Patent Description:
New techniques for adherent, mammalian cell selection address some of the challenges but remain limited for living cells. Laser capture microdissection (LCM) (Arcturus; Mountain View, CA) has enabled single cells or small groups of selected cells to be obtained from tissue sections for genetic and proteomic studies, although most applications utilize fixed or frozen specimens. <NUM> Protocols for use with live cells have been published, but are very low throughput and not suitable for isolating large numbers of single living cells. <NUM> Most applications of LCM utilize fixed or frozen specimens. <NUM>-<NUM> Thus, these techniques have only partially met the needs of investigators for the positive selection of adherent, mammalian cells. Microlaser Technologies (Bermied, Germany) markets an instrument that uses a laser to cut out a region of interest from a tissue section and then generate a shock wave that "catapults" the cells into an overlying collection device. <NUM> Again most of the work with this technique has utilized fixed specimens, but collection of living cells has been demonstrated. <NUM> Cells are subjected to stress due to the direct effects of the shock wave and desiccation from removal of fluid overlying the sample during collection. ClonePix (Genetix, Hampshire, UK) is an automated system that uses image recognition to guide a suction pipette that aspirates colonies of loosely adherent cells from plates. The system requires cells that grow in loosely adherent clusters or suspension-adapted versions of adherent cells growing in a semi-solid methylcellulose media, thus it is not applicable to the vast majority of mammalian cells.

Recently, the Allbritton group developed an array technology for sorting adherent cells. <NUM>-<NUM> This cell sorting strategy uses arrays of releasable, microfabricated elements, termed pallets, formed from the biocompatible epoxy photoresist, either formulated from EPON SU-<NUM> or 1002F epoxy resins. <NUM>, <NUM> The epoxy is photolithographically defined on a standard microscope slide to create the pallet array. The pallets can be varied in size from tens to hundreds of microns to provide an adequate growth area for single cells or large colonies. In addition, the pallet surfaces can be modified with proteins or gels to enhance cell attachment and growth. <NUM>, <NUM>, <NUM> To culture cells on these arrays, cells are initially placed in suspension, but are allowed to settle and grow on individual pallets prior to analysis. When cells are plated on the array, the virtual air wall or polyethylene glycol hydrogel wall limit the location for cell attachment to the upper pallet surface. <NUM>, <NUM> Since the array is transparent, cells can be analyzed by standard microscopy techniques during culture. Subsequent to analysis, individual pallets containing the desired cells are released from the array using a pulsed laser and are then collected. <NUM>, <NUM> Recent studies of the selection and expansion of single cells have demonstrated a high rate of viability after laser-based release and exceptional success in clonal expansion of individual, sorted cells. <NUM>, <NUM> The approach makes possible a range of cell selection criteria for determining cells of interest (e.g. phenotrypic and temporal criteria and other characteristics) not accessible by alternative methods. <NUM> The pallet array has recently been used as a platform for culturing and sorting stem cell, and sorting cells based on antibody affinity. <NUM>, <NUM>.

Although some unique advantages have been demonstrated for the pallet array over other cell sorting technologies, several limitations need to be overcome before it can be widely accepted by the biology research community. The most serious limitation is that an expensive optical system is required to release a target pallet from the array. The optical system (including pulsed laser, beam splitter, mirror and lens) must be precisely aligned and maintained. To effectively release a pallet from the glass surface on which it is formed, the beam of the laser must be focused precisely at the interface between pallet and glass within a distance of a few micrometers. <NUM> To assist the user to find the right laser focal plane, indicators need to be built on the pallet array which adds complexity to fabrication. The shock wave generated by the laser is detrimental to the viability of cells, and as a result the energy of each laser pulse must be restricted to be less than <NUM>µJ in order to maintain high post-sort cell viability. However, a very low energy of release requires precise control of the adhesion force between the pallet and glass to keep pallets attached to the array until released is desired. In addition to the limitations required for laser-based release, the pallet array itself has drawbacks. First, the pallet array is made from photoresist having autofluorescence in the range of <NUM>-<NUM>, which coincides with the range of wavelengths of the most frequently used dyes (e.g. FITC, Oregon green, Alexa Fluor <NUM>, etc) for fluorescence imaging. <NUM>, <NUM> Second, the fabrication of the pallet array is expensive and complicated, since the whole fabrication process needs a clean environment and expensive microfabrication tools including mask aligner, photoresist spin coater, metal evaporator, and plasma cleaner.

Accordingly, there is a need for new ways to construct microcarriers useful for cell sorting.

A first aspect of the invention is an apparatus for collecting or culturing cells or cell colonies. The apparatus comprises a common substrate formed from an elastomer and having a plurality of wells formed therein, wherein said wells are separated by walls, wherein the common substrate is an elastomeric substrate; and a plurality of rigid cell carriers releasably connected to said common substrate, with said carriers arranged in the form of an array, and with each of said carriers received in one of said wells, wherein said carriers are configured to release from said substrate upon mechanical distortion of said substrate.

A further aspect of the invention is a method of collecting cells or cell colonies, comprising: (a) providing an apparatus according to the first aspect of the invention, (b) depositing a liquid media carrying said cells on said apparatus so that said cells settle on or adhere to said cell carriers; and then (c) releasing at least one selected carrier having said cells thereon from the cavity in which it is received by mechanical pushing of said at least one carrier to apply gradual release energy to said at least one carrier; and then (d) collecting said at least one selected carrier in order to collect cells or cell colonies settled on or adhered to said carrier.

In some embodiments, the carriers are coated with a biologically active molecule (that is, one or more) on at least the top surface thereof (e.g., all of the top surface, a major or minor portion of the top surface, etc.), wherein the biologically active molecule is selected from the group consisting of a peptide, a protein, a carbohydrate, a nucleic acid, a lipid, a polysaccharide, a hormone, an extracellular matrix molecule, a cell adhesion molecule, a natural polymer, an enzyme, an antibody, an antigen, a polynucleotide, a growth factor, a synthetic polymer, polylysine, and a drug.

The present invention is explained in greater detail in the drawings herein and the specification set forth below.

The present invention is now described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown.

In the figures, the thickness of certain lines, layers, components, elements, or features may be exaggerated for clarity. Where used, broken lines illustrate optional features or operations unless specified otherwise.

As used herein, the singular forms "a," "an" and "the" are intended to include plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements components and/or groups or combinations thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or groups or combinations thereof.

As used herein, the term "and/or" includes any and all possible combinations or one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and claims and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

It will be understood that when an element is referred to as being "on," "attached" to, "connected" to, "coupled" with, "contacting," etc., another element, it can be directly on, attached to, connected to, coupled with and/or contacting the other element or intervening elements can also be present. In contrast, when an element is referred to as being, for example, "directly on," "directly attached" to, "directly connected" to, "directly coupled" with or "directly contacting" another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature can have portions that overlap or underlie the adjacent feature.

Spatially relative terms, such as "under," "below," "lower," "over," "upper" and the like, may be used herein for ease of description to describe an element's or feature's relationship to another element(s) or feature(s) as illustrated in the figures. For example, if the device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. The device may otherwise be oriented (rotated <NUM> degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly," "downwardly," "vertical," "horizontal" and the like are used herein for the purpose of explanation only, unless specifically indicated otherwise.

It will be understood that, although the terms first, second, etc., may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. Rather, these terms are only used to distinguish one element, component, region, layer and/or section, from another element, component, region, layer and/or section. Thus, a first element, component, region, layer or section discussed herein could be termed a second element, component, region, layer or section without departing from the teachings of the present invention. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

"Interdigitated" as used herein with respect to carriers or microcups in an array means that the pattern of the array is staggered or off-set (typically in a uniform or repeating pattern) so that gap intersections are reduced in size and the opportunity for cells to settle at such intersections is reduced. Interdigitation can be achieved by one or more of a variety of means. The microcups can be hexagonal or triangular in cross-section; the microcups, when square or rectangular, can be offset from one another in adjacent row. The microcups can be provided with one or more vertical ridges that, when arranged in an array, interdigitates with gaps between microcups in adjacent rows. Numerous variations on the foregoing will be apparent to those skilled in the art.

"Cells" for carrying out the present invention are, in general, live cells, and can be any type of cell, including animal (e.g., mammal, bird, reptile, amphibian), plant, or other microbial cell (e.g., yeast, gram negative bacteria, gram positive bacteria, fungi, mold, algae, etc.).

"Liquid media" for carrying out the present invention, in which cells are carried for depositing on an array as described herein (and specifically within the cavities of the microcups) may be any suitable, typically aqueous, liquid, including saline solution, buffer solutions, Ringer's solution, growth media, and biological samples such as blood, urine, saliva, etc. (which biological samples may optionally be partially purified, and/or have other diluents, media or reagents added thereto).

"Substrate" as used herein is, in general, a flexible or elastomeric substrate, and may be conveniently formed from a material in which cavities may be produced and the carrier molded directly therein. Examples include, but are not limited to, silicones (e,g. , polydimethylsiloxane (or "PDMS"), Silastic, Texin and ChronoFlex silicone materials), polyurethane substrates, styrene-butadiene copolymer, polyolefin and polydiene elastomers, thermoplastic elastomers, other biomedical grade elastomers, etc..

"Biodegradable polymer" as used herein includes biodegradable polyesters and biodegradable aliphatic polymers. Numerous examples are known, including but not limited to those described in <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>. Particular examples include, but are not limited to, polymers that includes poly(lactic acid) (including poly(L-lactide) and poly(DL-lactide)), polyglycolide, poly(lactide-co-glycolide) (PLGA) (including poly(DL-lactide-co-glycolide)), poly(caprolactone) (PCL), poly[(R)-<NUM>-hydroxybutyric acid (PLA), poly(glycolic acid) (PGA), poly(ethylene glycol) (PEG), poly(hydroxy alkanoates) (PHA), dendritic polymers with acidic, hydroxyl and ester functional groups, modified polyesters, acetylated cellulose, starch, a starch derivative, a co-polymer of PLA and a modified polyester, or a combination thereof.

"Hydrogel" as used herein refers to a composition comprising a network of natural or synthetic polymer chains that are hydrophilic, and in which a significant amount of water is absorbed. Numerous examples are known, including but not limited to those described in <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>.

As noted above, the present invention is generally comprised of a common substrate formed from an elastomer and having a plurality of wells formed therein, wherein said wells are separated by walls, wherein the common substrate is an elastomeric substrate; and a plurality of rigid cell carriers releasably connected to the common substrate, with said carriers arranged in the form of an array, and with each of the carriers received in one of said wells, wherein said carriers are configured to release from said substrate upon mechanical distortion of said substrate.

The cavities in said substrate are separated by walls. The walls may be uniform or non-uniform and of any suitable dimension. In some embodiments, the walls have an average width of at least <NUM> micrometers, up to <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> micrometers. In general, the. walls have an average height of at least <NUM> or <NUM> micrometers, up to <NUM>, <NUM>, or <NUM> micrometers.

The cavities in the substrate in some embodiments have floors. The floors can be uniform or non-uniform and of any suitable thickness. In some embodiments, the floors have an average thickness of from <NUM> or <NUM> to <NUM> or <NUM> micrometers.

In other embodiments, the floor is eliminated, and the cavity is a continuous opening from the top surface of the substrate to the bottom surface of the substrate. Such arrays can be made in accordance with known techniques by, for example, from the substrate with such continuous cavities on top of a release layer.

The array may be in any suitable uniform or non-uniform arrangement, including but not limited to interdigitated arrays and/or or tilings.

The substrate has a top surface, and the carriers are preferably positioned either below the top surface, or up at (that is, even with, or flush with) the top surface). Preferably the carriers do not protrude above the top surface of the substrate. This configuration can follow from one preferred way of making the array, by forming the substrate with the cavities and then casting the carriers in the cavities, as discussed further below.

The carriers are configured to release from said substrate upon mechanical distortion of said substrate: that is, the application of a gradual energy such as mechanical pushing or continuous vibration, in contrast to a "burst" of energy, as discussed further below. The carriers or rafts may be in any suitable geometry, including cylindrical, elliptical, triangular, rectangular, square, hexagonal, pentagonal, octagonal, etc., including combinations thereof. In some embodiments, the carriers have heights of at least <NUM> micrometers, up to <NUM> or <NUM> micrometers. In some embodiments, the carriers have maximum widths of at least <NUM> or <NUM> micrometers, up to <NUM> micrometers.

The substrate can be produced by any suitable technique, such as printing or microprinting. The carriers can likewise be produced by any suitable technique, such as by casting the carriers in the cavities or wells formed during printing of the substrate. In some embodiments, the carriers have a concave top surface portion. While any desired physical or structural feature can be incorporated into the carrier top portion, alone or in combination, a concave top surface portion is conveniently formed by meniscus coating of the side walls of said wells or cavities in the substrate during the process of casting said carriers in those cavities or wells.

The carriers (also referred to as "rafts" herein) can be formed of any suitable material. The rafts are, in some embodiments, preferably transparent or semitransparent (e.g., visually transparent, optically transparent, optically transparent at certain wavelengths, and/or optionally containing elements or features that magnifies, reflects, refracts, absorbs or otherwise distorts light or certain wavelengths of light as light passes therethrough, etc.) A variety of polymers and other materials can generally satisfy the requirements for the microcarriers or rafts. Currently polystyrene (including copolymers thereof) and epoxy are preferred. A wide range of epoxies can be used including the epoxy novolac resins such as EPON l00lF, 1009F, and 1007F. These resins can be used alone or with crosslinkers. Preformulated epoxies, such as Loctite Hysol and other medical device epoxies can also be used. Medical device polymers such as polystyrene (including copolymers thereof, such as poly(styrene-co-acrylic acid) (PS-AA)), poly(methyl methacrylate), polycarbonate, and cyclic olefin copolymer can also be used as raft materials. Sol-gel materials, ceramics, and glasses (e.g., sodium silicate) can also be used as raft materials. Biodegradable polymers and hydrogels can also be used as raft materials. The rafts may be formed of a single material, may be a composite of two or more layers of different materials, etc. The rafts may be "doped" with one or more additional agents, such as growth factors (e.g., as in matrigel), magnetic or ferromagnetic particles or nanoparticles, live feeder cells, etc..

Arrays of the present invention are, in some respects, used in like manner as previous arrays, subject to some of the modifications described further herein.

The present invention provides a method of collecting cells or cell colonies, generally involving the steps of (a) providing an apparatus according to the first aspect of the invention as described above; (b) depositing a liquid media carrying the cells (including but not limited to non-adherent cells) on apparatus so that the cells settle on or adhere to the cell carriers; and then (c) releasing at least one selected carrier having the cells thereon from the well in which it is received by mechanical pushing of said at least one carrier to apply gradual release energy to said at least one carrier; and then (d) collecting said at least one selected carrier in order to collect cells or cell colonies settled on or adhered to said carrier.

In general, any suitable device for applying a release energy gradually may be employed. Sudden "bursts" of energy are less preferred because the resilient engagement of the carrier in the generally elastic substrate tends to serve as a "shock absorber" that resist release of the carrier by application of all but very large energy bursts (which then tend to release, for some (but not all) embodiments, undesirably large numbers of carriers). Hence, in some embodiments, release energy is typically applied over a duration of at least <NUM> millisecond (ms), at least <NUM>, at least <NUM>, and at least <NUM> second to achieve carrier or raft release.

In some embodiments, mechanical pushing is carried out by positioning a probe (e.g., a blunt probe, a needle, micropipette tip, etc), adjacent (e.g. above, below) beneath the common substrate and oriented towards the at least one selected carrier, and then progressively contacting the probe to the substrate. Progressively contacting may be carried out at any suitable rate of speed (as a non-limiting example, at a rate of <NUM> or <NUM> to <NUM> or <NUM>/s) until the at least one carrier is released therefrom. In some embodiments the probe does not pierce the substrate; in other embodiments the probe pierces the substrate and contacts and dislodges the at least one carrier.

In some embodiments, the invention is configured and carried out so that cells are deposited on the apparatus at an efficiency of capture (that is, are received in carriers rather than on walls) of at least <NUM>, <NUM>, or <NUM> percent.

Control of probe movement. Probe or microneedle movement can be provided by any suitable means, such as a miniaturized piezoelectric driver (Physique Instrumente GmbH, P-<NUM>) (<FIG>) or similar piezoelectric device. Typically, these devices can travel up to <NUM> in the forward or reverse direction with velocities up to <NUM>/s and step sizes as little as <NUM>, while generating forces up to <NUM> N. The devices can be controlled by a 5V TTL signal. The microneedle is supported on the piezo-driven rod and an XYZ microstage by any suitable means, such as custom mounts or clamps. Movement of the microneedle is in some embodiments controlled using a standard digital board interfaced via Metamorph (Molecular Devices) or uManager (http://www. micro-manager. org/) software. If the piezomotor proves insufficient for a particular application, DC motor (for example, Pololu Robotics & Electronics, Las Vegas, NV) can be utilized using similar mounting and control software.

A third strategy is a commercially available microinjection system (Eppendorf) with the injection pipette replaced by the microneedle, since the required motions for the microneedle are similar to that of a microinjection pipette. Still other approaches for the application of release energy include an ultrasound transducer, which may be used to vibrate or gradually vibrate a carrier from its corresponding cavity.

Collection plate and scaffolding support for the microraft array. Since the substrate (which also serves as the mold for the microcarriers) is a flexible polymer, a scaffold may be used in some (but not all) embodiments to prevent sagging of the array during imaging and raft release. In addition, released rafts are generally collected for subsequent culture. A scaffold and collection plate are in some embodiments combined into a single unit. Support posts or walls are, for example, fabricated from 1002F photoresist or PDMS on a glass base using standard photolithography or soft lithography. If needed, high quality glass plates (Erie Scientific, Portsmouth, NH) that have a flatness with a variance of less than <NUM> micron over several centimeters of travel are utilized for the collection plate. Alternatively, a polycarbonate cassette is machined using a CNC tool to provide the scaffold as well as collection plate. A jig or clamp is provided to hold the raft array over the scaffolding during raft release. Special care can be paid to sterility of the array as necessary.

For the probe-based (e.g., needle-based) release, the amount of array sag can be large since needle movement in the z direction does not need to be precise; however, the constraints for imaging are much tighter even with low magnification objectives (<NUM>. 63X, numerical aperture (NA) <NUM>). The depth of field for this objective is <NUM>; therefore, the goal in some (but not all) embodiments is to limit the amount of sag in the array between support posts to ≤ <NUM>. PDMS is an example in the following discussions; however, similar strategies can be employed for other mold materials. Again, in other embodiments, some sag, or even considerable sag, is less problematic and no steps to avoid sag need be taken.

Three strategies can be utilized to reduce array sag. <NUM>) Increase Young's modulus of the mold. A PDMS formulation with reduced elasticity or a Young's modulus of <NUM>-<NUM> MPa (<NUM>-fold higher than that of Sylgard <NUM> PDMS) can be used. Simulations using Comsol suggest that array sag can be reduced to less than <NUM> with support posts <NUM> apart. <NUM>) In-plane stretching of the PDMS mold. The substrate can be stretched along the axes parallel to the array surface to offset the out-of-plane sag (z-axis). If necessary, a film laminating instrument will be used to stretch the array uniformly before it is attached to a scaffold. <NUM>) Decrease the scaffold spacing. The distance between the posts or walls for array support (<FIG>) can be varied to increase or decrease the degree of array sag as necessary.

Rafts released into the collection wells can be cultured in the collection plate or retrieved for culture in standard multiwell plates. If cells floating in the medium (not attached to a surface) act as a source of contamination, the array can be washed extensively prior to release or vias can be inserted on either side of the collection plate for washing the array (<FIG>).

In some embodiments, one or more biologically active molecules is applied to or coated on the rafts (particularly, the top surface or layer of the raft). Different rafts in the same device may be coated with the same, or a different, molecule. Such biomolecules include a peptide, a protein, a carbohydrate, a nucleic acid, a lipid, a polysaccharide, a hormone, an extracellular matrix molecule, a cell adhesion molecule, a natural polymer, an enzyme, an antibody, an antigen, a polynucleotide, a growth factor, a synthetic polymer, polylysine, a drug, including combinations thereof. Coating may be carried out by any suitable technique, including but not limited to simple adsorption and covalent coupling. See, e.g., <CIT>. More particular examples of biologically active molecules include, but are not limited to, fibronectin, laminin, thrombospondin, collagen including collagen IV, elastin, tenascin, vitronectin; carbohydrates, and lipids; fibrinogen, tenascin; bovine pituitary extract, epidermal growth factor, hepatocyte growth factor, keratinocyte growth factor, and hydrocortisone. (See, e.g., <CIT>; see also <CIT>); pharmaceutical preparations or compounds; substances which influence the properties of biological cells; messengers; growth factors (e.g., vascular endothelial growth factor, bone morphogenic factor beta, epidermal growth factor, endothelial growth factor, platelet-derived growth factor, neural growth factor, fibroblast growth factor, insulin growth factor, or transforming growth factor); differentiation factors (e.g., neurotrophin, colony stimulating factor, transforming growth factor); antigens; allergens; etc. (See, e.g., <CIT>; see also <CIT>).

Carriers of the present invention may be composites of two or more (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) layers, with each layer formed of a different material, or having a different composition, than the immediately adjacent layer or layers. This feature can be used to incorporate a variety of advantageous structural and/or functional features into the carrier.

For example, in some embodiments, the carriers may be made magnetic or ferromagnetic by incorporating magnetic or ferromagnetic particles or nanoparticles into one or more layers of the carrier. If desired, a barrier layer can be provided between the layer(s) in which such particles or nanoparticles are incorporated, and the cell-supporting surface, to inhibit the transfer of particles or nanoparticles from the carriers to the cells.

In some embodiments, the carriers, or one or more layers of the carriers, comprise polystyrene (including copolymers thereof). In some embodiments, the carriers, or one or more layers of the carriers, comprise an anionic transparent magnetic polystyrene (e.g., a polystyrene copolymer incorporating an anionic comonomer such as acrylic acid, and containing magnetic or ferromagnetic particles or nanoparticles).

In some embodiments, the carriers comprise a rigid lower layer (sufficiently rigid to facilitate the mechanical displacement of the carrier from the elastomeric support; e.g., formed of a rigid polymer such as polystyrene, ceramic or glass, etc.); optionally, one or more intervening layers; and a cell-growth compatible upper layer on which cells can be grown such as a gel layer (e.g., matrigel or hydrogel, containing growth factors, antibodies, or the like). For example, the cell growth-compatible upper layer may comprise polystyrene such as an anionic polystyrene), a hydrogel (optionally containing live feeder cells to facilitate the growth of cells thereon, in any suitable amount, e.g., from <NUM>, <NUM> or <NUM> to <NUM> or <NUM>,<NUM> cells per carrier, such as murine embryonic fibroblasts); a biodegradable polymer, a biologically active material or biomolecule as described above, etc..

The present invention is explained in greater detail in the following non-limiting Examples.

As one non-limiting example of the invention, we describe here an improved technology for creating an array of individually releasable elements which overcomes the above limitations. Instead of fabricating pallets on glass using photolithography and photoresist, we use an array of microwells made from PDMS as the template to micromold the rafts. The micromolded raft contains no photoinitiator and therefore has a low autofluorescence background. The micromolding process does not require any microfabrication tool, so the fabrication becomes extremely simple and inexpensive. Since the raft is located inside the microwell, cells can fall into the microwell and then attach, thus eliminating the necessity of using a virtual air wall or PEG hydrogel wall to localize cell attachment. The most important improvement is to replace the expensive optical system with a low-cost needle release system. A selected raft can be effectively released from the array by the action of a needle inserted through the PDMS substrate. The use of a needle eliminates the necessity of building laser focal indicators on the pallet array, and also eliminates the possibility of laser damage to cells and rafts.

Arrays of micromolded concave rafts were fabricated on a PDMS plate. Cells fell in the microwells and attached to the surface of rafts so that the cells could be readily viewed with conventional microscopy. Single rafts were released by the action of a needle inserted through the PDMS plate. Upon release of a raft with an attached cell, the cell remained adherent to the underlying raft. The feasibility of collecting and then cloning the cell on the released raft was demonstrated. Cell isolation based on fluorescence and creation of a pure fluorescent cell line was demonstrated.

SU-<NUM> photoresist was purchased from MicroChem Corp. (Newton, MA). The Sylgard <NUM> silicone elastomer kit was purchased from Dow Corning (Midland MI). Gamma-butyrolactone, octyltrichlorosilane, propylene glycol monomethyl ether acetate, rhodamine B, glutaraldehyde, L-glutamine were obtained from Sigma-Aldrich (St. Louis, MO). EPON epoxy resin 1009F and 1002F (fusion solids) were purchased from Miller Stephenson Chemical Co. (Sylmar, CA). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (PBS), and penicillin/streptomycin were obtained from Invitrogen (Carlsbad, CA). Polycarbonate plates (<NUM> inch × <NUM> inch × <NUM> inch) were purchased from McMaster-Carr (Los Angeles, CA). All other reagents were from Fisher Scientific (Pittsburgh, PA).

Fabrication of mold. The microwell array was fabricated by casting PDMS on a mold. The mold was fabricated by standard photolithography on a glass slide with <NUM>-<NUM> thick SU-<NUM> with an area of microstructures of <NUM> × <NUM>. Glass slides were first rinsed with deionized water and ethanol to remove dust, and dried with a stream of nitrogen. The slides were then cleaned with the air plasma cleaner (Harrick Plasma, Ithaca, NY) for <NUM> before use. SU-<NUM> films of <NUM>-µm thickness were obtained by spin-coating SU-<NUM> photoresist (formulation <NUM>) on the glass slides following the protocol provided by MicroChem Corp. <NUM> Briefly, approximately <NUM>-<NUM> of SU-<NUM> was dispensed to the center of glass slides, and then the resist was spin-coated at <NUM> rpm for <NUM> followed by <NUM> rpm for <NUM> on a WS-<NUM>-4NPP spin coater (Laurell Technologies Corp. , North Wales, PA). The coated slides were baked on a hot plate at <NUM> for <NUM> followed by a second bake at <NUM> for <NUM> to remove organic solvent. To prepare SU-<NUM> mold, the SU-<NUM> film was exposed to UV light at a dose of <NUM> mJ/cm<NUM> through a photomask with the designed features using an Oriel collimated UV source equipped with a <NUM> short pass filter (Omega Optical, Brattleboro, VT). The post-exposure baking was performed on a hot plate at <NUM> for <NUM> followed by a second bake at <NUM> for <NUM>. The SU-<NUM> samples were then developed in SU-<NUM> developer for <NUM>, rinsed with <NUM>-propanol, and dried by a stream of nitrogen. The mold is finally hard baked on a hotplate at <NUM> for <NUM>. Fabrication of SU-<NUM> molds of alternative thicknesses (<NUM>-<NUM> in this study) was performed using the same process, except that the appropriate time parameters for that thickness were substituted.

Fabrication of PDMS microwell array. The surface of the mold was treated to render it non-sticky to PDMS by spin coating <NUM> vol% octyltrichlorosilane in propylene glycol monomethyl ether acetate at <NUM> rpm for <NUM>, followed by baking at <NUM> hotplate for <NUM>. PDMS prepolymer (<NUM>: <NUM> mixture of base:curing-agent of Sylgard <NUM> kit) was spread on the mold, and degassed under vacuum to remove trapped air bubble. To control the thickness of PDMS to be around <NUM>, PDMS on the mold was spin-coated at <NUM> rpm for <NUM>. PDMS was cured by baking the mold on <NUM> hotplate for <NUM>. PDMS microwell array (<FIG>) was obtained by peeling it from the mold.

Micromolding of rafts on the microwell array. A solution composed of <NUM> wt% 1009F epoxy resin in gamma-butyrolactone was prepared. An approximate amount of the solution was spread on microwell array (<FIG>). The trapped air bubbles in microwells were removed by degassing under vacuum using an oil pump. The microwell array was then vertically hung on a rack using tape, and the excess polymer solution dewetted on the PDMS surface and slowly flew out of the microwell array. Thus, each microwell was filled with a convex polymer solution (<FIG>). The solvent (gamma-butyrolactone) in the polymer solution was evaporated by baking the microwell array in an oven at <NUM> for <NUM>. The film was then further baked in a vacuum oven at <NUM> for <NUM> to completely evaporate the solvent. At the same time l009F epoxy resin was solidified by thermally induced epoxy ring-opening and condensation reactions. <NUM> With the evaporation of solvent, polymer in each microwell shrank and finally solidified at the bottom of the well into a concave raft (<FIG>). The height of the raft was approximately <NUM>% of the total height of the well.

Cell culture on the raft array. A plastic chamber (<NUM> × <NUM> × <NUM>) was machined from a polycarbonate plate by a computer numerical controlled (CNC) machine. The plate of microwell array with detachable rafts was glued to the chamber by using PDMS and cured in an oven at <NUM> for <NUM>. The array and the chamber were treated with air plasma cleaner for <NUM>. The array was sprayed with <NUM>% ethanol for sterilization, and then dried in a biosafety cabinet. <NUM> of phosphate buffered saline (PBS) was added into the chamber. To remove the trapped air bubbles inside the microwells, the plate was placed in a sterile vacuum desiccator (catalog # <NUM>, Electron Microscopy Sciences, Hatfield, PA) and degassed for <NUM> at room temperature inside the biosafety cabinet. The plate was then taken out of the desiccators, PBS buffer was aspirated, and a suspension of HeLa cells (<NUM>,<NUM> cells) was added to the chamber. The cells were cultured on the array in DMEM supplemented with PBS (<NUM>%), and L-glutamine (<NUM>/L) at <NUM> in a humidified, <NUM>% CO<NUM> atmosphere. Penicillin (<NUM> units/mL) and streptomycin (<NUM>µg/mL) were added to the media to inhibit bacterial growth. Immediately prior to use, the growth medium was removed from the cell chamber and replaced with PBS.

Release of rafts by a needle. The concave raft composed of 1009F epoxy resin was readily dislodged from the well by the action of a needle inserted through the PDMS (<FIG>). Three types of needles were tested (<FIG>): the anodized steel needles (<NUM> base diameter, <NUM> tip diameter) and tungsten needles (<NUM> base diameter, <NUM> tip diameter) were purchased from Fine Science Tools (Foster City, CA), and tungsten carbide needles (<NUM> base diameter, tip angle = <NUM>°, tip radius = <NUM>) were purchased from Semprex Corporation (Campbell, CA). A needle was inserted into a small PDMS plate (length × width × height = <NUM> × <NUM> × <NUM>), and the PDMS plate was self-stuck to a polycarbonate plate (length × width × height= <NUM> × <NUM> × <NUM>) having a cavity of (length × width × height = <NUM> × <NUM> × <NUM>). A micromanipulator was installed on the stage of an inverted fluorescence microscope (TE300, Nikon). Then the polycarbonate plate with fixed needle was attached to the micromanipulator. The needle was moved to the center of imaging field by the x- and y-direction micrometers. The needle was lowered to punch through the PDMS by controlling the z-direction micrometer (<FIG>).

Cell collection after needle release of raft. A collection chamber (<NUM> × <NUM> × <NUM>) was machined from a polycarbonate plate by a CNC machine, and its bottom was glued with a glass plate. Prior to needle release, the microwell array was rinsed with fresh culture medium to remove nonadherent and dead cells. Then <NUM> of fresh culture medium was added to the cell culture chamber, so that the liquid was close to overflow and formed a convex surface. The collection plate was placed directly above the cell culture chamber, and the excess liquid squeezed out. In this manner an enclosed compartment was formed between microwell array and collection plate filled with culture medium. Then the assembly was inverted and placed on the microscope stage. The selected cells were released by the needle by detaching the rafts to which they were attached. The raft carried the cells to the collection plate by gravity force. The collection plate and microwell array were separated in a sterile environment. The collection plate containing the released cells/rafts was placed into a polystyrene Petri dish and transferred to a standard tissue culture incubator. The growth of the collected cells was observed over time by transmitted light microscopy.

Characterization of fluorescence with standard microscopy filter sets. 1002F photoresist was formulated according to a previous publication. <NUM> Films of SU-<NUM> photoresist (<NUM> thickness), 1002F photoresist (<NUM> thickness), 1009F resin (<NUM> thickness), PDMS (<NUM> thickness) were prepared on glass slides by spin coating at an approximate spin rate, and baked in an oven at <NUM> for <NUM> to remove solvent or to cure. The SU-<NUM> and 1002F film were exposed to UV at a dose of <NUM> and <NUM> mJ respectively, and baked at <NUM> for <NUM> to finish photoinduced crosslinking reaction. Finally, all four types of films were baked at <NUM> for <NUM>. The fluorescence of the films were examined by a Nikon Eclipse TE300 inverted fluorescent microscope equipped with three fluorescent filter sets: a fluorescein filter set (B-2A; Nikon Instruments; excitation filter <NUM>-<NUM>, dichroic <NUM> long pass, emission <NUM> long pass); a TRITC filter set (G-2E; Nikon Instruments; excitation filter <NUM>-<NUM> dichroic <NUM> long pass, emission <NUM>-<NUM>); and a Cy5 filter set (<NUM>; Chroma Technology, Rockingham, VT; excitation filter <NUM>-<NUM>, dichroic <NUM>-nm long pass, emission <NUM>-<NUM>). Data were collected by a cooled CCD camera (Photometrix Cool Snap; Roper Scientific, Tuscon, AZ) using Metafluor Software (Molecular Devices, Sunnyvale, CA).

Fluorescence Microscopy. Transillumination and fluorescence microscopy were performed using an inverted microscope (TE300, Nikon). Imaging of GFP-expressing cells was performed using a standard fluorescein filter set.

Scanning electron microscopy (SEM) of cells. Cells plated on microwell arrays were rinsed gently with PBS and then fixed with <NUM> wt % glutaraldehyde in PBS for <NUM>. This sample was washed with PBS, and dehydrated with a series of ethanol/water mixtures of increasing ethanol concentration (<NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>% ethanol, <NUM> in each mixture). The fixed cells were observed by SEM (FEI Quanta <NUM> ESEM, FEI Company).

Fabrication of microwell array with detachable bases. Microwell arrays with controlled depth and dimension were fabricated by casting PDMS against a mold. This molding process has been generally used in fabricating microfluidic channels and microdevices. <NUM>, <NUM> The fabricated PDMS microwell array has been used to pattern cells for a variety of applications including imaging cytometry,<NUM> hybridoma selection,<NUM> microenvironment for stem cell research,<NUM>, <NUM> etc. PDMS microwell array has been combined with optical tweezers or micropipette to isolate the selected non-adherent cells. <NUM>, <NUM>, <NUM> The mold was fabricated by using SU-<NUM> photoresist and the standard photolithography process. The microwell arrays with density of over <NUM> - <NUM> wells/cm<NUM> are used for the current experiments, and the dimension of wells is in the range of <NUM>-<NUM> (<FIG>).

A filling-dewetting process was used to mold pallets in the microwells (<FIG>). We observed that a polymer solution composed of <NUM> wt% of EPON epoxy 1009F resin in gamma-butyrolactone does not wet PDMS. When a drop of the solution was added to a PDMS plate and the plate was tilted, the solution gradually traveled out of the PDMS surface without leaving any residue. This dewetting phenomenon is caused by their mismatched surface tension. PDMS has a surface tension of <NUM>-<NUM> dyne/cm, while gamma-butyrolactone is a polar solvent with a relatively high surface tension of <NUM> dyne/cm, and EPON epoxy resin has a surface tension of <NUM>-<NUM> dyne/cm. The 1009F polymer solution was added the PDMS microwell array, and vacuum was used to remove the trapped air bubble inside each well (<FIG>). When the PDMS is tilted or hung vertically, the polymer solution slowly drained off the PDMS surface due to dewetting, leaving each well filled with polymer solution. As a result, an array of microwells individually filled with polymer solution was achieved on the PDMS plate (<FIG>). The polymer solution was found to be convex in each well (Figure 4B). The plate was then baked at elevated temperature to evaporate the solvent. The evaporation caused shrinkage of the polymer. A concave polymer pallet is generated inside each well at the end of solvent evaporation (<FIG>). The concave shape is caused by the mismatched surface tension between PDMS and 1009F resin/gamma-butyrolactone during solvent evaporation. The thickness of the pallet can be adjusted by the concentration of epoxy resin in solvent. By using <NUM> wt% resin concentration, the height of pallet is approximately <NUM>/<NUM> of the depth of the well. Gamma-butyrolactone was found to be compatible with PDMS with negligible swelling. <NUM> 1009F resin was used due to its high melting point (Tm = <NUM>-<NUM>) and its low autofluorescence. <FIG> shows the microwell array bottomed with molded rafts. The concave shape of each raft is clearly shown in a ruptured section (<FIG>). The raft has poor adhesion to the PDMS well so that it can be easily detached.

In the filling-dewetting process, the microwell array was used as the template for molding of pallets. The micromolding process does not require any microfabrication tool and a cleanroom facility; a small laminar flow bench is enough for the whole micromolding process. A mold for fabricating PDMS microwell array can be obtained from a microfabrication foundry service. As a result, the fabrication process becomes extremely simple and inexpensive after obtaining a mold.

Micromolding is a versatile process to fabricate rafts. It requires a simple polymer solution composed of resin and solvent, and it does not require inclusion of photocatalyst. In contrast, photocatalyst is an indispensable component of the photoresist for fabricating pallets using photolithography. On the other hand, the polymer solution can include other components (e.g. magnetic particles, color or fluorescent dye, pore generator, etc.), so that functional rafts (e.g. magnetic, color-coded or fluorescent, porous, etc.) can be easily molded. It is always difficult to fabricate functional pallets by photolithography, since the functional component usually interferes with or blocks the UV light needed for development.

Autofluorescence. Fluorescence-based assays are important tools for cell selection. SU-<NUM> and 1002F, the photoresist from which the pallets are constructed by photolithography, has strong autofluorescence in the range of <NUM>-<NUM>. <NUM>, <NUM> This wavelength range unfortunately coincides with the wavelength of the most frequently used dyes (e.g. FITC, Oregon green, Alexa Fluor <NUM>, etc.) for fluorescence imaging. 1002F photoresist has a lower level of autofluorescence than SU-<NUM>. The SU-<NUM> or 1002F photoresist contains about <NUM> wt% photoinitiator, triarylsulfonium hexafluoroantimonate. The autofluorescence comes from the photodecomposition by-products which have conjugated structure. <NUM> Using the micromolding method, the raft is composed of only 1009F resin, and as a result the autofluorescence is very low. To determine the level of auto-fluorescence, thin films of SU-<NUM> photoresist, 1002F photoresist, 1009F resin and PDMS were spin coated on glass slides, and their fluorescence intensity was obtained with commonly used filter sets in fluorescence microscopy (<FIG>). The thickness of film was <NUM>, except that PDMS has a film thickness of <NUM> and it is shown for comparison. Under FITC filter set, the autofluorescence of 1009F resin is only <NUM>% of that of SU-<NUM> photoresist, and <NUM>% of that of 1002F photoresist. The autofluorescence of 1009F resin is slightly higher than that of PDMS, which is generally considered one of the lowest autofluorescence polymers. <NUM> Under TRITC and CY5 filter sets, the autofluorescence of 1009F resin is almost negligible. Compared with SU-<NUM> and 1002F photoresist, the reduced autofluorescence of 1009F resin is due to the absence of photocatalyst. The reduced autofluorescence of molded pallets is particularly valuable for highly sensitive measurements.

Release of individual rafts from a large array with a needle. The micromolded rafts are seated at the bottom of PDMS microwells. Although rafts have shown poor adhesion with PDMS (<FIG>), they are not easily detached from the array since they are surrounded by PDMS wells. The selected raft can be detached from the array simply by the mechanical action of a needle pushed through the PDMS from the backside. PDMS is a flexible material, and a needle can easily penetrate a PDMS film of <NUM> thickness. <FIG> shows the needle system built on an inve1ied microscope. The needle was attached on the transparent plastic plate, and its movement at x, y, and z direction was controlled precisely by a micromanipulator. The needle was moved to the center of field of view by × and y micrometers. The raft to be released was the moved to the spot the needle would penetrate. The penetration depth was controlled by lowering the needle by z micrometer. Depending on the size of raft, a variety of needles can be used. <FIG> shows three types of needles used for releasing rafts. The tungsten carbide needle (top) with a tip diameter of <NUM> and the anodized steel needle (middle) with a tip diameter of <NUM> are suitable for releasing big rafts, while the tungsten needle (bottom) with a tip diameter of <NUM> is suitable for releasing small rafts. To demonstrate the release of individual rafts, a large array composed of <NUM>,<NUM> rafts/cm<NUM> (<NUM> size, <NUM> gap) was used. The raft was doped with <NUM> ppm of rhodamine B so that the detachment of rafts from the array could be clearly visualized by fluorescence microscopy. The selected rafts (marked with asterisk) were released by inserting the needle through the PDMS and punching the raft out of the microwell. The release of rafts was confirmed by watching the raft float away from the microwell array under the microscope. Most of the rafts were released from the array by only one punching action (<NUM>%, N=<NUM>). Sometimes additional penetrations were required before release was accomplished: two (<NUM>%, N=<NUM>), three (<NUM>%, N=<NUM>), or four (<NUM>%, N=<NUM>). The penetration site could be visualized in the PDMS after withdrawal of the needle (<FIG>). To confirm the release of rafts, rhodamine B doped rafts were observed under fluorescence microscopy before and after penetration of the PDMS with the needle (<FIG>). The images clearly show the four selected rafts were released without disturbing neighboring rafts. In this experiment, <NUM>% (N=<NUM>) of targeted rafts were released and <NUM>% of adjacent rafts were detached. Multiple rafts in an array could be released by moving the microscope stage to sequentially place rafts under the point of needle penetration. Larger rafts are more easily released from the array. The smallest rafts tested had a diameter of <NUM> (<FIG>,C,D). For small rafts, a gap of at least <NUM> prevented adjacent rafts from being disturbed by the needle release action. Since the rafts were individually addressable and releasable with the needle, the rafts were suitable candidates for the array-based scanning and cloning of adherent, mammalian cells.

Cell culture on microwell array with detachable rafts. To determine if rafts surrounded by a PDMS well could be used to create a cell-based array, arrays were oxidized by plasma cleaner for <NUM> to provide a surface suitable for cell attachment. HeLa cells were plated on the arrays. Most cells fell into the wells by gravity and settled near the center of rafts due to the concave surface shape of the rafts. Twenty minutes after cell plating, the array was gently rinsed with fresh medium to remove the cells that did not fall into the wells. <NUM> rafts were used to create an array of single cells per raft (<FIG>), and <NUM> rafts were used to create an array of multiple cells per raft (<FIG>). The arrays were examined bymicroscopy after <NUM>. <NUM>% of cells (N = <NUM> cells) were located inside the well and attached to the pallets. SEM images (<FIG>,<FIG>) corroborated these findings.

Release of individual rafts with cells. To determine the feasibility of releasing rafts with living cells, the pallets with cells on their surface were released using a needle as described above (<FIG>). To isolate single cells, an array of <NUM> rafts was used. The selected single cell (marked with asterisk) was separated from the array by detaching the raft on which it was attached. The release process is shown in <FIG>, C, D. After release, the cell stayed attached to the raft and was unharmed by the process. To isolate a small colony of cells (<NUM>-<NUM> cells), an array of <NUM> rafts was used (<FIG>, F, G). To isolate a larger colony of cells (><NUM> cells), an array of <NUM> rafts was used (<FIG>, I, J).

Proliferation of single cells from released rafts. To determine the feasibility of collecting single cells for culture and expansion, rafts (length × width × depth = <NUM> × <NUM> × <NUM>) with single HeLa cells were released, collected, and placed into a culture dish. The cells were imaged by microscopy within an hour of collection and then at varying times thereafter. At one hour after collection, the HeLa cell remained on the raft top (<FIG>). By <NUM> after collection, single cells divided into two daughter cells (<FIG>). The cells had migrated from the rafts onto the adjacent surface by <NUM> (<FIG>). By <NUM>, the single cell had expanded into a small colony to create a clonal population from the original single cell. Of the released single HeLa cells <NUM>% (N=<NUM>) proliferated into colonies. These data demonstrate the feasibility of collecting living, single cells from the raft array and producing clonal colonies. In similar experiments using rafts containing a colony of HeLa cells (number of cells> <NUM>), the proliferation rate was <NUM>% (N = <NUM>).

Cell sorting based on fluorescence. To demonstrate cell sorting based on fluorescence, a HeLa cell line stably transfected with the enhanced green fluorescent protein (eGFP) fused to the histone-H1 protein was used. Histone-H1 is tightly associated with cellular DNA so that transfected cells display green fluorescence localized to their nuclei. Wild-type HeLa cells were mixed with the eGFP-histone-H1 expressing cells at a ratio of <NUM>:<NUM>, respectively. The cells were then plated on an array of molded rafts (length × width × depth = <NUM> × <NUM> × <NUM>) at limiting dilution to yield <NUM> cell/pallet: i.e. <NUM>,<NUM> cells were plated on the array composed of <NUM>,<NUM> wells/rafts. The array was imaged by microscopy (transmitted light and fluorescence). Pallets with fluorescent cells were easily visualized amongst rafts containing nonfluorescent cells (<FIG>). Under these conditions, no background fluorescence from the rafts and PDMS was detectible. After <NUM>, a proportion of rafts on the array contained <NUM>-<NUM> fluorescent cells, which were the daughter cells from the single parental cells originally plated on the raft array. To demonstrate sorting of these clonal colonies, individual rafts containing fluorescent colonies were selected, released, collected, and placed in culture (<FIG>). Expansion of these fluorescent colonies for <NUM> days yielded clonal populations of cells expressing the fusion protein (<FIG>). These experiments demonstrate the ability to sort colonies of cells based on whether the individual cells retain the properties of the parental cell. This selection strategy may find utility in the molecular engineering of cells or the development of cell lines, for example, stem cells.

Comparison with the currently used cell sorting methods. Current methods for cell sorting of adherent cells rely on either re-suspending adherent cells so that they may be used in a flow cytometer, or the use of a time consuming process called "limiting dilution". Suspending cells is not desired because the suspending process damages the cells and places them in an unnatural state (not adhered to a surface). This process also causes the loss of morphologic features of the normally adherent cell. Limiting dilution is a time consuming and laborious assay, resulting in only an enriched sample of target cells. Sorting by flow cytometry is expensive as the instrument generally retails for several hundred thousand dollars and requires a trained and dedicated technician. As a result, shared cell sorting facilities are established in research universities and institutes. Operating, maintaining, and staffing a sorting facility is an expensive undertaking.

The micromolded raft array technology has a number of unique advantages over other cell sorting methods. First, in the raft array technology, individual cells of interest are identified then isolated by detaching the structure that supports the cells. Each cell remains fixed on the solid surface at all times. This simplicity and robustness allows one to rapidly isolate adherent cells without the need to re-suspend them, and without the need to perform a limiting dilution. In a single step, a researcher can quickly scan tens of thousands of cells and collect one or several cells from the initial population. Cells experience no stresses and are completely viable for further growth and expansion. Second, the cells can be rescanned multiple times, as the cells are completely unharmed in the scanning and isolation process, making this technology an extremely attractive alternative to flow sorting when adherent cell assays are desired. Third, cells can be separated based on new sorting criteria that other methods cannot do, for example, cell morphology, cell growth rate, and cell secretion. No other company (including industry leaders) offers a similar product. Finally, raft array technology is extremely simple, and it does not rely on any sophisticated equipment, making it affordable for any biology laboratory. It provides an inexpensive yet efficient method for biologists to perform cell sorting and creation of cell lines in their laboratory. The technology is especially valuable for sorting of very small samples (<NUM>,<NUM> - <NUM>,<NUM> cells), such as those obtained from animal models or biopsy specimens. The viability after sorting (whether cells are alive and able to grow) remains extremely high - well over <NUM>% of sorted cells survive the sorting process by this method, unlike other methods where many if not most cells die after sorting. This means that stem cells and other primary cells taken directly from a tissue sample can be effectively isolated in the laboratory. The micromolded pallet array technology creates the possibility of opening an entire market of adherent cell sorting.

Chamber dimension: <NUM> (length) × <NUM> (width) × <NUM> (height), total volume <NUM><NUM> » <NUM>. Array dimension: <NUM> (length) × <NUM> (width) = <NUM><NUM>.

The plating density for a particular cell line will depend upon the array used and optimal density for cell growth. A general guideline for a total number of cells to be plated to obtain a single cell/raft condition is to plate about ½ of the number of rafts on the array (Table <NUM>). However, cell density can be titrated for optimal results.

Raft array: <NUM> × <NUM> (LxW) wells. There are <NUM>,<NUM> wells on the entire array. All steps performed in a laminar flow hood.

An elastomeric PDMS mold (<NUM> × <NUM> × <NUM>) was fabricated by casting PDMS on an SU-<NUM> master fabricated by standard photolithography on a glass slide. The SU-<NUM> thickness was <NUM> - <NUM>. Approximately <NUM> of polystyrene solution (<NUM> wt% in GBL) was added to the PDMS mold (not shown). This amount of solution generates a film of approximately <NUM> thickness after baking. Polystyrene solution was found to be dewetting on PDMS surface during baking, causing the solution to shrink. To prevent the dewetting, the PDMS mold was treated with air plasma for <NUM> prior to the addition of the polystyrene solution. This treatment did not affect the mold release in the final step. A short (<NUM>) degas by oil pump was required to remove the trapped air bubbles in the PDMS mold. Since GBL has a high boiling point of <NUM>, polystyrene solution did not evaporate or boil during degassing. The polystyrene solution remained as a clear, viscous solution after degassing. The mold was then heated on a hotplate at <NUM> overnight (<NUM>) to completely evaporate the GBL solvent. Finally, the PDMS/polystyrene was cooled to room temperature and the PDMS mold was slowly peeled from the solidified polystyrene.

Solvents for other materials from which rafts or carriers are fabricated are described in the table below. Composite carriers are conveniently prepared by carrying out the process with a first material, and then sequentially repeating the process one or more times with a different material, until a composite of two or more materials is formed.

While deforming the PDMS frame during microraft release does not affect adjacent microrafts, we observed that loosely adherent cells can detach and contaminate the collected cell colonies. To overcome this limitation, this example utilizes magnetism to manipulate the microraft. Microrafts are doped with magnetic nanoparticles so they can carry the cell of interest to the collection dish via magnetic attraction. Due to the ability to fabricate microrafts with a variety of polymers outside those required for carrier development an anionic transparent magnetic polystyrene was developed which has better biocompatibility and a lower autofluorescence than magnetic 1002F or SU8 photoresists.

Magnetic microrafts were developed with these polymers and coated with a non-magnetic polymer to provide a barrier between the magnetic film and plated cells. Additional layers of polymer added over pre-existing microrafts remained isolated within the PDMS microwells even after the addition of a forth polymer. Magnetic microrafts were released from the PDMS frame and magnetically collected with an external magnet. Cells grown on magnetic rafts were imaged with traditional transmitted light and fluorescence microscope, as well as confocal microscope. The growth and localization of cells on these microrafts with untreated poly(styrene-co-acrylic acid) (PS-AA) surfaces was monitored. Finally, single cells attached to magnetic microrafts were sorted and magnetically collected.

The following materials were obtained from the Aldrich Chemical Company (St. Louis, MO): iron(II) chloride tetrahydrate (<NUM>%), iron(III) chloride anhydrous (<NUM>%), iron(III) nitrate nonahydrate (<NUM>+%), <NUM>% ammonium hydroxide solution, oleic acid (<NUM>%), toluene (reagent grade), triarylsulfoniumhexafluorophosphate salts, mixed, <NUM>% in propylene carbonate, y-butyralactone (GBL, <NUM>+%), <NUM>-methoxy-<NUM>-propanol (1002F developer, <NUM>%), glutaraldehyde, Rhodamine B, <NUM>,<NUM>'-azobisisobutyronitrile (AIBN, <NUM>%), styrene (>_99%) and acrylic acid (<NUM>%). EPON resin SU-<NUM> and EPON resin 1002F (phenol, <NUM>,<NUM>'-(l-methylethylidene)bis-, polymer with <NUM>,<NUM>'-[(<NUM>-methylethylidene) bis(<NUM>,<NUM>-phenyleneoxymethylene]bis-[oxirane]) were obtained from Miller-Stephenson (Sylmar, CA). Phenyl red free Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), 1X phosphate buffered saline (PBS), pH <NUM>, <NUM>% trypsin with EDTA solution and penicillin/streptomycin were received from Invitrogen (Carlsbad, CA). Sylgard <NUM> silicone elastomer kit (PDMS) was received from Dow Corning (Midland, MI). Fibronectin extracted and purified from human plasma was obtained from Chemicon International Inc. (Temecula, CA). Collagen I from rat tail tendon and Falcon™ Petri dishes were purchased from BD Biosciences (San Jose, CA). Polycarbonate plates (<NUM>" × <NUM>" × <NUM>") were purchased from McMaster-Carr (Los Angeles, CA). Wild-type HeLa cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All other chemicals were procured from Fisher Scientific (Pittsburgh, PA).

Magnetic polystyrene development. Magnetite nanoparticles were synthesized by the co-preciptation of iron salts in deionized water by the addition of ammonium hydroxide. <NUM> The nanoparticles were magnetically decanted and the fluid was replaced with fresh deionized water and iron nitrate. <NUM> mixing at <NUM> in the presence of iron nitrate allows for oxidation of nanoparticles to maghemite (<NPL>)). Magnetically decanting the nanoparticles and replacing the liquid with deionized water gives a magnetic ferrofluid. Maghemite nanoparticles were then made hydrophobic through extraction with oleic acid. The magnetic phase was magnetically decanted and excess oleic acid removed by three washes of ethanol. Oleic acid coated maghemite nanoparticles were then dissolved in toluene (<NUM> of maghemite/<NUM> toluene). Poly(styrene-co-acrylic acid) (PS-AA) was prepared by copolymerization of styrene and acrylic acid in gamma-butyrolactone (GBL), as described previously (see, e.g., <NPL>). Briefly <NUM> styrene, <NUM> acrylic acid, <NUM> AIBN and <NUM> GBL were mixed in a flask and heated in a <NUM> water bath for <NUM> to complete copolymerization. A <NUM>:<NUM> v/v mixture of PS-AA in toluene was slowly added to the maghemite ferrofluid. The toluene was then evaporated (Buchi R200 rotovapor, Flawil, Switzerland) until a thick gel remained. GBL was added to this magnetic polystyrene gel until the desired viscosity for efficient dip coating was achieved.

Fabrication of PDMS mold. PDMS molds for the arrays of microrafts were developed though soft lithography from an SU-<NUM> master. The SU-<NUM> masters were developed though typical photolithography experiments as described previously (<NPL>)). SU-<NUM> masters for raft release and cell culture experiments were composed of <NUM> thick <NUM>×<NUM> squares with <NUM> gaps. The SU-<NUM> master for the <NUM> layer rafts were composed of <NUM> thick <NUM>×<NUM> squares with <NUM> gaps which were developed by frontside exposure. Following development, the SU-<NUM> masters were made non-sticky to PDMS by spin coating <NUM>% vol. octyltrichlorosilane in propylene monomethyl ether acetate at <NUM> rpm for <NUM>, followed by baking at <NUM> on a hotplate for <NUM>. PDMS prepolymer (<NUM> : <NUM> mixture of base : curing-agent of Sylagard <NUM> kit) was poured over the SU-<NUM> master and degassed (house vacuum) to remove trapped air bubbles. Following degassing the sample was spin-coated at <NUM> rpm for <NUM> and baked at <NUM> for <NUM>. which gives a <NUM>-thick PDMS layer over the SU-<NUM> master. The PDMS was then gently peeled from the SU-<NUM> master leaving a PDMS mold containing an array of multiwells.

Fabrication of magnetic microrafts. Releasable magnetic microstructures were micromolded within the microwells of the PDMS mold, as described previously (see, e.g., <NPL>). For single-layer microraft arrays magnetic 1002F or magnetic poly(styrene-co-acrylic acid) were applied over the PDMS mold. Trapped air bubbles within the microwells were removed though degassing under vacuum (Oerlikon Leyboid pump). The PDMS mold was then strung to a DC motor and lowered into a solution of the magnetic polymer well side down. Slowly raising the PDMS mold gives a convex solution of polymer in each microwell. Placing the PDMS mold in a <NUM> oven for <NUM> evaporates the bulk of the GBL giving concave microstructures within the microwells. Further evaporation of the magnetic microstructures is achieved by a <NUM> bake at <NUM> vacuum oven (-<NUM> in. Multi-layer microrafts may be constructed through repeating the above process with different polymers dissolved in GBL.

Following fabrication of the microraft arrays the PDMS mold was placed onto a polycarbonate cassette, microraft array face side down, and the PDMS mold was stretched to reduce any sagging. While still attached to the cassette a second polycarbonate cassette (<NUM> × <NUM> × <NUM>/ top release or <NUM> × <NUM> inner × <NUM> mmo. × <NUM> height/ bottom release) was glued to the top of the PDMS mold using PDMS with a <NUM> bake in an oven for <NUM>.

Release and collection of magnetic microrafts. Microrafts on an inverted array were released from the top by means of previously used procedures (see, e.g.,<NPL>). Additionally, magnetic rafts were released with a needle from below the array and magnetically collected against gravity onto a collection plate. The microraft array attached to the release chamber with culture media enclosed within the chamber by a collection plate was directly placed upright on a microscope stage. The release needle, an anodized steel microneedle with a <NUM> base diameter and <NUM> tip diameter (Fine Science Tools, Foster City, CA) was either bound to a PDMS block or bent at a <NUM>° angle and attached to an XYZ micromanipulator with a polycarbonate brace. The needle tip was positioned between the center of the microscope objective and the microraft of interest. Individual microrafts were released from the PDMS mold by raising the needle to puncture the PDMS and eject the selected microraft. Following release the micromanipulator was lowered to its original position. Released microrafts were drawn to the collection plate by a permanent magnet held above the cassette. The magnet was kept over the collection substrate to retain microrafts as the collection plate is gently lifted off the microraft cassette.

Cell culture on magnetic microrafts. For quick (<NUM>) adhesion of cells onto microrafts the array was first treated in a plasma cleaner (Harrick Plasma, Ithaca, NY) for <NUM>. The microraft array and cassette holder were thoroughly sprayed with <NUM>% ethanol and allowed to dry in a tissue culture hood. Following sterilization and <NUM> rinses with sterile DI H<NUM>O, <NUM> type I collagen from mt tail (<NUM>µg mL -<NUM>) was added to the microraft array for <NUM> including a <NUM> degassing by vacuum to remove trapped air bubbles within the microwells. <NUM> rinses of DI H<NUM>O was followed by the addition of DMEM supplemented with FBS (<NUM>%), L-glutamine (<NUM> L -<NUM>), penicillin (<NUM> units mL -<NUM>), and streptomycin (<NUM>µg mL -<NUM>). A suspension of <NUM>,<NUM> cells was then added to the microraft array and allowed to adhere to the microrafts for <NUM> in a <NUM> incubator with a <NUM>% CO<NUM> atmosphere.

Prior to cell selection, loose cells were removed with <NUM> rinses of H<NUM>O and DMEM was added to the microraft chamber. A plasma cleaned and sterilized polystyrene petri dish was then mated to the microraft cassette which made a concealed chamber filled with cell culture media. Following single cell collection the petri dish was removed from the microraft cassette and filled with <NUM> conditioned media and allowed to culture in a <NUM> incubator with a <NUM>% CO<NUM> atmosphere. Conditioned media was made by culturing subconfluent cultures of GFP-HeLa cells in DMEM supplemented with FBS (<NUM>%), L-glutamine (<NUM> L -<NUM>), penicillin (<NUM> units mL -<NUM>), and streptomycin (<NUM>µg mL -<NUM>) for <NUM>. Cells were centrifuged (<NUM>,<NUM> × g, <NUM>) and the supernatant removed and stored at -<NUM> until ready for use.

Single-layer magnetic rafts. In the current work, microrafts were developed by dip-coating various polymers (SU-<NUM>, 1002F and PS-AA) containing <NUM> to <NUM> wt¾ uniformly distributed maghemite nanoparticles dissolved in <NUM> wt% GBL on a PDMS mold consisting of arrays of <NUM> × <NUM> squares isolated by <NUM> tall <NUM> wide PDMS walls. To assist in release of the microrafts the SU-<NUM> master was developed by using backside exposure. This creates a slightly bowed sidewall which decreases the sharp contact angle of the microrafts. These polymers showed successful dewetting on the PDMS and microraft construction. Magnetic rafts remain isolated within the PDMS wells and possess a slightly concave surface as monitored by SEM and TEM (not shown). TEM images of vertical slices through microrafts composed of <NUM>% yFe<NUM>O<NUM> in 1002F or PS-AA show that these structures have concave curvatures of <NUM>° and <NUM>°, respectively. The microraft thickness and curvature can be altered by adjusting the concentration of polymer in GBL that is dip-coated.

Transparency of the magnetic polymers is retained during microraft fabrication (not shown). It has previously been shown that magnetic nanoparticles will accumulate at the surface of the polymer during photolithographic processing of magnetic photoresists. Horizontal slices through the magnetic microrafts were imaged by TEM to identify the dispersion of magnetic nanoparticles throughout the microrafts. All microrafts composed of <NUM>% yFe<NUM>O<NUM> in 1002F showed evenly distributed nanoparticles throughout the polymer with a <NUM> thick layer of maghemite nanoparticles accumulated at the surface and bottoms of the microrafts (not shown). These results confirm the previous hypothesis that nanoparticles are carried to the extremities of the polymer by evaporating GBL molecules (<NPL>)). Microrafts developed with <NUM>% yFe<NUM>O<NUM> in PS-AA have uniformly distributed nanoparticles through the polymer, however, unlike the magnetic 1002F there is no accumulation of nanoparticles at the microraft surfaces (not shown). The retention of the nanoparticles within the microrafts is likely due to coordinative bonding between the magnetic nanoparticles and the PS-AA, a phenomenon hypothesized to occur in similar nanocomposites by previous researchers (<NPL>)).

Multi-layer magnetic rafts. In this work we tested the ability to fabricate microrafts containing multiple layers of different polymers. Successful layering is dependent on the should be capability of polymer of dewetting on the microraft surface between the PDMS wells. The surface tension of 1002F and PS-AA are <NUM> and <NUM> dyne cm-<NUM>, respectively, which is still significantly lower than that of the polymer solvent (GBL, <NUM> dyne cm-<NUM>). Second, the quantity of polymer coating the microrafts should be high enough to ensure that when the GBL is evaporated the polymer will uniformly coat the microraft.

To fabricate two layer magnetic rafts a magnetic raft array was constructed as described above using 1002F or PS-AA containing <NUM>% yFe<NUM>O<NUM>. A layer of PS-AA dissolved in <NUM> wt% GBL was then coated on the magnetic raft by repeating the fabrication procedure for making the first microraft layer. Following evaporation of solvent a uniform layer of PS-AA is coated on the magnetic raft. The polymer remains isolated within the PDMS wells and the microrafts retain smooth side walls as confirmed by transmitted light microscopy and SEM (<FIG>). Addition of a second layer did not cause any noticeable light scatter when imaged by transmitted light and fluorescence microscopy. TEM images of vertical sections through the two layer microrafts show the central thickness of the poly(styrene-co-acrylic acid) layer to be <NUM> with a concave curvature of <NUM>° (<FIG>). While the viscosities of the polymers used for the first and second layers are the same the second layer is much thinner due to less total polymer filling the PDMS microwells which have been previously filled with a magnetic polymer. Thicknesses of the microraft layers can be adjusted by controlling the volume of polymer within the GBL during dip coating. Addition of poly(styrene-co-acrylic acid) dissolved in <NUM> wt% GBL gives a second layer thickness of <NUM> with a concave curvature of <NUM>° (<FIG>).

Successes in two layer microraft fabrication demonstrate the capabilities of developing microrafts exhibiting multiple properties. To expand upon the fabrication capabilities; microrafts developed with four successive dip coating steps of different polymers were prepared. 1002F, 1002F containing <NUM>% Bodipy FL, 1002F containing <NUM>% maghemite nanoparticles and 1002F containing <NUM>% Rhodamine B, each dissolved in <NUM> wt% GBL, were each sequentially dip-coated onto a PDMS mold consisting of arrays of <NUM> × <NUM> squares isolated by <NUM> tall <NUM> wide PDMS walls (<FIG>). The polymer remained isolated within the PDMS walls and optical transparency was retained for these microrafts <FIG>). A cross-section of the microrafts imaged by light microscopy shows that the surface has a much less concave surface geometry than single or two layer microrafts with a concave curvature of only <NUM>°. Microrafts were also imaged by confocal fluorescence microscopy to analyze the segregations of each successive layer. A GFP filter set shows Bodipy FL fluorescence isolated at the second layer of the microraft and the mCherry filter set shows a very thin Rhodamine B fluorescent layer at the top of the microraft.

Cell culture on magnetic rafts. For magnetic microrafts to be proper platforms for sorting individual cells and cell colonies they should be capable of providing both good cellular adhesion and long term growth on the substrate. PS-AA, 1002F and magnetic 1002F have all been shown previously to be biologically compatible substrates (see, e.g., <NPL>);<NPL>); <NUM><NPL>)). These substrates along with the recently developed magnetic PS-AA have all been shown to be good substrates for modifying with extracellular matrices, such as fibronectin and collagen, which allow for quick attachment of cells (< 2hrs). Cells plated on microrafts coated with collagen adhere to the surface after an hour and begin to reach across the surface within <NUM> hours of plating as observed with transmitted light and SEM (not shown). Cells allowed to culture on these microrafts for <NUM> days will fill up the microraft and cross over the PDMS wall to adjacent microrafts. PS-AA and magnetic PS-AA have negative surface charges and allow for cellular adhesion without surface modification within <NUM> hrs of cell plating. Additionally, microrafts developed from these materials do not require plasma treatment or the addition of an extracellular matrix which also modifies the surface of the PDMS walls allowing for cell crossing to adjacent microrafts. Cell colonies grown on these surfaces remain isolated on the microraft surface and within the confines of the PDMS walls.

A layer of native polymer applied over magnetic micropallets was previously shown to provide a protecting layer to prevent nanoparticle uptake by cells (<NPL>)). Applying a thin layer of non-magnetic polymer over the magnetic microrafts would remove possible nanoparticle contamination within cells which could disrupt cellular functions important in sensitive biological assays. Furthermore, microrafts fabricated with numerous polymer layers have a much flatter geometry and surfaces flush with the PDMS side walls. These factors could make cells cultured on these microrafts more susceptible to crossing the PDMS gap to adjacent microrafts. Microrafts were developed with <NUM> successive dip-coating steps with 1002F to create a tall and flat microraft. Following plasma treatment and fibronectin coating, cells loaded on these microrafts showed good initial attachment, however, cells migrated between microrafts within <NUM> days of culture. Microrafts were also developed by <NUM> successive dip-coated steps using PS-AA. These microrafts also had flat tops which rose to the level of the PDMS wells. Cell colonies grown on these microrafts remained confined within the PDMS walls <NUM> days following plating.

Many biological assays rely on fluorescent markers to identify the cell lines of interest. The ability to perform sensitive fluorescence measurements on multi-layer magnetic rafts was demonstrated by examining cells loaded with fluorescence dyes with fluorescence and confocal microscopy. Cells stained with a nuclear dye (Hoechst <NUM>, excitation/ emission <NUM>/<NUM>) and a dye loaded in the cytoplasm (CellTracker Green, excitation/ emission <NUM>/<NUM>) were plated on the two-layer magnetic microrafts. The Hoechst <NUM> was clearly isolated within the nucleus of the cells and CellTracker Green exhibited good fluorescence with no background scatter or distortion caused by imaging through the microraft (not shown).

Release and collection of magnetic microrafts. Utility of magnetic microrafts relies upon the ability to selectively release and manipulate the microrafts with an external magnet. One example for collecting microrafts is schematically illustrated in <FIG>. Magnetic microrafts were prepared for release by attaching the microraft array to a polycarbonate chamber, as described previously (see, e.g., <NPL>)). The chamber was filled with DMEM supplemented with <NUM>% FBS and matted to a second polycarbonate cassette attached to a glass slide. Three methods were developed for releasing and magnetically collecting individual <NUM> × <NUM> square microrafts (<NUM> tall <NUM> wide PDMS walls) from the microraft array with a microneedle (<NUM> tip diameter). The efficiency of collection of these loose magnetic microstructures was then quantified by varying the magnetic field strength and the concentration of maghemite within the microrafts: data are given in Table <NUM> below.

Magnetism can provide a method for purifying magnetic microrafts from cell debris and other contamination that may fall down during the gravity based collection utilized during top-down release of microrafts. Microrafts were released and allowed to fall down to the initial collection plate with the same protocol as used previously (see, e.g., <NPL>)). As schematically illustrated in <FIG>, a permanent magnet was held under the collected microrafts as the microraft array was replaced with a glass slide attached to a polycarbonate cassette. The permanent magnet was then removed and placed over the collection glass. Gentle agitation of the glass holding the microrafts frees the magnetic microrafts and allows for magnetic collection against gravity onto the collection substrate if the magnetic force experienced by the microrafts is sufficient. Microrafts containing <NUM>% maghemite were collected with <NUM>% efficiency with magnet displacements up to <NUM> from the initial position, corresponding to a magnetic field of <NUM> mT. Increasing the height of the collection substrate to <NUM> (<NUM> mT) lowers the collection probability to <NUM> ± <NUM>. Decreasing the concentration of maghemite in the microrafts to <NUM>% results in collection efficiencies of <NUM> ± <NUM>, <NUM> ± <NUM> and <NUM> ± <NUM> with magnet separations of <NUM>, <NUM> and <NUM> (<NUM>, <NUM> and <NUM> mT), respectively. Furthermore, microrafts containing <NUM>% maghemite were collected with <NUM> ± <NUM> and <NUM> ± <NUM> % efficiencies at magnet separations of <NUM> and <NUM> (<NUM> and <NUM> mT), respectively. Likewise, <NUM>-layer microrafts composed of <NUM>% magnetic 1002F bottoms and PS-AA tops resulted in collection probabilities of <NUM>% at distances up to <NUM> (<NUM> mT) and <NUM> ± <NUM> % at <NUM> (<NUM> mT). This method shows the ability to obtain pure microrafts where an initial magnetic collection is not feasible.

Along with purifying collected microrafts, magnetism can be utilized to vertically collect magnetic microrafts immediately following release. Placing the microraft cassette in an upright orientation on an open microscope stage allows for access of the microneedle for release from the bottom-up. Two approaches were successfully applied to release the microrafts in this orientation. In the first method, the microneedle was attached to the XYZ micromanipulator with a U brace to position the needle beneath the microraft array. Bending the microneedle at a <NUM>° angle prior to attachment to the brace displaces the equipment from the objectives optical path and reduces light scatter by the equipment. This method allows for the integration of a motorized release system. As a low-cost alternative, the microraft needle was mounted onto a PDMS block which could be placed over the microscope objective of an inverted microscope. Raising the microscope objective provides the z-axis manipulation of the microneedle required to dislodge the microraft.

Placing the external magnet over the collection substrate allows for immediate collection following release of the selected microrafts (not shown). Microrafts containing <NUM>% maghemite were collected with <NUM>% efficiency at distances up to <NUM> (<NUM> mT) the maximum achievable collection plate separation with the current system. Microrafts with maghemite concentrations of <NUM>% were collected with <NUM> ± <NUM>, <NUM> ± <NUM> and <NUM> ± <NUM>% efficiencies at magnet separations of <NUM>, <NUM> and <NUM> (<NUM>, <NUM> and <NUM> mT), respectively. Microrafts with only <NUM>% maghemite exhibited a collection probability of <NUM> ± <NUM> % at a magnet separation of <NUM> (<NUM> mT). The addition of a PS-AA layer over the <NUM>% maghemite loaded microraft lowered the collection efficiency with a magnet displacement of <NUM> (<NUM> mT) to <NUM> ± <NUM>%. Slightly higher collection efficiencies were observed for the agitated microrafts with respect to the immediately collected microrafts. This could be a result of these agitated microrafts rising further up the collection plate prior to being caught in a high magnetic field. Releasing microrafts from the bottom has the advantage in that it allows for a one-step collection without requiring plate transfers which is a simpler method and lowers the stresses cells encounter during fluid exchanges.

Cell sorting and purification with magnetic microrafts. Utility of magnetic microrafts for bioanalytical applications was demonstrated by sorting single highly fluorescent HeLa cells from a heterogeneous population of GFP-HeLa cells exhibiting various degrees of fluorescence spiked with HeLa cells at a <NUM>:<NUM> ratio. In triplicate experiments, <NUM>,<NUM> cells were plated on an array of <NUM>,<NUM> two-layer microrafts (PS-AA top/ <NUM>% magnetic PS-AA bottom -<NUM> × <NUM> square <NUM> tall PDMS wells <NUM> gap) attached to a <NUM> tall polycarbonate cassette designed to fit a <NUM> round polystyrene petri dish bottom. Three hours following cell plating, <NUM> microrafts containing single cells exhibiting high fluorescence were released from the bottom and magnetically collected into individual petri dishes (not shown). Following microraft collection the chamber was transferred to a sterile environment where the petri dish could be removed from the microraft cassette and filled with fresh media and covered with the petri dish top. Keeping the magnet held under the petri dish during this process helps retain the microraft at the center of the petri dish during the separation and wash steps. Immediately following collection the petri dish was imaged for the presence of the collected microraft. All <NUM> microrafts were collected and retained their single cell following collection (not shown). Following a <NUM> day incubation period <NUM> ± <NUM>% of single cells grew into a colony (not shown).

Claim 1:
An apparatus for collecting or culturing cells or cell colonies, said apparatus comprising:
a common substrate formed from an elastomer and having a plurality of wells formed therein, wherein said wells are separated by walls, wherein the common substrate is an elastomeric substrate; and
a plurality of rigid cell carriers releasably connected to said common substrate, with said carriers arranged in the form of an array, and with each of said carriers received in one of said wells, wherein said carriers are configured to release from said substrate upon mechanical distortion of said substrate.