WO2018003443A1 - Heat transfer plate, well plate unit and device for stripping cells - Google Patents

Heat transfer plate, well plate unit and device for stripping cells Download PDF

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Publication number
WO2018003443A1
WO2018003443A1 PCT/JP2017/021209 JP2017021209W WO2018003443A1 WO 2018003443 A1 WO2018003443 A1 WO 2018003443A1 JP 2017021209 W JP2017021209 W JP 2017021209W WO 2018003443 A1 WO2018003443 A1 WO 2018003443A1
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WIPO (PCT)
Prior art keywords
heat transfer
light
container
transfer plate
cell
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PCT/JP2017/021209
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French (fr)
Japanese (ja)
Inventor
千恵 児島
武志 川野
瀧　優介
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公立大学法人大阪府立大学
株式会社ニコン
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Publication of WO2018003443A1 publication Critical patent/WO2018003443A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a heat transfer plate, a well plate unit, and a cell peeling apparatus.
  • a method for recovering a target cell by culturing and differentiating universal cells such as iPS cells is known.
  • a method for recovering a target cell by culturing and differentiating universal cells such as iPS cells is known.
  • a method is known in which cells are cultured on a collagen gel containing gold nanoparticles, and the cells are irradiated with light to solderize the collagen gel in the light irradiation region, and the cells contained in the light irradiation region are detached and recovered.
  • Patent Document 1 Japanese Unexamined Patent Application Publication No. 2012-39947 Japanese Unexamined Patent Application Publication No. 2012-39947
  • a heat transfer plate on which a container having a plurality of wells in which cells are accommodated is placed, and a main body part in contact with at least a part of the container, and a light transmission part provided corresponding to the bottom surface of the plurality of wells A heat transfer plate is provided.
  • a container having a plurality of wells in which cells are accommodated, a heat transfer plate on which the container is placed, a heater disposed in contact with the heat transfer plate, and an object in the well
  • a light irradiating unit that irradiates the region with light, and the heat transfer plate has a main body unit that is in contact with at least a part of the container and a light transmissive unit provided corresponding to the bottom surfaces of the plurality of wells.
  • the apparatus includes a container having a plurality of wells in which cells are accommodated, and a heat transfer plate on which the container is placed, and the heat transfer plate is in contact with at least a part of the container. And a well plate unit having a light transmitting portion provided corresponding to the bottom surfaces of the plurality of wells.
  • An example of the structure of the cell peeling apparatus 10 of this embodiment is shown.
  • An example of the structure of the heat-transfer plate 200 of this embodiment is shown.
  • An example of the structure of the well 110 of this embodiment is shown.
  • An example of the external appearance of the container 100 of this embodiment is shown.
  • An example of the external appearance of the heat-transfer plate 200 of this embodiment corresponding to FIG. 4 is shown.
  • An example of the well plate unit of this embodiment is shown.
  • An example of the processing flow of the cell peeling apparatus 10 of this embodiment is shown.
  • An example of the effect of the cell peeling method of this embodiment is shown.
  • FIG. 1 shows an example of the configuration of the cell peeling apparatus 10 of the present embodiment.
  • the cell peeling apparatus 10 selectively peels and collects target cells using light irradiation.
  • the cell peeling device 10 includes a container 100, a heat transfer plate 200, a heater 300, a driving unit 350, a light irradiation unit 400, an optical member 500, a mirror 600, a lens 700, an observation unit 800, and a cell acquisition unit 900.
  • the container 100 accommodates cells to be detached.
  • the container 100 has a plurality of wells 110 in which cells are accommodated, and the cells are accommodated in each of the wells 110.
  • the plurality of wells 110 are a plurality of indentations provided on the upper plate of the container 100.
  • the container 100 is formed of a transparent material such as resin or glass.
  • the container 100 stores a medium whose structure is changed by heating in each well 110, and holds cells on the medium.
  • the medium may include a gel that forms a sol by heating and gold fine particles.
  • the heat transfer plate 200 has the container 100 placed thereon, and transfers heat from the heater 300 to the container 100.
  • the heat transfer plate 200 includes a main body part 210 and a light transmission part, and mainly conducts heat in the main body part 210 and transmits light in the through part 220.
  • the light transmission part of the heat transfer plate 200 is provided corresponding to the bottom surfaces of the plurality of wells 110, and may be one or a plurality of penetration parts 220 as shown in FIG. 1, for example.
  • the main body 210 may have a higher thermal conductivity than the container 100.
  • the plurality of through portions 220 are respectively provided at positions corresponding to some or all of the plurality of wells 110 in the main body 210.
  • the penetration part 220 may be provided as a power of 2 (2, 4, 8, 16, 32, 64, etc.). Further, the plurality of through portions 220 may be provided at equal intervals in the main surface of the heat transfer plate 200. Thereby, the heat transfer plate 200 enables light irradiation to the cells in the well 110 while ensuring that the main body 210 contacts the container 100 and heat conduction to the container 100.
  • the main body 210 may be made of a metal having good thermal conductivity.
  • the main body 210 is made of aluminum (for example, the thermal conductivity as a bulk is 226 to 237 W / mK: the values in parentheses indicate the thermal conductivity as a metal bulk, and the unit is W / mK. ), Gold (318), silver (429), copper (386-402), iron (72-80.4), tungsten (169-176), and alloys containing parts thereof.
  • at least a part of the main body 210 may be formed of a non-metallic material such as graphite having a good thermal conductivity (for example, a thermal conductivity of 200 to 2000 W / mK).
  • the main body 210 may be made of a resin having a higher thermal conductivity as a bulk than the container 100.
  • acrylonitrile styrene resin for example, the thermal conductivity as a bulk is 0.15 to 0.16 W / mK: the value in parentheses indicates the thermal conductivity as the bulk of the resin, and the unit is W / mK.
  • One or more resins having higher thermal conductivity may be selected and used for at least a part of the main body 210.
  • the main body 210 is preferably formed of aluminum from the viewpoint of thermal conductivity, workability, cost, and the like.
  • the main body 210 may be in contact with at least a part of the outer bottom surface of each of the plurality of wells 110.
  • the main body 210 may be disposed so as to cover at least the bottom surfaces of the side walls of the plurality of wells 110 when the container 100 is mounted.
  • Each of the plurality of penetrating portions 220 may be an opening that transmits light and penetrates the main body portion 210 in the thickness direction.
  • the plurality of through portions 220 may be voids that are not filled with a material. In this case, the through portions 220 are through holes. Instead, the penetrating portion 220 may be filled with a light transmissive material (for example, a transparent material) such as glass or resin having good thermal conductivity (for example, a resin cited as a candidate for the main body portion 210). .
  • the penetrating portion 220 transmits at least a part of the wavelength included in the emitted light of the light irradiation unit 400, and preferably transmits visible light.
  • the through portion 220 may be provided below at least a part of the bottom surface inside each of the plurality of wells 110.
  • the heat transfer plate 200 may have a shape and thickness that match the shape of the container 100 so that the container 100 is in close contact with the heat transfer plate 200.
  • the heat transfer plate 200 has a predetermined size (for example, 0.01 to 1 mm) than the length that the legs protrude. It may have a large thickness.
  • the heat transfer plate 200 can prevent the main body other than the legs of the container 100 from floating from the heat transfer plate 200 while maintaining the heat transfer property.
  • the heat transfer plate 200 may have a thickness of 1 to 3 mm.
  • the heater 300 is disposed in contact with the heat transfer plate 200, generates heat, and transfers the heat to the container 100 through the heat transfer plate 200.
  • the heater 300 also functions as a stage for the container 100 and the heat transfer plate 200. That is, the heater 300 moves the container 100 and the heat transfer plate 200 by moving in the three-dimensional space. In addition, a stage for driving the heater 300 may be separately prepared.
  • the rectangular heater 300 may be fitted into a stage whose center is hollowed into a rectangular shape and driven together with the stage.
  • the heater 300 has a light transmission part 310 that transmits light to at least a part of a region where the heat transfer plate 200 is mounted.
  • the heater 300 may have a heat generating device (that is, a transparent heat generating device) in the light transmission part 310, and may transmit the heat generated in the light transmission part 310 to the heat transfer plate 200.
  • the heater 300 includes an opaque or transparent heat generating device around the light transmitting portion 310, and transfers heat from the heat generating device to the heat transfer plate 200 via the light transmitting portion 310. Also good.
  • the driving unit 350 drives the heater 300 and moves in a three-dimensional direction.
  • the drive unit 350 drives the heater 300 (or the stage in which the heater 300 is incorporated) in the main surface direction (XY direction) and the vertical direction (Z direction) of the container 100, the heater 300, and the like by power means such as a motor. You can do it.
  • the drive part 350 may rotate the heater 300 within a main surface plane.
  • the light irradiation unit 400 irradiates the target region in the well 110 of the container 100 with light.
  • the light irradiation unit 400 irradiates at least one cell to be exfoliated in the well through the penetration part 220 and the light transmission part 310 while heating the container 100 with the heater 300, and thereby the target.
  • the resulting cells may be detached from the medium.
  • the light irradiator 400 may be anything that irradiates light that gives energy to the extent that the medium fluidized by heating fluidizes, and a laser light source that emits light having a wavelength of 400 to 1200 nm and an intensity of 0.1 to 1000 mW. It may be.
  • the optical member 500 receives the light emitted from the light irradiation unit 400, and adjusts the optical path, intensity, and / or phase of the incident light beam.
  • the optical member 500 may include a digital mirror device (DMD), a spatial light modulator (SLM), and / or a galvanometer mirror.
  • the mirror 600 reflects the light emitted from the light irradiation unit 400 and passing through the optical member 500 toward the container 100.
  • the mirror 600 may transmit light other than the light emitted from the light irradiation unit 400 in order to allow the observation unit 800 to observe the cells in the container 100.
  • the mirror 600 may be a dichroic mirror that reflects light having the wavelength of light emitted from the light irradiation unit 400 and transmits light having other wavelengths.
  • the lens 700 is disposed on the optical path between the mirror 600 and the heater 300, enters the light reflected by the mirror 600, and emits the incident light toward the light transmission unit 310.
  • the lens 700 functions as an objective lens that enables the observation unit 800 to observe cells in the container 100.
  • the observation unit 800 is arranged on the same optical path as the observation target well 110 and the lens 700, and allows the observer to observe the well 110 in an enlarged manner.
  • the observation unit 800 includes an eyepiece lens and functions as a microscope in combination with the lens 700 to allow the observer to observe the enlarged cells.
  • the observation unit 800 may include an imaging element and output the enlarged image data in the well 110 that has been imaged to a display device or the like.
  • the cell acquisition unit 900 acquires the cells detached from the medium by the light irradiated from the light irradiation unit 400.
  • the cell acquisition unit 900 may include a capillary that can suck cells by a capillary effect.
  • the cell acquisition unit 900 may have a mechanism for recovering detached cells by moving the container 100 over the recovery container and inverting it.
  • the cell detachment apparatus 10 transmits the light emitted from the light irradiation unit 400 in the well 110 of the container 100 through the optical member 500, the mirror 600, the lens 700, the light transmission unit 310, and the penetration unit 220. And irradiate the cells to be detached with light.
  • the cell peeling apparatus 10 performs light irradiation on the cells by the light irradiation unit 400 while heating the container 100 with the heater 300.
  • the cell peeling apparatus 10 can peel the cells from the medium in a shorter time and with a weak light irradiation. As a result, the cell peeling device 10 can reduce the invasion to the cells and reliably collect the cells with less damage.
  • the cell peeling device 10 may include a graphite sheet between the heat transfer plate 200 and the container 100.
  • the container 100 may not be in surface contact with the heat transfer plate 200 because there are individual differences in the shape of the container 100 and the surface of the container 100 is not uniform. Even in such a case, at least a part of the space between the container 100 and the heat transfer plate 200 is filled with a graphite sheet having excellent thermal conductivity and flexibility, so that the heat from the heater 300 can be efficiently transferred to the container. 100.
  • an opening having a shape corresponding to the penetrating portion 220 may be provided in the graphite sheet so as not to obstruct observation by the observation unit 800.
  • FIG. 2 shows an example of the configuration of the heat transfer plate 200 of the present embodiment.
  • the main body 210 of the heat transfer plate 200 may have a multilayer structure, and the through-hole 220 may be a gap.
  • the main body 210 of the heat transfer plate 200 may include a core layer 212, a surface layer 214, and a surface layer 216.
  • the core layer 212 may be an aluminum plate having a thickness
  • the surface layer 214 and the surface layer 216 may be graphite sheets
  • the core layer 212, the surface layer 214, and the surface layer 216 may be bonded with an adhesive or the like. .
  • the heat transfer plate 200 can improve thermal conductivity while maintaining strength.
  • FIG. 3 shows an example of the configuration of the well 110 of the present embodiment.
  • the well 110 includes a medium 112, a cell 117, and a culture solution 118.
  • the medium 112 includes a gel 114 and fine particles (for example, gold fine particles 116).
  • the gel 114 may be a gel material whose structure changes when the temperature is equal to or higher than a predetermined temperature (for example, 40 ° C.), and may be, for example, a collagen gel or a gelatin gel.
  • the gold fine particles 116 generate heat by absorbing light having a wavelength emitted from the light irradiation unit 400.
  • the structure of only the gel 114 is changed by the heat generation of the gold fine particles 116 in the vicinity of the region where the light from the light irradiation unit 400 (indicated by the dotted arrow in the drawing) is incident.
  • Only the cells 117 that are lost lose the scaffold and are detached from the medium 112.
  • the observer may irradiate the target cell 117 with light while observing the inside of the well 110 with the observation unit 800.
  • the cell acquiring unit 900 can selectively acquire only the target cell 117 by peeling it out of the large number of cells 117 accommodated in the observation well 110.
  • FIG. 4 shows an example of the appearance of the container 100 of the present embodiment.
  • the container 100 may be a microplate in which a plurality of wells 110 are arranged in a matrix.
  • the part other than the part where the well 110 is formed in the container 100 may be filled with the forming material of the container 100.
  • at least a part may be a void.
  • FIG. 5 shows an example of the appearance of the heat transfer plate 200 of the present embodiment corresponding to FIG.
  • the heat transfer plate 200 may be a plate provided with a plurality of through portions 220 provided in a matrix corresponding to the wells 110 of FIG.
  • FIG. 5 shows a form in which each of the plurality of openings is provided at equal intervals in the row direction and / or column direction of the matrix and is larger than the bottom surface of the corresponding well.
  • each of the plurality of openings may be smaller than the bottom surface of the corresponding well 110. In this case, since the area of the main body 210 of the heat transfer plate 200 is increased, the thermal conductivity with respect to the well 110 is improved.
  • FIG. 6 shows an example of the well plate unit of the present embodiment.
  • the container 100 and the heat transfer plate 200 may be combined to form a well plate unit.
  • the well plate unit may have a form in which the containers 100 are stacked as they are on the heat transfer plate 200 as shown in FIG. 1, but as shown in FIG.
  • the heat transfer plate 200 may be incorporated on the side.
  • the container 100 includes a portion for forming the well 110, an upper plate portion, and a side wall portion. That is, the container 100 has an upper plate provided with a recess for forming the well 110 on the upper side of FIG. 6, but has no plate on the lower side and is a gap, and the through-hole 220 of the heat transfer plate 200 is also provided. It becomes a void.
  • each of the plurality of wells 110 is fitted into the plurality of openings of the corresponding penetration part 220.
  • each of the plurality of openings is larger than the bottom surface of the corresponding well 110. According to this embodiment, since the heat transfer plate 200 is also heated from the side portion of the container 100, heat can be transferred to the container 100 more efficiently.
  • FIG. 7 shows an example of the processing flow of the cell peeling apparatus 10 of the present embodiment.
  • the cell peeling device 10 executes the processes of S110 to S190.
  • a medium 112 that is fluidized by heating is supplied to a container 100 including a plurality of wells 110 as a substrate for cell culture.
  • the medium 112 may include a gel and gold fine particles that are fluidized by heating.
  • an acetylated collagen peptide-modified dendrimer solution is generated from an acetylated collagen peptide, mixed with a tetrachloroauric (III) acid solution, and then gold ions are reduced.
  • a collagen gel containing gold fine particles is generated, and this may be used as the medium 112 in this embodiment.
  • the medium 112 may be supplied to all or a part of the plurality of wells 110 of the container 100. Further, after the medium 112 is supplied, phosphate buffered saline (PBS ( ⁇ )) is supplied, and the solution is stored in the well 110 at a predetermined temperature (for example, 37 ° C.) for a predetermined time (for example, 6 hours). By holding this, the medium 112 may be cleaned.
  • PBS phosphate buffered saline
  • the cells 117 are fixed to the medium 112.
  • the medium 112. For example, 8.0 ⁇ 10 3 Hela cells may be seeded in the medium 112.
  • a culture solution 118 such as DMEM may be supplied to the fixed cells 117.
  • the fixed cells 117 are cultured.
  • the container 100 in which the cells 117 are fixed is held at a predetermined temperature (for example, 37 ° C.) for a predetermined time (for example, 24 hours), and incubation is performed.
  • the inside of the well 110 of the container 100 is washed.
  • the culture solution 118 in the well 110 is removed and washed with a washing solution such as PBS ( ⁇ ) at least once (for example, three times). Accordingly, the inside of the well 110 is washed before the cells 117 are detached from the medium 112, the separation work is easily performed, and impurities collected together with the cells are reduced. Note that S140 may be omitted.
  • At least a part of the solution in the well 110 is removed.
  • the cleaning of S140 is executed, at least a part of the cleaning liquid remaining in the well 110 is removed.
  • the culture medium 118 may be newly supplied after all the cleaning liquid in the well 110 is removed.
  • a part of the culture medium 118 may be removed.
  • the heat capacity of the contents of the well 110 is reduced, and the temperature of the medium 112 can be raised in a shorter time.
  • the culture medium 118 is partially removed without being removed, the cells 117 in the well 110 can be protected.
  • the container 100 is heated by the heater 300.
  • the heat transfer plate 200 is mounted on the light transmission part 310 of the heater 300, and the container 100 is placed so that the positions of the plurality of through parts 220 of the heat transfer plate 200 correspond to the positions of the plurality of wells 110.
  • the heater 300 generates heat. Thereby, the heat generated from the heater 300 is transmitted to the container 100 through the heat transfer plate 200.
  • the heating in S160 may be continued until a predetermined temperature or heating time is achieved.
  • the process may proceed to S170 when the solution such as the medium 112 or the culture medium 118 in the container has reached a predetermined temperature (for example, 36 ° C.).
  • the cells 117 to be peeled in the well 110 are irradiated with light from the light irradiation unit 400, and the cells 117 are removed from the medium 112. Peel off.
  • the cells 117 to be peeled are specified, and laser light is emitted from the light irradiation unit 400 toward the specified cells 117 to be peeled.
  • the light irradiation from the light irradiation unit 400 may be continued until a predetermined temperature or irradiation time is achieved.
  • a laser beam having a wavelength of 532 nm is irradiated for 10 seconds at an intensity of 3 mW to the cells 117 to be peeled.
  • the light irradiation unit 400 causes the emitted light to enter the heater 300 through the optical member 500, the mirror 600, and the lens 700, and is transmitted through the light transmission unit 310 of the heater 300 and the penetration unit 220 of the heat transfer plate 200. Then, the cells 117 to be peeled in the well 110 are irradiated with light.
  • the medium 112 in the vicinity of the cell 117 irradiated with light is heated and fluidized by light irradiation from the light irradiation unit 400 in addition to heat from the heater 300. As a result, the light irradiation unit 400 peels the cells 117 to be peeled from the medium 112.
  • the cell peeling device 10 may adjust the position and focus of the irradiation light.
  • the driving unit 350 may adjust the light irradiation position of the light irradiation unit 400 so as to coincide with the position of the cell 117 to be peeled by moving the heater 300 functioning as a stage in the main surface direction.
  • the position of light irradiation of the light irradiation unit 400 may be changed by operating the galvanometer mirror of the optical member 500.
  • the cell peeling apparatus 10 may adjust optical conditions, such as a focus of a laser beam, by adjusting the optical member 500, the lens 700, and / or other adjustment apparatuses.
  • light irradiation may be performed on a plurality of cells 117 to be separated at once.
  • the light irradiation pattern data corresponding to the cell 117 to be peeled is created in advance, the light irradiation pattern is realized by the DMD or SLM of the optical member 500, and the light irradiation unit 400 passes through the light irradiation pattern.
  • the well 110 may be irradiated with light. Thereby, the light irradiation part 400 can peel all the cells 117 of the peeling object in the well 110 by one light irradiation.
  • the target of light irradiation by the light irradiation unit 400 is changed.
  • the driving unit 350 may change the light irradiation position of the light irradiation unit 400 to another undetached cell 117 to be peeled by moving the heater 300 in the main surface direction.
  • the optical member 500 may operate the galvanometer mirror to change the light irradiation position of the light irradiation unit 400 to another undetached cell 117 to be peeled.
  • the cell acquisition unit 900 acquires the detached cell 117. For example, while confirming the position of the cell 117 peeled by the observation unit 800, the cell acquisition unit 900 approaches the peeled cell 117 at the tip of the capillary, sucks the cell 117 with the capillary, and then sucks the cell 117.
  • the cells 117 may be obtained by discharging the cells.
  • the cell acquisition unit 900 may acquire a plurality of detached cells 117 in one or a plurality of wells 110 at a time instead of collecting the cells 117 one by one. For example, all the detached cells 117 in the container 100 may be collectively collected in the collection container by moving the container 100 onto the collection container and covering the container 100.
  • the cell detachment method of this embodiment is realized by executing the processing of S110 to S200 using the cell detachment apparatus 10.
  • the medium 112 is rapidly heated by light irradiation while the medium 112 that is fluidized by heating through the heat transfer plate 200 is gently heated, so that the medium 112 is fluidized.
  • the time of light irradiation with high invasiveness to the cells 117 can be made shorter than before, and the target cells 117 can be obtained more reliably.
  • FIG. 8 shows an example of the effect of the cell detachment method of this embodiment.
  • the horizontal axis of the graph in FIG. 8 indicates the time (seconds) from the start of heating by the heater 300, and the vertical axis indicates the measurement result (° C.) of the surface temperature of the medium 112. The measurement was performed under the condition that only the medium 112 was accommodated in the well 110 and the culture solution 118 was not filled.
  • a laser beam of 532 nm was continuously irradiated with an intensity of 5 mW from about 180 seconds later.
  • the dotted line graph in FIG. 8 shows the measurement result obtained by placing the container 100 directly on the heater 300 without the heat transfer plate 200, and the straight line graph shows the aluminum heat transfer plate 200 between the heater 300 and the container 100. The result of having been measured between them is shown.
  • the temperature was raised relatively quickly before light irradiation, and reached 40 ° C. in several tens of seconds after the light irradiation.
  • the heat transfer plate 200 is used in this way, the light irradiation time can be made relatively short.
  • the light irradiation intensity can be reduced to 1/12 and the light irradiation time can be reduced to 1/6 as compared with the method not using the heat transfer plate 200. I was able to. Moreover, it has confirmed with respect to the Hela cell and MDCK cell that the cell has manufactured even after the cell exfoliation by applying the cell exfoliation method of this embodiment.
  • 10 cell peeling device 100 container, 110 well, 112 medium, 114 gel, 116 gold fine particle, 117 cell, 118 culture solution, 200 heat transfer plate, 210 main body, 212 core layer, 214 surface layer, 216 surface layer, 220 Penetration part, 300 heater, 310 light transmission part, 350 drive part, 400 light irradiation part, 500 optical member, 600 mirror, 700 lens, 800 observation part, 900 cell acquisition part

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Abstract

The present invention addresses the problem of harvesting cells under noninvasive conditions for the cells. Provided are: a heat transfer plate on which a container provided with a plurality of wells for housing cells is to be placed, said heat transfer plate comprising a main body part, which has a higher heat conductivity than the container and is in contact with at least a part of the outer bottom surface of each of the wells, and a plurality of transparent penetrating parts, which are positioned below at least a part of the inner bottom surface of each of the wells and penetrate through the main body part; and a device for stripping cells, said device being provided with the heat transfer plate.

Description

伝熱プレート、ウェルプレートユニット、及び、細胞剥離装置Heat transfer plate, well plate unit, and cell peeling device
　本発明は、伝熱プレート、ウェルプレートユニット、及び、細胞剥離装置に関する。 The present invention relates to a heat transfer plate, a well plate unit, and a cell peeling apparatus.
　ｉＰＳ細胞等の万能細胞を培養、分化させて目的の細胞を回収する方法が知られている。しかし、ｉＰＳ細胞の樹立及び樹立後の分化誘導が成功する確率は比較的低いので、培養後の細胞の中から分化能および安全性の面で最適な細胞を選択して回収する必要がある。金ナノ粒子を含むコラーゲンゲル上で細胞を培養し、細胞に光照射することで光照射領域のコラーゲンゲルをゾル化し、光照射領域に含まれる細胞を剥離して回収する方法が知られている（例えば、特許文献１及び２参照）。
　特許文献１　特開２０１２－３９９４７号公報 A method for recovering a target cell by culturing and differentiating universal cells such as iPS cells is known. However, since there is a relatively low probability that iPS cell establishment and differentiation induction after establishment will be successful, it is necessary to select and recover optimally selected cells in terms of differentiation ability and safety from the cultured cells. A method is known in which cells are cultured on a collagen gel containing gold nanoparticles, and the cells are irradiated with light to solderize the collagen gel in the light irradiation region, and the cells contained in the light irradiation region are detached and recovered. (For example, refer to Patent Documents 1 and 2).
Patent Document 1 Japanese Unexamined Patent Application Publication No. 2012-39947
　しかし、従来の方法によると大きな光照射強度及び／又は長い光照射時間が必要であり、細胞にとって侵襲性が高かった。 However, according to the conventional method, a high light irradiation intensity and / or a long light irradiation time are required, and the cell is highly invasive.
　細胞が収容される複数のウェルを有する容器が載置される伝熱プレートであって、容器の少なくとも一部が接する本体部と、複数のウェルの底面に対応して設けられた光透過部とを有する伝熱プレートを提供する。 A heat transfer plate on which a container having a plurality of wells in which cells are accommodated is placed, and a main body part in contact with at least a part of the container, and a light transmission part provided corresponding to the bottom surface of the plurality of wells A heat transfer plate is provided.
　本発明の第２の態様においては、細胞が収容される複数のウェルを有する容器と、容器が載置された伝熱プレートと、伝熱プレートに接して配置されたヒーターと、ウェル内の対象領域に光を照射する光照射部と、を備え、伝熱プレートは、容器の少なくとも一部に接する本体部と、複数のウェルの底面に対応して設けられた光透過部とを有する細胞剥離装置を提供する。 In the second aspect of the present invention, a container having a plurality of wells in which cells are accommodated, a heat transfer plate on which the container is placed, a heater disposed in contact with the heat transfer plate, and an object in the well A light irradiating unit that irradiates the region with light, and the heat transfer plate has a main body unit that is in contact with at least a part of the container and a light transmissive unit provided corresponding to the bottom surfaces of the plurality of wells. Providing equipment.
　本発明の第３の態様においては、細胞が収容される複数のウェルを有する容器と、容器が載置される伝熱プレートとを備え、伝熱プレートは、容器の少なくとも一部に接する本体部と、複数のウェルの底面に対応して設けられた光透過部とを有するウェルプレートユニットを提供する。 In the third aspect of the present invention, the apparatus includes a container having a plurality of wells in which cells are accommodated, and a heat transfer plate on which the container is placed, and the heat transfer plate is in contact with at least a part of the container. And a well plate unit having a light transmitting portion provided corresponding to the bottom surfaces of the plurality of wells.
　本発明の第４の態様においては、ゲルと微粒子とを含む媒体と、媒体上に配置された細胞とをウェル内に有する容器を準備する工程と、ウェルの底面を介して媒体に光を照射し、ゲルの少なくとも細胞に接触する領域をゾル化する工程と、を有する細胞剥離方法を提供する。 In the fourth aspect of the present invention, a step of preparing a container having a medium containing a gel and fine particles and cells arranged on the medium in the well, and irradiating the medium with light through the bottom surface of the well And a step of solling at least a region of the gel that contacts the cell.
本実施形態の細胞剥離装置１０の構成の一例を示す。An example of the structure of the cell peeling apparatus 10 of this embodiment is shown. 本実施形態の伝熱プレート２００の構成の一例を示す。An example of the structure of the heat-transfer plate 200 of this embodiment is shown. 本実施形態のウェル１１０の構成の一例を示す。An example of the structure of the well 110 of this embodiment is shown. 本実施形態の容器１００の外観の一例を示す。An example of the external appearance of the container 100 of this embodiment is shown. 図４に対応する本実施形態の伝熱プレート２００の外観の一例を示す。An example of the external appearance of the heat-transfer plate 200 of this embodiment corresponding to FIG. 4 is shown. 本実施形態のウェルプレートユニットの一例を示す。An example of the well plate unit of this embodiment is shown. 本実施形態の細胞剥離装置１０の処理フローの一例を示す。An example of the processing flow of the cell peeling apparatus 10 of this embodiment is shown. 本実施形態の細胞剥離方法の効果の一例を示す。An example of the effect of the cell peeling method of this embodiment is shown.
　以下、発明の実施の形態を通じて本発明を説明するが、以下の実施形態は請求の範囲にかかる発明を限定するものではない。また、実施形態の中で説明されている特徴の組み合わせの全てが発明の解決手段に必須であるとは限らない。 Hereinafter, the present invention will be described through embodiments of the invention. However, the following embodiments do not limit the invention according to the claims. In addition, not all the combinations of features described in the embodiments are essential for the solving means of the invention.
　図１は、本実施形態の細胞剥離装置１０の構成の一例を示す。細胞剥離装置１０は、光照射を利用して目的の細胞を選択的に剥離して回収する。細胞剥離装置１０は、容器１００、伝熱プレート２００、ヒーター３００、駆動部３５０、光照射部４００、光学部材５００、ミラー６００、レンズ７００、観察部８００、及び、細胞取得部９００を備える。 FIG. 1 shows an example of the configuration of the cell peeling apparatus 10 of the present embodiment. The cell peeling apparatus 10 selectively peels and collects target cells using light irradiation. The cell peeling device 10 includes a container 100, a heat transfer plate 200, a heater 300, a driving unit 350, a light irradiation unit 400, an optical member 500, a mirror 600, a lens 700, an observation unit 800, and a cell acquisition unit 900.
　容器１００は、剥離対象となる細胞を収容する。容器１００は、細胞が収容される複数のウェル１１０を有し、各ウェル１１０の各々において細胞を収容する。例えば、複数のウェル１１０は、容器１００の上板に設けられた複数のくぼみである。容器１００は樹脂またはガラス等の透明材料により形成される。容器１００は、各ウェル１１０内に加熱により構造が変化する媒体を収納し、媒体上で細胞を保持する。一例として、媒体は、加熱によりゾル化するゲルと金微粒子とを含んでよい。 The container 100 accommodates cells to be detached. The container 100 has a plurality of wells 110 in which cells are accommodated, and the cells are accommodated in each of the wells 110. For example, the plurality of wells 110 are a plurality of indentations provided on the upper plate of the container 100. The container 100 is formed of a transparent material such as resin or glass. The container 100 stores a medium whose structure is changed by heating in each well 110, and holds cells on the medium. As an example, the medium may include a gel that forms a sol by heating and gold fine particles.
　伝熱プレート２００は、容器１００が載置され、ヒーター３００からの熱を容器１００に伝える。伝熱プレート２００は、本体部２１０と光透過部とを有し、主に本体部２１０で熱を伝導し、貫通部２２０において光を透過する。伝熱プレート２００の光透過部は、複数のウェル１１０の底面に対応して設けられ、例えば図１に示すように、１又は複数の貫通部２２０であってよい。本体部２１０は、容器１００よりも熱伝導率が高くてよい。複数の貫通部２２０は、本体部２１０において、複数のウェル１１０の一部または全部に対応する位置にそれぞれ設けられている。例えば、貫通部２２０は、２の累乗数個（２個、４個、８個、１６個、３２個、６４個等）設けられてよい。また、複数の貫通部２２０は、伝熱プレート２００の主面内において均等な間隔で設けられてよい。これにより、伝熱プレート２００は、本体部２１０が容器１００に接触して容器１００への熱伝導を確保しつつ、ウェル１１０内の細胞への光照射を可能にする。 The heat transfer plate 200 has the container 100 placed thereon, and transfers heat from the heater 300 to the container 100. The heat transfer plate 200 includes a main body part 210 and a light transmission part, and mainly conducts heat in the main body part 210 and transmits light in the through part 220. The light transmission part of the heat transfer plate 200 is provided corresponding to the bottom surfaces of the plurality of wells 110, and may be one or a plurality of penetration parts 220 as shown in FIG. 1, for example. The main body 210 may have a higher thermal conductivity than the container 100. The plurality of through portions 220 are respectively provided at positions corresponding to some or all of the plurality of wells 110 in the main body 210. For example, the penetration part 220 may be provided as a power of 2 (2, 4, 8, 16, 32, 64, etc.). Further, the plurality of through portions 220 may be provided at equal intervals in the main surface of the heat transfer plate 200. Thereby, the heat transfer plate 200 enables light irradiation to the cells in the well 110 while ensuring that the main body 210 contacts the container 100 and heat conduction to the container 100.
　本体部２１０は、熱伝導性が良好な金属により構成されてよい。例えば、本体部２１０は、アルミニウム（例えば、バルクとしての熱伝導率は２２６～２３７Ｗ／ｍＫ：以下、括弧内の値は金属のバルクとしての熱伝導率を示し、単位はＷ／ｍＫである。）、金（３１８）、銀（４２９）、銅（３８６～４０２）、鉄（７２～８０．４）、タングステン（１６９～１７６）、および、これらの一部を含有する合金を含んでよい。また、本体部２１０は、少なくとも一部が、熱伝導性が良好なグラファイト（例えば、熱伝導率２００～２０００Ｗ／ｍＫ）等の非金属材料が形成されてもよい。また、本体部２１０は、容器１００よりもバルクとしての熱伝導率が高い樹脂により構成されてもよい。例えば、アクリロニトリル・スチレン樹脂（例えば、バルクとしての熱伝導率は０．１５～０．１６Ｗ／ｍＫ：以下、括弧内の値は樹脂のバルクとしての熱伝導率を示し、単位はＷ／ｍＫである。）、アクリロニトリル・ブタジエン・スチレン樹脂（０．１９～０．３６）、エポキシ樹脂（０．３）、四フッ化エチレン・エチレン共重合体（０．２５）、四フッ化エチレン・六フッ化プロピレン共重合体（０．２５）、ポリアミド６（０．２５～０．４３）、ポリアミド６６（０．２４～０．４３）、ポリアミド６１０（０．２２）、ポリブチレンテレフタレート（０．２５）、ポリカーボネート（０．１９）、低密度ポリエチレン（０．３３）、中密度ポリエチレン（０．３３～０．４２）、高密度ポリエチレン（０．４６～０．５２）、ポリエーテルエーテルケトン（０．２６）、ポリエーテルサルホン（０．１８～０．２４）、ポリエチレンテレフタレート（０．３１）、フェノール樹脂（０．１３～０．２５）、四フッ化エチレン・パーフルオロビニルエーテル共重合体（０．２５）、ポリイミド（０．２８～０．３４）、ポリメチルメタクリレート（０．１７～０．２５）、ポリアセタール（０．２３）、ポリプロピレン（０．１２５）、ポリフェニレンサルファイド（０．２９）、ポリフェニレンオキシド（０．１９）、ポリスチレン（０．１０～０．１４）、ポリサルホン（０．１２～０．１６）、四フッ化エチレン樹脂（０．２５）、ポリ塩化ビニル（０．１３～０．２９）、ポリ塩化ビニリデン（０．１３）、ポリフッ化ビニリデン（０．１３）、ポリ三フッ化塩化エチレン（０．１９～０．２９）、ポリ四フッ化エチレン（０．２５）、ポリオキシメチレン（０．２３～０．２９）、セルロースアセテート（０．２０）、セルロースアセテートブチレート（０．１７～０．３３）、不飽和ポリエステル樹脂（０．１７～０．２１）、ポリウレタン樹脂（０．２１）、及び、シリコーン樹脂（０．１５～０．１７）等の樹脂から、容器１００よりも熱伝導率が高い樹脂を１つ以上選択し、本体部２１０の少なくとも一部に用いてよい。容器１００は、例えば上述した樹脂から、本体部２１０よりもバルクとしての熱伝導率が低い樹脂を１つ以上選択し用いてもよい。特に熱伝導性、加工性、コスト等の観点から本体部２１０は、アルミニウムにより形成されることが好ましい。本体部２１０は、容器１００が搭載されると、複数のウェル１１０の各々の外側の底面の少なくとも一部に接してよい。一例として、本体部２１０は、容器１００が搭載されると、少なくとも複数のウェル１１０の側壁の底面を覆うように配置されてよい。 The main body 210 may be made of a metal having good thermal conductivity. For example, the main body 210 is made of aluminum (for example, the thermal conductivity as a bulk is 226 to 237 W / mK: the values in parentheses indicate the thermal conductivity as a metal bulk, and the unit is W / mK. ), Gold (318), silver (429), copper (386-402), iron (72-80.4), tungsten (169-176), and alloys containing parts thereof. Further, at least a part of the main body 210 may be formed of a non-metallic material such as graphite having a good thermal conductivity (for example, a thermal conductivity of 200 to 2000 W / mK). Further, the main body 210 may be made of a resin having a higher thermal conductivity as a bulk than the container 100. For example, acrylonitrile styrene resin (for example, the thermal conductivity as a bulk is 0.15 to 0.16 W / mK: the value in parentheses indicates the thermal conductivity as the bulk of the resin, and the unit is W / mK. ), Acrylonitrile / butadiene / styrene resin (0.19 to 0.36), epoxy resin (0.3), ethylene tetrafluoride / ethylene copolymer (0.25), ethylene tetrafluoride / hexafluoro Propylene copolymer (0.25), polyamide 6 (0.25 to 0.43), polyamide 66 (0.24 to 0.43), polyamide 610 (0.22), polybutylene terephthalate (0.25) ), Polycarbonate (0.19), low density polyethylene (0.33), medium density polyethylene (0.33 to 0.42), high density polyethylene (0.46 to 0.52), Reether ether ketone (0.26), polyether sulfone (0.18 to 0.24), polyethylene terephthalate (0.31), phenol resin (0.13 to 0.25), tetrafluoroethylene perfluoro Vinyl ether copolymer (0.25), polyimide (0.28 to 0.34), polymethyl methacrylate (0.17 to 0.25), polyacetal (0.23), polypropylene (0.125), polyphenylene sulfide (0.29), polyphenylene oxide (0.19), polystyrene (0.10 to 0.14), polysulfone (0.12 to 0.16), tetrafluoroethylene resin (0.25), polyvinyl chloride (0.13-0.29), polyvinylidene chloride (0.13), polyvinylidene fluoride (0.13), polytrifluoride Ethylene (0.19-0.29), polytetrafluoroethylene (0.25), polyoxymethylene (0.23-0.29), cellulose acetate (0.20), cellulose acetate butyrate (0 .17 to 0.33), unsaturated polyester resin (0.17 to 0.21), polyurethane resin (0.21), silicone resin (0.15 to 0.17), etc. One or more resins having higher thermal conductivity may be selected and used for at least a part of the main body 210. For the container 100, for example, one or more resins having a lower thermal conductivity as a bulk than the main body 210 may be selected and used from the above-described resins. In particular, the main body 210 is preferably formed of aluminum from the viewpoint of thermal conductivity, workability, cost, and the like. When the container 100 is mounted, the main body 210 may be in contact with at least a part of the outer bottom surface of each of the plurality of wells 110. For example, the main body 210 may be disposed so as to cover at least the bottom surfaces of the side walls of the plurality of wells 110 when the container 100 is mounted.
　複数の貫通部２２０のそれぞれは、光を透過し、本体部２１０を厚み方向に貫通するように設けられた開口であってよい。複数の貫通部２２０は、材料が充填されていない空隙であってよく、この場合、貫通部２２０は貫通孔となる。これに代えて、貫通部２２０には、熱伝導率が良好なガラス又は樹脂（例えば、本体部２１０の候補として挙げた樹脂）等の光透過材料（例えば透明材料）が充填されていてもよい。なお、貫通部２２０は、少なくとも光照射部４００の出射光に含まれる波長の一部を透過させ、好ましくは可視光を透過させる。貫通部２２０は、複数のウェル１１０の各々の内側の底面の少なくとも一部の下方に設けられていてよい。 Each of the plurality of penetrating portions 220 may be an opening that transmits light and penetrates the main body portion 210 in the thickness direction. The plurality of through portions 220 may be voids that are not filled with a material. In this case, the through portions 220 are through holes. Instead, the penetrating portion 220 may be filled with a light transmissive material (for example, a transparent material) such as glass or resin having good thermal conductivity (for example, a resin cited as a candidate for the main body portion 210). . The penetrating portion 220 transmits at least a part of the wavelength included in the emitted light of the light irradiation unit 400, and preferably transmits visible light. The through portion 220 may be provided below at least a part of the bottom surface inside each of the plurality of wells 110.
　ここで、伝熱プレート２００は、容器１００が伝熱プレート２００に密着するように、容器１００の形状に合わせた形状及び厚みを有してよい。例えば、容器１００が外縁部に容器１００の底面から突出する脚を有する場合、伝熱プレート２００は当該脚が突出する長さよりも予め定められた大きさ分（例えば、０．０１～１ｍｍ）、大きな厚みを有してよい。これにより、伝熱プレート２００は、伝熱性を維持しつつ容器１００の脚以外の本体が伝熱プレート２００から浮いてしまうのを防ぐことができる。一例として、伝熱プレート２００は１～３ｍｍの厚みを有してよい。 Here, the heat transfer plate 200 may have a shape and thickness that match the shape of the container 100 so that the container 100 is in close contact with the heat transfer plate 200. For example, when the container 100 has legs that protrude from the bottom surface of the container 100 at the outer edge, the heat transfer plate 200 has a predetermined size (for example, 0.01 to 1 mm) than the length that the legs protrude. It may have a large thickness. Thus, the heat transfer plate 200 can prevent the main body other than the legs of the container 100 from floating from the heat transfer plate 200 while maintaining the heat transfer property. As an example, the heat transfer plate 200 may have a thickness of 1 to 3 mm.
　ヒーター３００は、伝熱プレート２００に接して配置され、熱を発生し、伝熱プレート２００を介して容器１００に熱を伝える。ヒーター３００は、容器１００及び伝熱プレート２００のステージとしても機能する。すなわち、ヒーター３００は、３次元空間を移動することで、容器１００及び伝熱プレート２００を移動する。また、ヒーター３００を駆動するステージを別途用意してもよい。 The heater 300 is disposed in contact with the heat transfer plate 200, generates heat, and transfers the heat to the container 100 through the heat transfer plate 200. The heater 300 also functions as a stage for the container 100 and the heat transfer plate 200. That is, the heater 300 moves the container 100 and the heat transfer plate 200 by moving in the three-dimensional space. In addition, a stage for driving the heater 300 may be separately prepared.
　例えば、矩形形状のヒーター３００が、中央が矩形形状にくりぬかれたステージに嵌め込まれ、ステージと共に駆動されてもよい。ヒーター３００は、伝熱プレート２００を搭載する領域の少なくとも一部に光を透過する光透過部３１０を有する。ヒーター３００は、光透過部３１０において発熱装置（すなわち、透明な発熱装置）を有し、光透過部３１０で発生した熱を伝熱プレート２００に伝えてよい。これに代えて、ヒーター３００は、光透過部３１０の周囲に不透明又は透明な発熱装置を備え、当該発熱装置からの熱を光透過部３１０を介して、伝熱プレート２００に伝えるものであってもよい。 For example, the rectangular heater 300 may be fitted into a stage whose center is hollowed into a rectangular shape and driven together with the stage. The heater 300 has a light transmission part 310 that transmits light to at least a part of a region where the heat transfer plate 200 is mounted. The heater 300 may have a heat generating device (that is, a transparent heat generating device) in the light transmission part 310, and may transmit the heat generated in the light transmission part 310 to the heat transfer plate 200. Instead, the heater 300 includes an opaque or transparent heat generating device around the light transmitting portion 310, and transfers heat from the heat generating device to the heat transfer plate 200 via the light transmitting portion 310. Also good.
　駆動部３５０は、ヒーター３００を駆動して３次元方向に移動する。駆動部３５０は、モーター等の動力手段により、容器１００およびヒーター３００等の主面方向（Ｘ－Ｙ方向）および鉛直方向（Ｚ方向）にヒーター３００（またはヒーター３００が組み込まれたステージ）を駆動してよい。また、駆動部３５０は、ヒーター３００を主面平面内で回転させてもよい。 The driving unit 350 drives the heater 300 and moves in a three-dimensional direction. The drive unit 350 drives the heater 300 (or the stage in which the heater 300 is incorporated) in the main surface direction (XY direction) and the vertical direction (Z direction) of the container 100, the heater 300, and the like by power means such as a motor. You can do it. Moreover, the drive part 350 may rotate the heater 300 within a main surface plane.
　光照射部４００は、容器１００のウェル１１０内の対象領域に向けて光照射する。光照射部４００は、ヒーター３００で容器１００を加熱しながら、貫通部２２０及び光透過部３１０を介して、ウェル内の剥離対象となる少なくとも１の細胞に対して光を照射し、これにより対象となる細胞を媒体から剥離してよい。光照射部４００は、加熱により流動化する媒体が流動化する程度のエネルギーを付与する光を照射するものであればよく、波長４００～１２００ｎｍ、強度０．１～１０００ｍＷの光線を出射するレーザー光源であってよい。 The light irradiation unit 400 irradiates the target region in the well 110 of the container 100 with light. The light irradiation unit 400 irradiates at least one cell to be exfoliated in the well through the penetration part 220 and the light transmission part 310 while heating the container 100 with the heater 300, and thereby the target. The resulting cells may be detached from the medium. The light irradiator 400 may be anything that irradiates light that gives energy to the extent that the medium fluidized by heating fluidizes, and a laser light source that emits light having a wavelength of 400 to 1200 nm and an intensity of 0.1 to 1000 mW. It may be.
　光学部材５００は、光照射部４００から出射された光を入射し、入射した光線の光路、強度、及び／又は、位相を調整する。例えば、光学部材５００は、デジタルミラーデバイス（ＤＭＤ）、空間光変調器（ＳＬＭ）、及び／又は、ガルバノミラーを含んでよい。 The optical member 500 receives the light emitted from the light irradiation unit 400, and adjusts the optical path, intensity, and / or phase of the incident light beam. For example, the optical member 500 may include a digital mirror device (DMD), a spatial light modulator (SLM), and / or a galvanometer mirror.
　ミラー６００は、光照射部４００から出射されて光学部材５００を通過した光を、容器１００に向けて反射する。ミラー６００は、観察部８００による容器１００内の細胞の観察を可能にするために光照射部４００から出射された光以外の光を透過させてよい。例えば、ミラー６００は、光照射部４００の出射光の波長の光を反射し、それ以外の波長の光を透過するダイクロイックミラーであってよい。 The mirror 600 reflects the light emitted from the light irradiation unit 400 and passing through the optical member 500 toward the container 100. The mirror 600 may transmit light other than the light emitted from the light irradiation unit 400 in order to allow the observation unit 800 to observe the cells in the container 100. For example, the mirror 600 may be a dichroic mirror that reflects light having the wavelength of light emitted from the light irradiation unit 400 and transmits light having other wavelengths.
　レンズ７００は、ミラー６００とヒーター３００の間の光路上に配置され、ミラー６００により反射された光を入射し、入射した光を光透過部３１０に向けて出射する。レンズ７００は、観察部８００による容器１００内の細胞の観察を可能にするための対物レンズとして機能する。 The lens 700 is disposed on the optical path between the mirror 600 and the heater 300, enters the light reflected by the mirror 600, and emits the incident light toward the light transmission unit 310. The lens 700 functions as an objective lens that enables the observation unit 800 to observe cells in the container 100.
　観察部８００は、観察対象のウェル１１０およびレンズ７００と同一の光路上に配置され、観察者にウェル１１０を拡大観察させる。例えば、観察部８００は、接眼レンズを有し、レンズ７００との組み合わせにより顕微鏡として機能し、観察者に拡大された細胞を観察させる。また、例えば、観察部８００は、撮像素子を有し、撮像したウェル１１０内の拡大画像データを表示装置等に出力してよい。 The observation unit 800 is arranged on the same optical path as the observation target well 110 and the lens 700, and allows the observer to observe the well 110 in an enlarged manner. For example, the observation unit 800 includes an eyepiece lens and functions as a microscope in combination with the lens 700 to allow the observer to observe the enlarged cells. In addition, for example, the observation unit 800 may include an imaging element and output the enlarged image data in the well 110 that has been imaged to a display device or the like.
　細胞取得部９００は、光照射部４００から照射された光により媒体から剥離された細胞を取得する。例えば、細胞取得部９００は、毛細管効果により細胞を吸引可能な毛細管を有して良い。また、細胞取得部９００は、容器１００を回収容器の上に移動して反転させることにより、剥離した細胞を回収する機構を有して良い。 The cell acquisition unit 900 acquires the cells detached from the medium by the light irradiated from the light irradiation unit 400. For example, the cell acquisition unit 900 may include a capillary that can suck cells by a capillary effect. In addition, the cell acquisition unit 900 may have a mechanism for recovering detached cells by moving the container 100 over the recovery container and inverting it.
　以上のように、細胞剥離装置１０は、光照射部４００から出射した光を、光学部材５００、ミラー６００、レンズ７００、光透過部３１０、及び貫通部２２０を介して、容器１００のウェル１１０内に導き、剥離対象の細胞に光照射する。ここで、細胞剥離装置１０は、ヒーター３００で容器１００を加熱しながら、光照射部４００による細胞への光照射を実行する。 As described above, the cell detachment apparatus 10 transmits the light emitted from the light irradiation unit 400 in the well 110 of the container 100 through the optical member 500, the mirror 600, the lens 700, the light transmission unit 310, and the penetration unit 220. And irradiate the cells to be detached with light. Here, the cell peeling apparatus 10 performs light irradiation on the cells by the light irradiation unit 400 while heating the container 100 with the heater 300.
　これにより、細胞剥離装置１０は、より短い時間及び弱い光照射で、細胞を媒体から剥離することができる。この結果、細胞剥離装置１０は、細胞への侵襲を低減し、細胞をより少ないダメージで確実に回収することができる。 Thereby, the cell peeling apparatus 10 can peel the cells from the medium in a shorter time and with a weak light irradiation. As a result, the cell peeling device 10 can reduce the invasion to the cells and reliably collect the cells with less damage.
　また、細胞剥離装置１０は、伝熱プレート２００と容器１００の間にグラファイトシートを備えてもよい。容器１００の形状に個体差があり、容器１００の面が不均一である等の理由で、容器１００が伝熱プレート２００と面接触できない場合がある。このような場合であっても、容器１００と伝熱プレート２００の間の空間の少なくとも一部を熱伝導性と柔軟性に優れるグラファイトシートで充填することにより、効率よくヒーター３００からの熱を容器１００に伝えることができる。なお、グラファイトシートを設ける場合、観察部８００による観察の障害とならないように、グラファイトシートに貫通部２２０に準じた形状の開口が設けられてよい。 In addition, the cell peeling device 10 may include a graphite sheet between the heat transfer plate 200 and the container 100. The container 100 may not be in surface contact with the heat transfer plate 200 because there are individual differences in the shape of the container 100 and the surface of the container 100 is not uniform. Even in such a case, at least a part of the space between the container 100 and the heat transfer plate 200 is filled with a graphite sheet having excellent thermal conductivity and flexibility, so that the heat from the heater 300 can be efficiently transferred to the container. 100. In the case where a graphite sheet is provided, an opening having a shape corresponding to the penetrating portion 220 may be provided in the graphite sheet so as not to obstruct observation by the observation unit 800.
　図２は、本実施形態の伝熱プレート２００の構成の一例を示す。図示するように、伝熱プレート２００の本体部２１０は、複数層構成であり、貫通部２２０は空隙であってよい。例えば、伝熱プレート２００の本体部２１０は、コア層２１２、表面層２１４、および、表面層２１６を含んで良い。 FIG. 2 shows an example of the configuration of the heat transfer plate 200 of the present embodiment. As shown in the figure, the main body 210 of the heat transfer plate 200 may have a multilayer structure, and the through-hole 220 may be a gap. For example, the main body 210 of the heat transfer plate 200 may include a core layer 212, a surface layer 214, and a surface layer 216.
　一例として、コア層２１２は厚さアルミニウム板であり、表面層２１４及び表面層２１６はグラファイトシートであってよく、コア層２１２と表面層２１４及び表面層２１６とは粘着剤等により接着されてよい。これにより、伝熱プレート２００は、強度を維持しつつ熱伝導性を高めることができる。 As an example, the core layer 212 may be an aluminum plate having a thickness, the surface layer 214 and the surface layer 216 may be graphite sheets, and the core layer 212, the surface layer 214, and the surface layer 216 may be bonded with an adhesive or the like. . Thereby, the heat transfer plate 200 can improve thermal conductivity while maintaining strength.
　図３は、本実施形態のウェル１１０の構成の一例を示す。ここでは、容器１００の複数のウェル１１０のうち剥離対象の細胞を含むウェルを拡大した図を示す。図示するように、細胞剥離処理時に、ウェル１１０は、媒体１１２、細胞１１７、及び、培養液１１８を含む。 FIG. 3 shows an example of the configuration of the well 110 of the present embodiment. Here, the figure which expanded the well containing the cell of peeling object among the some wells 110 of the container 100 is shown. As shown in the figure, during the cell detachment process, the well 110 includes a medium 112, a cell 117, and a culture solution 118.
　媒体１１２は、ゲル１１４と微粒子（例えば、金微粒子１１６）とを含む。ゲル１１４は、予め定められた温度以上（例えば、４０℃）になると構造が変化するゲル材料であってよく、例えば、コラーゲンゲルまたはゼラチンゲル等であってよい。金微粒子１１６は、光照射部４００が出射する波長の光を吸光して発熱する。 The medium 112 includes a gel 114 and fine particles (for example, gold fine particles 116). The gel 114 may be a gel material whose structure changes when the temperature is equal to or higher than a predetermined temperature (for example, 40 ° C.), and may be, for example, a collagen gel or a gelatin gel. The gold fine particles 116 generate heat by absorbing light having a wavelength emitted from the light irradiation unit 400.
　これにより、光照射部４００からの光（図中で点線矢印で示す）が入射した領域近傍にゲル１１４のみが金微粒子１１６の発熱により構造が変化し、結果として、光照射された領域に含まれる細胞１１７のみが足場を失って媒体１１２から剥離される。観察者は、観察部８００でウェル１１０内を観察しながら、目的の細胞１１７に光照射をしてよい。この結果、細胞取得部９００は、観察ウェル１１０内に収容される多数の細胞１１７のうち、目的の細胞１１７のみ剥離して選択的に取得できる。 As a result, the structure of only the gel 114 is changed by the heat generation of the gold fine particles 116 in the vicinity of the region where the light from the light irradiation unit 400 (indicated by the dotted arrow in the drawing) is incident. Only the cells 117 that are lost lose the scaffold and are detached from the medium 112. The observer may irradiate the target cell 117 with light while observing the inside of the well 110 with the observation unit 800. As a result, the cell acquiring unit 900 can selectively acquire only the target cell 117 by peeling it out of the large number of cells 117 accommodated in the observation well 110.
　図４は、本実施形態の容器１００の外観の一例を示す。図示するように、容器１００は、複数のウェル１１０がマトリクス状に配置されたマイクロプレートであってよい。容器１００のうちウェル１１０の形成部分以外は、容器１００の形成材料で充填されていてよく、これに代えて、少なくとも一部が空隙であってよい。 FIG. 4 shows an example of the appearance of the container 100 of the present embodiment. As illustrated, the container 100 may be a microplate in which a plurality of wells 110 are arranged in a matrix. The part other than the part where the well 110 is formed in the container 100 may be filled with the forming material of the container 100. Alternatively, at least a part may be a void.
　図５は、図４に対応する本実施形態の伝熱プレート２００の外観の一例を示す。図示するように、伝熱プレート２００は、図４のウェル１１０に対応してマトリクス状に設けられた複数の貫通部２２０が設けられたプレートであってよい。図５は、複数の開口のそれぞれは、マトリクスの行方向及び／又は列方向において均等な間隔で設けられ、対応するウェルの底面よりも大きい形態を示す。 FIG. 5 shows an example of the appearance of the heat transfer plate 200 of the present embodiment corresponding to FIG. As shown in the drawing, the heat transfer plate 200 may be a plate provided with a plurality of through portions 220 provided in a matrix corresponding to the wells 110 of FIG. FIG. 5 shows a form in which each of the plurality of openings is provided at equal intervals in the row direction and / or column direction of the matrix and is larger than the bottom surface of the corresponding well.
　これにより、ウェル１１０と貫通部２２０の位置合わせが容易になる。これに代えて、複数の開口のそれぞれは、対応するウェル１１０の底面よりも小さくてもよい。この場合、伝熱プレート２００の本体部２１０の面積が大きくなるので、ウェル１１０に対する熱伝導性が向上する。 This facilitates the alignment of the well 110 and the through portion 220. Alternatively, each of the plurality of openings may be smaller than the bottom surface of the corresponding well 110. In this case, since the area of the main body 210 of the heat transfer plate 200 is increased, the thermal conductivity with respect to the well 110 is improved.
　図６は、本実施形態のウェルプレートユニットの一例を示す。本実施形態において、容器１００と伝熱プレート２００は、組み合わされてウェルプレートユニットを構成してよい。ウェルプレートユニットは図１に示すように伝熱プレート２００上にそのまま容器１００が積み重ねられた形態であってよいが、図６に示すように下側が開口された空隙となっている容器１００の下側に伝熱プレート２００が組み入れられた形態であってもよい。 FIG. 6 shows an example of the well plate unit of the present embodiment. In the present embodiment, the container 100 and the heat transfer plate 200 may be combined to form a well plate unit. The well plate unit may have a form in which the containers 100 are stacked as they are on the heat transfer plate 200 as shown in FIG. 1, but as shown in FIG. The heat transfer plate 200 may be incorporated on the side.
　この場合、容器１００は、ウェル１１０を形成する部分と上側プレート部分と側壁部分とからなる。すなわち、容器１００は、図６の上側にはウェル１１０を形成するくぼみが設けられた上側プレートを有するが、下側にはプレートがなく空隙となっていて、伝熱プレート２００の貫通部２２０も空隙となる。 In this case, the container 100 includes a portion for forming the well 110, an upper plate portion, and a side wall portion. That is, the container 100 has an upper plate provided with a recess for forming the well 110 on the upper side of FIG. 6, but has no plate on the lower side and is a gap, and the through-hole 220 of the heat transfer plate 200 is also provided. It becomes a void.
　これにより、複数のウェル１１０のそれぞれが、対応する貫通部２２０の複数の開口に嵌め込まれた形態となる。なお、本形態においては複数の開口のそれぞれは対応するウェル１１０の底面よりも大きくなる。この形態によると容器１００の側部からも伝熱プレート２００からの加熱がなされるので、より効率的に容器１００に熱を伝えることができる。 Thereby, each of the plurality of wells 110 is fitted into the plurality of openings of the corresponding penetration part 220. In the present embodiment, each of the plurality of openings is larger than the bottom surface of the corresponding well 110. According to this embodiment, since the heat transfer plate 200 is also heated from the side portion of the container 100, heat can be transferred to the container 100 more efficiently.
　図７は、本実施形態の細胞剥離装置１０の処理フローの一例を示す。本実施形態において、細胞剥離装置１０は、Ｓ１１０～Ｓ１９０の処理を実行する。 FIG. 7 shows an example of the processing flow of the cell peeling apparatus 10 of the present embodiment. In the present embodiment, the cell peeling device 10 executes the processes of S110 to S190.
　まず、Ｓ１１０において、加熱によりに流動化する媒体１１２を、細胞培養用の基材として、複数のウェル１１０を備える容器１００に供給する。媒体１１２は、加熱により流動化するゲルと金微粒子とを含んでよい。例えば、特許文献１～２に記載されるように、アセチル化コラーゲンペプチドからアセチル化コラーゲンペプチド修飾デンドリマー溶液を生成し、テトラクロロ金（ＩＩＩ）酸溶液を混合し、その後、金イオンを還元して、金微粒子を含むコラーゲンゲルを生成し、これを本実施形態における媒体１１２としてよい。 First, in S110, a medium 112 that is fluidized by heating is supplied to a container 100 including a plurality of wells 110 as a substrate for cell culture. The medium 112 may include a gel and gold fine particles that are fluidized by heating. For example, as described in Patent Documents 1 and 2, an acetylated collagen peptide-modified dendrimer solution is generated from an acetylated collagen peptide, mixed with a tetrachloroauric (III) acid solution, and then gold ions are reduced. A collagen gel containing gold fine particles is generated, and this may be used as the medium 112 in this embodiment.
　Ｓ１１０において、容器１００の複数のウェル１１０の全部また一部に対して媒体１１２を供給してよい。また、媒体１１２の供給後にリン酸緩衝生理食塩水（ＰＢＳ（－））を供給して、所定の温度（例えば、３７℃）で所定時間（例えば、６時間）の間、ウェル１１０内で溶液を保持することで、媒体１１２の洗浄を実行してもよい。 In S110, the medium 112 may be supplied to all or a part of the plurality of wells 110 of the container 100. Further, after the medium 112 is supplied, phosphate buffered saline (PBS (−)) is supplied, and the solution is stored in the well 110 at a predetermined temperature (for example, 37 ° C.) for a predetermined time (for example, 6 hours). By holding this, the medium 112 may be cleaned.
　次に、Ｓ１２０において、媒体１１２に細胞１１７を固定する。例えば、８．０×１０３個のＨｅｌａ細胞を媒体１１２に播種してよい。更に、Ｓ１２０において、固定された細胞１１７に対してＤＭＥＭ等の培養液１１８を供給してよい。 Next, in S120, the cells 117 are fixed to the medium 112. For example, 8.0 × 10 3 Hela cells may be seeded in the medium 112. Further, in S120, a culture solution 118 such as DMEM may be supplied to the fixed cells 117.
　次に、Ｓ１３０において、固定された細胞１１７を培養する。例えば、所定の温度（例えば、３７℃）で所定時間（例えば、２４時間）、細胞１１７が固定された容器１００を保持し、インキュベーションを実行する。 Next, in S130, the fixed cells 117 are cultured. For example, the container 100 in which the cells 117 are fixed is held at a predetermined temperature (for example, 37 ° C.) for a predetermined time (for example, 24 hours), and incubation is performed.
　次に、Ｓ１４０において、容器１００のウェル１１０内を洗浄する。例えば、ウェル１１０内の培養液１１８を除去し、ＰＢＳ（－）等の洗浄液で少なくとも１回（例えば３回）洗浄する。これにより、細胞１１７を媒体１１２から剥離する前にウェル１１０内を洗浄し、剥離作業を実行しやすくし、細胞とともに回収される不純物を低減する。なお、Ｓ１４０を省略してもよい。 Next, in S140, the inside of the well 110 of the container 100 is washed. For example, the culture solution 118 in the well 110 is removed and washed with a washing solution such as PBS (−) at least once (for example, three times). Accordingly, the inside of the well 110 is washed before the cells 117 are detached from the medium 112, the separation work is easily performed, and impurities collected together with the cells are reduced. Note that S140 may be omitted.
　次に、Ｓ１５０において、ウェル１１０内の溶液の少なくとも一部を除去する。例えば、Ｓ１４０の洗浄が実行された場合、ウェル１１０内に残存する洗浄液を少なくとも一部除去する。細胞１１７を保護する観点から、ウェル１１０に洗浄液を少なくとも一部残存させてもよい。また、ウェル１１０内の洗浄液を全て除去した後に培養液１１８を新しく供給してもよい。 Next, in S150, at least a part of the solution in the well 110 is removed. For example, when the cleaning of S140 is executed, at least a part of the cleaning liquid remaining in the well 110 is removed. From the viewpoint of protecting the cells 117, at least a part of the washing solution may remain in the well 110. Alternatively, the culture medium 118 may be newly supplied after all the cleaning liquid in the well 110 is removed.
　また、Ｓ１４０が省略されてウェル１１０内にＳ１２０で供給された培養液１１８が残存している場合、培養液１１８のすくなとも一部を除去してもよい。培養液１１８の一部を除去することで、ウェル１１０の内容物の熱容量が減少し、媒体１１２をより短時間で昇温することができる。また、培養液１１８を全て除去せずに一部残存させておくとウェル１１０内の細胞１１７を保護することができる。 Further, when S140 is omitted and the culture medium 118 supplied in S120 remains in the well 110, a part of the culture medium 118 may be removed. By removing a part of the culture solution 118, the heat capacity of the contents of the well 110 is reduced, and the temperature of the medium 112 can be raised in a shorter time. Further, if the culture medium 118 is partially removed without being removed, the cells 117 in the well 110 can be protected.
　次に、Ｓ１６０において、容器１００をヒーター３００で加熱する。例えば、ヒーター３００の光透過部３１０上に伝熱プレート２００を搭載し、伝熱プレート２００の複数の貫通部２２０の位置と複数のウェル１１０と位置が対応するように容器１００を伝熱プレート２００上に配置し、ヒーター３００を発熱させる。これにより、ヒーター３００から発生した熱が伝熱プレート２００を介して容器１００に伝えられる。 Next, in S160, the container 100 is heated by the heater 300. For example, the heat transfer plate 200 is mounted on the light transmission part 310 of the heater 300, and the container 100 is placed so that the positions of the plurality of through parts 220 of the heat transfer plate 200 correspond to the positions of the plurality of wells 110. Arranged above, the heater 300 generates heat. Thereby, the heat generated from the heater 300 is transmitted to the container 100 through the heat transfer plate 200.
　Ｓ１６０の加熱は、予め定められた温度又は加熱時間が達成されるまで継続されてよい。例えば、容器内の媒体１１２又は培養液１１８等の溶液が予め定められた温度（例えば、３６℃）になったことに応じてＳ１７０の処理に進んでよい。 The heating in S160 may be continued until a predetermined temperature or heating time is achieved. For example, the process may proceed to S170 when the solution such as the medium 112 or the culture medium 118 in the container has reached a predetermined temperature (for example, 36 ° C.).
　次に、Ｓ１７０において、伝熱プレート２００を介して容器１００を加熱しながら、ウェル１１０内の剥離対象の細胞１１７に対して光照射部４００からの光を照射し、当該細胞１１７を媒体１１２から剥離する。例えば、観察部８００によりウェル１１０内を拡大して観察しながら、剥離対象の細胞１１７を特定し、特定した剥離対象の１つの細胞１１７に向けて光照射部４００からレーザー光を照射する。 Next, in S <b> 170, while heating the container 100 via the heat transfer plate 200, the cells 117 to be peeled in the well 110 are irradiated with light from the light irradiation unit 400, and the cells 117 are removed from the medium 112. Peel off. For example, while observing the inside of the well 110 with the observation unit 800 being magnified, the cells 117 to be peeled are specified, and laser light is emitted from the light irradiation unit 400 toward the specified cells 117 to be peeled.
　光照射部４００からの光照射は予め定められた温度又は照射時間が達成されるまで継続されてよい。一例として、剥離対象の細胞１１７に対して、波長５３２ｎｍのレーザー光を３ｍＷの強度で１０秒間照射する。 The light irradiation from the light irradiation unit 400 may be continued until a predetermined temperature or irradiation time is achieved. As an example, a laser beam having a wavelength of 532 nm is irradiated for 10 seconds at an intensity of 3 mW to the cells 117 to be peeled.
　光照射部４００は、光学部材５００、ミラー６００、及び、レンズ７００を介してヒーター３００に出射光を入射させ、ヒーター３００の光透過部３１０及び伝熱プレート２００の貫通部２２０を介して透過してウェル１１０内の剥離対象の細胞１１７に光照射する。光照射された細胞１１７付近の媒体１１２は、ヒーター３００からの熱に加えて、光照射部４００からの光照射により加熱されて、流動化する。これにより、光照射部４００は、剥離対象の細胞１１７を媒体１１２から剥離する。 The light irradiation unit 400 causes the emitted light to enter the heater 300 through the optical member 500, the mirror 600, and the lens 700, and is transmitted through the light transmission unit 310 of the heater 300 and the penetration unit 220 of the heat transfer plate 200. Then, the cells 117 to be peeled in the well 110 are irradiated with light. The medium 112 in the vicinity of the cell 117 irradiated with light is heated and fluidized by light irradiation from the light irradiation unit 400 in addition to heat from the heater 300. As a result, the light irradiation unit 400 peels the cells 117 to be peeled from the medium 112.
　ここで、光照射の前／間に、細胞剥離装置１０は、照射光の位置及びフォーカスを調整してよい。例えば、駆動部３５０は、ステージとして機能するヒーター３００を主面方向に移動することで、光照射部４００の光照射の位置が剥離対象の細胞１１７の位置と一致するように調整してよい。 Here, before / during light irradiation, the cell peeling device 10 may adjust the position and focus of the irradiation light. For example, the driving unit 350 may adjust the light irradiation position of the light irradiation unit 400 so as to coincide with the position of the cell 117 to be peeled by moving the heater 300 functioning as a stage in the main surface direction.
　また、光学部材５００のガルバノミラーの操作を行うことで、光照射部４００の光照射の位置を変更してよい。また、細胞剥離装置１０は、光学部材５００、レンズ７００、及び／又はその他の調整装置を調整することにより、レーザー光のフォーカス等の光学条件を調整してよい。 Further, the position of light irradiation of the light irradiation unit 400 may be changed by operating the galvanometer mirror of the optical member 500. Moreover, the cell peeling apparatus 10 may adjust optical conditions, such as a focus of a laser beam, by adjusting the optical member 500, the lens 700, and / or other adjustment apparatuses.
　Ｓ１７０において、剥離対象の１つの細胞１１７に対して光照射を実行する代わりに、剥離対象の複数の細胞１１７に対して一括で光照射を実行してもよい。例えば、剥離対象の細胞１１７に対応する光照射パターンのデータをあらかじめ作成しておき、光学部材５００のＤＭＤまたはＳＬＭにより当該光照射パターンを実現し、光照射部４００は当該光照射パターンを介してウェル１１０に光照射を行ってよい。これにより、光照射部４００は、ウェル１１０内の剥離対象の全部の細胞１１７を一度の光照射で剥離することができる。 In S170, instead of performing light irradiation on one cell 117 to be detached, light irradiation may be performed on a plurality of cells 117 to be separated at once. For example, the light irradiation pattern data corresponding to the cell 117 to be peeled is created in advance, the light irradiation pattern is realized by the DMD or SLM of the optical member 500, and the light irradiation unit 400 passes through the light irradiation pattern. The well 110 may be irradiated with light. Thereby, the light irradiation part 400 can peel all the cells 117 of the peeling object in the well 110 by one light irradiation.
　次に、Ｓ１８０において全ての剥離対象の細胞１１７に対する光照射が終了したか否かを判断する。終了した場合、処理をＳ２００に進め、そうでない場合は処理をＳ１９０に進める。 Next, in S180, it is determined whether or not the light irradiation on all cells 117 to be detached has been completed. If completed, the process proceeds to S200, and if not, the process proceeds to S190.
　Ｓ１９０において、光照射部４００による光照射の対象を変更する。例えば、駆動部３５０は、ヒーター３００を主面方向に移動することで、光照射部４００の光照射の位置を別の未剥離の剥離対象の細胞１１７に変更してよい。また、光学部材５００がガルバノミラーの操作を行うことで、光照射部４００の光照射の位置を別の未剥離の剥離対象の細胞１１７に変更してよい。 In S190, the target of light irradiation by the light irradiation unit 400 is changed. For example, the driving unit 350 may change the light irradiation position of the light irradiation unit 400 to another undetached cell 117 to be peeled by moving the heater 300 in the main surface direction. Further, the optical member 500 may operate the galvanometer mirror to change the light irradiation position of the light irradiation unit 400 to another undetached cell 117 to be peeled.
　Ｓ２００において、細胞取得部９００が剥離された細胞１１７を取得する。例えば、観察部８００で剥離された細胞１１７の位置を確認しながら、細胞取得部９００が毛細管の先端を剥離された細胞１１７に近接し、毛細管で細胞１１７を吸引し、その後、吸引した細胞１１７を排出することで細胞１１７を取得してよい。 In S200, the cell acquisition unit 900 acquires the detached cell 117. For example, while confirming the position of the cell 117 peeled by the observation unit 800, the cell acquisition unit 900 approaches the peeled cell 117 at the tip of the capillary, sucks the cell 117 with the capillary, and then sucks the cell 117. The cells 117 may be obtained by discharging the cells.
　また、細胞取得部９００は、１個ずつ細胞１１７を回収することに代えて、１又は複数のウェル１１０における複数の剥離された細胞１１７を一度に取得してもよい。例えば、容器１００を回収容器上に移動し、容器１００を覆らすことで容器１００内の全部の剥離された細胞１１７を回収容器内に一括して回収してもよい。 In addition, the cell acquisition unit 900 may acquire a plurality of detached cells 117 in one or a plurality of wells 110 at a time instead of collecting the cells 117 one by one. For example, all the detached cells 117 in the container 100 may be collectively collected in the collection container by moving the container 100 onto the collection container and covering the container 100.
　このように細胞剥離装置１０を用いてＳ１１０～Ｓ２００の処理を実行することで、本実施形態の細胞剥離方法を実現する。本実施形態の細胞剥離方法によれば、伝熱プレート２００を介して加熱により流動化する媒体１１２をゆるやかに加熱しながら光照射により媒体１１２を急速に加熱して媒体１１２を流動化するので、細胞１１７への侵襲性が高い光照射の時間を従来よりも短くすることができ、より確実に目的の細胞１１７を取得することができる。 Thus, the cell detachment method of this embodiment is realized by executing the processing of S110 to S200 using the cell detachment apparatus 10. According to the cell detachment method of the present embodiment, the medium 112 is rapidly heated by light irradiation while the medium 112 that is fluidized by heating through the heat transfer plate 200 is gently heated, so that the medium 112 is fluidized. The time of light irradiation with high invasiveness to the cells 117 can be made shorter than before, and the target cells 117 can be obtained more reliably.
　図８は、本実施形態の細胞剥離方法の効果の一例を示す。図８のグラフの横軸はヒーター３００による加熱開始からの時間（秒）を示し、縦軸は媒体１１２の表面温度の測定結果（℃）を示す。測定は、ウェル１１０に媒体１１２のみを収容し、培養液１１８を充填しない条件で行われた。 FIG. 8 shows an example of the effect of the cell detachment method of this embodiment. The horizontal axis of the graph in FIG. 8 indicates the time (seconds) from the start of heating by the heater 300, and the vertical axis indicates the measurement result (° C.) of the surface temperature of the medium 112. The measurement was performed under the condition that only the medium 112 was accommodated in the well 110 and the culture solution 118 was not filled.
　また、光照射部４００からは、約１８０秒後の時点から５３２ｎｍのレーザー光が５ｍＷの強度で継続して照射された。図８中の点線グラフは、伝熱プレート２００が無くヒーター３００上に直接容器１００を載置して測定した結果を示し、直線グラフは、アルミニウム製の伝熱プレート２００をヒーター３００と容器１００の間に配置して測定した結果を示す。 Further, from the light irradiation unit 400, a laser beam of 532 nm was continuously irradiated with an intensity of 5 mW from about 180 seconds later. The dotted line graph in FIG. 8 shows the measurement result obtained by placing the container 100 directly on the heater 300 without the heat transfer plate 200, and the straight line graph shows the aluminum heat transfer plate 200 between the heater 300 and the container 100. The result of having been measured between them is shown.
　グラフに示されるように、伝熱プレート２００が無い条件では光照射するまでの間の昇温が遅く、光照射をしばらく継続した２５０秒付近でも媒体１１２の流動化温度である４０℃に達していない。従って、伝熱プレート２００がない条件では光照射の時間を長くし、光強度を大きくすることが必要で細胞１１７への侵襲性が高まる。 As shown in the graph, under the condition where there is no heat transfer plate 200, the temperature rise until the light irradiation is slow, and the fluidization temperature of the medium 112 reaches 40 ° C. even in the vicinity of 250 seconds when the light irradiation is continued for a while. Absent. Therefore, in the absence of the heat transfer plate 200, it is necessary to increase the light irradiation time and increase the light intensity, and the invasiveness to the cells 117 is increased.
　一方で、伝熱プレート２００を用いた場合、光照射をするまでの間に比較的早く昇温しており、光照射後数１０秒程度で４０℃に達した。このように伝熱プレート２００を用いると光照射の時間を比較的短くすることができる。 On the other hand, when the heat transfer plate 200 was used, the temperature was raised relatively quickly before light irradiation, and reached 40 ° C. in several tens of seconds after the light irradiation. When the heat transfer plate 200 is used in this way, the light irradiation time can be made relatively short.
　例えば、本実施形態の細胞剥離方法によると、伝熱プレート２００を用いない方法に比べて、光照射強度を１２分の１にすることができ、また、光照射時間を１／６にすることができた。また、本実施形態の細胞剥離方法を適用して細胞剥離後も細胞が製造していることをＨｅｌａ細胞及びＭＤＣＫ細胞に対して確認することができた。 For example, according to the cell detachment method of this embodiment, the light irradiation intensity can be reduced to 1/12 and the light irradiation time can be reduced to 1/6 as compared with the method not using the heat transfer plate 200. I was able to. Moreover, it has confirmed with respect to the Hela cell and MDCK cell that the cell has manufactured even after the cell exfoliation by applying the cell exfoliation method of this embodiment.
　以上、本発明を実施の形態を用いて説明したが、本発明の技術的範囲は上記実施の形態に記載の範囲には限定されない。上記実施の形態に、多様な変更または改良を加えることが可能であることが当業者に明らかである。その様な変更または改良を加えた形態も本発明の技術的範囲に含まれ得ることが、請求の範囲の記載から明らかである。 As mentioned above, although this invention was demonstrated using embodiment, the technical scope of this invention is not limited to the range as described in the said embodiment. It will be apparent to those skilled in the art that various modifications or improvements can be added to the above-described embodiment. It is apparent from the scope of the claims that the embodiments added with such changes or improvements can be included in the technical scope of the present invention.
　請求の範囲、明細書、および図面中において示した装置、システム、プログラム、および方法における動作、手順、ステップ、および段階等の各処理の実行順序は、特段「より前に」、「先立って」等と明示しておらず、また、前の処理の出力を後の処理で用いるのでない限り、任意の順序で実現しうることに留意すべきである。請求の範囲、明細書、および図面中の動作フローに関して、便宜上「まず、」、「次に、」等を用いて説明したとしても、この順で実施することが必須であることを意味するものではない。 The execution order of each process such as operations, procedures, steps, and stages in the apparatus, system, program, and method shown in the claims, the description, and the drawings is particularly “before” or “prior”. It should be noted that they can be implemented in any order unless the output of the previous process is used in the subsequent process. Regarding the operation flow in the claims, the description, and the drawings, even if it is described using “first”, “next”, etc. for the sake of convenience, it means that it is essential to carry out in this order. is not.
　１０　細胞剥離装置、１００　容器、１１０　ウェル、１１２　媒体、１１４　ゲル、１１６　金微粒子、１１７　細胞、１１８　培養液、２００　伝熱プレート、２１０　本体部、２１２　コア層、２１４　表面層、２１６　表面層、２２０　貫通部、３００　ヒーター、３１０　光透過部、３５０　駆動部、４００　光照射部、５００　光学部材、６００　ミラー、７００　レンズ、８００　観察部、９００　細胞取得部 10 cell peeling device, 100 container, 110 well, 112 medium, 114 gel, 116 gold fine particle, 117 cell, 118 culture solution, 200 heat transfer plate, 210 main body, 212 core layer, 214 surface layer, 216 surface layer, 220 Penetration part, 300 heater, 310 light transmission part, 350 drive part, 400 light irradiation part, 500 optical member, 600 mirror, 700 lens, 800 observation part, 900 cell acquisition part

Claims (26)

  1. 　細胞が収容される複数のウェルを有する容器が載置される伝熱プレートであって、
    　前記容器の少なくとも一部が接する本体部と、
    　前記複数のウェルの底面に対応して設けられた光透過部と
    を有する伝熱プレート。     A heat transfer plate on which a container having a plurality of wells for containing cells is placed,
    A main body part to which at least a part of the container contacts,
    A heat transfer plate having a light transmission portion provided corresponding to the bottom surface of the plurality of wells.
  2. 　前記光透過部は、前記本体部を貫通するように設けられた開口である請求項１に記載の伝熱プレート。 The heat transfer plate according to claim 1, wherein the light transmission part is an opening provided so as to penetrate the main body part.
  3. 　前記光透過部は、前記開口に光透過材料が充填されて形成されている請求項２に記載の伝熱プレート。 The heat transmission plate according to claim 2, wherein the light transmission portion is formed by filling the opening with a light transmission material.
  4. 　前記光透過材料は、樹脂またはガラスである請求項３に記載の伝熱プレート。 The heat transfer plate according to claim 3, wherein the light transmitting material is resin or glass.
  5. 　前記本体部は、少なくとも前記複数のウェルの側壁の底面を覆っている請求項１から４のいずれか１項に記載の伝熱プレート。 The heat transfer plate according to any one of claims 1 to 4, wherein the main body covers at least bottom surfaces of side walls of the plurality of wells.
  6. 　前記伝熱プレートを構成する材料の熱伝導率は、前記容器を構成する材料の熱伝導率よりも高い請求項１から５のいずれか１項に記載の伝熱プレート。 The heat transfer plate according to any one of claims 1 to 5, wherein a heat conductivity of a material constituting the heat transfer plate is higher than a heat conductivity of a material constituting the container.
  7. 　前記光透過部は、波長４００～１２００ｎｍの範囲のうちのいずれかの波長の光を透過する請求項１から６のいずれか１項に記載の伝熱プレート。 The heat transfer plate according to any one of claims 1 to 6, wherein the light transmitting portion transmits light having any wavelength within a wavelength range of 400 to 1200 nm.
  8. 　細胞が収容される複数のウェルを有する容器と、
    　前記容器が載置された伝熱プレートと、
    　前記伝熱プレートに接して配置されたヒーターと、
    　前記ウェル内の対象領域に光を照射する光照射部と、
    　を備え、
    　前記伝熱プレートは、
    　前記容器の少なくとも一部に接する本体部と、
    　前記複数のウェルの底面に対応して設けられた光透過部と
    を有する細胞剥離装置。     A container having a plurality of wells for containing cells;
    A heat transfer plate on which the container is placed;
    A heater disposed in contact with the heat transfer plate;
    A light irradiation unit for irradiating light to a target region in the well;
    With
    The heat transfer plate is
    A main body contacting at least a part of the container;
    A cell detaching device having a light transmission portion provided corresponding to the bottom surface of the plurality of wells.
  9. 　前記光透過部は、前記本体部を貫通するように設けられた開口である請求項８に記載の細胞剥離装置。 The cell detaching apparatus according to claim 8, wherein the light transmission part is an opening provided so as to penetrate the main body part.
  10. 　前記光透過部は、前記開口に光透過材料が充填されて形成されている請求項９に記載の細胞剥離装置。 The cell detaching device according to claim 9, wherein the light transmitting portion is formed by filling the opening with a light transmitting material.
  11. 　前記光透過材料は、樹脂またはガラスである請求項１０に記載の細胞剥離装置。 The cell peeling device according to claim 10, wherein the light transmitting material is resin or glass.
  12. 　前記本体部は、少なくとも前記複数のウェルを側壁の底面を覆っている請求項８から１１のいずれか１項に記載の細胞剥離装置。 The cell peeling device according to any one of claims 8 to 11, wherein the main body covers at least the plurality of wells on a bottom surface of a side wall.
  13. 　前記伝熱プレートを構成する材料の熱伝導率は、前記容器を構成する材料の熱伝導率よりも高い請求項８から１２のいずれか１項に記載の細胞剥離装置。 The cell exfoliation device according to any one of claims 8 to 12, wherein the heat conductivity of the material constituting the heat transfer plate is higher than the heat conductivity of the material constituting the container.
  14. 　前記光透過部は、前記光照射部から照射される光を透過する請求項８から１３のいずれか１項に記載の細胞剥離装置。 The cell detaching device according to any one of claims 8 to 13, wherein the light transmission unit transmits light emitted from the light irradiation unit.
  15. 　前記光照射部から照射される光は、波長４００～１２００ｎｍの範囲のうちのいずれかの波長の光を含む請求項８から１４のいずれか１項に記載の細胞剥離装置。 The cell peeling device according to any one of claims 8 to 14, wherein the light irradiated from the light irradiation unit includes light having any wavelength within a wavelength range of 400 to 1200 nm.
  16. 　前記細胞を取得する細胞取得部を更に備える、
    　請求項８から１５のいずれか１項に記載の細胞剥離装置。     A cell acquisition unit for acquiring the cells;
    The cell peeling apparatus according to any one of claims 8 to 15.
  17. 　前記ヒーターは、前記光照射部から照射される光を透過する光透過部を有する、
    　請求項８から１６のいずれか１項に記載の細胞剥離装置。     The heater has a light transmission part that transmits light emitted from the light irradiation part.
    The cell peeling apparatus according to any one of claims 8 to 16.
  18. 　前記ウェル内を観察するための観察部を更に備える、
    　請求項８から１７のいずれか１項に記載の細胞剥離装置。     An observation unit for observing the inside of the well;
    The cell peeling device according to any one of claims 8 to 17.
  19. 　細胞が収容される複数のウェルを有する容器と、
    　前記容器が載置される伝熱プレートと
    を備え、
    　前記伝熱プレートは、
    　前記容器の少なくとも一部に接する本体部と、
    　前記複数のウェルの底面に対応して設けられた光透過部と
    を有するウェルプレートユニット。     A container having a plurality of wells for containing cells;
    A heat transfer plate on which the container is placed;
    The heat transfer plate is
    A main body contacting at least a part of the container;
    A well plate unit having a light transmission portion provided corresponding to the bottom surface of the plurality of wells.
  20. 　前記光透過部は、前記本体部を貫通するように設けられた開口である請求項１９に記載のウェルプレートユニット。 The well plate unit according to claim 19, wherein the light transmission part is an opening provided so as to penetrate the main body part.
  21. 　前記光透過部は、前記開口に光透過材料が充填されて形成されている請求項２０に記載のウェルプレートユニット。 21. The well plate unit according to claim 20, wherein the light transmitting portion is formed by filling the opening with a light transmitting material.
  22. 　前記光透過材料は、樹脂またはガラスである請求項２１に記載のウェルプレートユニット。 The well plate unit according to claim 21, wherein the light transmitting material is resin or glass.
  23. 　前記本体部は、少なくとも前記複数のウェルの側壁の底面を覆っている請求項１９から２２のいずれか１項に記載のウェルプレートユニット。 The well plate unit according to any one of claims 19 to 22, wherein the main body covers at least the bottom surfaces of the side walls of the plurality of wells.
  24. 　前記伝熱プレートを構成する材料の熱伝導率は、前記容器を構成する材料の熱伝導率よりも高い請求項１９から２３のいずれか１項に記載のウェルプレートユニット。 24. The well plate unit according to any one of claims 19 to 23, wherein a thermal conductivity of a material constituting the heat transfer plate is higher than a thermal conductivity of a material constituting the container.
  25. 　前記光透過部は、波長４００～１２００ｎｍの範囲のうちのいずれかの波長の光を透過する請求項１９から２４のいずれか１項に記載のウェルプレートユニット。 The well plate unit according to any one of claims 19 to 24, wherein the light transmitting portion transmits light having any wavelength within a wavelength range of 400 to 1200 nm.
  26. 　ゲルと微粒子とを含む媒体と、前記媒体上に配置された細胞とをウェル内に有する容器を準備する工程と、
    　前記ウェルの底面を介して前記媒体に光を照射し、前記ゲルの少なくとも細胞に接触する領域をゾル化する工程と、
    　を有する細胞剥離方法。     Preparing a container having a medium containing a gel and fine particles and cells arranged on the medium in a well;
    Irradiating the medium with light through the bottom of the well, and solating at least a region of the gel that contacts the cells;
    A cell detachment method comprising:
PCT/JP2017/021209 2016-06-27 2017-06-07 Heat transfer plate, well plate unit and device for stripping cells WO2018003443A1 (en)

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