JP7282327B2 - CELL-FIXING DEVICE, CELL-FIXING METHOD, AND CELL-MANIPULATING METHOD - Google Patents

Description

TECHNICAL FIELD The present disclosure relates to cell fixation devices and cell fixation methods.

Patent Documents 1 and 2 disclose devices for fixing cells.

JP-A-2002-27969 WO2009/016842

The device described in Patent Document 1 has fine grooves between the liquid reservoir and the through-hole. When a sample containing cells is put into the liquid reservoir and the through-hole is made negative pressure, the cells are fixed at the inlet of the fine groove on the liquid reservoir side. The device described in Patent Document 2 uses micromesh to fix cells.

The devices described in Patent Documents 1 and 2 collectively fix a plurality of cells when trying to fix minute cells. Therefore, with the devices described in Patent Documents 1 and 2, it is difficult to separate and fix one minute cell from other cells. An object of one aspect of the present disclosure is to provide a cell-fixing device and a cell-fixing method that can separate and fix one minute cell from other cells.

One aspect of the present disclosure is a cell-fixing device for fixing cells, comprising: a first member having a groove formed on its surface; a second member that contacts the first member and covers the groove; and the groove. a channel formed by the second member; and a plurality of holes penetrating the first member or the second member and communicating with the channel, wherein the channel comprises the plurality of holes. a first channel extending from a portion communicating with a first hole, which is a part, to a portion communicating with a second hole, which is a part of the plurality of holes; and a second channel extending to a portion communicating with one of the holes, wherein the second channel has a narrower width than the first channel and includes a bottleneck portion capable of fixing the cells A cell fixation device.

Using the cell fixing device according to one aspect of the present disclosure, one minute cell can be separated and fixed from other cells.
Another aspect of the present disclosure is a cell fixation method of fixing cells using a cell fixation device. The cell fixing device includes a first member having grooves formed on its surface, a second member that contacts the first member and covers the groove, and a channel formed by the groove and the second member. , and a plurality of holes passing through the first member or the second member and communicating with the flow path.

a first flow channel extending from a portion communicating with a first hole, which is a part of the plurality of holes, to a portion communicating with a second hole, which is a part of the plurality of holes; a second flow path branched from the flow path and extending to a portion communicating with a third hole, which is one of the plurality of holes. The second channel has a narrower width than the first channel, and includes a bottleneck portion capable of fixing the cells.

In the cell fixing method, which is another aspect of the present disclosure, the liquid containing the cells is supplied to the first hole and sucked from the second hole and the third hole, whereby the liquid containing the cells is Flow from the first flow path to the second flow path stops the suction.
Using a cell fixation method that is another aspect of the present disclosure, one minute cell can be separated and fixed from other cells.

Fig. 2 is a perspective view showing the configuration of the cell fixing device 1 in which the first member 3 and the second member 5 are separated. 3 is a plan view showing the configuration of a first groove 9 and second grooves 11 and 13. FIG. Fig. 3 is a cross-sectional view of the cell fixing device 1 cut along the III-III cross section in Fig. 2; Fig. 3 is a cross-sectional view of the cell fixing device 1 cut along the IV-IV cross section in Fig. 2; FIG. 5A is an explanatory diagram showing the branch channel 43A to which the cells 45 are not fixed, and FIG. 5B is an explanatory diagram showing the branch channel 43A to which the cells 45 are fixed. It is a photograph showing 43 A of branch flow paths which fixed the cell. It is explanatory drawing showing the structure of 43 A of branch flow paths. FIG. 8A is an explanatory diagram showing a state in which a plurality of cells 45 with a size of W1 or larger are fixed to the upstream portion 49, and FIG. 8B shows a single cell 45 with a size of W1 or larger fixed to the upstream portion 49. FIG. 8C is an explanatory diagram showing a state in which one cell 45 whose size is smaller than W1 and equal to or larger than W2 is fixed to the downstream portion 51. FIG.

Exemplary embodiments of the present disclosure are described with reference to the drawings.
<First Embodiment>
1. Configuration of Cell Fixation Device 1 The configuration of the cell fixation device 1 will be described with reference to FIGS. 1 to 4. FIG. The cell fixing device 1 is a device used for fixing cells. As shown in FIG. 1, the cell fixation device 1 includes a first member 3 and a second member 5. As shown in FIG.

The first member 3 is a plate-shaped member that is rectangular in plan view. The first member 3 is made of polydimethylsiloxane (PDMS). The elastic modulus of the first member 3 is smaller than the elastic modulus of the cells to be fixed.

As shown in FIG. 1, the direction parallel to the short sides of the first member 3 is defined as the X direction. The direction parallel to the long side of the first member 3 is the Y direction. The thickness of the first member 3 is 0.5 mm. Note that the thickness of the first member 3 is preferably 1.0 mm or less.

As shown in FIG. 1, one surface 7 of the first member 3 is formed with a first groove 9 and second grooves 11 and 13 . The first groove 9 and the second grooves 11 and 13 are portions of the surface 7 that are recessed more than other portions. The first groove 9 is a linear groove parallel to the Y direction. The first groove 9 has an extended portion 15 at its Y-direction end. In addition, the first groove 9 has an extended portion 17 at the end on the side opposite to the Y direction. The expanded portions 15 and 17 are portions expanded in the XY plane more than other portions (hereinafter referred to as main body portion 19) of the first groove 9. As shown in FIG. The width and depth of the body portion 19 are constant at any position.

As shown in FIG. 2, the second groove 11 includes 16 branch portions 21, 8 primary confluence portions 23, 4 secondary confluence portions 25, 2 tertiary confluence portions 27, and 1 and a book quaternary junction 29 . The 16 branch portions 21 each branch from the main body portion 19 and extend along the X direction. The 16 branches 21 are arranged along the Y direction. The interval between two adjacent branch portions 21 is constant regardless of which two branch portions 21 are selected. The interval between two adjacent branches 21 is 500 μm or more. The branch portion 21 is narrower than the main body portion 19 . The branch portion 21 is shallower than the main body portion 19 . The width and depth of the bifurcation 21 are adjusted so that cells can be fixed. The width of branch 21 is preferably 80 to 99% of the width of the cells to be fixed.

Each of the eight primary confluence portions 23 is a portion where the two branch portions 21 merge. The primary confluence portion 23 extends along the X direction. The width and depth of the primary confluence portion 23 are greater than the width and depth of the branch portion 21 .

Each of the four secondary confluences 25 is a portion where the two primary confluences 23 are merged. The secondary confluence 25 extends along the X direction. The width and depth of the secondary confluence portion 25 are greater than the width and depth of the branch portion 21 .

Each of the two tertiary confluences 27 is a portion where the two secondary confluences 25 merge. The tertiary junction 27 extends along the X direction. The width and depth of the tertiary confluence portion 27 are greater than the width and depth of the branch portion 21 .

One quaternary junction 29 is a portion where two tertiary junctions 27 join. The quaternary confluence portion 29 reaches the suction portion 31 while bending. The suction portion 31 is a portion where the second grooves 11 and 13 join. The suction portion 31 is a portion recessed from the surrounding surface 7 . The suction portion 31 is located at a position moved in the direction opposite to the Y direction from the extension portion 17 . The suction section 31 is expanded in the XY plane compared to the quaternary junction section 29 .

The second groove 13 has a shape that is symmetrical with the second groove 11 with the body portion 19 as the axis of symmetry. A fourth junction 29 provided in the second groove 13 also reaches the suction portion 31 .
The second member 5 is a plate-shaped member that is rectangular in plan view. The size of the second member 5 in the X direction and the Y direction is the same as that of the first member 3 . The material of the second member 5 is the same as the material of the first member 3 . Therefore, the elastic modulus of the second member 5 is smaller than the elastic modulus of the cells to be fixed.

The second member 5 has a first hole 33 , a second hole 35 and a third hole 37 . The first hole 33, the second hole 35, and the third hole 37 each penetrate the second member 5 in the thickness direction. The shapes of the first hole 33, the second hole 35, and the third hole 37 are circular in plan view. The first holes 33, the second holes 35, and the third holes 37 are arranged in that order along the Y direction. The interval between the first hole 33 and the second hole 35 is wider than the interval between the second hole 35 and the third hole 37 . The first hole 33, the second hole 35, and the third hole 37 correspond to multiple holes.

One surface 39 of the second member 5 can abut the surface 7 of the first member 3 . Surface 39 is planar except for first hole 33 , second hole 35 and third hole 37 . At this time, the first groove 9 is covered by the surface 39 except for the extensions 15,17.

As shown in FIG. 3 , the first groove 9 and the surface 39 form a first flow path 41 . The first channel 41 is a channel with a closed periphery. The first hole 33 communicates with the extended portion 15 . The second hole 35 communicates with the extended portion 17 . Therefore, a path is formed from the outside of the cell-fixing device 1 to the outside of the cell-fixing device 1 through the first hole 33, the expanded portion 15, the first channel 41, the expanded portion 17, and the second hole 35 in order. be done.

The width of the first flow path 41 is preferably 50 μm or more and 5 mm or less. The width of the first channel 41 is the width in the X direction. When the width of the first channel 41 is 50 μm or more, the cells easily flow through the first channel 41 and are easily sucked into the branch channels 43A described later. When the width of the first flow channel 41 is 5 mm or less, the flow of cells in the first flow channel 41 is moderately slowed down, making it easier for the cells to access the branch flow channel 43A.

When the surface 39 of the second member 5 is brought into contact with the surface 7 of the first member, the second grooves 11 and 13 are covered with the surface 39 except for the suction portion 31 . As shown in FIGS. 3 and 4 , the second grooves 11 and 13 and the surface 39 form a second flow path 43 . The second channel 43 is a channel with a closed periphery. The second flow path 43 branches off from the first flow path 41 and extends to the suction portion 31 .

A portion of the second flow path 43 formed by the branch portion 21 and the surface 39 is referred to as a branch flow path 43A. A plurality of branch flow paths 43A are present. The plurality of branch flow paths 43A are flow paths branched at a plurality of positions in the first flow path 41, respectively. 43 A of branch flow paths are narrower than the 1st flow path 41. As shown in FIG. 43 A of branch channels are parts which fix a cell. 43 A of branch channels correspond to the bottleneck part which can fix a cell. The width of the branch flow path 43A is the same as the width of the branch portion 21 . The width of the branch channel 43A is preferably 80 to 99% of the width of the cells to be fixed.

A portion of the second flow path 43 formed by the primary confluence portion 23, the secondary confluence portion 25, the tertiary confluence portion 27, the quaternary confluence portion 29, and the surface 39 is referred to as a confluence flow path 43B. The confluence flow path 43B is a flow path in which a plurality of branch flow paths 43A are merged.

The third hole 37 communicates with the suction portion 31 . Therefore, a path is formed from the first flow path 41 to the outside of the cell fixation device 1 through the branch flow path 43A, the confluence flow path 43B, the suction portion 31, and the third hole 37 in sequence.

2. Example (2-1) Preparation of Cell Suspension Coconut KJ strain was cultured under aerobic conditions. The culture conditions were as follows.

Medium: MA5 medium Light-dark period: 12/12 hours _CO2 concentration: 2%
Cocomixa KJ strain was granted accession number FERM P to the National Institute of Technology and Evaluation Patent Organism Depository (NITE-IPOD) (Room 120, Kazusa Kamatari 2-5-8, Kisarazu City, Chiba Prefecture) on June 4, 2013. -22254. The short axis of the Cocomixa KJ strain is 1 to 2 μm, and the long axis is 2 to 5 μm.

Cultivation of Coco-Mixa strain KJ was started at _OD720 of 0.005 and continued until _OD720 reached approximately 0.5. After the culture was completed, a cell suspension containing Cocomixa KJ strain was prepared. A cell suspension corresponds to a liquid containing cells.

(2-2) Cell Fixation Method The surface 39 of the second member 5 was brought into contact with the surface 7 of the first member. Next, a cell suspension was supplied to the first hole 33 . Next, suction was performed from the second hole 35 and the third hole 37 using a syringe pump. The suction pressure was 118 Pa. The suction pressure is preferably 1 Pa or higher, more preferably 10 Pa or higher, and particularly preferably 100 Pa or higher.

The cell suspension was discharged to the outside of the cell fixing device 1 through the first hole 33 , the expanded portion 15 , the first channel 41 , the expanded portion 17 and the second hole 35 in order.
A part of the cell suspension flows from the first flow channel 41 into the branch flow channel 43A, passes through the confluence flow channel 43B, the suction part 31, and the third hole 37 in sequence, and exits the cell fixing device 1. expelled. When the cell suspension flowed through the branch channel 43A, some of the cells were fixed in the branch channel 43A.

FIG. 5A shows branch channel 43A in which cells 45 are not immobilized. FIG. 5B shows the branch channel 43A with the cells 45 immobilized thereon. The channel wall 46 of the branch channel 43A is spread by the cells 45 . FIG. 6 shows a photograph of the branch channel 43A with fixed cells. In FIG. 6, algae cells are fixed at positions indicated by triangular indicators. As shown in Figure 6, one cell is fixed separately from the other cells. It is preferable to stop the suction after some of the cells are fixed in the branch channel 43A.

(2-3) Implementation of Injection The second member 5 was separated from the first member 3 . Cells fixed to bifurcation 21 were injected under a microscope using a micromanipulator. The following samples were introduced into cells by injection. Injection corresponds to cell manipulation.

Sample: Alexa488-dextran 10kDa 0.6mM
(2-4) Collection and Culture of Cells A portion of the first member 3 containing fixed cells was cut out using a scalpel. The cut out portion includes a portion of the branch portion 21 and its peripheral portion. Next, the excised portion was put into the culture medium. The cells were then cultured in a _CO2 incubator with an internal temperature of 25°C. Cell tracking was performed while culturing the cells.

3. Effects of Cell Fixing Device 1 (1A) By using the cell fixing device 1, one minute cell can be separated from other cells and fixed.

(1B) The cell fixing device 1 has a plurality of branch channels 43A. The cell fixing device 1 can fix cells in each of the plurality of branch channels 43A. The cell fixing device 1 also includes a confluence channel 43B where a plurality of branch channels 43A join together. Therefore, it is possible to suppress variations in the flow rate of the cell suspension among the plurality of branch flow paths 43A.

(1C) The interval between the branch flow paths 43A is 500 μm or more. Therefore, it is easy to cut out a portion including only one branch portion 21 from the first member 3 and culture it.
(1D) The elastic modulus of the first member 3 is smaller than the elastic modulus of the cells. Therefore, as shown in FIG. 5B, the channel wall 46 of the branch channel 43A is easily spread by the fixed cells 45 . Therefore, the cells 45 can be firmly fixed.

(1E) The material of the first member 3 is polydimethylsiloxane. The elastic modulus of polydimethylsiloxane is generally less than that of cells. Therefore, as shown in FIG. 5B, the channel wall 46 of the branch channel 43A is easily spread by the fixed cells 45 . Therefore, the cells 45 can be firmly fixed.

(1F) In the first member 3, the thickness from the surface 7 to the back surface 47 is 1.0 mm or less. Therefore, it is easy to observe the fixed cells from the rear surface 47 side using a microscope.
<Second embodiment>
1. Differences from First Embodiment Since the basic configuration of the second embodiment is the same as that of the first embodiment, the differences will be described below. Note that the same reference numerals as in the first embodiment indicate the same configurations, and refer to the preceding description.

In the first embodiment described above, the width of the branch flow path 43A is constant at any position. On the other hand, the second embodiment differs from the first embodiment in that the branch flow path 43A has the form shown in FIG.

As shown in FIG. 7, the branch flow path 43A has an upstream portion 49 and a downstream portion 51. As shown in FIG. The upstream portion 49 is located on the side of the first flow path 41 . The downstream portion 51 is located closer to the confluence channel 43B than the upstream portion 49 is.

The width of the upstream portion 49 is W1. The width of the downstream portion 51 is W2. W1 and W2 are widths in the Y direction. W2 is less than W1. The value of W1 is, for example, 2 μm or more and 3 μm or less. The value of W2 is, for example, 3 μm or more and 5 μm or less.

A step is formed at the boundary between the downstream portion 51 and the upstream portion 49 . The width of the branch channel 43A changes abruptly at the boundary between the downstream portion 51 and the upstream portion 49. As shown in FIG.
2. Effects of Cell Fixation Device 1 According to the second embodiment described in detail above, the effects of the above-described first embodiment are obtained, and the following effects are also obtained.

(2A) The cell fixing device 1 can fix cells 45 of various sizes. For example, as shown in FIGS. 8A and 8B, the cell 45 can be fixed to the upstream portion 49 when the size of the cell 45 is W1 or larger. As shown in FIG. 8A, multiple cells 45 can accumulate in the upstream portion 49 .

Also, for example, as shown in FIG. 8C , when the size of the cell 45 is smaller than W1 and equal to or larger than W2, the cell 45 can be fixed to the downstream portion 51 .
<Other embodiments>
Although the embodiments of the present disclosure have been described above, the present disclosure is not limited to the above-described embodiments, and various modifications can be made.

(1) Grooves may be formed in portions of the surface 39 that face the first grooves 9 and the second grooves 11 and 13 . In that case, the first flow path 41 is formed by the first groove 9 and the groove on the surface 39 side. The second flow path 43 is formed by the second grooves 11 and 13 and the groove on the surface 39 side.

(2) At least some of the first holes 33 , the second holes 35 , and the third holes 37 may be holes penetrating from the rear surface 47 to the front surface 7 . That is, at least part of the first hole 33 , the second hole 35 and the third hole 37 may be formed in the first member 3 .

(3) The suction portion 31 may be co-located with the extension portion 17 . In that case, instead of the second hole 35 and the third hole 37 , a single hole communicating with the suction portion 31 and the expansion portion 17 can be formed in the second member 5 .

(4) The fourth confluence portion 29 may merge with the first groove 9 on the opposite side in the Y direction from all the branch portions 21 .
(5) The material of the first member 3 and the second member 5 may be a material other than polydimethylsiloxane.

(6) The number of branch flow paths 43A may differ from the number in the first and second embodiments. The number of branch flow paths 43A is preferably 1 or more and 4 or less, more preferably 2 or more and 4 or less, for one primary confluence flow path. The primary confluence channel is a portion formed by the primary confluence portion 23 and the surface 39 of the confluence channel 43B. When the number of branch channels 43A is one or more for one primary confluence channel, the structure of the cell fixing device 1 can be maintained and cells can be fixed to the branch channels 43A. When the number of branch flow paths 43A is two or more for one primary confluence flow path, the interval between the branch flow paths 43A increases. As a result, the plurality of cells fixed to the branch channel 43A are moderately separated from each other, so that it is easy to introduce the substance into the cells. When the number of branch channels 43A is four or less with respect to one primary confluence channel, the substances can be introduced into more cells at once while the intervals between the branch channels 43A are appropriately separated. It can be carried out.

The number of primary confluence channels may differ from the number in the first and second embodiments. The number of primary confluence channels is preferably two or more and four or less. When the number of primary confluence channels is two or more, a plurality of cells can be immobilized at once. If the number of primary confluence channels is four or less, a strong suction force can be maintained.

(7) A plurality of functions possessed by one component in the above embodiment may be realized by a plurality of components, or a function possessed by one component may be realized by a plurality of components. . Also, a plurality of functions possessed by a plurality of components may be realized by a single component, or a function realized by a plurality of components may be realized by a single component. Also, part of the configuration of the above embodiment may be omitted. Moreover, at least part of the configuration of the above embodiment may be added or replaced with respect to the configuration of the other above embodiment. It should be noted that all aspects included in the technical idea specified by the wording described in the claims are embodiments of the present disclosure.

(8) In addition to the cell fixation device described above, in various forms such as a system having the cell fixation device as a component, a method for fixing cells, a method for culturing cells, a method for observing cells, a method for introducing genes into cells, etc. The present disclosure can also be implemented.

DESCRIPTION OF SYMBOLS 1... Cell fixation device 3... First member 5... Second member 7... Surface 9... First groove 11, 13... Second groove 15, 17... Extension part 19... Body part 21... Branching portion 23 Primary confluence portion 25 Secondary confluence portion 27 Tertiary confluence portion 29 Quaternary confluence portion 31 Suction portion 33 First hole 35 Second hole 37 Third Hole 39 Front surface 41 First channel 43 Second channel 43A Branch channel 43B Merging channel 45 Cell 46 Channel wall 47 Back surface 49 Upstream part , 51 downstream

Claims (7)

A cell fixing device for fixing cells,
a first member having grooves formed on its surface;
a second member that contacts the first member and covers the groove;
a channel formed by the groove and the second member;
a plurality of holes passing through the first member or the second member and communicating with the flow path;
with
The flow path is
a first flow path extending from a portion that communicates with a first hole that is part of the plurality of holes to a portion that communicates with a second hole that is a portion of the plurality of holes;
a second flow path branched from the first flow path and extending to a portion communicating with one of the plurality of holes;
with
The second channel has a narrower width than the first channel and includes a bottleneck portion capable of fixing the cells ,
The cell fixing device, wherein the width of the bottleneck is 80 to 99% of the width of the cell, and the elastic modulus of the first member is smaller than the elastic modulus of the cell. The cell fixation device according to claim 1,
The second flow path includes a plurality of branch flow paths branched at a plurality of positions in the first flow path, and a confluence flow path in which the plurality of branch flow paths merge,
The cell fixing device, wherein the bottleneck part is provided in the branch channel. The cell fixation device according to claim 2,
The bottleneck is
upstream and
a downstream portion positioned closer to the confluence channel than the upstream portion and having a width narrower than that of the upstream portion;
A cell fixation device comprising: The cell fixation device according to claim 2 or 3,
The interval between the branch channels is 500 μm or more ,
The cell fixing device , wherein the material of the first member and the second member is polydimethylsiloxane . The cell fixation device according to any one of claims 1 to 4 ,
The material of the first member is polydimethylsiloxane,
The cell fixing device , wherein the first member has a thickness of 1.0 mm or less from the front surface to the back surface . A cell fixing method for fixing cells using a cell fixing device,
The cell fixation device is
a first member having grooves formed on its surface;
a second member that contacts the first member and covers the groove;
a channel formed by the groove and the second member;
a plurality of holes passing through the first member or the second member and communicating with the flow path;
with
The flow path is
a first flow path extending from a portion that communicates with a first hole that is part of the plurality of holes to a portion that communicates with a second hole that is a portion of the plurality of holes;
a second flow path branched from the first flow path and extending to a portion communicating with a third hole, which is one of the plurality of holes;
with
The second channel has a narrower width than the first channel and includes a bottleneck portion capable of fixing the cells,
the width of the bottleneck portion is 80 to 99% of the width of the cell, and the elastic modulus of the first member is smaller than the elastic modulus of the cell;
supplying the liquid containing the cells to the first hole;
Suctioning from the second hole and the third hole causes the liquid containing the cells to flow from the first flow path into the second flow path,
A cell fixation method that stops the aspiration. Performing the cell fixation method according to claim 6,
separating the second member from the first member;
Injecting into the fixed cells,
cell manipulation methods .

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