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WO2025178905A1 - Module de compartimentation d'échantillons - Google Patents

Module de compartimentation d'échantillons

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Publication number
WO2025178905A1
WO2025178905A1 PCT/US2025/016399 US2025016399W WO2025178905A1 WO 2025178905 A1 WO2025178905 A1 WO 2025178905A1 US 2025016399 W US2025016399 W US 2025016399W WO 2025178905 A1 WO2025178905 A1 WO 2025178905A1
Authority
WO
WIPO (PCT)
Prior art keywords
tissue sample
apertures
sample
insert
tissue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/016399
Other languages
English (en)
Inventor
Ville HÄRMÄ
Ognjen Golub
Austin Harvey
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Thermo Fisher Scientific Oy
Life Technologies Corp
Original Assignee
Thermo Fisher Scientific Oy
Life Technologies Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Thermo Fisher Scientific Oy, Life Technologies Corp filed Critical Thermo Fisher Scientific Oy
Publication of WO2025178905A1 publication Critical patent/WO2025178905A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • 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
    • B01L3/505—Containers for the purpose of retaining a material to be analysed, e.g. test tubes flexible containers not provided for above
    • B01L3/5055—Hinged, e.g. opposable surfaces
    • 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
    • B01L2200/16—Reagents, handling or storing thereof
    • 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
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/043—Hinged closures
    • 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
    • B01L2300/0822—Slides

Definitions

  • Microscopy of sectioned tissue samples stained with fluorescent dyes and/or immunostaining provides valuable histological, cellular and biomarker information.
  • fluorescent dyes and/or immunostaining e.g., direct or indirect staining with primary or secondary antibodies conjugated to fluorophores
  • spectral bleed-through i.e., detection of fluorescence from neighboring fluorescent channels in the channel of interest can hamper identification of actual targets.
  • One workaround would be to limit the number of targets by staining samples with a few spectrally separate fluorophores.
  • multiplexing the detection is often required since the availability of tissue samples can be limited, and the tissue and the detection reagents can be expensive.
  • tissue sample compartmentalization module comprising: a first member defining a first plurality of apertures; and a second member defining a second plurality of apertures, the second member pivotably connected to the first member, wherein the second member is configured to engage with the first member to form a closed configuration or pivot relative to the first member between an open configuration and a closed configuration, wherein in the closed configuration the first plurality of apertures and the second plurality of apertures are aligned.
  • the tissue sample compartmentalization module further comprises a locking arm coupled to the second member, the locking arm configured to rotate relative to the second member, wherein in the closed configuration the locking arm is configured to engage a portion of the first member to fasten the first and second members together.
  • the locking arm defines a central channel, wherein in the closed configuration the central channel of the locking arm is configured to receive a portion of the first member.
  • the tissue sample compartmentalization module further comprises an insert defining a plurality of third apertures, wherein in the closed configuration the insert is positioned between the first and second members and the third plurality of apertures are aligned with the first plurality of apertures and the second plurality of apertures.
  • Another embodiment described herein is a method for labeling one or more analytes in a tissue sample, the method comprising: (a) placing a substrate comprising the tissue sample onto the first member of the tissue sample compartmentalization module described herein; (b) aligning the tissue sample with the first member; (c) placing an insert on the tissue sample to align a plurality of third apertures defined by the insert with the plurality of first apertures defined by the first member; (d) engaging or pivoting the second member relative to the first member to sandwich the insert and the tissue sample between the first member and the second member, and to align the plurality of second apertures with the plurality of third apertures; (e) fastening the first member and the second member together by a locking arm, sealing insert to the tissue sample; and (f) incubating a solution comprising an affinity molecule in an aligned second and third aperture, wherein the affinity molecule labels an analyte in the tissue sample.
  • the sample is a tissue sample from a subject selected from humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof.
  • the analyte comprises one or more of metabolites, proteins, nucleic acids, carbohydrates, or lipids.
  • the method further comprises labeling two or more distinct analytes, wherein each aligned second and third aperture comprises a solution comprising a distinct affinity molecule for each analyte.
  • the affinity molecule is an antibody, a portion of an antibody, an antibody-like molecule, a ligand receptor, a ligand for a receptor, one member of a coupling pair, an aptamer, or an antigen.
  • the affinity molecule is conjugated to a fluorescent molecule, conjugated to an enzyme, bound by another affinity molecule that is conjugated to a fluorescent molecule, or bound by another affinity molecule that is conjugated to an enzyme.
  • the substrate is a microscope slide.
  • Another embodiment described herein is method for dividing a tissue sample into compartments for labeling two or more analytes in the tissue sample comprising: (a) placing a substrate comprising the tissue sample onto the first member of the tissue sample compartmentalization module described herein; (b) aligning the tissue sample with the first member; (c) placing an insert on the tissue sample to align a plurality of third apertures defined by the insert with the plurality of first apertures defined by the first member; (d) engaging or pivoting the second member relative to the first member to sandwich the insert and the tissue sample between the first member and the second member, and to align the plurality of second apertures with the plurality of third apertures; and (e) fastening the first member and the second member together by a locking arm, sealing insert to the tissue sample.
  • the sample is a tissue sample from a subject selected from humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof.
  • the substrate is a microscope slide.
  • step (c) further comprises overlapping as many of the plurality of first apertures defined by the first member; as possible with the tissue sample.
  • Another embodiment described herein is a method of analyzing a tissue sample for a plurality of analytes comprising: (a) placing a substrate comprising the tissue sample onto the first member of the tissue sample compartmentalization module described herein; (b) aligning the tissue sample with the first member; (c) placing an insert on the tissue sample to align a plurality of third apertures defined by the insert with the plurality of first apertures defined by the first member; (d) engaging or pivoting the second member relative to the first member to sandwich the insert and the tissue sample between the first member and the second member, and to align the plurality of second apertures with the plurality of third apertures; (e) fastening the first member and the second member together by a locking arm, sealing insert to the tissue sample; and (f) incubating a solution comprising an affinity molecule in an aligned second and third aperture, wherein the affinity molecule labels an analyte in the tissue sample, (g) detecting a signal from each affinity molecule that is bound to the plurality of
  • kits comprising: one or more tissue sample compartmentalization modules described herein; one or more affinity molecules; one or more signaling reagents; optionally, one or more reagents comprising deparaffinization reagents, enzyme activation reagents, antigen retrieval reagents, sample dilution reagents, reagent dilution buffers, blocking reagents, or endogenous enzyme activity blocking reagents; and optionally, one or more containers, packaging, instructions for use, MSDS sheets, reference or control tissue samples, or reference or control targets.
  • FIG. 1 A-B show an example of spatial transcriptomics.
  • FIG. 1 A shows the spectral emission profiles of 9 fluorophores illustrating the spectral overlap of different dyes (DAPI, eFluor 506, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 555, eFluor 615, Alexa Fluor 647, Alexa Fluor 700, Alexa Fluor 750).
  • FIG. 1 B shows a spectrally mixed (left) and unmixed (right) composite images of human tonsil tissue sample stained with 9 fluorophores.
  • FIG. 3 shows an exemplary illustration of a compartmentalized tissue sample on a microscope slide.
  • a tissue section is affixed to the slide and divided into multiple sample area.
  • the compartmentalization is a 4 x 4 array.
  • the shape of the tissue sample controls the number of usable compartments. In the example, only 8 individual sample area are compartmentalized in the tissue.
  • FIG. 4 A-B show an exemplary prototype tissue compartmentalization module.
  • FIG. 4A shows a typical size tissue samples drawn with a blue marker, with two tissues drawn per microscope slide.
  • FIG. 4B shows two prototype compartmentalization apparata attached on the slide from FIG. 4A.
  • FIG. 5 A-E show fluorescent microscopy data obtained using a prototype tissue sample compartmentalization apparatus with three fluorescent dyes and an unstained control.
  • FIG. 5A shows the compartmentalized tissue sample indicating the three dyes (eF506, AF532, AF488) and an unstained control.
  • FIG. 5B-E shows the spectral data for eF506, AF532, AF488, and the unstained control, respectively, as compared to standard methodology in which a whole tissue section is stained without compartmentalization.
  • FIG. 7A-D show a process workflow for using the tissue sample compartmentalization module.
  • FIG. 7A illustrates a microscope slide is shown with tissue sections.
  • FIG. 7B illustrates two tissue sample compartmentalization modules in an open configuration are centered on each tissue section to maximize the available tissue.
  • FIG. 7C illustrates the modules in a closed and locked configuration, and immunohistochemical (or other) assays are performed.
  • FIG. 7D illustrates the modules removed where the samples are imaged using fluorescence microscopy and spectral extraction is performed as needed.
  • FIG. 8A-B show fluorescence microscopy data for two fluorophores (AF488, AF555) obtained using a tissue sample compartmentalization module as compared to standard methodology.
  • FIG. 9A-D show a process workflow for using the tissue sample compartmentalization module with individual cell cultures.
  • FIG. 9A shows a cell attachment enhancing surface coated microscope slide.
  • FIG. 9B shows cell suspensions seeded in individual compartments created by the tissue sample compartmentalization module.
  • FIG. 90 shows experiments being performed in individual sample environments of the tissue sample compartmentalization module (e.g., drug treatments, immunofluorescence staining).
  • FIG. 9D shows a microscope slide ready for analysis after the issue sample compartmentalization modules have been removed. DETAILED DESCRIPTION
  • amino acid As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.
  • the term “substantially” means to a great or significant extent, but not completely.
  • the term “about” or “approximately” as applied to one or more values of interest refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system.
  • the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ⁇ 10% of the value modified by the term “about.”
  • “about” can mean within 3 or more standard deviations, per the practice in the art.
  • the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value.
  • the symbol means “about” or “approximately.”
  • All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ⁇ 10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”
  • an “analyte” or “antigen” as used herein refers to any substance recognized by an antibody, or another means of detection.
  • a “detectable label” as used herein refers to any molecule which may be detected directly or indirectly to reveal the presence of a target in the sample.
  • a direct detectable label may be used.
  • Direct detectable labels may be detected per se without the need for additional molecules. Examples include fluorescent dyes, radioactive substances, and metal particles.
  • Indirect detectable labels may be used, which require the employment of one or more additional molecules. Examples include enzymes that affect a color change in a suitable substrate, as well as any molecule that may be specifically recognized by another substance carrying a label or react with a substance carrying a label.
  • Other examples of indirect detectable labels thus include antibodies, antigens, nucleic acids and nucleic acid analogs, ligands, substrates, and haptens.
  • color labels include, but are not limited to, chromophores, fluorophores, chemiluminescent compounds, electrochemiluminescent labels, bioluminescent labels, and enzymes that catalyze a color change in a substrate. More than one type of color may be used, for instance, by attaching distinguishable color labels to a single detection unit or by using more than one detection unit, each carrying a different and distinguishable color label.
  • Fluorophore as used herein is a molecule that emits detectable electro-magnetic radiation upon excitation with electro-magnetic radiation at one or more wavelengths.
  • fluorophores A large variety of fluorophores are known in the art and are developed by chemists for use as detectable molecular labels and can be conjugated to affinity molecules described herein.
  • the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
  • a probe may react with a target, or directly bind to a target, or indirectly react with or bind to a target by directly binding to another substance that in turn directly binds to or reacts with a target.
  • the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, guinea pigs, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or archaea, among other life forms. In one embodiment, the subject is a primate. In one embodiment, the subject is a human. [0038] “Target,” also used interchangeably with “analyte,” as used herein refers to any substance present in a sample that is capable of being detected.
  • primates e.g., humans, male or female; infant, adolescent, or adult
  • nonhuman primates e.g., rats, mice, guinea pigs, rabbits, pigs, cows, sheep
  • the module 100 includes a first member 104 and a second member 108.
  • the second member 108 is pivotably connected to the first member 104 by a pivot assembly 112.
  • the pivot assembly 112 includes a first pivot member 116 coupled to the second member 108.
  • the first pivot member 116 is received by a first slot 120 defined by the first member 104.
  • the pivot assembly 112 is configured to facilitate pivoting movement of the second member 108 relative to the first member 104 between a first, open configuration (shown in FIG. 6A) and a second, closed configuration (shown in FIG. 6B). Stated another way, the second member 108 is configured to rotate relative to an axis defined by the first pivot member 116.
  • the first member 104 includes a planar surface 124.
  • a plurality of first apertures 128 are arranged on the planar surface 124.
  • the first apertures 128 extend through the planar surface 124, and entirely through the first member 104.
  • the first member 104 defines the plurality of first apertures 128.
  • the plurality of first apertures 128 are arranged in a grid pattern. More specifically, the plurality of first apertures 128 are arranged in a 5 x 4 grid pattern.
  • the plurality of first apertures 128 can be arranged in any suitable or desired pattern.
  • the plurality of first apertures 128 are illustrated as having a square shape. In other examples of embodiment, the plurality of first apertures 128 can have any suitable polygonal shape (e.g., a circle, a triangle, a rectangle, a pentagon, etc.).
  • the first member 104 includes a pair of first projections 132 that extend away from the planar surface 124.
  • the first projections 132 each define the slot 120.
  • the first member 104 also includes a second projection 136.
  • the second projection 136 extend away from the planar surface 124 such that the planar surface 124 is recessed relative to the projections 132, 136. Stated another way, the first member 104 is offset from the second member 108.
  • the projections 132, 136 are respectively positioned on opposing ends of the first member 104.
  • the projections 132, 136 also serve as guides to facilitate alignment of a microscope slide on the planar surface 124 of the first member 104.
  • each first, second, and third aperture 128, 140, 160 is aligned (or vertically aligned).
  • the aligned first, second, and third apertures 128, 140, 160 define a passage that extends entirely thought the module 100.
  • the first, second, and third apertures 128, 140, 160 can also be referred to as first, second, and third wells 128, 140, 160.
  • tissue sample compartmentalization modules described herein may be manufactured using standard manufacturing methods including injection molding, 3D-printing, casting, laser ablation, punching, other means, or combinations thereof.
  • the tissue sample compartmentalization modules may comprise materials such as plastics (polyethylene, polypropylene, polystyrene, polyoxymethylene, polytetrafluoroethylene, copolymers), metals (aluminum, titanium, stainless steel), glass, composite materials (graphite, carbon fibers, fiberglass), or combinations thereof.
  • the insert or gasket may comprise water-tight materials such as silicone, closed cell foams, open cell foams, polytetrafluoroethylene, rubber, fiberglass, aramid fibers, felt, cork, paper, or combinations thereof.
  • FIG. 7B a plurality of tissue sample compartmentalization modules 100 are placed into engagement with the microscope slide 200.
  • a pair of modules 100 are positioned into engagement with the slide 200, such that one module 100 is associated with each tissue sections 204a, 204b.
  • Each module 100 is positioned in an open configuration, as shown in FIG. 6A.
  • the microscope slide 200 is positioned on the planar surface 124 of the first member 104 of each module 100.
  • the projections 132, 136 of each member facilitate alignment of the slide 200 relative to the first member 104.
  • the slide 200 can be laterally moved relative to the first member 104 to align the respective tissue section 204a, 204b over the plurality of first apertures 128.
  • the insert 156 is then positioned onto the slide 200.
  • the insert 156 is positioned over each respective tissue section 204a, 204b with the plurality of third apertures 160 being vertically aligned with the plurality of first apertures 128.
  • the insert 156 can be manually positioned on the slide 200 or can be configured to be positioned on the slide 200 in response to pivoting movement of the second member 108 relative to the first member 104 from the open configuration (see FIG. 6A) to the closed configuration (see FIG. 6B).
  • each module 100 is actuated into the closed configuration and locked.
  • the second member 108 of each module 100 is pivoted relative to the first member 104 to align the plurality of second apertures 140 with the plurality of third apertures 160 of the insert 156.
  • the locking arm 144 can then pivot relative to the second member 108 into engagement with the first member 104, locking the members 104, 108 together.
  • the slide 200 and the insert 156 are sandwiched (or positioned between) the members 104, 108.
  • the second and third apertures 140, 160 cooperate to form a plurality of wells that are separated from the first apertures 128 by the slide 200.
  • IHC provides a method of detecting targets in a sample or tissue specimen in situ. See e.g., Mokry, Acta Medica 39(4): 129-140 (1996). The overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the targets of interest.
  • a sample is fixed with formalin, embedded in paraffin, and cut into sections for staining and subsequent inspection by light microscopy.
  • Current methods of IHC use either direct labeling or secondary antibody-based or hapten-based labeling.
  • Samples may comprise a cell sample, such as a cell smear or colony, or a tissue specimen derived from a living organism, such as a tissue sample from an organ. Samples may also comprise other naturally obtained samples such as plant tissue samples, and synthetically derived samples such as chemical or industrial products and food products.
  • Treatments may be performed to reduce nonspecific binding.
  • carrier proteins, carrier nucleic acid molecules, salts, or detergents may reduce or prevent non-specific binding.
  • Non-specific binding sites may be blocked in some embodiments with inert proteins like, HSA, BSA, ovalbumin, with fetal calf serum or other sera, or with detergents like polyoxyethylene sorbitan monolaurate (TWEEN®20), octylphenoxypolyethoxyethanol (Nonidet P-40), t- octylphenoxypolyethoxyethanol (TRITONTM X-100), triterpene glycosides (Saponin), nonionic polyoxyethylene surfactants (BRIJ®-35), or nonionic triblock copolymers (PLURONICS®).
  • non-specific binding sites may be blocked with unlabeled competitors for the recognition event between the target and the affinity molecule.
  • non-specific binding may be reduced by adding unlabeled competitor nucleic acids or nucleic acid analogs such as digested, total human DNA, or unlabeled versions of the affinity molecule.
  • repetitive sequences may be blocked, for example, using nucleic acids or nucleic acid analogs that specifically recognize those sequences, or sequences derived from a total DNA preparation. Salt, buffer, and temperature conditions may also be modified to reduce non-specific binding.
  • Cross reactivity of different components of the detection methods may be avoided, for example, by using antibodies derived from different species.
  • combinations of, for example, secondary antibodies against primary antibodies and haptens may also be used to avoid unwanted cross reactivity.
  • Endogenous biotin binding sites or endogenous enzyme activity for example phosphatase, catalase, or peroxidase
  • Endogenous biotin and peroxidase activity may be removed as a step in the staining procedure.
  • Endogenous biotin and peroxidase activity may be removed by treatment with peroxides, while endogenous phosphatase activity may be removed by treatment with levamisole. Heating may destroy endogenous phosphatase and esterase activity.
  • the method may comprise: placing a substrate comprising the tissue sample onto the first member of the tissue sample compartmentalization module, wherein the tissue sample compartmentalization module comprises a first member defining a first plurality of apertures; and a second member defining a second plurality of apertures, the second member pivotably connected to the first member, the second member is configured to engage with the first member to form a closed configuration or pivot relative to the first member between an open configuration and a closed configuration, wherein in the closed configuration the first plurality of apertures and the second plurality of apertures are aligned; aligning the tissue sample with the first member; placing an insert on the tissue sample to align a plurality of third apertures defined by the insert with the plurality of first apertures defined by the first member; engaging or pivoting the second member relative to the first member to sandwich the insert and the tissue sample between the first member and the second member, and to align the plurality of second apertures with the
  • the method may also include labeling two or more distinct analytes.
  • Each aligned second and third aperture of tissue sample compartmentalization module described herein may comprise a solution comprising a distinct affinity molecule for each analyte.
  • the methods described herein may further comprise multiplexing performed for two or more targets simultaneously, wherein the two or more target molecules are each separately bound by affinity molecules. Multiplexing may be performed for 10 or more targets simultaneously, wherein the 10 or more target molecules are each separately bound by affinity molecules. Multiplexing may be performed for 100 or more targets simultaneously, wherein the 100 or more target molecules are each separately bound by affinity molecules.
  • the method may include: placing a substrate comprising the tissue sample onto the first member of the tissue sample compartmentalization module, wherein the tissue sample compartmentalization module comprises a first member defining a first plurality of apertures; and a second member defining a second plurality of apertures, the second member pivotably connected to the first member, the second member is configured to engage with the first member to form a closed configuration or pivot relative to the first member between an open configuration and a closed configuration, wherein in the closed configuration the first plurality of apertures and the second plurality of apertures are aligned; aligning the tissue sample with the first member; placing an insert on the tissue sample to align a plurality of third apertures defined by the insert with the plurality of first apertures defined by the first member; engaging or pivoting the second member relative to the first member to sandwich the insert and the tissue sample between the first member and the second member, and to align the plurality of second apertures with the plurality
  • An analyte or plurality of analytes described herein may include one or more of metabolites, proteins, nucleic acids, carbohydrates, or lipids.
  • An affinity molecule described herein may be an antibody, a portion of an antibody, an antibody-like molecule, a ligand receptor, a ligand for a receptor, one member of a coupling pair, an aptamer, or an antigen.
  • the affinity molecule may comprise a primary antibody.
  • the affinity molecule may comprise a secondary antibody.
  • the affinity molecule may be conjugated to a fluorescent molecule, conjugated to an enzyme, bound by another affinity molecule that is conjugated to a fluorescent molecule, or bound by another affinity molecule that is conjugated to an enzyme.
  • the method may comprise use of more than one affinity molecule, for example, two affinity molecules, three affinity molecules, four affinity molecules, five affinity molecules, six affinity molecules, seven affinity molecules, eight affinity molecules, nine affinity molecules, or 10 affinity molecules. More than 10 affinity molecules may be used in any of the methods described herein.
  • An affinity molecule described herein may be detected by a signal from a detectable label associated with the affinity molecule which may be directly or indirect associated with the affinity molecule.
  • a detectable label used herein may be a fluorophore, polymer particle, metal particle, hapten, enzyme, luminescent label, radioactive label, and the like.
  • fluorophores examples include fluorescein or its derivatives, such as fluorescein-5- isoth iocyanate (FITC), 5-(and 6)-carboxyfluorescein, 5- or 6-earboxyfluorescein, 6-(fluorescein)- 5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate, rhodamine or its derivatives such as tetramethylrhodamine and tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC).
  • fluorescein or its derivatives such as fluorescein-5- isoth iocyanate (FITC), 5-(and 6)-carboxyfluorescein, 5- or 6-earboxyfluorescein, 6-(fluorescein)- 5-(and 6)-carboxamido hexanoic acid, fluorescein isothiocyanate, rhodamine or its derivatives such as
  • fluorophores useful herein include, but are not limited to, fluorescent proteins such as green fluorescent protein (GFP) and its analogs or derivatives, fluorescent amino acids such as tyrosine and tryptophan and their analogs, fluorescent nucleosides, and other fluorescent molecules such as Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, Cy 7, IR dyes, Dyomics dyes, phycoerythrins, Oregon green 488, pacific blue, rhodamine green, and Alexa dyes.
  • fluorescent labels which may be used herein include conjugates of phycoerythrin, inorganic fluorescent labels such as particles based on semiconductor material like coated CdSe nanocrystallites.
  • polymer particles labels include, but are not limited to, microparticles, beads, or latex particles of polystyrene, PMMA or silica, which can be embedded with fluorescent dyes, or polymer micelles or capsules which contain dyes, enzymes, or substrates.
  • metal particles include, but are not limited to, gold particles and coated gold particles, which can be converted by silver stains.
  • haptens include, but are not limited to, fluorophores, myc, nitrotyrosine, biotin, avidin, strepavidin, 2,4-dinitrophenyl, digoxigenin, bromodeoxyuridine, sulfonate, acetylaminofluorene, mercury trinitrophenol, and estradiol.
  • HRP horse radish peroxidase
  • examples of commonly used substrates for horse radish peroxidase include, but are not limited to, Alexa FluorTM 350, Alexa FluorTM 488, Alexa FluorTM 546, Alexa FluorTM 555, Alexa FluorTM 568, Alexa FluorTM 594, and Alexa FluorTM 647 Tyramide Reagents or Biotin-XX Tyramide Reagent (Thermo Fisher), 3,3'-diaminobenzidine (DAB), diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole (AEC), Benzidine dihydrochloride (BDHC), Hanker- Yates reagent (HYR), Indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1 -naphtol (CN), a-naphtol pyronin (a-NP), o-dianisidine (OD), 5-bromo-4-chlor
  • Both colors can be detected simultaneously, such as by fusion or juxtaposition of the signals, signal enhancement or quenching, or detection of multiple colors in the sample.
  • the exact choice of detectable label or combinations of detectable labels may be based on personal preferences in combinations with restrictions of the sample type, sample preparation method, detection method and equipment, and optional contrasting labels used in the sample.
  • the apparatus and methods disclosed herein can be applied to a variety of targets. Any target which can be recognized by a suitable affinity molecule is compatible with the apparatus and methods described herein.
  • the recognition may be direct or indirect, via another affinity molecule, such as at least one primary, secondary, or higher order affinity molecule.
  • a target or analyte may comprise a protein, such as a glycoprotein or lipoprotein, phosphoprotein, methylated protein, or a protein fragment, a peptide, or a polypeptide.
  • a target or analyte may comprise a nucleic acid segment or a nucleic acid analog segment.
  • a target or analyte may comprise one or more of lipids; glyco-lipids; carbohydrates; polysaccharides; salts; ions; or a variety of other organic and inorganic substances.
  • a target or analyte may be expressed on the surface of the sample, such as on a membrane or interface.
  • a target or analyte may be contained in the interior of the sample.
  • an interior target or analyte may comprise a target or analyte located within the cell membrane, periplasmic space, cytoplasm, or nucleus, or within an intracellular compartment or organelle.
  • An approximate amount of a target in a sample may be determined.
  • a control target within the sample may be assayed as well as an experimental target.
  • a nucleic acid target for example, a chromosomal paint or counterstain may be used.
  • the intensity of a contrasting label for the plasmid or chromosome or a neutral locus thereon may be compared to the intensity of the target locus.
  • the intensity of the label from the sample may also be compared to that of a known standard or control sample.
  • Immunohistochemical staining is used for detecting specific antigens in tissues.
  • tissue samples are formalin fixed to preserve the integrity of the tissue.
  • a tissue section that was embedded in paraffin must be deparaffinized (i.e., dewaxed) and then rehydrated before applying the primary antibody.
  • a tissue section that was frozen and embedded in optimal cutting temperature compound (OCT) must be rehydrated before applying the primary antibody.
  • Enzyme- conjugated secondary antibodies are then applied, and the specific staining can be visualized after adding the enzyme-specific substrate.
  • an antigen may be unmasked or the signal enhanced by means described herein, such as by enzyme digestion or microwave antigen retrieval.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un appareil de compartimentation d'échantillons tissulaires et des procédés de marquage et d'identification d'un ou de plusieurs analytes dans un échantillon tissulaire. Dans un mode de réalisation, l'appareil comprend un module de compartimentation d'échantillons tissulaires comprenant un premier élément définissant une première pluralité d'ouvertures ; et un second élément définissant une seconde pluralité d'ouvertures, le second élément étant relié de manière pivotante au premier élément, le second élément étant conçu pour venir en prise avec le premier élément afin de former une configuration fermée ou pivoter par rapport au premier élément entre une configuration ouverte et une configuration fermée, dans la configuration fermée, la première pluralité d'ouvertures et la seconde pluralité d'ouvertures étant alignées et formant une pluralité de compartiments d'échantillons. Le module de compartimentation d'échantillons tissulaires permet des environnements d'échantillons individuels pour des lames de microscope tissulaire sectionnées.
PCT/US2025/016399 2024-02-22 2025-02-19 Module de compartimentation d'échantillons Pending WO2025178905A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010062310A1 (fr) * 2008-10-28 2010-06-03 Millipore Corporation Ensemble pour culture biologique
US20230017773A1 (en) * 2020-06-10 2023-01-19 10X Genomics, Inc. Fluid delivery methods
US20230242976A1 (en) * 2018-12-10 2023-08-03 10X Genomics, Inc. Imaging system hardware

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010062310A1 (fr) * 2008-10-28 2010-06-03 Millipore Corporation Ensemble pour culture biologique
US20230242976A1 (en) * 2018-12-10 2023-08-03 10X Genomics, Inc. Imaging system hardware
US20230017773A1 (en) * 2020-06-10 2023-01-19 10X Genomics, Inc. Fluid delivery methods

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Methods in Molecular Biology", vol. 80, 1998, HUMANA PRESS
LARSSON: "Immunocytochemistry: Theory and Practice", 1988, CRC PRESS
MOKRY, ACTA MEDICA, vol. 39, no. 4, 1996, pages 129 - 140
SHI ET AL., J. HISTOCHEM. CYTOCHEM., vol. 45, no. 3, 1997, pages 327 - 343

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