CELL CULTURE CHIP
20220033749 · 2022-02-03
Assignee
Inventors
Cpc classification
B01L2300/0829
PERFORMING OPERATIONS; TRANSPORTING
B01L3/502
PERFORMING OPERATIONS; TRANSPORTING
International classification
Abstract
A cell culture chip from which a liquid retained in an opening is able to be sucked by a simple operation procedure while a liquid in a channel remains is provided.
A cell culture chip according to the present invention includes a bottom portion; a base portion formed on an upper surface of the bottom portion; a first well provided by opening the base portion in a first direction extending from a portion of a main surface of the base portion toward the bottom portion and having a shape such that a capillary force thereof is smaller than that of the chamber; a second well provided by opening the base portion in the first direction at a position separated from the first well in a second direction parallel to the main surface; and a tubular chamber that is defined by a region sandwiched between the bottom portion and the base portion and that provides communication between the first and second wells in the second direction. The first well has a shape with which a capillary force of the first well is smaller than a capillary force of the chamber.
Claims
1. A cell culture chip comprising: a bottom portion; a base portion formed on an upper surface of the bottom portion; a first well provided by opening the base portion in a first direction extending from a portion of a main surface of the base portion toward the bottom portion, the main surface being a surface opposite to the bottom portion; a second well provided by opening the base portion in the first direction at a position separated from the first well in a second direction parallel to the main surface; and a tubular chamber that is defined by a region sandwiched between the bottom portion and the base portion and that provides communication between the first well and the second well in the second direction, wherein the first well has a shape such that a capillary force of the first well is smaller than a capillary force of the chamber.
2. The cell culture chip according to claim 1, wherein the first well has a reduced-diameter region of which an opening diameter continuously decreases without increasing toward the bottom portion at a position closer to the bottom portion than the main surface of the base portion.
3. The cell culture chip according to claim 2, wherein the first well has an inner wall defined by the base portion, and wherein the inner wall includes a curved surface or a flat surface non-parallel to the main surface in the reduced-diameter region of the first well.
4. The cell culture chip according to claim 2, comprising: a communication well that is formed continuously with the first well in the first direction and that provides communication between the reduced-diameter region of the first well and the chamber in the first direction, wherein the communication well has a bottom surface defined by the bottom portion and has an opening diameter smaller than the opening diameter of the reduced-diameter region of the first well.
5. The cell culture chip according to claim 3, wherein a bottom surface of the first well is defined by the bottom portion.
6. The cell culture chip according to claim 1, wherein the second well has a shape such that a capillary force of the second well is smaller than a capillary force of the chamber.
7. The cell culture chip according to claim 6, wherein the second well has a reduced-diameter region of which an opening diameter continuously decreases without increasing toward the bottom portion at a position closer to the bottom portion than the main surface of the base portion.
8. A cell culture chip comprising: a bottom portion; a base portion formed on an upper surface of the bottom portion; a first well provided by opening the base portion in a first direction extending from a portion of a main surface of the base portion toward the bottom portion, the main surface being a surface opposite to the bottom portion; a second well provided by opening the base portion in the first direction at a position separated from the first well in a second direction parallel to the main surface; and a tubular chamber that is defined by a region sandwiched between the bottom portion and the base portion and that provides communication between the first well and the second well in the second direction, wherein the first well has a reduced-diameter region of which an opening diameter continuously decreases without increasing at a position closer to the bottom portion than the main surface of the base portion.
9. The cell culture chip according to claim 8, wherein the first well has an inner wall defined by the base portion, and wherein the inner wall includes a curved surface or a flat surface non-parallel to the main surface in the reduced-diameter region of the first well.
10. The cell culture chip according to claim 9, wherein the second well has a reduced-diameter region in which an opening diameter continuously decreases without increasing at a position closer to the bottom portion than the main surface of the base portion.
11. The cell culture chip according to claim 3, comprising: a communication well that is formed continuously with the first well in the first direction and that provides communication between the reduced-diameter region of the first well and the chamber in the first direction, wherein the communication well has a bottom surface defined by the bottom portion and has an opening diameter smaller than the opening diameter of the reduced-diameter region of the first well.
12. The cell culture chip according to claim 2, wherein a bottom surface of the first well is defined by the bottom portion.
13. The cell culture chip according to claim 8, wherein the second well has a reduced-diameter region in which an opening diameter continuously decreases without increasing at a position closer to the bottom portion than the main surface of the base portion.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0057]
[0058]
[0059]
[0060]
[0061]
[0062]
[0063]
[0064]
[0065]
[0066]
[0067]
[0068]
[0069]
[0070]
[0071]
[0072]
[0073]
[0074]
[0075]
[0076]
DESCRIPTION OF EMBODIMENTS
[0077] An embodiment of a cell culture chip according to the present invention will be described with reference to the drawings. It should be noted that the following drawings are merely schematically illustrated. That is, the dimensional ratios on the drawings and the actual dimensional ratios do not necessarily coincide with each other, and the dimensional ratios do not necessarily coincide with each other between the drawings.
[0078]
[0079] The cell culture chip 1 includes a bottom portion 3 and a base portion 5. The base portion 5 includes a first well 10 and a second well 20 that are open in the Y direction toward the bottom portion 3 from a portion of a surface (a main surface 5a) opposite to the bottom portion 3. That is, the first well 10 has an opening area 10a on the main surface 5a side of the base portion 5 and is open in the Y direction toward the bottom portion 3. Similarly, the second well 20 has an opening area 20a on the main surface 5a side of the base portion 5 and is open in the Y direction toward the bottom portion 3. That is, the Y direction corresponds to a “first direction”.
[0080] The opening area 10a of the first well 10 and the opening area 20a of the second well 20 are disposed at positions separated from each other in a direction parallel to the main surface 5a of the base portion 5. Here, description is given based on the assumption that both are disposed at positions separated from each other in the X direction. In this case, the X direction corresponds to a “second direction”. Alternatively, the opening area 10a of the first well 10 and the opening area 20a of the second well 20 may be separated from each other in the Z direction, or may be separated from each other in the X direction and the Z direction. The “second direction” corresponds to a direction extending from the opening area 10a of the first well 10 toward the opening area 20a of the second well 20.
[0081] The base portion 5 has a tubular recessed portion at a position on the bottom portion 3 side, and a chamber 7 is formed by a region sandwiched between the recessed portion and the bottom portion 3. In the present embodiment, the chamber 7 constitutes a space in which cells are cultured.
[0082] In the present embodiment, one end of the chamber 7 communicates with the first well 10 via a communication well 19, and the other end of the chamber 7 communicates with the second well 20 via a communication well 29. Note that the communication well 19 communicates with the first well 10 in the Y direction, and the bottom portion 3 constitutes a bottom surface of the communication well 19. Similarly, the communication well 29 communicates with the second well 20 in the Y direction, and the bottom portion 3 constitutes a bottom surface of the communication well 29.
[0083] In the present embodiment, the first well 10 and the second well 20 have regions of which opening diameters decrease without increasing toward the bottom portion 3 at positions closer to the bottom portion 3 than the main surface 5a. This point will be described with reference to
[0084] As illustrated in
[0085]
[0086] To fill the cell culture chip 1 with the culture solution 30, the culture solution 30 is injected from the first well 10 side or the second well 20 side. For example, when the culture solution 30 is injected from the first well 10 side, the culture solution 30 flows to the second well 20 side via the communication well 19 and the chamber 7. By injecting a predetermined amount or more of the culture solution 30 into the cell culture chip 1, the chamber 7 located between the first well 10 and the second well 20 is filled with the culture solution 30. Thus, cells can be cultured in the chamber 7.
[0087] As illustrated in
[0088]
[0089] When the tip of the pipet 31 is inserted into the first well 10 and a suction operation is started, the liquid level of the culture solution 30 starts being gradually lowered (see
[0090] That is, according to the configuration of the present embodiment, the culture solution 30 does not flow in from the chamber 7 to the first well 10 side at a time point immediately after the suction of the culture solution 30 retained in the first well 10 is completed. Even when the suction operation from the pipet 31 is continued with the constant suction force, the suction of the culture solution 30 does not progress.
[0091] The inventor of the present invention considers the reason why such a phenomenon occurs as follows.
[0092]
[0093] When ϕ denotes an angle of a corner portion of an inflow port 111 (hereinafter, referred to as “opening 111” in this case), and D1 denotes a distance between both ends of a portion of a culture solution 130 (130a) where the meniscus of the culture solution 130a retained in the corner portion is in contact with the corner portion, a capillary force P1 generated at the meniscus of the culture solution 130a is expressed by Expression (1) as follows. In the following Expression (1), γ indicates a surface tension, and θ indicates a contact angle of the culture solution 130 (130a).
P1≈2.Math.γ cos (θ+ϕ/2)/(D1/2) (1)
[0094] In contrast, when D2 is an inner diameter of a groove 110, a capillary force P2 generated at the meniscus of a culture solution 130 (130b) retained in the groove 110 is expressed by the following expression similarly using γ and θ. However, in the following Expression (2), since the groove 110 has inner wall surfaces facing each other in parallel and the angle between both ends of a portion of the culture solution 130b where the meniscus of the culture solution 130b is in contact with the inner wall surfaces is 0°, the calculation is performed on the basis of that the component of ϕ is 0.
P2≈2.Math.γ cos (θ)/(D2/2) (2)
[0095] For example, in a case where the contact angle θ of the culture solution 130 is 20°, and the inner diameter D2 of the groove 110 is 400 μm, P1=P2 is satisfied at the distance D1 being nearly equal to 180 μm between both ends of the portion where the meniscus of the culture solution 130a is in contact with the corner portion of the opening 111. In other words, at the distance D1 being smaller than 180 μm, the capillary force P1 generated at the meniscus of the culture solution 130a is larger than the capillary force P2 generated at the meniscus of the culture solution 130b retained in the groove 110, and is P1>P2.
[0096] This means that, in the conventional microchannel chip 100 illustrated in
[0097] In view of this fact, in the structure illustrated in
[0098] Here, as described above with reference to
[0099] Consequently, the capillary force P1 generated at the meniscus of the culture solution 30 retained in the corner portion in the reduced-diameter region 11 becomes smaller than the capillary force P2 of the meniscus of the culture solution 30 in the chamber 7. Accordingly, even when the suction of the culture solution 30 is continued by the pipet 31 from the state illustrated in
[0100] The cell culture chip 1 of the present embodiment includes the communication well 19, and the corner angle of the corner portion of the communication well 19 may be, for example, approximately 90°, similarly to the opening 111 of the conventional microchannel chip 100. In this case, when the suction operation is continued from the state of FIG. 5B, the culture solution 30 (30a) is retained in the corner portion of the communication well 19. However, since at least the culture solution 30 in the first well 10 has been completely removed in a state before the state of
[0101] In the case where the contact angle θ of the culture solution 30 and the height of the chamber 7 are predetermined, the preferable minimum value of the length D of the inclined surface (the length of a chamfered portion) when the inner wall 10c in the reduced-diameter region 11 of the first well 10 is viewed in the Z direction is as provided in Table 1 below. By providing the inner wall 10c in the reduced-diameter region 11 as an inclined surface having a value larger than the value described in the table, the capillary force of the corner portion in the first well 10 can be made smaller than the capillary force of the chamber 7.
TABLE-US-00001 TABLE 1 Length of inclined Height of chamber 7 [μm] surface D [μm] 200 300 400 500 600 900 Contact 5 129 194 258 323 387 516 angle 10 116 175 233 291 349 466 θ[°] 20 90 135 180 225 270 360 40 23 34 46 57 68 91
[0102] Specific examples of dimensions of the cell culture chip 1 are as follows.
[0103] The height (the length in the Y direction) of the base portion 5 is 1 mm or more and 20 mm or less, and is 3 mm as an example.
[0104] The height (the length in the Y direction) of the bottom portion 3 is 0.1 mm or more and 5 mm or less, and is 1 mm as an example.
[0105] The height (the length in the Y direction) of the chamber 7 is 200 μm or more and 2000 μm or less, and is 400 μm as an example.
[0106] The opening diameter 10b on the opening area 10a side of the first well 10 is 0.5 mm or more and 40 mm or less, and is 2 mm as an example.
[0107] The minimum value of the opening diameter 10b in the reduced-diameter region 11 of the first well 10 is 0.5 mm or more and 40 mm or less, and is 1.75 mm as an example. Also, the length of the inclined surface when the inner wall 10c in the reduced-diameter region 11 is viewed in the Z direction is 20 μm or more and 2000 μm or less, and is 180 μm as an example.
[0108] The opening diameter of the communication well 19 is 0.2 mm or more and 39 mm or less, and is 1.75 mm as an example. Also, the height (the length in the Y direction) of the communication well 19 is 0.2 mm or more and 3 mm or less, and is 0.6 mm as an example.
[0109] The separation distance between the central axis of the first well 10 and the central axis of the second well 20 is 2 mm or more and 40 mm or less, and is 9 mm as an example.
[0110] The opening diameter on the opening area 20a side of the second well 20 is 0.5 mm or more and 40 mm or less, and is 1 mm as an example.
[0111] The minimum value of the opening diameter in the reduced-diameter region of the second well 20 is 0.5 mm or more and 40 mm or less, and is 0.75 mm as an example. Also, the length of the inclined surface when the inner wall 10c in the reduced-diameter region 11 is viewed in the Z direction is 20 μm or more and 2000 μm or less, and is 180 μm as an example.
[0112] The opening diameter of the communication well 29 is 0.2 mm or more and 39 mm or less, and is 0.7 mm as an example. Also, the height (the length in the Y direction) of the communication well 19 is 0.2 mm or more and 2000 mm or less, and is 0.6 mm as an example.
(Modification)
[0113] The cell culture chip 1 of the present embodiment can be variously modified. This will be described below.
[0114] <1> The cell culture chip 1 of the above-described present embodiment has the structure in which the second well 20 side also has the reduced-diameter region similar to the reduced diameter region 11 of the first well 10. Thus, even when the culture solution 30 is sucked from the second well 20 side by the pipet 31, the culture solution in the second well 20 can be removed while the culture solution 30 is retained in the chamber 7 for the same reason. However, the present invention does not exclude a structure having a reduced-diameter region only on one well (the first well 10/the second well 20) side.
[0115] <2>
[0116] <3>
[0117] <4> As illustrated in
[0118] However, as illustrated in
[0119] <5> As illustrated in
[0120] Note that, in the structure of the cell culture chip 1 illustrated in
OTHER EMBODIMENTS
[0121] Other embodiments will be described below.
[0122] <1> In the above-described embodiment, the bottom portion 3 and the base portion 5 included in the cell culture chip 1 are described as being provided by separate members, however the bottom portion 3 and the base portion 5 may be formed by integral molding into a single member.
[0123] <2> The first well 10 included in the cell culture chip 1 described with reference to
[0124] <3> According to the cell culture chip 1 of each embodiment described above, it has been described that the culture solution 30 filled inside can be extracted from the first well 10 side or the second well 20 side while the culture solution 30 is retained in the chamber 7. However, the substance to be extracted from the cell culture chip 1 while being retained in the chamber 7 is not limited to the culture solution 30, and may be any liquid.
[0125] <4> In the above-described embodiment, the case where the pipet 31 is used when the culture solution 30 is sucked from the cell culture chip 1 has been described as an example, however the suction method is not limited to the pipet 31. The cell culture chip 1 of the present invention can employ another typical method of disposing the tip of a suction instrument on one well (for example, the first well 10) side and sucking the culture solution 30 retained in the well.
[0126] <5> In the above-described embodiment, the cell culture chip 1 in which the pair of wells (10, 20) communicate with each other through the chamber 7 has been described. However, in the cell culture chip 1 of the present invention, the number of wells and the number of chambers are not limited.
[0127]
[0128] In the cell culture chip 1 illustrated in
[0129] A cell culture chip 1 illustrated in
[0130] A cell culture chip 1 illustrated in
[0131] The cell culture chip 1 illustrated in each of
[0132] <6> In the cell culture chip 1 described with reference to
REFERENCE SIGNS LIST
[0133] 1: cell culture chip [0134] 3: bottom portion [0135] 5: base portion [0136] 5a: main surface of base portion [0137] 7: chamber [0138] 7a: culture chamber [0139] 7b, 7c: communication channel [0140] 10: first well [0141] 10a: opening area of first well [0142] 10b: opening diameter of first well [0143] 10c: inner wall of first well [0144] 11: reduced-diameter region [0145] 19: communication well [0146] 20: second well [0147] 20a: opening area of second well [0148] 29: communication well [0149] 30, 30a: culture solution [0150] 31: pipet [0151] 41, 42, 43, 44: well [0152] 100: conventional microchannel chip [0153] 101: base [0154] 102: resin film [0155] 110: groove [0156] 111: inflow port [0157] 112: outflow port [0158] 120: pipet [0159] 130, 130a: culture solution