WO2024211897A2 - Composant de transfert cellulaire pour dispositif de dosage multi-puits - Google Patents

Composant de transfert cellulaire pour dispositif de dosage multi-puits Download PDF

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Publication number
WO2024211897A2
WO2024211897A2 PCT/US2024/023594 US2024023594W WO2024211897A2 WO 2024211897 A2 WO2024211897 A2 WO 2024211897A2 US 2024023594 W US2024023594 W US 2024023594W WO 2024211897 A2 WO2024211897 A2 WO 2024211897A2
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WO
WIPO (PCT)
Prior art keywords
cell
assay
emc
well
detectable
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Ceased
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PCT/US2024/023594
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English (en)
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WO2024211897A3 (fr
Inventor
Kandaswamy Vijayan
Yi Zhang
Aurelien LAGUERRE
Chulmin Choi
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Zafrens Inc
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Zafrens Inc
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Publication of WO2024211897A3 publication Critical patent/WO2024211897A3/fr
Anticipated expiration legal-status Critical
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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Rigid containers without fluid transport within
    • B01L3/5085—Rigid containers without fluid transport within for multiple samples, e.g. microtitration plates
    • B01L3/50853—Rigid containers without fluid transport within for multiple samples, e.g. microtitration plates with covers or lids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/54—Labware with identification means
    • B01L3/545—Labware with identification means for laboratory containers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/02—Identification, exchange or storage of information
    • B01L2300/021—Identification, e.g. bar codes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B01L2400/049—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics vacuum
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/56—Labware specially adapted for transferring fluids
    • B01L3/563—Joints or fittings; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors

Definitions

  • This disclosure relates to devices that facilitate high throughput assays and more particularly to encoded micro-components configured to specifically associate individual wells with the contents of the individual well.
  • the present disclosure provides devices and methods for transferring single cells via a transfer component into an assay well.
  • Also described herein may be a system configured to facilitate the identification of a specific cell from a population of cells, such as a population of heterogeneous cells, where a single cell from the population of heterogeneous cells may be subject to testing in a single assay well.
  • the ability to evaluate different cells originating from a population of cells, such as a heterogeneous population of cells, and subject to the same stimulus or perturbation may provide a useful stratification that may elucidate how different cell types express individual functionality.
  • functionality may translate differentials of cells into specific classes of cells (specific cancer cells, neurons, epithelial cells, adipose cells, and the like).
  • specific cancer cells specifically cancer cells, neurons, epithelial cells, adipose cells, and the like.
  • the present disclosure provides encoded micro-components (EMCs) configured to associate the identity of a living cell within a multi-well assay with a particular well, where each well in the multi-well assay holds a single unique cell originating from a heterogenous population of cells.
  • EMCs encoded micro-components
  • the transfer component may be a stand-alone device affixed to, and/or a component of (e.g., integral with) a multi-well assay device, which may employ at least one EMC.
  • the present disclosure provides a transfer component configured to transfer a single viable cell to a single well of the multi-well assay device, the transfer component comprising: an enclosure having a base and/or bottom portion, the bottom portion including a floor with side walls extending upward; at least one inlet port positioned in at least one side wall and at least one outlet port positioned in at least one side wall Attorney Docket No.009775.00019 ⁇ WO a vacuum port; and a transfer surface (e.g., tray), wherein the transfer surface comprises at least one cell capturing element, and wherein the transfer surface is configured to engage with the enclosure such that the transfer surface is positioned between the floor of the bottom portion of the device and the top of side walls so as to form a sealed compartment (e.g., flow cell) below the transfer surface and an open volume above transfer surface.
  • the transfer component may include a top portion having side walls that may be configured to engage with the side walls of the bottom portion to form a sealed cell transfer device.
  • the top portion and the bottom portion are integral with each other.
  • the transfer surface of the transfer component may include at least one cell capturing element that may comprise at least one adhesion site (e.g., cell adhesion site).
  • Each of the at least one cell capturing element may comprise at least four adhesion sites, allowing for at least four points of contact with a single cell to the at least four adhesion sites of the capturing element.
  • the adhesion site(s) may be, for example, a number of hole(s) (e.g., a number of through holes through the transfer surface) in a pattern/distribution and having a size and/or shape (e.g., having one or more diameters and/or areas) such that the adhesion sites are configured to contact the cell surface over an area such that when a vacuum (negative pressure) is applied across the hole(s), the cell may stably adhere to all or part of the pattern of holes.
  • the adhesion sites may also, or alternatively, comprise wells having diameters and depths similar to a diameter of a single cell (e.g., 0.5-2x the diameter of a single cell in diameter and approximately 0.5-2x the diameter of a single cell deep).
  • the cell transfer component may be configured to cause a flow of cells in media over the adhesion sites, such that single cells adhere to single adhesion sites either by vacuum force or by gravity allowing the single cell to settle into a single well. Further, the transfer component and the assay device may interlock such that the adhesion sites of the at least one cell capturing element (e.g., a plurality of cell capturing elements) align with the assay wells of the assay device.
  • the at least one cell capturing element e.g., a plurality of cell capturing elements
  • the transfer component is integral with the assay device as a single assay device in which the capturing elements in the transfer surface of the transfer component are aligned with and face corresponding assay wells of the assay device (e.g., aligned with centers of corresponding assay wells in a one-to-one relationship).
  • the assay wells may be arbitrarily sized.
  • the assay wells may have a diameter that is at least 2x that of the adhesion site(s)/wells as adhesion sites.
  • an assay device comprising a plurality of assay wells such that each assay well accommodates at least one unique viable cell, wherein the unique viable cell arises from a population of heterogenous cells and wherein each assay well includes at least one Attorney Docket No.009775.00019 ⁇ WO encoded micro-component (EMC), the EMC comprising: a detectible code on at least one surface of the EMC, the detectible code including an identifiable pattern of at least one label and/or image, wherein the detectible code on the EMC is unique to the assay well and the detectible code links the identity of the unique viable cell to the assay well from which the unique viable cell originated.
  • EMC Attorney Docket No.009775.00019 ⁇ WO encoded micro-component
  • the disclosure also provides for a system that may facilitate the identification of a specific cell from a population of heterogeneous cells, where a single cell from the population of heterogeneous cells may be subject to testing in a single assay well.
  • a system for conducting an assay starting with a heterogenous population of cells comprising: a) a cell transfer component configured to transfer a single unique cell originating from the composition of heterogenous population of viable cells to a single assay well, the cell transfer component including at least one cell capturing element; and b) an assay device comprising a multiplicity of assay wells, wherein at least one well in the assay device includes a single unique cell that is identifiable by a readable code, and wherein at least one well in the assay device comprises: an encoded micro-component configured to identify at least one characteristic of the contents of the well and/or at least one aspect of the assay conducted in the well.
  • the plurality of assay wells may include, for example, microwells, nanowells, and/or picowells.
  • the detectable EMC code may be capable of detection via a variety of means.
  • the detectable EMC code may be visually detectible.
  • the visually detectable EMC code may, for example, be ascertained using a microscope.
  • the detectable EMC code may be mechanically (e.g., physically/optically) detectible, for example, the detectable EMC code may be mechanically detectible by means including but not limited to fluorescence intensity.
  • the detectable EMC code may be chemically detectable, such chemically detectable means may include, among other suitable means, deoxyribonucleic acid (DNA) sequencing (see, e.g., U.S. Patent Application No. 17/687,376 filed on March 4, 2022 to Lance et al., incorporated by reference herein in its entirety).
  • the detectable EMC code may include at least one detectable dot (e.g., light-emitting/electromagnetically detectable dot/chip) and/or detectable color (e.g., a dye).
  • a detectable dot may be a quantum dot.
  • each detectable dot may be detected by the same means of detection.
  • each detectable dot may be discerned using visual means, chemical means, biochemical means, mechanical means, or spectroscopic means.
  • Attorney Docket No.009775.00019 ⁇ WO each detectable dot may be discerned using one or more detection means (e.g., a combination of different detection means).
  • a non-limiting example may be that each detectable dot may be discerned using a combination of both visual and chemical means.
  • the EMC may include an orientation marker. The orientation marker may be included within the detectable EMC code located on at least one surface of the EMC.
  • the orientation marker may be a unique label and/or image that may be used, for example, to properly orient the reading of the detectable EMC code.
  • the EMC provided in each assay well may include three or more surfaces.
  • the identical detectible EMC code may be included on more than one surface of the EMC.
  • the EMC may be implanted into a bead such that at least one surface of the EMC is exposed on the surface of the bead.
  • the at least one surface of the EMC that is exposed on the surface of the bead may include the detectable EMC code, and thus the detectable EMC code on the surface of the EMC may still be readable while embedded in the bead.
  • the assay device and/or system described herein may allow for the identification of additional features of an assay, including but not limited to such features as the identity of the technician that conducted an assay, as well as assay conditions (i.e., temperature, pH, atmospheric pressure, concentration, etc.), date and/or time the assay was conducted, one or more (e.g., any/all) synthetic steps used to generate a test compound, and/or one or more (e.g., any resulting) reaction products of the assay.
  • the devices, methods, and systems provided herein may facilitate high throughput assay procedures. Such assay procedures may include but are not limited to eliciting cellular activity in response to a perturbing event.
  • the devices, methods, and systems disclosed may include capturing, culturing, perturbing, and/or performing assays on single cells.
  • the devices, methods, and systems provided may facilitate a cellular assay.
  • the devices, methods, and systems may correlate information obtained from the cellular assay to an individual cell subjected to the assay, based on the assay well from which the cell originated. For example, captured genomic information (e.g.
  • FIGS. 1A to 1E are illustrations of exemplary EMC embodiments.
  • FIG. 1A shows a circular EMC and
  • FIG. 1B shows the same circular EMC from a different angle.
  • FIG. 1C shows an EMC having the shape of a pyramid.
  • FIG. 1D shows an EMC taking the shape of a multi-surface wedge-like object.
  • FIG. 1E shows a hemisphere-shaped EMC.
  • FIGS. 2A and 2B are illustrations of exemplary assay wells.
  • FIG. 2A shows a partial view of an assay well within an assay device in which an EMC is located.
  • FIG. 2B shows an assay well in an assay device having a recessed portion such that the recessed portion is sized to hold the EMC but too small for a cell;
  • FIG. 3A shows a bead with at least one EMC embedded on the surface of the bead. As shown in FIG.
  • FIG. 3 shows an EMC partially implanted within the bead and the detectable EMC code visible on the surface of a fully implanted EMC.
  • FIGS. 3B-D show examples of EMCs embedded in/attached to beads; [0023]
  • FIG. 4A and 4B show an EMC configuration and EMC coding component.
  • FIG. 4A shows an EMC configured by two distinct shapes joined together.
  • FIG. 4B shows an EMC coding component employed to identify a reagent used to make a compound;
  • FIGS. 5A to 5C illustrate iterative examples of a cell capturing element.
  • FIG. 5A shows a cell capturing element in a transfer surface of a transfer component configured to impart minimal stress on the cell during cell capturing.
  • FIG. 5B shows a transfer surface of a transfer component having a cell capturing element located within a depressed region of a transfer component. The cell capturing element is configured to provide more surface contact between the cell and the capturing element thereby reducing stress on the cell during cell capturing.
  • FIG. 5C shows a cell capturing element with at least four holes/adhesion sites and a cell that is adhered to the cell capturing element via a vacuum force sufficient to retain the cell in place; [0025] FIG.
  • FIG. 6 shows a schematic of a cell transfer component comprising a cell capturing element in the transfer surface within the transfer component that allows for capture of a single cell from a heterogenous population of cells.
  • FIG. 7 shows an example implementation of the transfer component as part of a multi-well assay device;
  • FIG. 8 shows another example implementation of a transfer component as part of a multi-well assay device;
  • FIG. 9 shows an example multi-well assay device comprising a transfer device; Attorney Docket No.009775.00019 ⁇ WO [0029]
  • FIG. 10 shows a brightfield image of cells after being transferred via the example multi-well assay device comprising the transfer device.
  • EMC embedded micro-components
  • transfer components and methods configured to transfer a single cell from a specific site in the transfer component into an individual corresponding assay well. Systems employing these components in an integrated approach are also disclosed herein. Definitions [0031] Terms not defined herein have their accepted scientific and medical meaning. [0032] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting to the described embodiments.
  • the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
  • the term “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur and that the description includes instances where the event or circumstance occurs and instances where it does not occur.
  • the term “about” when used with regard to a dose amount means that the dose may vary by +/- 5%.
  • the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others.
  • the term “consisting essentially of” when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the term “consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.
  • Attorney Docket No.009775.00019 ⁇ WO [0038]
  • the term “encoded” refers to a code comprising one or more labels and/or images that are found on one or more surfaces of a micro-component.
  • the terms “encoded micro-component” also referred to as “EMC” and/or “EMS device” may relate to an encoded object having any shape, such as a sphere, a cube, a cylinder, or any other shape.
  • the shape may have any shape that includes a surface and/or at least 2 surfaces/faces that point in multiple directions.
  • the shape may be a hemisphere having a flat surface and/or a curved surface, and, preferably, at least 3 surfaces/faces that are pointed in multiple directions.
  • an EMC may be a structure having more than 3 surfaces/faces, for example, the EMC may take the shape of a pyramid, a pentagonal shape, or any other suitable shape having more than 3 surfaces/faces.
  • an EMC may be embedded in and/or attached to a bead.
  • An EMC may, for example, be attached to a bead via magnetic forces.
  • At least one surface of the EMC is encoded with a detectable code.
  • a hemisphere EMC may be encoded with a detectable code on its flat surface and/or curved surface.
  • the definition is not so limited, however, as in some aspects, a plurality of EMC surfaces/faces may be encoded with the same detectable code (e.g., such that the detectable code is detectable from various perspectives/for various orientations of the EMC).
  • the different surfaces/faces of the EMC may be encoded with different detectable codes.
  • an EMC may include 2 or more codes located on at least one surface of the EMC.
  • detectable EMC code may refer to a code present on at least one surface of an EMC. Further, more than one surface of an EMC may include the same detectable EMC code. [0041] Alternatively, or in addition, a different detectable EMC code may be present on different (e.g., each) surface of the EMC.
  • a detectable EMC code is capable of relaying to the user a single or multiple pieces of information for a given well comprising the detectable EMC code and/or a bead comprising the detectable EMC code.
  • the code may be initiated with a specific prefix unique to (e.g., indicating) the technician conducting the assay, the remainder of the code providing one or more pieces of additional information.
  • a specific prefix unique to e.g., indicating
  • an EMC having a single EMC code or multiple detectable EMC codes may also encode for such information as the date and/or time the assay was conducted, the conditions of a particular assay, and/or any other relevant information having to do with an assay performed on living or viable cells.
  • a second or subsequent code present on one or more surfaces/faces of the EMC may indicate particular assay conditions and thus may indicate the presence of a particular chemical compound, cytokines, or immune cells in the well containing Attorney Docket No.009775.00019 ⁇ WO the EMC.
  • a detectable EMC code may be a collection of patterns and/or labels. The definition is not so limited, however, as a detectable EMC code may include symbols, characters, shapes, colors, quantum dots, fluorescent tags, or any other discernable medium.
  • a detectable EMC code may comprise one type of code, e.g. include only a series or number of shapes, or a combination of different code types, e.g. shapes, colors, quantum dots, fluorescent tags, etc.
  • a discernable code refers to a code that is visibly, chemically, or spectroscopically readable. So, for example, a visibly discernable code includes one that may read with magnification such as a microscope.
  • a chemically discernable code may include DNA and/or ribonucleic acid (RNA) that is bound to the micro-component, provided that where the code contains two or more labels or images, each label or image is a separate, unique strand of DNA.
  • RNA ribonucleic acid
  • a spectroscopically readable code refers to separate labels which may include, for example, fluorescent labels distinct from each other on the micro-component.
  • the discernable code can use a mixture of such readable components.
  • the orientation marker in the code may be fluorescent and the remainder of the code may be visibly readable.
  • code includes any set of images, numbers, lines, and/or any combination thereof, where at least one of the images is not based on a nucleic acid. Images include structural images such as pictures, drawings, colors, quantum dots, Morse code, Roman numerals, or other visual patterns recognized by a reader of the code (e.g., a detector/interpreter of the code). Lines may include, but are not limited to, computer-readable barcodes or any other computer-readable codes.
  • the term “orientation marker” is defined as a component in the detectable EMC code that instructs the user where to start reading the code.
  • a detectable EMC code can be etched through the EMC and as a result, the underside of the EMC will read differently from the top side of the EMC.
  • the detectable EMC code may contain a common feature or “orientation marker” as the starting point for the code.
  • Non-limiting examples of such a feature and/or marker may include a capital C, a dollar sign ($), a hashtag (#), and/or any other feature that is uniformly used to identify where to start reading the code.
  • orientation marker will typically be found at the start of the detectable EMC code
  • the orientation marker can also be inserted at a point intermediate in the code, to be used, for example, as a security measure whereby only certain individuals are able to read the code.
  • the orientation marker associated with the code may be an image or label selected from a group of unique images and/or labels that are used either alone or in combination to identify the start of the code. For example, a single $ by itself may not indicate the starting point of the code but two $$ may serve as the orientation marker.
  • the different types of orientation Attorney Docket No.009775.00019 ⁇ WO markers are endless.
  • orientation markers may define the starting point for the detectable EMC code may include the dollar sign adjacent to a particular number such as $7 or a combination of numbers such as 77$.
  • the orientation marker may be further protected by allowing for intervening “nonsense” labels or images. In such a case, the orientation marker may direct the reader to a first code such as “$!” from which the technician is instructed (separately) to a series of non-sense images and/or labels such as the next occurrence of “Z”. When a specific non-sense image is reached, for example, the technician may read and decode the code following the non-sense symbol.
  • the critical feature of the orientation marker is that it is orientable (asymmetrical in at least one direction) and has a known orientation (e.g., is known and can be used to orient the code of the EMC).
  • micro in “micro-component” refers to any structure having its longest axis which is less than 1,000 microns (i.e., a micron and/or sub-micron structure).
  • micro-component refers to a micron and/or sub-micron materials that are structured, inert, retrievable in an aqueous medium, and labeled with a discernable code on at least one of the micron and/or sub-micron structures.
  • the exact size of the micro-component is dependent on the size of the micro-well. In one exemplary embodiment, the size of the micro-component occupies no more than 50% of the volume of the assay micro-well, preferably no more than 25% of the assay micro-well; and more preferably less than 10% of the volume of the assay micro-well.
  • the shape of the micro-component is not critical and can include, by way of example only, cubical, cylindrical, and elliptical shapes. [0046] In some embodiments, the encoded micro-component is generated as a discrete, stand-alone, structure.
  • the encoded micro-component is sized and shaped to be implanted into and/or attached to a bead used in the assay provided that the implantation retains an exposed surface with a discernable code.
  • multiple encoded micro-components with the same discernable code can be implanted into the bead to ensure that at least one of the implanted components retains a discernable code.
  • the term “assay bead” or “bead” refers to a bead that may comprise a compound releasable from the bead when placed in a well.
  • the bead may allow for the change in functionality and/or one or more features of a cell to be measured either alone or in the presence of additional components in the well.
  • additional components may include antibodies, cytokines, chemokines, and the like.
  • the assay beads may typically include an encoded structure such as DNA that correlates to and/or codes for the compound released from the bead. Additional features for such beads are well known in the art including an RNA-capturing element incorporated thereon. Attorney Docket No.009775.00019 ⁇ WO [0048]
  • the term “perturbation” refers to a disturbance or interruption to a system or process that may cause it to deviate from its normal state or path of development.
  • a “perturbation” may refer to a physical, chemical, or biological change that affects the functioning of a system, such as a change in temperature, osmolality, pH, the removal of a compound or feature (e.g., removal of a single nutrient), or the introduction of a new species (e.g., chemical species).
  • Cell perturbation may be employed to measure and/or analyze the effects of altering the normal functioning of cells. As such, perturbation may be facilitated by introducing a foreign substance into the cell, such as a drug, hormone, or genetic material, that disrupts the normal functioning of the cell. The effects of the perturbation can then be measured and analyzed to gain insight into the role of the perturbed gene or protein in the cell’s normal functioning.
  • stimulation refers to the process of applying external stimulus to a cell and/or cell culture in order to induce or enhance a physiological response.
  • cell stimulation may involve the release of hormones, neurotransmitters, and/or other molecules that cause a change in the cell’s activity which in turn may alter gene expression, protein production, cell cycle progression, and other cellular processes.
  • Cell stimulation may damage a cell by causing changes in the cells’ structure, metabolism, or physiology. Further, stimulation of a cell may lead to cell death, either directly or indirectly, by activating certain pathways or proteins that induce cellular damage.
  • Damage can also be caused by overstimulation, which can lead to an increase in intracellular calcium, oxidative stress, and cell death. Additionally, certain chemicals used in the stimulation process can directly damage a cell’s membrane, leading to lipid peroxidation, membrane disruption, and cell death.
  • cell stimulation involves the addition of an external agent, such as a growth factor or an agonist, to the cell environment.
  • an external agent such as a growth factor or an agonist
  • Cell perturbation is typically an experimental technique used to alter or disrupt a cell’s natural processes. Cell perturbation may be used to investigate the effects of a compound or other external agent on the cell’s behavior.
  • cell perturbation usually does not involve the addition of an external agent, but instead involves disrupting the cell’s environment in some way, such as through temperature and/or pressure changes.
  • An intended cell perturbation at one level may be desired whereas extending the perturbation to a different level may be undesired.
  • undesired perturbations are to be avoided as such undesired perturbations may result in inaccurate, confounded, or unusable experimental results.
  • split pool split generation may refer to a technique used to generate test compounds in a systematic way. Split pool split generation involves breaking down a larger Attorney Docket No.009775.00019 ⁇ WO pool of compounds into smaller pools, and then combining these smaller pools into new random combinations.
  • a test compound may be generated by a single precursor molecule.
  • the precursor may be split into two parts, the “split pool” and the “split product”.
  • the split pool is further reacted with reagents to yield a range of possible reactions and products.
  • the split product is then used to synthesize the desired test compound.
  • This technique may be useful for creating a library of compounds from a single precursor molecule, making it an efficient method for drug discovery.
  • split pool split generation may allow for the efficient testing of a large number of compounds in a shorter period of time and using fewer resources.
  • the EMC may comprise one part of a multi-variate device having multiple functionalities (see FIG. 4A).
  • the EMC may include two separate portions where one portion of the multi-variate device may have a surface comprising a reactive functionality that allows for the generation of a releasable test compound and a different part of the EMC may comprise a recordation portion.
  • a different portion of the same EMC may detect changes in reaction conditions such as pH, or temperature.
  • a reactive functionality may be triggered by a specific stimulus.
  • Non-limiting examples of a stimulus that may trigger the reactive functionality of an EMC may include physical, biochemical, and/or genetic stimuli.
  • a detectable code may be added to and/or generated onto the EMC for each step in the synthesis of the compound, for example, in a split-pool-split generation of the test compound.
  • the EMC may have a T-shape that may use two different surfaces (FIG. 4A).
  • the T-shaped EMC may have one surface capable of compound synthesis and another surface that may identify the synthetic steps that were employed to create the compound.
  • the EMCs that were split into a separate pool may be isolated and a colored magnetic particle or particles may be added to the pool.
  • the specific color of the magnetic particle may indicate the specific step in the reaction sequence and the particle may be coated with a unique identifier such as a unique fluorescent molecule having a characteristic fluorescent peak to indicate the specific reagent used. Based on the combination of a unique color and a unique identifier of a particle, the technician may deconvolute a particular step used in the synthesis of the compound and also identify the particular reagent used.
  • the term “population of cells” refers to a plurality of cells, which may be of and/or derived from any immortalized cell line and/or any primary cells.
  • a “heterogeneous population of cells” refers to a mixture of cells from two or more cell lines, cells of two or more cell types, Attorney Docket No.009775.00019 ⁇ WO of two or more distinct morphologies and/or functionalities, etc.
  • the bead may be magnetic and the EMC may be ferromagnetic, and as such the EMC will attach to the bead via magnetic interaction.
  • the EMC and bead may attach by other means, such as van der Waals attraction and the like.
  • the bead may be made of a soft composition where one or more copies of the EMC may be embedded into the outer layer of the bead while exposing at least one surface of the EMC, such that the exposed surface of the EMC presents a discernable code.
  • the EMC may be fully embedded if the bead is transparent.
  • FIG. 1A shows an EMC (100a) having a cylindrical shape with a first or top side (102a) and a second or bottom side (104a). The EMC (100a) shown in FIG.
  • a discernible and readable EMC code (110a) is located on the top side (102a) of EMC (100a).
  • the same EMC code (110a) may be located on both the top side (102a) and bottom side (104a) of the EMC (100a), such that the two sides of the EMC (100a) may be interchangeable.
  • the identical EMC code (110a) will be face up regardless of whether the top side (102a) or the bottom side (104a) is exposed in the well of the assay device.
  • the EMC (100b) has a similar cylindrical shape to the EMC (100a) in FIG.1A, however, a discernible and readable EMC code (110b) is instead located on the side (112b) of the EMC (100b). In this configuration, it is irrelevant which side, the top side (102b) or the bottom side (e.g., (104a) in FIG. 1A), faces down when placed into the well.
  • the readable EMC code (110b) located on the side (112b) of the EMC (100b) eliminates the possibility that the only encoded surface of the EMC (100b) will orient with that surface facing the bottom of the assay well and/or another surface (e.g., a bead).
  • the EMC (100c) may be pyramidal in Attorney Docket No.009775.00019 ⁇ WO shape thereby allowing for four sides or surfaces/faces.
  • the advantages of a pyramidal-shaped EMC (100c) may be that, when added to an assay well, three of the four surfaces/faces of the pyramid may expose a discernable and readable EMC code (110c).
  • an EMC (100d) may have a wedge- like form. Accordingly, FIG. 1D shows an EMC (100d) having one side of an otherwise cubic structure (114d) having a tapered edge (116d). The configuration shown in FIG.
  • 1D may be used to partially embed the EMC (100d) into one or more beads.
  • a bead impregnated with an EMC (100d) may also have the capability to deliver a candidate compound to a given well (see, e.g., FIG. 3).
  • the EMC (100d) can be entirely embedded.
  • the discernable and/or readable EMC code (110d) is shown in FIG.
  • FIG. 1D may be embedded into a bead such that the EMC code (110d) as seen on a bottom or exposed surface (118d) of the EMC (100d) will allow for the EMC code (110d) to be read from the surface (118d) that protrudes from the bead.
  • FIG. 1E depicts an EMC (100e) having only two surfaces/faces.
  • FIG. 1E shows a 3D semi-circle shaped EMC (100e).
  • Such an EMC (100e) configuration may also be referred to as hemispherical in shape.
  • the EMC (100e) has 2 surfaces/faces, a first flat surface (120) and a second round or curved surface (122e) forming a semi-sphere or hemisphere.
  • the hemispherical EMC (100e) may, for example, include a code (110e) on the first flat surface (120e) as shown in FIG. 1E and/or on the round or curved surface (122e) (not shown).
  • a hemispherical EMC (100e) having a code (110e) positioned on the flat surface (120e) may be located on a flat surface (120e) down in an assay well such that the EMC code (110e) is detectable through the bottom of a clear assay well (see, e.g., FIG. 2A).
  • FIGS. 1F-1G show EMCs fabricated by etching silicon discs. To fabricate the EMCs in FIGS. 1F-1G, 10 or 15-micron silicon discs, respectively, were etched using nanofabrication techniques such that each EMC has a unique pattern. A photoresist layer was deposited on the top surface of a 100 mm diameter silicon-on-oxide wafer purchased from University Wafer (top Si layer 220 nm; middle oxide layer 3 micron; bottom Si layer 725 micron).
  • EMC code (110a-e) may be placed on the EMC (100a-e).
  • the EMC (100a-e) may contain multiple different EMC codes (110a-e).
  • a first EMC code (110a-e) may include an orientation marker and/or that is specific for a first condition (e.g., cell type);
  • a second EMC code (110a-e) may use a different orientation marker and/or be specific for a second condition (e.g., the identity of the assay operator);
  • a third EMC code (110a-e) may use an orientation marker that may be the same as and/or different from the first and second orientation markers and/or may identify the specific reaction conditions of the corresponding well, and so on.
  • FIGS. 2A-2B depict two assay well configurations.
  • FIG. 2A illustrates an assay well (200a) having an opening (202a), and a floor (204a), with a two-surface hemisphere- shaped EMC (100e) device positioned on the floor (204a) of the well.
  • the EMC as depicted in FIG. 2A also includes an orientation marker (210a).
  • the orientation marker (210a) in the figure is represented as $$, it is noted that the orientation marker (210) may be any one or a collection of images and/or labels that may include but are not limited to, numbers, symbols, pictures, etc.
  • any EMC such as EMCs (100a-100e) may be applied to an assay well(200a) and/or assay well device as disclosed herein and/or may be configured with an orientation marker (210a).
  • FIG. 2B depicts an assay well (200b) with an opening (202b) and a floor (204b) and lacking the EMC (100e) device, for illustrative purposes/clarity only.
  • the assay well (200b) may also, or alternatively, include a recess (206b), in the floor (204b) of the assay well (200b), that may be specifically shaped and sized to hold the EMC (100e) but not a cell (208b).
  • the EMC (100a-e) may be designed to occupy minimal space in the assay well (200b), thereby not interfering with the cell (208b) or the assay being conducted while the EMC code is still readable.
  • the EMC (100a-b) may be situated in the recess (206b) of the assay well (200b) and a removable cover (not shown) may secure the EMC (100a-e) in place.
  • other alternatives to secure the EMC in the recess (206b) of the assay well (200b) to ensure the EMC (100a-e) does not interfere with an assay may include snaps and/or protrusions positioned within a side wall of the recess (206b).
  • snaps and/or protrusions positioned within a side wall of the recess (206b).
  • FIG. 3A in some embodiments, one or more copies of the EMC (100d) device as shown in the wedge-like shape (see FIG.
  • FIGS. 3B-D show examples of EMCs embedded in/attached to beads.
  • FIG. 3B shows bright-field images of PEG-polyacrylamide hydrogel beads having attached silicon disc EMCs.
  • the silicon disc EMCs were prepared similar to those in FIGS. 1F-1G.
  • the silicon disc EMCs were embedded within PEG-polyacrylamide hydrogel beads prior to crosslinking inside a cylindrical microwell.
  • the pre-polymerized mixture comprised 187.5mM AAm-PEG2K-NH3 and 62.5mM AAm-PEG3.7K-Aam and was crosslinked with 1%[v/v] 2-hydroxy-2- methylpropiophenone at appropriate UV dosage.
  • FIG. 3C shows beads randomly embedded with micro-components, such that the randomly embedded micro-components cause each bead to be unambiguously distinguishable from each other.
  • FIG. 3D shows beads with EMCs attached thereto and imaged in wells of a multi- well assay device.
  • the EMCs were 40 micron diameter silicon discs coated with a thin layer of silicon oxide in a PECVD chamber.
  • the silicon disc EMCs were incubated with 160 micron TENTAGEL®-amine beads.
  • FIG. 3D shows single beads visible in single wells, and the blow- up images show the EMCs attached to the beads, with visible and distinguishable codes.
  • FIG. 4A shows an embodiment of an EMC (400a) having both a reactive portion (402a) and a recordation portion (404a). As seen in FIG.
  • an EMC (400a) may include a portion that synthesizes a test compound--the reactive portion (402a)--as well as a portion configured to record the synthetic steps involved in the particular procedure and, accordingly, Attorney Docket No.009775.00019 ⁇ WO the compound generated--the recordation portion (404a).
  • a coded set of low micron e.g., 100 microns or less, 50 microns or less, 10 microns or less, etc.
  • sub-micron (less than 1 micron) particles having bound detectible labels may adhere to the recordation portion (404a).
  • FIG. 4A depicts an EMC (400a) having the two portions (i.e., the reactive portion (402a) and the recordation portion (404a)) represented by different parts of the T-shaped EMC (400a) with the understanding that there may include a separate place to do the synthesis in the reactive portion (402a) and a separate portion configured to record each step involved the particular synthesis procedure and/or reaction in the recordation portion (404a).
  • an EMC (400b) may be employed in the generation of compounds.
  • the EMC (400b) may be, for example, the same as the EMC (400a) in FIG. 4A. As such, FIG.
  • EMCs (400b) depicts how an EMC (400b) allows for the generation of test compounds with increased efficiency using a split-pool-split procedure.
  • EMCs (400b) are split into 5 groups (Pools 1-5).
  • a 1 , A 2 ... A 5 are employed in separate vessels to document the appropriate recordation portion (404b) of the EMC (400b).
  • the label used is a micro- or sub- microbead (406b) with a common label that identifies the first step of the reaction sequence.
  • Non-limiting examples of such labels may include different colors, different fluorescent molecules, different patterns of quantum dots, and/or any other suitable label.
  • the label on the micro- or sub-microbeads (406b) used in the first reaction vessels (A 1 -A 5 ) may have a pattern of 3 quantum dots arranged to distinguish each reaction vessel from each other. Examples would include: horizontal line . .. Triangle . vertical lines
  • the label on the beads (408b) used in the second reaction vessels (B 1 -B 5 ) may have a pattern of 4 quantum dots arranged to distinguish each reaction vessel from each other.
  • the label on the beads used in each of these may have 5, 6, and 7 quantum dots respectively.
  • the beads used in each of the 5 separate reaction steps may have a unique discernible color for each step.
  • the beads used in the first step may be red
  • the beads used in the second step may be blue
  • Attorney Docket No.009775.00019 ⁇ WO on there are an infinite number of possible labels that can be used on the beads including separate images and separate codes (e.g., 1001, 1002, 1003, 1004, 1005 for the first 5 reaction vessels; e.g., 2001, 2002, 2003, 2004, 2005 for the second 5 reaction vessels; and so forth).
  • each of these 5 labeled microbeads (406b) may be bound to a different detectable marker, 1, 2, 3, 4, and 5.
  • the detectable marker may be a unique antibody (e.g. Ab-1 through Ab-5) or alternatively, the detectable marker may be a defined pattern of quantum dots that denote each separate reaction step.
  • Other examples of detectable markers may include different fluorescent compounds or any other suitable form of detectable marker.
  • the next step in the process may entail recording the synthetic conditions employed in the first reaction step of sub-pool 1 (Rx1). In some aspects, such synthetic conditions are recorded by attaching a microbead (406b) A 1 to the EMC (400b) as seen in FIG.
  • the EMC (400b) will acquire a label to denote Rx2 (A 2 ), which similarly records the reaction steps. In the embodiment disclosed, the process is continued until Rx5 is reached. [0075] Following the completion of the first step, all of the EMCs (400b) are combined or pooled and subsequently homogenized. As shown in FIG.4B, the EMCs (400b) are again split into 5 groups, B 1 , B 2 ... B 5 , and 5 different reaction conditions and/or reagents may be used. Afterward, the first vessel may include 5 different compounds, all with 5 different A (A 1 -A 5 ) substituents and 1 (one) B substituent (of B 1 -B 5 ).
  • the process is continued for 3 additional steps to provide for the following distribution of 3,125 different compounds in 5 vessels each containing 625 compounds where each the population of 5 compounds with unique E 1 -E 5 labels, 25 compounds with unique D 1 -D 5 labels, 125 compounds with unique C 1 -C 5 labels, 625 compounds with unique B 1 -B 5 labels and 3,125 compounds with unique A 1 -A 5 labels.
  • the last step in the process described herein may include homogenizing the compounds and thereby distributing the resulting beads into individual wells, preferably 1 bead per well. An assay may then be conducted and, following completion of the assay, any bead showing activity may be extracted from its well.
  • the extracted bead may then be analyzed to determine the structure of the active compound.
  • the process of deconvoluting the structure on each bead is preferably done in the inverse of the reaction steps. For example, because Step E is the last reaction step, there are only 5 possible matches for the final step in the reaction. [0078] Once that reaction step is ascertained, only 5 possible matches remain for the fourth step of the reaction. If the fourth step is coded by a fluorescent compound, for example, Attorney Docket No.009775.00019 ⁇ WO then identification of that compound is made spectroscopically. The process is repeated until a determination of the reaction conducted in the first step (i.e., Step A in FIG. 4B) is completed.
  • Cell Transfer Component for transferring single cells to single wells of multi-well assay device [0080] Also described herein is a high throughput assay device containing tens, hundreds, thousands, tens of thousands, hundreds of thousands, or millions of wells. Each well may be configured to include one or more EMCs along with at least one cell originating from a population of cells of interest, such as a substantially heterogeneous population of cells. To avoid having two different cells in a single well, a cell transfer component with the capability of transferring a single cell into a single well is required. The cell transfer component is configured to maintain cell viability and avoid cell damage during cell capture and transfer to a well of the multi-well assay device.
  • Single-cell transfer components may therefore be configured to accommodate an aqueous solution, optionally comprising cell nutrients, throughout cell capture and transfer.
  • aqueous solution optionally comprising cell nutrients
  • FIGS. 5A to 5C depict a portion of a cell transfer component that is configured to capture only a single cell. As seen in FIG.
  • the portion of the single-cell transfer component (500a) includes at least one cell capturing element (502a), each of which may be aligned to and/or positioned to be aligned to a single well in an assay device (see, e.g., FIGS. 6-8).
  • each cell capturing element (502a) may be positioned to face and be aligned opposite to the center of an opening of a single corresponding assay well in an assay device comprising and/or affixed to the cell transfer component.
  • the cell capturing element (502a) is configured to capture a single cell in a manner that reduces or eliminates unintended stress on the cell (504a) (shown in FIG. 5C).
  • the perturbation may be induced by exposing a cell (504c) to be assayed (“assay cell”) to another type of cell (e.g., an immune cell) or exposing the assay cell to an antibody, a bacterium, a virus, a test compound, a physical perturbation, and/or any other suitable means of perturbation.
  • the perturbation is conducted under controlled conditions, and therefore constitutes an “intended perturbation.”
  • Unintended perturbations may arise when a cell capturing element (e.g., cell capturing element (502a)) imparts substantial unintended stress on the cell. In such cases, the cell will become perturbed by the capturing process, and that perturbation may interfere with the accuracy of the experiment results.
  • Previous single-cell capture devices employ capturing elements having one or two holes in each well of an assay device, but the wells of the assay device are necessarily approximately single-cell-sized to avoid capturing multiple cells in the well. Such capture devices do not allow for transferring each captured cell simultaneously to a single corresponding assay well of arbitrary/desired size. There remains a need for cell transfer devices and/or components that allow for transferring single cells into arbitrarily sized assay wells while reducing/avoiding stress to the cell during capture and transfer.
  • the present transfer component comprises cell capturing elements that allow for transferring single captured cells to assay wells large enough to perform desired assays, such as proliferation assays, motility assays, etc.
  • the cell capturing element (502a) provided comprises one or more holes/adhesion sites (506a) (e.g., one or more through holes through the transfer surface (508a) comprising the cell capturing element (502a)), each designed to form one or multiple points of contact with a cell.
  • the one or multiple points of contact may amount to a total area of contact with the cell that is subject to a negative pressure applied to the one or more holes/adhesion sites (506a).
  • the negative pressure applied over the total area of contact amounts to a total vacuum force sufficient to retain a cell at the cell capturing element (502a), for example, in the face of a flow applied over the cell and/or cell capturing element (502a).
  • the size and/or area of the one or more holes/adhesion sites (506a) may be selected such that the resulting total vacuum force that is minimally perturbing/non-damaging to the cell.
  • the number of holes/adhesion sites (506a) required to retain the cell is at least 2 or more, 3 or more, 4 or more, and preferably, at least 5 or more.
  • the holes may be sized such that a cell aligning over 2 or fewer holes/adhesion sites will likely suffer from a lack of sufficient contact points/area to stably remain on that cell capturing element (502a) without using potentially damaging vacuum pressure that may induce undesired perturbations in the cell (504a).
  • the number of holes/adhesion sites may be 1, and the hole/adhesion site may be sized such that an area of contact with the cell on the hole(s) is sufficient for a total vacuum force to be largely non- perturbing/non-damaging to avoid undesired perturbations.
  • the hole/adhesion site is also small enough that the cell does not flow through the hole, if present, and/or to avoid a second cell from also adhering to the hole/adhesion site.
  • the hole diameter may be less than 1/3, less than 1/4, or less than 1/5 the diameter of a target cell and/or average cell of a population of cells.
  • the transfer component described herein includes a transfer surface comprising a cell capturing element (e.g., 502a) comprising one or more holes/adhesion sites (506a).
  • the cell capturing element e.g., 502a
  • the cell capturing element comprises multiple holes/adhesion sites (e.g., 4 or more) and consequently, includes a greater number of points of contact/adhesion with the cell.
  • the cell capturing element (502a) is located on a portion of a top surface of a cell transfer surface (508a) of the cell transfer component. As shown in FIG. 5A, the cell capturing element (502a) includes a multiplicity of holes/adhesion sites (506a) that form the capturing element (502a).
  • multiplicity of holes/adhesion sites (506a-c) depicted in Figures 5A-5C are illustrative only and not intended to be limiting.
  • the number of holes/adhesion sites (506a) that make up a cell capturing element (502a) may range from 1 to about 4,000 and are sized and selected based on the size of the cells and the number of contact points/amount of contact area desired in light of a total vacuum force sufficient to overcome fluid flow forces (e.g., of an applied fluid flow over the transfer surface (508a)).
  • the number of holes/adhesion sites (506a) in each cell capturing element (502a) ranges from 4 to about 400, or from about 10 to about 100.
  • the term “stably adhere” and similar terms refer to the fact that under the vacuum pressure applied, which is selected to reduce/avoid Attorney Docket No.009775.00019 ⁇ WO perturbation in the captured cell, and the loading conditions used, cells that have insufficient contact points/area are subject to dislodgement from the cell capturing element (502a). In general, the vacuum to be applied is reduced such that the cell does not evidence a level of perturbation that adversely affects its value in the assay.
  • the number of holes may be selected such that a size of each hole, for example, may be no more than about 5% of the surface area of the cell in contact with the hole and preferably no more than about 3% and even more preferably no more than about 1% of the surface area of the cell.
  • vacuum pressures may be about 0.7 atmospheres or less and, preferably, about 0.5 atmospheres or less.
  • the cell capturing element (502b) features holes/adhesion sites (506b) which are located throughout a scalloped and/or recessed region (510b) of the cell transfer surface (508b).
  • the scalloped and/or recessed region (510b) may be recessed from the cell transfer surface (508b) by vertical walls, as shown, and/or scalloped/recessed by a gradual/sloped/bowl shape (e.g., that better conforms to the unstrained shape of a cell).
  • the width/diameter of the scalloped and/or recessed region (510b) is, in some embodiments, 0.5-2x the width/diameter of a target cell and/or of an average cell of a heterogeneous population (e.g., an average width/diameter of the heterogeneous population). In some embodiments, 1-2x the width/diameter of a target cell and/or of an average cell. In some embodiments, the scalloped and/or recessed region (510b) has a depth (e.g., maximum depth) that is 0.5-2x the diameter (e.g., average diameter) of the target cell and/or the average cell.
  • a depth e.g., maximum depth
  • the scalloped and/or recessed region (510b) has a depth of about 0.5-1.5x the height of cell (504c) at its maximum point and has a width/diameter no wider than about 1 to about 1.5 times the diameter of the target cell and/or the average cell. In some embodiments, the scalloped and/or recessed region (510b) is configured to be no deeper than about 1x the height of target cell and/or average cell, the diameter is about 1.1-1.3x the width/diameter of target cell Attorney Docket No.009775.00019 ⁇ WO and/or average cell.
  • the scalloped and/or recessed regions (510b) can serve as cell capturing elements (502b) without any hole/vacuum/adhesion force -- cells can be flowed over the scalloped and/or recessed regions (510b) and allowed to settle into the scalloped and/or recessed regions (510b) without any applied vacuum and allowed to settle by gravity into individual scalloped and/or recessed regions (510b).
  • the scalloped and/or recessed regions (510b) having a size similar to that of the target cells/cell population reduces the likelihood that more than one cell will settle into and/or be sucked into the scalloped and/or recessed region (510b).
  • FIG. 5C illustrates an embodiment where a cell (504c) is captured by the cell- capturing element (502c).
  • the scalloped and/or recessed region (510c) is configured to be no deeper, at its deepest point, than about 0.5-2x the height of cell (504c) and no wider than about 1-2x the width/diameter of cell (504c).
  • the depth of the scalloped and/or recessed region (510c) is about 0.5-1.5x the height of cell (504c), the width/diameter is about 1-1.5x the width/diameter of cell (504c). In an embodiment, the depth of the scalloped and/or recessed region (510c) is about 1x the height of cell (504c), the width/diameter is about 1.1-1.3x the width/diameter of cell (504c). Such depths and widths are configured to allow substantially a single cell (504c) to settle into the scalloped and/or recessed region (510b-c). As discussed herein, and shown in FIG.
  • a cell transfer component (600) comprises a bottom portion (602) and a top portion (604).
  • the cell transfer component (600) may include a lid and/or cover (612) that may be positioned so that a sealed container is achieved.
  • the lid and/or cover (612) may be a multi-well assay component, having a plurality of assay wells aligned with and/or configured to be aligned with cell capturing elements (502a-c) of the cell transfer component (600).
  • side walls (616) of the lid and/or cover (612) may extend down to at least the top of side walls (614) extending upward from the bottom portion (602) of the transfer component to form a sealed cell capturing device (600).
  • the side walls (614) may comprise a four sidewalls, with at least a first side wall Attorney Docket No.009775.00019 ⁇ WO opposing a second side wall.
  • the lid and/or cover (612) may be integral with the top of the sidewalls (614).
  • the cell capturing device (600) depicted in FIG. 6 includes at least one inlet port (606) for fluid and an outlet port (607) for the fluid. In some aspects, more than one inlet port (606) may be used.
  • a first inlet port (606) and first outlet port (607) e.g., on the first side wall and the second side wall, respectively
  • the first flow may be selected to cause cells to move across the transfer surface at a rate configured to populate the cell capturing elements.
  • a second inlet port (606) and second outlet port (607) may also, or alternatively, facilitate the introduction of a second flow of fluid beneath the transfer surface (508a-c).
  • the first flow and second flow may be selected to create a desired flow differential over the cell capturing elements (502a-c) that results in a negative pressure sufficient to capture a cell on the cell capturing element (502a-c). In some embodiments, for instance in Fig.
  • the cell capturing device (600) includes at least one outlet port (607) and, in some aspects more than one outlet port (607).
  • a cell transfer surface (e.g., a tray) (508a-c) positioned in the cell transfer component (600) may include a number of cell capturing elements (502a-c), only four of which are shown for illustrative purposes.
  • the bottom portion (602) of the cell transferring component (600) optionally in combination with the top portion (604) is fitted to the inlet port (606) with a fluid delivery system (not shown) which initially contains cell culture media and/or a population of cells.
  • a fluid delivery system not shown
  • the fluid intake rate and the fluid outlet rate are set to be equal, which will result in a constant fluid level.
  • the bottom portion (602) and the top portion (604) may be filled with the cell culture media.
  • a first flow may be established in the bottom portion (602) and a second flow established in the top portion (604).
  • the fluid intake rate may then be adjusted slightly higher and a vacuum port (610) may be activated.
  • the intake flow rate, the outlet flow rate, and the vacuum suction rate may be adjusted to be in equilibrium (the fluid level is maintained at a substantially constant level).
  • the fluid delivered through the fluid inlet port (606) may thereby be adjusted to include a heterogeneous population of cells.
  • the heterogenous population of cells may include living, viable, human cells.
  • the vacuum pressure required to maintain equilibrium increases. The process is continued until the increase in pressure indicates that Attorney Docket No.009775.00019 ⁇ WO substantially all of the cell capturing elements contain a cell stably adherent thereto.
  • the procedures for cell capturing involve placing a transfer surface (e.g., tray) (508a-c) having a number of cell capturing elements (502a-c) thereon.
  • the cell capturing device (600) is a 3-dimensional closed surface when the top portion (604) is engaged.
  • the inlet port (606), the outlet port (607), and the vacuum port (610) include locks/plugs/stoppers or other structures that may function to completely close the system.
  • the fluid provided through the inlet port (606) may include an aqueous solution comprising necessary cell nutrients maintained at physiological viscosity and temperature that may be used to transport the cells into the cell transfer component (600) (e.g., by providing a flow to cause the cells to pass over the cell transfer component (600)).
  • the wells within the assay may individually provide nutrients and therefore the nutrients may not be present in the aqueous solution introduced into the cell transfer component (600).
  • a pressure (less than about 1 atmospheres) is applied below through hole(s) (506a-c) of cell capturing element (502a- c).
  • a differential flow is established in the bottom portion (602) using one or more inlet ports (606) and outlet ports (607) in the bottom portion (602), wherein the differential flow in the bottom portion (602) relative to the fluid flow in the top portion (604) generates the required vacuum force through the cell capturing element (502a-c) holes/adhesion sites (506a-c).
  • the fluid flow in the top portion (604) is continued until the flow rate and pressure generated indicate that substantially all of the cell capturing elements contain cells, and/or until it is visually confirmed that substantially all of the cell capturing elements contain cells and/or substantially all cells in the top portion are adhered to a cell capturing element (502a-c).
  • the top portion (604) of the cell transfer component (600) is removable from the bottom portion (602).
  • the side walls (614) of the bottom portion (602) may extend above the tray (508a-c) such that the removal of the top portion (604) of the cell transfer component (600) will not expose the cells to the atmosphere.
  • the fluid level is reduced by draining (with or without a vacuum) to the bottom of the inlet port(s) (606)/ outlet port(s) (607) which lie above the tray (508a-c).
  • the top portion (604) of the cell transfer component (600) is or includes the assay device, which is configured to have the assay wells (e.g., 200a-b, not shown in FIG. 6 for clarity of illustration of the elements in FIG. 6) aligned with, and opening towards, the cell capturing elements (502a-c).
  • inverting the cell transfer component (600) will result in a transfer of the cells from the cell transfer component (600) into the assay device while being maintained, substantially at all times, in an aqueous environment.
  • the top portion (604) of the cell transfer component (600) includes the assay device, the top portion (604) may slide downward from a first lock position above the inlet port (606) to a second lock position below the inlet port (606), to minimize the distance between the cell capturing elements (502a-c) and the assay wells.
  • the distance between the assay wells in the top portion (604) of the cell transfer component (600) and the tray (508a-c), in the first locked position is, preferably, no more than about 50 microns and, more preferably, no more than about 25 microns.
  • the distance between the assay wells and ⁇ or the tray (508a-c) may be less than that prior to being slidably engaged.
  • a space formed by the top portion (604) and the assay wells may be filled with liquid, such as cell media, such that no air remains.
  • the cells Upon inverting (flipping) the locked device, the cells may be released by gravity into their respective wells of the assay device. If necessary, the vacuum line can be reversed so that a small positive pressure may be created above the cell transfer elements (502a-c) to facilitate the deposit of the cells into the respective assay wells.
  • the cell transfer elements (502b-c) are scalloped and/or recessed regions (510b-c) without hole(s)/adhesion sites (see, e.g., FIG.
  • the transfer device does not comprise a bottom portion as described above, but constitutes a transfer surface (508b-c) with scalloped and/or recessed regions (510b-c) located to face and align with centers of corresponding wells of a multi-well assay device comprising and/or attached to the cell transfer element.
  • FIG. 7 shows an example implementation of the transfer component 600 as part of a multi-well assay device.
  • the cell capturing elements (502a-c) are through holes.
  • the cell capturing elements (502a-c) are positioned to match a pitch of the assay wells (200a-b) (i.e., to align on a one-to-one basis).
  • the inlet ports 606 and outlet ports 607 are used to set up a flow of cells through the top portion (604) and a differential flow through the bottom portion (602) to establish a vacuum/negative pressure through the through holes to capture the cells.
  • the device is inverted and flows stopped to allow the cells to fall into corresponding wells.
  • FIG. 8 shows an example implementation of a transfer component 600 as part of a multi-well assay device.
  • the cell capturing elements (502b-c) are scalloped and/or recessed regions (510b-c), as disclosed herein.
  • the lid and/or cover comprises a multi-well surface comprising assay wells (200a-b).
  • the cell capturing elements (502a-c) are positioned to match the pitch of the assay wells (200a-b) (i.e., to align with on a one-to-one basis).
  • the inlet port 606 and outlet port 607 are used to set up a flow of cells through the top portion (604) so that the cells pass slowly over the scalloped and/or recessed regions (510b-c). The device is inverted, allowing the cells to fall into corresponding wells.
  • FIG. 9 shows an example multi-well assay device comprising a transfer device, as described herein.
  • the transfer component comprises a transfer surface configured as a through hole array.
  • the multi-well device is configured with ports to and from a flow cell above the through hole array and ports to and from a flow cell below the flow cell array, configured to generate a differential flow across the top and bottom of the through-hole array.
  • a difference in flow rate set up a pressure differential that traps cells as illustrated in the time-lapse images below.
  • a flow over the transfer surface i.e., between the transfer surface and assay wells
  • the flow below the transfer surface occurred at a rate of 10 microliters per minute, setting up a pressure differential drawing cells and media across the cell capturing elements.
  • Arrows indicate the direction of the flow of cells.
  • FIG. 10 shows a brightfield image of cells after being transferred from the through holes (e.g., as demonstrated in FIG. 9, to assay wells (e.g., as in the bottom portion of FIG. 7, after inverting the device).
  • the majority of wells have single cells, illustrating a low error rate of multiple cells per well or no cells per well.
  • the device of FIGS. 9-10 included 120,000 assay wells and corresponding through holes. After applying a flow with T-cells to the device and capturing with the through holes, and then inverting as in Attorney Docket No.009775.00019 ⁇ WO FIG. 7, 81% of assay wells (97,200 wells) had a single cell, 10% of wells were empty, and 9% of cells had multiple cells. These results were achieved after a total time of 10 minutes from the introduction of the T-cells to the transfer surface as in FIG. 9.
  • System for identifying a cell from a population As above, this disclosure provides for a system that allows the identification of a specific cell from a population of heterogeneous cells that are being tested in an assay well.
  • the disclosed system also encompasses the ability to identify other features used in the assay such as the technician doing the assay, the conditions being used in the assay, the date and optionally the time that the assay was conducted, the synthetic steps used to generate a test compound, and the like.
  • the system described herein may include a cell transfer component as disclosed herein, e.g., that transfers a single cell to the assay device via a vacuum force created within the cell capturing element.
  • the cell capturing element may include a number and/or size of points/area of contact configured to stably adhere the single cell to the capturing element.
  • the cell capturing element may include a multiplicity of points of contact with the vacuum force and/or an area of contact with the vacuum force such that the amount of vacuum force required to stably adhere to the single cell decreases as the points/area of contact increases.
  • the points of contact may be a number of holes/adhesion sites having a predetermined diameter. The predetermined diameter may be based on a number of the holes/adhesion sites as described herein.
  • the system so disclosed comprises a high through-put assay device (greater than 3,000 assay wells and, preferably, greater than 30,000 wells, and, more preferably, greater than 300,000 wells), an EMC, and a transfer component, each of which is described above.
  • the phrases mean any combination of one or more of the elements A, B, ... or N including any one element alone or the one element in combination with one or more of the other elements which may also include, in combination, additional elements not listed.
  • "a” or “an” means “at least one” or “one or more.”
  • Embodiment 1 A transfer component configured to transfer a single cell to a single well of a multi-well assay device.
  • Embodiment 2. The transfer component of embodiment 1, comprising a floor with side walls extending upward to form a volume.
  • Embodiment 3 The transfer component of any one of embodiments 1-2, comprising a tray positioned to separate the volume into a bottom portion comprising the floor and a top portion.
  • each cell capturing element of the plurality of cell capturing elements comprises one or more through holes between the bottom portion and the top portion, such that a flow established via the first inlet port and the first outlet port causes negative pressure through the one or more through holes in a direction from the top portion to the bottom portion, and
  • Embodiment 8 The transfer component of any one of embodiments 1-7, wherein the plurality of cell capturing elements are positioned on the tray to be configured to each align with and face a single corresponding assay well of the multi-well assay device when the multi- well assay device is engaged with the transfer component.
  • the transfer component of any one of embodiments 1-8 wherein, a second inlet port is formed in the first side wall above the tray; a second outlet port is formed in the second side wall above the tray, and the second inlet port and the second outlet port are configured to establish a flow through the top portion of the transfer component that is less than the flow established via the first inlet port and the first outlet port.
  • Attorney Docket No.009775.00019 ⁇ WO [0116]
  • Embodiment 10 The transfer component of any one of embodiments 1-9, wherein at least one cell capturing element of the plurality of cell capturing elements comprises at least four through holes. [0117] Embodiment 11.
  • Embodiment 12 The transfer component of any one of embodiments 1-11, wherein a cell capturing element of the plurality of cell capturing elements is configured to generate a vacuum force on a single cell to transfer the single cell to the assay device.
  • Embodiment 14 The transfer component of any one of embodiments 1-3, wherein the transfer device is integral to the multi-well assay device.
  • Embodiment 15 The transfer component of any one of embodiments 1-2, comprising a plurality of cell capturing elements on a surface of the floor facing into the volume.
  • each of the plurality of cell capturing elements comprises at well having a diameter within 0.5-2x an average diameter of the population of cells and a depth within 0.5-2x an average height of the population of cells, and [0126] Embodiment 20.
  • a system for conducting an assay on a heterogenous population of cells comprising: an assay device comprising a multiplicity of assay wells, wherein an assay well of the multiplicity of assay wells comprises an encoded micro-component configured to identify at least one characteristic of contents of the assay well and/or at least one aspect of an assay conducted in the assay well; and a cell transfer component configured to transfer a single cell originating from the heterogenous population of cells to the assay well, the cell transfer component comprising at least one cell capturing element aligned with and facing the assay well.
  • an assay device comprising a multiplicity of assay wells, wherein an assay well of the multiplicity of assay wells comprises an encoded micro-component configured to identify at least one characteristic of contents of the assay well and/or at least one aspect of an assay conducted in the assay well
  • a cell transfer component configured to transfer a single cell originating from the heterogenous population of cells to the assay well, the cell transfer component comprising at least one
  • Embodiment 25 The system of any one of embodiments 22-24, wherein the at least four adhesion sites are configured to facilitate release of the single cell into the assay well by applying a positive pressure.
  • Embodiment 26 The system of any one of embodiments 22-23, wherein the cell capturing element comprises at least four adhesion sites and wherein the at least four adhesion sites are configured to capture the single cell by applying negative pressure to the single cell.
  • the encoded micro-component is configured to identify one or more of: an identity of a technician conducting the assay, a temperature, pH, or atmospheric pressure in the assay well during the assay; a date and/or time the assay was conducted; one or more synthetic steps used to generate a test compound in the assay well; or one or more reaction products of the assay in the assay well.
  • Embodiment 27 The system of any one of embodiments 22-26, wherein the assay well comprises a surface that forms a recess configured to accommodate the encoded micro- component.
  • Embodiment 28 The system of any one of embodiments 22-27, wherein the recess is configured to exclude the single cell.
  • Embodiment 29 A method for performing an assay on a heterogeneous population of cells, the method comprising capturing each cell, of the heterogeneous population of cells, via negative pressure applied via a one or more adhesion sites of a respective cell capturing element of a plurality of cell capturing elements.
  • Embodiment 30 The method of embodiment 29, further comprising aligning the plurality of cell capturing elements with a plurality of assay wells. Attorney Docket No.009775.00019 ⁇ WO [0137] Embodiment 31.
  • each assay well comprising an encoded micro-component configured to identify at least one characteristic of contents of the assay well and/or at least one aspect of the assay conducted in the assay well.
  • Embodiment 32 The method of any one of embodiments 29-31, comprising releasing the captured cells from respective cell capturing elements, of the plurality of cell capturing elements, into a respective assay well, of the plurality of assay wells, aligned with the respective cell capturing element, such that a majority of the released cells each become is a single cell in a respective assay well; and [0139] Embodiment 33.
  • Embodiment 34 The method of any one of embodiments 29-33, wherein each encoded micro-component comprises a respective detectable code configured to identify the at least one characteristic and/or the at least one aspect of the assay conducted in an assay well comprising the encoded micro-component.
  • Embodiment 35 The method of any one of embodiments 29-34, comprising imaging the assay well comprising the encoded micro-component and a single cell, of the released cells, to detect the detectable code and one or more features of the single cell.
  • Embodiment 36 Embodiment 36.
  • Embodiment 37 The method of any one of embodiments 29-36, wherein the encoded micro-component is one or more of a quantum dot or a dye.
  • Embodiment 38 An assay device comprising a plurality of assay wells such that each assay well accommodates at least one cell, wherein the cell is from a population of heterogenous cells and wherein each assay well includes at least one encoded micro-component (EMC).
  • EMC encoded micro-component
  • Embodiment 40 The assay device of any one of embodiments 38-39, wherein the detectible code comprises an identifiable pattern of at least one label and/or image.
  • Embodiment 41 The assay device of any one of embodiments 38-40, wherein the detectible code on the EMC is unique to the assay well.
  • Embodiment 42 The assay device of any one of embodiments 38-41, the detectible code links the identity of the cell to an assay well from which the cell originated. Attorney Docket No.009775.00019 ⁇ WO [0149] Embodiment 43.
  • the assay device of any one of embodiments 38-42, wherein the surface of the EMC comprises an orientation marker.
  • Embodiment 44 The assay device of any one of embodiments 38-43, wherein the orientation marker is a label and/or image configured to be used to orient reading of the detectable code.
  • Embodiment 45 The assay device of any one of embodiments 38-44, wherein the detectable code on the EMC is visually detectable.
  • Embodiment 46 The assay device of any one of embodiments 38-45, wherein the detectable code on the EMC is detectable by physical means.
  • Embodiment 47 Embodiment 47.
  • Embodiment 48 The assay device of any one of embodiments 38-46, wherein the physical means includes inducing and measuring fluorescence generated on the EMC.
  • Embodiment 48 The assay device of any one of embodiments 38-47, wherein the detectable code on the EMC is detectable by chemical means.
  • Embodiment 49 The assay device of any one of embodiments 38-48, wherein at least one label included on the EMC is a detectable dot or color.
  • Embodiment 50 The assay device of any one of embodiments 38-49, wherein each detectable dot is detected by different detection means.
  • Embodiment 51 Embodiment 51.
  • Embodiment 52 The assay device of any one of embodiments 38-50, wherein the plurality of assay wells includes microwells, nanowells, and/or picowells.
  • Embodiment 52 The assay device of any one of embodiments 38-51, wherein the EMC is embedded in a bead.
  • Embodiment 53 The assay device of any one of embodiments 38-52, wherein the bead is translucent or at least one surface of the EMC is exposed from the surface of the bead, wherein the exposed surface comprises the detectible code.
  • Embodiment 54 Embodiment 54.
  • a system for conducting an assay starting with a population of cells comprising a cell transfer device configured to transfer a single cell originating from a population of cells to a single assay well of an assay device, the cell transfer device including at least one cell capturing element; and the assay device comprising the assay well.
  • the assay device comprises a multiplicity of assay wells, wherein at least one assay well in the assay device comprises the single cell transferred via the cell transfer device.
  • Embodiment 57 The system of any one of embodiments 54-55, wherein at least one assay well in the assay device comprises: an encoded micro component (EMC) comprising a detectable code configured to identify at least one characteristic of contents of the assay well and/or at least one aspect of the assay conducted in the well.
  • EMC encoded micro component
  • Embodiment 57 The system of any one of embodiments 54-56, wherein the cell transfer device comprises the transfer component of any one of embodiments 1-21.
  • Embodiment 58 The system of any one of embodiments 54-57, wherein the assay device comprises the assay device of any one of or embodiments38-52.
  • Embodiment 59 Embodiment 59.
  • Embodiment 60 The system of any one of embodiments 54-59, wherein the cell capturing element includes at least four points of contact configured to stably adhere a single cell to the capturing element.
  • Embodiment 61 The system of any one of embodiments 54-60, wherein the cell capturing element includes a multiplicity of points of contact such that a vacuum force used to stably adhere the single cell decreases as the points of contact increase.
  • Embodiment 62 The system of any one of embodiments 54-61, wherein the points of contact are holes having a predetermined diameter.
  • Embodiment 63 The system of any one of embodiments 54-62, wherein at least one surface of the EMC includes an orientation marker configured to orient reading of the detectable code.
  • Embodiment 64 The system of any one of embodiments 54-63, wherein the orientation marker is a unique label and/or image that is used to orient the reading of the detectable code.
  • Embodiment 65 The system of any one of embodiments 54-64, wherein the detectable code on the EMC is visually detectable.
  • Embodiment 66 The system of any one of embodiments 54-65, wherein the detectable code on the EMC is detectable by physical means.
  • Embodiment 67 Embodiment 67.
  • Embodiment 71 The system of any one of embodiments 54-66, wherein the physical means include inducing and measuring fluorescence generated on the EMC.
  • Embodiment 68 The system of any one of embodiments 54-67, wherein a detectable code on the EMC is detectable by chemical means.
  • Embodiment 69 The system of any one of embodiments 54-68, wherein at least one label included on the EMC is a detectable dot or color.
  • Embodiment 70 The system of any one of embodiments 54-69, wherein each detectable dot is detectable by a combination of different detection means.
  • Embodiment 71 Embodiment 71.
  • Embodiment 72 The system of any one of embodiments 54-71, wherein the EMC is embedded into a bead, and at least one surface of the EMC is visible from a surface of the bead. [0179] Embodiment 73. The system of any one of embodiments 54-72, wherein the at least one surface of the EMC exposed on the surface of the bead includes the detectible code.

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Abstract

L'invention concerne des dispositifs qui permettent des procédés de dosage décrits ici par codage de puits individuels avec un dispositif qui identifie un composant unique (par exemple, une cellule) qui doit être inclus dans chacun des puits remplis. L'invention concerne également des dispositifs et des procédés qui sont conçus pour transférer une cellule unique d'un site spécifique dans un composant de transfert dans un puits de dosage.
PCT/US2024/023594 2023-04-06 2024-04-08 Composant de transfert cellulaire pour dispositif de dosage multi-puits Ceased WO2024211897A2 (fr)

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US12577556B2 (en) 2022-05-09 2026-03-17 Zafrens Inc. Perturbation beads for use in assays

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DE19948473A1 (de) * 1999-10-08 2001-04-12 Nmi Univ Tuebingen Verfahren und Vorrichtung zum Messen an in einer flüssigen Umgebung befindlichen Zellen
EP3155396A4 (fr) * 2014-06-12 2017-12-27 Wafergen, Inc. Capture de cellules individuelles avec des films de capture en polymère
AU2020345847A1 (en) * 2019-09-10 2022-04-07 Orca Biosystems, Inc. Mesh for cell layer preparation
EP4351972A4 (fr) * 2021-06-07 2025-04-30 Plexium, Inc. Distributeurs de transfert pour dispositifs de dosage à exclusion de taille par billes

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US12577556B2 (en) 2022-05-09 2026-03-17 Zafrens Inc. Perturbation beads for use in assays

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