Method for freezing fine droplets and device used therefor

Abstract

Provided are a method for freezing fine droplets with which it is possible to more efficiently and appropriately freeze a larger number of fine droplets 15, and a device used therefor. A method and a device for freezing fine droplets whereby fine droplets 15, discharged from a discharge head 11 while including fine matter, are frozen into a vitrified state by cooling rapidly, the fine droplets 15 being frozen by passage through a gas-phase cooling space 20 cooled by a cooling means 30. The cooling space 20 is an interior space in which two ends in the direction of passage of the fine droplets 15 are provided as an inlet and an outlet. A generation device related to the fine droplets 15 including the discharge head 11 is provided by inkjet technology or cell sorter technology. The discharge head 11 is disposed on the inlet 21 side of the cooling space 20 facing the cooling space 20, and a frozen fine droplet receiving part 50 in which the frozen fine droplets are stored is disposed on the outlet 22 side of the cooling space 20.

Description

Method for freezing microdroplets and apparatus therefor

[0001] The present invention relates to a method for freezing microdroplets, which are ejected from a discharge head while enclosing micro-objects such as cells, and rapidly cooled to be frozen in a vitrified state, and an apparatus therefor.

[0002] The cryopreservation technique of cells is one of the indispensable techniques in many research fields and clinical medicine that handle cells. When cells are frozen at a normal speed as they are, water molecules inside and outside the cells crystallize during the freezing process, which damages cell membranes and cell organelles, thereby causing lethal damage to the cells. Therefore, in existing freezing methods, cryoprotective agents such as dimethyl sulfoxide and glycerol are added to suppress the crystallization of water molecules inside the cells and achieve cryopreservation by making them in a vitrified state. However, the cytotoxicity of cryoprotective agents and the dehydration effect due to the osmotic pressure difference inside the cells during the freezing process have been regarded as problems.

[0003] In contrast, the present inventor has developed an apparatus that uses inkjet technology to enclose cells in microdroplets and discharges them onto a substrate cooled with liquid nitrogen, thereby cryopreserving the cells instantaneously without a cryoprotective agent. In this apparatus, water molecules inside and outside the cells are brought to the glass transition temperature (Tg: -137°C) before crystallization, thereby making them in a vitrified state. When this apparatus is used for general animal cells, it shows almost the same survival rate as the conventional method, and in undifferentiated cells, the undifferentiated state was confirmed after thawing (see Non-Patent Document 1).

[0004] Further, the present inventor has disclosed a method for handling frozen cell-containing droplets and an apparatus therefor (see Patent Documents 1 and 2), in which cell-containing droplets (microdroplets) are discharged onto the surface of a cooled carrier member by an apparatus using inkjet technology, and the frozen cell-containing droplets frozen and adhered to the surface of the carrier member are separated and peeled off from the surface of the carrier member by applying an impact to the carrier member, and the frozen cell-containing droplets separated from the carrier member by the impact are stored in a container that opens facing the surface of the carrier member to which the frozen cell-containing droplets are adhered and is cooled, i.e., in a cooled cooling container.

[0005] In the conventional inkjet technology described above, tiny droplets ejected from the nozzle of a glass inkjet head were deposited onto an aluminum substrate cooled with liquid nitrogen, or onto a glass substrate placed on top of that substrate. This method required managing and processing the droplets via the substrate to which they were attached, making it difficult to increase processing speed.

[0006] Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing. Yoshitake Akiyama, Masato Shinose, Hiroki Watanabe, Shigeru Yamada, and Yasunari Kanda. PNAS April 16, 2019 116 (16) 7738-7743; first published April 1, 2019. https: / / doi.org / 10.1073 / pnas.1808645116. Edited by Robert H. Austin, Princeton University, Princeton, NJ, and approved March 4, 2019 (received for review June 27, 2018). Japanese Patent Publication No. 2022-71350 (p. 1). Japanese Patent Publication No. 2024-123475 (p. 1).

[0007] The problem that we aim to solve with regard to methods for freezing microdroplets and the apparatus used therefor is that, in conventional methods, rapid freezing is performed by depositing microdroplets ejected by inkjet technology onto a substrate such as a cooled glass substrate, making it impossible to perform the freezing operation continuously. In other words, in conventional methods, the management and processing of the microdroplets is done via the substrate to which they are attached, resulting in a slow processing speed and making it difficult to freeze a large number of microdroplets.

[0008] Therefore, the object of the present invention is to provide a method for freezing microdroplets and an apparatus for using the same, which can freeze a larger number of microdroplets more efficiently and appropriately.

[0009] To achieve the above objective, the present invention has the following configuration. According to one example of a method for freezing microdroplets according to the present invention, a method for freezing microdroplets is characterized by rapidly cooling microdroplets discharged from a discharge head containing minute objects to freeze them into a vitrified state, wherein the microdroplets are frozen by passing them through a cooling space of a gas phase cooled by a cooling means.

[0010] According to an example of a method for freezing minute droplets according to the present invention, the cooling space is characterized by being an internal space with both ends serving as inlets and outlets in the direction of passage of the minute droplets.

[0011] Furthermore, according to an example of a microdroplet freezing apparatus according to the present invention, the apparatus used for the method of freezing microdroplets comprises the discharge head, the cooling means, and the cooling space, wherein the generating apparatus for the microdroplets, including the discharge head, is provided by inkjet technology or cell sorter technology, the discharge head is positioned on the side of the inlet of the cooling space so that the discharged microdroplets can pass through the cooling space from the inlet to the outlet, and a frozen microdroplet receiving section for storing the frozen microdroplets is positioned on the side of the outlet of the cooling space from which the frozen microdroplets are discharged.

[0012] Furthermore, according to an example of a microdroplet freezing apparatus according to the present invention, the cooling means is characterized by comprising a cooling space forming wall portion that forms the cooling space, and a liquid nitrogen holding portion that is in contact with the periphery of the cooling space forming wall portion.

[0013] Furthermore, according to an example of a microdroplet freezing apparatus according to the present invention, the liquid nitrogen holding section is characterized by comprising a liquid nitrogen storage tank-like section in which liquid nitrogen is stored, and a liquid nitrogen impregnation section provided in contact with the periphery of the cooling space forming wall section and into which the liquid nitrogen is impregnated.

[0014] Furthermore, according to an example of a microdroplet freezing device according to the present invention, the liquid nitrogen impregnation portion is provided by a member made of a fibrous material or a sponge-like member that is capable of absorbing liquid nitrogen.

[0015] Furthermore, according to an example of a microdroplet freezing apparatus according to the present invention, the cooling means is characterized by comprising a cooling space forming wall portion that forms the cooling space, and a Peltier element in contact with the periphery of the cooling space forming wall portion.

[0016] Furthermore, according to an example of a microdroplet freezing apparatus according to the present invention, it may be characterized by comprising a micro-vibration device for micro-vibrating the frozen microdroplet receiving section, or a micro-movement device for micro-moving the frozen microdroplet receiving section.

[0017] Furthermore, according to an example of a microdroplet freezing apparatus according to the present invention, the discharge head is positioned downward so that the discharge direction of the microdroplets is downward along the vertical direction, and the cooling space is a passage that extends vertically.

[0018] Furthermore, according to an example of a microdroplet freezing device according to the present invention, the cooling space forming wall portion may be a straight tubular member or an array forming member made of a metal material.

[0019] The method for freezing microdroplets and the apparatus used therefor according to the present invention offer the particularly advantageous effect of being able to freeze a larger number of microdroplets more efficiently and appropriately.

[0020] This is a schematic cross-sectional view showing an example of the configuration of the microdroplet freezing device according to the present invention. This is a schematic cross-sectional view showing an example of the configuration forming the cooling space of the microdroplet freezing device according to the present invention. This is a schematic perspective view showing an example of the configuration forming an array-shaped cooling space of the microdroplet freezing device according to the present invention. This is a schematic cross-sectional view showing an example of the configuration of the frozen microdroplet receiving section of the microdroplet freezing device according to the present invention. This is a graph showing the relationship between cooling time and microdroplet temperature for microdroplets of a specific size passing at a specific speed through a cooling space cooled to a required cooling temperature when using the microdroplet freezing device according to the present invention. This is a schematic cross-sectional view showing an example of the configuration of the discharge head of the microdroplet freezing device according to the present invention.

[0021] Examples of the microdroplet freezing method and apparatus used therefor according to the present invention will be described in detail based on the attached drawings (Figures 1 to 6). The microdroplet freezing apparatus according to the present invention shown in Figure 1 and others can be used in a microdroplet freezing method that rapidly cools microdroplets 15, which are discharged from a discharge head 11 containing fine particles (microorganisms) such as cells 15a to be frozen, and freezes them into a vitrified state.

[0022] The method for freezing microdroplets according to the present invention is characterized by freezing the microdroplets 15 by passing them through a cooling space 20 of the gas phase cooled by a cooling means 30, as shown in Figure 1 and the like. In other words, the present invention proposes a method and apparatus for freezing microdroplets 15 ultra-instantaneously during passage by passing them from one end (inlet) to the other end (outlet) of a cooled cooling space 20 (for example, the internal space of a hollow metal pipe (tube) or array forming member that is open at the top and bottom) in the air (gas phase) along the vertical direction.

[0023] In other words, the starting point of the present invention lies in the observation that when minute droplets 15 discharged from the discharge head 11 pass through the gas phase cooling space 20, they become granular droplets close to spheres due to surface tension, making them easily cooled uniformly. The essence of the present invention lies in the discovery that such minute droplets 15 can be cooled to a temperature lower than the temperature at which they can freeze in a vitrified state, by passing them through a temperature space (cooling space 20) of appropriate length that is appropriately cooled, at an appropriate passage speed, and for an appropriate amount of time (appropriate cooling time), thereby enabling appropriate freezing and appropriate recovery. Furthermore, as will be explained below, it has been confirmed that the appropriate passage speed, appropriate passage time (appropriate cooling time), and passage distance related to the freezing of minute droplets 15 can be appropriately set in an appropriate cooling space 20.

[0024] This method allows for the continuous collection of frozen droplets at the outlet 22 of the cooling space 20 (directly below in this embodiment) without the need for a conventional freezing substrate (such as a glass substrate). Therefore, it offers the particularly advantageous effect of enabling the more efficient and appropriate freezing of a larger number of microdroplets 15. Experiments using animal cultured cells confirmed that a survival rate nearly equivalent to that achieved with conventional glass substrate methods was obtained. Furthermore, aggregation ability was evaluated for human platelets, yielding favorable results.

[0025] Furthermore, in the method for freezing microdroplets according to the present invention, the cooling space 20 can be appropriately and rationally configured by being an internal space with both ends serving as inlets and outlets in the direction of passage of the microdroplets 15. That is, for example, the internal space (cooling space 20) can be appropriately and easily formed by a hollow metal pipe open at the top and bottom (see Figures 1 and 2) or an array forming member (see Figure 3).

[0026] Furthermore, according to the microdroplet freezing apparatus of the present invention, the apparatus comprises a discharge head 11, a cooling means 20, and a cooling space 20, and the generating apparatus for microdroplets 15 including the discharge head 11 is provided by inkjet technology or cell sorter technology, the discharge head 11 is positioned on the side of the inlet 21 of the cooling space 20 so that the discharged microdroplets 15 can pass through the cooling space 20 from the inlet 21 to the outlet 22, and a frozen microdroplet receiving section 50 for storing the frozen microdroplets 15 is positioned on the side of the outlet of the cooling space from which the frozen microdroplets are discharged (see Figure 1, etc.).

[0027] In other words, in this embodiment, the discharge head 11 is positioned facing the inlet 21 of the cooling space 20. The frozen microdroplet receiving section 50 is provided in the form of a tray-shaped container 51 with an open top so that the frozen microdroplets 15 can be collected, and is cooled by a cooling means 52. As the cooling means 52, liquid nitrogen or a Peltier element can be used, similar to the cooling means 30 of the cooling space 20 (details will be described later). In this embodiment, the discharge head 11 is positioned so as not to come into contact with the cooling space forming wall 31 that forms the inlet 21 of the cooling space 20, in order to prevent it from freezing.

[0028] According to this, the microdroplet freezing device according to the present invention can be rationally configured, and it has the particularly advantageous effect of being able to freeze a larger number of microdroplets 15 more efficiently and appropriately. Furthermore, since the microdroplets 15 can be ejected by spraying using inkjet technology, the ejection speed of the microdroplets 15 can be increased, and the microdroplets 15 fly straight and do not come into contact with the inner wall surface 31a of the member forming the cooling space 20, but are frozen stably and instantaneously in the cooling space 20 and can reach the frozen microdroplet receiving section 50. As a result, a large number of frozen microdroplets 15 can be continuously generated, and this large number of frozen microdroplets 15 can be appropriately stored in the frozen microdroplet receiving section 50.

[0029] In this embodiment, the cooling space 20 is a long and narrow internal space (passage) in the direction through which the microdroplets 15 pass. This long and narrow internal space (cooling space 20) means that the length in the direction through which the microdroplets 15 pass is longer than the width in at least one direction perpendicular to the direction through which the microdroplets 15 pass (for example, the inner diameter of a tube (cylindrical body) or the width of the narrowest part of a flattened array). In other words, the required length in the direction through which the microdroplets 15 pass in the cooling space 20 is determined in relation to the passage speed and time of the microdroplets 15 and the cooling rate, but usually the length in the direction through which the microdroplets 15 pass is set to, for example, at least 5 times, and usually 10 times or more, the width in the aforementioned direction. This allows the cooling space 20 to be cooled efficiently and appropriately.

[0030] Furthermore, the cooling means in this embodiment includes a cooling space forming wall portion 31 that forms a cooling space 20, and a liquid nitrogen holding portion 40 that is in contact with the periphery of the cooling space forming wall portion 31. This allows the cooling space 20 to be appropriately provided between the discharge head 11 and the frozen microdroplet receiving portion 50. As shown in Figure 2, the liquid nitrogen holding portion 40 in this embodiment includes a liquid nitrogen storage tank-like portion 41 in which liquid nitrogen (refrigerant 43) is stored, and a liquid nitrogen impregnation portion 42 that is in contact with the periphery of the cooling space forming wall portion 31 and into which the liquid nitrogen is impregnated. In the embodiment shown in Figure 2, the cooling space forming wall portion 31 (metal pipe) and the liquid nitrogen storage tank-like portion 41 are integrally provided in a liquid-tight seal state.

[0031] Furthermore, the liquid nitrogen-impregnated portion 42 in this embodiment is preferably a member made of a fibrous material or a sponge-like member that can absorb liquid nitrogen. As a specific example, as shown in Figure 2, a polypropylene nonwoven fabric is wrapped around the outer circumference of a metal pipe with good thermal conductivity (cooling space forming wall portion 31) that forms a long, narrow cooling space 20, thereby forming the liquid nitrogen-impregnated portion 42. This allowed the inside of the metal pipe (cooling space 20) to be stably maintained at a low temperature of approximately -180°C. In addition, finite element analysis allowed for the calculation of the fall time required for freezing of each size of minute droplet 15, and the required length of the metal pipe could be determined.

[0032] According to this, the long and narrow cooling space 20 can be cooled rationally and appropriately, and a larger number of minute droplets can be frozen more efficiently and appropriately. In other words, in this liquid nitrogen impregnation section 42, liquid nitrogen 43 can be drawn up by capillary action, so even if the amount of liquid nitrogen 43 stored decreases, the entire cooling space forming wall section 31 can be cooled stably, and the cooling space 20 can be maintained at an extremely low temperature.

[0033] Furthermore, the cooling space forming wall portion 31 may be a straight tubular member (see Figure 1, etc.) or an array forming member (see Figure 3) made of a metal material such as copper. In other words, the cooling space 20 is not limited to the form of an elongated internal space formed by a straight tubular member that opens vertically, but may also be a flat, long, and narrow internal space formed by an array forming member as shown in Figure 3.

[0034] Next, an example of the configuration of a microdroplet freezing device when the cooling space 20 is arrayed will be described with reference to Figure 3. In this example configuration of Figure 3, multiple inkjet ejection heads 11 are arranged in parallel in a straight line, thus forming an array. Specifically, 10 ejection heads 11 are arranged in parallel. Note that the arraying of ejection heads 11 is not limited to a configuration in which multiple ejection heads 11, each having one ejection hole 11a on a single head member, are arranged in parallel, as in the example configuration of Figure 3. A multi-nozzle head with multiple ejection holes 11a formed on a single head member may also be used.

[0035] In the cooling space forming wall portion 31, which is formed in the shape of a flat rectangular frame, the width W (width in one direction perpendicular to the direction of passage of the minute droplets 15) that forms the narrow array should be 20 mm or less, and preferably 10 mm or less, because if it is too wide the cooling efficiency will be poor. The minimum width W should be set so that the minute droplets 15 ejected from the inkjet ejection head 11 reach the frozen minute droplet receiving portion 50 without hitting the inner wall surface 31a of the cooling space forming wall portion 31, for example, it should be about 1 mm.

[0036] Furthermore, in this embodiment, the width W forming this narrow array is set to a dimension that is the width (diameter) of the inkjet ejection head 11 plus a little extra, and the ejection head 11 and the inner wall surface 31a of the cooling space forming wall 31 (the inner edge of the inlet 21 of the cooling space 20) are set not to come into contact. Although not shown in Figure 3, the cooling space forming wall 31 can be cooled with liquid nitrogen (refrigerant 43) or the structure shown in Figure 2.

[0037] In this embodiment of the present invention, a hollow structure (cooling space forming wall 31) with high thermal conductivity, such as copper, is cooled to an extremely low temperature using an extremely low-temperature liquid such as liquid nitrogen or a Peltier element as a cooling medium, so as to provide an extremely low-temperature cooling space 20. This allows for the instantaneous freezing of minute droplets 15 within the cooling space 20. In other words, with the cooling space 20 configured in this way, minute droplets can be instantly frozen in a vitrified state, containing biological samples such as cells 15a, proteins, and allergens. It should be noted that the material forming the cooling space forming wall 31 is not limited to a metal, but may be any other material with high thermal conductivity, for example, graphite, which is a carbon crystalline material, may be used.

[0038] As an example of the temperature distribution of the cooling space 20, in a configuration where a liquid nitrogen-impregnated section 42 is provided as shown in Figure 2, and liquid nitrogen (-196°C) is used as the refrigerant 43, a copper pipe as the cooling space forming wall 31, and expanded polystyrene as the insulating material 45, it was confirmed that the temperature at the top of the copper pipe was -80°C, and the temperature in the internal space of the copper pipe below 5 mm from the end of the copper pipe was around -180°C.

[0039] The cooling rate should be 5000°C / s or higher, and preferably 10000°C / s or higher, if no protective agent is added. When a protective agent is used, the cooling rate depends on the concentration, but if no protective agent is added, it is desirable that the surface of the microdroplet 15 cools to -137°C or lower during falling (passing through the cooling space). This allows the frozen (vitrified) microdroplet 15 to be properly recovered at extremely low temperatures. The recovered frozen microdroplet 15 may be deposited or suspended in liquid nitrogen.

[0040] Furthermore, according to the present invention, the cooling means 30 may be provided with a cooling space forming wall portion 31 that forms the cooling space 20 and a Peltier element that is in contact with the periphery of the cooling space forming wall portion 31. This also allows for rational and appropriate cooling of the long and narrow cooling space 20, enabling the freezing of a larger number of minute droplets 15 more efficiently and appropriately.

[0041] Furthermore, in this embodiment, the discharge head 11 is positioned downward so that the discharge direction of the microdroplets 15 is downward along the vertical direction, and the cooling space 20 is a passage that extends vertically. That is, the microdroplets 15 are arranged to fall along gravity through the gas (air) of the narrow cooling space 20, for example, in at least one direction. As a result, the microdroplets 15 fall straight down without contacting the inner wall surface 31a of the member forming the cooling space 20, and are frozen stably and instantaneously in the cooling space 20, and can reach the frozen microdroplet receiving section 50 appropriately. Therefore, a large amount of frozen microdroplets containing cells 15a, etc., can be stored in the frozen microdroplet receiving section 50, and a large amount of these cells 15a, etc., can be frozen and preserved appropriately.

[0042] Furthermore, in this embodiment, it is preferable to provide a micro-vibration device (not shown) that causes the frozen micro-droplet receiving section 50 to vibrate slightly, or a micro-movement device 60 (see Figure 4) that causes the frozen micro-droplet receiving section 50 to move slightly. This prevents the frozen micro-droplets 15 from accumulating vertically in the frozen micro-droplet receiving section 50 upon droplet placement, and allows for the stable and large-scale storage of a larger number of frozen micro-droplets 15 in an evenly distributed state within the frozen micro-droplet receiving section 50. Note that Figure 4 shows a state in which frozen micro-droplets 15 accumulate vertically in the frozen micro-droplet receiving section 50 upon droplet placement, but this state can be avoided by using the micro-vibration device or the micro-movement device 60.

[0043] As the above-mentioned micro-vibration device, a vibration motor can be used, and by appropriately attaching it to a base on which the frozen micro-droplet receiving section 50 is placed, it can be easily configured to cause the frozen micro-droplet receiving section 50 to vibrate slightly. Furthermore, a biaxial stage (XY stage) can be used as the micro-movement device 60. In Figure 4, the frozen micro-droplet receiving section 50 is placed on the micro-movement device 60, and the micro-movement device 60 is placed on the base stage 61. By employing such a micro-vibration device and micro-movement device 60, it is possible to generate micro-vibrations and micro-movements, which eliminates the accumulation of micro-droplets 15 in the vertical direction, and allows for the large and stable storage of micro-droplets 15 in an evenly distributed state within the frozen micro-droplet receiving section 50. In this embodiment, since the frozen microdroplet receiving section 50 vibrates or moves as described above, and because the containment section 51, which constitutes a part of the frozen microdroplet receiving section 50, is detached and used to store frozen microdroplets 15 in each of the containment sections 51, the containment container 51 is positioned so as not to be fixed to the cooling space forming wall section 31 that forms the outlet 22 of the cooling space 20. Furthermore, in order to cool the cooling space 20 and maintain a low temperature, it is desirable to seal the outlet 22 and the containment container 51 so as not to communicate with the outside space in order to prevent warm air from flowing in from above and raising the temperature of the cooling space 20.

[0044] Next, we will describe an example that includes specific numerical values ​​(an example in which minute droplets 15 are ejected downwards along the vertical direction and fall using inkjet technology).

[0045] First, the falling speed and falling distance of the micro-droplets 15 will be described. For micro-droplets 15 of, for example, 21.9 pL generated by a micro-droplet generator using inkjet technology (hereinafter simply referred to as "inkjet 10"), the falling speed when discharged from the inkjet 10 is preferably about 8 m / s or less, and 6 m / s or less, so that the heat of the micro-droplets 15 is appropriately cooled by heat conduction when passing through the cooling space 20. On the other hand, since the mass of the micro-droplets ejected at high speed by the inkjet 10 is very small, they decelerate rapidly. Therefore, the minimum speed at which the micro-droplets can fall linearly and appropriately is preferably 1 m / s or more, and 2 m / s or more.

[0046] In the analysis results of the embodiment shown in FIG. 5, the cases where the falling speeds are 2 m / s and 4 m / s are shown. However, since the cooling rate of the micro-droplets 15 (the volume of this embodiment is set to 21.9 pL) varies according to the falling speed of the micro-droplets 15, the falling distance is determined according to the falling speed. At this time, at the end of the fall in the cooling space 20, the droplet temperature is preferably about -100°C or less, and -130°C or less. As shown in FIG. 5, in order to make the droplet temperature -100°C at the end of the fall, the falling time of the micro-droplets 15 must pass through the cooling space 20 for at least 20 ms, and preferably 40 ms or more, taking into account the safety factor. Also, the falling distance is calculated as the falling speed × falling time.

[0047] Next, the appropriate size of the micro-droplets 15 according to the present invention will be described. Vitrification of the micro-droplets 15 can be achieved at a lower freezing rate compared to when no protective agent is added by adding a protective agent such as sucrose or trehalose. Therefore, depending on the added concentration of the cryoprotectant, when using a protective agent, it is preferably 1 μL or less, and 100 nL or less. When not using a protective agent, it is preferably 100 pL or less, and 50 pL or less. Note that the minimum size of the micro-droplets 15 depends on the capabilities of the inkjet 10 and the size of particles such as the cells encapsulated therein. For example, the minimum size of the micro-droplets 15 can be set to the minimum volume that can appropriately encapsulate one microparticle (micro-object).

[0048] Here, the differences between inkjet technology, cell sorter technology, and the spray method will be described. There may also be a method of generating minute droplets by spraying and dropping them in an appropriate cooling space. However, in the case of spraying, warm air drops together with the minute droplets, which inhibits the cooling of the minute droplets. Therefore, it is preferable to generate minute droplets 15 by inkjet technology or cell sorter technology, etc., and the frequency (droplet ejection frequency) for generating minute droplets 15 is preferably 10 kHz or less.

[0049] Next, assuming that ideally one cell 15a is included in one minute droplet 15 obtained by inkjet technology, the total number of cells 15a that are vitrified, recovered, and stored while being included in the minute droplet 15 will be described.

[0050] The apparatus in which the cooling space 20 is formed by a tube as shown in FIG. 1 is an apparatus that provides high-throughput freezing of cells. The number of cells that can be frozen in one operation time is, as an experimental value, about 300 cells / operation (3 minutes) in the conventional method of freezing minute droplets 15 (cells 15a) on the substrate surface, whereas in the method according to the present invention (the embodiment shown in FIG. 1) of freezing cells 15a during the fall in the cooling space 20, even when the droplet ejection frequency is 100 Hz, an experimental result of about 1000 cells / 50 seconds = 3600 cells / operation (3 minutes) could be obtained. That is, according to the method of the present invention, 10 times or more of cells 15a could be cryopreserved.

[0051] And in the inkjet technology according to this embodiment, when the droplet ejection frequency at the time of drop freezing is improved up to 1 kHz as an example, the number of cells included in the minute droplet 15 can be improved up to 1 cell / droplet. That is, in this case, theoretically, high-throughput up to 180,000 cells / operation (3 minutes) is possible. Also, as in the embodiment shown in FIG. 3, since further efficiency improvement is possible by parallelizing the minute droplet generation mechanism section, high-throughput can be increased infinitely in calculation.

[0052] In reality, it is not always the case that one cell 15a is reliably encapsulated in a single microdroplet 15. Depending on the concentration of the cells 15a in the solution in which they are dispersed, if the concentration is set low to avoid clogging of the dispensing head 11, then, for example, one cell 15a may be encapsulated in several to five microdroplets 15. Also, if the microorganisms (e.g., platelets) encapsulated are small compared to the size of the microdroplet 15, then multiple microorganisms may be encapsulated in the microdroplet 15.

[0053] The viability of cells 15a encapsulated in rapidly frozen microdroplets 15 using the method of the present invention described above will now be explained. Although it depends on the volume of cells 15a encapsulated in the microdroplets 15 and the low-temperature tolerance of the cells 15a, in general mammalian cells (mouse fibroblasts in the example), verification by cell viability staining after freeze-thawing showed that a viability of approximately 80% was obtained after freeze-thawing. Furthermore, it has been confirmed that even relatively small biological samples such as platelets maintain their function after thawing. In addition, germ cells such as bovine sperm can also be frozen and have been confirmed to maintain their normal morphology after thawing.

[0054] Next, an example of the form of the discharge head 11 will be described based on Figure 6 (enlarged view of the area around the discharge head). In addition, a heater 12 may be attached to the discharge head 11, as disclosed in Patent Document 2. The tip of the head unit portion (discharge head 11) to which the heater 12 is attached may or may not be inserted into the inside of the cooling space forming wall portion 31 (the portion corresponding to the inlet 21 of the cooling space 20), as shown in Figure 6. By attaching the heater 12 to the discharge head 11 in this way, freezing of the discharge head 11 can be prevented, and more appropriate freezing of the minute droplets 15 can be achieved, as disclosed in Patent Document 2.

[0055] Although various preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments, and many modifications can be made without departing from the spirit of the invention.

[0056] 10 Inkjet 11 Discharge head 11a Discharge port 12 Heater 15 Microdroplet 15a Cell 20 Cooling space 21 Inlet 22 Outlet 30 Cooling means 31 Cooling space forming wall 31a Inner wall surface 40 Liquid nitrogen holding part 41 Liquid nitrogen storage tank-like part 42 Liquid nitrogen impregnation part 43 Refrigerant (liquid nitrogen) 45 Insulating material (expanded polystyrene) 50 Frozen microdroplet receiving part 51 Container 52 Cooling means 60 Microtransfer device 61 Base stage W Width for forming a narrow array

Claims

1. A method for freezing microdroplets, wherein microdroplets discharged from a discharge head containing minute particles are rapidly cooled to freeze them into a vitrified state, characterized in that the microdroplets are frozen by passing them through a cooling space of a gas phase cooled by a cooling means.

2. The method for freezing microdroplets according to claim 1, characterized in that the cooling space is an internal space with both ends provided as inlets and outlets in the direction of passage of the microdroplets.

3. A device for use in a method for freezing microdroplets according to claim 2, comprising the ejection head, the cooling means, and the cooling space, wherein the generating device for the microdroplets, including the ejection head, is provided by inkjet technology or cell sorter technology, the ejection head is positioned on the inlet side of the cooling space so that the ejected microdroplets can pass through the cooling space from the inlet to the outlet, and a frozen microdroplet receiving section for storing the frozen microdroplets is positioned on the outlet side of the cooling space from which the frozen microdroplets are discharged.

4. The microdroplet freezing apparatus according to claim 3, characterized in that the cooling means comprises a cooling space forming wall portion that forms the cooling space and a liquid nitrogen holding portion that is in contact with the periphery of the cooling space forming wall portion.

5. The microdroplet freezing apparatus according to claim 4, characterized in that the liquid nitrogen holding section comprises a liquid nitrogen storage tank-like section in which liquid nitrogen is stored, and a liquid nitrogen impregnation section provided in contact with the periphery of the cooling space forming wall section and into which the liquid nitrogen is impregnated.

6. The microdroplet freezing device according to claim 5, characterized in that the liquid nitrogen impregnation portion is provided by a member made of a fibrous material or a sponge-like member that is capable of absorbing liquid nitrogen.

7. The microdroplet freezing apparatus according to claim 3, characterized in that the cooling means comprises a cooling space forming wall portion that forms the cooling space and a Peltier element in contact with the periphery of the cooling space forming wall portion.

8. The microdroplet freezing apparatus according to claim 3, characterized by comprising a micro-vibration device for micro-vibrating the microdroplet receiving section, or a micro-movement device for micro-moving the microdroplet receiving section.

9. The microdroplet freezing apparatus according to claims 3 to 8, characterized in that the discharge head is positioned downward so that the discharge direction of the microdroplets is downward along the vertical direction, and the cooling space is a passage that extends vertically.

10. The microdroplet freezing apparatus according to claim 4 or 7, characterized in that the cooling space forming wall portion is a straight tubular member or array forming member made of a metal material.

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