JP7097310B2 - Temperature control device and cell culture device - Google Patents

Description

The present invention relates to a temperature control device and a cell culture device using the same.

Applied research such as drug discovery and environmental analysis using cell arrays as an evaluation system in the rapidly expanding field of regenerative medicine is being actively conducted, and there is an increasing need for methods for finely manipulating and analyzing individual cells. Therefore, not only simple cell culture but also a technique of adhering cells to an arbitrary place on a substrate material and performing patterning is required.

Patent Document 1 describes that cells are patterned by forming a pattern of a hydrophilic region and a hydrophobic region on the surface of a substrate to give strength or weakness to the adhesive strength of the cells to the substrate.

Patent Document 2 describes a heating / cooling device in which a plurality of thermoelectric modules are arranged in a matrix.

Japanese Unexamined Patent Publication No. 2014-103857 US Patent Application Publication No. 2003/097845

According to the study of the inventor of the present application, there is room for study in the technique of Patent Document 1 from the viewpoint of temporal control of cells in an arbitrary region on the surface of the substrate.

Further, the technique of Patent Document 2 has room for study from the viewpoint of efficient heating / cooling. FIG. The thermoelectric modules disclosed in 3A to 3C include panels 12 and 14 sandwiching semiconductor pellets and conductors 20 and 22 in contact with the panels 12 and 14, and a voltage is applied between the conductors 20 and 22 to make a semiconductor. An electric current is passed through the pellets, resulting in, for example, increasing or decreasing the temperature of the panel 12. However, since the panel 12 or 14 and the conductor 20 or 22 are interposed between the object to be heated / cooled and the semiconductor pellet, the efficiency of heating / cooling is lowered.

That is, it is desired to improve the performance of the temperature control device and the cell culture device using the temperature control device.

Other issues and novel features will become apparent from the description and accompanying drawings herein.

The temperature control device according to one embodiment includes a plurality of cells arranged in a matrix, each cell including a first end portion and a second end portion, and the first end portion is connected to a row electrode. An n-type semiconductor including a p-type semiconductor, a third end portion and a fourth end portion, the third end portion connected to a column electrode, and a common electrode connecting the second end portion and the fourth end portion. , Have.

According to one embodiment, the performance of the temperature control device and the cell culture device can be improved.

It is a top view which shows the temperature control apparatus which concerns on one Embodiment. It is a perspective view which shows the cell array area of the temperature control apparatus which concerns on one Embodiment. It is a perspective view which shows the cell of the temperature control apparatus which concerns on one Embodiment. It is sectional drawing which follows the AA line and the BB line of FIG. It is sectional drawing in the manufacturing process of the temperature control apparatus which concerns on one Embodiment. It is sectional drawing in the manufacturing process of the temperature control apparatus following FIG. It is sectional drawing in the manufacturing process of the temperature control apparatus following FIG. It is sectional drawing in the manufacturing process of the temperature control apparatus following FIG. It is sectional drawing in the manufacturing process of the temperature control apparatus following FIG. It is a top view which shows the cell culture apparatus which concerns on one Embodiment. It is sectional drawing which follows the CC line of FIG. It is sectional drawing of the main part of the cell culture apparatus which concerns on one Embodiment. It is a plan view which shows the cultured cell in the cell culture apparatus which concerns on one Embodiment. It is a plan view which shows the cultured cell in the cell culture apparatus which concerns on one Embodiment. It is sectional drawing of the main part of the cell culture apparatus which concerns on modification 1 of FIG. It is sectional drawing of the main part of the cell culture apparatus which concerns on modification 2 of FIG.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In all the drawings for explaining the embodiment, the members having the same function are designated by the same reference numerals, and the repeated description thereof will be omitted. Further, in the following embodiments, the same or similar parts will not be repeated in principle unless it is particularly necessary.

(Embodiment)
<Temperature control device>
Hereinafter, the temperature control device of this embodiment will be described with reference to FIGS. 1 to 4.

FIG. 1 is a plan view showing a temperature control device according to the present embodiment. As shown in FIG. 1, the temperature control device TCD of the present embodiment has a substrate SB, a plurality of terminal TAs, a plurality of wiring WRs, and a plurality of cells CL arranged in a matrix. The substrate SB has a rectangular main surface SBa, and a plurality of terminal TAs are arranged along each side on the main surface SBa. A cell array region CLA is arranged in the central portion of the main surface SBa of the substrate SB, and the cell array region CLA is surrounded by a plurality of terminals TA.

The cell array region CLA is provided with a plurality of cell CLs arranged in a matrix. A wiring WR extends from each of the plurality of terminals TA toward the cell array region CLA, and each wiring WR connects the terminal TA and the cell CL in the cell array region CLA.

As will be described later, each cell CL has a common electrode COM. The main surface SBa of the substrate SB is covered with an insulating film, and a plurality of common electrode COMs arranged in a matrix and a part of each terminal TA are exposed from the surface of the insulating film.

FIG. 2 is a perspective view showing a cell array region CLA of the temperature control device according to the present embodiment. The insulating films IF1 to IF6, which will be described later, are omitted. As shown in FIG. 2, on the main surface SBa of the substrate SB, a plurality of row electrodes (first electrodes) RE extending in the X direction and a plurality of column electrodes extending in the Y direction orthogonal to the X direction. (Second electrode) CE is arranged. The row electrode (first electrode) RE is arranged in the Y direction with a width of 2 μm and an interval of 13 μm, for example, and the column electrode (second electrode) CE is arranged in the X direction with a width of 2 μm and an interval of 10 μm, for example. A cell CL is arranged at the intersection of the plurality of row electrodes (first electrode) RE and the plurality of column electrodes (second electrode) CE. That is, the plurality of cells CL are arranged in a matrix in the X direction and in the Y direction orthogonal to the X direction.

FIG. 3 is a perspective view showing the cell CL of the temperature control device according to the present embodiment. The insulating films IF1 to IF6, which will be described later, are omitted. The cell CL has an n-type semiconductor NS, a p-type semiconductor PS, and a common electrode COM. The n-type semiconductor NS is composed of, for example, an n-type thermoelectric conversion material made of Fe ₂ TiVSi, and has the shape of a quadrangular prism or a cylinder. The n-type semiconductor NS is mounted on the row electrode CE, one end thereof is connected to the row electrode CE, and the other end is connected to the common electrode COM. The p-type semiconductor PS is composed of, for example, a p-type thermoelectric conversion material made of Fe ₂ TiVSiAl, and has the shape of a quadrangular prism or a cylinder. The p-type semiconductor PS is mounted on the row electrode RE, one end thereof is connected to the row electrode RE, and the other end is connected to the common electrode COM.

That is, the cell CL is a Pelche element having a π (pi) type structure. The sizes of the n-type semiconductor NS and the p-type semiconductor PS are, for example, a square having a bottom surface of 2 μm × 2 μm in the case of a quadrangular prism, and a circle having a diameter of 2 μm in the case of a cylinder. The separation distance between the n-type semiconductor NS and the p-type semiconductor PS is 1 μm. Therefore, the common electrode COM has a rectangular or race track shape of 2 μm × 5 μm. As shown in FIG. 1, the common electrodes COM are arranged in a matrix with an interval of 10 μm in the X direction and an interval of 10 μm in the Y direction. However, the spacing between the common electrode COM, the row electrode RE, and the column electrode CE can be changed, and is not limited to the above example.

Then, as shown in FIG. 3, when a current Ia is passed from the row electrode RE to the column electrode CE via the cell CL, the common electrode COM is cooled (heat absorbed), and conversely, the common electrode COM is cooled (heat absorbed), and conversely, the column electrode CE is passed through the cell CL. When the current Ib is passed through the row electrode RE, the common electrode COM is heated (radiated). In this way, the cooling or heating of the common electrode COM of the cell CL can be controlled by passing a current from one of the row electrode RE and the column electrode CE to the other. Further, by adjusting the amount of current Ia or Ib, the degree of cooling or heating can be adjusted.

As a modification, the n-type semiconductor NS may be mounted on the row electrode RE and connected to the row electrode RE, and the p-type semiconductor PS may be mounted on the column electrode CE and connected to the row electrode CE.

FIG. 4 is a cross-sectional view taken along the lines AA and BB of FIG. 3, wherein the cross-sectional view along the line AA is the AA region and the cross-sectional view along the line BB is the BB region. The AA line is the direction along the column electrode CE, and the BB line is the direction along the row electrode RE.

As shown in FIG. 4, the column electrode CE is formed on the substrate SB via the insulating film IF1. As shown in the BB region, the column electrode CE is embedded in the opening OP1 formed in the insulating film IF2, and the side surface of the column electrode CE is covered with the insulating film IF2. The insulating film IF3 is formed on the column electrode CE and the insulating film IF2, and the row electrode RE is formed on the insulating film IF3. That is, the column electrode CE and the row electrode RE are electrically separated by the insulating film IF3. As shown in the AA region, the row electrode RE is embedded in the opening OP2 formed in the insulating film IF4, and the side surface of the row electrode RE is covered with the insulating film IF4. Further, an insulating film IF5 is formed on the row electrode RE and the insulating film IF4.

An opening OP3 that exposes the surface of the column electrode CE is formed in the laminated insulating films IF3 to IF5, and an n-type semiconductor NS is embedded in the opening OP3. One end of the n-type semiconductor NS is electrically connected to the column electrode CE. An opening OP4 that exposes the surface of the row electrode RE is formed in the insulating film IF5, and a p-type semiconductor PS is embedded in the opening OP4. One end of the p-type semiconductor PS is electrically connected to the row electrode RE.

An insulating film IF6 having an opening OP5 is formed on the insulating film IF5, and a common electrode COM is embedded in the opening OP5. The common electrode COM has a main surface COMa and a back surface COMb, and the back surface COMb is electrically connected to the other end of the n-type semiconductor NS and the other end of the p-type semiconductor PS, and the main surface COMa is an insulating film. It is exposed from the main surface IF6a of IF6.

The substrate SB is composed of, for example, a single crystal silicon substrate or a ceramic substrate, and the insulating films IF1 to IF6 are composed of, for example, an inorganic insulating film such as a silicon oxide film. Further, the row electrode RE, the column electrode CE and the common electrode COM are preferably composed of a metal film having good electrical conductivity and thermal conductivity, and for example, copper (Cu), silver (Ag), and gold (Au). , Platinum (Pt) or chromium (Cr) in the metal or alloy. The n-type semiconductor NS is composed of Fe ₂ TiVSi, but may be composed of other n-type thermoelectric conversion materials. The p-type semiconductor PS is made of Fe ₂ TiVSiAl, but may be made of other p-type thermoelectric conversion materials. For example, the thermoelectric conversion materials include bismuth (Bi), tellurium (Te), antimony (Sb), lead (Pb), tin (Sn), selenium (Se), silicon (Si), germanium (Ge), and manganese (Mn). ), Magnesium (Mg), Cobalt (Co), Iron (Fe), Titanium (Ti), Oxygen (O), and contains at least one element, Bi-Te alloy, Pb-Te alloy. , Scutteldite compounds, clathrate compounds, chalcogenide compounds, silicide compounds, full whisler compounds, half whistler compounds, oxides, sulfides, and organic material-based semiconductors may be used.

According to this embodiment, the following effects can be obtained.

Since a plurality of common electrode COMs are arranged in a matrix in the cell array region CLA and the common electrode COM can be cooled or heated by the Pelche element, the temperature control of a minute region can be selectively performed in the cell array region CLA. ..

Further, the column electrode CE and the row electrode RE are connected to one end of the n-type semiconductor NS and the p-type semiconductor PS, and the common electrode COM is connected to the other end of the n-type semiconductor NS and the p-type semiconductor PS. Can be cooled or heated, so that the efficiency of cooling or heating can be improved.

<Manufacturing method of temperature control device>
A method of manufacturing the temperature control device of the present embodiment will be described with reference to FIGS. 4 to 9. 5 to 9 are cross-sectional views during the manufacturing process of the temperature control device of the present embodiment. As described above, FIG. 4 is a cross-sectional view of the cell CL, and FIGS. 5 to 9 are also cross-sectional views of the cell CL during the manufacturing process.

As shown in FIG. 5, after preparing the substrate SB, the insulating film IF1 is formed on the substrate SB. Next, the insulating film IF2 having the opening OP1 is formed on the insulating film IF1. Then, for example, after forming a metal film ML made of copper (Cu) in the opening OP1 and on the insulating film IF2, the metal film ML is subjected to a polishing treatment such as CMP (Chemical Mechanical Polishing). Then, as shown in FIG. 6, the metal film ML on the insulating film IF2 is removed, and the metal film ML is selectively left in the opening OP1 to form the column electrode CE. Further, the insulating films IF3 and IF4 are sequentially formed so as to cover the insulating film IF2 and the column electrode CE.

Next, as shown in FIG. 7, the insulating film IF4 is provided with the opening OP2, and the row electrode RE is formed in the opening OP2. The row electrode RE is formed by using a metal film deposition and polishing treatment in the same manner as in the above-mentioned method for manufacturing a column electrode CE.

Next, as shown in FIG. 8, an n-type semiconductor NS is formed. First, the insulating film IF5 is formed so as to cover the insulating film IF4 and the row electrode RE, and then the insulating films IF3, IF4 and IF5 are formed with an opening OP3 that exposes the surface of the column electrode CE. Then, an n-type thermoelectric conversion material is formed in the opening OP3 and on the insulating film IF5 in the same manner as in the above-mentioned manufacturing method of the column electrode CE, and then the n-type thermoelectric conversion material is polished and insulated. The main surface IF5a of the film IF5 is exposed. In this way, the n-type thermoelectric conversion material on the insulating film IF5 is removed, and the n-type semiconductor NS is formed by selectively leaving the n-type thermoelectric conversion material in the opening OP3.

Next, as shown in FIG. 9, the p-type semiconductor PS is formed in the opening OP4 provided in the insulating film IF5 in the same manner as in the method for manufacturing the n-type semiconductor NS.

Further, as shown in FIG. 4, an insulating film IF6 having an opening OP5 is formed on the insulating film IF5, and a common electrode COM is selectively formed in the opening OP5. The common electrode COM is the same as the method for manufacturing the column electrode CE described above. The common electrode COM is connected to the n-type semiconductor NS and the p-type semiconductor PS, and its main surface COMa is exposed from the main surface IF6a of the insulating film IF6.

Through the above steps, the cell CL of the temperature control device TCD according to the present embodiment is formed. The wiring WR and the terminal TA shown in FIG. 1 can be appropriately formed of a metal film constituting the row electrode RE, the column electrode CE, and the common electrode COM.

<Cell culture device>
Hereinafter, the cell culture device CCD, which is an application example of the temperature control device TCD of the present embodiment, will be described with reference to FIGS. 10 to 14. 10 is a plan view of the cell culture apparatus according to the present embodiment, FIG. 11 is a sectional view taken along the line CC of FIG. 10, and FIG. 12 is a sectional view of a main part of the cell culture apparatus according to the present embodiment. FIGS. 13, 13 and 14 are schematic plan views showing cultured cells in the cell culture apparatus according to the present embodiment. In FIG. 12, the insulating films IF1 to IF6 of FIG. 4 are collectively referred to as an insulating film IF.

As shown in FIGS. 10 and 11, the cell culture apparatus CCD includes a temperature control apparatus TCD, a container RS arranged on the temperature control apparatus TCD, a culture solution CF arranged in the container RS, and a culture solution CF. It is composed of a cell CEL placed therein. The container RS is arranged on the insulating film IF6 shown in FIG. 4 so as to cover the cell array region CLA. The common electrode COM of the cell CL, which is the temperature control unit, is configured to be in direct contact with the culture solution CF and the cell CEL in the container RS.

As shown in FIG. 12, a temperature-responsive material TRM is formed on the upper surface of the common electrode COM of the cell CL, which is the temperature control unit of the cell culture apparatus CCD. Then, the temperature-responsive material TRM can be made hydrophobic or hydrophilic by controlling the temperature in the cell CL to heat or cool the common electrode COM.

Then, the cells are easy to adhere to the hydrophobic surface and difficult to adhere to the hydrophilic surface. As a result, for example, in the cell culture apparatus CCD, when any common electrode COM is heated, the temperature-responsive material TRM on all the common electrode COM becomes hydrophobic, and the temperature-responsive material TRM on all the common electrode COM becomes hydrophobic. Cell CELs can adhere and form a pattern as shown in FIG.

On the other hand, when only a part of the common electrode COM is heated, only the temperature responsive material TRM on the heated common electrode COM becomes hydrophobic, and the temperature responsiveness on the unheated (or cooled) common electrode COM. The material TRM remains hydrophilic. Therefore, the cell CEL adheres only to the temperature-responsive material TRM on some common electrodes COM, and the cell CEL does not adhere to the other temperature-responsive material TRM. As a result, the pattern as shown in FIG. 14 can be formed.

Incidentally, examples of the temperature-responsive material TRM include a temperature-responsive polymer whose solubility in water dramatically changes due to a temperature change. For example, a temperature-responsive polymer is soluble in water at a low temperature, but when the temperature is raised to the lower critical solution temperature (LCST), it becomes insoluble, becomes cloudy and precipitates, and is cooled. Then, there is a polymer showing a reversible behavior of redissolving. Further, the temperature-responsive polymer is, for example, soluble in water at a high temperature, but insolubilizes below the upper critical solution temperature (HCST) and becomes cloudy and precipitates. There is a polymer showing. Examples of temperature-responsive polymers showing LCST include poly (N-isopropylacrylamide), poly (N-alkylacrylamide), and poly (N-vinylalkyl ether). In particular, poly (N-isopropylacrylamide) is preferable as a temperature-responsive material because its LCST is 32 ° C. and its properties change in the vicinity of room temperature.

In poly (N-isopropylacrylamide), at temperatures below LCST, the polymer chain is hydrated and stretched by the strong interaction between the amide bond site and water, and has a random coil-like conformation (hydrophilicity). ). On the other hand, poly (N-isopropylacrylamide) undergoes dehydration at a temperature higher than that of LCST, and becomes a globule state in which polymer chains are aggregated due to hydrophobic interaction (hydrophobicity).

In this way, in the cell culture apparatus CCD of the present embodiment, the cell CEL can be patterned, and further, the cell CEL is adhered by changing the position and number of the common electrode COM to be heated on the time axis. The pattern can be changed. From the above, in the cell culture apparatus CCD of the present embodiment, the patterning of the cell CEL can be dynamically changed.

Further, as described with reference to FIG. 3, a column electrode CE and a row electrode RE are connected to one end of the n-type semiconductor NS and the p-type semiconductor PS, and a common electrode COM is connected to the other end. Since the common electrode COM and the temperature-responsive material TRM are in contact with each other, the efficiency of cooling or heating can be improved.

FIG. 15 is a cross-sectional view of a main part of the cell culture apparatus according to the modified example 1 of FIG. As shown in FIG. 15, the surface of the common electrode COM is covered with a protective layer PL that does not react with the culture solution CF, and a temperature-responsive material TRM is formed on the upper surface of the protective film PL. The protective layer PL is made of a chemically resistant material so that the common electrode COM can be protected from the culture solution CF. Therefore, the protective layer PL is preferably made of gold (Au) or platinum (Pt).

FIG. 16 is a cross-sectional view of a main part of the cell culture apparatus according to the second modification of FIG. As shown in FIG. 16, for example, a monomolecular film MF is formed on the surface of the protective layer PL made of gold (Au), and the temperature responsiveness is obtained by reacting the monomolecular film MF with the temperature responsive material TRM. The adhesiveness of the material TRM to the common electrode COM can be improved.

The crystal structure of the temperature-responsive material TRM or the like can be easily confirmed by X-ray diffraction (XRD). In addition, observe the lattice image with a transmission electron microscope (TEM), and confirm the single crystal or polycrystal crystal structure and laminated structure from the spot-like pattern or ring-like pattern in the electron beam diffraction image. You can also.

The composition distribution of the temperature-responsive material TRM and the like includes XPS, Electron Probe Micro Analyzer (EPMA), Energy Dispersive X-ray Spectroscopy (EDX), and secondary ion mass analysis. It can be confirmed by a method (Secondary Ion Mass Spectrometry: SIMS) or the like. Further, the composition distribution of the magnetic material portion and the like can also be confirmed by using a method such as emission analysis or mass spectrometry by inductively coupled plasma (ICP).

Further, the arrangement of the Pelche elements can be confirmed by using a scanning electron microscope (SEM), an optical microscope, or the like.

Although the invention made by the present inventor has been specifically described above based on the embodiment, the present invention is not limited to the above embodiment and can be variously modified without departing from the gist thereof. Needless to say.

For example, a transistor may be connected in series to the cell CL between the column electrode CE and the row electrode RE, and the current flowing through the cell CL may be controlled by turning the transistor on / off.

CCD cell culture device CE row electrode (second electrode)
CEL cell CF culture solution CL cell CLA cell array region COM common electrode (third electrode)
COMa Main surface COMb Back surface IF, IF1 to IF6 Insulation film IF5a, IF6a Main surface MF Monomolecular film ML Metal film NS n-type semiconductor OP1 to OP5 Opening PL protective film PS p-type semiconductor RE row electrode (first electrode)
RS container SB board SBa main surface TA terminal TCD temperature control device TRM temperature responsive material WR wiring

Claims (9)

With the board
A plurality of first electrodes arranged on the substrate and extending in the first direction in a plan view,
A plurality of second electrodes arranged on the substrate and extending in a second direction orthogonal to the first direction in a plan view,
A plurality of cells arranged at the intersection of the plurality of first electrodes and the plurality of second electrodes, and a plurality of cells.
Equipped with
Each of the plurality of cells
A p-type semiconductor including a first end portion and a second end portion, wherein the first end portion is connected to one first electrode among the plurality of first electrodes.
An n-type semiconductor including a third end portion and a fourth end portion, wherein the third end portion is connected to one second electrode among the plurality of second electrodes.
A third electrode connecting the second end and the fourth end,
Have,
The plurality of first electrodes are composed of a plurality of first copper wirings.
The plurality of second electrodes are composed of a plurality of second copper wirings.
The third electrode is a temperature control device including a third copper wiring . In the temperature control device according to claim 1,
A temperature control device in which each of the plurality of cells is a Pelche element. In the temperature control device according to claim 1,
The p-type semiconductor is arranged on the one first electrode.
The n-type semiconductor is a temperature control device arranged on the one second electrode. In the temperature control device according to claim 1,
moreover,
A plurality of first terminals arranged on the substrate and electrically connected to the plurality of first electrodes.
A plurality of second terminals arranged on the substrate and electrically connected to the plurality of second electrodes,
Equipped with a temperature control device. With the board
A plurality of first electrodes arranged on the substrate and extending in the first direction in a plan view,
A plurality of second electrodes arranged on the substrate and extending in a second direction orthogonal to the first direction in a plan view,
A plurality of cells arranged at the intersection of the plurality of first electrodes and the plurality of second electrodes, and a plurality of cells.
A container placed on top of the plurality of cells and holding a culture medium containing the cells,
Equipped with
Each of the plurality of cells
A p-type semiconductor including a first end portion and a second end portion, wherein the first end portion is connected to one first electrode among the plurality of first electrodes.
An n-type semiconductor including a third end portion and a fourth end portion, wherein the third end portion is connected to one second electrode among the plurality of second electrodes.
The second end and the third electrode connected to the fourth end,
Have,
A cell culture device provided with a temperature-responsive material on the third electrode. In the cell culture apparatus according to claim 5 ,
moreover,
A cell culture apparatus having a protective layer disposed between the third electrode and the temperature-responsive material. In the cell culture apparatus according to claim 6 ,
The protective layer is a cell culture device made of gold or platinum. In the cell culture apparatus according to claim 6 ,
moreover,
A cell culture apparatus having a monolayer, which is disposed between the protective layer and the temperature responsive material. In the cell culture apparatus according to claim 8 ,
The monolayer is a cell culture device made of a thiol compound.

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