WO2023229988A1

WO2023229988A1 - Tissue sample mold

Info

Publication number: WO2023229988A1
Application number: PCT/US2023/023087
Authority: WO
Inventors: Meng Zhang; Fernandino VALDECANAS; Janine Hensel; Albert Kim; Monica NAGENDRAN; Seayar Mohabbat
Original assignee: 10X Genomics, Inc.
Priority date: 2022-05-23
Filing date: 2023-05-22
Publication date: 2023-11-30

Abstract

A mold for processing a tissue sample includes a receptacle having a surface surrounded by at least one sidewall to define an interior space for receiving the tissue sample, and one or more guides extending from the surface in a direction perpendicular to a plane of the surface. The one or more guides are positioned within the receptacle to designate areas for receiving a portion of the tissue sample within the interior space, and the plurality of areas each comprise a predetermined area of the surface. A tissue specimen slide can be prepared by positioning one or more tissue samples in the designated areas on the surface of the mold, filling the mold with an embedding material to embed the tissue samples in the embedding material, cooling the embedding material in the mold to produce a tissue-embedded block, and removing the tissue-embedded block from the mold after cooling.

Description

TISSUE SAMPLE MOLD

This patent application claims the benefit of U.S. provisional patent application

63/344,790, filed May 23, 2022, the entire contents of which is incorporated herein by reference in its entirety.

BACKGROUND

Cells within a tissue of a subject have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell’s position relative to neighboring cells or the cell’s position relative to the tissue microenvironment) can affect, e.g., the cell's morphology, differentiation, fate, viability, proliferation, behavior, and signaling and crosstalk with other cells in the tissue.

Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provides substantial analyte data for dissociated tissue (i.e., single cells), but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample).

Preparation of tissue samples can involve embedding tissue in an embedding material such as a wax. Thin slices of the embedded tissue are prepared and placed on a microscope slide for analysis.

SUMMARY

Using a base mold having guides for placing the tissue samples can advantageously improve the positioning of the tissue in embedding material so that the tissue samples can be properly aligned on a microscope slide, for example, in the form of slices of embedded tissue placed on a slide. After placement of the tissue samples in the designated sections or areas of the base mold, the tissue samples can be embedded in embedding material in the base mold. When the tissue samples are placed in positions in the base mold designated by guides, the resulting slices of embedded tissue will be aligned with visual guides or arrays on the microscope slide for improved analysis. Using a base mold with guides can improve the accuracy of positioning the tissue samples on microscope slides, and the efficiency of producing quality slides for analysis, for example for analysis in spatial transcriptomics, in situ spatial analyte analysis, and pathology and forensics. The tissue samples can be positioned on and/or within an embedding material so that the samples will be appropriately positioned on a substrate (e.g., a microscope slide). Described herein are apparatuses (e.g., molds) and methods of preparing tissue samples with an embedding material so that the tissue samples can be properly positioned on a substrate, such as the microscope slide, for accurate tissue analysis.

In an aspect, a mold for processing a tissue sample includes a receptacle having a surface surrounded by at least one sidewall to define an interior space for receiving the tissue sample, and one or more guides extending from the surface in a direction perpendicular to a plane of the surface. The one or more guides are positioned within the receptacle to provide or designate a plurality of areas for receiving a portion of the tissue sample within the interior space, and the plurality of areas each comprise a predetermined area of the surface.

In some implementations, the plurality of areas include tw o. three, four, six, eight, or ten areas (also referred to as sections herein). In some implementations, the predetermined area of the surface of the plurality of areas are equally sized and shaped.

In some implementations, the at least one sidewall includes a first sidewall and a second sidew all opposite the first sidewall, and a third sidewall and a fourth sidewall opposite the third sidewall, and the one or more guides are positioned in at least one line extending in a direction parallel to a first longitudinal axis extending from the first sidewall to the second sidewall. In some implementations, additionally or alternatively, at least a portion of the one or more guides are positioned in a line extending in a direction parallel to a second longitudinal axis of the receptacle from the third sidewall to the fourth sidewall. In some implementations, additionally or alternatively, the one or more guides are positioned equidistant from the first sidewall and the second sidewall.

In some implementations, the one or more guides include one or more of pins, plates, posts, and walls. In some implementations, the one or more guides define walls extending from the surface in a direction perpendicular to the plane of the surface within the interior space. In some implementations, the one or more guides comprises a biocompatible metal or metal alloy, for example, medical grade stainless steel. In some implementations, the one or more guides comprise a medical grade polymer. In some implementations, each of the one or more guides has a height of 0.5 mm to 50 mm. In some implementations, each of the one or more guides has a height of about 0.5 mm to 1 mm. In some implementations, the mold is configured to receive one or more portions of the tissue sample and an embedding material in one or more of the predetermined areas of the surface of the receptacle. In some implementations, the interior space of the mold has a length of about 24 mm and a width of about 12 mm. In some implementations, the interior space of an arrayed area for a spatial transcriptomics array has a length of about 6.5 mm or about 11 mm and a width of about 6.5 mm or about 11 mm. In some implementations, the mold is used to prepare two or more tissue samples for use on a spatial transcriptomics microscope slide. In some implementations, the mold is used to prepare tissue samples for use in a pathology -related assay. In some implementations, the mold is used to prepare tissue samples for use in a determination of the location of analytes in the tissue samples.

In another aspect, a method of preparing a tissue specimen slide includes disposing one or more tissue samples on a surface of a mold, the mold including the surface surrounded by at least one sidewall to define an interior space and one or more guides extending from the surface, where the one or more guides are positioned within the interior space to designate a plurality of areas, and the areas each define a predetermined area of the surface for receiving a tissue sample within the interior space. The method also includes filling the mold with an embedding material to embed the one or more tissue samples in the embedding material, cooling the embedding material in the mold to produce a tissue-embedded block, and removing the tissue-embedded block from the mold after cooling.

In some implementations, disposing the one or more tissue samples in the mold includes determining a placement of each of the one or more tissue samples based on a visual indication of the plurality of areas designated by the one or more guides, and placing each of the one or more tissue samples on the surface of the mold within one of the plurality of predetermined areas designated by the one or more guides. In some implementations, the method also includes sectioning the tissue-embedded block into multiple tissue sections, where the sectioning is parallel to a plane of the surface, and positioning one of the plurality of tissue sections on a microscope slide. Each tissue section includes a portion of each of the one or more tissue samples surrounded by the embedding material, and the embedding material includes an aperture formed through the embedding material at a position corresponding to at least one of the one or more guides in the mold. In some implementations, positioning one of the tissue sections onto a microscope slide includes floating a tissue section on a water surface, positioning the microscope slide beneath the tissue section, and contacting the microscope slide with a surface of the tissue section. In some implementations, the method also includes aligning the aperture with a fiducial on the microscope slide before the contacting of the microscope slide with the surface of the tissue section. In some implementations, the fiducial is formed as a printed shape on the microscope slide. In some implementations, the fiducial is positioned relative to an array on the microscope slide. In some implementations, the method also includes preserving the one or more tissue samples before placing in the mold.

In another aspect, a method for preparing a tissue sample for tissue analysis includes disposing one or more tissue samples on a surface of a mold, the surface of the mold surrounded by at least one sidewall to define and interior space, the interior space having a predetermined length and a predetermined width. The method also includes filling the mold with an embedding material to embed the one or more tissue samples in the embedding material, cooling the one or more tissue samples in the embedding material to form a tissue- embedded block, and removing the tissue-embedded block from the mold. The method also includes sectioning the tissue-embedded block into multiple tissue sections and positioning at least one of the multiple tissue sections in a designated sample area of a microscope slide.

In some implementations, the one or more tissue samples includes a plurality of tissue samples each representing a different tissue sample type. In some implementations, disposing one or more tissue samples on a surface of a mold includes determining a placement of each of the one or more tissue samples based on a visual indication of a plurality of predetermined areas of the surface designated by one or more guides extending from the surface of the mold, the one or more guides positioned within the interior space to designate predetermined areas of the surface for receiving a tissue sample of the one or more tissue samples within the interior space, and placing each of the one or more tissue samples on the surface of the mold within one of the predetermined areas designated by the one or more guides.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

DESCRIPTION OF DRAWINGS

The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.

FIG. 1 shows an exemplary mold with guides.

FIGs. 2A-E show five exemplary arrangements of guides within a base mold.

FIGs. 3A-C show an exemplary mold with guides corresponding to a microscope slide with locations for placement of eight tissue sections. FIG. 3A shows a mold with guides defining predetermined areas. FIG. 3B shows a microscope slide with arrayed areas corresponding to the predetermined areas of FIG. 3A. FIG. 3C shows the sample area of the microscope slide of FIG. 3B with tissue samples positioned in the arrayed areas through the use of the mold of FIG. 3A.

FIGs. 4A-E show an exemplary process for sectioning and placement of tissues on a microscope slide for tissue analysis. FIG. 4A shows embedded tissue blocks prepared using a base mold with guides. FIG. 4B shows sectioning via microtomy of embedded tissues of an exemplary embedded tissue block of FIG. 4A. FIG. 4C shows a slice or section of an exemplary embedded tissue block of FIG. 4A. FIG. 4D shows the placement of the tissue slice or section of FIG. 4C on an exemplary microscope slide, and FIG. 4E shows the placement of the exemplary slice or section on an exemplary microscope slide.

FIGs. 5A-5C show an exemplary process for preparing embedded tissue blocks for tissue analysis. FIG. 5A shows an exemplary base mold. FIG. 5B shows an embedded tissue block prepared using the mold of FIG. 5A and including multiple tissue samples. FIG. 5C shows an embedded tissue block prepared using the mold of FIG. 5A and including a single tissue sample.

FIG. 6 shows an exemplary arrangement of multiple tissue samples within a sample area of a microscope slide or assay device.

FIGs. 7A-E show an exemplary process for sectioning and placement of multiple tissues on a microscope slide for tissue analysis. FIG. 7A shows an embedded tissue block prepared using a base mold. FIG. 7B shows removal of a slice or section of the exemplary embedded tissue block of FIG. 7A. FIG. 7C shows the placement of the slice or section of FIG. 7B on an exemplary microscope slide, and FIG. 7D shows the placement of the exemplary slice or section on an exemplary microscope slide with other tissue sections. FIG. 7E shows a micrograph of the exemplary microscope slide of FIG. 7D.

DETAILED DESCRIPTION

Placement of sectioned tissue samples for analysis, for example when a tissue section requires specific placement on a substrate for analysis, can be difficult. Often times, tissue samples to be analyzed by certain methodologies require very small tissue sections. For example needle biopsies, or tissue related assays have size constraints that are related to the size of tissue to be assayed. As such, the sectioning of tissue samples can yield very small tissue sections that can be difficult to place on a microscope slide at a desired location for further analysis.

As provided by this disclosure, the solution to the problem of tissue sectioning and placement for further analysis are detailed herein. Tissue samples can be positioned on and/or within an embedding material so that the samples will be appropriately positioned when finally placed on a substrate (e.g., a microscope slide). Described herein are apparatuses (e.g., molds) and methods of preparing tissue samples with an embedding material so that the tissue samples can be properly sectioned and positioned on a substrate, such as a microscope slide, for downstream tissue analysis.

The apparatuses and methods described herein are directed to positioning tissue samples in a modified mold within embedding material so that the samples will be appropriately positioned for placement on the microscope slide, which can be otherwise difficult to achieve due to sample migration during the embedding process and/or microscope placement process, the small size of the sample, or delicate state of the samples that may be easily damaged during handling. In some embodiments, tissue samples are placed or intended to be placed on microscope slides that include fiducials designating specific placement locations of the tissue for analysis purposes. In such embodiments, apparatuses and methods provided herein can prepare tissue samples embedded in the embedding material that align the samples on a particular microscope slide within a fiducial pattern or design, resulting in samples are positioned to produce more accurate tissue analysis results. Poor alignment between the tissue samples and the slide fiducials can otherwise result in samples that are difficult or impossible to analyze, or the analysis from poorly aligned samples leads to inaccurate results.

I. Introduction

While the methods, apparatuses, devices and molds described herein are suited to histology and cytology generally, specific utility is also found when practicing the present disclosure for spatial analysis technologies, such as spatial transcriptomics and in situ spatial analyte analysis where tissue section placement can be highly constrained.

Histology, cytology, and pathology methodologies described herein can provide sectioned tissue samples for analysis by microscopy, assay, or other techniques to assess aspects and pathologies of a tissue. For example, histological samples can be formed according to the techniques described herein for analysis of composition, structure, and function of tissues. Cytology refers to the testing of cells within a tissue, often for the screening or diagnosis of cancer or other disease states. Pathology generally refers to the examination of tissue samples for diagnostic purposes. The sectioning of tissue samples and their fixation (if desired) and assembly of the tissue sections into a wax or other embedding material enables the analysis of very thin tissue samples for these and other purposes, that can then be positioned on microscope slides or arrayed slides using the methods described below. Tissue samples prepared for the purposes of histological, cytological, or pathological analysis can be used in combination with other methods, including spatial analysis methodologies.

The tissue sample described herein can be a biological sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. The tissue sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a histology sample, a histopathology sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, cultured tissues or cells. The tissue sample can be harvested from a subject (e.g., via surgical biopsy, whole subject sectioning), grown in vitro on a growth substrate or culture dish as a population of cells, or prepared as a tissue slice or tissue section. Biological samples include tissue samples such as biopsies (e.g., liquid and solid biopsies) and tissue sections (e.g., fresh frozen or FFPE). The sample can include a patient derived sample, for example a patient derived organoid (PDO) or patient derived xenograft (PDX).

The thickness of the biological sample, for use in the disclosed methods may be dependent on the method used to prepare the sample and the physical characteristics of the tissue in combination with the experiment to be run on the tissue section. Thus, any suitable section thickness may be used, depending on the what the tissue section is to be used for. In some embodiments, the thickness of the tissue sample section will be at least 0. 1, 0.2, 0.3, 0 4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 micrometers. In other embodiments, the thickness of the tissue sample section is at least 10, 12, 13, 14, 15, 20, 30, 40 or 50 micrometers. Thicker samples may be used if desired or convenient e.g. 70 or 100 micrometers or more. Typically, the thickness of the tissue sample section is between 1-100 micrometers, 1-50 micrometers, 1-30 micrometers, 1-25 micrometers, 1-20 micrometers, 1- 15 micrometers, 1-10 micrometers, 2-8 micrometers, 3-7 micrometers or 4-6 micrometers, but as mentioned above thicker samples may be used. Grown samples may be sufficiently thin for analy sis without further processing steps.

Alternatively, grown samples, and samples obtained via biopsy or sectioning, can be prepared as thin tissue sections using a mechanical cutting apparatus such as a vibrating blade microtome or a cryostat instrument. As another alternative, in some embodiments, a thin tissue section can be prepared by applying a touch imprint of a biological sample to a suitable substrate material. Tissue sections as used herein can include whole animal sectioning and tissue biopsies. Tissue sections can be fresh tissue such as primary tissue sections, or tissue sections can be preserved samples such as frozen samples or fixed (e.g., formalin or paraformalin) and/or paraffin-embedded samples. For example, fresh, froze, fixed, unfixed or expanded tissues can be used.

The biological sample may be prepared in any convenient or desired way and it is not restricted to any particular type of tissue preparation. Fresh, frozen, fixed, unfixed or expanded tissues may be used. Any desired convenient procedure may be used for fixing (e.g. crosslinking), embedding or expanding the biological specimen, e.g. tissue sample, as described and known in the art. Thus, any known fixatives or embedding materials may be used. Exemplary' details of processes for partitioning and embedding biological samples for analysis are described in U.S. Patent Publication No. 2021/0247316A1, the contents of which is incorporated herein by reference in its entirety.

Spatial analysis methodologies and compositions described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods and compositions can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte. For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.

The molds described here can be used to generate tissue sections that will align with arrays areas on slides where spatial transcriptomics methods are practiced. Use of arrayed slides comprising a tissue section generated from one or more embedded tissues in the molds described herein can be used in spatial transcriptomics methods as described below.

Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Patent Nos. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent Application Publication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641, 2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709, 2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322, 2018/0245142, 2017/24191 1 , 2017/08981 1 , 2017/067096, 2017/029875, 2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO 2020/176788, Rodriques et al., Science 363(6434): 1463-1467, 2019; Lee et al., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE 14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36: 1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020), both of which are available at the 1 Ox Genomics Support Documentation website, and can be used herein in any combination, and each of which is incorporated herein by reference in their entireties. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein.

Some general terminology that may be used in this disclosure can be found in Section (I)(b) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. A “biological sample” that can be embedded in medium and used in a mold described herein is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In some embodiments, a biological sample can be a tissue section. In some embodiments, a biological sample can be a fixed and/or stained biological sample (e.g., a fixed and/or stained tissue section). Non-limiting examples of stains include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e g., fluorescent stains). In some embodiments, a biological sample (e.g., a fixed and/or stained biological sample) can be imaged. Biological samples are also described in Section (I)(d) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, sequencing (e.g., of a cleaved extended capture probe and/or a cDNA molecule complementary to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and/or proximity capture, is described in Section (II)(g) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Some quality control measures are described in Section (IT)(h) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

Exemplary methods for identifying spatial information of biological and/or medical importance can be found in U.S. Patent Application Publication No. 2021/0140982A1, U.S. Patent Application No. 2021/0198741A1, and/or U.S. Patent Application No. 2021/0199660.

During analysis of spatial information, sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample. Various methods can be used to obtain the spatial information. In some embodiments, specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate. For example, specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location. The molds described herein are used to align a tissue section with the described specific array locations on a substrate, thereby maximizing efficiencies of tissue placement on an arrayed slide.

Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See, for example, the Exemplar}⁷ embodiment starting with “In some nonlimiting examples of the workflow s described herein, the sample can be immersed.. . ” of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020).

Another application of the molds described herein is where a mold is created to fit a different assay dimension that that used for some spatial transcriptomics assays, for example for use in an in situ spatial analyte analysis workflow such as is practiced by the Xenium instrument (lOx Genomics, Inc.). The Xenium instrument analyzes analytes in situ in a tissue, and the footprint for the tissue section to be placed for assaying is a different dimension than that for some spatial transcriptomics methods. As such, a mold as exemplified in FIG. 5, FIG. 6 and FIG. 7 is of a different dimension than those exemplified in FIG. 1, FIG. 2 and FIG. 3. The in situ sequencing methods that might use the mold designed for use in generating a tissue section that aligns with a substrate used for in situ sequencing methods is described in more detail below.

Molds described herein can provide tissues for practicing methods involving analyzing, e.g., detecting or determining, one or more sequences present in the probes or probe sets or products thereof (e.g., rolling circle amplification products thereol). In some embodiments, the detecting is performed at one or more locations in the biological sample. In some embodiments, the locations are the locations of RNA transcripts in the biological sample. In some embodiments, the locations are the locations at which the probes or probe sets hybridize to the RNA transcripts in the biological sample and are optionally ligated and amplified by rolling circle amplification.

Methods for binding and identifying the one or more sequences that relate to the location of RNA transcripts in a sample by using various probes or oligonucleotides have been described in, e.g., US2003/0013091, US2007/0166708, US2010/0015607, US2010/0261026, US2010/0262374, US2010/0112710, US2010/0047924, and US2014/0371088. Detectably-labeled probes can be useful for detecting multiple target nucleic acids and be detected in one or more hybridization cycles (e.g., sequential hybridization assays, or sequencing by hybridization).

In some embodiments, the assay comprises in situ sequencing. In situ sequencing ty pically involves incorporation of a labeled nucleotide (e.g., fluorescently labeled mononucleotides or dinucleotides) in a sequential, template-dependent manner or hybridization of a labeled primer (e.g., a labeled random hexamer) to a nucleic acid template such that the identities (e.g., nucleotide sequence) of the incorporated nucleotides or labeled primer extension products can be determined, and consequently, the nucleotide sequence of the corresponding template nucleic acid. Aspects of in situ sequencing are described, for example, in Mitra et al., (2003) Anal. Biochem. 320, 55-65, and Lee et al., (2014) Science, 343(6177), 1360-1363. In addition, examples of methods and systems for performing in situ sequencing are described in US 2016/0024555, US 2019/0194709, and in US 10,138,509, US 10,494,662 and US 10,179,932, all of which are herein incorporated by reference in their entireties. Exemplary' techniques for in situ sequencing or in in situ sequence detection comprise, but are not limited to, STARmap (described for example in Wang et al., (2018) Science, 361(6499) 5691), MERFISH (described for example in Moffitt, (2016) Methods in Enzymology, 572, 1-49), hybridization-based in situ sequencing (HyblSS) (described for example in Gyllborg et al., Nucleic Acids Res (2020) 48(19):ell2, and FISSEQ (described for example in US 2019/0032121, the content is herein incorporated by reference in its entirety).

A sample for use in any of the methods and devices described herein can be derived from any biological sample. A biological sample may be obtained from a subject using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In addition to the subjects described above, a biological sample can be obtained from a prokaryote such as a bacterium, an archaea, a virus, or a viroid. A biological sample can also be obtained from non-mammalian organisms (e.g., a plant, an insect, an arachnid, a nematode, a fungus, or an amphibian). A biological sample can also be obtained from a eukaryote, such as a tissue sample, a patient denved organoid (PDO) or patient derived xenograft (PDX). A biological sample from an organism may comprise one or more other organisms or components therefrom. For example, a mammalian tissue section may comprise a prion, a viroid, a virus, a bacterium, a fungus, or components from other organisms, in addition to mammalian cells and non-cellular tissue components. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., a patient with a disease such as cancer) or a pre-disposition to a disease, and/or individuals in need of therapy or suspected of needing therapy.

The biological sample which can be used in the methods and devices described herein can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can include nucleic acids (such as DNA or RNA), proteins/polypeptides, carbohydrates, and/or lipids. The biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions. In some embodiments, the biological sample may comprise cells which are deposited on a surface. In some embodiments, the biological sample may comprise cells from a portion of a cell block or a cell pellet.

Biological samples can further be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms. Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. Biological samples can also include fetal cells and immune cells.

A substrate used in methods and devices described herein can be any support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and/or reagents (e.g., probes) on the support. In some embodiments, a biological sample can be attached to a substrate using the methods and devices described herein. Attachment of the biological sample can be irreversible or reversible, depending upon the nature of the sample and subsequent steps in the analytical method. In certain embodiments, the sample can be attached to the substrate reversibly by applying a suitable polymer coating to the substrate and contacting the sample to the polymer coating. The sample can then be detached from the substrate, e.g., using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers that are suitable for this purpose. In some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

A variety of steps can be performed to prepare or process a biological sample for and/or during an assay. Except where indicated otherwise, the preparative or processing steps described below can generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and/or analysis.

A biological sample can be harvested from a subject (e.g., via surgical biopsy, whole subject sectioning) or grown in vitro on a growth substrate or culture dish as a population of cells and prepared for analysis as a tissue slice or tissue section and used in the methods and devices disclosed herein.

The thickness of the tissue section can be a fraction of (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximum cross-sectional dimension of a cell. However, tissue sections having a thickness that is larger than the maximum cross-section cell dimension can also be used. For example, cryostat sections can be used, which can be, e.g., 10-20 pm thick. More generally, the thickness of a tissue section typically depends on the method used to prepare the section and the physical characteristics of the tissue, and therefore sections having a wide variety of different thicknesses can be prepared and used. For example, the thickness of the tissue section can be at least 0. 1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 pm. Thicker sections can also be used if desired or convenient, e.g., at least 70, 80, 90, or 100 pm or more. Typically, the thickness of a tissue section is between 1-100 pm, 1-50 pm, 1-30 pm, 1-25 pm, 1-20 pm, 1-15 pm, 1- 10 pm, 2-8 pm, 3-7 pm, or 4-6 pm, but as mentioned above, sections with thicknesses larger or smaller than these ranges can also be analysed.

Multiple sections can also be obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy sample by embedding the tissue in embedding medium using the methods and devices disclosed herein and performing serial sectioning of the biopsy sample using a sectioning blade. Spatial information among the serial sections can be preserved in this manner, and the sections can be analysed successively to obtain three-dimensional information about the biological sample.

In some embodiments, the biological sample can be prepared using formalin-fixation and paraffm-embedding (FFPE), using the devices disclosed herein. In some embodiments, cell suspensions and other non-tissue samples can be prepared using formalin-fixation and paraffin-embedding using the devices disclosed herein. Following fixation of the sample and embedding in a paraffin or resin block, the sample can be sectioned as previously described and mounted on the appropriate substrate that aligns with the device mold used. Prior to analysis, the paraffin-embedding material can be removed from the tissue section (e.g., deparaffinization) by incubating the tissue section in an appropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes).

As an alternative to formalin fixation described above, a biological sample can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analy sis. For example, a sample can be fixed via immersion in ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof.

In some embodiments, the biological sample can be embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this manner typically involves contacting the biological sample with a hydrogel such that the biological sample becomes surrounded by the hydrogel in a device of the present disclosure. For example, the sample can be embedded by contacting the sample with a suitable polymer matenal, and activating the polymer material to form a hydrogel in the presence of a tissue sample disposed in a device of the present disclosure. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample. Biological samples can include analytes (e.g., protein, RNA, and/or DNA) embedded in a 3D matrix. In some embodiments, amplicons (e g., rolling circle amplification products) derived from or associated with analytes (e g., protein, RNA, and/or DNA) can be embedded in a 3D matrix. In some embodiments, a 3D matrix may comprise a network of natural molecules and/or synthetic molecules that are chemically and/or enzymatically linked, e.g., by crosslinking. In some embodiments, a 3D matrix may comprise a synthetic polymer. In some embodiments, a 3D matnx comprises a hydrogel.

In some aspects, a biological sample can be embedded in any of a variety of other embedding materials in a device of the present disclosure to provide structural substrate to the sample prior to sectioning and other handling steps. In some cases, the embedding material can be removed e g., prior to analysis of tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylate resins), epoxies, and agar.

In some aspects, a substrate upon which are found tissue sections generated by practicing the methods and devices of the present disclosure can further be placed into a cassette configured to receive a substrate (e.g., a glass microscope slide that is arrayed for spatial transcriptomics, in situ gene expression analysis, etc.). In preferred aspects, the cassette includes a bottom portion and a top portion, wherein the top portion has one or more snap joints (e.g., a cantilevered snap joint) configured to couple to lugs (cantilevered lugs) of the bottom portion. In some aspects, the bottom portion has the snap joints while the top portion has the lugs. The slide substrate is positioned on the bottom portion and then the top portion is coupled to the bottom portion such that the slide is sandwiched therebetween. The substrate has one or more sample regions defined by the inclusion of fiducial marks that surround the sample region, with a gasket that surround the fiducials. The substrate used, for example a slide, has a length of about 75 mm and a width of about 25 mm. In various embodiments, the substrate has an imageable area of about 24 mm by about 12 mm for in situ gene expression analysis. However, the sample positioning area is less than the imageable area and is about is about 10.45 mm by about 22.45 mm. For a spatial transcriptomics arrayed area, the slide dimensions are the same at 75 mm x 25 mm. However, the arrayed areas are larger, depending on which arrayed area is utilized. For example, one arrayed area is 8 mm x 8 mm, which includes the fiducials surrounding the sample positioning area. The sample positioning area is 6.5 mm x 6.5 mm. A second example is a larger arrayed area which includes fiducials of 12.5 mm x 12.5 mm, wherein the sample positioning area is 11 mm x 11 mm.

TI. Apparatus and Methods for Preparing a Biological Sample

As discussed, the ability to position tissue samples on and/or within an embedding material during sample preparation can be an important aspect of biological analysis. While biological samples can be prepared on a substrate (e.g., a slide) for further analysis (e.g., microscopy, histology, cytology, pathology, spatial analysis, in situ analyte analysis, and/or immunohistochemistry), appropriately positioning the samples on a substrate at a desired location can affect accuracy and quality of the analy sis, and this type of positioning is oftentimes difficult to achieve. Thus, described herein are apparatuses or devices (e.g., molds) and methods of preparing tissue samples within an embedding material so that the tissue samples upon sectioning can be properly positioned on a substrate, such as a microscope slide or one or more particular locations on a microscope slide, for accurate tissue analysis.

In some embodiments, tissue samples such as tissue sections or whole or portions thereof from tissue samples are placed or intended to be placed on microscope slides that include fiducials designating specific placement locations of the tissue for analysis purposes. In some implementations, the tissue samples are preserved or otherwise treated prior to use. In some embodiments, apparatuses and methods provided herein can provide tissue sections or slices of tissue samples embedded in embedding material at specific locations that align the samples to a particular microscope slide fiducial pattern or design, resulting in samples that produce accurate tissue analysis results. Poor alignment between the tissue samples and the slide fiducials can otherwise result in samples that are difficult to analyze or produce inaccurate results.

In some embodiments, using a base mold having one or more guides for placing the tissue samples can improve the positioning of the tissue in the embedded material so that the tissue samples upon sectioning will be properly aligned with fiducials or desired areas on the microscope slide when the sections or slices of embedded tissue are placed on the slide. The guides in the base mold visually designate areas in the mold for placement of the tissue samples. The number of designated areas in the mold indicated by the guides can correspond to fiducials or desired areas on commercial microscope slides.

After placement of the tissue samples in the designated areas of the base mold, embedding material can be added to the base bold to embed the tissue samples in embedding material in the base mold. In some implementations, the guides help prevent movement of the tissue samples within the base mold during the addition of the embedding material to the base mold. After the embedding material is set and/or cured, the embedded tissue block is removed from the base mold. The guides used to designate the areas in the base mold can leave imprints or cavities in the embedding material surrounding the embedded tissue. Following sectioning of the embedded tissue block, a section of the embedded tissue block can be positioned on a microscope slide by floating the section on the surface of a water bath and contacting the floating section with the microscope slide. The imprints or cavities in the embedding material surrounding the tissue in the floating section can be used as a guide for positioning of the section on the microscope slide.

The base mold guides described herein can facilitate the proper placement of the tissue samples in the mold to produce embedded tissue sections that correspond to arrays or specific desired locations on commercial microscope slides. Using a base mold with guides can improve the accuracy of positioning the tissue sections on microscope slides, and the efficiency of producing quality slides for analysis.

FIG. 1 shows an exemplary mold 101 with guides 106. Mold 101 includes a surface 102 surrounded by at least one sidewall 104 A-D, and guides 106. The surface 102 is surrounded by first sidewall 104A, second sidewall 104C opposite first sidewall 104A, third sidewall 104B and fourth sidewall 104D opposite third sidewall 104B. The sidewalls 104 A- D surround the surface 102 to form a receptacle of the mold having an interior space. In some embodiments, the mold is made from stainless steel, or other material useful in the histology and cytology arts. The guides 106 extend from the surface 102 in a direction perpendicular to a plane of the surface 102. In some embodiments, the guides 106 can be a height of about 0.5 mm to about 50 mm. In some embodiments, the guides 106 are between 0.5-1 mm in height. In some implementations, the guides 106 have a height of 0.5 mm to 5 mm, a height of 1 mm to 10 mm, a height greater than 10 mm, or any other suitable height. In some embodiments, the guides 106 are pins, plates, posts, or walls.

The guides 106 are positioned within the receptacle to designate a plurality of predetermined areas 108 of the surface 102. Designate, as used herein, can mean positioned to partially outline a perimeter of each area 108 within the interior space of the mold 101. The positioning of the guides 106 provides a visual indication of comers or edges of each predetermined area 108 of the surface 102. The guides 106 designate the predetermined areas 108 for receiving a portion of the tissue sample within the interior space of the mold 101. The guides 106 allow a technician placing tissue or other samples in the mold 101 to visualize the predetermined areas 108 and place each tissue in one of the predetermined areas 108 as designated by the guides 106. In some embodiments, the guides can designate two predetermined areas, three predetermined areas, four predetermined areas, five predetermined areas, six predetermined areas, seven predetermined areas or eight predetermined areas 108. In some embodiments, the guides can designate more than eight predetermined areas, however at some point the number of predetermined areas is limited by the ability to handle and manipulate smaller and smaller tissue samples which can be challenging.

In some implementations, the mold 101 is a base mold, or a tissue embedding mold. The mold 101 is configured to receive one or more portions of the tissue sample and an embedding material in one or more predetermined areas of the receptacle surface 102. The mold 101 can be designed to receive one or more tissue samples and to be filled with an embedding material, such as OCT, a wax, or other embedding material known in the art for preparation of tissue sections. In some implementations, the embedding material is paraffin wax, a resin (e.g., a methacrylate resin), an epoxy, or an agar. Non-limiting details of processes for partitioning and embedding biological samples for analysis are described in U.S. Patent Publication No. 2021/0247316A1, the contents of which is incorporated herein in its entirety. Each predetermined areas 108 defines an area on the surface 102. The predetermined area 108 of the surface 102 can have a shape, such as a square or a rectangle. In some implementations, the predetermined area may be another shape such as a triangle, circle, rectangle with rounded edges, or any other suitable shape. In some implementations, each predetermined area 108 defines an area of the surface that has the same shape and size. In some implementations, at least a portion of the guides 106 are positioned in at least one line extending in a direction parallel to a first longitudinal axis 110 extending from the first sidewall 104A to the second sidewall 104C. In some implementations, at least a portion of the guides 106 are positioned in a line extending in a direction parallel to a second longitudinal axis 112 of the receptacle from the third sidewall 104B to the fourth sidewall 104D. In some implementations, the guides are positioned along a center-line of the surface 102, equidistant from the first sidewall 104A and the second sidewall 104C, or equidistant from the third sidewall 104B and the fourth sidewall 104D.

In some implementations, the guides 106 are formed as protrusions or markers. In some implementations, the guides 106 are merely visual indications on the surface 102 of the mold 101. In some implementations, the guides 106 protrude from the surface 102 of the mold 101. In some implementations, the guides 106 are formed as one or more pins, plates, posts, and walls. In some implementations, the guides 106 define walls extending from the surface 102 in a direction perpendicular to the plane of the surface 102 within the interior space of the mold 101. In some implementations, the one or more guides 106 comprises a biocompatible metal or metal alloy, for example, stainless steel or aluminum alloy. In some implementations, the one or more guides 106 comprise a medical grade polymer. In some implementations, each of the one or more guides 106 has a height of 0.5 mm to 50 mm, preferably 0.5-1 mm, measured from the surface 102 of the mold to an end of the guide 106. In some implementations, the guides 106 have a height of 0.5 mm to 5 mm, a height of 1 mm to 10 mm, a height greater than 10 mm, or any other suitable height.

In some implementations, the surface 102 has a length of 40 mm and width of 20 mm. In some implementations, the surface 102 has a length of 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, or any other suitable length. In some implementations, the surface 102 has a width of 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm or any other suitable width. In some implementations, the guides 106 designate predetermined areas 108 on the surface which are square in shape. In some implementations, the square predetermined areas 108 each have sides of 5 mm, 6.5 mm, 7.5 mm, 10 mm, 11 mm, 12 mm, or any other suitable length. In some implementations, the predetermined areas 108 each have a surface area of 6.5 mm². In some implementations, the predetermined areas 108 each have a surface area of 11 mm². In some implementations, each of the predetermined areas 108 for a spatial transcriptomics array has a length of about 6.5 mm or about 11 mm and a width of about 6.5 mm or about 11 mm.

In some implementations, the guides 106 are colored or otherwise differentiated from the mold 101 by visual markers. In some implementations, the guides 106 are both formed as structures extending from the surface 102 within the mold 101 receptacle, and are differentiated from the mold 101 by visual markings such as colors In some implementations, one or more of the guides 106 extend from or abut the sidewalls 104A-D surrounding the surface 102.

The mold 101 can be manufactured with a number of guides 106 chosen and arranged to designate a desired number of predetermined areas 108 of the mold 101. In some implementations, the number of guides 106 arranged within the mold 101 is between one and 20. FIG. 1 shows a mold 101 with 20 guides 106 indicating eight predetermined areas 108. A guide 106 forms the comer of each predetermined area 108. As will be described in greater detail in FIGS. 2A-E, in some implementations, a guide 106 is at less than all comers of each predetermined area 108, such that the guides 106 visually designate the predetermined area 108 but do not extend about the perimeter of the predetermined area 108.

FIGS. 2A-E show exemplary arrangements of guides within a base mold. As described above, a mold can be manufactured with a number of guides 206 chosen and arranged to indicate a desired number of areas 208 of the mold. In some implementations, the number of guides 206 arranged within the mold is between one and 20. In some implementations, the plurality of areas 208 include two, three, four, six, eight, or ten predetermined areas. In some implementations, the predetermined areas 208 designated by the guides 206 are equally sized and shaped.

FIG. 2A shows a mold having guides 206 to form eight predetermined areas 208. The guides 206 are formed as walls protruding from the surface 202 of the mold. A portion of the guides 206 are arranged in a line parallel to a longitudinal axis 210 extending in a direction from the first sidewall 204A to the opposite second sidewall 204C. Another portion of the guides 206 are arranged in a line parallel to a longitudinal axis 212 extending in a direction from the third sidewall 204B to the opposite fourth sidewall 204D. Flat surfaces of the guides 206 formed as walls visually designate at least a portion of the predetermined area 208 perimeter to facilitate placement of tissue samples within the areas 208. The eight predetermined areas 208 are designated by 10 guides 206, oriented to form cross-like assemblies through the middle of the interior space of the mold.

FIGS. 2B and 2C show molds having guides 206 formed as circular pins extending from the surface 202. At least some of the guides 206 are arranged to form lines parallel to the longitudinal axis 210 of the mold extending in a direction from the first sidewall 204A to the second sidewall 204C, and to form lines parallel to the longitudinal axis 212 of the mold extending in a direction from the third sidewall 204B to the fourth sidewall 204D. FIG. 2B shows nine guides 206 designating 16 predetermined areas 208 on the surface 202. In some implementations, the nine guides 206 can be understood to designate eight predetermined surfaces 208, or four predetermined areas 208 in the middle of the surface 202 of the mold. FIG. 2C shows five guides 206 designating four predetermined areas 208. Alternatively, the five guides 206 can be understood to designate eight predetermined areas 208.

FIGS. 2D and 2E show molds having guides 206 formed as cross-shaped structures extending from the surface 202. In FIG. 2D, the single guide 206 designates four predetermined areas 208 of the surface 202. The guide 206 has walls forming the first arms of the cross which extend in directions parallel to the longitudinal axis 210 of the mold extending in a direction from the first sidewall 204A to the second sidewall 204C, and walls forming the second arms of the cross parallel to the longitudinal axis 212 of the mold extending in a direction from the third sidewall 204B to the fourth sidewall 204D. Additionally, the guide 206 is positioned equidistant from the first sidewall 204A and the second sidewall 204C, and also equidistant from the third sidewall 204B and the fourth sidewall 204D. In some implementations, the guide 206 need not be aligned with the longitudinal axes of the mold. In some implementations, the guide 206 can be positioned on the surface 202 in a location so as to not be equidistant between the sidewalls 204 A-D of the mold. FIG. 2E shows mold having multiple cross-shaped structures as guides 206 forming predetermined areas 208 of the surface 202. Each of the guides 206 have walls forming the first arms of the cross which extend in directions parallel to the longitudinal axis of the mold extending in a direction from the first sidewall 204A to the second sidewall 204C, and walls forming the second arms of the cross parallel to the longitudinal axis of the mold extending in a direction from the third sidewall 204B to the fourth sidewall 204D. The guides 206 are positioned to form a line parallel to the longitudinal axis 212 of the mold extending in a direction from the first sidewall 204A to the second sidewall 204C. The three guides 206 designate eight predetermined areas 208 of the surface 202 of the mold. A base mold for use in preparation of an embedded tissue block can be selected based on the desired number of tissue samples to be included in the areas and/or a desired microscope slide to be used. The number of predetermined areas of the surface of the mold designated by the guides can be chosen to accommodate the desired number of tissue samples. In some cases, a mold can be chosen having a particular shape or size of guides positioned on the surface to accommodate larger or smaller tissue samples or to aid in positioning of small tissue samples on the surface of the mold. After choosing the desired mold for the preparation, the desired placement of one or more tissue samples is determined based on the visual indication of the areas designated by the guides. One or more tissue samples are then disposed on the surface of a mold within one of the predetermined areas designated by the guides. Placement of the tissue samples in the predetermined areas positions the tissue samples so as to align with fiducials, arrays, or desired positions on a microscope slide. Fiducials can be etched or printed on a surface of the microscope slide, or inset in the microscope slide. In some implementations, the fiducial is formed as a printed shape on the microscope slide. In some implementations, the fiducial is positioned relative to an array on the microscope slide. In some implementations, the arrays are formed as a number of marked areas on the microscope slide. In some implementations, the arrays are formed as an arrangement of raised portions of the microscope slide. In some implementations, the arrays can include additional substances arranged on the microscope slide for interaction with the tissue samples or to facilitate analysis of the samples.

FIGS. 2A-E illustrate possible arrangements of guides 206 on the surface 202 of the mold to designate predetermined areas 208. As described above, other numbers, orientations, shapes and arrangements of guides 206 are possible. The mold may be designed and manufactured with any suitable number, orientation, shape, and arrangement of guides so as to designate the desired number and arrangement of the predetermined areas of the mold. In some implementations, the desired number and arrangement of predetermined areas of the mold correspond to arrays, desired areas, or fiducials on commercial or other microscope slides. For example, FIG. 3A shows an exemplary mold 301 with guides 306 corresponding to arrays 314 A-H on an exemplary microscope slide 305 shown in FIG. 3B, allowing for placement of tissue samples within the arrays 314A-H as shown in FIG. 3C.

Referring to FIG. 3A, the mold 301 includes a surface 302 surrounded by sidewalls 304 A-D, and multiple guides 306 protruding from the surface 302 in a direction perpendicular to a plane of the surface 302. The guides 306 designate multiple predetermined areas 308 A-H of the interior space of the receptacle formed by the surface 302 and sidewalls 304 A-D. The 20-guide 306 of the mold 301 designate eight predetermined areas 308 A-H, each extending over an area of the surface 202.

The predetermined areas 308 A-H of the mold correspond to eight arrayed areas 314 A-H on a microscope slide 305, shown in FIG. 3B. The microscope slide 305 may be a commercially available slide, or any microscope slide. The microscope slide can include additional information 316, for example identifying the type of slide, the identification number of the slide for analy sis purposes, or other information. In some implementations, the array is indicated by a series of one or more fiducials etched or printed on a surface of the microscope slide, or inset in the microscope slide. In some implementations, the array is an arrangement of raised portions of the microscope slide. The array can include additional substances arranged on the microscope slide for interaction with the tissue samples or to facilitate analysis of the samples.

As shown in FIG. 3C, when an embedded tissue sample section is prepared using the mold 301, the arrangement of the tissue samples 318 A-E align with the predetermined areas 308 A-H on the surface 302 of the mold 301 to produce an embedded tissue section with the tissue samples 318 A-E positioned within the section for alignment with the arrayed areas 314 A-E on the microscope slide 305.

FIGS. 4A-4E illustrate the method of preparing an embedded tissue sample slide from an embedded tissue block prepared using a base mold with guides (for example mold 101 in FIG. 1, molds of FIGS. 2A-E, mold 301 of FIG. 3). FIG. 4A shows a first embedded tissue block 422 having eight embedded tissue samples, and a second embedded tissue block 420 having four embedded tissue samples. The second embedded tissue block 420 can be produced from a mold having guides designating only four predetermined areas, or alternatively, can be produced from a mold having guides designating eight predetermined areas by only placing four tissue samples in the mold. After the tissue samples are placed in the predetermined areas on the surface of the mold, the mold is filled with embedding material to embed the one or more tissue samples in the embedding material. The embedding material may be a wax, including paraffin wax or any other suitable wax. The embedding material is cooled in the mold to produce a tissue-embedded block, such as first embedded tissue block 422 or second embedded tissue block 420. The tissue embedded block is removed from the mold after the cooling.

The first embedded tissue block 422 and second embedded tissue block 420 each can include one or more cavities corresponding to the position of the guides in the mold from which the block was formed. The protruding guide structures leave cavities in a surface of the embedding material when the embedded tissue block is removed from the mold.

FIG. 4B shows microtome or cryostat sectioning of the exemplary embedded tissue block 422 into multiple sample sections. Sectioning of the embedded tissue block 424 is accomplished by the use of a sectioning instrument 424, such as a microtome or cryostat, capable of slicing thin sections of the embedded tissue block. The sectioning instrument 424 sections the embedded tissue block 422 in a direction parallel to the surface of the embedded tissue block 422 that w as adjacent to the surface of the mold. FIG. 4C shows a section 426 of the embedded tissue block 422. The section 426 of the embedded tissue block 422 includes a section of each tissue sample 428 and surrounding embedding material 430. The section 426 also can include one or more apertures 429 where the cavities formed by the guides were in the first embedded tissue block 422.

FIG. 4D shows the placement of the section 426 on a microscope slide 432. The tissue section with surrounding embedding material 426 can be floated in a w ater bath, where the tissue section with surrounding embedded material floats on the surface of the water. The microscope slide 432 is positioned below the floating tissue sample and embedded matenal, and is and brought up under the section 426 to align with the arrayed areas of the slide 432. When the tissue section 426 is contacted with the microscope slide 432, the tissue section 426 adheres to the slide upon contact. By positioning the microscope slide 432 beneath the tissue section 426 and aligning the slide 432 with the tissue samples of the tissue section 426 prior to contact, when the microscope slide 432 and attached tissue section 426 are removed from the water, the tissue samples in the attached tissue section 426 will be aligned with the fiducial and/or array on the microscope slide 432. Positioning the tissue sections within the predetermined areas of the mold designated by the guides during preparation provides portions of the tissue sample 428 in each section 426 that can be easily aligned with the arrayed areas on the microscope slide 432.

In some implementations, the microscope slide 432 can include a fiducial marker that is etched or printed on the surface of the microscope slide, or inset in the microscope slide 432. In some implementations, the fiducial is formed as a printed shape on the microscope slide 432. In some implementations, the fiducial is positioned relative to one or more arrays on the microscope slide 432.

The fiducial can aid in positioning the section 426 on the microscope slide 432, so that each tissue sample 428 is positioned in a desired location on the microscope slide 432. Additionally, the embedding material 430 may include one or more apertures 429 through the embedding material 430 formed by one or more of the guides during preparation of the embedded tissue block in the mold. The one or more apertures 429 can be used to facilitate alignment of the section 426 and the microscope slide 432. The FIG. 4E shows the placement of a tissue section 434 corresponding to the second embedded tissue block 420 on a microscope slide 432, with the four tissue samples aligned with the arrayed areas of the microscope slide 432. The aligned tissue sections 426 on the microscope slide 432 provides a good quality sample for histological analysis.

FIGs. 5A-5C illustrate a method for preparing embedded tissue blocks for tissue analysis. FIG. 5A shows an exemplary base mold 500. Mold 500 includes a surface 502 surrounded by at least one sidewall 504. The sidewalls 504 surround the surface 502 to form a receptacle of the mold 500 having an interior space. In some embodiments, the mold is made from stainless steel, or other material useful in the histology and cytology arts. In some embodiments, the mold is made from a polymer material.

Mold 500 includes a second surface 507 extending from the sidewalls 504 and second sidewalls 505 surrounding the second surface 507. Mold 500 also includes first wings 508A and 508B and second wings 509 (only one second wing is visible in FIG. 5A). The first wings 508A and 508B extend upwards from the second sidewalls 505, and second wing 509 extends downwards from the second sidewalls 505. First wings 508A and 508B and second wing 509 provide surfaces for grasping the mold 500 or for balancing the mold 500 on a surface such as a lab bench. In some embodiments, mold 500 includes guides positioned on the surface 502 or marked in the sidewalls 504, as described above with regard to FIG. 1 (not shown).

In some embodiments, the surface 502 of the mold 500 has a length (L) and width (W) sized to be equivalent to or smaller than a sample area of a microscope slide for use with tissue analysis equipment. In some embodiments, the surface 502 of the mold 500 has a width of 12 mm and a length of 24 mm. In some embodiments, the mold for embedding the tissue sample is about 10 mm by about 22 mm. In some embodiments, a depth of the sidewalls 504 is 5 mm. In some embodiments, a depth of the sidew alls 504 is 10 mm. In some embodiments, a depth of the sidewalls 504 is 15 mm. In some embodiments, the width of the surface 502 is 5 mm, 10 mm, 12 mm, 15 mm, 18 mm, 20 mm or any other suitable length. In some embodiments, the length of the surface 502 is 5 mm, 10 mm, 15 mm, 18 mm, 20 mm, 24 mm, 25 mm, 28 mm, 30 mm, or any other suitable length. In some embodiments, the depth of the sidew alls 504 is 1 mm, 2 mm, 3 mm, 4 nun, 5 mm, 6 mm, 7 mm, 8, mm, 9 mm, 10 mm, 12 mm, 15 nun, or any other suitable depth. A mold with a width of 12 mm and a length of 24 mm can be used to prepare tissue samples for efficient placement on a microscope slide with a sample area of 12 mm by 24 mm.

By matching the footprint of the mold receptacle to a sample area of a microscope slide for use with tissue analysis equipment, efficient placement of tissue samples within the sample area of the microscope slide can be achieved. Use of molds with other dimensions could result is a tissue sample too big or too small for the sample area of the microscope slide, resulting in poor placement of the tissue samples; interference with frames, fiducials, or labels on the slides; or increased time required for placement of the tissue sample within the sample area. For example, using a mold sized for a particular microscope sample area, a user utilizing microscope slides used in spatial gene expression methodologies (e.g., microscope slides having spatially -barcoded arrays) may be able to efficiently align the tissue sample with a region of interest in the microscope slide (e.g., the region containing the spatially- barcoded array) for tissue analysis. The use of a mold with both a particular size receptacle and guides for positioning the tissue samples within the receptacle so as to match regions of interest in a microscope slide further increase efficiency in preparation of microscope slides for tissue analysis. Such microscope slides can be used for analyzing and visualizing spatial gene expression data, or for measuring the total mRNA in tissue sections. The microscope slides for such purposes may require intact tissue sections to be positioned in particular regions of the sample area for tissue analysis.

FIG. 5B shows a first embedded tissue block 501 A prepared using the mold 500 of FIG. 5A. First embedded tissue block 501 A has three embedded tissue samples 512A-C arranged to form a tissue microarray. FIG. 5C shows a second embedded tissue block 501B prepared using the mold 500 of FIG. 5A and having one embedded tissue sample 513. First embedded tissue block 501 A and second embedded tissue block 501B each include a base portion 516 and a raised portion 514, with the tissue samples embedded in the raised portion 514.

The raised portion 514 has the dimensions of the receptacle formed by the surface 502 and surrounding sidewalls 504 of the mold 500. In some embodiments, the dimensions of the raised portion 514 are matched to a sample area of a microscope slide, based on the dimensions of the surface 502 of the mold 500. The base portion 516 has larger dimensions than the raised portion 514, the dimensions of the base portion defined by the size of the second surface 507 and the depth to which the mold 500 is filled with embedding material. By providing a raised portion 514 for embedding tissues 512A-C and 513 with dimensions matching a sample area of a microscope slide, multiple tissue samples (e.g., tissue samples 512A-C) in a tissue microarray can be efficiently positioned in the sample area of a microscope slide without interfering with fiducials, frames, or labels of the microscope slide. Positioning multiple tissue samples within the mold 500, such that they are arranged in the raised portion 514 of the first embedded tissue block 501 A allows for multiple tissue sample types or tissue sample sources to be placed on a single microscope slide for tissue analysis.

In some embodiments, the positioning of the embedded tissues 512A-C and 513 is aided by the use of guides in the mold 500 to designate predetermined areas for placement of the tissue samples. For example, each of the embedded tissues 512A-C can be different tissue sample type. Embedded tissue 512A can be placed in a first area of the mold 500 demarcated by guides (not shown), and embedded tissues 512B and 512C can also be placed in second and third areas of the mold 500 demarcated by guides. Positioning embedded tissues 512A-C and 513 in the mold 500 can reduce the occurrence of overlapping tissue samples on microscope slides for improved tissue analysis results.

FIG. 6 illustrates an exemplary arrangement of multiple tissue samples 612A-G within a sample area 615 of a microscope slide 622. Seven tissue samples 612A-G are arranged within the sample area 615 of microscope slide 622. Sample area 615 is defined by a width and length. In some embodiments, the width is 12 mm and the length is 24 mm. In some embodiments, the mold for embedding tissues is about 10 mm by about 22 mm. In some embodiments, the width is 5 mm, 10 mm, 12 mm, 15 mm, 18 mm, 20 mm or any other suitable length. In some embodiments, the length is 5 mm, 10 mm, 15 mm, 18 mm, 20 mm, 24 mm, 25 mm, 28 mm, 30 mm, or any other suitable length.

The sample area 615 of the microscope slide 622 is surrounded by frame 617 and includes fiducials 620. Fiducials 620 can be used for alignment of tissue samples on the microscope slide 622 sample area 615, or can be used for positioning or orienting the microscope slide within tissue analysis equipment.

The seven tissue samples 612A-G can be arranged on the microscope slide 622 sample area 615 by embedding the seven tissue samples 612A together using a mold (for example, mold 101 of FIG. 1, molds of FIGs. 2A-E, mold 301 of FIG. 3, mold 500 of FIG. 5A) to provide an embedded tissue sample (for example embedded tissue sample 501A) including multiple tissue samples that are then sectioned into slices or sections as described in FIGs. 4A-4E. In some embodiments, the seven tissue samples 612A-G are individually embedded and sectioned and are positioned on the microscope slide 622 sample area 615 as illustrated in FIGs. 7A-E, described below. FIGs. 7A-E illustrate a method for sectioning and placement of multiple tissues on a slide (for example slide 305 in FIG. 3, slide 622 in FIG. 6) for tissue analysis. FIG. 7A shows an embedded tissue block 722 having one embedded tissue sample 713. The embedded tissue block 722 can be produced from a mold having guides, or from a mold without guides. In some embodiments, the embedded tissue block 722 is produced from mold 500 in FIG. 5A. After the tissue sample is placed on the surface of the mold, the mold is filled with embedding material to embed the one or more tissue samples in the embedding material. The embedding material may be a wax, including paraffin wax or any other suitable wax. The embedding material is cooled in the mold to produce a tissue-embedded block, such as embedded tissue block 722. The tissue embedded block is removed from the mold after the cooling.

FIG. 7B shows removal 724 of a slice or section of the embedded tissue block 722 by sectioning using a microtome or cryostat into multiple sample sections. FIG. 7C shows the placement 726 of the sectioned slice or section 712 of the embedded tissue block 722 on a slide. The section 712 of the embedded tissue block 722 includes a section of each tissue sample 713 and surrounding embedding matenal. The section 712 can be positioned on a slide by using tweezers, which can allow for positioning of multiple tissue samples 713 on a single slide. FIG. 7D shows the positioning 728 of the slices or sections on an exemplary slide with other tissue sections. The slices or sections are positioned next to one another on the slide without overlapping the sections or slices with each other or with fiducials on the slide. FIG. 7E shows a micrograph of the exemplary slide of FIG. 7D, including the multiple slices or sections.

In some embodiments, the mold and/or slide can be included in a kit. The kit can also include cassettes, slide pry bars, and a cassette block. The kit can include any suitable accessories for use with tissue analysis equipment. The methods described above can be used to prepare slides suitable for tissue analysis in the tissue analysis equipment, including positioning of tissue samples within a sample area of the slide for analysis by the tissue analysis equipment.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

We claim: A mold for processing a tissue sample comprising: a receptacle having a surface surrounded by at least one sidewall to define an interior space for receiving the tissue sample; and one or more guides extending from the surface, wherein the one or more guides are positioned within the receptacle to designate a plurality of predetermined areas of the surface configured to receive a portion of the tissue sample within the interior space. The mold of claim 1, wherein the plurality of predetennined areas of the surface comprise at least two, at least four, at least six, or at least eight predetermined areas. The mold of claim 2, wherein the plurality of predetermined areas of the surface are equally sized and shaped. The mold of claim 3, wherein the at least one sidewall comprises a first sidewall and a second sidewall opposite the first sidewall, and a third sidewall and a fourth sidewall opposite the third sidewall, and wherein the one or more guides are positioned along at least one line extending in a direction parallel to a first longitudinal axis extending from the first sidewall to the second sidewall. The mold of claim 4, wherein at least a portion of the one or more guides are positioned along a line extending in a direction parallel to a second longitudinal axis of the receptacle from the third sidewall to the fourth sidewall. The mold of claim 4, wherein the one or more guides are positioned equidistant from the first sidewall and the second sidewall. The mold of claim 1, wherein the one or more guides are pins, or plates, or posts, or walls. The mold of claim 1, wherein the one or more guides define walls extending from the surface in a direction perpendicular to a plane of the surface within the interior space. The mold of claim 1, wherein the one or more guides comprises a biocompatible metal or metal alloy. The mold of claim 9, wherein the biocompatible metal is stainless steel. The mold of claim 1, wherein the one or more guides comprises a medical grade polymer. The mold of claim 1, wherein each of the one or more guides has a height of about 0.5 mm to about 50 mm. The mold of claim 12, wherein the height is about 0.5 mm to about 1 mm. The mold of claim 1, wherein the mold is configured to receive one or more portions of the tissue sample and an embedding material in one or more of the plurality of predetermined areas of the surface of the receptacle. The mold of claim 1, wherein the interior space has a length of about 24 mm and a width of about 12 nun. The mold of claim 1, wherein the interior space of an arrayed area for a spatial transcriptomics array has a length of about 6.5 mm or about 11 mm and a width of about 6.5 mm or about 11 mm. The mold of claim 1, wherein the mold is configured to prepare two or more tissue samples for use on a spatial transcriptomics microscope slide. The mold of claim 1, wherein the mold is configured to prepare tissue samples for use in a pathology-related assay. The mold of claim 1, wherein the mold is configured to prepare tissue samples for use in a determination of the location of analytes in the tissue samples. A method of preparing a tissue specimen slide, the method comprising: disposing one or more tissue samples on a surface of a mold, the mold comprising the surface surrounded by at least one sidewall to define an interior space and one or more guides extending from the surface, the one or more guides positioned within the interior space to designate a plurality of predetermined areas of the surface configured to receive a tissue sample of the one or more tissue samples within the interior space; filling the mold with an embedding material to embed the one or more tissue samples in the embedding material; and removing a tissue-embedded block from the mold after cooling.

21. The method of claim 20, wherein disposing the one or more tissue samples in the mold comprises: determining a placement of each of the one or more tissue samples based on a visual indication of the plurality of predetermined areas of the surface designated by the one or more guides; and placing each of the one or more tissue samples on the surface of the mold within one of the plurality of predetermined areas designated by the one or more guides.

22. The method of claim 21, further comprising: sectioning the removed tissue-embedded block into a plurality of tissue sections, wherein the sectioning is parallel to a plane of the surface; and positioning one of the plurality of tissue sections on a microscope slide; wherein each tissue section of the plurality of tissue sections includes a portion of each of the one or more tissue samples surrounded by the embedding material, and wherein the embedding material includes an aperture formed through the embedding material at a position corresponding to at least one of the one or more guides in the mold.

23. The method of claim 22, wherein positioning one of the plurality of tissue sections on a microscope slide comprises: floating a tissue section on a water surface; positioning the microscope slide beneath the tissue section; and contacting the microscope slide with a surface of the tissue section.

24. The method of claim 23, further comprising: aligning the aperture with a fiducial on the microscope slide before contacting of the microscope slide with the surface of the tissue section.

25. The method of claim 24, wherein the fiducial is formed as a printed shape on the microscope slide.

26. The method of claim 24, wherein the fiducial is positioned relative to one or more arrayed areas on the microscope slide.

27. The method of claim 20, further comprising: preserving or fixing the one or more tissue samples before placing in the mold.

28. A method for preparing a tissue sample for tissue analysis, the method comprising: disposing one or more tissue samples on a surface of a mold, the surface being surrounded by at least one sidewall to define an interior space, the interior space having a predetermined length and predetermined width; filling the mold with an embedding material to embed the one or more tissue samples in the embedding material; cooling the one or more tissue samples in the embedding material to form a tissue- embedded block; removing the tissue-embedded block from the mold; sectioning the tissue-embedded block into a plurality of tissue sections; and positioning at least one of the plurality of tissue sections in a designated sample area of a microscope slide.

29. The method of claim 28, wherein the one or more tissue samples comprise a plurality of tissue samples each representing a different tissue sample type.

30. The method of claim 28, wherein disposing one or more tissue samples on a surface of a mold further comprises: determining a placement of each of the one or more tissue samples based on a visual indication of a plurality of predetermined areas of the surface designated by one or more guides extending from the surface of the mold, the one or more guides positioned within the interior space to designate a plurality of predetermined areas of the surface configured to receive a tissue sample of the one or more tissue samples within the interior space; and placing each of the one or more tissue samples on the surface of the mold within one of the plurality of predetermined areas designated by the one or more guides.