Cell-seeding and -culturing device
|発明の名称 （英語）||Cell-seeding and -culturing device|
|発明の概要（英語）||Provided is a device for seeding cells in a plurality of cell arrangement areas in a simple manner and a short period of time. A seeding and culturing device (1) for cells capable of forming a nerve network, the device comprising a cell-culturing substrate (2) having a plurality of cell arrangement areas (8) enclosed by a plurality of projecting parts, and a flow channel substrate (3) arranged on the cell-cultivating substrate (2) and having a plurality of through-holes (14), wherein the through-holes (14) are configured so as to provide flow channels in which the upper surface side of the substrate is an entrance (15) and the lower surface side of the substrate is an exit, and the exit (16) of the flow channels is positioned above any of the cell arrangement areas.|
Research has recently become increasing active in the area of intercellular networks, and proposals have been made for constructing intercellular networks in vitro. Cell networks that have been constructed in vitro are useful in imaging research and electrophysiological research using the patch-clamp method. In order to construct an intercellular network in vitro, it is necessary to culture cells arranged at prescribed locations. For example, in Non-Patent Document 1, an experiment was conducted to detect the electrical potential of nerve cells by providing a region surrounded by a plurality of projections on a silicon substrate having a transistor arranged thereon, and arranging peripheral nerve cells of Lymnaea stagnalis in the form of large ganglia thereon. In addition, Non-Patent Document 2 discloses a neurochip obtained by forming a plurality of roughly disk-shaped enclosures referred to as "cages" on a substrate, arranging nerve cells in a space in the center of the cages, and allowing axons of the nerve cells to extend through several tunnels provided in the cages towards nerve cells in adjacent cages. However, Non-Patent Documents 1 and 2 do not disclose nerve cell seeding systems for efficiently seeding nerve cells in a large number of cell establishment areas. Methods involving the manual seeding of cells in individual cell arrangement areas using equipment such as ordinary pipettes, pipettes equipped with a measuring function or micro-injectors make it difficult to accurately seed cells in extremely minute cell arrangement areas, thereby resulting in inferior seeding efficiency and making these methods undesirable.
On the other hand, the patch-clamp method is an electrophysiological technique for examining signal transduction between cells by measuring membrane potential and membrane current, and is carried out using ordinary pipettes. However, in the case of the pipette patch-clamp method, since the technique involves capturing (clamping) individual cells with a pipette followed by measuring their electrical changes, it is unable to accommodate high-throughput screening by multi-point measurement. The inventors of the present invention developed a planar patch-clamp method for the purpose of using the patch-clamp method for high-throughput screening (Patent Documents 1 and 2). This planar patch-clamp method consists of providing a plurality of microscopic through holes in an electrically insulated substrate and then measuring electrical changes, such as changes in membrane potential or membrane current, in cells arranged on these through holes, thereby enabling high-throughput screening by multi-point measurement. However, in order to construct an intercellular network in vitro, similar to the difficulty encountered in seeding cells into a large number of cell establishment areas, the difficulty remained in accurately arranging cells on a plurality of the through-holes in the electrically insulated substrate.
Thus, in order to construct an intercellular network in vitro, it is additionally required to be able to accurately and rapidly arrange cells in prescribed arrangement areas in order to study electrical properties using the planar patch-clamp method.
In addition to methods using pipettes, an automated cell seeding method that mechanically automates a cell seeding method using pipettes by utilizing the principle of an inkjet printer has been developed for use as a technology for seeding cells in prescribed cell arrangement areas (Patent Document 3). In addition, as another example of a technology for cell seeding, a technology has been implemented for seeding cells based on the principle of embedding cells in sequential cell arrangement areas in the form of depressions by transporting cells to a prescribed depression using microchannels and then transporting the cells to the next depression when that depression has been filled (Non-Patent Document 3).
Non-Patent Document 4 discloses a device according to the preamble of claim 1.
Prior Art Documents
Patent Document 1: Japanese Unexamined Patent Publication No. 2009-204407
Patent Document 2: Japanese Unexamined Patent Publication No. 2013-146261
Patent Document 3: Japanese Unexamined Patent Publication No. 2009-131240
Patent Document 4: International Publication No. WO 2013/094418
Non-Patent Document 1: G. Zeck, et al., PNAS 98 (2001), 10457-10462
Non-Patent Document 2: J. Erickson, et al., J. Neurosci. Methods, 175 (2008), 1-16
Non-Patent Document 3: W.H. Tan, et al., PNAS 104 (2007), 1146-1151
Non-Patent Document 4 : MIHO SAITO ET AL., "Control of neuronal cell -migration using cell -cage patterns on culture substrates", 74TH JSAP AUTUMN MEETING, 2013 KOEN YOKOSHU, abstract no. 17a-C4-5, page 12-150
1. A cell seeding and culturing device (1) capable of forming a nerve cell network, the device comprising:
a cell culturing substrate (2) capable of forming a nerve cell network, having a plurality of cell arrangement areas (8) surrounded by a plurality of projections (12), which inhibit cell moving and loss;
the device being characterized in that it comprises a flow channel substrate (3) having a plurality of through holes (14) through which cells are allowed to pass, and being arranged on the culturing substrate (2),
wherein each of the through holes (14) defines a flow channel, wherein the upper side of the substrate is an entrance (15) and the lower side of the substrate is an exit (16), wherein the flow channel is tapered from an entrance to an exit so as to seed cells, the exit of each flow channel is located above the cell arrangement areas (8), and the size of the exit (16) is smaller than that of the cell arrangement area, and
wherein a spacer member (9) having a thickness greater than the height of the projections (12) is arranged between the culturing substrate (2) and the flow channel substrate (3).
2. The cell seeding and culturing device (1) according to claim 1, wherein the entrance of the flow channel for seeding cells is 100 - 500 µm, the exit is 20 - 50 µm, and the thickness of the flow channel substrate is 0.2 mm - 5 mm.
3. The cell seeding and culturing device (1) according to claim 1 or 2, wherein the internal diameter of cell retainer which is configured from a plurality of projections (12) which inhibit cell moving and loss is 10 - 25 µm.
4. The cell seeding and culturing device according to claim 1, wherein the height of the projections (12) is 5 - 15 µm.
5. The cell seeding and culturing device (1) according to any one of claims 1 to 4, which enables to place one to several cells on a cell arrangement area (8) .
6. The cell seeding and culturing device (1) according to any one of claims 1 to 5, wherein the flow channel substrate (3) is removable from the culturing substrate (2) after cell establishment.
7. The cell seeding and culturing device (1) according to any one of claims 1 to 6, having a first gap between the culturing substrate (2) and the flow channel substrate (3) and which corresponds to the thickness of the spacer member (9), wherein said first gap enables to form a nerve cell network between the cell arrangement areas (8).
8. The cell seeding and culturing device (1) according to any one of claims 1 to 7, wherein a second gap through which cells flow out is not present between the exits (16) of the flow channels and the projections (12) .
9. The cell seeding and culturing device (1) according to any one of claims 1 to 8, further comprising a liquid storage substrate (4) that defines a liquid storage area (18) on the upper surface of the flow channel substrate (3).
10. The cell seeding and culturing device (1) according to any one of claims 1 to 9, wherein the culturing substrate (2) is an electrically insulated substrate used in a planar patch-clamp method, and has through holes (21) for the planar patch-clamp method in the cell arrangement areas (8) that do not allow the passage of cells but allow the attaining of electrical continuity.
11. The cell seeding and culturing device according to any one of claims 1 to 10, wherein the culturing substrate (2) is a substrate for imaging a nerve cell network.
|指定国||Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR|
|参考情報 （研究プロジェクト等）||CREST Creation of Nanosystems with Novel Functions through Process Integration AREA|
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