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WO2024145528A1 - Dispositifs, systèmes et procédés pour enregistrements électrophysiques de cultures en suspension - Google Patents

Dispositifs, systèmes et procédés pour enregistrements électrophysiques de cultures en suspension Download PDF

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WO2024145528A1
WO2024145528A1 PCT/US2023/086315 US2023086315W WO2024145528A1 WO 2024145528 A1 WO2024145528 A1 WO 2024145528A1 US 2023086315 W US2023086315 W US 2023086315W WO 2024145528 A1 WO2024145528 A1 WO 2024145528A1
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Prior art keywords
ring
organoid
assembloid
kirie
electrodes
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Sergiu P. PASCA
Bianxiao Cui
Xiao Yang
Csaba Forro
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture

Definitions

  • Devices, systems, and methods are provided for long-term, minimally invasive electrophysical recordings of 3D multicellular systems, which can be leveraged to develop neuropsychiatric disease models.
  • the data provided herein provides flexible electronics (i.e. the device, also referred to as kirigami electronics or Kiri E) that transition in suspension cell culture from a flat 2D pattern to a 3D basket-like configuration that integrates into the organoids and assembloids to accommodate the long-term culture.
  • the device integrates into a recording system to allow for electrophysical recordings of the organoid or assembloid, where the multicellular assembloids can be cultured in a chamber of the recording system.
  • the device comprises (i) at least four concentric rings comprising a first inner most ring, a second inner most ring, a third inner most ring and an outer most ring, wherein the first inner most ring, the second inner most ring, the third inner most ring, and the outer most ring are operably joined to each other by at least ten latches, wherein each latch is operably joined to each ring at a connection point on each ring, wherein the at least ten latches extend from the first inner most ring to the outermost ring, wherein the latches form a spiral pattern; (ii) at least 24 electrodes wherein the first inner most ring comprises 14 of the total electrodes, the second inner most ring comprises 14 of the total electrodes, and the third inner most ring comprises 14 of the total electrodes; and (iii) a tail member attached to the outmost
  • the devices of the present disclosure measure an electrophysical property of a 3D organoid or assembloid.
  • the organoid or assembloid may be any organoid or assembloid that can be cultured in suspension cell culture.
  • the organoids or assembloids are neural organoids or neural assembloids.
  • the neural organoids or assembloids may be any neural organoids or assembloids deemed useful.
  • the neural organoid may be a cortical organoid, midbrain organoid, striatal organoid, spinal cord/hindbrain organoid, ventral forebrain organoid, or organoids comprising any combination thereof.
  • an organoid comprises a combination of different organoids it is referred to as an assembloid.
  • the latches of the present disclosure are operably joined to all rings in the device at connection points.
  • the length of the latch between any given ring may have any length that allows for the elongation of the device. In some embodiments, the length of the latch between each ring is 0.2 to 2 mm.
  • the latches of the devices may have a particular shape. The shape may be any shape that assists in reducing the overall strain of the device. In some embodiments, the latches have the highest curvature closer to an inner ring than an outer ring. In some embodiments, the latches of the present device may form a spiral pattern in the device. In some embodiments, the devices of the present disclosure have a maximal strain in the range of 0.01 -0.1% strain.
  • the device comprises electrodes located in a center portion of the ring.
  • the center portion of the ring has a specified diameter.
  • the diameter may be any diameter deemed useful.
  • the center portion of the ring has a diameter of 0.5 mm to 2 mm.
  • the device is nanofabricated by photolithography.
  • the methods of the present disclosure comprise culturing an organoid or assembloid in the circular concave region of the recording system. In some embodiments, the culturing results in the growth in size of the organoid or assembloid while maintaining spherical morphology of the organoid or assembloid.
  • the device of the present disclosure integrates into the organoid or assembloid. In some embodiments, the device is embedded 200-800y.m into the organoid or assembloid. In some embodiments, the methods of the present disclosure further comprise comprising contacting the center of the device in the recording system the organoid or assembloid before step (i).
  • one or more cells in the organoid or assembloid express a light-activatable channel.
  • the organoid or assembloid may be stimulated with light prior to step (ii).
  • the organoid or assembloid is derived from an individual suffering from a neuropsychiatric disorder.
  • FIG. 1A-FIG. 1J Design and optimization of KiriE.
  • FIG. 1A Schematics of the generation of neural organoids from hiPS cells, the concept of vertically deformable KiriE, and the integration of neural organoids with KiriE.
  • FIG. 1 B The spiral KiriE design consists of concentric rings connected by spiral latches with 32 electrodes (gold circles) in the central area.
  • FIG. 11 Strain distribution in a simplified spiral KiriE under its own weight and the weight of a 1 .2 mm-diameter organoid in the medium. The maximum strain is 0.06%, well below the strain at the elastic limit of SU-8.
  • FIG. 1 J The plot of strain along the metal interconnect of a microelectrode (the blue latch in FIG. 11). There is minimal strain between the first and the second rings, where the organoid is located.
  • the vertical dashed lines indicate the positions of five concentric rings.
  • FIG. 2A-FIG. 2K Platform for long-term KiriE-hCO integration and monitoring.
  • FIG. 2A A schematic of the KiriE platform and its functional modules. FFC, flat flexible cable.
  • FIG. 2B An image of the contact pads on the KiriE tail aligned with the electrode pads of the FFC through for direct contact interfacing.
  • FIG. 2C Photographs of a spiral KiriE-hCO assembly. The zoomed-in photograph shows a spherical hCO on a spiral KiriE at day 120 of differentiation.
  • FIG. 2D Images of an hCO integrated with a spiral KiriE at different stages of differentiation. These images show that the same hCO continued to grow in size when it was cultured on KiriE.
  • FIG. 2A A schematic of the KiriE platform and its functional modules. FFC, flat flexible cable.
  • FIG. 2B An image of the contact pads on the KiriE tail aligned with the electrode pads of the FFC through for direct contact interfacing.
  • FIG. 2E Images of an hCO integrated with a honeycomb KiriE at different times.
  • Data are presented as mean ⁇ s.e.m.
  • FIG. 22 discloses an illustration of an exemplary embodiment of the circular chamber and the rectangular region of the recording system.
  • the KiriE design and optimization To chronically integrate with and record from 3D neural organoids in suspension, the kirigami concept was employed to design an ultra-thin 2D kirigami pattern that spontaneously transforms into a specific 3D geometry upon release in suspension (FIG. 1 A).
  • the 3D geometry is a basket with a diameter of 1 cm and 32 microelectrodes located in a 1 mm central area.
  • KiriE patterns are nanofabricated by photolithography with two SU-8 insulation layers encapsulating metal connections for a total thickness of 0.9 pm. In the central area integrating with organoids, SU-8 and metal interconnects are 9 pm and 3 pm wide respectively, and the platinum microelectrodes are 25 pm in diameter.
  • a pipeline was developed combining scripted design with a finite element method (FEM) simulation framework that quantifies deformation under a load and stress accumulations at different locations of the kirigami patterns (Methods).
  • FEM finite element method
  • hCO was gently pipetted at day 20 of differentiation into the center of KiriE.
  • hCOs were continuously monitored (FIG. 2C), and observed that hCOs cultured on either spiral KiriE (FIG. 2D) or honeycomb KiriE (FIG. 2E) grew in size (FIG. 2F) and maintained their spherical morphology over 44 days (FIG. 2G).
  • fluorescent KiriE was nanofabricated by covalently incorporating a Rhodamine fluorophore within the SU-8 polymer (18).
  • hCOs were derived from a hiPS cell line that has been genetically engineered to express agreen fluorescent protein (CAG::eGFP). Confocal imaging demonstrated that, as expected, both spiral and honeycomb KiriEs are highly deformable in the vertical direction (FIG. 2, H and I). It was found that, after 26 days, KiriE was embedded in hCOs as deep as 300- 500 pm (FIG. 13, A and B). Fluorescence intensity of CAG::eGFP cells remained constant in relation to the distance from KiriE (FIG. 2J) , suggesting that the ultra-thin and flexible KiriE does not perturb the overall cell distribution. Channel-indexing barcodes in the KiriE design allowed us to identify individual electrodes (FIG. 2K and FIG.
  • KiriE does not interfere with hCO development.
  • hCOs were plated on KiriE at day 20 of differentiation and examined them up to day 190.
  • KiriE-hCOs were incised out of the culture chamber and sectioned into thin slices for immunofluorescence.
  • SOX2+ neural progenitors organized in the ventricular zones (FIG. 3A) as well as expression of neuronal (MAP2) and glial cell lineage (GFAP) markers (FIG. 3B). Fluorescence analysis as a function of distances from KiriE showed relatively uniform distribution of SOX2+, MAP2+, and GFAP+ cells in hCO at the KiriE-hCO interface (FIG. 3, C and D).
  • KiriE-hCO Detecting disease-related phenotypes in KiriE-hCO.
  • KiriE was integrated with hCO derived from an hiPS cell line carrying a heterozygous loss of the DGCR8 gene. It was previously shown that cortical neurons derived from this line (or from patients carrying the 22q1 1 .2 deletion that encompasses DGCR8) display increased spontaneous activity using both calcium imaging and patch clamp (21 ). Electrophysiological recordings of spontaneous activity showed that the firing rate of DGCR8+/- KiriE-hCOs at day 133-141 was approximately three times of the isogenic control KiriE-hCOs at day 137-138 (FIG.
  • FIG. 14C 1 .02 ⁇ 0.16 Hz for DGCR8+/- hCOs and 0.38 ⁇ 0.04 Hz for control hCOs.
  • About 30% of the channels probing DGCR8+/- hCOs showed firing rates greater than 1 Hz (in contrast to just 2% in control hCOs), and some DGCR8+/- hCO neurons fired as high as ⁇ 7 Hz. Therefore, KiriE can capture disease-related phenotypes in intact hCOs without the need to dissociate or slice for recordings.
  • hCOs can be integrated with human striatal organoids (hStrOs) to form assembloids and study cortico-striatal circuits (22).
  • hStrOs human striatal organoids
  • the basket-like geometry of KiriE provides an ideal platform to support the in situ fusion of organoids into assembloids and long-term monitoring of electrical activity.
  • hStrOs were differentiated from hiPS cells and integrated them with KiriE using the same procedure for hCOs.
  • Immunofluorescence staining of MAP2+ neurons, GFAP+ glial lineage cells in KiriE-hStrO cryosections demonstrated a seamlessly integrated neural interface similar to KiriE-hCO assembly.
  • Single-unit recording was successfully obtained from KiriE-hStrOs that were stable across days (FIG. 14, D and E).
  • hCOs and hStrOs were separately derived and infected hStrOs with AAV- hSynl ::mCherry for fluorescence visualization, and infected hCOs with AAV-hSyn1 ::eYFP and AAV-hSyn1 ::ChrimsonR-tdTomato for fluorescence visualization and optogenetic stimulation (FIG. 5D).
  • AAV-hSynl ::mCherry for fluorescence visualization
  • infected hCOs with AAV-hSyn1 ::eYFP and AAV-hSyn1 ::ChrimsonR-tdTomato for fluorescence visualization and optogenetic stimulation (FIG. 5D).
  • an hStrO was placed at day 80-82 of differentiation in the center of KiriE. Two days later, and after the hStrO had adhered to the electrode area, an hCO was placed at the same stage adjacent to the hStrO and allowed them to
  • the KiriE platform has several advantages compared to prior technologies (3-12, 24).
  • Second, the multimodal KiriE culture platform enables long-term medium perfusion and in situ functional assays including longitudinal morphology monitoring of hCOs, live-cell imaging, real-time electrophysiology, and optogenetic and pharmacological modulation, without the need to transfer across multiple setups that could perturb development or induce cellular stress.
  • KiriE detects the disease-associated electrophysiological phenotypes and interregional circuit connectivity in assembloids integrated in situ. This platform could incorporate pH, oxygen and neurotransmitter sensors to probe metabolic and neural activity. KiriE can be designed into complex 3D geometries such as interconnected baskets for probing multi-synaptic neural transmission in linear or loop assembloids.
  • NURBS curve which outlines the general shape of the latch. Normal vectors to the polygons’ outline were used to ensure that the latch connects to the polygons without any mechanically unfavorable kinks. Furthermore, NURBS curves are at least twice continuously derivable, ensuring smoothness of the outline, which is important for mechanical stability.
  • the final layout is built by a generator script that takes parameters as input such as shape controls of the latch, number of rings, latches and electrodes (FIG. 6H).
  • Non-linear Naghdi-shell formulation was employed to simulate the deformation of KiriE, as it is suited for large displacement simulation of shell mechanics.
  • the weak formulation of the mechanical equations and the choice of functional space were inspired by a previous work using Python’s FEniCS package (FEniCS-Shells) (27).
  • FeniCS is an open-source finite element method solver package (28).
  • KiriE The deformation of KiriE was simulated when it is subjected to gravitational loading plus the weight of a 1 .2 mm-diameter organoid in medium. The bending, shear, and membrane strain tensors were retrieved to verify whether the compounded strain was within the elastic limit of SU- 8.
  • Fabrication of KiriE The fabrication of KiriE is achieved by multi-layer photolithography (PL) as previously described (17, 18) with minor optimizations. The key fabrication steps are as follows: (1 ) A 80-nm thick Ni sacrificial layer was evaporated (AJA International Inc) onto a 100 mm Si wafer (p-type 0.1 -0.9 Q cm, Pure Wafer).
  • Negative photoresist SU-8 2000.5 was mixed with Lissamine rhodamine B ethylenediamine (RhBen; -10 pg/ml; Thermo Fisher Scientific) and placed in the dark at room temperature (RT) for at least 3 days to afford stable fluorescence labeling (18).
  • PL using the completely dissolved SU-8/RhBen solution was performed by repeating steps 4 and 5 to pattern the bottom SU-8 layer.
  • Steps 2 and 3 were repeated for PL patterning of the interconnect layer consisting of 5 nm Ti, 100 nm Au and 50 nm Pt.
  • Step 6 was repeated for PL patterning of the top SU-8 layer as the insulating layer of the interconnect lines.
  • the Si wafer was hard baked at 195 °C for 1 .5 h to allow interdiffusion of the bottom and top SU-8 layers.
  • the preparation of the 3D culture chamber includes gluing a #1 .5 glass coverslip (VWR) at the bottom and an FFC (Digi-Key) on the top surface.
  • the glass coverslip was glued to the bottom of the culture chamber using PDMS, and cured at 55 °C for 24 h.
  • An FFC was mounted onto the 3D culture chamber with Metabond cement (Metabond quick luting cement; Parkell).
  • the whole chamber was treated for 1 min by oxygen plasma (Basic Plasma Cleaner, PDC-32G, Harrick Plasma) and sterilized in 70% ethanol.
  • KiriE was transferred to and aligned with the 3D culture chamber.
  • the culture chamber was initially immersed under water to avoid accidental tear of KiriE. Then, the liquid level was slowly reduced. Electrical connection of the KiriE direct contact interfacing pads to the FFC was performed using a previously described method (17).
  • the interfacing pads of KiriE were aligned with the FFC leads using a transfer pipette. Aligned interfacing pads were fully dried by surgical spears (Braintree Scientific) and fixed in place using epoxy adhesive (5 minute epoxy, Devcon). The interface was covered with Metabond cement. Metabond cement was also used to fix the PDMS support of KiriE to the culture chamber.
  • a 3D printed bar was glued to the opening of the circular chamber with Metabond cement to seal the chamber.
  • the culture chamber was housed individually in the 10 cm tissue culture dish.
  • CAG::eGFP hiPS cell line was generated as previously described (29).
  • the parental hiPS cell line (8858-3) was maintained in 6-well plates using StemFlex medium (Life Technologies, A3349401 ).
  • Cas9, gRNA and donor plasmids were obtained from Addgene (plasmids #42230, #41818 and #52344, respectively).
  • 3 pg Cas9, 1 pg gRNA and 1 pg donor plasmids were used.
  • the cells were immediately seeded into a well of a 6-well plate that was pre-coated with vitronectin and contained pre-warmed Essential 8 medium supplemented with the ROCK inhibitor Y-27632 (10 pM; Selleck Chemicals, S1049). While the nucleofected cells recovered to reach 70%-80% confluency, 1 g ml— 1 of puromycin was applied for 5 days, after which the media was switched back to StemFlex media. Puromycin- resistant clones became visible after 7 days. Clones were pooled together, expanded, cryopreserved and later sorted into 96-well plates to ensure single clone formation by seeding one cell per well.
  • hiPS cells were exposed to 1 % DMSO (Sigma-Aldrich, 472301) in Essential 8 medium.
  • 1 % DMSO Sigma-Aldrich, 472301
  • Essential 8 medium For aggregation into organoids, approximately 3 x 106 single cells were added per well in AggreWell 800 plates in Essential 8 medium supplemented with the ROCK inhibitor Y27632 (10 pM), centrifuged at 100g for 3 min and then incubated at 37 °C with 5% CO2.
  • organoids consisting of approximately 10,000 cells were collected from each microwell by pipetting the medium up and down in the well with a cut P1000 pipette tip and transferred into ultra-low attachment dishes (Corning, 3262) in Essential 6 medium (Thermo Fisher Scientific, A1516401 ) supplemented with the SMAD pathway inhibitors dorsomorphin (2.5 pM; Sigma- Aldrich, P5499) and SB-431542 (10 pM; R&D Systems, 1614).
  • Essential 6 medium supplemented with dorsomorphin and SB-431542 was changed every day.
  • the Wnt pathway inhibitor XAV-939 (1 .25 pM, Tocris, 3748) can be added with the two SMAD pathway inhibitors.
  • neural medium containing Neurobasal-A medium (Thermo Fisher Scientific, 10888022), B-27 supplement minus vitamin A (Thermo Fisher Scientific, 12587010), GlutaMAX (1 :100; Thermo Fisher Scientific, 35050079), and penicillin-streptomycin (10,000 U ml-1 , 1 :100; Thermo Fisher Scientific, 15140122).
  • Neurobasal-A medium Thermo Fisher Scientific, 10888022
  • B-27 supplement minus vitamin A Thermo Fisher Scientific, 12587010
  • GlutaMAX (1 :100; Thermo Fisher Scientific, 35050079
  • penicillin-streptomycin 10,000 U ml-1 , 1 :100; Thermo Fisher Scientific, 15140122.
  • the neural medium was supplemented with 20 ng ml-1 epidermal growth factor (EGF; R&D Systems, 236-EG) and 20 ng ml-1 basic fibroblast growth factor (FGF2; R&D Systems, 233-FB) for 16 d, with medium changed daily in the first 10 d and every other day for the following 6 d.
  • EGF epidermal growth factor
  • FGF2 basic fibroblast growth factor
  • the neural medium was supplemented with brain-derived neurotrophic factor (BDNF; 20 ng ml-1 ; PeproTech, 450-02) and NT-3 (20 ng ml-1 ; PeproTech, 450-03), L-ascorbic acid 2- phosphate trisodium salt (AA; 200 pM; FUJIFILM Wako Chemical Corporation, 323-44822), N6,2’-O-dibutyryladenosine 3',5’-cyclic monophosphate sodium salt (cAMP; 50 pM, MilliporeSigma, D0627) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA; 10 pM, MilliporeSigma, D2534), with medium changes every other day. From day 46, neural medium containing B-27 plus supplement (Thermo Fisher Scientific, A3582801 ) was used for medium changes every 4 d.
  • BDNF brain-derived neurotrophic factor
  • NT-3
  • neural organoids were transferred to neural medium containing Neurobasal-A medium, B-27 supplement minus vitamin A, GlutaMAX, penicillinstreptomycin, and supplemented with WNT pathway inhibitor IWP-2 (2.5 pM; STEMCELL Technologies, 72124) and recombinant Human/Murine/Rat Activin A (50 ng ml-1 ; PeproTech, 120-14P).
  • WNT pathway inhibitor IWP-2 2.5 pM; STEMCELL Technologies, 72124
  • recombinant Human/Murine/Rat Activin A 50 ng ml-1 ; PeproTech, 120-14P
  • neural medium was additionally supplemented with retinoid X receptor agonist SR 11237 (100 nM; Tocris, 3411 ).
  • neural medium was supplemented with BDNF, NT-3, AA, cAMP and DHA.
  • DAPT 2.5 pM; STEMCELL Technologies, 72082
  • neural medium containing B-27 plus supplement was
  • RT-qPCR Real-time qPCR
  • 3-4 hCOs were pooled per sample.
  • mRNA from day 25 hCOs was isolated using the RNeasy Mini Kit (Qiagen, 74106).
  • Template cDNA was prepared by reverse transcription using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Thermo Fisher Scientific, 11752250).
  • RT-qPCR was performed using the SYBR Green PCR Master Mix (Thermo Fisher Scientific, 4312704) on a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific, 4485689). Primers used in this study are listed in Table 1 .
  • Table 1 List of genes and primer sequences used for RT-PCR
  • viruses used in this study are AAV-DJ-hSyn1 ::eYFP (Stanford University Neuroscience Institute Gene Vector and Virus Core, GVVC-AAV-16, 6597), AAV-DJ- hSynl ::mCherry (Stanford University Neuroscience Institute Gene Vector and Virus Core, GVVC- AAV-17, 5297), and AAV1 -Syn::ChrimsonR-tdT (Addgene, 59171 -AAV1 ) (20).
  • the multifunctional culture chamber allows for live-cell confocal imaging of neural organoids, assembloids, and KiriE through the glass bottom.
  • the culture chamber was incubated in an environmentally controlled chamber on a Leica Stellaris 5 confocal microscope with a motorized stage for 15 min before imaging.
  • Imaging data was analyzed as previously described (18). Briefly, Imaris 9 software (Bitplane) was used for visualization and segmentation. A combination of Imaris, Imaged (National Institutes of Health) and MATLAB (MathWorks) was used for quantitative analysis of fluorescence intensity as a function of the distance from the KiriE. Imaris was used to define the KiriE structures and the 3D volume of hCOs. Imaged was used to export fluorescence intensities, distances from the KiriE and associated 3D coordinates. MATLAB was used to calculate fluorescence intensity as a function of the distance from the KiriE. The average fluorescence intensity values for all voxels with distances binned over an interval of 10 pm were normalized against the baseline value as the average fluorescence intensity of all voxels 150-160 pm away from KiriE.
  • anti-SOX2 (rabbit, Cell Signaling Technology, 3579S, 8, 1 :300 dilution), anti-PAX6 (mouse, DSHB, 1 :50 dilution), anti-MAP2 (guinea pig, Synaptic Systems, 188004, 3-37, 1 :200 dilution), anti-GFAP (rabbit, Dako, Z0334, 41259205, 1 :500 dilution).
  • Imaging data was analyzed in the same way as live-cell confocal imaging data.
  • the average fluorescence intensity values for all voxels with distances binned over an interval of 1 pm were normalized against the baseline value as the average fluorescence intensity of all voxels 50-51 pm away from KiriE.
  • hCOs Single cell RNA sequencing and data analysis. Dissociation of hCOs into single cells for scRNA-seq was performed as previously described (30) with minor optimizations. 3-4 hCOs at day 100 were pooled from KiriE-integrated condition or control condition from each hiPS cell line. hCOs were transferred to a 6-well plate (Corning, 3506) and incubated for 45-60 min at 37 °C with 3 ml enzymatic dissociation solution.
  • a device for measuring an electrophysical property of a 3D organoid or assembloid comprising:
  • At least four concentric rings comprising: a first inner most ring, a second inner most ring, a third inner most ring, and an outer most ring, wherein: the first inner most ring, the second inner most ring, the third inner most ring, and the outer most ring are operably joined to each other by at least ten latches, each latch is operably joined to each ring at a connection point on each ring, the at least ten latches extend from the first inner most ring to the outer most ring, and the latches form a spiral pattern;
  • a tail member attached to the outmost ring wherein the tail member extends outward from the outmost ring wherein the tail member comprises at least 24 contact interfaces pads equal in number electrodes; wherein the device has a two-dimensional shape when not in suspension and a three- dimensional shape in the form of a basket when in a liquid medium.
  • organoid or assembloid is a neural organoid or neural assembloid.
  • the latches have highest curvature closer to an inner ring than an outer ring
  • the device of any of clauses 1 -10 has a maximal strain of 0.01 %-0.1 %.
  • a tail member attached to the ring wherein the tail member extends outward from the ring wherein the tail member comprises at least 24 contact interfaces pads that are equal in number to the number of electrodes; wherein the device has a two dimension shape when not in suspension and a three dimensional shape in the form of a basket when in a liquid medium.
  • the device of any of clauses 12-19 has a maximal strain of 0.01 %-0.1 %.
  • a recording system comprising:

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Abstract

Sont proposés des dispositifs, des systèmes et des procédés pour des enregistrements électrophysiques à long terme minimalement invasifs de systèmes multicellulaires 3D qui peuvent être exploités pour développer des modèles de maladie neuropsychiatrique. Les données de la présente divulgation démontrent une électronique flexible (c'est-à-dire le dispositif, également appelé électronique de kirigami ou KiriE) qui effectue une transition dans une culture de cellules en suspension d'un motif 2D plat à une configuration de type panier 3D qui s'intègre dans les organoïdes et les assembloïdes pour s'adapter à la culture à long terme. Le dispositif s'intègre au système pour permettre des enregistrements électrophysiques de l'organoïde ou de l'assembloïde. Le système peut être intégré à une stimulation optogénétique et pharmacologique des organoïdes et des assembloïdes. Les dispositifs et les systèmes offrent l'avantage particulier de permettre l'enregistrement chronique d'organoïdes et d'assembloïdes tout en préservant leur morphologie, leur cytoarchitecture et leur composition cellulaire. Les procédés divulgués utilisent les dispositifs et les systèmes de la présente divulgation pour la manipulation et l'enregistrement d'organoïdes et d'assembloïdes.
PCT/US2023/086315 2022-12-29 2023-12-28 Dispositifs, systèmes et procédés pour enregistrements électrophysiques de cultures en suspension Ceased WO2024145528A1 (fr)

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