EP1751267A1 - Interface de substrat de reseau cellulaire a motif et ses procedes et utilisations - Google Patents

Interface de substrat de reseau cellulaire a motif et ses procedes et utilisations

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Publication number
EP1751267A1
EP1751267A1 EP05741336A EP05741336A EP1751267A1 EP 1751267 A1 EP1751267 A1 EP 1751267A1 EP 05741336 A EP05741336 A EP 05741336A EP 05741336 A EP05741336 A EP 05741336A EP 1751267 A1 EP1751267 A1 EP 1751267A1
Authority
EP
European Patent Office
Prior art keywords
substrate
microhole
membrane
cell
backing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05741336A
Other languages
German (de)
English (en)
Other versions
EP1751267A4 (fr
Inventor
Geoffrey Mealing
Christophe Py
Mike Denhoff
Reza Dowlatshahi
Karim Faid
Raluca Voicu
Mahmud Bani
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Research Council of Canada
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National Research Council of Canada
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Filing date
Publication date
Application filed by National Research Council of Canada filed Critical National Research Council of Canada
Publication of EP1751267A1 publication Critical patent/EP1751267A1/fr
Publication of EP1751267A4 publication Critical patent/EP1751267A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • 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
    • C12N5/0068—General culture methods using substrates
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • 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
    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • C12N2535/10—Patterned coating

Definitions

  • the invention relates to methods and materials suitable for use in growing and monitoring two-dimensional networks of living cells on a substrate.
  • a number of methods for studying cell-to-cell communication are known, including: conventional patch-clamp techniques (glass micropipette coupled to peripheral electronics); sharp electrode intracellular recording; field potential recordings; and using ion or voltage-sensitive fluorescent dyes.
  • a substrate comprising: a microhole containing layer having microholes extending through it; a guidance layer of substantially inert material sealably engaging portions of a first side of the microhole containing layer; said guidance layer in combination with the microhole containing layer defining a series of troughs extending substantially parallel to the microhole containing layer surface, wherein the trough walls are formed at least in part by the guidance layer and the trough base is defined at least in part by a region of the microhole containing layer defining a microhole.
  • a method of producing a substrate suitable for use in attaching and/or growing cells so as to promote development of structured cell networks in two or more dimensions comprises: a) obtaining a film on a first side of a substantially inert backing; b) creating microholes in the film; c) bonding the second side of the backing to a carrier; d) obtaining a mask in the first side of the backing and creating windows in the thin film mask, said windows being aligned so as to connect to a microhole; f) etching the backing through the windows in the mask, to create an inverted pyramid structure resulting in a membrane including the micro-hole; g) obtaining a second chip defining channels; h) bonding the second chip to the backing such that a channel is positioned over a microhole in substantially sealing engagement; i) releasing the backing from the carrier; j) applying a patterned growth cell guidance region on the first side of the membrane in alignment with micro-holes such that a micro-
  • a method of producing a substrate suitable for use in growing cells so as to promote growth of structured networks in two or more dimensions comprises: a) obtaining a film on a first side of a Si wafer with a crystalline orientation; b)creating microholes in the SiN/Au thin film; c) bonding the second side of the wafer to a carrier with wax or another sacrificial layer; d) obtaining a mask in the back of the wafer and creating windows in the thin film mask, said windows being aligned so as to connect to a microhole; f) etching the Si wafer through the windows in the mask, thereby creating an inverted pyramid structure resulting in a membrane including the micro-hole; g) obtaining a second chip defining channels with a defined pitch; h) bonding the second chip to the Si chip such that a channel is positioned over a microhole in substantially sealing engagement; i) releasing the Si chip from the carrier; j) defining
  • a method of producing a substrate suitable for use in growing cells so as to promote growth of structured networks in two or more dimensions comprises: a) obtaining a tip connected to a beam; b)obtaining a backing having a carrier bonded to a first surface, said backing defining towers and walls along a second surface surface; c) positioning the tip such that apex of the tip in contact with the top of a tower on the backing and the beam extends to and edge of the backing; d) filing the space between the tip and the backing with a material which is fluid when applied but can be converted to a solid form; e) converting the material of step d into a solid form; f) removing the tip and the backing from the cured material to reveal a well structure with microholes and channels therein; g) where the tip was positioned such that its removal results in openings to the outside air in regions formed by the tip or the beam, closing off such openings to form closed channels except at the end of the channels defined by
  • a method of forming an interface between a biological membrane and a substrate comprises: a) obtaining a substrate of claim 5; b) culturing cells on the microhole containing layer/guidance layer surface of the substrate; c) creating a patch-clamp connection between the cell and the substrate at a microhole.
  • a method of producing a system suitable for use in studying whole-cell electrical responses to a stimulus comprises: obtaining a substrate as described herein; culturing cells on the membrane/guidance layer surface of the substrate in a culture medium such that at least one cell grows over a microhole; creating a patch-clamp connection between the membrane and the substrate at a microhole; and, rupturing a portion of the membrane over the microhole.
  • FIGURE 1 is schematic representation of an embodiment of a patterned cell network substrate interface and a use thereof in patch clamp investigations.
  • FIGURE 2 is a schematic representation of an embodiment of an approach to the fabrication of a substrate for use as described in Figure 1.
  • FIGURE 3 depicts scanning electron micrograph pictures of embodiments of fluorinated PDMS stamps with (a) channels and (b) pillars used as secondary moulds and (c) and (d) their respective replicated PDMS microstxuctured substrates.
  • FIGURE 4 is a depiction of cells, microstructures and surface chemistry modifications relating to Example 1.
  • FIGURE 5 is a further depiction of cells, microstructures and surface chemistry modifications relating to Example 1.
  • FIGURE 6 is a depiction of results of Example 1 part C showing excitable neurons functionally connected as assessed by calcium imaging and electrophysiology.
  • FIGURE 7 is a schematic depiction of an embodiment of a substrate for a patterned cell network.
  • FIGURE 8 depicts (A) a schematic representation of an embodiment of a substrate, and (B) an embodiment of a Si-based substrate fabrication process.
  • FIGURE 9 is a series of depictions of embodiments the substrate.
  • FIGURE 10 is a depiction of an embodiment of a PDMS based fabrication approach in a sectional view.
  • FIGURE 11 is a depiction of an embodiment of a PDMS based fabrication approach in a plan view.
  • FIGURE 12 is a depiction of a prior art AFM tip.
  • FIGURE 13 is a depiction of alternative tip configurations useful in certain embodiments of the invention.
  • FIGURE 14 is a depiction of an embodiment of the process for fabrication of the substrate using a tip.
  • the present invention provides, in one aspect, an apparatus and method to grow cells, including neurons, on substrates with patterned guidance pathways printed on their surface such that they form structured 2-D networks.
  • the apparatus and method permits study of cell interfaces and/or a means to interrogate cell function and intercellular communication.
  • the structured 2-D cell networks may be interfaced with a patch-clamp platform that allow simultaneous recording, or stimulation of individual cultured cells in the network. This is accomplished by using an alternative to recent "patch-on-chip" technology that has been applied to isolated cells in suspension, but which is not suitable for use with cells growing on a substrate.
  • Traditional/conventional patch pipettes are constructed from various types of glass
  • Gigaseal formation is accomplished by, either physically positioning the patch pipette in very close proximity to the cell, or by positioning the suspended cell over an aperture in the planar chip, and then applying suction to draw the cell membrane to the pipette or chip substrate such that molecular forces are exerted over a nanometer distance.
  • physical alignment and suction are required for gigaseal formation.
  • Planar patch-clamp technology has not been applied to cells grown in culture, in organized networks. This would be tremendously useful, since ion channel activity or membrane potential could be monitored simultaneously in multiple cells connected in a well-defined circuit for extended durations. For example, in the case of neurons, pre- and post-synaptic events can be monitored.
  • Patterned microstructures have been used as tools to position cells (Ratner & Bryant, 2004), and alterations to nano-scale surface topography, made by etching silicon wafers, have been used to guide growth through interactions with growth cone filopodia (Fan et al., 2002).
  • Microcontact printing methods such as lithographically applied polylysine- conjugated laminin patterns, have also been used to guide neuron attachment and axonal outgrowth (James et al., 2000).
  • Figure 1 depicts a schematic drawing of novel planar patch-clamp interface for neurons in a synthetic network grown on a patterned substrate.
  • Neurons are positioned using locating wells (similar to those shown in Figure 4a) over 2-4 ⁇ m diameter orifices (O) which individually communicate with a specific subterranean fluidic channel housing an electrode.
  • a high resistance seal between the cell membrane and the perimeter of the orifice ensures detection of current flow through ion channels in the membrane patch covering the orifice.
  • Neurite growth is directed towards neighbours using guidance pathways (dotted lines), similar to the patterned channels shown in Figure 6a.
  • Electrodes in the subterranean microfluidic array are connected to a multichannel voltage controller/current amplifier and referenced to an electrode in the upper perfusion chamber. The above cues can be used to manipulate cell/extracellular matrix substrate interactions and guide cell positioning and attachment in the substrate and subsequent growth and connectivity in culture.
  • a high resistance (gigaohm) electrical seal that "partitions off' a small circular area (e.g. 0.5-10 ⁇ m diameter) of the membrane surface (area in left side circle in Figure 1) is achieved.
  • the apertures are connected to individual microfluidic perfusion channels and electrodes. This allows the recording of currents resulting from ion channel activity in the region of membrane over the orifice (essentially cell- attached patch-clamp).
  • a pore-forming antibiotic may be perfused into the area beneath the orifice to create a "perforated patch", permitting the recording of "whole-cell” currents resulting from ion channel activity through the entire cell membrane.
  • the subterranean architecture is constructed such that each orifice (and each cell) is connected to a different microfluidic channel and electrode. This permits the study of multiple cell-cell communications through gap junctions, or synapses (area shown in right side circle in Figure 1).
  • the surface for cell surface attachment, the size of the orifices, the perfusion channel dimensions and lengths and the microfluidic perfusion system may be selected to optimize use of the apparatus for particular cell types, for particular growth conditions, and/or to study particular phenomena.
  • the surface for cell attachment will be poly-dimethylsiloxane (PDMS) or any other suitable silicone derivative, laminin, collagen, structural extracellular matrix proteins, nanopattemed surfaces, proteins that modulate cellular interaction with the extracellular matrix, and/or polylysine.
  • PDMS poly-dimethylsiloxane
  • laminin laminin
  • collagen structural extracellular matrix proteins
  • nanopattemed surfaces proteins that modulate cellular interaction with the extracellular matrix
  • proteins that modulate cellular interaction with the extracellular matrix and/or polylysine.
  • the orifice size will be between about 1 to 10 ⁇ m. In some instances 2-8, in some instances 3-7 ⁇ m.
  • the orifice may be fitted with a sieve structure to provide support to the cell membrane, while still allowing membrane perforation.
  • the apparatus is described primarily with reference to the example of mammalian cells or mammalian-derived cells as the cells for examination, it will be appreciated that the invention includes embodiments useful in the study of other cell types, including bacterial cells. Dimensions and coating of the apparatus is preferably adjusted in light of the known preferences and size of the cells to be examined. For example, to study signalling within a bacterial colony, smaller orifices would be employed and an appropriate substrate coating such as polystyrene or agar would be employed. In light of the disclosure herein, one skilled in the art of the bacteria in question could readily select appropriate parameters.
  • the medium or other fluid employed within the perfusion channels will be selected based on known features of the medium or other fluid, the cells to be examined, and the nature of the investigation.
  • exogenous signalling messengers or other materials to induce or trigger a cellular response may be included in or added to the medium or other fluid to allow examination of the resulting cellular response or to manipulate (genetically or otherwise) cell development.
  • Ion- or voltage-sensitive dyes will in some instances be introduced in the chip perfusion channels and changes (eg. Fluorescence) in the dyes are monitored in parallel to electrical activity resulting from ion channel activity.
  • a cell adhesion surface adapted to permit the attachment and growth of cells of interest; • the surface defining a plurality of discrete orifices; • channels each having a cell end and a fluidic circuit bypassing the orifice; • the cell end of each channel in sealed connection with a single orifice; • the fluidic circuit being adapted to permit operative connection of an electrode so as to permit the taking of measurements within the channel, as well as the circulation of fluids without going through the orifice.
  • Hybrid silicon-polymer chips with microscale topography and contrasting surface chemistries were created using a novel combination of soft lithography techniques, and evaluated for their suitability as a platform to guide cell attachment, growth and differentiation. These capabilities are all desirable to synthesize organized neural networks in vitro. Neurons developed on these chips exhibit patterned growth and functional communication, evidenced by spontaneous and stimulated action potentials and intracellular calcium oscillations.
  • FIG. 1 A. Microstructure Fabrication and Surface Chemistry Modification The general protocol used for the rapid and efficient fabrication of the polymer microstructures and their subsequent chemical patterning is outlined in Figure 2.
  • Figure 2 depicts the fabrication and chemical patterning of PDMS microstructures. a) PDMS stamp with 10 ⁇ m deep channels hydrophilized by air plasma to introduce silanol groups on the surface, b) Chemisorption of a fluoro- siloxane derivative on the surface and curing in aqueous solution to form a highly hydrophobic surface, c) Spin coating of thin layers of uncured PDMS precursor on glass substrate, d) Imprinting of the microstructures by the fluorinated stamp and curing by heating, e) Hydrophilic microstructures created by air plasma, f) Chemical patterning of the PDMS microstructures through the introduction of hydrophobic functional groups, g) Transfer of the fluorinated siloxane and curing, h) PDMS microstructures with a dual hydrophobic-hydrophilic
  • a flexible stamp was made by replicating PDMS, using Sylgard 184 kit and a silicon wafer patterned with an SU8 negative photoresist (Microchem), having microsized features as a master mold following published procedures (Xia, 1998; Bensebaa, 2004).
  • the microstructures on the master mold consist of, either 5, 10, 25, 50 and 100 ⁇ m wide recessed lines spaced by the same width or of square pillars of 5, 10, 25, 50 and 100 ⁇ m spaced by the same dimensions.
  • the thickness of the SU8 photoresist was set to 10 ⁇ m.
  • the replicated PDMS stamp ( Figure 3 a and 3b) exhibits features that are complementary to those of the master SU8-silicon mold.
  • the PDMS stamp was subsequently washed in a Soxhlet setup using ethanol for 3 hours to remove any unreacted oligomers.
  • the washed PDMS stamp was characterized by contact angle (112.7°), XPS and ATR-FTLR.
  • the PDMS stamp was rendered hydrophilic by creating -OH groups on the surface in an air plasma reactor for 1 minute at 2 x 10- 1 mbar.
  • the PDMS-OH stamp shows a very low contact angle (6.4°) and was stored in de-ionized water prior to further modification in order to preserve its hydrophilic character.
  • the patterned PDMS-OH substrate was immersed overnight in 100 mM heptadecafluoro- 1,2,2,2,- tetrahydrodecyl-triethoxysilane (HFS) solution in ethanol.
  • the fluorosilane-modified patterned PDMS (PDMS-CF3) ( Figure 2b) was rinsed thoroughly with ethanol after incubation and immersed in H 2 O for polycondensation of the siloxane groups, yielding highly hydrophobic substrates with a contact angle of 112.8°.
  • XPS and ATR- FTIR studies are in agreement with those reported in the literature for similar materials and indicate that the fluoro-silane derivative is covalently attached to the surface of the secondary PDMS mold.
  • This non-sticking PDMS stamp was put in conformal contact (Figure 2c-d) with an uncured polymer film spin-coated on a glass cover slip (a polystyrene Petri- dish or a silicon wafer also worked) and cured thermally.
  • a film of uncured PDMS prepolymer was spin-coated at 2000 rpm to yield a thickness of 25-30 ⁇ m, imprinted by the secondary PDMS mold and cured at 90°C for 2 h. After curing, the fluorinated stamp was easily removed from the substrate. The features transferred to the polymer substrate were found to be complementary and virtually identical to those of the stamp ( Figure 3c and d). The imprinted samples were characterized by optical microscopy, scanning electron microscopy, and profilometry. It was found that the fluorinated stamp could be used multiple times, without mechanical or chemical degradation. The master mold, made by a standard lithography technique, could also be re-used. The imprinted PDMS was rendered hydrophilic in an air plasma reactor.
  • the imprinted PDMS-OH was stored in de-ionized water prior to further modification in order to preserve its hydrophilic character.
  • a flat PDMS obtained by thermal polymerization of a PDMS prepolymer in a polystyrene Petri-dish, was inked with HFS for 30 minutes, dried with nitrogen and then put in conformal contact with the top of the imprinted PDMS-OH for 90 minutes using a modification of a published methodology (Pfohl, 2001). After removing the stamp, the imprinted PDMS now had a dual character: hydrophobic (fluorinated) on top of the channels and hydrophilic (silanols) inside the wells or the bottom of the channels.
  • the samples were stored in water insuring the polycondensation of the siloxane groups on top of the imprinted substrate and the conservation of the silanol groups in the bottom and the walls.
  • Micro-XPS imaging indicated that only the tops of the imprinted microstructures contain fluorine (see Figure 2).
  • a flexible stamp was made by replicating PDMS, over a master mold following published procedures (Bensebaa et al., 2004).
  • the microstructures on the master mold consisted of either 5, 10, 25, 50 or 100 ⁇ m wide recessed lines spaced by the same width or square pillars of 5, 10, 25, 50 or 100 ⁇ m spaced by the same dimensions.
  • the replicated PDMS stamp (Fig. 3a,b) exhibited features complementary to those of the master SU8-silicon mold.
  • the PDMS stamp was rendered hydrophilic by creating -OH groups on the surface in an air plasma reactor for 1 min at 2 x 10 "1 mbar.
  • the patterned PDMS-OH substrate was immersed overnight in 100 mM heptadeca-fluoro-l,2,2,2,-tetrahydro-decyl-triethoxysilane (HFS) solution in ethanol.
  • FFS fluorosilane-modified patterned PDMS
  • the features transferred to the polymer substrate were complementary and virtually identical to those of the first stamp (Fig. 3c,d).
  • the imprinted PDMS was rendered hydrophilic in an air plasma reactor as shown.
  • the imprinted PDMS-OH was stored in de-ionized water prior to further modification in order to preserve its hydrophilic character.
  • a flat PDMS surface obtained by thermal polymerization of a PDMS prepolymer in a polystyrene Petri-dish, was inked with HFS for 30 minutes, dried with nitrogen, and then put in conformal contact with the top of the imprinted PDMS-OH for 90 min (Fig. 2f,g) using a modified methodology (Li et al., 2001). After removing the stamp, the imprinted PDMS now had a dual character: hydrophobic (fluorinated) on the upper surface and hydrophilic (silanols) inside the wells or channels (Fig. 2h).
  • N2a neuroblasts ATCC, Manassas, VA
  • cortical neurons from embryonic day 13 or 17 mice or embryonic day 17 rats were isolated and plated accordingly.
  • Figure 4 depicts microstructures and surface chemistry modifications effectively position N2a cells and guide proliferation, a) Hoffman contrast image showing that 50 ⁇ m square hydrophilic wells locate N2a cells and promote rapid attachment, b) Cells undergo division within 10 h and c) a colony has formed within 48 h.
  • N2a cells position and proliferate in hydrophilic channels 50, 25, and 10 ⁇ m wide (top to bottom). Channels narrower than the cell diameter alter cell shape and attenuate proliferation after a few divisions, e) F-actin immunostaining shows N2a cells extend processes along the edge of a 25 ⁇ m channel as they differentiate. Inset: deconvolved image of a growth cone guiding the neurite within the channel. Dashed lines represent the boundary of the channel. Topographic features of the substrate effectively positioned N2a neuroblasts in squares or channels, (Fig. 4a,d) and the hydrophilic nature of these microstructures promoted selective cell attachment after plating within the boundary of the microstructure.
  • a minimum ratio of 1.5:1 (channel width : cell diameter) was required for N2a cells to proliferate.
  • Cells seeded in 10 ⁇ m channels displayed attenuated proliferation and oval morphology.
  • Histology using F-actin antibody showed neurites guided by their growth cone within the confines of the channel (Fig. 4e).
  • Neural progenitors from E13 mouse cortex were also cultured on microchannel- patterned PDMS substrates. Neurons developed within the confines of the hydrophilic channels and displayed organized parallel architecture similar to that seen in brain substructures (Fig. 5). Unlike N2a cells, they were not hindered in the 10 ⁇ m wide channels (Fig.
  • FIG. 5c Remarkably, in contrast to neurons, astrocytes were not influenced by topological or chemical patterning features and grew randomly (Fig. 5d).
  • Figure 5 depicts microchannels and surface chemistry modifications effectively position cultured E13 neurons and guide growth, a) Hoffman contrast image showing neurons grown on 50, 25, and 10 ⁇ m wide hydrophilic channels (top to bottom), b) MAP-2 staining showing neurons (red) growing in 50 ⁇ m, c) or 25 ⁇ m wide channels. Dashed lines represent the boundary of the channel, d) GFAP- positive astrocytes are not guided on the same patterned substrate. C.
  • FIG. 6 depicts simple synthetic neural networks display excitability and connectivity on patterned PDMS substrates, a) Phase contrast image of a PDMS substrate, showing 25 ⁇ m wide hydrophylic channels to guide neural growth. Oriented E17 neurons can be observed in these channels, b) Fluorescence images taken 20 s apart show neurons loaded with calcium-sensitive dyes.
  • a stimulating electrode was positioned at one end of the PDMS channels (arrows). Traces 1-4 show relative changes in intracellular calcium concentration at numbered regions of interest identified in the top left image. Stimulation was applied at 30 s intervals (indicated by arrows below traces) to induce calcium oscillations, c) Voltage oscillations recorded from an E17 neuron, using whole-cell patch-clamp. Spontaneous, cyclical waves, composed of multiple action potentials, were observed. It was possible to briefly synchronize this activity using current stimulation (indicated by arrows) through the patch pipette. Scale bar: 10 mV, 1 s. Membrane potential oscillations were recorded in E17 cortical neurons using whole-cell patch-clamp.
  • This apparatus and method enables researchers to simultaneously monitor ion channel function in multiple, synaptically-connected cells in a well-defined circuit for extended durations. This provides a powerful research tool to investigate synaptic function and network signaling. Furthermore, from a pharmacological screening perspective, it presents an attractive alternative to fluorescence intensity plate reader assays, or to electrophysiological assays using isolated cells in suspension.
  • Biochip fabrication methods In an embodiment of the invention there is provided a method of studying cell membrane related activities comprising: (a) obtaining a cell adhesion surface having discrete orifices therein with attached channels;
  • FIG. 7 is a schematic description of an embodiment of a substrate such as a chip for a patterned cell network such as a neural network (7a), an MEA interface electrode array (7b) a microfluidic array (7c), a synthetic neural network MEA interface (7di), a PDMS chip with guidance pathways and electrical contacts (7dii), and electrical contacts (7dii), and a patch-on-chip interface (7e).
  • a substrate such as a chip for a patterned cell network such as a neural network (7a), an MEA interface electrode array (7b) a microfluidic array (7c), a synthetic neural network MEA interface (7di), a PDMS chip with guidance pathways and electrical contacts (7dii), and electrical contacts (7dii), and a patch-on-chip interface (7e).
  • Agents of interest may include pharmaceuticals, pharmaceutical candidates, small molecules, oligopeptides, polypeptides and derivatives thereof, DNA's, RNA's carbohydrate-derived compounds, hormones and hormone derivatives, neurotransmitters and their derivatives, agonists and/or antagonists of cell surface or internal cellular receptors or proteins of interest found in or on the cultured cells.
  • a cell-growth substrate comprising: an electrically insulating membrane supported by a solid wafer having microholes extending across it; - said solid wafer having apertures defined therein such that channels are provided across said support; - the channels in the inert substrate being located in substantial alignment with microholes in the membrane so as to provide a passage across both the membrane and the support; - a substantially rigid enclosing layer of substantially inert material sealably engaging portions of the substrate; - said enclosing layer having defined therein thicker and thinner regions such that, in combination with the enclosing layer, a series of channels extending substantially parallel to the membrane are defined; - cell guidance regions of substantially inert material secured to the exposed surface of the membrane such that at least some microholes with aligned channels remain open.
  • the membrane may be formed on the desired support or formed elsewhere and transferred onto the desired support.
  • the general purpose of the membrane is to facilitate the fabrication of a precise microhole. Thus, any suitable structure for that purpose will suffice.
  • a "cell-suitable membrane” is a membrane which is capable of supporting the growth of adherent cells of at least one type.
  • the membrane will be a thin film such as silicon nitride (SiN) or a heavily boron doped layer of an Si substrate, polyimide, etc.
  • a "cell-suitable membrane” is a membrane which is capable of supporting the growth of adherent cells of at least one type. In many instances, coating the cell contact portion of the membrane with a combination of a parylene thin film and polylysine treatment will make it cell-suitable even if it was not cell- suitable prior to coating.
  • microholes may vary in size depending on the cell type of interest.
  • microholes in a given membrane will preferably have a diameter of between about 0.5 to 10 ⁇ m. In some instances a microhole diameter of between about 1 and 7 ⁇ m will be desired. In some instances, a microhole diameter of between about 2 and 6 ⁇ m will be desired.
  • the enclosing layer may be produced from any one or combination of materials which is substantially inert to the medium and conditions intended for use with the cell system to be studied.
  • the enclosing layer is preferably made from a material which is substantially rigid or insufficiently elastomeric to collapse during the intended use. In some instances a curable polymer will be desired. In other instances a material which must be formed into the desired shape by machining, chemical etching, or another process whereby a portion of an original whole is removed, will be preferred.
  • the guidance regions may be made from any single or combination of materials which is substantially inert in the culture medium and conditions intended for use with the cell type of interest. In some instances the guidance regions provide a less favorable surface for adhesion by that cell type than is provided by the membrane. Guidance regions of this type are called “patterned growth guidance regions.” In some instances guidance regions will be formed as "wells” and “trenches” on the membranes. An example of an embodiment of this is in Figure 8.
  • the range of ratios (cell volume : channel volume) will be between about 1 x 10 "19 liters (cell): 0.2 mm 3 (channel) and about 1 x 10 "14 liters (cell): 0.001 mm 3 (channel). In some cases the range of ratios (cell volume : channel volume) will be between about 1 x 10 "18 liters (cell): 0.1 mm 3 (channel) and about 1 x 10 ⁇ 15 liters (cell): 0.01 mm 3 (channel). These ranges are provided by way of example only and it will be appreciated that a wide range of ratios are possible, depending on the cells, conditions and particular substrate configurations employed and the objects and duration of the study to be conducted.
  • Figure 8A depicts a schematic representations of an embodiment of substrates for use with a cell network such as a neural cell network.
  • Wells and trenches are conducive to selected implantation of neurons and directed growth of neurites (figure 8A(i) is of a pattern with 20 ⁇ m square wells, 3 ⁇ m wide trenches, all being 70 ⁇ m deep).
  • Micro-hole membranes have been described in the literature as allowing monitoring the electrophysiological activity of ion channels in neurons.
  • Subterranean microfluidics channel allow both recording this activity and delivery of drugs or other compounds of interest to the cell to allow chemical patch-clamping and other studies.
  • Jh Figure 8 A the top layer (solid lines): cell placement and directed growth (wells and trenches network). Micro-holes (black dots): join top to subterranean network. In this embodiment the holes are very small (3-5um) and are formed in a membrane (dashed lines).
  • Figure 9(a) is a side-view of an embodiment of the membrane micro-hole and explains how cell activity is monitored. It contains 4 parts (all within Figure 9(a); a) a substrate carrying a membrane with micro-hole, b) cell container, c) microfluidic channels, and d) electric connections.
  • Si wafer based neurochip 2.1.1 describes the general method of fabrication and an actual recipe.
  • Paragraphs 2.1.2 to 2.1.8 describe possible variations to the process, and their advantages.
  • a method of producing a chip suitable for use in growing cells so as to promote growth of structured two- dimensional networks comprising: a) obtaining a SiN/Au thin film on a Si wafer with a (100) crystalline orientation; b) creating microholes in the SiN/Au thin film (in some instances preferably having a diameter of between about 1 ⁇ m and about 5 ⁇ m) (in some instances a single hole may be desired, in other instances a sieve structure may be desired); c) bonding the front of the wafer to a carrier with wax or another sacrificical layer (that can later be released); d) thinning down the wafer by lapping to preferably a thickness of between about 25 and 75 ⁇ m; e) obtain a SiO thin film mask in the back of the wafer and create (preferably about 75-125 ⁇ m) more preferably about 100 ⁇ m windows in the SiO 2 thin film mask, said windows being aligned
  • the term "tower” refers to a solid structure which, when used as a mold, results in a well in the resulting product. In some instances a tower may have a square cross-section. However, it will be apparent that other shapes are possible including rectangular, circular, elliptical, and irregular shapes.
  • the term "wall” refers to a solid structure which, when used as a mold, will result in the connection of two wells in the resulting product. In some instances a wall will have a narrow rectangular cross-sectional area. However, it will be understood that other shapes are possible. Well size and shape may be selected based on the cells and conditions for study and, in light of the disclosure herein, it will be within the ability of one skilled in the art to do so.
  • step a Au is thought to be advantageous as a mask for etching SiN and as a protection against damage in subsequent steps, but it's use is optional and it could advantageously be replaced by Ni, Al, Cr and many other metals used in semiconductor technology; its nature is not critical to the application since the chip can be passivated with a bio-compatible plastic film in step g).
  • the SiN layer itself can be replaced by silicon dioxide (SiO 2 ), a metal film, or polymers such as polyimide, as long as the material of the substrate can be etched selectively to it.
  • the Si wafer itself can be replaced by other types of substrates, regardless of their electronic properties or bio-compatibility (glass, metal foils), but Si is thought to be particularly advantageous as fabrication processes are well-known to the semiconductor industry with that material.
  • the SiO 2 thin film mask in step e) can be replaced by other material known to be selective to Si in a KOH solution: for example SiN itself.
  • the KOH etching step may be replaced by other solutions known to etch Silicon, such as hydrazine, tetramethylammonium hydroxide or other solutions known to the state of the art (see Thin Film Processes, J.L.Vossen and W.Kern,Academic Press, NY), or by dry etching techniques such as reactive ion etching employing sulfur hexafluoride (SF6) as the reactive gas (same ref). For all those techniques, a different thin film mask as defined in step e) will be appropriate.
  • PDMS in step g) can be replaced by any curable polymer, such as the UV-curable epoxy 1191-M provided by Dymax Corp.
  • PDMS is thought to be advantageous as it is flexible and will bond easily with the rigid Si substrate.
  • SU8 in step j) is thought to be advantageous as tall structures capable of effectively guiding the implantation of neurons and growth of neurites can be obtained in a single lithographic step, but it may be replaced by a film etched by methods similar as in step e).
  • Figure 8B depicts an embodiment of this method with reference to the lettered steps.
  • the membrane in which microholes are formed is produced by imaging a lithography mask on the membrane. In some instances it will be desired to put designs on the membrane.
  • SiN membrane instead of using a SiN membrane one may use boron doped Si (Si:B) as a KOH etch stop.
  • Si:B boron doped Si
  • a Si membrane can have better physical properties than SiN. It is a strong, single crystal material and is a perfect match with the substrate. This process also avoids the use of PECVD film growth and ICP etching. See Figure 9(b) and Table II for a schematic description.
  • a specific fabrication process can be given by replacing the first two steps in section 2.1.1 General description of fabrication method with the following two steps.
  • Steps c) to k) could be the same as described in the general process description, section 2.1.1.
  • Table H
  • step h involves bonding two layers. These layers could be aligned optically, but the sloped (111) surfaces formed in the Si wafer during anisotropic etching could be used to allow mechanical alignment. In this case, the PDMS fluid channel layer would be shaped to exactly match features in the Si wafer, which will guide the positioning as the two pieces are brought together. See Figure 9(d). 2.1.5) - Wiring on Wafer Front Side
  • This approach provides an alternative "up-side-down" version of the membrane.
  • a microfluidics part is bonded onto that.
  • a SU-8 or similar layer is patterned on top in cases where it is necessary or desirable to confine the cells.
  • the three parts are all micromachined in a Si wafer. Also, as shown by the dashed line, it is possible to make trenches in the top surface to connect the cell pits.
  • processing of the above can be achieved by: 1) etch from back. 2) oxidize and pattern oxide for boron doping. This would preferably be accomplished by projection lithography (or electron beam lithography).
  • a SiN layer could be grown and used as the membrane. 3) etch from the top.
  • a single etch step can be used to form pits plus connecting trenches.
  • Figure 9(f) A possible layout of the micro-fluidic channels is sketched in Figure 9(h). The fluid channels would be completed by bonding a flat sheet of suitable material to the bottom of the Si wafer. This bottom layer could include wiring for electrical connections. See Figure 9(g).
  • the assembled substrate in some instances it will be desirable to coat the assembled substrate in a substantially non-conductive coating, such as parylene.
  • Alignment slots can be etched completely through the Si wafer, which, if desired, can match with pegs in a PDMS section to act as an alignment guide during bonding. See, for example, Figure 9h.
  • polyimide may be desirable to replace the more expensive and more complex membrane processes involving SiN or Si:B. Using polyimide will still allow the formation of an accurate micro-hole.
  • Polyimide is tough and dimensionally quite stable. When fully cured it is resistant to most solvents and acids. It is also stable at temperatures up to 400°C.
  • the thickness of the polyimide could be, for example, 2 ⁇ m. This would be coated with a metal film, which would be patterned to define the microholes.
  • the metal could be Ti, Ni, Au, Cr, or others.
  • Step f) would change as follows: f) etch the Si wafer in a KOH solution through the windows in the SiO2 mask that will reveal (111) facets in the Si crystals, thereby creating an inverted pyramid structure.
  • the KOH etch is preferably stopped with a thin layer of Si remaining before reaching the polyimide layer. This is because the KOH will etch the polyimide.
  • the etching process can be completed with a short isotropic etch to remove the final Si and exposing the polyimide membrane.
  • a suitable isotropic etchant would be a mixture of hydrofluoricacid, nitric acid, and acetic acid. Steps from g) to k) could remain the same.
  • Figures 8 depicts an embodiment of a basic 8-orifice chip design with flow-through channels.
  • Figures 10 and 11 and related Table III set out steps for an embodiment of the production and fusing of two PDMS chips. Table III
  • Steps (a) to (c) and (g) are omitted and a tip connected to a beam is employed to form the microhole (the apex of the tip in contact with a tower defines the microhole) and the beam typically defines an open channel to be closed in an assembly step, which will typically not require alignment.
  • the tip and beam may be formed from any suitable material. A material will be suitable if it is sufficiently firm to define the microhole and channel and can be removed once these structures have been formed. It will be understood that the "beam" may be contoured and/or bendable to permit formation of channels in various directions and/or dimensions.
  • AFM Atomic Force Microscope. This example relates to an AFM tip because such tips are readily available. However, it will be understood that any suitably-sized tip having an extension thereof will suffice, provided that the tip dimensions is suitable to form the desired size of microhole and the extension is of a suitable size and shape to define the desired channel.
  • An AFM tip is composed of a tall sharp tip, usually in Si, at the end of a cantilever. This general shape can be used to form a membrane micro-hole using the 3D PDMS molding process disclosed herein, by which complementary shapes to the masters are formed. The apex of the tip, in contact with a pattern conducive to the placement of cells, forms the micro-hole; the cantilever forms a subterranean microfluidic channel.
  • AFM tips fabrication processes are now in an industrial phase), but many different processes can be derived from Field-Emission Displays fabrication processes (see proceedings of International Vacuum Microelectronic Conference in JVST B, for example JVST B 15(2) (1997)).
  • a picture of a type of AFM tip is shown in Figure 12. The tip is 15um tall, its apex has a radius of curvature under 15nm; it is mounted on a 7um thick, 33um wide and 200um long cantilever.
  • the apex of the tip need only have a radius of curvature of a few microns and 2) the cantilever is not self standing but etched as a wall supported by the bulk of the Si wafer (see Figure 13 comparing a standard AFM tip with the one preferred in this case).
  • the tip will preferably be taller (50 ⁇ m); since its base rests on the cantilever-wall, that will in such cases preferably be wider (100 ⁇ m) and it would benefit from being taller for a better flow of the fluids (20 ⁇ m). All these changes are possible without changing the fabrication process.
  • the tip is preferably coated with an anti-sticking layer such as Teflon or Ti/Au, the thickness of which would be controlled such that the apex of the tip would preferably be no more than 5 ⁇ m, when 3 ⁇ m holes are desired, (coating thickness and tip size and shape can readily be adapted to a desired application, in light of the disclosure herein).
  • an anti-sticking layer such as Teflon or Ti/Au
  • One or several cantilever tips is aligned to a pattern of towers and walls patterns (ie, the complement of wells and trenches) in SU8-50 on a glass or flexible plastic sheet, such that the tip is centered on the top of the tower (see Figure 14 where the tips and cantilevers are indicated by “a” and the towers and walls by “b”).
  • the channels are preferably not blind ( Figure 14 is provided merely to describe a possible arrangement using an AFM tip or equivalent).
  • the Si chip is flooded with PDMS such that it immerses the AFM tip (alternatively, the alignment can be performed if the PDMS is poured first).
  • the bottom Si chip is removed first, and then the PDMS is fused to a second glass or PDMS sheet to close the microfluidics channel. Finally, the top glass or plastic sheet is removed. This simple process forms a micro-hole membrane at the apex of the tip, aligned with a wells and trenches network on top and connected to a microfluidic channel as the complement of the cantilever.
  • a commercial AFM tip can be broken from its cantilever and fused to a PDMS or glass sheet by spinning a PDMS film, laying the tip on it, and curing the PDMS.
  • the resulting mount is covered by a film 3 ⁇ m thick to blunt the tip and reinforce it.
  • microhole can be formed during the molding process.

Abstract

La présente invention a trait à un procédé et un appareil appropriés pour une utilisation dans l'étude d'activités liées à la membrane cellulaire. Des activités d'intérêt comprennent des études liées à la méthode patch-clamp de réseaux de cellules sur un substrat solide. On réalise la croissance de cellules, de préférence en configuration à motif, sur un substrat présentant des micro-trous. On forme des joints entre les cellules et les micro-trous. Chaque micro-trou est fixé à un canal. Dans plusieurs cas un seul trou va être fixé à un canal unique, permettant l'examen des effets d'un stimulus à une pluralité de points différents dans le réseau d'un ou de plusieurs types de cellules. L'invention peut présenter un intérêt, par exemple, pour ceux désirant étudier des interactions entre neurones ou entre jonctions neuromusculaires.
EP05741336A 2004-05-06 2005-05-05 Interface de substrat de reseau cellulaire a motif et ses procedes et utilisations Withdrawn EP1751267A4 (fr)

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