Method for measuring electric signal of cell tissue, and sensitizer

Abstract

The purpose of the present invention is to easily and efficiently obtain an electric signal generated in cell tissue by increasing the electric signal obtained from the cell tissue in a state in which there is no damage to cells and observation with a microscope or the like is also possible.　The present invention pertains to: a method for measuring an electric signal of cell tissue by adding a perfluorocarbon compound having at least 5 carbon atoms; and an electric signal sensitizer for cell tissue, comprising a perfluorocarbon compound having at least 5 carbon atoms. Moreover, provided are: a method for measuring an electric signal of cell tissue, wherein the perfluorocarbon compound is preferably perfluorodecalin, the measurement of the electric signal of the cell tissue is preferably performed by means of an electrode array, and the addition of the perfluorocarbon compound is preferably an addition caused by substituting a culture solution; and an electric signal sensitizer for cell tissue.

Description

Method for measuring electrical signals in cell tissue and sensitizer

[0001] The present invention relates to a method for measuring electrical signals in cellular tissue and a sensitizer.

[0002] One method for elucidating the function of cellular tissues is to measure electrical signals generated in the cellular tissue. Two well-known methods for measuring electrical signals generated in cellular tissues are the patch clamp method, which measures electrical signals outside the cellular tissue, and the method of measuring electrical signals by inserting electrodes into the cellular tissue. Recently, a method for measuring electrical signals in cellular tissues using microelectrode arrays (MEAs) has become widely known (Patent Documents 1 and 2). This method is a non-invasive method that allows measurements to be made outside the cellular tissue, and it also allows for simultaneous observation of multiple locations in the tissue over long periods of time. It is possible to measure electrical signals generated in the cellular tissue using densely packed electrodes on the array. The development of high-density microelectrode arrays (HD-MEAs), which are based on solid-state imaging device technology using complementary metal oxide semiconductors (CMOS), has made a significant contribution to elucidating the function of cellular tissues (Non-Patent Document 1). This electrode array has extremely high temporal and spatial resolution, making it possible to observe the activity of cellular tissue in detail.

[0003] While various cell tissues can be targeted for electrical signal measurement, representative examples include neurons and cardiomyocytes. Recently, in vitro electrical signal measurement has become increasingly important in primary cells derived from human induced pluripotent stem cells (hiPSCs) and neurons, among other cell tissues for which electrical signal measurement is useful. These cell tissues range from simple two-dimensional cell tissues consisting of a single cell to complex three-dimensional cell tissues with complex cellular structures. Electrical signal measurement is possible regardless of the shape of the cell tissue.

[0004] Planar MEAs designed for single-cell tissues have a limited contact area with the measurement surface when measuring 3D tissues. Therefore, various electrode designs have been reported to enable and further improve 3D recording, including shell-shaped electrodes, stretchable mesh nanostructured electrodes, and kirigami-structured electrodes that transition from 2D to 3D basket-like structures, for neural organoids, which are generally round in shape. Thus, continuous improvements in the fabrication techniques for neural tissues such as organoids and advances in electrode technology for cellular research are enhancing the utility of electrophysiological testing. These advances are expected to elucidate fundamental principles of brain function, provide insights into neurological diseases, and facilitate the discovery of novel therapeutics. In particular, 3D tissues such as neural organoids have attracted great interest because they can be induced from human induced pluripotent stem cells and can recapitulate important brain conditions. However, these electrodes are complex, making them difficult to fabricate, difficult to handle, and impossible to observe under a microscope. Therefore, there has been a demand for a method for measuring electrical signals that is simple, causes minimal damage to cell tissue, and can be observed using a microscope, but such a convenient method has not existed until now.

[0005] On the other hand, from a completely different perspective, a substance called perfluorodecalin (hereinafter referred to as "PFD") is known in the biotechnology field. PFD consists of 10 carbon atoms and 18 fluorine atoms, but its properties are highly hydrophobic and chemically stable. Because it is fully fluorinated, PFD is immiscible with water and is known to function as an effective oxygen carrier. Furthermore, PFD is colorless and chemically and biologically inert. These unique properties make PFD a promising candidate for a wide range of applications in biotechnology. For example, PFD-based compositions have been studied to locally increase oxygen supply to promote wound healing, such as in second-degree burns on pig skin and acute eye burns. They have also been shown to enhance short-term tissue preservation during organ isolation and transplantation. Furthermore, they are widely used in biomedical imaging as a contrast agent for magnetic resonance imaging (MRI) and ultrasound imaging. Furthermore, PFD nanoparticles are expected to be applied to drug delivery systems, and they are used to encapsulate hydrophobic drugs, attracting attention for their solubility and controlled release. Furthermore, PFD-like substances such as perfluorohexane (hereinafter referred to as "PFH"), perfluoroheptane (hereinafter referred to as "PFHept"), and perfluorooctane (hereinafter referred to as "PFO") are well known. It is preferable that they are liquid at room temperature or the incubation temperature of around 37°C.

[0006] International Publication No. 2015 / 012955 International Publication No. 2020 / 189172

[0007] Muller, J. et al. High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels. Lab Chip 15, 2767-2780 (2015)

[0008] An object of the present invention is to easily and efficiently obtain electrical signals generated in cell tissue.

[0009] The electrical signals emitted by cellular tissues are weak, and the measurable electrical signals are also small. Furthermore, existing electrodes for obtaining a large number of electrical signals are difficult to handle and do not allow observation under a microscope or the like. The present inventors have surprisingly discovered that by adding a perfluorocarbon compound having 5 or more carbon atoms, which is a chemical substance, for measuring the electrical signals of cellular tissues, it is possible to increase the electrical signals obtained from cellular tissues with minimal damage to cells, while still allowing observation under a microscope or the like, and thus to easily and efficiently obtain the electrical signals generated in cellular tissues, thereby completing the present invention.

[0010] That is, the present invention is characterized by the following: 1. A method for measuring electrical signals in cellular tissue, comprising adding a perfluorocarbon compound having 5 or more carbon atoms and measuring the electrical signals in the cellular tissue. 2. The method for measuring electrical signals in cellular tissue according to 1 above, wherein the perfluorocarbon compound is perfluorodecalin. 3. The method for measuring electrical signals in cellular tissue according to 1 or 2 above, wherein the measurement of the electrical signals in cellular tissue is performed using an electrode array. 4. The method for measuring electrical signals in cellular tissue according to 1 or 2 above, wherein the perfluorocarbon compound is added by replacing the culture medium. 5. An electrical signal sensitizer for cellular tissue, comprising a perfluorocarbon compound having 5 or more carbon atoms. 6. The electrical signal sensitizer for cellular tissue according to 5 above, wherein the perfluorocarbon compound is perfluorodecalin.

[0011] As shown in the examples of the present application, by adding a perfluorocarbon compound having 5 or more carbon atoms, the sensitivity of measuring electrical signals generated in cellular tissue can be increased, and electrical signals generated in cellular tissue can be measured easily and efficiently, without damaging the cellular tissue and in a state that allows observation using a microscope or the like.

[0012] Comparative images of mouse primary hippocampal cells (7 days in vitro) after PFD addition. Scale bar: 250 μm. "Control" is a comparative example before PFD addition. Cell surface area (μm 2) comparison. Increased with PFD addition. 3D reconstruction images of Z-stacks of primary hippocampal cells stained with calcein AM, acquired using a confocal imaging system, comparing PFD addition. Scale bar: 20 μm. Cell body height (μm) is flattened under PFD conditions. From top to bottom: comparison of active electrode maps of chips with PFD addition (one side active), comparison of average spike amplitude maps of chips with PFD addition (μV), and comparison of average spike rate maps of chips with PFD addition (Hz). The number of detected active electrodes is significantly higher with PFD addition. "Washed" indicates the time after PFD removal. A histogram of spike amplitude (μV) of active electrodes with PFD addition shows a distribution shifted to higher amplitudes under PFD conditions. The average spike amplitude (μV) of active electrodes is significantly higher under PFD conditions. A comparison of histograms of average spike rate (Hz) of active electrodes with PFD addition. Significantly higher under PFD conditions. The average spike rate (Hz) of active electrodes with PFD addition is significantly higher under PFD conditions. Changes in hippocampal cell activity with PFD addition. Comparative images of cortical organoids with PFD addition. The organoid diameter (mm) increases with the addition of PFD, and recovers after washing the PFD. Comparative confocal images of organoids at the focal plane of the culture dish bottom with PFD addition. Scale bar: 100 μm. The contact area of ​​the organoid with the bottom surface (mm 2 ) comparison. The area increased with the addition of PFD. Comparative map of spike amplitude (μV) of organoids with and without PFD. Scale bar is 0.5 mm. Functional contact area (mm) based on spike amplitude map. 2) increased with PFD addition. The bars in each graph, from left to right, represent comparisons between before and after PFD addition, immediately after washing, and the day after washing. The graphs, from left to right, show (i) interburst interval, (ii) mean firing rate, and (iii) burst duration. No significant differences were observed in interburst interval and burst duration, while a significant difference was observed in mean firing rate. Functional connectivity maps based on pairwise spike correlations between spike-sorted units in organoids. (i) is a control without PFD addition. (ii) is with PFD addition. The dots in the figure represent the spatial location of single units on the HD-MEA. The dot size indicates the mean spike amplitude, and the density indicates the number of functional connections found. Gray lines represent functional connectivity between single units, and the thickness of the line indicates the strength of the correlation. The right panels of (i) and (ii) plot the extracellular spike waveform trajectories of spike-sorted units selected from the boxed area in the lower left of the left panel. Changes in spike amplitude maps as a function of PFD dose. Spike amplitude maps of organoids without PFD and with different PFD doses. Scale bar: 500 μm. Changes in histograms of mean spike amplitude of active electrodes as a function of PFD dose. Histograms of mean spike amplitude of organoids without PFD and with different PFD doses. Recorded when PFD was added to organoids immediately after mounting on an HD-MEA. (A) Active electrode maps with and without PFD. Active electrodes are white, and inactive electrodes are black. Scale bar: 0.5 mm. (B) The number of detected active electrodes is significantly higher with PFD than under the control condition without PFD. (C) Spike raster plot. This plot shows the time at which spike activity was detected for each electrode (channel) over time. More electrodes detected neural activity with PFD than under the control condition without PFD. Activity map (center) and its enlarged view (right) from axon bundle recording analysis of PFD-treated motor neuron organoids. Bandpass filtered signal from axon bundle recording on channel 11 in the right panel of Figure 4A. The vertical axis is amplitude (μV). Example of action potential waveform from channel 11 in the right panel of Figure 4A. The center line is the average. The vertical axis is amplitude (μV).Active potentials propagate along the axon bundle due to neural activity in selected channels. Channels 1 to 11 are shown from top to bottom. Each division on the horizontal axis is 50 ms, and each division on the vertical axis is 50 μV. Histogram of spike counts versus stimulation timing (gray area) during optogenetic stimulation. The vertical axis represents spike counts. (i) Map of active electrodes (scale bar 0.5 mm) and (ii) distribution of mean spike amplitude (μV) when different perfluorocarbon compounds with 5 or more carbon atoms were applied to cortical organoids. The different perfluorocarbon compounds with 5 or more carbon atoms tested were perfluorohexane (PFH, C6F). 14 ), perfluoroheptane (PFHept, C7F 16 ), perfluorooctane (PFO, C8F 18 ), perfluorododecalin (PFD, C 10 F 18 Comparison of the number of active electrodes between different perfluorocarbon compounds with 5 or more carbon atoms. Comparison of the average spike amplitude at the active electrodes between different perfluorocarbon compounds with 5 or more carbon atoms.

[0013] The present invention will be described in detail below.

[0014] The present invention relates to a method for measuring electrical signals from cellular tissue, which involves adding a perfluorocarbon compound having 5 or more carbon atoms to the cell. The cellular tissue to be measured can be any cellular tissue, regardless of whether it emits an electrical signal or not. As described in the Background Art section, there are various methods for measuring electrical signals. In any method, the addition of a perfluorocarbon compound having 5 or more carbon atoms improves the sensitivity of the electrical signal measurement. Furthermore, it is preferable that the electrical signals from the cellular tissue be measured using an electrode array. It is also more effective and preferable that the perfluorocarbon compound having 5 or more carbon atoms is added by replacing the culture medium. Furthermore, the present invention relates to an electrical signal sensitizer for cellular tissue, which comprises a perfluorocarbon compound having 5 or more carbon atoms. The hydrocarbon portion of the perfluorocarbon compound may be linear, branched, or alicyclic, and may be saturated or unsaturated. There are no particular limitations as long as the number of carbon atoms is 5 or more, but examples of linear or branched perfluorocarbon compounds having 5 or more carbon atoms that are easily available include perfluorohexane, perfluoroheptane, and perfluorooctane, and examples of alicyclic perfluorocarbon compounds having 5 or more carbon atoms that are easily available include perfluorodecalin.

[0015] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0016] In the examples, three types of cell tissues were prepared: hippocampal neurons, cortical organoids, and motor neuron organoids, and the electrical signals of each cell tissue were measured.In addition, the electrical signals of cell tissues were measured using perfluorohexane, perfluoroheptane, perfluorooctane, and perfluorodecalin as sensitizers as perfluorocarbon compounds with 5 or more carbon atoms.It is estimated that the prepared tissue cells can obtain a lot of knowledge by analyzing the results of measuring electrical signals, but the improvement of measurement sensitivity by measuring electrical signals is not limited to the cell tissue, and can be applied to any cells.

[0017] <Fabrication of PDMS Culture Chip> A polydimethylsiloxane (hereinafter referred to as "PDMS") culture chip was fabricated using a well-known procedure. First, a master mold was fabricated, and a PDMS device was fabricated using a mask. The master mold was fabricated by pouring negative photoresist, SU-8 2100 or 2075 (manufactured by Nippon Kayaku Co., Ltd.), onto a silicon wafer and spin-coating at 1200-1500 rpm for 30 seconds. Next, the wafer was baked on a hot plate at 65°C for 9 minutes and at 95°C for 40 minutes. Afterwards, the chip was exposed to UV light (365 nm, 2.5-3.0 mW / cm). 2 ) for 60–75 seconds using a photomask with the desired pattern. The wafer was baked on a hotplate at 65°C for 7 minutes and then at 95°C for 13 minutes. After cooling, it was developed with SU-8 developer for 15 minutes and washed three times with isopropyl alcohol (IPA). The wafer was then baked in an oven at 150°C for 3 minutes. Culture chips were fabricated using the PDMS silicone elastomer kit Sylgard 184 (Dow Corning). The silicone elastomer and curing agent were mixed in a 10:1 weight ratio, degassed, poured into a master mold, and cured overnight in an oven at 80°C. PDMS structures were cut out with a scalpel, and holes for organoids were punched with a 1.5 mm biopsy punch.

[0018] <Cell plating> For primary hippocampal neurons, HD-MEA chips were precoated with 0.07% polyethyleneimine (PEI) in 50 mM borate buffer (pH 8.2) and cultured at 37°C and 5% CO2 for 1 hour. The chips were washed three times with sterile water and incubated in 0.02 mg / ml laminin (feeding medium (FM)) at 37°C and 5% CO2 for 1 hour. 100k dissociated cells were plated at 2000 cells / mm3. 2Cells were plated onto the chip area at a cell density of 1000 kJ / cm. Cells were cultured in FM at 37°C with 5% CO2, with medium changes every 3–4 days during the culture period. For motor neuron organoids (MNOs) and cortical organoids, the sensing area of ​​the HD-MEA chip was coated with Matrigel in DMEM / F12 (1:50) medium supplemented with HEPES (hydroxyethylpiperazine ethanesulfonic acid) buffer and incubated at room temperature for 1 hour. Organoids were placed on the center of the electrode surface and cultured in NMM (neuronal maintenance medium) at 37°C with 5% CO2. Cortical organoids were plated onto the MEA after 3–4 months of maturation, and motor neuron organoids were plated after 2 weeks. Medium changes were performed every 3–4 days during the culture period. For recording, HD-MEAs were also coated with Matrigel in DMEM / F12 (1:50) supplemented with HEPES and incubated at room temperature for 1 hour. After replacing the coating solution with RM (recording medium), the cortical organoids were placed on the MEA. For motor neuron organoid recording, the PDMS culture chip in the well was peeled off from the bottom and held with forceps. To detach the MNOs from the PDMS structure, a stream of culture medium was added to the well by pipetting. Once the MNOs were floating, they were picked up using a wide-bore pipette tip coated with 2% serum albumin (BSA) in phosphate-buffered saline (PBS) and transferred to the MEA surface.

[0019] <Electrophysiological recording> For the experiment, a 3.85 x 2.10 mm 2We used a CMOS-based HD-MEA (MaxOne, Maxwell Biosystems) containing 26,400 platinum electrodes covering a sensing area of ​​1000 μm. The microelectrodes have a diameter of 7.5 μm and a center-to-center distance of 17.5 μm. The system has 1,024 configurable low-noise readout channels and records at a sampling rate of 20 kHz. All recordings were performed using MaxLab Live software (v.20.1.6, Maxwell Biosystems) in a cell culture incubator at 37°C and 5% CO2 for 24 h after medium change. First, we performed an activity scanning assay, recording multiple defined areas of the MEA 365 times for 30 seconds. For 2D primary cell cultures, we scanned seven electrode configurations with a total of 6,600 electrodes at a 35 μm pitch covering the entire sensing area. For organoid recordings, we acquired images of the organoid position on the chip and manually selected the electrodes covered by the organoids at a 17.5 μm pitch to scan the electrodes. The results of the scan were used to identify active electrodes based on spike frequency and spike amplitude, and readout channels were routed to the 1024 most active electrodes.

[0020] <Addition of PFD> The medium was slowly removed until the meniscus of the medium touched the culture surface or the bottom of the cells. The removed conditioned medium was saved so that it could be added to the cells again after PFD addition. 100 μl of prewarmed PFD was slowly and gently added to the side of the well using a 200 μl pipette tip. After the experiment, the PFD was slowly removed from the edge of the chip while keeping the pipette tip perpendicular to the bottom of the chip surface. After removing the PFD, the saved conditioned medium was returned to the cells.

[0021] Optogenetic stimulation was used to stimulate organoids. At least 1 week before the experiment, 1 μl of AAVCAG-hCHR2-tdTomato (VectorBuilder Inc.) was added to 1 ml of culture medium. For stimulation, a 470 nm fiber-coupled LED (M470F3 - 470 nm, 17.2 mW (min) fiber-coupled LED, 1000 mA, Thorlabs) was used, controlled by a T-cube LED driver (LEDD1B, 1.2 A, Thorlabs). A multimode fiber (0.22 NA, High-OH, 105 μm diameter core, 250–1200 nm, Thorlabs) was connected to the LED and passed through a coin-shaped PDMS lid. The lid was placed on the electrode chip and aligned so that the tip of the fiber was positioned above the organoid. A 200ms TTL pulse at 0.1Hz was generated and sent by an Arduino Uno Rev3 device to control the LED driver.

[0022] Data analysis: The collected data were analyzed using MATLAB 2018b (MathWorks). The recorded signals were band-pass filtered in the frequency range of 300-3000 Hz, and nine positive and negative peaks with absolute values ​​greater than or equal to five times the standard deviation were identified as spikes.

[0023] Immunohistochemistry and Microscopy: Cells and tissues were fixed with 4% paraformaldehyde for 1 hour and washed three times with PBS. Tissues were permeabilized with 0.1% Triton-X in PBS for 30 minutes and washed three times with PBS. Afterwards, cells were blocked with 2% goat serum in PBS for 1 hour at room temperature. Tissues were then incubated overnight at 4°C in primary antibodies in 2% goat serum in PBS. After rinsing three times with PBS, cells were incubated with secondary antibodies in 2% goat serum in PBS for 2 hours at room temperature. Cell nuclei were labeled by incubating cells in 1:1000 Hoechst solution in PBS for 10 minutes, followed by rinsing three times with PBS.

[0024] Statistics: All analyses were performed in a minimum of three replicates. All statistical tests and graphical displays were performed using MATLAB® 2018b. Comparisons between two sample groups were performed using the Student's t-test with the ttest() function. Comparisons between more than two sample groups were performed using one-way analysis of variance (ANOVA) with the anova1() function, followed by a Tukey-Kramer post-hoc test for pairwise comparisons with the multicompare() function.

[0025] Hippocampal neurons were obtained from embryonic day 17 ICR mouse embryos (CLEA Japan). Specifically, hippocampal cells were extracted from mice, washed three times with HBSS 085-09355 (Hanks' Balanced Salt Solution, Wako), and then incubated with trypsin and DNase for 10 minutes at room temperature. The cells were then washed three times with HBSS and dissociated in Neurobasal Medium (Thermo Fisher Scientific 21103049) supplemented with 2% (v / v) B27 supplemented with vitamin A, 0.25% (v / v) GlutaMAX (Thermo Fisher Scientific 35050061), and 1% (v / v) PSA (Sigma A5955). Single cells were then centrifuged at 250×g for 5 minutes and resuspended in FM. 300k cells were seeded per well in a culture-treated 24-well plate and treated with PLL in 50mM borate buffer (pH 8.2) at 37°C and 5% CO2 for 1 hour. Cells were cultured in FM. Cells were maintained at 37°C and 5% CO2, and medium was changed every 3-4 days during the culture period.

[0026] Primary hippocampal neurons from mice were cultured in vitro. After removing the culture medium and casting a PFD on the neurons, the surface area of ​​the cell bodies, as observed microscopically, significantly increased (see Figures 1A and 1B). Furthermore, labeling the cells with calcein-AM and observing them by confocal imaging further supported these observations. 3D reconstructions of neurons imaged under PFD conditions showed a decrease in cell body height (see Figures 1C and 1D), providing further evidence of the flattening effect induced by PFD.

[0027] Primary hippocampal neurons were cultured on HD-MEAs for 3 weeks and stained with neuronal markers, such as NeuN and βIII-tubulin. Cells were recorded under both standard and PFD-treated conditions. After recording, the PFD was washed and reintroduced with the conditioned medium of the control group. Electrical readout of recordings with the PFD showed enhanced activity compared to the pre-PFD condition (see Figures 2A–G). Specifically, scanning the entire MEA surface revealed significantly more active electrodes under the PFD condition than under the control group (see Figures 2A-B). Furthermore, removing the PFD and reintroducing the conditioned medium used before PFD recordings revealed a similar number of active electrodes to the pre-PFD condition (see Figure 2B). MEA maps depicting the mean spike amplitude of electrodes showed more and higher amplitudes under the PFD condition compared to the control condition (see Figures 2A-C). The distribution of mean spike amplitude of electrodes was skewed toward higher values ​​under the PFD condition, with the mean value significantly higher than that of the control (see Figure 2C). The mean spike rate was also significantly higher under the PFD condition (Fig. 2E, F). Collectively, these results suggest that the addition of a PFD improves recording, and its effect is reversible, as all parameters returned to baseline levels after washing.

[0028] <Cortical Organoids> Human iPS cell (hiPSC) lines were obtained from Kyoto University through the RIKEN BioResource Center Cell Bank (409B2, HPS0076). hiPSCs were cultured in mTeSR plus medium on 12-well tissue culture plates coated with human embryonic stem cell-compatible Matrigel. The medium was changed every other day and the cells were passaged at 80% confluence. Briefly, hiPSCs were washed with calcium- and magnesium-free PBS, lifted from the plate with ReLeSR reagent (Stemcell Technologies), and seeded into new Matrigel-coated wells.

[0029] Cortical organoids were cultured according to previously reported procedures. HiPS cells were dissociated from 70-80% confluence by incubating in TrypLE Express at 37°C and 5% CO2 for 3 minutes. Cells were resuspended in HEPES-supplemented DMEM / F12 and centrifuged at 250xg for 5 minutes. 10,000 cells per well were seeded into a U-bottom 96-well plate (Prime surface, Sumitomo Bakelite) containing mTeSR plus ultra-low binding medium supplemented with 10 μM Y-23632. After 24 hours, the medium was changed to neural induction medium (NIM) consisting of HEPES-containing DMEM-F12, 15% (v / v) knockout serum replacement, 1% (v / v) minimal essential medium non-essential amino acids (MEM-NEAA), and 1% (v / v) Glutamax, supplemented with 100 nM LDN-193189, 10 μM SB431542, and 5% (v / v) heat-inactivated FBS. NIM was replaced without FBS on day 2 and every other day until day 10. From days 10 to 18, the medium was replaced every other day with neural differentiation medium 1 (NDM1), which consisted of a 1:1 mixture of DMEM / F12 with HEPES and Neurobasal medium, 0.5% (v / v) N2 supplement, 1% (v / v) B27 supplement without vitamin A, 1% (v / v) Glutamax, 0.5% (v / v) MEM-NEAA, 0.25 mg / ml human insulin solution, and 1% (v / v) penicillin / streptomycin / aphotericin (PSA) (Sigma, A5955). On day 18, the medium was replaced with neural differentiation medium 2 (NDM2). The medium consisted of Neurobasal medium, 0.5% (v / v) N2 supplement, 1% (v / v) B27 supplement with vitamin A, 1% (v / v) Glutamax, 0.5% (v / v) MEM-NEAA, 0.25 mg / ml human insulin solution, 200 mM ascorbic acid, and 1% (v / v) PSA, supplemented with 20 ng / ml brain-derived neurotrophic factor (BDNF).From day 28 onwards, at each medium change, the medium was gradually (25% steps) switched to recording medium (RM) consisting of Brainphys medium supplemented with 2% (v / v) B27 supplement with vitamin A, 1% (v / v) Glutamax, 1% (v / v) PSA, and 20 ng / ml BDNF. Cells were maintained at 37°C and 5% CO2, and NMM medium changes were performed every 3–4 days during the organoid culture period.

[0030] We evaluated the effects of PFD on three-dimensional neural tissue models, particularly cortical organoids. These organoids were generated using hiPSCs and cultured for 4 months before and after plating on HD-MEAs. Experiments were performed with or without PFD. The flattened morphology observed in two-dimensional cultures was also evident in three-dimensional tissues. Comparing the diameter of organoids observed from the top of the chip before and after PFD treatment revealed a significant increase in size, and removal of the PFD restored their size to pre-PFD size (Figures 3A and 3B). To examine changes in the contact area with the MEA, organoids were generated from an iPS cell line expressing mCherry and plated in well plates. Confocal microscopy images of fluorescent organoids taken from below the culture plate revealed an increased surface diameter at the focal plane where the cells contacted the bottom of the culture dish, indicating an increased contact area with the surface in the presence of PFD (Figure 3C). Electrophysiological recordings of organoid electrical footprints confirmed that the contact area between the organoid and the MEA was increased upon application of a PFD (see Figure 3D). Organoids exhibited consistent network-wide bursting behavior, with silent periods between each burst, both under normal conditions and in the presence of a PFD. Recordability and network parameters were assessed before the experiment, during PFD application, after PFD removal, and 1 day after the experiment (see Figure 3G). Recordability parameters, including the change in the number of active electrodes, the change in the number of spikes during a burst, and the mean spike amplitude, were significantly elevated under the PFD condition compared with pre-PFD recordings (see Figure 3B). These values ​​returned to baseline levels after PFD removal and 1 day after the experiment, demonstrating the reversibility of the enhanced recording effect.

[0031] Furthermore, analysis of functional connectivity between spike-sorted units, calculated based on pairwise spike correlations using the Spike Time Tiling Coefficient (STTC; see Cutts et al., Detecting Pairwise Correlations in Spike Trains: An Objective Comparison of Methods and Application to the Study of Retinal Waves, Journal of Neuroscience 34, 14288-14303 (2014)), revealed the emergence of new spike-sorted units and increased correlations between spike pairs of single units, revealing many connections between cells within the organoid (see Figure 3H).

[0032] Furthermore, under the same conditions as above, we also examined the effects of adding PFD in amounts of 10, 25, 50, 100, and 200 μl. The HD-MEA used was the same MaxWell Biosystems MaxOne (https: / / www.mxwbio.com / wp-content / uploads / 2024 / 10 / MaxOne-Brochure-online-version.pdf), which is circular and measures 19 mm in diameter and 8 mm in height. The amount of PFD needed should be sufficient to cover the organoids; adding at least 10 μl, preferably 50 μl, and even more preferably 100 μl, will maximize cell activity (see Figures 3I and 3J).

[0033] We also performed measurements under the same conditions as above, but without any time delay, after placing the organoids on the HD-MEA and immediately adding PFD. Even when PFD was added immediately after placing the organoids on the MEA, we were able to measure cell activity significantly better than when no PFD was added (see Figure 3K).

[0034] <Motor neuron organoids> Motor neuron spheroids were formed using a known procedure. hiPSCs were dissociated from 70-80% confluence by incubating in TrypLE Express at 37°C and 5% CO2 for 3 minutes. Cells were resuspended in HEPES-supplemented DMEM / F12 and centrifuged at 250xg for 5 minutes. 40,000 cells per well were seeded into a U-bottom, ultra-low-attachment 96-well plate (Prime surface, Sumitomo Bakelite) in mTeSR plus 7 medium supplemented with 10 μM Y-23632. After 24 hours, the medium was replaced with NIM supplemented with 100 nM LDN-193189 and 10 μM SB431542 for 2 days. The basal medium was gradually switched to N2 medium (N2M), consisting of Neurobasal Medium, 1% (v / v) N2 supplement, 1% (v / v) GlutaMAX, and 1% (v / v) PSA. Specifically, the basal medium ratio NIM:N2M was changed every other day on days 2 (4:0), 4 (3:1), 6 (1:1), 8 (1:3), and 10 (0:4). From days 2 to 6, the basal medium was supplemented with 10 μM SB431542, 100 nM LDN-193189, 5 μM DAPT, 5 μM SU5402, 1 μM RA, and 1 μM SAG. From days 6 to 12, the medium was supplemented with 5 μM DAPT, 5 μM SU5402, 1 μM RA, and 1 μM SAG. After 12 days of culture, the medium was replaced with neural maintenance medium (NMM), consisting of Neurobasal medium supplemented with 2% (v / v) B27 supplement containing vitamin A, 1% (v / v) Glutamax, 1% (v / v) PSA, and 20 ng / ml BDNF. Cells were maintained at 37°C and 5% CO2, and NMM medium was replaced every 3–4 days during the culture of 305 spheroids.

[0035] A custom-made PDMS culture chip was disinfected by immersion in 70% ethanol, allowed to dry, and then attached to the bottom of a well in a culture-treated 12-well plate. The internal channels of the chip were coated with Matrigel in DMEM / F12 with HEPES (1:50) for 1 hour at room temperature. The coating solution was replaced with NMM, and a d12 motor spheroid was placed into one of the wells of the culture device using a wide pipette tip. Axons from the spheroid began to extend to the surface. Due to the physical constraints of the PDMS culture chip, axons could only extend along the channels, and after 2 weeks of culture, they spontaneously formed axon bundles. Successful axon bundle formation resulted in a motor neuron organoid.

[0036] To examine the ability of this method to capture axonal signals from bundled axons, we generated motor neuron organoids and measured electrical signals upon addition of PFD. The generated motor neuron organoids were then removed from the chip and placed on an MEA for acute recording. The MEA was scanned to determine active electrodes. Most of the detected active electrodes overlapped with organoid structures containing axon bundles, suggesting that the detected activity originated from neural tissue. Spiking activity was detected at multiple electrodes beneath the axon bundles. In particular, the propagation of axonal activity could be visually tracked along the axon bundles. Motor neuron organoids were virally transfected to express channelrhodopsin (ChR2[H134R]) and then subjected to optogenetic stimulation. The organoid bodies of plated motor neuron organoids were stimulated every 10 seconds with 200-ms pulses of blue light (470 nm) using an optical fiber. Overlaying spike counts at the time of stimulation revealed increased spike activity during the light-illuminated period. Furthermore, by examining specific recording channels, we easily observed that some stimuli induced spikes in the organoids, followed by propagation of activity along the axon bundles (see Figure 4A-E).

[0037] <Perfluorocarbon compounds with 5 or more carbon atoms> Next, using the cortical organoids of Example 2, perfluorohexane (PFH, CF 14 ), perfluoroheptane (PFHept, C7F 16 ), perfluorooctane (PFO, C8F 18 ), perfluorododecalin (PFD, C 10 F 18 We measured electrical signals in tissues using perfluorohexane (PFH), perfluoroheptane (PFHept), perfluorooctane (PFO), and perfluorododecalin (PFD) as sensitizers. All compounds, including perfluorohexane (PFH), perfluoroheptane (PFHept), perfluorooctane (PFO), and perfluorododecalin (PFD), produced significantly larger electrical signals than those produced by the addition of culture medium (control). In particular, perfluoroheptane (PFHept) and perfluorooctane (PFO) produced larger electrical signals than perfluorohexane (PFH), and perfluorododecalin (PFD) produced larger electrical signals than perfluoroheptane (PFHept) and perfluorooctane (PFO) (see Figures 5A-C). More specifically, significant results were obtained in the number of active electrodes, the distribution of the number of active electrodes, and the average spike amplitude of the active electrodes. These results demonstrate that perfluorocarbon compounds with five or more carbon atoms are effective for measuring electrical signals in tissues and are useful as sensitizers for electrical signals in tissues.

[0038] As described above, the increased number of active electrodes, spike amplitude, and mean spike rate under the five-carbon or higher perfluorocarbon compound condition demonstrate improved signal detection and reliability. This allows for monitoring axonal signal recordings from axon bundles, which are typically difficult to record. Furthermore, these effects are reversible upon removal of the PFD, making it safe to use without adversely affecting subsequent recordings. Furthermore, this method is not limited to permanently plated cells; it can also be used to temporarily stabilize tissues for recording. Simply casting perfluorocarbon compounds with five or more carbons causes minimal damage to cells and is colorless, allowing for clear visualization of the tissue. Visualization of the tissue is also important when combining electrophysiology with optical techniques such as fluorescence imaging and optogenetic stimulation. In particular, optogenetic stimulation demonstrated in channelrhodopsin-expressing motor neuron organoids demonstrates the versatility of this approach and promises to be highly applicable to studying neural activity in complex three-dimensional structures.

[0039] Measuring the electrical activity of cell tissue using perfluorocarbon compounds with five or more carbon atoms can be used for analyzing cell tissue.

Claims

1. A method for measuring electrical signals in cellular tissue, comprising adding a perfluorocarbon compound having 5 or more carbon atoms.

2. The method for measuring electrical signals in cellular tissue according to claim 1, wherein the perfluorocarbon compound is perfluorodecalin.

3. A method for measuring electrical signals in cellular tissue according to claim 1 or 2, wherein the measurement of electrical signals in cellular tissue is performed using an electrode array.

4. The method for measuring electrical signals from cell tissue according to claim 1 or 2, wherein the perfluorocarbon compound is added by replacing the culture medium.

5. An electrical signal sensitizer for cell tissues, consisting of a perfluorocarbon compound with five or more carbon atoms.

6. The electrical signal sensitizer for cell tissue according to claim 5, wherein said perfluorocarbon compound is perfluorodecalin.

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