News Column

"Hanging Drop Devices, Systems And/Or Methods" in Patent Application Approval Process

July 15, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventors Takayama, Shuichi (Ann Arbor, MI); Tung, Yi-Chung (Ann Arbor, MI); Hsiao, Amy Yu-Ching (Ann Arbor, MI); Jan, Edward (Ann Arbor, MI), filed on February 25, 2014, was made available online on July 3, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also 3d Biomatrix, Inc.).

This patent application is assigned to 3d Biomatrix, Inc.

The following quote was obtained by the news editors from the background information supplied by the inventors: "In vitro cellular and tissue models for various drug testing and screening experiments are often central to the development of novel therapeutics in the pharmaceutical industry. Currently, however, most in vitro studies are still performed under conventional two-dimensional (2D) cell culture systems, which are often not physiological models for functional tissues and tumors. Therefore, drug studies involving such models may not produce accurate readouts. To obtain more meaningful results, in vivo studies involving animals are often utilized. However, one obvious drawback of in vivo studies is the time-consuming and expensive nature of these experiments. To bridge this gap between the non-physiological conventional 2D models and in vivo experiments, three-dimensional (3D) in vitro models that provide more therapeutically predictive and physiologically relevant results for drug testing and screening in the pharmaceutical industry are needed. One way to create 3D cell culture models is through the formation of spheroids, or 3D clusters or aggregates of cells.

"Scaling up of spheroid culture in a manner suitable for certain applications such as high-throughput screening and testing has several drawbacks. Traditional spheroid formation involves cultivation of suspended cells in hanging drops on the underside of a Petri dish lid. This process requires inverting of the lid following placement of the drops. As a result, the drops are susceptible to perturbation, resulting in falling, spreading, and merging with neighboring drops. Although inexpensive, this method is labor-intensive, does not permit efficient scalable production, and is not compatible with automated instruments for high-throughput screening. Because it is difficult to perform media exchange without damaging the spheroids, this method usually requires another labor-intensive step of transferring the spheroids manually, one by one, to a multi-well culture plate for longer-term culture, treatment, analysis, and harvest.

"An alternative is to induce the formation of spheroids under continuous agitation of cell suspension in bioreactors, such as spinner flasks and rotary culture vessels. This method requires the consumption of large quantities of culture media. It also requires specialized equipment and the size and uniformity of the spheroids are hard to control. The high variability in spheroids prohibits their use in many applications.

"Methods are also available to produce spheroids using 3D microwell structures and planar micropatterns. However, these methods require specialized and expensive equipment for generating the microwell structures and micropatterns. Moreover, since a plurality of spheroids is cultured within one fluid compartment, the spheroids cannot be individually monitored, manipulated, and treated with testing compounds. The difficulty of performing analysis on individual spheroids before and after treatment also makes these methods unsuitable for certain applications, for example, drug testing and screening applications.

"Other recent advances include microfluidic devices designed to generate and manipulate spheroids. However, these devices are expensive to design and produce. In addition, these devices are not suitable for long-term culture of spheroids, not chemically compatible with certain drugs, and not compatible with automated instruments for performing high-throughput screening.

"To address problems in the art, there is a need for the devices, methods and/or systems disclosed herein."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "The present disclosure relates generally to devices, systems, and methods of using such devices in creating and handling hanging drops of fluid. The present disclosure also relates generally to cell culture devices, systems and methods of using such devices. The present disclosure also relates generally to the use of cell culture devices for research and high throughput screening.

"For example, in some embodiments, the disclosure provides a system, comprising: a) at least one array plate, the at least one array plate comprising a top surface and a bottom surface and a plurality of holes therein, wherein each of the plurality of holes comprises a top and a bottom and wherein the bottom surface of said array plate comprises a at least one plateau substantially adjacent to the bottom of at least one of the plurality of holes; and b) wherein the at least one array plate is configured to accommodate a plurality of hanging drops, wherein each drop hangs from a corresponding one of the plurality of said holes and extends beneath the hole, wherein the number of hanging drops the that at least one array plate can accommodate is equal to or less than the number of holes in the at least one array plate. In certain embodiments, the system further comprises at least one second plate positioned below said at least one array plate. In certain embodiments, the at least one array plate further comprises at least one reservoir. In certain embodiments, one or more of the plurality of hanging drops contains one or more of the following: a plurality of cells; at least one complex tissue or organisms; an aqueous fluid containing biological and/or chemical entities; one or more proteins; one or more nanoparticles, one or more test compounds; one or more drugs; solid or gel formed by aqueous liquid; or combinations thereof. In certain embodiments, the at least one array plate, the at least one second plate, and/or the at least one lid is treated in order to modify to properties of the corresponding treated surface. In certain embodiments, the system may comply with American National Standards Institute and/or Society for Biomolecular Sciences standards. In certain embodiments the system is compatible with high-throughput screening. In certain embodiments, the at least one plateau on the bottom surface of the at least one array plate is configured to stabilize a geometry of said plurality of hanging drops. In certain embodiments, the at least on plateau on the bottom surface of the at least one array plate is configured to stabilize a position of said plurality of hanging drops. In certain embodiments, the at least one array plate is configured to stabilize and maintain measurable properties of said plurality of hanging drops. In certain embodiments, the at least one array plate further comprises at least one plateau on the top surface substantially adjacent to the top of at least one of the plurality of holes, wherein said at least one plateau on the top surface of said at least one array plate is configured to improve a transfer of liquids in and/or out of the holes. In certain embodiments, the system is configured to maintain a substantially stable humidity. In certain embodiments, the system is configured to maintain measurable properties of the environment of the plurality of hanging drops. In certain embodiments, the system is configured to handle small volumes of fluid. In certain embodiments, the system is configured to permit long term culturing of a plurality of cells within the one or more plurality of hanging drops. In certain embodiments, the system is configured to permit one or more of the following: long terms culturing, maintaining, analysis and/or testing of a plurality of cells; long term culturing, maintaining, analysis and/or testing of at least one complex tissue or organisms; long term culturing, maintaining, analysis and/or testing of an aqueous fluid containing biological and/or chemical entities; long term culturing, maintaining, analysis and/or testing of one or more proteins; long term culturing, maintaining, testing and or analysis of one or more nanoparticles; long term culturing, maintaining, analysis and/or testing of one or more test compounds; long term culturing, maintaining, analysis and or testing of one or more drugs; or combinations thereof.

"For example, certain embodiments are directed to method(s), comprising: inserting a plurality of hanging drops into a system, comprising: a) at least one array plate, the at least one array plate comprising a top surface and a bottom surface and a plurality of holes therein, wherein each of the plurality of holes comprises a top and a bottom and wherein the bottom surface of said array plate comprises a at least one plateau substantially adjacent to the bottom of at least one of the plurality of holes; and b) wherein the at least one array plate is configured to accommodate a plurality of hanging drops, wherein each drop hangs from a corresponding one of the plurality of said holes and extends beneath the hole, wherein the number of hanging drops the that at least one array plate can accommodate is equal to or less than the number of holes in the at least one array plate; and performing on one or more of the hanging drops culturing, maintaining, analysis, testing, or combinations thereof.

"For example, certain embodiments are directed to device(s), comprising: an array plate, comprising a top surface and a bottom surface, wherein the array plate comprises a plurality of holes therein, wherein each hole comprises a top surface and a bottom surface and wherein the bottom surface of said array plate comprises at least one plateau either adjacent, or substantially adjacent, to the bottom surface of one or more of said holes.

"For example, certain embodiments provide a device, comprising: a) one or more array plates comprising a top surface and a bottom surface, wherein each of the array plates comprises a plurality of rows and columns of holes therein, wherein each hole comprises a top surface and a bottom surface and wherein the bottom surface of the array plate comprises a plateau adjacent to the bottom surface of each of the holes; and b) a reservoir plate (e.g., a 96 well plate) located below the array plate, wherein the reservoir plate contacts the edges of the array plate (e.g., only the edges), and wherein the reservoir plate does not contact the holes. In some embodiments, the device further comprises a cover for the device, wherein the cover is placed on top of the array plate and wherein the cover does not contact the holes. In some embodiments, the reservoir comprises an aqueous liquid. In some embodiments, the device is fabricated from a polymeric plastic (e.g., polystyrene). In some embodiments, the array plate comprises 384 holes. In some embodiments, the holes are approximately 1.6 mm in diameter. In some embodiments, the holes are approximately 4.5 mm apart. In some embodiments, the device further comprises additional plateaus, or ring structures, adjacent, or substantially adjacent, to the top and/or bottom of at least one side (e.g., both sides) of the holes. In some embodiments, the edge of the plateau comprises a ring structure (e.g., to stabilize droplets). In some embodiments, surface treatment (e.g. coatings, plasma treatment, etc.) is performed on one or more elements of the devices.

"Certain embodiments provide a system, comprising: a) one or more array plates comprising a top surface and a bottom surface, wherein each of the array plates comprises a plurality of rows and columns of holes therein, wherein each hole comprises a top surface and a bottom surface and wherein the bottom surface of the array plate comprises a plateau substantially adjacent to the bottom surface of each of the holes; b) a reservoir plate located below the array plate, wherein the reservoir plate contacts the edges of the array plate (e.g., only the edges), and wherein the reservoir plate does not contact the holes; and c) a plurality of hanging drops of fluid, wherein the drops hang from one or more of the holes and extend beneath the hole. In some embodiments, the hanging drops contain a plurality of cells. In some embodiments, the cells remain in suspension. In other embodiments, the cells form aggregates or clusters or spheroids. In some embodiments, the cells are complex tissues or organisms, for example, embryos, tissues, small organisms, worms, etc. In other embodiments, the hanging drops are aqueous fluids containing biological and/or chemical entities or combinations thereof. Examples of the said entities include proteins, nanoparticles, and hydrogels. In some embodiments, the cells are cancer cells (e.g., growing in a spheroid). In some embodiments, the system further comprises a test compound (e.g., an anti-cancer drug). In some embodiments, the system further comprises a lid, wherein the lid covers the array plate but does not contact the cells. In some embodiments, the array plate, reservoir and cover are wrapped with a film that prevents, or inhibits, moisture loss. In some embodiments, the system further comprises one or more high throughput sample handling devices (e.g., robotic sample handling devices or plate readers).

"The present disclosure additionally provides methods, comprising: a) inserting a plurality of hanging drops of fluid into a device comprising i) one or more array plates comprising a top surface and a bottom surface, wherein each of the array plates comprises a plurality of rows and columns of holes therein, wherein each hole comprises a top surface and a bottom surface and wherein the bottom surface of the array plate comprises a plateau substantially adjacent to the bottom surface of each of the holes; and ii) a reservoir plate located below the array plate, wherein the reservoir plate contacts the edges of the array plate (e.g., only the edges), and wherein the reservoir plate does not contact the holes, wherein the drops hang one or more of the holes and extend beneath the hole of the array plate; and b) culturing cells in the hanging drops under conditions such that the cells grow and/or maintain viability In some embodiments, the cells are cancer cells, embryonic stem cells, hepatocytes, etc. (e.g., growing in a spheroid). In some embodiments, the method further comprises the step of contacting the cells with a test compound (e.g., a drug, chemical, vapor, biomolecule or nanoparticle) and assaying the effect of the test compound on the growth or other properties of the cells. In some embodiments, hanging drops are placed from the top or bottom of the array plate through the hole or at one opening of the hole. In some embodiments, the method further comprises the step of adding additional liquid and/or cells to the hanging drops by dispensing the liquid into the hole or at one opening of the hole. In some embodiments, the method further comprises the step of removing the liquid and/or cells through the holes. In some embodiments, different portions of the array plate (e.g., different hanging drops or populations of cells) are exposed to different test compounds and/or growth conditions.

"Certain embodiments are direct to a system, comprising an array plate, a lid, and a tray, wherein the array plate comprises a top surface and a bottom surface, a reservoir and a plurality of holes or access holes therein, wherein each access hole comprises a top surface and a bottom surface, wherein the bottom surface of the array plate comprises one or more plateau structures either adjacent, or substantially adjacent, to the bottom surface of the plurality of access holes, and wherein the top surface of the array plate comprises a second plateau structure or structures either adjacent, or substantially adjacent, to the top surface of the plurality of access holes. In certain system embodiments, the lid and the tray enclose, or substantially enclose the array plate to isolate the cell culture from external environment and substances. In certain embodiments, the array plate, the lid, and the tray are made of the same material. In other embodiments, one or more of the array plate, the lid and the tray are made of different materials. In some embodiments, the system is substantially airtight. In other embodiments, the system is sufficiently air tight to allow gas exchange between inside and outside of the system and/or maintain humidity inside the system. In some embodiments, either or both the array plate and the tray contain a reservoir that comprises of an aqueous liquid. In some embodiments, the aqueous liquid provides vapor to maintain the humidity inside the system. In other embodiments, the reservoir comprises other substances. In some embodiments, the bottom surface of the tray is substantially optically transparent. In some embodiments, the substantially optically transparent surface is substantially flat and provides a substantially unobstructed view of the cell culture for optical imaging and analysis, such as microscopic, colorimetric, fluorescence, and luminescence imaging and measurements. In some embodiments, the system (or systems) has geometries and measurements that comply with standards, for example present standards set by ANSI/SBS (American National Standards Institute/Society for Biomolecular Sciences), thus making the system compatible with mainstream imaging systems and automated equipment used in research and development (e.g. high-throughput screening).

"Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

"The accompanying figures facilitate an understanding of the various, non-limiting embodiments of this technology.

"FIG. 1 shows exemplary devices according to certain embodiments of the present disclosure. (a) Illustration of a 384-well formatted cell spheroid culture array plate used in embodiments of the present invention, and its cross-sectional view. (b) Photo and dimensions of the array plate. Photo of the array plate operated with liquid handling robot capable of simultaneously pipetting 96 cell culture sites. (d) Diagram of humidification chamber.

"FIG. 2 shows (a) Osmolality of COS7, mES, and A431.H9 cell spheroids with various cell populations over 7 to 12 days of culture, according to certain embodiments. (b) Fluorescence images of live/dead stained COS7 and mES cell spheroids over a 12-day culture. A431.H9 Spheroid Size (Diameter) vs. Initial Cell Number. (d) A431.H9 Spheroid Size (Volume) vs. Time for various initial number of cells/spheroid, according to certain embodiments.

"FIG. 3 shows TPZ results, time-lapse images of A431.H9 spheroids at various concentrations, bar graph outlining percent of control cell viability at various concentrations for all spheroid sizes and conventional 2D culture condition 96 h after drug treatment, according to certain embodiments.

"FIG. 4 shows 5-FU results, time-lapse images of A431.H9 spheroids at various concentrations, bar graph outlining percent of control cell viability at various concentrations for all spheroid sizes and conventional 2D culture condition 96 h after drug treatment, according to certain embodiments.

"FIG. 5 shows a schematic of an exemplary device used in certain embodiments.

"FIG. 6 shows a schematic of an exemplary device used in certain embodiments.

"FIG. 7 shows a detailed view of an exemplary device used in certain embodiments.

"FIG. 8 shows a detailed view of an exemplary device used in certain embodiments.

"FIG. 9 shows a view of a plateau region, according to certain embodiments.

"FIG. 10 shows a schematic of exemplary methods for adding and removing cells and liquids from devices of certain embodiments.

"FIG. 11 shows mES spheroids cultured using methods of certain embodiments.

"FIG. 12 shows mES spheroids cultured using methods of certain embodiments.

"FIG. 13 shows hepatocyte spheroids cultured using methods of certain embodiments.

"FIG. 14 shows a schematic of an exemplary device used in certain embodiments. A. Overview. B. close up of ring structures.

"FIG. 15 shows Z-factors for fluorescence-based and absorbance-based assays calculated at various different concentrations, according to certain embodiments.

"FIGS. 16A-F illustrates an exemplary array plate, according to certain embodiments.

"FIG. 16A shows a top view; FIG. 16B shows an isomeric view from top; FIG. 16C shows a side view; FIG. 16D shows an end view; FIG. 16E shows a cross section view along the section line M-M shown in FIG. 16A; and FIG. 16F shows a cross section view of the array plate along the section line L-L shown in FIG. 16C.

"FIGS. 17A-D illustrates exemplary plateau structures, according to certain embodiments. FIG. 17A shows a top view; FIG. 17B shows an isomeric view from top; FIG. 17C shows a bottom view; FIG. 17D shows an isomeric view from bottom.

"FIG. 18 shows a top, cross-sectional, and bottom view of an exemplary access hole structure, according to certain embodiments.

"FIG. 19 shows cross section and isomeric 3D representations, from top and bottom, of an exemplary array of access holes, according to certain embodiments.

"FIGS. 20 A-F illustrate an exemplary tray that the array plate shown in FIG. 16 may be used with, according to certain embodiments. FIG. 20A shows a top view; FIG. 20B shows an isomeric view from top; FIG. 20C shows a side view; FIG. 20D shows an end view; FIG. 20E shows a cross section view along the section line P-P shown in FIG. 20A; and FIG. 20F shows a cross section view of the tray along the section line N-N shown in FIG. 20C.

"FIGS. 21A-F illustrate an exemplary lid that the array plate shown in FIG. 16 may be used with, according to certain embodiments. FIG. 21A shows a top view; FIG. 21B shows an isomeric view from top; FIG. 21C shows a side view; FIG. 21D shows an end view; FIG. 21E shows a cross section view along the section line R-R shown in FIG. 21A; and FIG. 21F shows a cross section view of the lid along the section line T-T shown in FIG. 21C.

"FIGS. 22A-F illustrates an exemplary assembly of combined array plate, tray and lid, according to certain embodiments. FIG. 22A shows a top view; FIG. 22B shows an isomeric view; FIG. 22C shows a side view; FIG. 22D shows an end view; FIG. 22E shows a cross section view along the section line V-V shown in FIG. 22A; and FIG. 22F shows a cross section view of the assembly along the section line U-U shown in FIG. 22C.

"FIGS. 23 A and B illustrate an exemplary stacking of the assemblies shown in FIGS. 22A-F, according to certain embodiments. FIG. 23A shows a side view and FIG. 23B shows an end view.

"FIGS. 24A-D illustrate exemplary variations of access hole structure, according to certain embodiments. FIG. 24A shows an exemplary access hole structure with a tall and thin plateau structure on the top surface. FIG. 24B shows an exemplary access hole structure with a short and thin plateau structure on the top. FIG. 24C shows an exemplary access hole structure with a tall and thick plateau structure on the top. FIG. 24D shows an exemplary access hole structure with a tall and thin plateau structure on the top, with a different split line for injection molding."

URL and more information on this patent application, see: Takayama, Shuichi; Tung, Yi-Chung; Hsiao, Amy Yu-Ching; Jan, Edward. Hanging Drop Devices, Systems And/Or Methods. Filed February 25, 2014 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2477&p=50&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626

Keywords for this news article include: Nanoparticle, Nanotechnology, 3d Biomatrix Inc., Emerging Technologies.

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Source: Life Science Weekly


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