WO2013024817A1 - Dispositif d'expansion planaire de cellules, et procédé d'expansion de cellules mettant en œuvre celui-ci - Google Patents

Dispositif d'expansion planaire de cellules, et procédé d'expansion de cellules mettant en œuvre celui-ci Download PDF

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Publication number
WO2013024817A1
WO2013024817A1 PCT/JP2012/070497 JP2012070497W WO2013024817A1 WO 2013024817 A1 WO2013024817 A1 WO 2013024817A1 JP 2012070497 W JP2012070497 W JP 2012070497W WO 2013024817 A1 WO2013024817 A1 WO 2013024817A1
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WIPO (PCT)
Prior art keywords
cell
groove
cells
plane
width
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PCT/JP2012/070497
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English (en)
Japanese (ja)
Inventor
淳吾 荒木
久美子 星
恒子 千代田
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コニカミノルタホールディングス株式会社
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Priority to JP2013529006A priority Critical patent/JP5929915B2/ja
Publication of WO2013024817A1 publication Critical patent/WO2013024817A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions

Definitions

  • the present invention relates to a cell plane deployment device and a cell deployment method using the cell plane deployment device. More specifically, the present invention relates to a cell plane deployment device in which a specific groove is engraved on the surface thereof, and a cell plane deployment device capable of closely aligning cells along the groove, and the cell plane deployment device The present invention relates to a cell expansion method using the.
  • Cytology which is one of the means for applying cell information to medicine, includes peel cytology and abrasion cytology, and puncture cytology that more actively collects lesion cells.
  • a preparation is prepared by performing a series of operations described above, and it is microscopically examined with a microscope or the like to diagnose a lesion.
  • the cell suspension containing the target cells is separated to some extent under conditions such as differences in cell weight and cell size, making it easier to find the target cells, and then observing the separated cell population
  • a method for examining the presence or absence of target cells has been attempted.
  • Patent Documents 1 and 2 impart physical / chemical and / or physiological activity to at least cancer cells contained in a cell dispersion such as blood.
  • a cell treatment device that can be used is disclosed.
  • this cell treatment device has a first surface 301 and a second surface 302, and has a slit shape that penetrates from the first surface 301 to the second surface 302 and extends along one direction.
  • the slit member 300 is formed with 310.
  • the through hole 310 of the slit member 300 has a tapered portion 312, and the width in a cross section orthogonal to the direction in which the tapered portion 312 is formed decreases from the first surface 301 toward the second surface 302 side. And it is smaller than the average diameter of the cancer cell g at the end (width Wte) on the second surface 302 side. Cells other than cancer cells g contained in the cell dispersion are passed from the first surface 301 side to the second surface 302 side through the through hole 310.
  • CTC circulating cancer cells
  • a plurality of grooves 104 having the same size are arranged on the bottom surface 106.
  • a flow channel member 102 is prepared, and blood is allowed to flow as a cell suspension 150 from one side 110 to the other side 112 so as to cross the plurality of grooves 104 of the flow channel member 102.
  • the red blood cells 150A in the blood are captured in the blood 104, and the blood is separated into red blood cells 150A and other tissue-derived stem cells 150B.
  • the cell treatment device 200 disclosed in Patent Document 4 includes a plurality of columns 202 arranged in a line and an interval between the columns 202 and 202.
  • a container 208 composed of two upper and lower members 204 and 206 that are gradually narrowed for each row, and the distance between the column 202 and the column 202 from the one side 210 where the distance between the column 202 and the column 202 is the largest.
  • cells larger than the interval between the pillars 202 and 202 are captured between the pillars 202 and 202, and large cells are captured.
  • the cells are separated from 250A in the order of small cells 250C.
  • Reference numeral 250B in the figure is a cell having a size between the large cell 250A and the small cell 250C.
  • Reference numeral 214 denotes an inlet for supplying the cell suspension into the container.
  • JP 2010-227011 A JP 2010-227087 A JP 2008-212022 A US Patent Application Publication No. 2006/0051265
  • the cell treatment devices described in Patent Documents 1 and 2 are for imparting at least one of a physical action, a chemical action, and a physiological action to cancer cells and immune cells. It is not a device optimized for the purpose of observing a plurality of target cells including cancer cells by spreading the suspension in a flat shape.
  • the cell processing device is required to form the through-hole 310 having a width smaller than the “average diameter” of the cancer cells g, but the diameter of a rare CTC that is particularly important for cytology is larger than the average diameter. If it is small, there is a risk that it will be lost through such a through hole 310.
  • the cell suspension basically passes through the tapered portion 312 on the cell treatment device using a liquid delivery system such as a pump, and the cell suspension is treated with the cell without using the liquid delivery system.
  • a liquid delivery system such as a pump
  • Non-Patent Document 1 discloses a method of observing cells on an analog record board in which a plurality of grooves are carved, but there is no description or suggestion of the concept of controlling the width of the grooves.
  • the cell processing device 100 disclosed in Patent Document 3 described above is an apparatus for separating the tissue-derived stem cells 105B from the blood that is the cell suspension 150, but only the unnecessary red blood cells 150A are simply placed in the grooves 104.
  • tissue-derived stem cells 105B In order to observe tissue-derived stem cells 105B from a group of tissue-derived stem cells 150B that also contain leukocytes such as leukocytes. In such a case, it is necessary to first develop a group of tissue-derived stem cells 150B on, for example, a glass substrate and observe each cell of the cell population, and eventually identify the tissue-derived stem cells 105B that are target cells accurately. It was necessary to repeat a complicated process until it was done.
  • the large cells 250A are contained in the cell suspension. If there are many, clogging will occur and smaller cells 250B and 250C will not flow to the downstream side in some cases, and the cells may not be separated accurately.
  • the present invention separates a target cell and a cell having a diameter smaller than that when a cell suspension composed of a plurality of types is spread on a plane, and the target cell and a diameter of the same size as the target cell.
  • An object of the present invention is to provide a cell plane development device capable of closely aligning cells in a plane and a cell deployment method using the same.
  • the present invention was invented in order to achieve the problems and objects in the prior art as described above,
  • the cell plane deployment device of the present invention A cell plane deployment device having at least one groove capable of spreading target cells contained in a cell suspension in a plane,
  • the maximum width of the opening of the groove is equal to or larger than the diameter of the target cell
  • the groove has a width smaller than the diameter of the target cell in the depth direction so that the target cell can be held in the middle of the depth direction
  • the groove has a slit having a width of 1 ⁇ m or less or a hole having a diameter of 1 ⁇ m or less at the bottom of the groove, or does not have them.
  • the target cell is separated from cells and dust having a smaller diameter than the target cell, and the target cell and a cell having the same diameter can move and align along the groove.
  • the groove has a slit having a width of 0.4 ⁇ m or more and 1 ⁇ m or less at the bottom thereof.
  • the bottom of the groove is closed.
  • This configuration is preferable because it is easy to manufacture and inexpensive.
  • the capillary force can be increased as compared with the case where slits are formed, which is preferable in expanding cells.
  • the cell plane deployment device of the present invention When the groove is a tapered type having an inclined surface, the inclination angle is 45 degrees or less.
  • Such a configuration facilitates formation of grooves in which the target cells and non-target cells are properly aligned and retained.
  • the depth of the groove is smaller than twice the diameter of the target cell.
  • the maximum width of the groove opening or the distance between the peaks is not excessively widened, and a sufficient number of cells that can be processed with one cell plane deployment device can be secured and the capillary force is also easy to work. This is preferable.
  • the surface of the groove has a contact angle with water of 20 degrees or less.
  • Such a configuration is preferable from the viewpoints of, for example, that the cell suspension deployment speed is high and that the cell suspension can be prevented from rising (increasing thickness) due to surface tension more than necessary.
  • the cell plane deployment device of the present invention has a ceiling structure at a height of 20 ⁇ m to 100 ⁇ m from the upper surface of the opening of the groove.
  • the cell plane deployment device of the present invention A cell plane deployment device used when spreading cells of various sizes mixed in a cell suspension into a planar shape,
  • the cell plane deployment device It consists of a plate-shaped main body with a cell spreading part on the upper surface, In the cell deployment part of the main body part, A plurality of grooves are juxtaposed in a row from the one side to the other side of the main body, and the plurality of grooves are composed of a plurality of groove groups having different groove widths.
  • the cell plane deployment device is configured in this way, when the cell suspension is caused to flow into the cell deployment portion of the cell plane deployment device, cells of various sizes in the cell suspension are It can be deployed in alignment in a groove of a size that fits.
  • the plurality of groove groups are A groove having the same groove width is juxtaposed for each groove group.
  • the plurality of groove groups are The groove width is set so as to gradually increase for each groove group from one side of the main body portion to the other side.
  • the cells can be surely expanded in the groove in the order of the size of the cells.
  • the plurality of groove groups are The groove depth is gradually increased as the groove width increases.
  • the cells can be reliably held in the groove.
  • the groove width is in the range of 1 to 100 ⁇ m
  • the groove depth is in the range of 1 to 100 ⁇ m.
  • Such a size can reliably sort large cells to small cells according to cell size.
  • the groove has a V-shaped cross section.
  • the main body is formed by arranging a plurality of divided plate-like bodies in parallel.
  • segmentation plate-shaped body can be provided in combination suitably according to the number and the magnitude
  • the plurality of divided plate-like bodies are For each of the divided plate-like bodies, one kind of groove group composed of a plurality of grooves having the same groove width and the same groove depth is provided in parallel.
  • the cell suspension is a biological sample.
  • Such a biological sample is suitable for use in a test for cells in medicine, for example, a test for rare cells such as cancer cells in blood.
  • the cell expansion method of the present invention comprises: It includes a step of adding the cell suspension to the groove of the cell plane development device according to any one of the above.
  • the cell deployment method using such a cell plane deployment device not only when the cell suspension is added directly to the site where the groove of the cell plane deployment device is formed, but also the cell suspension is suspended at the site communicating with the groove. Even when the suspension is added, the cells can be aligned by spreading the cell suspension into the groove by capillary force.
  • the cell expansion method of the present invention comprises: In the step of contacting the cell suspension, The cell suspension is supplied into the groove so as to cross the plurality of groove groups from one side to the other side of the main body of the cell plane deployment device, and the cells in the cell suspension are reduced in size. Expanding the cells in order from the largest cells to the larger cells; It is characterized by having at least.
  • the cells in the cell suspension can be successively deployed in the groove of the cell plane deployment device in the order of small cells to large cells.
  • the cell expansion method of the present invention comprises: After the step of aligning and expanding the cells in the cell suspension in the groove from the small cells to the large cells, Furthermore, a step of observing the cells expanded in a planar shape with an observation means, It is characterized by having.
  • the cells in the cell suspension are deployed in the groove of the cell plane deployment device in order from the small cell to the large cell in the previous process. In this state, the cells can be observed.
  • target cells and cells and dust having a smaller diameter are separated from each other, and target cells and cells having a diameter equal to that are separated into the grooves. It is possible to provide a cell plane deployment device that moves and aligns along the cell and a cell deployment method using the same.
  • the ceiling structure is formed at a height of about twice the cell diameter at the upper part of the groove of the cell plane deployment device, the capillary force acts on the cells across the plurality of grooves, and a cell is used using a pump. Even if the suspension is not fed, a large amount of cells can be easily observed and aligned on the surface of the device.
  • the target cells in the cell suspension can be aligned in a plane, the cells can be observed in this state.
  • FIG. 1 is a perspective view of a cell plane deployment device of the present invention.
  • FIG. 2 is a cross-sectional view taken along line AA of the cell plane development device of the present invention shown in FIG.
  • FIG. 3 is a perspective view of a divided plate-like body of the cell plane deployment device of the present invention.
  • FIG. 4 is a perspective view for explaining a modification of the cell plane deployment device of the present invention.
  • FIG. 5 is a schematic diagram of a cross section of the cell plane development device of the present invention, wherein the groove formed on the surface is a tapered type (a) or a parallel type (b), and the target cell and the diameter of the target cell are shown. This is an embodiment in which smaller cells (non-target cells) are in contact with the groove.
  • FIG. 1 is a perspective view of a cell plane deployment device of the present invention.
  • FIG. 2 is a cross-sectional view taken along line AA of the cell plane development device of the present invention shown in FIG.
  • FIG. 3 is
  • FIG. 6 is a schematic diagram of a cross section of the cell plane deployment device of the present invention, where the groove formed on the surface is a tapered type (a) or a tapered type (a ′) having a flat peak, or The case of the parallel type (b) is shown.
  • FIG. 7 is a perspective view for explaining a modification of the cell plane deployment device of the present invention.
  • FIG. 8 is a state diagram showing a state in which cells are expanded in the groove of the cell plane expansion device of the present invention.
  • 9A and 9B show images obtained in Examples and Comparative Examples.
  • FIG. 9A shows Example 1
  • FIG. 9B shows Example 2
  • FIG. 9C shows Example 3
  • FIG. d) corresponds to Comparative Example 1.
  • FIG. 9A shows Example 1
  • FIG. 9B shows Example 2
  • FIG. 9C shows Example 3
  • FIG. d corresponds to Comparative Example 1.
  • FIG. 9A shows Example 1
  • FIG. 9B shows Example 2
  • FIG. 9C shows Example 3
  • FIG. 10 is an explanatory diagram of a cell plane deployment device according to an embodiment of the present invention
  • FIG. 10 (a) is a top view of the cell plane deployment device
  • FIG. 10 (b) is a sectional view of a groove of each divided plate-like body
  • FIG. 10 (c) is a view showing a state in which cells are developed in the grooves of each divided plate-like body.
  • FIG. 11 is the same as FIG. 5 of Patent Documents 1 and 2, and is a schematic cross-sectional view of the cell treatment device disclosed in Patent Documents 1 and 2.
  • 12A and 12B show a conventional cell treatment device, in which FIG. 12A is a perspective view and FIG. 12B is an explanatory view for explaining a cell separation state.
  • FIG. 13 shows a conventional cell processing device
  • FIG. 13 (a) is a perspective view
  • FIG. 13 (b) is an explanatory diagram for explaining a cell separation state.
  • a cell plane development device and a cell deployment apparatus using the same closely align various cells in a cell suspension in a planar shape for each size, and keep the aligned cells as they are. It can be observed.
  • deployment method which concerns on this invention and its embodiment is a method of aligning the various cells in a cell suspension reliably planarly for every magnitude
  • the “groove group” refers to a group of grooves having the same groove width, but includes a case where the groove group is composed of only one groove.
  • a cell deployment device 10 as shown in FIG. 1 and a liquid supply means (a cell suspension 30 for supplying a cell suspension 30 onto the cell plane deployment device 10) (Not shown).
  • the cell plane deployment device 10 includes a plate-like main body 12, and a plurality of grooves 16 are arranged in a row in the cell development part 14 on the main body 12.
  • the structure of the cell plane deployment device 10 may be any structure as long as grooves having the same dimensions, that is, grooves having the same width and depth are arranged in a plurality of rows.
  • the groove structure adopts a characteristic structure as described below.
  • the cell flat surface deployment device 10 of the present invention shown in FIG. 1 is composed of a plate-shaped main body 12, and the upper surface of the main body 12 has a cell deployment section 14 for deploying a cell suspension 30. It has become.
  • a plurality of grooves 16 are arranged side by side from the one side 20 to the other side 22 of the main body part 12.
  • the grooves 16a, 16b, and 16c have different groove widths and groove depths, and a group of grooves having the same dimensions constitute one groove group.
  • the group of grooves 16a constitutes a groove group 18A
  • the group of grooves 16b constitutes a groove group 18B
  • the group of grooves 16c constitutes a groove group 18C.
  • the cell plane deployment device 10 has a cross section that gradually increases in size (groove width and groove depth) from the one side 20 toward the other side 22 (from the right side to the left side in the drawing).
  • the three groove groups 18A, 18B, and 18C are arranged side by side so as to increase.
  • such a cell plane development device 10 creates individual divided plate-like bodies 12A, 12B, and 12C for each groove group 18 having different groove sizes. It is preferable that the main body portion 12 is configured by providing a plurality thereof in parallel.
  • the grooves of the divided plate-like bodies 12A, 12B, and 12C can be processed by a known fine processing technique, but for example, a commercially available prism sheet can be substituted.
  • the prism sheet is formed by arranging grooves of the same size in a plurality of rows, and is suitable for the divided plate-like body of the present invention.
  • the size (area) of the divided plate-like bodies 12A, 12B, and 12C can be changed in accordance with the ratio.
  • the size of the cell plane deployment device 10 of the present invention is not particularly limited, and the size can be appropriately adjusted in consideration of the number of cells to be deployed.
  • a maximum of a plurality of divided plate-like bodies 12A, 12B, and 12C can be combined to have a size of about A5 (150 mm ⁇ 210 mm).
  • the cell plane deployment device 10 when the cell plane deployment device 10 is about A5 (150 mm ⁇ 210 mm), about 10 ml of blood (cell suspension 30) is densely packed in the plurality of grooves of the cell deployment section 14 of the cell plane deployment device 10. Can be aligned.
  • the amount of cells in the cell suspension 30 is large, it may be necessary to make the cell plane deployment device 10 larger than A5 (150 mm ⁇ 210 mm). It is also possible to divide the divided plate-like bodies 12A, 12B, and 12C and observe one plate-like body at a time.
  • such a cell plane deployment device 10 is much larger than a standard size (about 25 mm ⁇ 75 mm) used for a conventional cell observation device such as a glass slide,
  • the cells 30A, 30B, and 30C can be observed, and the inspection accuracy can be improved by increasing the number of cells to be detected.
  • any method can be used as long as it can be ensured that the gap between the divided plate-like bodies and the divided plate-like bodies does not open. It ’s good.
  • a frame member (not shown) is prepared in advance, and the divided plate-like bodies 12A, 12B, 12C are fitted into the frame member (not shown). Examples include a method of bonding the plate-like body 12A and the divided plate-like body 12B with a removable adhesive (not shown).
  • the groove 16 is preferably tapered from the opening 50 to the entire bottom 52, or at least in the vicinity of the opening 50.
  • the groove 16 may have any shape even if it is not tapered. Can be.
  • the bottom 52 side having a width smaller than the diameter of the target cell 60 and deeper than the portion where the target cell 60 is held may also be a tapered type or a parallel type as shown in FIG. Or any shape.
  • the shape of the bottom 52 of the groove 16 when the groove 16 is viewed in a cross section, even if it is an inverted triangle (FIG. 6A), a semicircle (FIG. 6B) or an arc is drawn. Also, it may be rectangular and is not particularly limited.
  • a slit may be formed at the bottom 52 (near the valley) of the groove 16. Further, the side surface of the groove 16 may be a straight line, a broken line, or a curved line when viewed from the cross section of the groove 16, and is not particularly limited.
  • the maximum width T of the opening 50 of the groove 16 is usually equal to the diameter of the target cell 60 or larger than the diameter of the target cell 60.
  • the target cell 60 is held substantially at the position of the opening 50 in both the tapered type and the parallel type.
  • the target cell 60 enters the groove 16, and the target cell 60 is held at a position in the middle from the position of the opening 50 to the bottom.
  • the cell is the target cell 60 regardless of whether the bottom side is tapered or parallel to the position where the target cell 60 is held. It is held at the bottom side or the bottom (the bottom surface of the groove 16).
  • the target cell 60 can be moved and aligned along the groove 16 together with the cell suspension 30 developed by the capillary force acting between the walls of the groove 16.
  • the maximum width of the opening 50 is smaller than the diameter of the target cell 60, a part of the target cell 60 may enter the groove 16, but generally above the opening 50 (opposite to the bottom). Will be held in the position. As a result, the target cell 60 becomes difficult to move together with the cell suspension 30, or the target cell 60 may be moved to the adjacent groove 16 beyond the crest 54 that is the boundary of the groove 16. It is not preferable in order to align.
  • the “diameter of the target cell 60”, which serves as a reference for the width of the groove 16, can be set as “average diameter of the target cell 60” if necessary, if sufficient data on the diameter of the target cell 60 is obtained in advance.
  • “Average diameter of target cell 60 + 3 ⁇ ” ( ⁇ is a standard deviation), and the like, and the cell plane deployment device 10 having the groove 16 having a width based on the reference can be manufactured. The same applies to the “diameter of the target cell 60” related to the slit width and the depth H of the groove 16, which will be described later.
  • the cell plane deployment device 10 can target the cell suspension 30 including the target cell 60 and one or more types of non-target cells 70 other than the target cell whose average diameter is smaller than the target cell 60.
  • the width of the groove 16 is The target cell 60 is held at a site closer to the opening 50 of the groove 16, and It is preferable that the non-target cell 70 is held at a portion closer to the bottom of the groove 16 or the non-target cell 70 is held in a state of being in contact with the bottom (valley) of the groove 16.
  • the target cell 60 having a larger diameter is grounded to the side surface near the uppermost portion of the groove 16, while the non-target cell 70 having a smaller diameter is positioned from below the target cell 60 to the lowermost portion of the groove 16.
  • the groove shape is preferably such that the distance between the lower end of the target cell 60 and the upper end of the non-target cell 70 does not contact.
  • the inclination angle ⁇ is preferably 45 degrees or less.
  • the inclination angle ⁇ is in such a range, it becomes easy to form the groove 16 in which the target cell 60 and the non-target cell 70 are properly aligned and held.
  • the maximum width T of the opening 50 of the groove 16 or the distance between the peaks 54 and 54 is not excessively widened, and one cell plane It is preferable that the number of cells that can be processed by the deployment device 10 can be sufficiently secured and that the capillary force can be easily worked.
  • a horizontal or inclined top surface (upper surface of the opening 50) may not be formed between the grooves 16 (in this case, peaks).
  • 54 is the maximum width T of the opening 50), and the top surface may be formed as shown in FIGS. 6A 'and 6B (in this case, the peak The distance between 54 and the crest 54 is greater than the maximum width T of the opening 50).
  • a tapered type or a flat surface as shown in FIG. 8B may be formed.
  • the width depends on the density of the grooves 16 on the cell plane deployment device 10 (that is, the number of cells that can be processed), the degree of alignment of the target cells 60 in adjacent grooves, and the like. And can be adjusted as appropriate.
  • the depth H is preferably smaller than twice the diameter of the target cell 60.
  • the size, thickness, and material of the cell plane deployment device 10 of the present invention are not particularly limited as long as the size, thickness, and material can be observed under a microscope.
  • deployment device 10 the prism sheet etc. which have a magnitude
  • prism sheet (tip angle 90 degrees, pitch 0.02, width (pitch direction) 300 ⁇ length 300, manufactured by Organic Optical Co., Ltd.”, standard plate thickness 2 mm) "is preferable.
  • the cell plane development device 10 of the present invention for example, arranges an ultraviolet curable resin in a mold having a prism shape, arranges a transparent weight (for example, glass) or a transparent substrate on the ultraviolet curable resin, It can be produced by a method of irradiating light having a curing wavelength of the ultraviolet curable resin from the weight or the upper surface of the transparent substrate to cure the ultraviolet curable resin (see JP-A-2005-31658).
  • the surface of the groove 16 included in the cell plane development device 10 of the present invention has a contact angle with respect to water of 20 degrees or less, for example, by performing a hydrophilic treatment.
  • the contact angle of water on the surface of the cell plane development device 10 with respect to water can be achieved by a known hydrophilization treatment such as a treatment using a UV ozone cleaner or the like.
  • the cell suspension 30 When the contact angle of the surface of the cell plane deployment device 10 with respect to water is within such a range, the cell suspension 30 is developed at a high speed, and the cell suspension 30 rises due to surface tension more than necessary (the thickness is increased). From the viewpoint of being able to suppress the increase).
  • the cell suspension 30 includes one type of 60, and may further include one or more types of non-target cells 70 other than the target cell 60.
  • Examples of such cell suspension 30 include body fluids of animals such as humans, that is, blood, lymph fluid, tissue fluid, body cavity fluid, and the like. Moreover, it is not limited to the thing derived from a living body, The dispersion liquid of the cell prepared by suspending the cell artificially for a test, research, etc. may be sufficient.
  • the cell suspension 30 preferably contains cancer cells as the target cells 60.
  • cells other than cancer cells may be dispersed as non-target cells 70.
  • the cell suspension 30 may contain immune cells.
  • immune cells such as white blood cells (immune cells), red blood cells, and platelets (non-target cells 70) may be dispersed as in blood.
  • the cancer cell refers to a cell constituting cancer (malignant tumor). Cancer has the property of metastasizing, and cancer cells are mixed into body fluids such as blood described above when the cancer metastasizes. Cancer cells that are mixed in blood and can circulate in the living body are called circulating cancer cells [CTC] (or circulating tumor cells).
  • CTC circulating cancer cells
  • the cell suspension 30 may be blood containing circulating cancer cells.
  • immune cells refer to leukocytes, that is, cells such as granulocytes, lymphocytes, and monocytes.
  • Optical micrographs were taken of multiple types of circulating cancer cells, and the average projected area of various circulating cancer cells was determined to be 396 ⁇ m 2 to 796 ⁇ m 2 . Therefore, assuming that the shape of various circulating cancer cells is a sphere, the diameter of the circulating cancer cells is calculated to be 22 to 32 ⁇ m.
  • the slit refers to a hole extending long in the bottom 52 (near the valley) along the longitudinal direction of the groove 16 and can discharge a substance unnecessary for cell observation.
  • the cell plane development device 10 of the present invention may have such slits and holes as necessary.
  • the capillary force may be slightly weakened, but there is no problem in developing the cell suspension 30.
  • such a slit had a width (length in a direction perpendicular to the longitudinal direction of the groove 16) of 1 ⁇ m or less, and a hole having a diameter of 1 ⁇ m or less.
  • the capillary force can be increased as compared with the case where the slit is formed, which is more preferable in spreading the cells.
  • the slit is preferably formed in a part including a valley (deepest part), but may be formed in a part not including a valley as long as it does not hinder the discharge of unnecessary materials. Moreover, the slit may be formed over the whole bottom part 52 along the longitudinal direction of the groove
  • the length of the slit groove 16 in the longitudinal direction is not particularly limited.
  • the width of the slit is preferably such that the solvent (such as water) of the cell suspension 30 can overcome the surface tension only by the action of its own weight and fall off due to its function.
  • a width may vary depending on the type of the solvent, the material of the cell plane developing device 10, and the surface treatment (hydrophilization treatment), but it is preferable that the lower limit is, for example, 0.4 ⁇ m.
  • the lower limit value of the width of the slit can be made smaller.
  • the slit width is 0.4 ⁇ m or more and 1 ⁇ m or less, the debris such as cell debris is removed from the cell plane deployment device 10 together with the solvent of the cell suspension 30 without losing any cells. It is suitable for observation after deployment.
  • the staining solution can be easily removed when cells are stained with a fluorescent substance or the like during or after expansion.
  • the solvent and the staining solution of the cell suspension 30 are removed, the movement of the target cell 60 stops and the target cell 60 can be observed in a stationary state.
  • the cell suspension 30 uses a pump or the like due to the force acting between the groove 16 and the wall surface of the groove 16 even when the groove 16 has no ceiling structure and is open.
  • the target cell 60 can be aligned on the surface of the cell plane development device 10 by being developed in the groove 16 without feeding.
  • capillary force acts on the cell suspension 30, so that the cell suspension 30 is deployed more quickly and the target cells 60 are aligned. Will be able to do.
  • This ceiling structure is preferably provided at a height of 20 ⁇ m or more and 100 ⁇ m or less from the upper surface (top surface) of the opening 50, and more preferably at a height of about twice the diameter of the target cell 60.
  • Such a ceiling structure can be formed by, for example, a cover glass through a spacer having a thickness of 20 ⁇ m to 100 ⁇ m.
  • a cell deployment method using the above-described cell plane deployment device 10 will be described below.
  • the cell deployment method of the present invention includes a step of bringing the cell suspension 30 into contact with the groove 16 of the cell plane deployment device 10 of the present invention.
  • the capillary tube is used even when the cell suspension 30 is added to the site communicating with the groove 16.
  • the cells can be aligned by developing the cell suspension 30 in the groove by force.
  • the target cell 60, the non-target cell 70, dust, and the like are separated, and only the target cell 60 is densely planarly formed. Can be aligned.
  • the cell structure of the cell plane deployment device 10 of the present invention has a ceiling structure at a height of about twice the cell diameter, a capillary force acts on the cells, and the cell plane deployment device 10 can be more quickly installed. This is preferable because only the target cells 60 can be aligned on the surface.
  • a developing means such as a spin coater in combination.
  • the target cells 60 After aligning the target cells 60 as described above, the target cells 60 can be observed by focusing the microscope at a position (height) where the target cells 60 are held in the grooves 16. . At this time, even if the non-target cell 70 is present in the cell suspension 30, the observation of the target cell 60 is not hindered because the non-target cell 70 is held on the bottom 52 side of the target cell 60.
  • the cell plane development device 10 of the present invention described above as shown in FIG. 1 is prepared.
  • the cell plane deployment device 10 is configured such that the size of each groove 16 gradually increases from one side 20 to the other side 22 of the main body 12.
  • the cell suspension is directed from one side 20 to the other side 22 of the main body 12 so as to cross the plurality of groove groups 18 of the cell plane developing device 10, preferably so that the plurality of groove groups 18 are orthogonal to each other.
  • 30 is supplied. That is, the cell suspension 30 is supplied to the cell deployment part 14 so as to cross each groove group from the side where the small groove 16a is provided.
  • a bar arrow in FIG. 1 is a flow direction of the cell suspension 30.
  • the liquid supply means (not shown) for the cell suspension 30 is not particularly limited. For example, after the cell plane development device 10 is placed on a flat surface, it is used from one side 20 using a syringe. The cell suspension 30 can be supplied toward the other side 22.
  • the cell plane deployment device 10 is tilted, and the cell suspension 30 is allowed to flow from one side 20 of the main body 12 to the cell deployment unit 14 so that the cells 30A, 30B, and 30C in the cell suspension 30 are developed. You may make it let it.
  • FIG. 10 When the cell plane deployment device 10 is tilted, the cell plane deployment device 10 itself is tilted, and when the cell plane deployment device 10 is placed as shown in FIG. It is good also as a form which becomes the surface 40.
  • FIG. 10 When the cell plane deployment device 10 is tilted, the cell plane deployment device 10 itself is tilted, and when the cell plane deployment device 10 is placed as shown in FIG. It is good also as a form which becomes the surface 40.
  • each cell 30A, 30B, 30C in the cell suspension 30 is a liquid supply means (not shown) that moves across the groove using a gentle flow or its own weight. It does not matter.
  • the preferred flow rate of the cell suspension 30 is 10 to 300 ⁇ l / sec.
  • the cells 30A, 16A, and 16C in the cell suspension 30 are placed in the grooves 16a, 16b, and 16c.
  • 30B and 30C are developed in a planar shape.
  • the small cells 30A are sequentially increased from the small groove 16a to the large groove 16c.
  • a large cell 30C is expanded.
  • both the small cells 30A and the large cells 30C do not enter the large groove 16c, and the cells can be expanded densely while being surely sorted according to size.
  • the cell plane development device 10 in which the cells are developed in this plane is observed with an observation means (not shown).
  • observation means usually used means such as a CCD camera and a microscope can be used.
  • the cell observation can be performed immediately after the cell is expanded, so that the target cell 60 is found from the cell suspension 30 and this is detected.
  • a series of operations until the observation of the target cell 60 can be accelerated compared to the conventional method, and a large number of cells can be developed without omission, so that the detection accuracy of the target cell 60 can be easily increased.
  • the target cell 60 is, for example, a cancer cell in the blood
  • the cell suspension 30 is supplied from the smaller size of the groove 16 to the larger size, but in some cases, the reverse may be possible. is there.
  • the cell suspension 30 is supplied from the smaller one of the grooves 16 of the cell plane deployment device 10 to the larger one. In either case, it is possible to sort the large cells aligned and developed above the cell development part (upper surface side).
  • the groove 16 is formed in the cell plane deployment device 10 until the groove 16 reaches both side surfaces of the main body 12, the cell suspension 30 may flow out of the groove 16.
  • separate walls may be provided on both side surfaces to prevent the cell suspension 30 from flowing out from both ends of the groove 16.
  • deployment device 10 consists of three division
  • a plurality of types of grooves having different sizes may be formed on one divided plate-like body, and the number of divided plate-like bodies and the number of types of groove sizes are equal. You don't have to.
  • Pitch 0.02, width (pitch direction) 300 ⁇ length 300, standard plate thickness 2 mm) ”; taper type) surface was subjected to a hydrophilic treatment using a UV ozone cleaner.
  • the contact angle of the sheet surface with water was 20 degrees.
  • FIG. 9A shows a cell development image acquired under a fluorescence microscope.
  • Example 2 The surface of the prism sheet used in Example 1 was subjected to a surface treatment in the same manner as in Example 1.
  • a cover glass was placed on the surface of the prism sheet through a film having a thickness of 50 ⁇ m as a spacer.
  • FIG. 9B shows a cell development image acquired under a fluorescence microscope.
  • Example 3 In Example 1, as a prism sheet, a prism sheet having a width (pitch) of 15 ⁇ m between the peaks 54 and the peak 54 and an inclination angle ⁇ of 70 degrees (“Prism sheet tip angle 140 degrees, manufactured by Organic Optical Co., Ltd.,” Cells were developed in the same manner as in Example 1 except that pitch 0.015, width (pitch direction) 300 ⁇ length 300, standard plate thickness 2 mm ”(tapered type) was used.
  • FIG. 9C shows a cell development image acquired under a microscope.
  • Example 1 In Example 1, instead of the prism sheet, cells were developed in the same manner as in Example 1 except that a slide glass having no grooves (“Large Slide White Edge Polish No. 1” manufactured by Matsunami Glass Co., Ltd.) was used. did.
  • FIG. 9D shows a cell development image acquired under a microscope.
  • FIGS. 9 (a) to 9 (c) corresponding to Examples 1 to 3 when the cell plane development device 10 having the groove structure of this embodiment is used, the cells are uniform without overlapping. It was confirmed that the flat surface was developed. On the other hand, in FIG. 9D corresponding to Comparative Example 1, it was confirmed that the cells were partially biased and the cells were dense.
  • Example 4 Using peripheral blood collected from a cancer patient, this is developed on the cell plane deployment device 10 of the present invention to detect various cells in the peripheral blood, in particular, blood circulating cancer cells (CTC). went.
  • CTC blood circulating cancer cells
  • the cell suspension 30 here is a peripheral blood itself diluted with an appropriate buffer such as phosphate buffered saline (PBS) so as to be easily developed on the cell plane deployment device 10.
  • PBS phosphate buffered saline
  • a solution diluted with phosphate buffered saline (PBS) was used.
  • CTC blood circulation cancer cells
  • leukocytes were fluorescently labeled in advance.
  • the method for preparing the cell suspension 30 is as follows.
  • phosphate buffered saline PBS
  • PBS phosphate buffered saline
  • Tween 0.1% Tween
  • 1 ml and 10 ⁇ l of Alexa Fluor 647-labeled anti-CK antibody (from Micromet) for CTC labeling are added and allowed to react for 30 minutes in the dark at room temperature with gentle mixing.
  • 10 ⁇ l of DAPI (manufactured by Dojin Chemical Co., Ltd.) solution was added and reacted.
  • the antibody reagent not bound to the cells is removed by centrifugation, and newly added with phosphate buffered saline (PBS) and resuspended, whereby the cell suspension used in this example and did.
  • PBS phosphate buffered saline
  • FIG. 10A In this embodiment, as shown in FIG. 10A, four divided plate-like bodies (12A to 12D) each having a groove group composed of one type of groove are prepared, The cell plane development device 10 in which the four divided plate bodies (12A to 12D) are arranged in the order of the size of the groove 16 was used.
  • grooves 16a having a groove width T of 5 ⁇ m and a groove depth H of 5 ⁇ m are arranged side by side in a plurality of rows.
  • grooves 16b having a groove width T of 10 ⁇ m and a groove depth H of 10 ⁇ m are arranged side by side in a plurality of rows.
  • Grooves 16c having a width T of 15 ⁇ m and a groove depth H of 15 ⁇ m are arranged in a plurality of rows
  • the fourth divided plate 12D has a groove 16d having a groove width T of 20 ⁇ m and a groove depth H of 20 ⁇ m. Are arranged in a plurality of rows.
  • the groove of the divided plate-like body 12A having the groove 16a having a groove width T of 5 ⁇ m and a groove depth H of 5 ⁇ m platelets having a diameter of about 2 to 4 ⁇ m, CTC cell debris reduced to about several ⁇ m, thickness of 2 ⁇ m It is thought that some red blood cells are captured. While normal cells are spherical, erythrocytes have a flat shape with a diameter of 7 to 8 ⁇ m and a thickness of about 2 ⁇ m, so it is considered that they are also captured in the groove 16a of this size.
  • a part of red blood cells having a diameter of 7 to 8 ⁇ m and a thickness of about 2 ⁇ m, and a small lymph of about 6 to 9 ⁇ m.
  • the ball is thought to be captured.
  • lymphocytes having a diameter of about 9 to 15 ⁇ m and granulocytes having a diameter of about 10 to 17 ⁇ m are captured in the grooves of the divided plate-like body 12C having the groove 16c having a groove width T of 15 ⁇ m and a groove depth H of 15 ⁇ m. It is thought that.
  • CTC blood circulation cancer cells
  • the cell plane development device 10 composed of the four divided plate bodies 12A to 12D is tilted, and the size of the groove 16 of the cell plane development device 10 is changed from the small groove 16a to the large groove 16d from the one side 20 to the other.
  • the cell suspension 30 was supplied onto the cell spreading part 14 so that the groove 16 was orthogonal to the side 22, and each cell was spread in the groove.
  • the inclination of the cell plane development device 10 was adjusted so that the flow rate of the cell suspension was about 10 to 300 ⁇ l / sec.
  • a He—Ne laser (excitation wavelength: 633 nm) is applied to the divided plate-like body 12D having a groove 16d having a groove width T of 20 ⁇ m and a groove depth H of 20 ⁇ m located on the most downstream side.
  • Alexa Floor 647 fluorescence bound to CK specifically expressed in blood circulating cancer cells (CTC) was detected, and blood circulating cancer cells (CTC) were identified. did it.
  • the same He—Ne laser (as described above) is used for the divided plate-like body 12A having the groove 16a located on the most upstream side and having a groove width T of 5 ⁇ m and a groove depth H of 5 ⁇ m.
  • an excitation wavelength of 633 nm CTC cell debris was also detected.
  • CTCs blood circulation cancer cells
  • the cell suspension 30 is developed using the cell plane development device 10 of the present invention, cells of different sizes can be surely aligned and sorted in a groove having a size suitable for the cells, and as it is. It was confirmed that it could be observed.
  • non-blood cells existing in peripheral blood include circulating vascular endothelial cells (CEC) and circulating endothelial progenitor cells (CEP), which are generally about 10 to 15 ⁇ m in diameter. Have a size of
  • the divided plate-like body 12B having the groove 16b having the second groove width T of 10 ⁇ m and the groove depth H of 10 ⁇ m and the third groove width T of 15 ⁇ m and the groove depth H of 15 ⁇ m It is considered that these cells can be detected by observing the divided plate-like body 12C having the grooves 16c.
  • CTC blood circulating cancer cells
  • fetal nucleated red blood cells, peripheral blood, bone marrow fluid, tissues in the peripheral blood were also similarly detected. It can be used for detection of rare cells such as stem cells and cells detached in body fluids (ascites, saliva, sweat, urine, feces, spinal fluid, milk, etc.).
  • the cell plane development device of the present invention When cells are observed using the cell plane development device of the present invention, when the cell suspension is, for example, blood (10 mL) collected from a subject, a large number of cells are aligned at high density within a narrow field of view of the microscope. For example, cells with a small number such as cancer cells can be efficiently discovered.
  • Channel member 104 ⁇ groove 106 ⁇ ⁇ ⁇ bottom surface 110 ⁇ ⁇ ⁇ one side 112 ⁇ ⁇ ⁇ other side 150 ⁇ ⁇ ⁇ cell suspension 150A ⁇ ⁇ red blood cell 150B ⁇ ⁇ tissue-derived stem cell 200 ⁇ ⁇ ⁇ cell processing device 202 ⁇ ⁇ ⁇ pillar 204... Member 206... Member 208.
  • One side 212 ... the other side 214 ... the inlets 250A to 250C ... the cell 300 ... the slit member 301 ... the first surface 302 ... the second surface 310 ... the through hole 312 ... Tapered part g ... Cancer cell t ... Thickness dt ... Depth Wte ... Width

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Abstract

L'invention fournit un dispositif d'expansion planaire de cellules ainsi qu'un procédé d'expansion de cellules mettant en œuvre ce dispositif, permettant ainsi, lors de l'expansion planaire d'une suspension de cellules de diverses sortes, de séparer des cellules cibles et des cellules de plus petit diamètre que celles-ci, et d'aligner sous forme de plan et de manière serrée les cellules cibles et les cellules dont le diamètre est équivalent à celles-ci. Plus précisément, l'invention concerne un dispositif d'expansion planaire de cellules permettant une expansion sous forme de plan des cellules cibles contenues dans la suspension de cellules, et possédant au moins une rainure. La largeur maximale d'une face supérieure de partie ouverture de ladite rainure, est égale ou supérieure au diamètre desdites cellules cibles. En outre, ladite rainure est caractéristique en ce qu'afin de maintenir lesdites cellules cibles à mi-chemin dans la direction de sa profondeur, elle possède une largeur inférieure au diamètre desdites cellules cibles dans la direction de sa profondeur, et possède de manière facultative dans sa partie fond une fente de largeur de 1µm au plus, ou un orifice de diamètre de 1µm au plus.
PCT/JP2012/070497 2011-08-18 2012-08-10 Dispositif d'expansion planaire de cellules, et procédé d'expansion de cellules mettant en œuvre celui-ci WO2013024817A1 (fr)

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JPWO2015093420A1 (ja) * 2013-12-16 2017-03-16 コニカミノルタ株式会社 細胞検出方法および細胞検出装置

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