Patent Application
20140322806
The disclosure generally relates to a cell culture well article and methods of making and using the article
In embodiments, the disclosure provides a well article for culturing and assaying, for example, spheroidal cell masses. The well article includes at least one chamber having an opaque side wall; a transparent, round or concave bottom surface; and the transparent bottom is gas-permeable.
In embodiments, the disclosure provides methods for making the well article, and methods of using the well article in spheroid cell culture or in cellular assays.
In embodiments, the disclosure also provides a multi-well plate article having wells or chambers with opaque walls and a gas-permeable, transparent round-bottom, and methods for making the round-bottom multi-well plate article. The transparent round-bottom, such as an optically clear bottom window, permits convenient microscopic visualization or examination of the cultured spheroid cell mass.
In embodiments, the disclosure provides a well plate having a base, the base having a conical or tapered geometry, such as a forty five degree (45°) angle, or like taper angles, from the sidewall to a radius at the apex (i.e., the very bottom of the well) to provide a round-bottom well or multiwell plate having an opaque sidewall and an optically clear window or base for optical visualization, such as with a microscopic or like devices, of a spheroid cell culture.
In embodiments of the disclosure:
Various embodiments of the disclosure will be described in detail with reference to drawings, if any. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not limiting and merely set forth some of the many possible embodiments of the claimed invention.
In embodiments, the disclosed apparatus and the disclosed method of making and using the apparatus provide one or more advantageous features or aspects, including for example as discussed below. Features or aspects recited in any of the claims are generally applicable to all facets of the invention. Any recited single or multiple feature or aspect in any one claim can be combined or permuted with any other recited feature or aspect in any other claim or claims.
“Include,” “includes,” or like terms means encompassing but not limited to, that is, inclusive and not exclusive.
“About” modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, viscosities, and like values, and ranges thereof, or a dimension of a component, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example: through typical measuring and handling procedures used for preparing materials, compositions, composites, concentrates, component parts, articles of manufacture, or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term “about” also encompasses amounts that differ due to aging of a composition or formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture.
“Optional” or “optionally” means that the subsequently described event, circumstance, or structure, can or cannot occur, and that the description includes instances where the event, circumstance, or structure, occurs and instances where it does not.
“Consisting essentially of” or “consisting of” in embodiments can refer to, for example:
a spheroid cell culture article having:
a method of making the abovementioned spheroid cell culture article including:
a method of using the article for culturing spheroids including:
The article, the method of making the article, and the method of using the article, of the disclosure can include the components or steps listed in the claim, plus other components or steps that do not materially affect the basic and novel properties of the compositions, articles, apparatus, or methods of making and use of the disclosure, such as a particular article configuration, particular additives or ingredients, a particular agent, a particular structural material or component, a particular irradiation or temperature condition, or like structure, material, or process variable selected.
The indefinite article “a” or “an” and its corresponding definite article “the” as used herein means at least one, or one or more, unless specified otherwise.
Abbreviations, which are well known to one of ordinary skill in the art, may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” for gram(s), “mL” for milliliters, and “rt” for room temperature, “nm” for nanometers, and like abbreviations).
Specific and preferred values disclosed for components, ingredients, additives, dimensions, conditions, and like aspects, and ranges thereof, are for illustration only; they do not exclude other defined values or other values within defined ranges. The apparatus and methods of the disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values described herein, including explicit or implicit intermediate values and ranges.
The formation of uniformly-sized spheroids of, for example, tumor cells, can be facilitated if the cells are cultured in a round-bottom vessel that has low-adhesion properties. Researchers from the Institute of Cancer Research in Sutton, UK, used Corning, Inc.'s, ultra-low-adhesion (ULA)-coated 96-well clear, round-bottom, polystyrene microplates to produce tumor spheroids that enabled target validation and drug evaluation (see Vinci et al., BMC Biology, 2012, 10:29). The spheroids enhanced the biological relevance of the tumor cell cultures and facilitated a range of functional assays. The round-bottom well shape along with gravity and the ultra-low attachment coat or coating, encouraged the cells to come together and self-assemble into the spheroid shape rather than to form a monolayer of cells.
While the clear, round-bottom, ULA-coated wells of the 96-well plate were utilized with good results for migration and invasion assays due to the optical clarity of the structural polymer, evaluation of anti-cancer agents using fluorescent or luminescent detection in this plate were less optimal since the clear sidewalls added to the noise in the detected signal. While the tumor cells could be moved into opaque 96-well plates, this calls for pipetting the spheroids, which can be delicate, difficult, and time-consuming.
In embodiments, the present disclosure provides a spheroid cell culture article comprising:
The disclosed well plate having a well base having a conical or tapered geometry is superior for injection molding of the well's opaque side wall since it is unnecessary to alter existing flat pins that have been previously used to over-mold on a flat insert (i.e., base). Injection molding with existing flat pins can also be accomplished on a well-bottom geometry having a full radius (i.e., a hemispherical or untapered base). An optional distortion collar feature on the base can enhance the ability of an existing flat pin to achieve shut-off on the well-bottom geometry.
A well plate having the disclosed tapered or chevron shaped (e.g., a 45 degree angle from the sidewall to a radiused apex) well-bottom geometry has been successfully demonstrated for use in the formation of cell spheroids. The tapered well-base or well-bottom geometry also enables over-molding of the opaque well wall onto an insert (base) pre-formed with the tapered geometry using existing flat pins that would ordinarily be used to over-mold on a flat insert. The optional distortion collar feature integral with or attached to the insert (base) can facilitate resin flow shut-off of the injection molding pin. The well-bottom geometry can alternatively be generated by thermal-forming a polymer film prior to over-molding, or after over-molding using a thermal reforming process. Thermal reforming permits rapid change-over to generate well geometries for alternative multiple formats.
The disclosed well plate having a tapered or chevron shaped base (e.g., an approximate 45 degree angle) is advantaged by, for example, permitting over-molding onto the insert using the same pin equipment to over-mold on a flat insert. This over-molding can be facilitated by the addition of a distortion collar that can flex to permit the pin to shut-off the polymer flow. Using the flat pin for molding results in saving time and reducing cost for the generation of this well plate product since the mold-cavity pins do not need to be modified for different well geometries or different well count formats.
Thermal reforming permits rapid change-over to generate well geometries for many different well formats. Thermal reforming also permits design flexibility for assessment of different well geometries for each format and the ability to rapidly produce prototype or production samples for customer evaluation or specification.
A well base having an approximate 45 degree tapered angle also allows for a smaller radiused apex, which apex centers the collection of cells that settle by gravity to form a cell spheroid, and enhances optical imaging.
Referring to the Figures,
Well plates having opaque sidewalls and gas permeable, rounded-bottom wells having clear windows can provide significant advantages including, for example: no need to transfer the spheroid from one multiwell plate (in which spheroids can be formed and visualized) to another plate for conducting assays to, for example, evaluate drug compounds; avoiding the spheroid transfer step can save time and avoid potential loss or disruption of the spheroid; and the spheroids can receive superior oxygenation with well-bottoms made from a polymer having gas permeable properties at a given wall thickness. Increased oxygen availability to the cells in the spheroid culture is particularly helpful for cells with high oxygen requirements such as hepatocytes.
In embodiments, the article having a chamber having at least one concave arcuate surface can include, for example, a plurality of adjacent concave arcuate surfaces having, for example, from 1 to about 1,000 concave arcuate surfaces on the bottom of the same chamber, see for example,
In embodiments, the article can be, for example, a single well or multi-well plate configuration having numerous “spheroidal wells”, such as a plurality of dimples or pits in the bottom or base of each well. The plurality of spheroids or spheroid wells per chamber can preferably accommodate, for example, a single or one spheroid per spheroid well.
In embodiments, the gas permeable, transparent well bottom having the at least one concave arcuate surface or “cup” can be, for example, a hemi-spherical surface, a conical surface having a rounded bottom, and like surface geometries, or a combination thereof. The well and well bottom ultimately terminates, ends, or bottoms-out in a spheroid “friendly” rounded or curved surface, such as a dimple, a pit, and like concave frusto-conicial relief surfaces, or combinations thereof.
In embodiments, the opaque side wall surface (i.e., a surround) can be, for example, a vertical cylinder or shaft, a portion of a vertical conic of decreasing diameter from the chamber top to the chamber bottom, a vertical square shaft or vertical oval shaft having a conical transition, i.e., a square or oval at the top of the well, transitioning to a conic, and ending with a bottom having at least one concave arcuate surface, i.e., rounded or curved, or a combination thereof. Other illustrative geometric examples include holey cylinders, holey conic cylinders, first cylinders then conics, and other like geometries, or combinations thereof.
In embodiments, the article can further comprise, for example, a low-adhesion or no-adhesion coating on a portion of the chamber, such as on the at least one concave arcuate surface.
In embodiments, the article can further comprise, for example, a chamber annex, chamber extension area, or an auxiliary side chamber, for receiving a pipette tip for aspiration, the chamber annex or chamber extension (e.g., a side pocket) can be, for example, an integral surface adjacent to and in fluid communication with the chamber. The chamber annex can have a second bottom spaced away from the gas-permeable, transparent bottom. The chamber annex and the second bottom of the chamber annex can be, for example spaced away from the gas-permeable, transparent bottom such as at a higher elevation or relative altitude. The second bottom of the chamber annex deflects fluid dispensed from a pipette away from the transparent bottom to avoid disrupting or disturbing the spheroid.
In embodiments, the article can further comprise a porous membrane, such as a liner or membrane insert, situated within a portion of the chamber, situated within a portion of the chamber annex, or both the chamber and the chamber annex portion. The porous membrane can provide isolation or separation of a second cellular material, such as a different cell type or different cell state, situated in an upper portion of the chamber, in an upper portion of the chamber formed by the porous membrane, or both chambers, from first cellular material in a lower portion of one or both chambers near the transparent bottom.
In embodiments, the at least one concave arcuate surface can be, for example, a hemisphere, or a portion of a hemisphere, such as a horizontal section or slice of a hemisphere, having a diameter of, for example, from about 250 to about 5,000 microns (i.e., 0.010 to 0.200 inch), including intermediate values and ranges, depending on, for example, the well geometry selected, the number of concave arcuate surfaces within each well, the number of wells in a plate, and like considerations. Other concave arcuate surface can have, for example, parabolic, hyperbolic, chevron, and like cross-section geometries, or combinations thereof.
In embodiments, the spheroid can be, for example, substantially a sphere, having a diameter of, for example, from about 100 to about 500 microns, more preferably from about 150 to about 400 microns, even more preferably from about 150 to about 300 microns, and most preferably from about 200 to about 250 microns, including intermediate values and ranges, depending on, for example, the types of cells in the spheroid. Spheroid diameters can be, for example, from about 200 to about 400 microns, and the upper diameters being constrained by diffusion considerations (for a review of spheroids and spheroid vessels see Achilli, T-M, et. al. Expert Opin. Biol. Ther. (2012) 12(10)).
In embodiments, the disclosure provides a perfusion plate apparatus comprising:
at least one cell culture article comprising:
the at least a portion of the bottom comprises at least one concave arcuate surface, that is, a rounded or curved surface,
a media source well in fluid communication with at least one chamber of the cell culture article that controllably provides a source of fresh media to at least one chamber; and
a waste well in fluid communication with the at least one chamber of the cell culture article that controllably receives waste media from cell metabolism in the at least one chamber.
In embodiments, the aforementioned perfusion plate apparatus can further comprise, for example: a source liquid (not shown), a perfusion plug (655), i.e., liquid permeable material, situated in a liquid connection member (635), such as a tube, between a source well (630) and the cell culture rounded-bottom well of the apparatus (600), a perfusion plug (655) situated in a liquid connection member (645) between the cell culture rounded-bottom well and the waste well (640), or both situations. The waste well (640) preferably can have an arcuate bottom surface to facilitate removal of waste liquid medium (not shown) using, for example, a pipette, a vacuum pump, or like liquid removal equipment.
A related but distinct cell culture article (i.e., FloWell™ plate) is disclosed in commonly owned and assigned copending application, U.S. Provisional Patent Application Ser. No. 61/594,039, filed on Feb. 2, 2012, now PCT/US13/24030, to Goral, et al., entitled “Automatic continuous perfusion cell culture microplate consumables”. The related application mentions, for example, a microplate for culturing cells with automatic, continuous perfusion of a liquid medium, and can include, for example, a well frame which defines a plurality of cavities therethrough. The microplate can further include a planar substrate connected with the well frame. The planar substrate can provide a bottom surface to the plurality of cavities, forming a plurality of wells. The plurality of wells may include a first well, a second well fluidly connected with the first well, and a third well fluidly connected with the first well. The first well may be employed for culturing the cells in the liquid medium. The second well may be employed for providing an outflow or source of the liquid medium to the first well. The third well may be employed for receiving an inflow or waste stream of the liquid medium from the first well. The second well may be fluidly connected with the first well with a first perfusion membrane. A first perfusion membrane can be disposed in between the well frame and the planar substrate and may extend from an outlet section of the second well to an inlet section of the first well. The third well may be fluidly connected with the first well with a second perfusion membrane. The first and second perfusion membranes can have a porosity of, for example, from about 0.2 to about 200 microns. The second perfusion membrane can be disposed in between the well frame and the planar substrate and extend from an outlet section of the first well to an inlet section of the third well. Upon introduction of a perfusion-initiating amount of the liquid medium into the second well, the liquid medium flows from the second well through the first perfusion membrane to the first well and from the first well through the second perfusion membrane to the third well (see for example
In embodiments, the article having a chamber can include, for example, from 1 to about 2,000 chambers, from 10 to about 1,500 chambers, including intermediate values and ranges, and each chamber is physically separated from any other chamber, and each chamber has a single concave arcuate surface. In embodiments, such a multi-well plate product configuration can have, for example, one spheroid per chamber.
In embodiments, the at least one concave arcuate surface can have, for example, a plurality of adjacent concave arcuate bottom surfaces within the same well. In embodiments, a single well or multi-well plate configuration can have numerous spheroidal wells, such as dimples or pits in the bottom or base of each well. In embodiments, the article can accommodate a plurality of isolated spheroids in separated spheroid wells in a single chamber, preferably having, for example, one spheroid per spheroid well.
In embodiments, the disclosure provides a method of making an article, the article comprising:
a frame having a chamber, for example, a well, comprising:
the method of making comprising:
combining the gas-permeable, transparent arcuate bottom surface portion and an opaque side wall surface portion to form the article.
The method of making the article can further comprise, for example, inserting a porous membrane in at least one of the chambers.
There are several methods that can be used to make an opaque round-bottom multiwell plate having opaque sidewalls and a base with a clear window. The multiwell plate made by Examples 1 or 2, and having flat well bottom geometry can be subsequently thermally-reformed to create an alternative well-bottom geometry. A multiwell plate having a flat well-bottom geometry can be, for example, positioned in a nest that has at each well-bottom position areas removed from the nest that have a circular perimeter in roughly the same dimensions as the well format, or the nest can have a tapered angle, for example, from about 30 to about 60 degrees, including intermediate values and ranges, such as 40 to 50, and 45 degrees, turning into a radius at the apex, that is, rounded at the very bottom of the well. A heated platen, heated for example at 180 to 400 degrees F., more preferably 250 to 300 degrees F., having an array of pins that have, for example, a matching 45 degree tapered angle from the sidewall sloping into a radius at the apex, is held stationary, with a pin positioned over each well. The pin-platen is actuated so that the pins descend into the wells of the multiwell plate to contact the clear flat well-bottom surface. Time, temperature, and pressure can change the well-bottom material geometry as the pins advance until the reformed well-bottom material meets the matching nest geometry. The array of pins may be coated with a release agent, such as PTFE, to prevent the reformed well-bottom geometry from sticking to the heated pins, or the pins can be uncoated. The nest may also have a vacuum assist on the nest to help draw the polymer into the well geometry. Cooling air can be injected into the top of the wells above the heated pins to cool the hot pins slightly and facilitate their release from the polymer of the reformed well-bottom geometry. The platen with the pin array and the nest with the matching geometry can be rapidly changed to comply with many multiple well formats, including 384- and 1536-wells. Any number of wells may be reformed at the same time. In embodiments, the pin-platen can be held stationary while the nest holding the insert can be actuated to be brought into contact with the stationary pin-platen. In embodiments, both the pin-platen and the nest holding the insert can be actuated to provide relative motion that achieves contact and closure between the pins and the wells of the insert.
A v-shaped well base geometry having, for example, a tapered angle of 45 degrees, sloping from the well sidewall to a radius at the apex, is superior for spheroid formation and superior for optical measurement compared to a hemispherical well-bottom geometry. The v-shaped well base geometry also permits overmolding on the well base geometry using the same flat pins that can be used to over-mold opaque polymer onto a flat insert. The 45 degree well base tapered angle permits the pins to contact each well base on the bottom tapered sidewall. While this can also be done with a hemispherical geometry, it is more difficult. If the pin is not able to shut-off on the sidewall, the opaque polymer can leak into or onto the clear well bottom. Alternatively, if the pin cannot achieve shut-off on the sidewall, the pin can be made larger than the well, and achieve shut-off on the flat area surrounding each well at the top of the insert. This alternative solution is not desired because it can create a flat ledge within each well upon which cells can settle. Cells that settle on the ledge cannot contribute to the spheroid in the center of the well bottom, but can cause large variation in experimental results.
In embodiments, the post-forming equipment can have, for example, a heated pin, such as a single pin, a row of pins, or an array of pins, that can match the number and alignment of, for example, a single well or a plurality of wells in a row or an array of wells of an injection molded microwell plate. The geometry at the end of each pin can have, for example, a 45 degree angle or taper from the sidewall and have a radius at or near the apex.
In embodiments, an injection molded plate having a flat well-bottom geometry can be situated and secured in a nest for the post-forming step where the heated pin or pin array assembly is pressed, for example, into the flat well-bottoms of an injection molded microplate. The nest can have a matching 45 degree angle at each well position to match and receive the pin shape. In embodiments, a platen can hold the heated pin or pins and the nest holds the matching well-bottom geometry. The plane of the pin platen and the plane of the nest can be aligned in parallel and both can be perpendicular to a common axis for proper reversible and reproducible pressing.
The platen, the nest, or both, can be rapidly interchanged to accommodate other multiwell plate formats such as 384- and 1536-well plates. The platen bearing the heated pin(s) can be pressed into a matching well(s) of the multiwell plate to form the selected geometry defined by the geometry or cross-section of the pin's tip, such as hemispherical, conical, v-shaped, w-shaped, wedged shaped, and like geometries, or combined geometries, in the clear base of the well.
In embodiments, the disclosure provides a method of culturing spheroids comprising, for example:
charging any of the aforementioned articles with culture media; and
adding spheroid forming cells to the culture media.
There are several methods that can be used to make the disclosed article or articles. In embodiments, the disclosed article can be, for example, a multiwell plate having an opaque side wall for the wells and having an arcuate or concave rounded base or bottom surface, the base or bottom surface can be gas permeable and transparent or clear to visible light.
Commonly owned and assigned U.S. Pat. No. 7,674,346, mentions methods of making well plates such as bonding a polymeric holey plate to a glass or polymeric base and using, for example, heat or radiation to weld a gas permeable thin film to a holey plate to form the bottom of microplates (see e.g., col 2, lines 4 to 44).
Commonly owned and assigned U.S. Pat. No. 5,759,494, mentions methods of making well plates having opaque sidewalls by, for example, over-molding inserts to prevent cross-talk. Polymethylpentene (TPX) of the presently disclosed gas permeable spheroid plate can be selected as a suitable base or wall material that can be used in methods of making in the '494 patent and the present disclosure.
In embodiments, the structure of the presently disclosed gas permeable spheroid plate can also include one or more ridges arranged in one or more grid patterns as disclosed in the '494 patent, see for example, col. 4, line 30 to col. 5, line 54, to further reduce or prevent cross-talk. In over-molded plates without a grid, the perimeter of the well, a film, tended to peel away from the molded plate body. The grid appears to keep the film from peeling away from the plate body as it is reforming. The grid also appears to prevent stress marks from appearing in the film, since stress marks do appear in the plates without the grid.
U.S. Pat. No. 6,811,752, mentions methods of making a device having micro-chambers and microfluidic flow, and gas permeable, liquid impermeable membranes.
In one method (insert molding), the multiwell plate bottom or base portion having at least one rounded, cupped, or dimpled impression in the base is injection molded, or thermoformed using a clear polymer. Next, the molded or thermoformed clear multiwell bottom plate is inserted into a vertical press, then an opaque polymer is injected forming the remainder of the multiwell plate onto the clear, round well bottoms. An alternative to insert molding uses a 2-shot molding process.
Referring again to the Figures,
The following examples serve to more fully describe the manner of using the above-described disclosure, and to further set forth best modes contemplated for carrying out various aspects of the disclosure. These examples do not limit the scope of this disclosure, but rather are presented for illustrative purposes. The working example(s) further describe(s) how to prepare the disclosed cell culture well articles.
Injection Mold or Thermoform Methods
In another method of making, one can mold or thermoform an opaque multiwell plate that has no well-bottoms (i.e., make an opaque “holey plate”). Next, one can injection mold, or thermoform the multiwell plate bottom or base portion using a clear polymer. Then one can attach the holey plate and base components together using any of various assembly processes, including, for example, adhesive, ultrasonic, IR, thermal, laser welding, and like processes.
In another method of making, one can injection mold, or thermoform the entire well of a multiwell plate (without a skirt) using a clear polymer. Next, one can injection mold, or thermoform another multiwell plate (including the skirt) but without well bottoms using an opaque polymer. The well diameters of the opaque plate are molded to be slightly larger than the well diameters of the clear plate. The opaque plate acts as a sleeve to receive the clear plate and thus form opaque sidewalls and with a clear window at the base of the well.
In another method of making, one can distortion print an opaque coating on the exterior of a clear polymer sheet, leaving a clear zone in the area that will be thermoformed with, for example, a cup or dimple, or multiples thereof, into the center of the well bottom. Once thermoformed into a multiwell plate, all of the plate will appear opaque except for the clear zone that forms a window for optical viewing.
Non-adhesive coating The interior well-bottom surface, walls, or both can optionally be made non-adhesive to cells by coating those surfaces with a polymer that does not have any characteristics that promote cell attachment, such as a perfluorinated polymer, olefin, or like polymers, and mixtures thereof. Alternatively, following assembly of the opaque multiwell plate with clear, round or cupped well-bottoms, the interior well-bottom may be coated with a non-binding material such as an ultra-low attachment material, agarose, nonionic hydrogel, or like materials, that can inhibit cell attachment. Coatings can also be applied to the clear well bottom portion of the multiwell plate prior to joining with the opaque portion. The combination of, for example, a low-attachment substrate, the well curvature in the body and the base portions, and gravity, can induce cells to self-assemble into spheroids, which cell clusters are known to maintain differentiated cell function indicative of a more in-vivo like response (see Messner, et al., Archives of Toxicology, Nov. 11, 2012). Perfluorinated polymers or polymers such as poly 4-methylpentene also provide gas permeability at thicknesses normally used in molding processes, which gas permeability can be beneficial for metabolically active cell types. Representative thickness and ranges of gas permeable polymer can be, for example, from about 0.001 inch to about 0.025 inch, from 0.0015 inch to about 0.03 inch, including intermediate values and ranges (where 1 inch=25,400 microns; 0.000039 inches=1 micron). Additionally or alternatively, other materials having high gas permeabilities, such as polydimethylsiloxane polymers, can provide sufficient gas diffusion at a thickness, for example, of up to about 1 inch.
Post Forming Method Another method of making a well plate article of the disclosure is accomplished by thermal reforming. In the thermal reforming method a commercially available plate, for example, having standard black (opaque) side-walls and a clear flat plastic bottom is selected. Using a combination of vacuum on the outside and pressure supplied by a hot forming tool with a full radius pushed into the interior of the well(s) on the opposite side of the plate and further into the bottom of the well, the concave arcuate base or tapered base having a round bottom radius is formed in the well bottom. The outside bottom area can be, for example, preheated with IR radiation to facilitate forming the cupped or internal concave arcuate bottom radius, or the tapered base.
Method of Use; Culturing of Cells
Image 11A shows a spheroid in an all clear control 96-well plate (i.e., the walls of this comparative plate are transparent) having a full hemisphere well-bottom geometry.
Image 11B shows a spheroid in a prototype thermally reformed 96-well plate having a v-shaped or 45 degree tapered well-bottom geometry.
Image 11C shows a spheroid in an injection-molded single well with the v-shaped or 45 degree well-bottom geometry.
Image 11D shows a spheroid in an injection-molded single well having a full hemisphere well-bottom geometry.
HT-29 cells were seeded into wells including the comparative well (11A) and inventive v-shaped wells (11B and 11C) and hemisphere shaped well (11D) at a concentration of 10,000 cells per well, and incubated for 96 hours at 37 degrees C. in an incubator with 5% CO2 and 85% humidity. The
The cell seeding procedure generally included the following steps: trypsinize; count the cells; centrifuge to remove medium from the cells; and re-suspend the cells. Seeding densities can be, for example: 10 k, 20 k or 30 k cells per well. The seeding volume can be, for example, 200 microliters per well. Monitoring images can be recorded, for example, at 30 minutes, at 24 hrs, and at 96 hrs after seeding, including intermediate values and ranges.
The spheroids in images 11B and 11C are in the center of the v-shaped well base. The spheroids in images 11A and 11D are not in the center of the hemisphere. The v-shaped base geometry appears to promote centering of the spheroids in the center of the well base, which centering facilitates optical visualization.
In embodiments, one can form a gas permeable, rounded well-bottom using the thermal reforming process. In existing methods, the plates can be molded on flat inserts of, for example, polystyrene that is 0.005″, which thickness is considered not gas permeable. However, during the thermal reforming process, one can decrease the thickness of the insert at the radiused apex to, for example, about 0.003″ (photograph not included), which makes the well-bottom as gas permeable as the HYPER-product film (which is also 0.003″ thick polystyrene).
The disclosure has been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications are possible while remaining within the scope of the disclosure.
This application claims the benefit of priority to U.S. Provisional Application No. 61/817,539 filed on Apr. 30, 2013, the content of which is relied upon and incorporated herein by reference in its entity. The entire disclosure of any publication or patent document mentioned herein is incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61817539 | Apr 2013 | US |
