a cooling member for substrate cooling and a cell freezing device (Comparative Example) using a conventional aluminum base were evaluated and compared to confirm the effectiveness and effect of the present invention. The device used in the Example was the same as the device shown in Figures 1 and 2, and the device used in the Comparative Example was the device shown in Figures 1 and 2, except that the cooling member was replaced with an aluminum base instead of the liquid nitrogen adsorbent.

[0024] Fig. 4 is a photograph of the liquid nitrogen adsorbent 13 of the cell freezing device according to the embodiment of the present invention, the transporter 14 and carrier 15 for the object to be cooled that are placed on top of it, and the lock plate 23. For the liquid nitrogen adsorbent 13, a polypropylene nonwoven fabric commercially available as an oil adsorption pad (Monotaro, Internet retailer, product number: MOEP4050-4) was cut and rolled into a spiral shape as shown in Fig. 10, with the center part wrapped with wire to prevent it from spreading. The liquid nitrogen adsorbent 13 has excellent elasticity, and the transporter 14 was brought into close contact with the surface of the liquid nitrogen adsorbent 13.

[0025] FIG. 5 is a photograph of the aluminum base 22 of a conventional cell freezing device as a comparative example, the transporter 14 and carrier 15 for the object to be cooled that are placed on top of it, and the lock plate 23. Springs were attached to the four legs at the bottom of the aluminum base 22, and the inclination of the base was adjusted so that the transporter 14 would contact the base 22 and transfer heat. In the cell freezing device as a comparative example, although not shown, the aluminum base 22 is immersed in liquid nitrogen stored in a container and cooled to liquid nitrogen temperature. Other than using the aluminum base to cool the object to be cooled, the same cell freezing device as the example of the present invention was used (i.e., the liquid nitrogen, the container, the thawing medium, the transporter rotation mechanism, etc. were the same).

[0026] (Low temperature performance evaluation) The cooling performance was evaluated and compared for the case where a liquid nitrogen adsorbent was used (Example) and the case where a conventional aluminum base was used (Comparative Example).

[0027] Figure 6 shows the results of measuring and comparing the cooling preparation time, which is the time required to cool the device from room temperature to a temperature at which droplets can be frozen. A K-type thermocouple was used to measure the temperature of the top surface of the carrier 15 of each device in the example and comparative example. The cooling preparation time was defined as the time from the start of cooling to the completion of cooling, with the completion of cooling being when the temperature reached -185°C. The cell freezing device was installed in a clean booth to suppress temperature fluctuations due to air flow.

[0028] When the aluminum base of the comparative example was used, the cooling preparation time was 167 to 211 seconds, and the cooling rate was -1.0°C / sec to -1.2°C / sec. In contrast, when the liquid nitrogen absorbent of the embodiment was used, the cooling preparation time was 47 to 54 seconds, and the cooling rate was -3.9°C / sec to -4.3°C / sec. Therefore, it was found that when the liquid nitrogen absorbent was used, the cooling rate was about four times faster than when the conventional aluminum base was used, and the cooling preparation time could be shortened to within one minute.

[0029] FIG. 7 shows the results of measuring the time change in temperature of the upper surface of the carrier for the cell freezing devices of the embodiment and the comparative example after filling the carrier with liquid nitrogen and sufficiently cooling the carrier until the temperature of the upper surface of the carrier is stabilized at a low temperature of about -190°C for a certain period of time. The temperature was measured for 10 minutes by attaching a T thermocouple to the upper surface of the carrier. As a result, when the aluminum base of the comparative example was used, the low temperature was maintained from 93 seconds to 141 seconds, but when the liquid nitrogen adsorbent of the embodiment was used, a temperature rise of 0.9°C to 1.4°C was confirmed in 10 minutes. The reason why the temperature rose in the comparative example from 93 seconds to 141 seconds is thought to be because the liquid nitrogen evaporated over time, the height of the liquid level decreased, and the upper part of the aluminum base became higher than the height of the liquid level and emerged (exposed) from the liquid level, resulting in a decrease in cooling efficiency. On the other hand, the reason why such a sudden temperature rise does not occur in the examples is believed to be that even if the height of the liquid nitrogen level is lower than the top of the liquid nitrogen adsorbent (polypropylene nonwoven fabric) and the top of the liquid nitrogen adsorbent is exposed from the liquid level, the liquid nitrogen that has soaked into the liquid nitrogen adsorbent rises through the liquid nitrogen adsorbent by capillary action and is supplied to the top surface of the liquid nitrogen adsorbent, where it can cool the transporter and carrier of the cooled object above it. Therefore, it was confirmed that the cell freezing device of the present invention using the liquid nitrogen adsorbent can significantly improve the low temperature maintenance performance.

[0030] (Cell viability assessment) Next, we actually performed cell freezing using cultured NIH3T3 cells to confirm the effect of improving low temperature maintenance performance.

[0031] First, the density-adjusted culture medium of cells (NIH3T3) was used to suspend the cells (5.0 × 10 6 The cell concentration (cells / mL) was adjusted. Next, the conditions were set so that the volume of the ejected droplets would be about 40 pL, and droplets containing the cells were ejected from the inkjet head and landed on a glass substrate cooled with liquid nitrogen, where they were instantly frozen. Here, two methods were examined for cooling the substrate: one using the liquid nitrogen adsorbent according to the present invention (Example) and the other using a conventional aluminum base (Comparative Example).

[0032] After freezing, a 5 mL tube containing 4.5 mL of culture medium warmed to 37°C was placed as a thawing medium, and the lock plate of the device was pulled vigorously to instantly transport and immerse the substrate into the tube containing the 37°C culture medium to rapidly thaw the cells. The tube was then centrifuged at 20°C and 2000×g for 10 seconds, and the glass substrate was removed from the tube and centrifuged again at 20°C and 2000×g for 30 seconds. The supernatant was aspirated so that 100 to 200 μL remained, and after pipetting, it was seeded into 96 wells.

[0033] Then, using the cytofluorescent dye Double Staining (DOJINDO), the cytoplasm in sperm cells was stained green and the nuclei in dead cells were stained red. Fluorescence observation was then performed using an inverted microscope (ECLIPSETi, Nikon) under the conditions of phase contrast, Texas Red (excitation wavelength (EX) 596 nm, emission wavelength (EM) 615 nm), and GFP (EX 488 nm, EM 507 nm). The images obtained were stacked and the numbers of live and dead cells were counted, and the percentage of cells stained green out of the total number of cells stained was calculated as the viability rate.

[0034] Table 1 shows the results of counting the cell survival rate when the liquid nitrogen adsorbent was used and when the conventional aluminum base was used. Also, Fig. 8 and Fig. 9 are fluorescent images obtained by combining a fluorescent image and a phase contrast image after 3 hours of culture. Fig. 8 shows an image when the liquid nitrogen adsorbent of the embodiment was used, and Fig. 9 shows an image when the conventional aluminum base of the comparative example was used.

[0035] [Table 1]

[0036] From the results in Table 1, it can be seen that the cell survival rate was 46.7±3.1% when the aluminum base was used, while it increased to 57.1±4.3% when the liquid nitrogen adsorbent was used. Therefore, the effectiveness of the cell freezing device of the present invention using the liquid nitrogen adsorbent was confirmed. [Explanation of symbols]

[0037] 11 Liquid Nitrogen 12 containers 13 Liquid nitrogen absorbent 14. The Transporter 15 Career 16 Substrate 17 Inkjet head 18 Intracellular encapsulation droplets 19 Cryovial 20 Liquid Nitrogen Tank 21 Thawing medium 22 Aluminum base 23 Lock plate 24 Embryo-encapsulated droplets

Claims

1. A substrate onto which droplets containing cells are deposited; a cooling member that comes into contact with an object to be cooled including the substrate and cools the object to be cooled, the cooling member is a liquid nitrogen absorbent that has absorbed liquid nitrogen; The liquid nitrogen absorbent is a fiber material or sponge foam having elasticity and wettability with respect to liquid nitrogen, and deforms to adhere closely to the object to be cooled; A cell freezing device characterized in that the object to be cooled is placed in contact with the upper part of the liquid nitrogen absorbent so as to be capable of conducting heat therethrough.

2. The cell freezing device described in Claim 1, characterized in that the liquid nitrogen absorbent is a rectangular sponge foam, and the body to be cooled is placed on the flat upper part of the sponge foam.

3. The cell freezing device described in Claim 1, characterized in that the liquid nitrogen absorbent material is a nonwoven fabric sheet wound into a spiral shape, the nonwoven fabric sheet is positioned so that the spiral surfaces form the upper and lower surfaces, and the body to be cooled is placed on top of the spiral surfaces.

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