JP2006042671A - Cell-culturing microarray having electrode and method for electrically measuring cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell-culturing microarray installed with electrodes in grooves or tunnels connecting in a plurality of fine compartments capable of holding one cell each, and a method of measurement by utilizing the same. <P>SOLUTION: This cell-culturing microarray is provided by having in the plurality of compartment walls for confining the cells in a specific spacial arrangement and grooves or tunnels connecting the compartments, installing in the plurality of electrode patterns for measuring the electric potential change of the cells in each of the grooves or tunnels, and arranging an optically transparent semi-permeable membrane and a vessel for culturing liquid on the compartment wall. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a new cell culture microchamber capable of culturing nerve cells in a single cell unit while observing the state of cells under a microscope and simultaneously measuring potential changes related to cell activity.

  Conventionally, in order to observe changes in the state of cells and the response of cells to drugs, the average value of the cell population has been observed as if it were a single cell characteristic. In reality, however, cells rarely have a synchronized cell cycle in the population, and each cell expresses a protein in a different cycle.

  In order to solve these problems, techniques such as synchronized culture have been developed, but since the origin of the cultured cells is not from the same single cell, the difference in the genes of each of the derived cells before the culture is Differences in protein expression may be generated, and when analyzing the results of responses to stimuli, whether the fluctuations are derived from the fluctuations of the responses that the cell reaction mechanism itself has universally It was difficult to clarify whether the fluctuation originated from the difference (that is, the difference in genetic information).

  For the same reason, cell lines are generally not completely cultured from a single cell, so it was difficult to clarify whether the reproducibility of responses to stimuli fluctuates due to differences in the genes of each cell. .

  In addition, because there are two types of stimulation (signal) for cells, which are given by the amount of signal substances, nutrients, dissolved gas contained in the solution around the cell, and by physical contact with other cells, It was actually difficult to judge fluctuations.

  Furthermore, in order to monitor cell activity, detection methods in which cells themselves are damaged are often used, such as observing fluorescence images obtained by modifying proteins and sugar chains exposed on the cell surface with fluorescently labeled antibodies. .

  As a method that does not damage cells so much, a method of measuring intracellular calcium and pH with these indicators has been developed. In any case, an indicator (generally a fluorescent reagent) is generally inserted into the cell and measured. Alternatively, there is a measurement method devised so that a change in potential can be traced by bringing an electrode into contact with a cell.

  In measuring the state of cells, especially in neurons, a relatively simple neural network consisting of a small number of neurons is artificially constructed, and the cell network reveals information processing functions in a fully controlled environment. Research to try to do is also actively conducted (Non-Patent Documents 1-3).

  Multi-point simultaneous measurement technology and cell network pattern control technology are important for the measurement of information processing models in which each neuron is the smallest structural unit. Neuronal action potential measurement technology However, at the initial stage, the method of damaging cells such as the patch clamp method was mainly used, so it was not possible to measure at multiple points of 3 or more at the same time. In recent years, the development of a method for measuring and culturing nerve cells on an electrode array (MEAS) substrate has made it possible to overcome the above-mentioned problems and perform long-term culture for several weeks. ing.

  In addition, many researches have been made on techniques for controlling the network pattern of nerve cells using chemical or physical techniques. For example, in chemical methods, Letourneau et al. Succeeded in drawing a pattern with a cell-adhesive substrate such as laminin on the surface of a substrate on which neurons are cultured, and extending neurites along the pattern (for example, non- Patent Document 4).

  In the physical method, the growth and movement of nerve cells are restricted if the height of the barrier is about 10 μm or more by culturing on a substrate on which a step that becomes a barrier for the extension of nerve cells is constructed on the substrate surface. There is a report that this is possible (for example, Non-Patent Documents 5-6).

  The inventors' group selects only one specific cell, cultures that single cell as a cell line, and controls the solution environmental conditions of the cell when observing the cell, and A technology for controlling the cell concentration at a constant level or a technology for culturing and observing the cells while identifying interacting cells has been developed (Patent Document 1). Furthermore, a cell culture microchamber has been developed that can freely change the shape of a cell culture container in a region heated by irradiation with focused light while performing cell culture (Patent Document 2).

JP 2004-81086 A JP 2004-81085 A Dichter, M.A.Brain Res., 149, 279-293 (1978) and Mains R.E., Patterson P. H. J. Cell. Biol., 59, 329-345 (1973) Potter S.M., DeMarse T.B., J. Neurosci.Methods, 110, 17-24 (2001) Jimbo Y., Tateno T., Robinson H.P.C., Biophys. J. 76, 670-678 (1999) Letourneau P.C .: Dev. Biol., 66, 183-196 (1975) Stopak D. et al .: Dev. Biol., 90, 383-398 (1982) Hirono T., Torimitsu K., Kawana A., Fukuda J., Brain Res., 446, 189-194 (1988)

  The conventional electrode array substrate technology has a structure in which cells are in direct contact with electrodes. For this reason, it is possible to measure changes in the potential of the cell itself. For example, in a cell group that constitutes a network such as a nerve cell, information on which cell is interacting with it is lost, and all the averages are averaged. There is a problem that only the experimental result of value can be obtained. In a system that extends neurites and axons and interacts with multiple cells like a nerve cell, if you do not take data by strictly defining the relationship between the electrode and the direction and orientation of the cell, you can obtain useful data. Difficult to get.

  The present invention eliminates the above-mentioned problems of the prior art and clarifies the function of the cell, and changes the stimulation response of the cell network over a long period of time while completely controlling the shape of the intercellular network. Therefore, it is an object to provide a new technical means capable of performing electrical measurement.

  In order to solve the above problems, the present invention has a plurality of cell culture compartments for confining cells in a specific spatial arrangement, and each compartment is connected to each other by a groove through which cells cannot pass. In this groove, a structure having a plurality of electrode patterns for measuring changes in cell potential is provided.

  The electrode pattern is installed in a groove between adjacent cell culture sections, and has a structure capable of measuring a potential difference between cells. When the measurement cell is a nerve cell, the cell stretches the axon, and the potential change of the axon itself between the cells that couple with adjacent cells at the synapse is measured.

  According to the present invention, it is possible to continuously measure morphological changes and changes in electrical characteristics while culturing for a long time while controlling the spatial arrangement of a cell network in units of one cell. Since the electrodes for detecting the change in electrical characteristics are arranged between the cells, it is possible to obtain only information on the cells sandwiching the electrodes.

Example 1
1 is a top view schematically showing an example of the structure of a cell culture microarray with electrodes according to the present invention according to Example 1, and FIG. 2 is a cross-sectional view taken in the direction of the arrow at the position AA in FIG. It is. Reference numeral 1 denotes a substrate, and all the structures are constructed on the substrate 1. Reference numeral 2 denotes a cell culture section, which is periodically constructed at a predetermined interval. 3 is a groove that connects adjacent cell culture compartments. Reference numeral 10 denotes a resin layer, which is formed on the substrate 1, and the cell culture compartment 2 and the groove 3 communicating between them are formed by removing the resin layer 10. Reference numeral 4 denotes an electrode for potential measurement, which is provided in all of the grooves 3. The electrode 4 is deposited on the surface of the substrate 1 with gold having a thickness of 100 nm. Reference numeral 5 denotes an external terminal, which is provided in the vicinity of the substrate 1 corresponding to the electrode 4. Reference numeral 6 denotes a wiring for connecting the electrode 4 and the external terminal 5.

  Reference numeral 9 denotes a semipermeable membrane, which is provided in close contact with the upper surface of the resin layer 10 in which the cell culture compartment 2 and the groove 3 communicating between them are formed. An upper housing 22 covers the entire surface of the resin layer 10 with an appropriate space on the semipermeable membrane 9. Reference numeral 22-1 denotes a falling portion of the upper housing 22. Reference numeral 21 denotes a culture solution tank formed between the semipermeable membrane 9 and the upper housing 22. Reference numeral 23 denotes an opening provided in the upper housing 22, through which a culture solution is supplied to the culture solution tank 21. A common electrode 14 is provided in the culture solution tank 21.

  In Example 1, each cell culture section 2 has a side of 30 μm and a depth of 25 μm for the purpose of measuring cells derived from human cells one by one. The groove 3 has a width of 5 μm and a depth of 25 μm. The distance between the cell culture compartments 1 is 30 to 200 μm.

  A specific preparation method is described. The substrate 1 is a 0.18 mm non-fluorescent glass that can be observed with a 100 × objective lens. First, layers of electrodes 4, wirings 6, and terminals 5 are formed on the upper surface by vapor deposition. Next, the region of the electrode 4 and the terminal 5 is masked to form an insulating layer on the entire surface. Next, the photocurable resin SU-8 (epoxy-based photoresist material: manufactured by Micro Chem Inc., US Pat. No. 4,882,245) having a viscosity adjusted so that the layer thickness is 25 μm, the electrode 4, The resin layer 10 is formed by covering the insulating layer and the surface of the terminal 5. Next, positions corresponding to the cell culture section 2 and the groove 3 of the resin layer 10 are locally removed. Thereby, the cell culture section 2 and the groove 3 are formed. The exposed electrode 4 is disposed in the groove 3, but the wiring 6 crossing the groove 3 is isolated by an insulating layer. Also, the terminal 5 is exposed.

  A cell affinity substance such as laminin or collagen is applied to the surfaces of the cell culture compartment 2 and the groove 3, and the cell culture compartment 2 and the groove 3 are filled with a culture buffer, and the cells are put into the cell culture compartment 2. Next, in order to encapsulate the cells in the cell culture compartment 2, a cover of the semipermeable membrane 9 is put on the upper surfaces of the areas of the cell culture compartment 2 and the groove 3. Thereafter, an upper housing 22 that covers the entire lid of the semipermeable membrane 9 (the entire surface of the resin layer 10) is attached. At this time, the upper housing 22 has an appropriate falling portion 22-1, covers an appropriate space on the semipermeable membrane 9, and forms a culture bath 21. A common electrode 14 is formed on the inner surface of the upper housing 22 of the culture bath 21 using an optically transparent electrode such as ITO. Reference numeral 23 denotes an opening of the culture solution tank 21, and fresh culture solution is always supplied to and discharged from the culture solution tank 21 through this opening.

  The above-mentioned semipermeable membrane 9 uses a transparent cellulose membrane so as not to interfere with optical observation. Here, the molecular weight cut off is 30,000 daltons. The upper housing 22 is also made of a transparent material such as plastic so as not to interfere with optical observation.

  In this way, a cell culture microarray containing a plurality of cells can be prepared. Here, regarding the method of putting cells into the cell culture compartment 2, several methods are conceivable. For example, there is a method in which a microcapillary is inserted into a solution containing cells, one cell is caught at the tip, and this is put into the cell culture section 2. Alternatively, when the cell size is approximately the same as that of the cell culture compartment 2, a droplet containing cells is dropped on the upper surfaces of the cell culture compartment 2 and the groove 3 region, and the upper surface is traced so as to push out excess liquid. Thus, cells can be placed in the cell culture compartment 2.

  In order to stably fix the cells placed in the cell culture compartment 2, self-supporting bond formation by biotin-avidin reaction is used. Since the photocurable resin SU-8 has a reactive epoxy group, after pre-baking before light irradiation to form the substrate SU-8 layer, immediately apply a solution containing biotin hydrazide, The biotin is immobilized by reacting the epoxy group and hydrazide group of the resin. By irradiating light to solidify the resin to form a structure, a SU-8 pattern with biotin introduced on the surface can be obtained.

  The state in which the axon extends in the groove 3 is measured by measuring the impedance between the terminals 5 connected to the electrode 4 arranged in the groove 3 and the common electrode 14 or connected to the electrode 4. 5 can be detected by measuring the electromotive force due to the cell itself detected at 5 using the potential of the common electrode 14 as a reference value. All of this can be done while observing the cells with a microscope. Moreover, in the cell culture microarray with electrodes of the present invention, it is possible to stimulate or measure a specific cell with the same electrode by selecting a large number of electrodes.

  For example, as a result of culturing rat cerebellar granule cells with the cell culture microarray with electrodes of the present invention, cells placed in the cell culture compartment 2 form a network without escaping from the cell culture compartment 2. Observed. Before and after the axon is extended and the cells are in contact with each other, the electrode 4 arranged in the groove 3 between the cell culture compartments 2 where the axon is extended and the cells are in contact with each other, and the groove in which the axon on the opposite side of the cell is not extended It is confirmed that an electromotive force is generated between the electrodes 4 arranged on the line 3. From these, it can be seen that the structure of the cell culture microarray with electrode of the present invention exhibits the expected performance.

  In addition, biological responses such as peptides and amino acids, endocrine disrupting substances, and chemical substances suspected of toxicity can be added, and the cellular response can be measured by potential change.

(Example 2)
Example 2 describes an example in which the cell culture compartment 2 and the groove 3 are formed with an agarose gel 100 instead of the photocurable resin SU-8.

  FIG. 3 is a plan view of the second embodiment of the present invention, and FIG. 4 is a cross-sectional view seen in the arrow direction at the position BB in FIG. 1 and 2 and FIGS. 3 and 4, the groove 3 connecting the cell culture compartments 2 is changed to a tunnel 3, and a plurality of electrodes 4 are provided in the tunnel 3 and the tunnel 3 is Except for the fact that it is made only on the bottom surface of the agarose gel 100, the structure is essentially the same as in Example 1. However, 1-1 is a wall, provided on the substrate 1, and the peripheral portion of the agarose gel 100 is formed and held by this wall.

  In Example 2, after forming the electrode 4, the wiring 6, and the terminal 5 on the board | substrate 1 similarly to Example 1, the wall 1-1 is affixed on the upper surface of the board | substrate 1, and the agarose gel 100 is put in the wall 1-1. Insert. A 2% agarose gel (melting temperature 65 ° C.) is heated in a microwave to melt. A melted agarose solution is added to the inside of the outer wall of the lower substrate 1 heated to 65 ° C., and immediately spread to a uniform thickness using a spin coater. Here, the addition amount of the agarose gel and the rotation speed of the spin coater are adjusted so that the agarose gel film becomes 1 mm thick. Although the thickness varies depending on the device and the lot of agarose gel, good results are obtained at 50 rpm for 15 seconds and then at 200 rpm for 10 seconds. The agarose gel film 100 is formed by leaving it in a wet box at 25 ° C. for 1 hour. At this time, the agarose gel film is formed on the entire inner surface of the outer wall of the substrate 1. Next, in order to form the cell culture compartment 2 with the agarose gel 100, after the agarose gel 100 is formed, the portion of the cell culture compartment 2 is removed.

  FIG. 4 schematically shows a cross-sectional view of an agarose electrode-attached cell culture microarray having the cell culture compartment 2 created, and an optical system and a control system for creating the tunnel 3 in the agarose gel 100. The upper surface of the agarose gel is used by attaching a cellulose film in the same manner as in Example 1. For example, heat-melted agarose is applied to the surface of the cellulose film with a spin coater, and an agarose thin film is formed on one side in advance. After the cells are placed in the compartment 2, the agarose-coated surface comes into contact with the agarose gel 100. Just place it on Alternatively, when the agarose gel 100 is formed, a small amount of streptavidin-conjugated agarose is added and hardened. Streptavidin is exposed on the surface of the agarose gel derivative. Separately, as in Example 1, a biotin-modified cellulose membrane obtained by reacting biotin hydrazide with a cellulose membrane into which an aldehyde group has been introduced by periodate oxidation and reducing by a hydroboration reaction is prepared. By fixing the agarose gel derivative and the biotin-modified cellulose membrane using a biotin-avidin reaction, a structure in which cells are enclosed in an agarose structure can be formed.

  As the laser to be irradiated, a laser 1480 nm that is absorbed by water is used. The laser beam 142 passes through an expander 143, reflects infrared light of 740 nm or more, passes through a filter 144 that transmits light of 1480 nm (± 20 nm), passes through a vapor deposition filter 145 that transmits light of 700 nm or more, and collects the light. The optical lens 146 focuses on the upper surface of the substrate 1. The convergent light of 1480 nm is absorbed by the water contained in the agarose layer, and the temperature in the vicinity rises to near the boiling point. When the laser power is 20 mW, the agarose melts with a line width of about 20 μm in the vicinity where the focused light hits and is removed by thermal convection. The problem is that the intensity of the convergent light absorbed by the agarose changes without the substrate 1 electrode. Therefore, the agarose gel temperature is estimated and the laser power is controlled by feedback control so that the temperature can always be controlled by convergent light irradiation. The convergent light reaching the agarose part is converted into heat and emits infrared light. The infrared light passes through the filter 145, is reflected by the filter 144, and reaches the infrared camera 160-1. The image data of the infrared camera 160-1 is taken into the arithmetic device with a video recording mechanism 161, the temperature is estimated from the light detection intensity, and the power of the laser 141 is prepared. When it is difficult to control the temperature only with the laser power, the moving speed of the stage 164 is controlled by the output from the arithmetic unit so that the agarose temperature of the convergent light irradiation unit is always maintained. That is, the rotation of the stepping motor 162 is controlled by the arithmetic device 161, and the rotation of the stepping motor is a mechanism for moving the stage 164 by the power transmission device 163.

  The substrate 1 is mounted on the stage 164, and the tunnel 3 can be freely formed in the agarose gel. An ITO transparent electrode is formed in the tunnel 3 in advance. In addition, an optical system for detecting transmitted light from the light source 170 is also incorporated in order to monitor the progress of cell observation and agarose processing. Light from the light source 170 passes through the transparent upper housing 22, passes through the objective lens 146 while being scattered at the agarose portion, and is captured as an image by the CCD camera 160-2 by a vapor deposition filter (mirror) that reflects visible light. The image data is sent to the arithmetic device 161, overlapped with the infrared camera 160-1, and used for confirmation of a temperature rise portion and a structure pattern by laser irradiation.

  On the upper surface of the agarose gel, the culture solution supplied and discharged from the opening 23 is constantly circulating. Alternatively, various chemical substances such as a cell stimulating substance and an endocrine disrupting substance can be added from the opening 23, and the state of the cell can be monitored by an electrode or combined observation.

  In the second embodiment, since two electrodes are provided in one tunnel 3, it is easy to detect variations in impedance and inductance in the tunnel 3. Each electrode is coupled to a terminal 5 by a wiring 6 so that electrical measurement can be performed independently or as a pair. For example, when a nerve cell extends an axon and couples with a neighboring cell, the potential of the electrode 5 adjacent to one cell can be measured using another electrode as a reference potential.

  Moreover, in Example 2, since the tunnel 3 can be dug additionally, the activity condition of the cell which changed the tunnel structure can be evaluated, seeing the condition of research.

(Example 3)
FIG. 5 is a plan view showing Example 3 in which a plurality of cell culture sections 2 that are most practically important are adjacent to each other as a one-dimensional array. As is clear by comparing FIG. 5 and FIG. 3, in Example 3, the cell culture section 2 using the agarose gel 100 and the tunnel 3 connecting them are formed in the horizontal direction, and the others are performed. Same as Example 2. Even in Example 3, the tunnel 3 that connects the cell culture sections 2 does not necessarily have to be formed from the beginning. For example, only the direction in which the nerve cell wants to extend the axon is opened when the axon is formed. be able to. The electrodes 4 are all prepared in advance at positions where tunnels or future tunnels will be created.

  The cross-sectional view of the third embodiment is the same as that of the second embodiment, and is omitted.

(Other)
All of the above examples have been described as completed cell culture microarrays. However, the cell culture microarray requires researchers or the like who use the cell culture microarray to put cells or the like into the cell culture compartment 2. Accordingly, in Example 1, the substrate 1 on which the electrode 4, the external terminal 5, the wiring 6, the cell culture section 2 and the groove 3 are formed, the semipermeable membrane 9 and the upper housing 22 are supplied as a cell culture microarray kit. It is practical that a researcher or the like who has purchased this prepares a culture solution, puts cells or the like into the cell culture section 2, overlaps the semipermeable membrane 9 and the upper housing 22, and completes and uses it. is there. Similarly in Examples 2 and 3, the substrate 1 in which the agarose gel in which the cell culture compartment 2 and the necessary tunnel are formed, such as electrodes, is arranged on the substrate 1, the semipermeable membrane 9 and the upper housing 22 are arranged in the cell culture micro. It is practical that it is supplied as an array kit and used for the purpose of research.

The top view which showed typically one example of the structure of the cell culture microarray with an electrode of this invention concerning Example 1. FIG. Sectional drawing seen in the arrow direction in the AA position of FIG. The top view of Example 2 of this invention. Sectional drawing seen in the arrow direction in the BB position of FIG. The top view which shows Example 3 which made the some cell culture division 2 most practically important adjoined by using one-dimensional array.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Board | substrate, 1-1 ... Wall, 2 ... Cell culture division, 3 ... Groove, 4 ... Electrode for electric potential measurement, 5 ... External terminal, 6 ... Wiring, 9 ... Semipermeable membrane, 10 ... Resin layer, 14 ... Common electrode, 21 ... culture bath, 22 ... upper housing, 22-1 ... falling part of upper housing, 23 ... opening, 100 ... agarose gel, 141 ... laser, 142 ... laser beam, 143 ... expander, 144 ... Filters, 145 ... deposition filters, 146 ... condensing lenses, 160-1 ... infrared cameras, 160-2 ... CCD cameras, 161 ... calculation devices, 163 ... power transmission devices, 164 ... stages, 170 ... light sources.

Claims (9)

  A cell culture microarray in which electrodes are provided in grooves or tunnels that connect multiple microcompartments that can hold cells one by one.   A plurality of partition walls for confining cells in a specific spatial arrangement are provided on a substrate, and a plurality of electrode patterns for measuring cell potential changes are provided between the cells. A neuron cell culture microchamber characterized in that an optically transparent semipermeable membrane and a culture solution tank are arranged on the top.   A cell culture microarray in which a material is made of agarose and has a plurality of compartments for confining cells in a specific spatial arrangement, and electrodes are provided in the tunnels connecting the compartments.   On the substrate, the material is made of agarose or an agarose derivative, and there are multiple compartments for confining the cells in a specific spatial arrangement, and each compartment holds substantially one cell, In order to obtain interaction, a cell culture microarray in which agarose is locally heated with convergent light in any direction to form tunnels, and at least one electrode is arranged in each tunnel.   The cell culture microarray according to claim 3 or 4, wherein a culture solution tank capable of arbitrarily changing the solution is arranged on the upper surface of the agarose.   On the substrate, the material is made of agarose or an agarose derivative, and there are multiple compartments for confining cells in a specific spatial arrangement. Each compartment holds substantially one cell, and each cell In order to arbitrarily define the direction to secure the interaction between cells by extending axons etc., the agarose is locally heated with convergent light to form tunnels, connect the compartments, and use the electrodes placed in each tunnel to interact between cells An electrical cell measurement method that measures the potential change caused by aging.   On the substrate, the material is made of agarose and there are multiple compartments to keep the cells confined in a specific spatial arrangement. Each compartment holds substantially one cell, and each cell carries In order to arbitrarily define the direction to ensure the interaction between extended cells, agarose is locally heated and heated with convergent light to form tunnels, connect the compartments, and use the electrodes placed in each tunnel to stimulate electrical stimulation between the cells. An electrical cell counting method that measures changes in potential to which cells respond.   8. An electrical cell measurement method using a cell culture microarray according to claim 6 or 7, wherein a biological substance such as a peptide or amino acid, an endocrine disrupting substance, or a chemical substance suspected of toxicity is added, and the cell response is measured by a potential change. . On the substrate, there are tunnels connecting between the partition walls and a plurality of partition walls for confining cells in a specific spatial arrangement, and a plurality of electrode patterns for measuring cell potential changes are provided in each tunnel. An electrode placed in each tunnel is used using a nerve cell culture microchamber characterized in that an optically transparent semipermeable membrane and a culture bath are arranged on the partition wall. An electrical cell measurement method that measures electrical potential changes in response to electrical stimulation between cells.

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