WO2006135258A1 - Moulage - Google Patents

Moulage Download PDF

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Publication number
WO2006135258A1
WO2006135258A1 PCT/NZ2006/000150 NZ2006000150W WO2006135258A1 WO 2006135258 A1 WO2006135258 A1 WO 2006135258A1 NZ 2006000150 W NZ2006000150 W NZ 2006000150W WO 2006135258 A1 WO2006135258 A1 WO 2006135258A1
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WO
WIPO (PCT)
Prior art keywords
cell
cells
cellular material
cellular
mould
Prior art date
Application number
PCT/NZ2006/000150
Other languages
English (en)
Inventor
Maan Mustafa Alkaisi
James Johan Muys
Original Assignee
Advanced Nano Imaging Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Advanced Nano Imaging Limited filed Critical Advanced Nano Imaging Limited
Publication of WO2006135258A1 publication Critical patent/WO2006135258A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G01N2001/366—Moulds; Demoulding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G01N2001/368—Mounting multiple samples in one block, e.g. TMA [Tissue Microarrays]

Definitions

  • This invention relates to a process for creating replicas of cellular materials using soft lithography techniques.
  • the process is particularly applicable to cells in a near living state.
  • the process is termed "BioimprintingTM”.
  • Lithographic techniques are generally not applicable to biological materials. The techniques require the use of elevated temperatures and pressure, or give low resolution patterning at the nanoscale (US 2005/019503). Polymers employed in lithography such as PDMS have also been regarded as unsuitable for patterning of biological materials due to their cytophilic properties (US 6,770,721).
  • the imprinted polymers are at micrometer scale. No nanoscale images of cells are included or suggested.
  • the present invention provides a process for producing an imprint of cellular material, the process comprising: a) placing cellular material on a substrate; b) incubating the cellular material in media under near physiological conditions to promote attachment to the substrate; c) aspirating the media from the cellular material; d) coating the cellular material on the substrate with nanoscale moulding material; e) curing the nanoscale moulding material; and separating the mould produced in step e) from the cellular material.
  • Curing may be partial or total.
  • curing may be initiated prior to the application of the moulding material to the cells.
  • the curing is a rapid curing step.
  • the cured moulding material is removed from the cellular material to provide the mould or Bioimprint.
  • the moulding material is removed from the cells without further processing before removal.
  • a further curing or hardening step may be effected once the Bioimprint is removed from the cellular material.
  • a washing step may optionally be effected to remove any residual biological material in the Bioimprint.
  • the cellular material in step c) may be subjected to further treatment such as drying and/or washing with buffer, and optionally a further incubation step before stepd).
  • the nanoscale moulding material is polymethylsiloxane (PDMS) (methacryloxypropyl) methylsiloxane (mcpms-DMS), or isobornylacrylate (IBA) monomer.
  • PDMS polymethylsiloxane
  • mcpms-DMS methacryloxypropyl methylsiloxane
  • IBA isobornylacrylate
  • the nanoscale moulding material is degassed prior to use. Where rapid curing such as UV curing is effected, the moulding material may further comprise a photoinitiator.
  • the invention provides a cast produced by subjecting a Bioimprint or mould of the invention, to coating, curing and separation steps as set out herein, or similar.
  • the invention also provides a mould or cast when produced by a process of the invention.
  • the invention also provides a kit for use in analysis of cellular material the kit comprising: nanoscale moulding material; a substrate for the cellular material; and optionally instructions for use.
  • the kit is for cytological analysis.
  • the nanoscale moulding material may be preapplied to the substrate.
  • a preferred substrate is a biochip.
  • the nanoscale moulding material may also be pre-photoinitiated for curing. In that instance, the moulding material is packed in light impenetrable packaging.
  • kit components may be present in one or more containers.
  • the kit may further include patient data cards.
  • the kit preferably includes instructions for use.
  • the present invention also provides a process for nanoscale imaging of cellular material, the process comprising:
  • Microscopy at nanoscale is conveniently effected using atomic force, scanning electron or scanning tunnelling microscopy.
  • the image obtained by microscopy may be permanently recorded, for example as a digital photograph.
  • the moulds and casts are themselves a permanent record also.
  • the invention also provides a process for analysing function or interaction of cellular material in response to an agent or stimuli the process comprising:
  • the cellular material is a cell sample.
  • the cellular material is protein
  • Agents useful in this process include cells, drugs, ligands, proteins, amino acids, virus, bacteria, toxins and endocytotic molecules.
  • Preferred ligands include antibodies, proteins, DNA and cell receptors.
  • Stimuli useful in this process include electrical, chemical, magnetic sound and radiation stimuli.
  • the invention provides a cytological screening system comprising: a cellular Bioimprint or cast produced according to a process of the invention; and a cell analyser.
  • the analyser examines the Bioimprint for abnormal cell in particular.
  • Another aspect of the present invention is a cytological screening method, the method comprising: a) producing a cellular Bioimprint or cast according to a process of the invention; and b) examining the Bioimprint or cast to identify features indicative of cellular abnormality, variation from a range or threshold level in a control cell sample indicates that cellular abnormality exists.
  • the invention also provides a screening method, the method comprising: a) producing a mould or cast of a cellular material according to a process of the invention; and b) examining the mould or cast to identify features indicative of abnormality, variation from a range or threshold level in a control sample of cellular material indicating that abnormality exists.
  • Examination is preferably by AFM, SEM or STM microscopy.
  • a quantitative report can be generated by comparing the results of the cytological screening method with reference values for normal cells. It will be appreciated that the method may be automated with the examination and report generation effected using an appropriately programmed computer system.
  • Bioimprint process Transfer of cell membrane data into PDMS casting's and molds using soft lithography. Components are not drawn to scale and are exaggerated for clarity. Figure 2 - Optical Images of Molds
  • Optical images showing PDMS molds and castings taken of pituitary cells at curing temperatures ranging from (a) 95 0 C, (b) 37°C and (c)-(f) 65-75°C. Arrows indicate nucleus dehydration effects replicated from cells into the PDMS during imprint process.
  • Figure 4 AFM Image of Cell in Fluid AFM micrograph of a 20 ⁇ m, 5 day old cultured pituitary cell in liquid taken in tapping mode.
  • BioimprintTM pattern transfer scheme for fabrication of cellular impressions using flash imprint lithography
  • AFM BioimprintTM (a) impression of a cell fabricated in polymer and its (b) mirror inversion.
  • Figure 11 Molecular imprint of an impression focused on a dimple site imaged in AFM phase mode, illustrates the underlying 'scaffold' protein structure.
  • a 5 ⁇ m amplitude image shows the varying stages of pit convergence, where arrows 1 and 2 show the beginning and final phases of merging, respectively. Further illustrated in the 3 ⁇ m (b) phase image are two pits (3, 4) beginning to blend.
  • AFM image focused on an area of membrane of endometrial cancer cell showing numerous pits with a mean diameter of 375 nm.
  • AFM images showing Rapid cell division (a) Deformed/irregular membrane (b,c)
  • Figure 17(b) Photograph showing pituitary cells captured within lO ⁇ m cavities by a pointed interdigitated microsystem using positive dielectrophoresis.
  • the suspending medium was Tris-Gly-Dex of conductivity 1.7 mS/m, with an AC applied voltage of 8 Vp-p at a frequency of 930 kHz.
  • a magnified region of the array clearly shows the cells trapped within the cavities.
  • Figure 18 AFM image of the positive replica of a 40 ⁇ m endometrial cancer cell created by digitally inverting its corresponding polymeric impression.
  • the micrograph shows numerous dimple depressions scattered and concentrated around a nucleus (N) form, which is visible by the conformation of the membrane around it.
  • N nucleus
  • a scope trace focused on the membrane above the nucleus details the indentation (1),(2) profiles at locations on the membrane surrounding the nucleus, indicating cell dehydration.
  • Figure 19 AFM image of the positive replica of a 40 ⁇ m endometrial cancer cell created by digitally inverting its corresponding polymeric impression.
  • the micrograph shows numerous dimple depressions scattered and concentrated around a nucleus (N) form, which is visible by the conformation of the membrane around it.
  • a scope trace focused on the membrane above the nucleus details the indentation (1),(2) profiles at locations on the membrane surrounding the nucleus, indicating cell dehydration.
  • Figure 19 AFM image of the positive replica of a 40
  • AFM scan of a positive replica made from an endometrial cancer BioimprintTM impression illustrates a rounded nucleus (N) beneath a membrane containing numerous dimple depressions of varying sizes
  • a 40 ⁇ m wide magnification of the membrane reveals two types of depressions; deep and wide (1) as well as more abundant smaller and shallower (2) pits.
  • a BioimprintTM positive replica of the impression made from a 50 ⁇ m endometrial cancer cell shows the membrane extending leftwards from a 1 ⁇ m tall rounded nucleus (N) body, which is seen to contain two large ruptured depressions (1),(2).
  • the present invention provides a process for producing an imprint or mould of cellular material at the nanoscale.
  • BioimprintingTM refers to the process of forming an impression of cellular material in a nanoscale moulding material.
  • BioimprintTM should be similarly understood.
  • cellular material refers to cellular materials suitable for imprinting including cells and subcellular or intra-cellular structures.
  • cells cell surfaces, tissues, tissue samples, membranes, fusion pores, dimples, enzymes, proteins, antibodies, nucleic acid molecules (eg RNA, DNA), protein-DNA complexes, organelles such as nuclei, mitochondria, chromatin, vacuoles, vesicles, transduction channels, and ion channels, but are not limited thereto.
  • the cellular materials may include toxins such as toxic proteins, spores or pathogens such as virus or bacteria. Any biomaterial which is intended for examination at the nanoscale can be used in the process.
  • the process aims to provide an imprint of the cellular material in a near living state. This may be achieved by imprinting the material in a near-physiological conditions, which maintain the material in a near living state. Any suitable physiological or preservative conditions known in the art may be employed.
  • cellular material may be cultured in medium 199
  • Dulbecco's Modified Eagle Medium Dulbecco's phosphate buffered saline, minimal essential media and the like, at physiological temperatures, for example 20°C to 40°C, more usually 30 0 C to 38°C, and preferably 37 0 C for human cells. Culture may be under normal atmospheric conditions, or 5% CO 2 and 95%.O 2 .
  • near physiological conditions means as close as practical to the conditions the cellular material is in its normal living state, and includes under physiological and preservative conditions.
  • the cellular material is attached to a substrate.
  • Attachment of cellular materials to a substrate prevents features from being completely submersed within the moulding material, as well as facilitating entity separation after moulding.
  • Known art techniques and materials can be used to achieve this attachment.
  • coating the substrate with poly-1-lysine, collagen, gelatin cell-tak, or using PEG crosslinkers can facilitate cell attachment.
  • Silanes carrying amino groups such as amino-propyl triethoxy silane (APTES), or multivalent cations such as Ni(II) and CO(II) can be used to promote protein, DNA and RNA binding to substrates.
  • APTES amino-propyl triethoxy silane
  • multivalent cations such as Ni(II) and CO(II) can be used to promote protein, DNA and RNA binding to substrates.
  • attachment may be effected by incubation on a substrate for a period of a few hours, days, or even weeks.
  • attachment incubation is carried out forat least one day (24 hours) to ensure adequate attachment.
  • Typical incubations are in the range of from 1 day to 10 days, preferably 3 to 7 days, and more preferably 5 days.
  • Substrates for cells include a range of well known surfaces to which cellular materials may adhere.
  • glass surfaces such as glass slides and cover slips, Petri dishes, mica, silicon or quartz and including microarrays and biochips.
  • the microarrays may be well containing plates used in high throughput screening techniques. DNA, RNA and virus molecules bind well to mica and protein substrates.
  • the substrate may include markings to divide the substrate into discrete areas, as well as including unique identifiers for the discrete areas. For example alpha-numeric grid references. Capture may be effected from a fluid containing cells flowed over the array or chip, by submersion of the array or chip in such fluid or by other known art processes. The cell is held inside the well by gravity. Cells may also be directed to unique cell addresses using processes such as dielectrophoresis. (US 6,692,952; James Muys, M.M. Alkaisi, J. J. Evans, J. Nagase, Biochip: Cellular analysis by Atomic Force Microscopy using Dielectrophoretic Manipulation. J. JAP, 44 7B: 5717-5723, 2005).
  • a particle in a uniform electric field, a particle will experience no net force due to the formation and subsequent cancellation of equal and opposite Coulomb charges at the particle surface.
  • the field is non-uniform, the varying magnitude of field intensity across the region occupied by the particle will result in the generation of non-equilibrium Coulomb charges in the particle, creating a net dielectrophoretic force.
  • Dipole moments induced from the AC field combined with the field gradients generated from the nonuniform electrode structures result in polarisation of the spherical particle.
  • the electric fields then interact with the dipole moment to generate an electrostatic force.
  • biological cells when subjected to an electric field they will polarise, with the inherent charges separating to form a dipole moment.
  • Polarisation refers to the positive and negative charge alignment on a given body.
  • the greater the field strength across one side of a particle the greater the force induced on the opposing side of the particle. Resulting in a force toward the region of highest electric field.
  • Non-uniform AC electric fields generated by interdigitated microelectrode arrays can be used to manipulate and control biological cells by dielectrophoresis. This technique is used in conjunction with an integrated BiochipTM platform designed as a non-destructive system for trapping and immobilising living cells into cavities. Using dielectrophoresis-based single particle traps, cells have been directed into cavities aligned between the interdigitated electrodes by both positive and negative dielectrophoresis. Non-uniform electric fields generated by the electrodes on the BiochipTM provides a means of discriminating and manipulating cellular systems using a force known as dielectrophoresis.
  • Cellular dielectric properties act as an invisible fingerprint; composition and structure rich data, which is the cell itself, are the fundamental drivers for characterisation by dielectrophoresis (DEP).
  • DEP dielectrophoresis
  • a dielectrophoretic force induced in the structure directs the particle to high and low field gradients generated by the irregular electrode structures.
  • Cavities etched within the surface or created on the electrode above the BiochipTM act as incubators, trapping particles at known positions on the substrate surface. Each cavity preferably has a unique locator, enabling single- cell handling, addressing and identification throughout experimentation.
  • AC field generation to microelectrodes on a BiochipTM may be delivered by a sinusoidal wave from a function generator operating in a voltage and frequency range of 2-6 V (peak-to-peak) at 500-700 kHz, respectively, in a cell suspended in a solutions of varying conductivity for 5-15 minutes.
  • a solution used for trapping endometrial cancer cells by positive dielectrophoresis is a sugar-based solution of 8.3/0.3% sucrose/glucose in de-ionised water.
  • Other operating conditions and solutions such as are known in the art may also be used.
  • the BiochipTM may additionally comprise functional wells or cavities to enhance selectivity and sorting of cellular material for example, the wells may be functionalised by coating with a range of materials including antibodies, dyes, stains, fluorophores or labels designed to identify the type or state of cellular material trapped within the well or cavity.
  • the cellular material may be suspended, immersed or otherwise contacted with growth and/or maintenance culture media such as are known in the art to maintain near physiological conditions. Some suitable media are discussed above and detailed in the accompanying examples.
  • the material is attached to the substrate such that any suspending media can be removed without the material dissociating from the substrate.
  • a preferred embodiment shown in Figure 1 illustrates the case of a culture of single biological cells attached to a Petri dish and suspended in culture media.
  • the culture media if present is aspirated from the cellular material prior to treatment with the moulding material. Aspiration may be effected by pouring, blotting, pipetting, syringing or similar.
  • the substrate may be completely dried, washed in a buffer solution (eg phosphate buffered saline) or a thin film of culture media or buffer may remain depending on the cellular material and desired end use of the bioimprint.
  • the thin film maintains cell hydration.
  • a thin film measured in micrometers (usually O.l ⁇ m to lO ⁇ m) is contemplated in one embodiment.
  • the media should be rapidly exchanged for the moulding material.
  • the cellular material in the imprinting process of the invention is coated with a nanoscale moulding material.
  • the moulding material should completely cover the exposed surfaces of the substrate and cellular materials.
  • the thickness of the moulding material will be 30 ⁇ m to lcm, more usually lOO ⁇ m to 5mm. This may be achieved by pouring, spraying, painting, spin coating, dipping or otherwise applying the moulding material to the cellular material.
  • a typical thickness for this moulding material applied by these techniques is 1 to 3mm, preferably 2mm.
  • the cellular material and substrate is inverted and lowered into or onto the moulding material to coat it (a stamping process).
  • the moulding material should be lO ⁇ m to 600 ⁇ m thick, preferably lOO ⁇ m to 400 ⁇ m, more preferably 10 to 400 ⁇ m thick, preferably 100 to 200 ⁇ m.
  • the cellular material and in particular cells may be pressed down. This depresses or indents the material. When indenting is present visualisation of subcellular structures is facilitated.
  • the cellular material is held in a receptacle for receiving the moulding material, and the moulding material poured on.
  • a Petri dish is a suitable example for cell moulding or imprinting.
  • Nanoscale moulding materials suitable for use in the process of the invention include any known art materials capable of producing a high resolution image. These are generally polymeric materials with a low viscosity. This ensures complete cover of the cellular material and substrate. Fluid viscosity ranges are typically in the order of 0.01 cps to 100 cps, measured at room temperature (22 degrees Celsius).
  • the moulding material may be de-gased prior to application. This may be achieved by placing the entire fluid in a vacuum to expel any gas present.
  • Moulding materials useful herein include polymerisable compounds known to those skilled in the art.
  • the material may consist of polymerisable monomers, oligomers, or a combination thereof. These include for example organic materials and composites such as epoxies, acrylates (including methyl acrylates), acrylamides, acrylic acids, vinyls, and ketene acetyl groups containing monomers or oligomers.
  • Organic materials and composites such as epoxies, acrylates (including methyl acrylates), acrylamides, acrylic acids, vinyls, and ketene acetyl groups containing monomers or oligomers.
  • Polycarbonates, polyvinyl resins, polyamides, polyimides, polyurethanesl, polysiloxanes, cyclic olefin copolymers, polyesters, polyethers, perfluoropolyethers and the like are also contemplated for use herein. Such materials are commonly used in lithography and are
  • silicone and silicone containing materials such as polydimethylsiloxane (PDMS), and (methyacryloxypropyl)methylsiloxane- dimethylsiloxane (mcpms-DMS).
  • PDMS and mcpms-DMS elastomers are biocompatible and transparent materials, well suited for applications in bio- and nanotechnology but not limited thereto.
  • Other examples of useful moulding materials include epoxies such as SU-8 (Microchem, MA, USA), poly(methylmethacrylate) (PMMA), and isobornylacrylate (IBA) monomer. These materials can all transfer features at the nanometer level. Additionally, a low surface roughness provides a flat substrate necessary for nanoscale imaging. Combinations of nanoscale moulding materials in the form of layers are also contemplated. Use of two or three layers of different materials most usually with different viscosities may be employed to facilitate imaging.
  • the moulding material may include the polymerisation initiators (curing agents). These initiators may be selected from known art initiators or curing agents.
  • the curing agents are generally supplied with the moulding material and used according to manufacturers instructions. For example Sylgard 184 system (Dow Corning, USA) Photoinitiators are currently preferred for use.
  • initiators examples include 2,2-Dimethoxy-2-phenylacetophenone, (DMPA), Sigma Aldrich, USA), acetophenone, (Benzene) hydroxybenzophenone, 1-hydroxy-cyclohexylphenyl ketone, methyl phenylglyoxylate, 4-(4-methylphenylthiophenyl)-phenylmethanone, blend of poly(2-hydroxy-2-methyl- 1 - [4-( 1 -methylvinyl)phenylpropan- 1 -one, 2,4,6- trimethylbenzoyldiphenylphosphine oxide and methylbenzophenone derivatives irgacure-819 (phosphine oxide, phenylbis(2,4,6-trimethylbenzoyl)) and -651 (2,2-dimethoxy-l,2- diphenylethan-1-one) (Ciba Specialty Chemicals, USA) H-NU-640 and H-NU-660 and H-NU - 470 (5,2-
  • the initiators can be mixed with the moulding material directly or in a solvent depending on the initiator selected. For example, 2,2-Dimethoxy-2-phenylacetophenone may be dissolved in xylene and then mixed with the moulding material.
  • Other polymers, catalysts and photo- initiators may be selected from "Silicon compounds: Silanes or Silicones, Gelest Catalog 3000- A, ABCR GmbH & Co, KG, Düsseldorf, Germany.
  • the photoinitiator is usually included in amounts according to manufactures instructions. This may be at from about 0.01% to about 5% weight of the moulding material.
  • Cross-linking agents may also be used.
  • cross-linkers for use with acrylates and methacrylates include tetraethyleneglycol dimethacrylate (TeGDMA), ethyleneglycol dimethyacrylate (EGDMA), bis-acrylates and bis-methacrylates of polyethylene glycols of molecular weights in the order of 200-600, glycerol di-, and triacrylate, diethyleneglycol diacrylate, hexanediol dimethyl acrylate, pentaerythritol triacrylate, 1,3-propanediol, diacrylate and 1,3 proponediol dimethacrylate amongst others.
  • Combinations of moulding materials and initiators may also be used. For example IBA/TeGDMA 92:5% weight ratio with 3% weight ratio of initiator.
  • the moulding material may also optionally include one or more additional agents such as photosensitisers (eg ketones, dyes such as azines and thiazoles); adhesion agents (such as silanes); viscosity controllers (eg acid derivatives such as acetyltriethyl citrate, dicapryladipate, diethylene glycol dibenzoate, dibutyl fumarate and the like); synergisers and release agents.
  • photosensitisers eg ketones, dyes such as azines and thiazoles
  • adhesion agents such as silanes
  • viscosity controllers eg acid derivatives such as acetyltriethyl citrate, dicapryladipate, diethylene glycol dibenzoate, dibutyl fumarate and the like
  • synergisers and release agents may be present in from about 0% to 5%, preferably 0.1% to 5% by weight of the moulding material composition.
  • an acrylated amine synergist to overcome oxygen inhibition - amine synergists act as a reducing agent when used in conjunction with photoinitiators to enhance surface curing.
  • amine synergists greatly increase the polymerisation rate by acting as an oxygen scavenger via formation of free radicals in U. V light.
  • Acrylated amine oligomers used at the amine synergist in the invention include methyldiethanol amine - CN386 Sartomer Company (PA, USA).
  • Other oxygen scavengers used as a reducing agent component in the polymer include iodonium salts.
  • the moulding material is degassed prior to application. This may be achieved by subjecting the material to vacuum conditions for a suitable period, most usually for a minimum of 10 minutes.
  • the curing or hardening step is preferably carried out rapidly to minimise damage to the cellular material.
  • Curing techniques may be selected which are appropriate for the nanoscale moulding material employed in the process. This may involve thermal curing by cooling or heating, light curing (for example a UV flashstep), chemical curing, or use of other forms of radiation sufficient to cause curing. Combinations of curing techniques may also be employed. As will be appreciated curing will depend on the polymer type of the nanoscale moulding material, intensity of the UV lamps and the like. Thermal curing at temperatures from 30°C to 150°C, preferably 35 to 75°C% and from a few (two) minutes up to 6 hours are contemplated. As noted above, rapid cure times in the order of 2 to 10, preferably 4 to 6 minutes at high temperatures (7O 0 C to 110 0 C, preferably 9O 0 C to 100 0 C) are feasible.
  • the curing is preferably carried out at physiological temperature or room temperature, usually, 5 0 C to 4O 0 C, preferably 35°C to 38°C). For human cells 37°C is appropriate.
  • Rapid curing and variations thereof means curing in less than 10 minutes, preferably less than 5 minutes, and most usually in 1 to 5 minutes. Most preferred are cure times in the order of 10 to 180 seconds, preferably 20 to 50 seconds. For example IBA/TeGDMA with DMPA in the ratios discussed above cures in approximately 40 seconds in UV light (365) intensity of 6.5mW/cm 2
  • the curing process generally operates by causing cross-linking resulting in a solidified material.
  • Partial or full curing is contemplated.
  • Full curing may be advantageous if modification of the cellular material is sought.
  • Full curing increases the level of binding between the cellular material and the moulding material.
  • a cell from which a PDMS imprint is removed after full curing may remove the membrane layer from the cell body. This allows examination of sub-membrane regions of a cell.
  • Partial curing refers to the polymer being in a condition in which it is still able to be polymerised for example by light or heat. Partial curing is conveniently effected by subjecting the polymer and composition to curing conditions for a fraction of the recommended full curing conditions. For example, one quarter, one third, or one half of the recommended full cure time.
  • the partially cured mould may be removed and curing or hardening completed using conventional techniques noted above.
  • the moulding material may be subjected to a partial curing step prior to application to the cellular material and substrate. This decreases the time required to cure the material after application and maintains the physiological integrity of the cellular material.
  • the mould produced by the process of the invention may be cleaned if required. Residual material, for example protein, may be transferred to the mould from the cellular material, or media. This is particularly the case where UV exposure is prolonged, for example for about 5 to 15 minutes. In that instance the imprint becomes a molecular imprint and functional material from the biomaterial may be transferred and imaged. This process may be controlled by controlling cure times and degree of cleaning. Residual material will usually be removed by application of a cleaning agent.
  • Application may conveniently be by submersion or rinsing in a solvent.
  • suitable solvents include water, saline and PBS but are not limited thereto. More rigorous cleaning may be employed where molecular imprints with functional material are not required. For example acids such as HCl at 33% concentration can be used for three minutes or until the material is removed, in instances where solvents fail to remove the biological materials or where excess material is transferred into the polymer. Ultrasonic can also be used in conjunction with solvents to facilitate cleaning. Where molecular imprints with functional material are required this will usually involve acid and ultrasound free cleaning, and short wash times in the order of 10 to 60 seconds.
  • the final result is an inverted impression or replica of the cellular material topographic surface image permanently cast in the mould. If the substrate contains markings as discussed above, then these markings will also have been transferred to the mould.
  • the invention also provides casts produced from the Bioimprint of the invention. Casts are most usually produced by inverting the mould and repeating the coating, curing and separation steps of a process of the invention or similar.
  • the moulding material selected in creating the cast may be the same or different from that used to create the Bioimprint. It will be appreciated that in this situation no cellular material is present. Therefore, the key rapid cure times are not critical. Any nanoscale moulding material may be selected. Again different nanoscale moulding materials can be used in the form of multiple layers, preferably two or three layers only.
  • kits useful for analysis of cellular material and more particularly, cytological analysis.
  • the kit comprises nanoscale moulding material, and a substrate for the cellular material. Most usually instructions for use will be provided in or on the kit.
  • the platform substrate may be composed of one or more distinct substrates.
  • a preferred platform for use is a biochip array.
  • the chip array may be a single chip or multiple chips. The chips can be packed separately or with another substrate for example a petri dish.
  • the moulding material may in one embodiment be preapplied to the substrate. It may also be pre- photoinitiated. In that case the kit or the container in which the moulding material is packaged needs to be impenetrable to light. Foil packaging conveniently achieves this result. Other options known in the art can be employed.
  • kit components may be present in one or more containers.
  • the kit may further include a patient data card. This can be a unique identifier for the patient, and imaging results added to same.
  • the BioimprintsTM and casts of the invention enable nanoscale visualisation of cellular materials.
  • the moulds and casts may be treated with a conductive material such as gold, aluminium or other metallic layers to enable electron imaging.
  • the BioimprintsTM and casts are a non- degrading, and non-biohazardous record for high resolution topographic analysis. The resolution of the BioimprintTM is almost unlimited. Ultimately, only the size of the moulding material limits the process.
  • BioimprintsTM are also cost effective as a cell culture potentially contains millions of cells, a biological sample has many data points that are often unanalyzed due to expiry. Potentially large amounts of time and money are wasted in repeated experiments which often introduce inter- experiment deviations. By creating long-life imprints of the complete sample in a single-shot impression, samples can be efficiently utilised.
  • Analytical tools include Optical, Confocal, Scanning Electron Microcopes (SEM' s), Tunnelling Electron Microscopes (TEM), Scanning Tunnelling Microscopes (STMs) and Atomic Force Microscopes (AFM' s) or combinations of these such as AFM and optical or confocal microscopy.
  • SEM' s Scanning Electron Microcopes
  • TEM Tunnelling Electron Microscopes
  • STMs Scanning Tunnelling Microscopes
  • AFM' s Atomic Force Microscopes
  • Optical and Confocal microscopy can be used for example to select a subset of cellular material for examination before subjecting that subset to AFM imaging or similar. Subsets may be labelled, dyed or stained subsets such as live cells, labelled antibodies or the like discussed above.
  • the AFM enables living biological systems and their components to be imaged in a liquid environment and at high resolution.
  • BioimprintingTM presents a simpler, alternative method that utilizes the nanoscale capabilities to create high resolution imprints.
  • Use of multiple AFM tips can be used to facilitate high throughput screening. Oxide sharpened silicon-nitride DNP-S tips, nanotube imaging probes, and electron beam deposited tips (eg Sting probes, MikroMasch, Estonia).
  • the present invention also provides a process for nanoscale imaging of cellular material which comprises examining a BioimprintTM or cast produced by the invention using atomic force microscopy or scanning election microscopy.
  • a permanent record of the imaged BioimprintTM or cast may be made. For example by taking a digital photograph.
  • the Bioimprints and casts are of course also permanent records in their own right.
  • BioimprintsTM of the invention and AFM or other imaging techniques enables examination of cellular materials and more particularly cells and intra-cellular structures and processes.
  • bioimprinting include monitoring transfection processes in cells and selecting stably transfected cells for example transfected with a viral vector, or monitoring the production of monoclonal antibodies. This could conveniently be achieved by sorting cells into wells, conveniently 1-5 cells per well, on a microarray plate or BiochipTM and BioimprintingTM same to confirm transfection. In a similar manner, cells can be examined to determine cell health, particularly of cell lines, stem cells and the like where long term viability and storage is required, or to identify the presence of disease. Whole tissue samples can also be analysed using Bioimprinting to examine cell-cell interactions and cell-extracellular matrix interactions. Response to drugs over time can be monitored by BioimprintingTM and imaging biopsied cells.
  • the present invention also provides a process for analysing function or interaction of cellular material in response to an agent or stimuli.
  • the process comprises imaging a first sample of cellular material using a process of the invention, contacting the cellular material with an agent or stimuli and imaging a second sample of the cell for material.
  • the second image can be compared to the first image to identify any changes in the cellular material in response to the agent or stimuli.
  • Agents contemplated for use herein include one or more of cells, drugs, ligands, proteins, amino acids, virus, bacteria or endocytotic molecules, but are not limited thereto. These agents are particularly useful for monitoring cell responses to cells, drugs, virus or bacterial adhesion and penetration, or monitoring endocytotic processes.
  • Endocytotic molecules may be any molecule capable of being taken up by a cell. Such molecules include oxygen, carbon dioxide, ethanol, proteins, amino acids, polynucleotides, polysaccharides and the like.
  • Proteins (including hormones and peptides) of interest include low density lipoprotein (LDL), gonadotrophin releasing hormone (GnRH), insulin, growth hormone, leutinizing hormone, interferon, transferrin IgG, IgE and the like.
  • LDL low density lipoprotein
  • GnRH gonadotrophin releasing hormone
  • insulin insulin
  • growth hormone leutinizing hormone
  • interferon transferrin IgG, IgE and the like.
  • Ligands encompass molecules which bind to the cellular material and includes antibodies, proteins (as discussed above and including hormones), viruses and toxins but are not limited thereto.
  • Preferrred ligands are antibodies, including monoclonal, polyclonal, humanized, and single chain, antibodies and immunologically active fragments of antibodies.
  • Toxins contemplated for use herein include lectins, ricin, and diphtheria toxin, but again are not limited thereto.
  • ligands or agents may be included in the moulding material or on the AFM tip.
  • AFM tips may be functionalised with a variety of agents, particularly ligands and receptors. This facilitates mapping of cell surfaces for example for receptor density.
  • agents may be labelled using any conventional techniques and labels such as radioisotopes, fluorescent, fluorophore, luminescent and bioluminescent labels, quantum dots, or metallic labels such as gold, platinum and palladium but not limited thereto.
  • labels are discussed in more detail above. See also Handbook of fluorescent Probes and Research Products, Molecular Probes Inc. 9 th Ed, 2002. Antibodies in particular are amenable to such labelling.
  • Stimuli contemplated for use herein include chemical, magnetic, sound, radiation and electircal stimuli.
  • chemotherapy or the addition of chemicals such as potassium chloride, calcium (particularly (Ca +), zinc, neurotransmitters (including amino acids such as glutamate, aspartate, glycine, GABA, serotonin, and acetylcholine, but not limited theretoUltrasound, radiation such as radiotherapy or x-ray treatment
  • the cellular material is protein and agents used are ligands such as cells, receptors, proteins and DNA.
  • the invention provides a cytological screening system comprising a BioimprintTM or cast produced by the invention and a cell analy.
  • the analyser can be used to examine the print for abnormal cells.
  • a related cytological screening method examines the BioimprintTM or cast for features indicative of cellular abnormality. Variation in features compared to a normal control cell sample indicates that cellular abnormality exists. Conversely, the absence of cellular abnormalities indicates that the cells are healthy.
  • features indicative of cancer include disorganised arrangement of cells, loss of normal specialised cell features, large numbers of cells dividing, large variable shaped nuclei, small cytoplasmic volume relative to nuclei, large variation in cell size and shape, poorly defined tumor boundary, chromosomes on the outside of the nuclei.
  • neoplastic cells Most indicative of neoplastic cells are abnormalities in cell volume, nuclear volume and nuclear to cytoplasmic volume ratios.
  • Measured values for one or more features such as those described above can be compared to reference values for normal cells. These values may be normal ranges, or threshold below or above which are regarded as normal. The variation from the ranges or threshold level should be statistically significant. Quantitative reports may then be generated establishing the number of cells examined, and the number of cells, or percentage of cells likely to be neoplastic within the total cell population.
  • the cytological screening processes may be carried out by an operator using visual analysis. However, this is time consuming, and subject to individual interpretation and human error.
  • the screening process is automated and uses computer analysis to examine the BioimprintTM or cast, to measure set features, and to calculate deviations from standard ranges or means, or variance from a predetermined value or threshold.
  • This process is facilitated by the use of biochips on which individual cells have a unique address or identifier.
  • Systems for cell analysis as are known in the art may be used, for example dielectrophoretic cell sorting systems. See also for example US 5,235,522 and WO95/34050.
  • the cytological screening method of the invention may be used alone or in conjunction with other known cell screening techniques.
  • the advantage with the present technique is that it provides direct cell analysis under near physiological conditions. It reduces errors introduced through other processes involving staining, or measurement of electrical resistance or light transmission to identify abnormal cells.
  • the method can be used to screen for a wide range of cellular abnormalities indicative of a range of diseases or disorders. Viral infection and cancers are a few examples. Cancers which may be screened for include lung, blood, ovarian, breast, throat, prostate, skin, bone, endometrial and cervical cell cancers. The method of the invention may have particular application in cervical cell screening where the level of false negatives resulting from human analysis is high.
  • the screening processes are equally applicable to examination of other cellular material such as proteins, DNA, RNA and the like.
  • AU rat pituitary cells in the following examples were cultured in accordance with the requirements of the Animal Ethics Committee of the Wales School of Medicine and Health Sciences (CSMHS), University of Otago. Cells from the anterior pituitary gland were collected from adult female Sprague-Dawley rats. Initially, cells were cultured in media consisting of 500 ml DMEM including GlutaMaxI with high glucose and 110 mg/1 sodium pyruvate (Gibco BRL Products) supplemented with 1.8 g HEPES, 1.5 g BSA, 800 U penicillin and 800 ⁇ g streptomycin (Sigma-Aldrich, Australia), and which contained oestradiol at physiological level (300 pg/ml).
  • DMEM including GlutaMaxI with high glucose and 110 mg/1 sodium pyruvate (Gibco BRL Products) supplemented with 1.8 g HEPES, 1.5 g BSA, 800 U penicillin and 800 ⁇ g streptomycin (Sigma-Aldrich,
  • cells were diluted 10 fold in dispersion media to yield a final concentration of 100,000-200,000 cells/ml.
  • cells were incubated at 37°C, 5% CO % until required for experimentation.
  • FBS fetal bovine serum
  • Stimulation of LH hormone in the gonadotroph cell type was done using GnRH at 10 "7 concentration, in dispersion media. Cells were washed and incubated in pure dispersion media for an hour, prior to an additional hour long incubation in the GnRH solution, after which, cell molds were taken.
  • Fig. 1 In one method using a heat curable polymer the pattern transfer for fabrication of molds and corresponding casts are illustrated in Fig. 1 : Firstly, a relatively high ratio of PDMS silicone elastomer to curing agent (Sylgard 184, Dow Corning) was mixed at a respective mass ratio of 10:3, to ensure rapid curing. Air was then removed from the solution using a vacuum chamber for 25 mins. Immediately after aspirating all suspension media, approximately 5 grams of PDMS was poured into the petri dish and thermally cured either on a hotplate, oven or in a 5% CO2 incubator at temperatures ranging from 37-140°C.
  • PDMS silicone elastomer to curing agent Sylgard 184, Dow Corning
  • the mask was peeled off and hardened by placing in a 95 0 C oven for 20 mins. Finally, casts were produced by inverting the mold, and repeating the process again. Essentially, all analytical information is held in the original mold, however, casting illustrates the capability of replica molding and facilitates comprehension of cell structure.
  • DNP-S oxide sharpened silicon-nitride
  • DI Veeco Instruments
  • Flexibility was important for probe-based selection, as substrate properties have enormous impact on cell deformation and contamination that can occur to expensive tips during scanning.
  • AFM is used as the primary imaging source for the ability to compare images of actual cells to those replicated using the Bioimprint process.
  • Results Micrographs are compared with those taken of cultured pituitary cells by AFM (fluid-tap) as well as published work by other groups. Motivation for the comparison is to relate the resolution and structural integrity of the PDMS castings to how they adequately mirror, or even enhance, cells imaged in a near-physiological, probing-based environment.
  • Fig. 2 (a) Low temperature (37°C) and extended curing time (180 mins), (b) high temperature (95°C) and extensively shorter curing time (4.5 mins) and (c) mid- temperature (70 0 C) and time (20 mins), potentially illustrating the cell cytoplasm collapsing around the nucleus. Further, optical images (d)-(f) illustrate the normality of hydrated cells that are seen across the population.
  • FIG. 3 Micrographs show cell dehydration caused by using a critical curing temperature of 95°C compared to those in Fig. 8, where no visible drying effects such as submersed nucleated areas within the membrane are cast in the (a) mold, and transferred through to the (b) cast.
  • FIG. 4 A cell imaged using the AFM' s fluid tapping mode is shown in Fig. 4, whereby cellular structure and membrane coverage of a cell at a near-physiological condition 5 days after culture is illustrated.
  • replicas using the Bioimprint process can be made both at short ( ⁇ 1 day) and longer times of cell culture and unlike fluid imaging methods, cells don't require prolonged culture periods for a strong cell-surface adhesion.
  • the forces from sharp probes can damage the soft membrane surface as well as misrepresent the true structure.
  • DNase I Deoxyribonuclease I
  • Fig. 5 a structure believed to be DNA based on experimental findings and observations by groups [1] imaging similar complexes by AFM was discovered.
  • the solitary DNA strand was imaged using PDMS molding, illustrating the adaptability of the method to a range of material.
  • the designated width of the strand is highly dependent on the AFM tip-apex, which in this case is approximately 30 nm, making the structure appear dimensionally larger than actuality.
  • the AFM 's ability to distinguish between double-stranded DNA, which is formed when an overhanging pyrimidine strand folds back on the double helix and triple-stranded DNA, which sits higher on the substrate, enables tentative identification of triple-stranded DNA shown by the highlighted regions at the strand ends and middle areas.
  • the relatively weak attachment of DNA on the substrate allows it to be physically imbedded in the PDMS and be removed from the surface.
  • strongly bound DNA is essential for good imaging: Using this technique, molds could be re-transferred to a liquid environment for further observation, such as studies on enzymatic reactions or protein-DNA interactions. Section analysis reveals the DNA molecule having a vertical height of 24 nm, excluding submersion within the PDMS, and an approximate length of 3.5 ⁇ m.
  • a crucial factor limiting the AFM resolution in fluid is the apex of the imaging probe. Nominal tip radii of the DNP-S probes are roughly 30 nm, whereas the Sting probe apex is typically less than 10 nm. Though availability of specialized tips for fluid imaging are limited, sharper probes are expensive and often less reliable. Even still sharper tips can cause damage to the soft membrane, often puncturing or even removing the thin lipid layer surrounding the cell. A technique envisioned would be to utilize the adaptability of the PDMS imprints to be analyzed in multi-imaging environments, such as scanning or tunnelling electron microscopy after metallic coating.
  • the membrane is fairly robust to the process and retains much of its characteristic features. Efforts have concentrated on establishing conditions that exploit the nanoscale resolution of PDMS as well as keeping the cell in a preservative and representable state for curing. Clearly, as PDMS cures exponentially with temperature; a temperature increase will cause an exponentiate decrease in cure time. However, regarding cell condition and viability there is a trade-off between heat and time in the transitional, non-physiological environment. Other polymer materials that retain similar resolution to PDMS but cure at room temperature, or integrating a rapid flash-UV step into the process are possible solutions to any dehydration effects observed.
  • DNA molecules bind well to mica and protein surfaces, however, the AFM tip interacts strongly with samples and suffers damping effects when imaging in fluid and requires further immobilization methods.
  • methods used to image biological objects by AFM rely on surface modification to sufficiently bind objects onto the substrate in order to overcome the strong tip interaction.
  • a potential use for Bioimprint lies in the ability to hold samples in place while imaging, without the need for additional modification.
  • PDMS is extremely flat and offers a low surface roughness.
  • a novel soft lithographic technique for creating cell replica molds using the nanoscale capabilities of PDMS has been introduced with transfer of high resolution data potentially illustrating membrane mechanics.
  • Cells cast using Bioimprint molding show defined features at the membrane and can be compared to the resolution achieved in vitro.
  • the adaptability of this technique on hardy biomaterials such as DNA presents a novel immobilization method for AFM imaging.
  • nanoscale moulding material was produced as follows:
  • Fig. 6 The pattern transfer scheme for BioimprintTM fabrication and molecular imprinting is illustrated in Fig. 6: Initially, air was removed from the polymer solution using a vacuum chamber and 2 grams of liquid polymeric photo-activated composite was placed on top of a transparent mask substrate. The mask was then imprinted on top of the cells such that only a thin layer 30 ⁇ m of polymer was formed and the material polymerized under a 350 W Mercury UV Lamp (with an intensity of 6.7 mW/cm 2 ) for 5 minutes. In preparation for AFM imaging, samples were washed in detergent in order to detach any biomaterial that may have stuck to the polymer during curing. This technique produced cell impressions as shown by the micrographs in Fig. 7; after imaging the original (a) impression, a digital mirror inversion (b) creates an orientated image matching that of the original cell.
  • a localized region of high tension is created at the end of the dimple, which fuses with the secretory granule when in proximity.
  • membrane depressions are caused by a scaffold of proteins formed on the intercellular wall.
  • Chandler et al. ref [7] the transient formation states of the exocytotic fusion pore were captured at scaffold induced sites using freeze-fracture and fixation techniques combined with EM. Since then, this technique has been used to study fusion pores in numerous cell types enabling a common picture of the fusion pore.
  • a primary component of this mechanism is the protein structure that can be envisioned as a 'scaffold', built into the cytoplasmic face of the inter-cellular plasma membrane designed to dilate through polymerization in order to form membrane dimples.
  • BioimprintTM the molecular imprinting capabilities of BioimprintTM.
  • the molecular construct of relevant sites can be investigated.
  • a less rigorous cleaning process acid free cleaning, no ultrasound used, short wash times of 10-60 seconds
  • U. V exposure a molding process is altered into a molecular imprinting and transfer technique.
  • the ability to transfer cellular material into molded impressions is illustrated in Fig. 11 : Shown is an AFM phase image of a raw cell impression at a 'dimple' site made in polymer.
  • molecule structures have been transferred to the polymer, providing insights into the underside composition and form of the membrane. Clear molecule-rich zones with linking architecture are seen beneath the 'dimple'. The molecules measure approximately 8 nm wide matching the dimensions of protein structures such as actin micro-filaments, which are active in intracellular vesicle traffic. If indeed the molecules seen are actin filaments, which is largely a protein predominate in the cytoskeleton, the plasma membrane would also be captured in the polymer and the view is of the cytoplasmic face of the plasma membrane. However, it is difficult to determine the extent and effectiveness that the imprinting technique had in capturing the full molecular structure.
  • dimples are reported to be approximately 100-300 nm wide and not around 600 nm as seen in images observed in this study. This could be explained by effects resulting from the imprinting technique, such as a collapse in either the underlying secretory granule or plasma membrane.
  • the lack of features located within dimples are possibly explained in the previous statement or due to their un-stimulated states but more than likely due to the rejection of the polymer or pore closure upon application.
  • Rat Pituitary cells Fusion pore construct Rat Pituitary cells Fusion pore construct.
  • Figures 1 and 6 re-iterate the two BioimprintTM pattern transfer schemes used, (a) In Figure 1, the first method used Poly(dimethylsiloxane) (PDMS) (Dow Corning, USA) solution was mixed at a ratio of 10:3 of polymer to curing agent, at room temperaturethe air was removed from the solution in vacuum and pre-cured for 2 mins at 95°C. Approximately 5 grams of this composite was applied above the cells and immediately cured in a 37°C incubator for 2 hours.
  • PDMS Poly(dimethylsiloxane)
  • the polymer For the BioimprintTM process to accurately replicate cells the polymer must be able to conform to the soft biological structure rapidly to minimize biological response to the polymer. Cell adherence to the substrate prevents complete submersion of the cell within the polymer. After hardening, the polymer is peeled off and a 30 minute oven bake at 95 °C completes polymerization.
  • Regulated discharge of hormones via exocytosis proceeds by formation and coupling of a fusion pore, or porosome, where the vesicle lumen connects the extracellular space.
  • BioimprintTM is introduced as an alternative technique, providing superior resolution of the cell membrane in an attempt to characterize membrane topology.
  • the hardened polymer composite shows a low surface roughness and excellent conformation to the extra-cellular structure.
  • a 32 ⁇ m scan of a pituitary cell illustrates the distribution of pits at the cell membrane and their dynamic formation around what seems to be a nucleus-form within the cell.
  • Most notable are the larger pits, which have mean widths and depths with standard errors (S.E) of 953 ⁇ 173 nm S.E and 200 ⁇ 26 nm S.E, respectively, which in this case are observed around the nucleus and not on the plasma membrane directly above the nucleus.
  • S.E standard errors
  • a further characteristic observed is the submersed nuclei and uniform indentation profile across the cell surface, an effect that is associated with cell dehydration.
  • Fig. 13 Illustrated in Fig. 13 are the impression (a) amplitude and phase images from two different pituitary cells: (a) At 5 ⁇ m magnification, pit sites are seen across the entire surface region of the membrane, (b) Separately a 3 ⁇ m scan demonstrates the depressional morphology showing considerable variability in sizes and different degrees of overlap with neighboring pits. This interaction could be a result of either expansion of pits within proximity of each other or pits adjoining following movement within the plasma membrane. Different degrees of interaction are seen and there are cases where adjacent features seem to overlap, shown by arrows at points 1 and 3 on Fig. 13 (a) and (b), respectively. In some cases, remnants of what appears to be a complete convergence of multiple pits as are observed at location 2 in Fig. 13 (a).
  • FIG. 16 shows the ability to make accurate imprint impressions of (a) cells even after they have been cultured for lengthy (5 days) periods and the interaction of cell extrusions can be seen.
  • One cell is seen to contain numerous pits, which at higher magnification (b) is further detailed. These pits had a calibrated mean width and depth of 719 ⁇ 90 nm S.E and 144 ⁇ 29 nm S.E, respectively.
  • a 35 ⁇ m image of a cell shown in Fig. 10 exhibits much smaller membrane pits that saturate most areas of the membrane. Though not shown, as in Fig. 10 locations immediately above the nucleus did not contain as many pits as the rest of the membrane. Mean widths and depths of the pits measure 375 ⁇ 66 nm S.E and 55 ⁇ 18 nm S.E, respectively.
  • Using a rapidly U.V-curable polymer is believed to produce an impression akin to that of a living cell.
  • Endometrial cancer cells were cultured in accordance with institutional guidelines of the Wales School of Medicine and Health Sciences, University of Otago, New Zealand, after ethical approval and appropriate informed consent.
  • the preparation of cells were as follows: Endometrial adenocarcinoma tissues were harvested from women undergoing hysterectomy, and non-myometrial biopsies were taken from the opened uterus tumor area. Tissues were then digested in collagenase-A (1 mg/ml), and the cells dispersed, and cultured overnight in medium consisting of alpha-MEM containing 1 % penicillin/streptomycin, 0.1 % BSA and 10 % fetal calf serum.
  • Fig. 1 Prior to polymer application all incubation media was aspirated and samples were washed in physiological phosphate-buffered saline (PBS).
  • PBS physiological phosphate-buffered saline
  • Fig. 1 The pattern transfer scheme for impression fabrication is illustrated in Fig. 1: Initially, Poly(dimethylsiloxane) (PDMS) (Dow Corning, USA) solution was mixed at a ratio of 10:3 of polymer to curing agent, the air was removed from the solution in vacuum and pre-cured for 2 mins at 95°C. Approximately 5-8 grams of composite was applied above the cells attached on a 5 cm plastic Petri-dish and immediately incubated in a 37°C oven for 2 hours. The thickness of the resulting polymer above the cells was typically between 2.5 and 5 mm.
  • PDMS Poly(dimethylsiloxane)
  • the attachment of cells to the substrate prevent features from being submersed completely within the polymer material, enabling an impression of the exposed surface of the cells to be made in the polymer.
  • the mask was peeled off, washed in DIW ultra- sonic bath to remove any biological material attached and a final polymerization stage was completed in a 95°C oven for 2 hours.
  • the hardened BioimprintTM impressions were analyzed by an AFM (DI 3100, Veeco Instruments, Santa Barbara, CA) in tapping mode using triangular non-contact cantilevers (NSCI l 5 MikroMasch, Estonia), which were typically operated between 0.6-1 Hz at a resonant frequency of -315 kHz with a nominal sub- 10 nm radius of curvature and a force constant 48 N/m.
  • AFM Dynamicon Micronasch
  • FIG. 18 shows a BioimprintTM replica of a malignant endometrial cell, which is positively inverted to achieve a digital transpose of the negative replica or 'impression' made by the cell during imprinting.
  • the replica presents visible cellular features on both micron and nanoscales. Throughout the image, numerous dimple depressions, which have a mean width and depth of 820 nm and 360 nm, respectively, are seen located on the membrane. Though these features appear to be too large to be fusion pores, they are potentially associated with exocytosis.
  • FIG. 18 An additional feature depicted in Fig. 18 is the outline of a spherical form impacting on the cell membrane, which is assumed to be the nucleus (N).
  • a scope trace reveals the impact of the underlying nucleus on the membrane, causing a distorted effect indicated by points (1),(2).
  • Atomic force microscopy influence of air drying and fixation on the morphology and viscoelasticity of cultured cells. J Microscopy.
  • Fig. 18 The impact of the location of the nucleus on the formation of dimple depression sites on the membrane is further evident in Fig. 18, where they are seen predominantly concentrated at areas around the nucleus. This is reinforced in the AFM positive replica of the endometrial cancer cell shown in Fig.19, where the majority of dimple depressions are scattered around the nucleus (N).
  • the nucleus appears well hydrated and as a uniform rounded structure with no indentation profile or submersed effect.
  • Fig. 19 a 10 ⁇ m. image selectively focused on an area of the membrane is seen saturated by both spherical larger and more numerous smaller depressions, as shown by points (1) and (2), respectively. This illustrates the significant variation in the size of depression sites seen at the membrane.
  • Figure 16 shows additional features of rapid cell division (a), deformed/irregular membrane (bic), small cytoplasmic volume relative to nucleus (d), chromosomes above nuclei (eif) protruding nucleic (git), and large variation in membrane/nucleic cell shape (a-h) which may be indicative of cancer mutations.
  • An additional benefit of the AFM is its ability to accurately sense 3-D topography with a high degree of contrast.
  • the contrast is weak and the Z-dimension is often disregarded as analytical and quantitative evidence in diagnosis, or when evaluating cellular function. This is illustrated by the scope trace in Fig. 20 measuring the cross-section of 3 smaller dimples in (a). The dimple depressions are seen having an average diameter of 600 nm and depth of 100 nm, whereas, the larger pits appear much deeper.
  • Figure 21 reinforces this by showing a positive BioimprintTM replica of an endometrial cancer cell bearing a different appearance and nucleus arrangement from those presented previously.
  • a rounded nucleus (N) is seen clearly offset to the right of the cell, with the membrane extending leftwards. Again, numerous depressions are seen located on the membrane around the nucleus, but especially apparent are two large pits located on the membrane directly above the nucleus.
  • the scope trace in Fig.21 shows the depressions formed as ruptures, approximately 3 ⁇ m wide and extending at least 700 nm deep within the cell. The shape and actual depth of the rupture is difficult to accurately measure due to limitations associated with the imaging tip, which has resulted in an image that reflects the profile of the imaging tip rather than of the rupture.
  • FIG. 22 An AFM image of a BioimprintTM positive replica shows a 40 ⁇ m malignant endometrial cell with a unique nucleus (N) form, which appears to be distinctly separated from the membrane.
  • a scope trace in Fig. 22 (b) quantitatively illustrates the 18 ⁇ m wide nucleus, which is seen extending sharply by -300 nm above the surrounding membrane level. While other cell types imaged do not display such variation in nuclei form and membrane structure, without non-malignant controls it remains uncertain whether these are an artifacts induced from the BioimprintTM process or cellular properties that are characteristics of cancer.
  • a soft lithographic technique for creating replica cell impressions with nanoscale information transfer has been introduced and tested on human endometrial cancer cells.
  • BioimprintTM overcomes many of the inherent difficulties associated with cellular imaging by AFM and advances their integration as investigative tools in biology. With visual verification ultimately being the mainstay for cancer diagnosis, a method facilitating the use of imaging at potentially atomic resolution could be used more to characterize morphological abnormalities at the nanoscale
  • Fabrication of the electrode microsystems and microcavities are done in a two-stage process using standard photolithography, wet/dry etching and metallic deposition techniques. Shown in Fig 23., first a thin 250 nm layer of gold along with a 40 nm adhesive film of nichrome, are thermally evaporated onto a PECVD 200 nm nitride-coated silicon 10 cm wafer (Silicon Quest Int'l). The nitride layer is used as a thermally stable, biocompatible, electrical insulator to isolate the electrode structures.
  • the series of patterned interdigitated microelectrode structures are formed after gold and nichrome wet-etching through a photolithography fabricated 1.5 ⁇ m positive resist mask (AZ 1500, Clariant, USA) that has been developed for 15s (AZ 300MIF, Clariant, USA). Particular attention has been directed toward achieving anisotropic electrode side- walls and uniformity of structure.
  • a mask defining an array of lO ⁇ m single-cell traps in the form of microcavities are precisely aligned between the interdigitated electrodes. Vacuum contact mode using a mask aligner (MA6, Karl Suss, Germany) ensures accurate positioning of the mask alignment procedure.
  • the system's exposing light source is an unfiltered 350 W Mercury Lamp, with an lamp intensity at the centre of the stage being 6.7 mW/cmz.
  • the cavity size has been designed to incubate a mature lO ⁇ m diameter pituitary cell. After photolithography and development, the resist is hard-baked (185 °C, 30 min) and cavities 3.7 ⁇ m deep are etched through the silicon nitride layer and into the silicon substrate by two reactive-ion-etching (RIE) runs.
  • RIE reactive-ion-etching
  • Table II Process parameters for the fabrication of cavities by reactive ion etching of silicon nitride and silicon.
  • Fig. 24(a) The pointed electrode structures in Fig. 24(a) and 24(b) is designed to attract cells that respond to positive DEP, with the cavities located between the electrode tips to trap the cells. Whereas, the electrode in Fig. 24(b), combines traps located at both positive and negative DEP regions to separate cells that respond opposingly to either the intense or weak field regions.
  • Rat pituitary cells were supplied by the Laboratory for Cell and Protein Regulation, Wales School of Medicine and Health Sciences. Cells from the anterior pituitary gland were collected from pro-oestrus adult female Sprague-Dawley rats and cultured in dispersion medium: Consisting of 500 ml DMEM including GlutaMaxl with high glucose and 110 mg/1 sodium pyruvate (Gibco BRL Products) supplemented with 1.8 g HEPES, 1.5 g BSA, 800 U penicillin and 800 ⁇ g streptomycin (Sigma-Aldrich, Australia), and which contained oestradiol at physiological level (300 pg/ml). Cells were then triturated and kept at 4 0 C until required for experimentation.
  • Tris-Gly-Dex tris-glycine-dextrose
  • FIG 25 illustrates the two separate methods used for trapping cells on the Biochip.
  • a purposely built holding device shown in Fig. 25(a)
  • a limited volume of cell suspension is placed on top of the Biochip while needles deliver the desired signal to the contact pads.
  • Heat generated from the electrodes forms variable heating gradients in and around the substrate surface. Typically resulting in an intracellular localised temperature rise below 2 "C per 100 mW electrode power, causing a slight deviation of the DEP spectra G. Fuhr et al., Biochim. Biophys. Acta 353 (1994) 1201.
  • Ohmic heating can cause hydrodynamic effects that result in formation of an inhomogeneous liquid media, allowing the creation of space charges in the bulk that interact with the externally generated electric field.
  • Fig. 25(b) Using 'dip-stick' technique illustrated in Fig. 25(b), the entire Biochip was submersed in the cellular suspension at a 30° angle; once the cells became attached to within the cavities, the Biochip was removed, rinsed and suspended in dispersion media.
  • a further method utilising a Biochip holder was developed to apply voltage to the contact pads while steadying the platform in horizontal configuration during experimentation. 70-100 ⁇ l of the pituitary cell suspension was pipetted onto the surface and after a settling period of 3 mm, power was applied to the electrodes.
  • Field generation to the microelectrodes was delivered by a sinusoidal AC wave between 1 kHz and 13 MHz with a voltage range of 1 - 25 using a function generator (HP3312A, HP, USA).
  • Biochip Signals applied to the Biochip were continually monitored with an oscilloscope (HP4523, HP, USA) and the electric field was applied between 20 min and 3 h.
  • the generator leads were connected to the contact pads using a set of miniature aluminium clips that were then submersed in solution.
  • the Biochip holder used a set of pointed needle-contacts to apply power to the electrodes, which were connected to the generator using a set of crocodile clips.
  • Figure 217 illustrates trapping of pituitary cells by positive DSP using the pointed interdigitated microelectrodes.
  • Cells were suspended in a solution complete of Tris-Gly-Dex and the voltage set-point was reduced to 8 V p-p to ensure minimal cell damage from the high field gradients.
  • cells became attached within the hydrophillic exposed silicon cavities between a period of 30 - 60 min, whereby DEP was then discontinued.
  • Prolonged suspension of cells in the low conductive Tris- Gly-Dex solution exclusively, resulted in deformation, and after capture, physiological dispersion media was added to enhance cell viability.
  • Fig. 17(a) Demonstrating the suitability of the AFM as an analysis tool for the Biochip is illustrated in Fig. 17(a): Where a trapped cell within a cavity was imaged by an AFM (Dimension 3100, DI, USA) in air tapping mode. The cell was captured by positive DEP using the Tris-GlyDex solution at a frequency of 900 kHz and 8 V p-p using the planar electrode structure. Mechanical contact can cause non-reversible physiological change and damage in the cell structure. Utilizing the AFM' s non — contact imaging mode of cells in fluid, the Biochip platform and arrangement provides imaging of cells that ensure extended viability and lifetime.
  • Fluid movement and temperature increases induced by thermal heating from the electrode microsystems was reduced using the 'dip-stick' method.
  • the advantages of this technique were in the small quantity of culture medium used and the increased number of living cells trapped from exposure to a larger cell population.
  • Novelty of the Biochip device lies in the ability to rapidly trap single cells in defined wells for analysis by atomic force microscopy.
  • DEP is typically an unorganized process whereby an aggregation of particles are pushed toward a 'general' area of high and low electric field intensities, without individual identification or addressing of exact particle position or location.
  • Utilizing the combined DEP force in conjunction with the cells affinity for migration into the hydrophilic exposed silicon cavities we have demonstrated that individual cell-to-cavity capture can be achieved by positioning patterned cavity arrays between the electrodes.
  • DEP is a well-defined positioning tool that is ideal for biological studies. This work has shown that it is possible to manipulate biological cells by dielectrophoresis using the electric fields generated by a series of inicrofabricated electrodes. By rapidly creating cellular arrays using single cavity traps, cells can be individually identified from an entire culture population having further potential in bioanalytical research.
  • the interdigitated microelectrode systems required to produced the non-uniform electric field gradients can be fabricated in parallel with inexpensive lithographic methods. No complex MEMS systems are required and current platforms can easily be interfaced with existing electrical systems.
  • the present invention provides a way of creating replicas of cellular material in a near living state, and with nanoscale resolution capabilities. Imaging may be conveniently carried out at ambient conditions. Forces greater than those which can be employed directly on living cells can be used as there is no risk of damaging or destroying biological material.
  • Bioimprinting technology extend beyond cellular structure, morphology, imaging and study of cellular functions and interactions. Large numbers of cells from a definitive time frame can be examined and re-examined at a later date. For example if new information about the example is discovered, higher resolution imaging techniques become available, or different areas of the sample need to be exampled.
  • the moulds and casts produced by the invention may be transported non-hazardously, and without putting biological materials at risk.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé de création de moules et de modèles de matériau cellulaire au moyen de techniques de lithographie molle. Des cellules sont incubées dans un milieu sur un substrat pour favoriser la liaison au substrat. Les cellules sont nettoyées pour éliminer le milieu et sont revêtues par un matériau de moulage à l'échelle nanométrique qui est entièrement durci de façon à créer une empreinte complète ou partiellement durci de façon à éliminer la couche supérieure des cellules à mesure que le moule est retiré pour permettre une visualisation interne des cellules. Une reproduction négative peut également être créée. Les modèles ou moules sont imagés par microscopie à force atomique, par microscopie électronique à balayage ou par microscopie à effet tunnel, les images pouvant être stockées de manière permanente.
PCT/NZ2006/000150 2005-06-13 2006-06-13 Moulage WO2006135258A1 (fr)

Applications Claiming Priority (4)

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NZ54071305 2005-06-13
NZ540713 2005-06-13
NZ54346305 2005-11-09
NZ543463 2005-11-09

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010260032A (ja) * 2009-05-11 2010-11-18 Toyota Central R&D Labs Inc ターゲット分子の選択的吸着領域を有する吸着用担体及びその製造方法
CN102373406A (zh) * 2010-08-13 2012-03-14 鸿富锦精密工业（深圳）有限公司 镀膜方法
WO2019246623A1 (fr) * 2018-06-22 2019-12-26 Allevi, Inc. Systèmes et procédés pour une distribution, une stratification et un dépôt améliorés d'hydrogels réticulables

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004114016A2 (fr) * 2003-06-09 2004-12-29 Princeton University Office Of Technology Licensing And Intellectual Property Lithographie d'impression a surveillance et controle ameliores, et appareil associes
GB2406543A (en) * 2003-10-04 2005-04-06 Agilent Technologies Inc Masters for imprint lithography
WO2005101466A2 (fr) * 2003-12-19 2005-10-27 The University Of North Carolina At Chapel Hill Procede de fabrication de microstructures et de nanostructures au moyen de la lithographie molle ou d'impression

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004114016A2 (fr) * 2003-06-09 2004-12-29 Princeton University Office Of Technology Licensing And Intellectual Property Lithographie d'impression a surveillance et controle ameliores, et appareil associes
GB2406543A (en) * 2003-10-04 2005-04-06 Agilent Technologies Inc Masters for imprint lithography
WO2005101466A2 (fr) * 2003-12-19 2005-10-27 The University Of North Carolina At Chapel Hill Procede de fabrication de microstructures et de nanostructures au moyen de la lithographie molle ou d'impression

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FORSLIND B.: "Replication Techniques for dry and wet biological surfaces", SCANNING MICROSCOPY INTERNATIONAL, CHICAGO, vol. 13, no. 1, 1999, pages 133 - 139, XP002306368 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010260032A (ja) * 2009-05-11 2010-11-18 Toyota Central R&D Labs Inc ターゲット分子の選択的吸着領域を有する吸着用担体及びその製造方法
CN102373406A (zh) * 2010-08-13 2012-03-14 鸿富锦精密工业（深圳）有限公司 镀膜方法
WO2019246623A1 (fr) * 2018-06-22 2019-12-26 Allevi, Inc. Systèmes et procédés pour une distribution, une stratification et un dépôt améliorés d'hydrogels réticulables
US11872745B2 (en) 2018-06-22 2024-01-16 3D Systems, Inc. Systems and methods for improved dispensing, layering, and deposition of cross-linkable hydrogels

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