Method for measuring electrical signals in cell tissue and sensitizer

Abstract

The present invention aims to increase the electrical signals obtained from cell tissue without damaging the cells and in a state that allows observation under a microscope, etc., and to easily and efficiently obtain the electrical signals generated in the cell tissue. The present invention provides a method for measuring electrical signals from cellular tissue by adding a perfluorocarbon compound having 5 or more carbon atoms, and an electrical signal sensitizer for cellular tissue comprising a perfluorocarbon compound having 5 or more carbon atoms. The perfluorocarbon compound is preferably perfluorodecalin, the electrical signals from the cellular tissue are preferably measured using an electrode array, and the perfluorocarbon compound is preferably added by replacing the culture medium.

Description

[Technical Field]

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

[0002] One method for elucidating the function of cellular tissues is to measure electrical signals generated in the cellular tissues. There are two well-known methods for measuring electrical signals generated in cell tissue: the patch clamp method, which measures electrical signals from cell tissue outside the cell tissue, and the method of measuring electrical signals by inserting electrodes inside the cell tissue. Recently, a method for electrical measurement of cellular tissue using microelectrode arrays (MEA) has become widely known (Patent Document 1, Patent Document 2). This method is a non-invasive method that enables measurements outside the cellular tissue and allows simultaneous observation of many locations in the tissue over a long period of time. It is possible to measure electrical signals generated in the cellular tissue using electrodes densely arranged on the array. This method has further contributed to elucidating the functions of cellular tissues with the development of high-density microelectrode arrays (HD-MEA) based on solid-state imaging device technology using complementary metal oxide semiconductors (CMOS) (Non-Patent Document 1). This electrode array has extremely high temporal and spatial resolution, enabling detailed observation of cellular tissue activity.

[0003] Although all cell tissues can be used to measure electrical signals, 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 three-dimensional cell tissues with complex cellular structures, making electrical signal measurement possible regardless of the cell tissue's shape.

[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 generally have round morphologies. Thus, the continued improvement of neural tissue fabrication techniques 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 treatments. In particular, three-dimensional tissues such as neural organoids have attracted great interest because they can be derived from human induced pluripotent stem cells and can recapitulate important aspects of the brain. However, the above-mentioned electrodes are complex and therefore difficult to manufacture, are difficult to handle, and furthermore, cannot be observed under a microscope, etc. Therefore, there has been a demand for a method for measuring electrical signals that is simple, causes little damage to cell tissue, and allows observation under a microscope, etc., but such a convenient method has not existed until now.

[0005] On the other hand, from a completely different perspective, in the field of biotechnology, a substance called perfluorodecalin (hereinafter referred to as "PFD") is known as follows. PFD, consisting of 10 carbon atoms and 18 fluorine atoms, is characterized by its highly hydrophobic and chemically stable nature. Being 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 components have been investigated 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 in 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-based nanoparticles are expected to be used for drug delivery systems, where their solubility and controlled release have been of interest. Furthermore, perfluorohexane (hereinafter referred to as "PFH"), perfluoroheptane (hereinafter referred to as "PFHept"), perfluorooctane (hereinafter referred to as "PFO"), etc. are well known as similar substances to PFD. It is preferable that the medium is liquid at room temperature or at a culture temperature of around 37°C. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2015 / 012955 [Patent Document 2] International Publication No. 2020 / 189172 [Non-patent literature]

[0007] [Non-Patent Document 1] Muller, J. et al. High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels. Lab Chip 15, 2767-2780 (2015) Summary of the Invention [Problem to be solved by the invention]

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

[0009] The electrical signals emitted by cellular tissues are weak, and the measurable electrical signals are also small. Furthermore, existing electrodes for obtaining large electrical signals are difficult to handle and cannot be observed using a microscope. The inventors have surprisingly found that by adding a perfluorocarbon compound having 5 or more carbon atoms, which is a chemical substance, to measure electrical signals from cellular tissue, it is possible to increase the electrical signals obtained from cellular tissue with minimal damage to cells and in a state that allows observation under a microscope, etc., and to easily and efficiently obtain electrical signals generated in cellular tissue, thereby completing the present invention.

[0010] That is, the present invention is characterized by the following points. 1. A method for measuring electrical signals from cellular tissue, which comprises adding a perfluorocarbon compound having 5 or more carbon atoms and measuring the electrical signals from the cellular tissue. 2. The method for measuring electrical signals from cellular tissue according to 1 above, wherein the perfluorocarbon compound is perfluorodecalin. 3. The method for measuring electrical signals in cell tissue according to 1 or 2 above, wherein the measurement of electrical signals in cell tissue is performed using an electrode array. 4. The method for measuring electrical signals from cellular tissues according to 1 or 2 above, 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 5 above, wherein the perfluorocarbon compound is perfluorodecalin. [Effects of the Invention]

[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. [Brief explanation of the drawings]

[0012] [Figure 1A] Comparative images of mouse primary hippocampal cells (in vitro, day 7) after PFD addition. Scale bar: 250 μm. "Control" shows a comparative example before PFD addition. [Figure 1B] Comparison of cell surface area (μm2). Increased by the addition of PFD. [Figure 1C] 3D reconstructions of confocal z-stacks of primary hippocampal cells stained with calcein AM, with PFD added. Scale bar 20 μm. [Figure 1D] The height (μm) of the cell body is flattened under PFD conditions. [Figure 2A] From top to bottom: comparison of active electrode maps of the chip with and without PFD (one side is active), comparison of average spike amplitude maps of the chip with and without PFD (units: μV), comparison of average spike rate maps of the chip with and without PFD (units: Hz). [Figure 2B] The number of detected active electrodes was significantly higher with the addition of PFD. "Washed" indicates the results after the PFD was removed. [Figure 2C]The histogram of spike amplitude (µV) of the active electrode with PFD addition shows a distribution shifted to higher amplitudes under the PFD condition. [Figure 2D] The mean spike amplitude (µV) of the active electrodes is significantly higher in the PFD condition. [Figure 2E] Histogram comparison of the average spike rate (Hz) of the active electrode with and without PFD. The spike rate was significantly higher under the PFD condition. [Figure 2F] The average spike rate (Hz) of the active electrode with PFD addition is significantly higher in the PFD condition. [Figure 2G] Changes in hippocampal cell activity with the addition of PFD. [Figure 3A] Comparative images of cortical organoids with PFD addition. [Figure 3B] The addition of PFD increased the organoid diameter (mm), which recovered after washing out the PFD. [Figure 3C] Confocal comparison images of organoids at the focal plane of the culture dish bottom with PFD. Scale bar: 100 μm. [Figure 3D] Comparison of contact area (mm2) with the bottom surface of the organoid. The area increases with the addition of PFD. [Figure 3E] Comparison map of spike amplitude (µV) in organoids treated with PFD. Scale bar: 0.5 mm. [Figure 3F] The functional contact area (mm2) based on spike amplitude maps increased with the addition of PFD. [Figure 3G] The bars in each graph compare, from left to right, 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 the interburst interval and burst duration, but a significant difference was observed in the mean firing rate. [Figure 3H]Functional connectivity maps based on pairwise spike correlations between spike-sorted units in organoids. (i) is a comparative example without PFD. (ii) is with PFD. The dots in the figure represent the spatial location of single units on the HD-MEA. The size of the dots indicates the mean spike amplitude, and the density indicates the number of functional connections found. The gray lines represent functional connections between single units, and the thickness of the lines 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. [Figure 3I] Changes in spike amplitude maps depending on the amount of PFD. Spike amplitude maps of organoids without PFD and with different amounts of PFD. Scale bar: 500 μm. [Figure 3J] 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. [Figure 3K] Recordings taken when a PFD was added to organoids immediately after placement on an HD-MEA. (A) Active electrode maps with and without a 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 when a PFD is added compared to the control condition without a PFD. (C) Spike raster plot. This plot shows the time at which spike activity was detected for each electrode (channel) over time. When a PFD was added, more electrodes detected neural activity compared to the control condition without a PFD. [Figure 4A] Activity map (center) and its enlarged view (right) from axon bundle recording analysis of motor neuron organoids with PFD addition. [Figure 4B] The right panel of Figure 4A shows the bandpass-filtered signal of the axon bundle recording on channel 11. The vertical axis is amplitude (μV). [Figure 4C] An example of an action potential waveform for channel 11 in the right panel of Figure 4A. The center line is the average. The vertical axis is amplitude (μV). [Figure 4D] The neural activity of the selected channels causes active potentials to propagate along the axon bundle. From top to bottom, channels 1 to 11. The horizontal axis scales 50 ms, and the vertical axis scales 50 μV. [Figure 4E] Histogram of spike counts versus stimulation timing (gray area) during optogenetic stimulation. The vertical axis represents spike counts. [Figure 5A] (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, C6F14), perfluoroheptane (PFHept, C7F16), perfluorooctane (PFO, C8F18), and perfluorododecalin (PFD, C10F18). [Figure 5B] Comparison of the number of active electrodes between different perfluorocarbon compounds with carbon numbers of 5 or more. [Figure 5C] Comparison of average spike amplitudes at active electrodes between different perfluorocarbon compounds with carbon numbers of 5 or more. DETAILED DESCRIPTION OF THE INVENTION

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

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

Examples

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

[0016] In the examples, three types of cell tissues, hippocampal neurons, cortical organoids, and motor neuron organoids, were prepared, and the electrical signals of each cell tissue were measured. In addition, as perfluorocarbon compounds having 5 or more carbon atoms, perfluorohexane, perfluoroheptane, perfluorooctane, and perfluorodecalin were used as sensitizers to measure the electrical signals of the cell tissues. Although it is presumed that many findings can be obtained by analyzing the results of measuring the electrical signals of the prepared tissue cells, the improvement in the measurement sensitivity by measuring the electrical signals in the cell tissue is not limited, and it can be applied to all cells.

[0017] <Production of PDMS culture chip>[ Polydimethylsiloxane (PDMS) culture chips were fabricated using a well-known procedure. First, a master mold was fabricated, and PDMS devices were fabricated using a mask. The master mold was created by pouring negative photoresist, SU-8 2100 or 2075 (Nippon Kayaku Co., Ltd.), onto a silicon wafer and spin-coating it at 1200-1500 rpm for 30 seconds. The wafer was then baked on a hotplate at 65°C for 9 minutes and then at 95°C for 40 minutes. Then, UV light (365 nm, 2.5–3.0 mW / cm 2 ) was irradiated 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 incubated at 37°C and 5% CO 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% CO for 1 hour. 100k dissociated cells were cultured at 2000 cells / mm. 2 The cells were plated onto the chip area at a cell density of 1000 kJ / cm. The cells were cultured in FM at 37°C with 5% CO2, and the medium was changed 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 (hydroxyethylpiperazineethanesulfonic acid) buffer and cultured at room temperature for 1 hour. The organoids were placed on the center of the electrode surface and cultured in NMM (neural maintenance medium) at 37°C and 5% CO2. Cortical organoids were plated on the MEA after 3-4 months of maturation, and motor neuron organoids were plated after 2 weeks. During the culture period, medium changes were performed every 3-4 days. For recording, HD-MEAs were also coated with Matrigel in DMEM / F12 (1:50) supplemented with HEPES and cultured for 1 hour at room temperature. After replacing the coating solution with RM (recording medium), 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 with forceps and held. To detach the MNOs from the PDMS structure, a stream of culture medium was added to the well by pipetting. Once the MNOs floated, 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, 3.85 × 2.10 mm 2 We 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 1024 configurable low-noise readout channels and records at a sampling rate of 20 kHz. All recordings were performed in a cell culture incubator at 37°C and 5% CO2 for 24 hours after medium exchange using MaxLab Live software (v.20.1.6, manufactured by MaxWell Biosystems). First, an active scan assay was performed, and multiple defined regions of the MEA were recorded 365 times for 30 seconds. In two-dimensional primary cell culture, seven electrode configurations with a total of 6600 electrodes with a 35-μm pitch covering the entire sensing area were scanned. In the case of organoid recording, an image of the position of the organoid on the chip was obtained, and the electrodes covered by the organoid were manually selected at a 17.5-μm pitch and scanned. Using the results of the scan, active electrodes were identified based on spike frequency and spike amplitude, and the 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 stored so that it could be added back to the cells after PFD application. 100 μl of warmed PFD was slowly and gently added from the side of the well using a 200-μl pipette tip. After the experiment, with the pipette tip kept in vertical contact with the bottom of the chip surface, the PFD was slowly removed from the edge of the chip. After removing the PFD, the stored conditioned medium was returned to the cells.

[0021] <Optogenetic stimulation> We used optogenetic techniques 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, 1.2A, (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. After permeabilization with 0.1% Triton-X in PBS for 30 minutes and washing three times with PBS, cells were blocked with 2% goat serum in PBS for 1 hour at room temperature. Next, tissues were incubated overnight at 4°C in primary antibody in 2% goat serum in PBS. After rinsing three times with PBS, cells were incubated with secondary antibody 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 (registered trademark) 2018b. Comparisons between two sample groups were performed using a Student's t-test with the function ttest(). Comparisons between more than two sample groups were performed using one-way analysis of variance (ANOVA) with the function anova1(), followed by a Tukey-Kramer post-hoc test for pairwise comparisons with the function multcompare(). [Example]

[0025] <Hippocampal neurons> 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 x 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. When the culture medium was removed and a PFD was cast onto the neurons, the surface area of ​​the cell bodies as observed microscopically significantly increased (see Figure 1A and Figure 1B (Control refers to a comparison before PFD addition; similarly below).) Furthermore, these observations were further supported by labeling the cells with calcein-AM and observing them by confocal imaging. 3D reconstructions of neurons imaged under PFD conditions showed a decrease in cell body height (see Figure 1C and Figure 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 normal 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 in the PFD condition than in the control group (see Figures 2A-C and 2B). Furthermore, when the PFD was removed and the conditioned medium used before PFD recording was reintroduced, a similar number of active electrodes was detected as in the pre-PFD condition (see Figure 2B). MEA maps depicting the mean spike amplitude of the electrodes showed more and higher amplitudes in the PFD condition compared to the control condition (see Figures 2A-D). The distribution of mean spike amplitude of the electrodes was skewed toward higher values ​​in the PFD condition, with the mean value being significantly higher than in 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. [Example]

[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. Medium was changed every other day and 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) minimum 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), consisting 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). This 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 four months before plating on HD-MEAs. Experiments were performed with and 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 administration revealed a significant increase in size, and removal of the PFD resulted in a return to the pre-PFD size (Figure 3A, Figure 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 showed 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 a PFD (Figure 3C). Electrophysiological recordings of the organoid electrical footprints confirmed that the application of a PFD increased the contact area between the organoid and the MEA (Figure 3D). Organoids exhibited consistent network-wide bursting behavior, with silent periods between bursts, 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 PFD conditions compared to 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 recordability.

[0031] Furthermore, analysis of functional connections 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)). The addition of PFD 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, the amount of PFD added was changed to 10, 25, 50, 100, and 200 μl, and the effect was also examined. The HD-MEA used was the same MaxOne manufactured by MaxWell Biosystems as above (https: / / www.mxwbio.com / wp-content / uploads / 2024 / 10 / MaxOne-Brochure-online-version.pdf), which is circular with a diameter of 19 mm and a height of 8 mm. Basically, the amount of PFD should be sufficient to cover the organoids, and adding at least 10 μl, preferably 50 μl, and even more preferably 100 μl or more will enable more efficient cell activity (see Figure 3I and Figure 3J).

[0033] In addition, under the same conditions as above, measurements were also performed after adding PFD immediately after placing the organoids on the HD-MEA. Even when PFD was added immediately after placing the organoids on the MEA, cell activity was significantly higher than when no PFD was added (see Figure 3K). [Example]

[0034] <Motor neuron organoids> Motor spheroids were formed as described previously. 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 DMEM / F12 supplemented with HEPES 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 of 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 as described above, we generated motor neuron organoids and measured electrical signals upon addition of PFD. The motor neuron organoids prepared as described above were 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, including axon bundles, suggesting that the detected activity originated from neural tissue. Spiking activity was detected at multiple electrodes located 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 organoids were stimulated every 10 seconds with 200-ms pulses of blue light (470 nm) using an optical fiber. Overlapping spike counts at each stimulation interval revealed increased spike activity during the light exposure period. Furthermore, by examining specific recording channels, we observed that some stimulations induced spikes within the organoids, followed by readily observed activity propagating along the axon bundles (see Figure 4A–E). [Example]

[0037] <Perfluorocarbon compounds with 5 or more carbon atoms> Next, using the cortical organoids from Example 2 above, perfluorohexane (PFH, CF) was used. 14 ), perfluoroheptane (PFHept, C7F 16 ), perfluorooctane (PFO, C8F 18 ), perfluorododecalin (PFD, C 10 F 18 ) was used as a sensitizer to measure electrical signals in cell tissue. All compounds, including perfluorohexane (PFH), perfluoroheptane (PFHept), perfluorooctane (PFO), and perfluorododecalin (PFD), produced significantly larger electrical signals than the control compound, which was culture medium. In particular, perfluoroheptane (PFHept) and perfluorooctane (PFO) produced larger electrical signals than perfluorohexane (PFH), and perfluorododecalin (PFD) produced larger 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 indicate that perfluorocarbon compounds with five or more carbon atoms are effective in measuring electrical signals in cellular tissues and are also useful as sensitizers for electrical signals in cellular 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. [Industrial Applicability]

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

4. 3. The method for measuring electrical signals from cell tissue according to claim 1, 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 the perfluorocarbon compound is perfluorodecalin.

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