Hanging-drop three-dimensional cell culture device and methods employed therewith

Abstract

A slide-like hanging-drop cell culture device (100) is disclosed for generating, maintaining, observing, and processing three-dimensional tissues. The device provides an array of culture cavities (102) with concave bottoms (103) configured to promote aggregation and, after re-inversion, to seat formed tissues for repeatable positioning. An underside imaging cavity (104) with an inner step (105) accommodates an immersion medium (162) to enable high-magnification observation from below, while the upper face supports coverslip-based imaging using a coverslip (160), thereby permitting bidirectional microscopy without transfer. A detachable cover (120) forms a closed arrangement and incorporates hydration features (124, 126) to reduce droplet evaporation during incubation. Optional accessories include a wash array (140) arranged to deliver fluids via conduits (142) and a wash-filter array (150) with conduits (152), extensions (154) and an internal filter (155) to retain fragments during processing. Methods of use include dispensing cell suspensions, forming hanging-drops, incubating, re-inverting for positioning, washing or permeabilizing on the slide, imaging from either side, and retrieving (182) or embedding (170) tissues for downstream analysis with optional lift- out using forceps (180).

Description

[0001]HANGING-DROP THREE-DIMENSIONAL CELL CULTURE DEVICE AND METHODS EMPLOYED THEREWITH PRIORITY STATEMENT The present application claims priority to Indian Patent Application No. 202441073130, filed on 27 September 2024. The disclosure of the foregoing application is incorporated herein by reference in its entirety. FIELD OF INVENTION The present disclosure relates to the field of cell culture and tissue engineering, and more particularly to a histology slide-based hanging-drop culture device for the generation, maintenance, imaging, washing, retrieval, and processing of three-dimensional (3D) tissues. BACKGROUND OF THE INVENTION The following description of related art is intended to provide background information pertaining to the field of the disclosure. This section may include certain aspects of the art that may be related to various features of the present disclosure. However, it should be appreciated that this section be used only to enhance the understanding of the reader with respect to the present disclosure, and not as admissions of prior art. Three-dimensional (3D) cell culture refers to the in vitro cultivation of cells within a three-dimensional environment. The 3D culture of cells is superior to classical 2D culture on flat surfaces as it allows the cell to interact and organize in all directions, closely mimicking the in vivo conditions found within living tissues. The 3D cultures offer better data compatibility with natural tissues in terms of genetic, protein and histological data. The 3D cultures can be of single cell type or multiple cell types.3D cultures are prepared by using a cell in suspension from a trypsin digest of cells cultured in a monolayer. Among known approaches, the hanging-drop technique is frequently employed. In this method, cell suspensions are deposited on an inverted surface, such as the underside of a Petri dish lid, to form droplets that serve as self-contained microenvironments for cell aggregation. Although effective for initiating spheroid formation, this approach is prone to evaporation, mechanical shock, and variability. It also necessitates subsequent transfer of tissue constructs to other substrates for imaging, which can lead to rolling or displacement during microscopy. Alternative devices based on arrays of tubular spaces have also been described. In these configurations, droplets form at the open lower ends of the tubes, while a lid and holder provide a sterile environment. While such systems allow parallel culture, they present limitations including complex handling, low throughput, restrictions on construct size, and the need for centrifugation or additional equipment to retrieve tissues. Imaging in these systems remains cumbersome, and integration with histology workflows is not straightforward. The main drawbacks of hanging-drop culture in Petri dishes are that they are susceptible to mechanical shock, sample intermixing from shock, and 3D tissue rolling during imaging due to improper levelling of the microscope stage. In the hollow tube array configuration, the downstream protocol requires additional centrifugation equipment for 3D tissue retrieval. Existing hanging-drop and multi-well plate methods for 3D tissue culture are further limited by low throughput, restrictions on the size and uniformity of the resulting tissue constructs, and incompatibility with direct histology embedding. These limitations reduce scalability and constrain analytical workflows. Accordingly, there exists a need for a compact and integrated hanging-drop culture device that permits scalable generation of three-dimensional tissues, reduces evaporation, provides stable handling during imaging, enables washing and retrieval without centrifugation, and is compatible with histology embedding. Such a device and associated methods address limitations of conventional Petri dish and tubular array techniques while supporting reliable analysis of 3D cultures. OBJECTS OF THE INVENTION The principal object of the present invention is to provide a compact, microscope-compatible hanging-drop cell culture device that enables scalable formation and maintenance of three-dimensional (3D) tissues with reduced evaporation and handling sensitivity. Another object of the present invention is to permit direct on-slide microscopic imaging of 3D tissues without transfer, including high-magnification observation facilitated, in certain implementations, by underside oil-receiving features, thereby minimizing rolling or displacement during imaging. A further object of the present invention is to facilitate on-slide washing and permeabilization and retrieval of 3D tissue constructs without centrifugation. Another object of the present invention is to provide compatibility with histology workflows, including options for on-slide embedding or transfer for sectioning and staining. Another object of the present invention is to offer modularity and scalability, including single-well and multi-well arrays to accommodate different construct sizes and experimental requirements. These and other objects will become evident from the detailed description that follows. SUMMARY OF THE INVENTION The present disclosure provides a hanging-drop cell culture device arranged on a microscope-compatible substrate to generate, maintain, observe, and process three-dimensional (3D) tissues. In one aspect, the hanging-drop cell culture device defines one or more culture cavities configured to stably support hanging-droplets for cell aggregation and 3D tissue formation, and a cooperating cover forms a closed environment suited for incubation. The device is configured to enable direct, repeatable imaging on the same substrate and to support on-slide washing and retrieval of tissue constructs, thereby reducing transfers and handling steps. In certain implementations, each culture site includes a concave bottom region to assist gravity-based centering of the 3D aggregate upon re-inversion, promoting consistent positioning for microscopy and handling. The slide body may further include underside cavities with an inner step dimensioned to receive an immersion medium to facilitate high-magnification, high-numerical-aperture observation from below, while the top surface accommodates standard coverslip-based imaging. The cover incorporates hydration provisions adapted to mitigate droplet evaporation during incubation, and retention features configured to engage the slide for a closed arrangement. In another aspect, the device is operable with wash accessories that interface with the culture sites to direct washing or permeabilization solutions without dislodging the 3D tissue, and optional wash-filter accessories that retain fragments or small constructs during processing. The device further provides ergonomic and alignment features to assist handling namely grips, supports, guides and to facilitate controlled placement and removal of coverslips. The architecture is modular and scalable, enabling single-well and multi-well arrays, including higher-density formats to accommodate different droplet volumes, construct sizes, and throughput requirements. The device may be manufactured from glass or polymeric materials compatible with cell culture and optical imaging, using processes such as micromachining, molding, or additive fabrication, as appropriate for the desired tolerances and optical quality. The disclosure also provides method including, dispensing a cell suspension to define hanging droplets at the culture cavities; incubating to form 3D tissues; re-inverting to position the tissues for observation; performing on-slide washing; retrieving the tissues by pipetting for molecular analysis; imaging directly on the substrate; histology processing. In related aspects, kits comprising the slide-like device and one or more covers, wash accessories, and consumables are contemplated to support the foregoing methods. The foregoing summary is provided to introduce selected aspects in a simplified form and is not intended to identify essential features nor to limit the scope of the claimed subject matter, which is defined solely by the appended claims. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS The nature, scope and operation of the present invention will be better understood with reference to the accompanying drawings, which illustrate a preferred embodiment of the invention. It is important to note that these drawings are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Skilled artisans will recognize that the elements depicted in these figures though may be drawn to scale; various modifications and substitutions may be made without departing from the spirit of the invention. Figure 1 illustrates a top-side perspective view of the hanging-drop cell culture device (100) showing culture cavities (102) with concave regions (103), a peripheral groove (106), support studs (107), finger grips (108), a central platform (109), coverslip positioning guides (L-brackets) (110), and a triangular lift-off recess (112); Figure 2 illustrates a bottom-side view of the device (100) showing underside imaging cavities (104) with finger grips (108); Figure 3 illustrates a sectional view through a culture site showing the concave-bottom region (103) of the culture cavity (102) and the corresponding underside cavity (104) with inner step (105); Figure 4 illustrates a perspective view of a detachable cover (120) showing finger grips (122), a retaining collar (125), and hydration provisions including an inward step (124) and a hydration chamber (126). Figure 5 illustrates the detachable cover (120) in an inverted orientation, showing finger grips (122), the retaining collar (125), and the inward step (124); Figure 6 illustrates a perspective view of the assembled, closed arrangement, with the hanging-drop cell culture device (100) oriented bottom-side facing up to show the underside imaging cavities (104) having circular inner steps (105) and the finger grips (108). The slide (100) is shown engaged with and resting on the retaining collar (125) of the detachable cover (120), which includes finger grips (122); Figure 7 illustrates magnified view of the upper face of the hanging-drop cell culture device (100) with the L-bracket (110); Figure 8 illustrates a magnified view of the upper face of the histology slide (100) with a triangular recess (112); Figure 9 illustrates a perspective view of a wash accessory (140) with wash conduits (142); Figure 10 illustrates a perspective diagram of the wash-filter array (150) with washing conduits (152) on the top side that continue to the underside as extended portions (154) Figure 11 illustrates a perspective diagram with the histology slide (100) assembled to the underside of the wash array (140) and the wash-filter array (150), wherein the washing conduits (152) continue as underside extensions (154) that interface with the top side of the wash array (140); Figure 12 illustrates a cross-section assembly showing the histology slide (100) with the culture cavity floor (103) seating a post–hanging-drop 3D tissue (134), positioned beneath a wash array conduit (142) having an internal funnel surface (144) and a wash-filter array conduit (154) including an internal filter (155); Figure 13 illustrates the device (100) prepared for imaging, with a coverslip (160) overlaid on the culture cavities (102) and immersion oil (162) received within the underside cavities (104) to permit high-magnification imaging from the underside. The configuration enables bidirectional observation of samples; Figure 14 illustrates the device (100) generating an embedded 3D tissue (134) within paraffin wax (170), the embedded construct being oriented outward for retrieval using forceps (180); Figure 15 illustrates the device (100) generating a 3D tissue (134) that can be manually retrieved by suction using a micropipette system (182); Figure 16 illustrates stepwise method flowchart for 3D tissue formation; and Figure 17 illustrates a use-workflow variant depicting microscopy procedure decisions namely top-side coverslip imaging and underside immersion oil imaging—together with live-imaging and drug-treatment sequences, sample storage, and re-inversion for embedding and sectioning. DETAILED DESCRIPTION OF THE INVENTION The following is a detailed description of embodiments of the present disclosure depicted in the accompanying drawings. Like reference numerals refer to like elements across the figures. The embodiments are described in such detail as to clearly communicate the disclosure; however, the details provided herein is not intended to limit the scope of the invention. Rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims. In the following description, numerous specific details are set forth to provide a thorough understanding of embodiments of the present invention. It will be apparent to one skilled in the art that embodiments of the present invention may be practiced without some of these specific details. If the specification states that a component “may,” “can,” “could,” or “might” be included or possess a characteristic, such language is not intended to be limiting. As used herein, “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise, and “in” is to be interpreted as “in” or “on” as contextually appropriate. The present invention is not limited to the embodiments herein, and various modifications and substitutions may be made without departing from the spirit and scope of the invention. For clarity and consistency, the device described herein is referred to as a “hanging-drop cell culture device (100)”; unless the context indicates otherwise, the expressions “slide- like device,” “histology slide,” and “culture slide device” are used interchangeably to refer to device (100). The present invention discloses a hanging-drop cell culture device (100) configured to generate, maintain, image, process, and retrieve three-dimensional (3D) tissues on a microscope-compatible substrate. The device (100) generally comprises a slide-like body bearing a plurality of culture cavities (102) with concave bottom regions (103), corresponding underside imaging cavities (104) with inner steps (105), a peripheral groove (106) for cover engagement, support studs (107), finger grips (108), a central platform (109), coverslip positioning guides (L-brackets) (110), and a triangular lift-off recess (112). A detachable cover (120) may include finger grips (122), a retaining collar (125) arranged to couple with the groove (106), and hydration provisions (124, 126). Optional processing accessories include a wash array (140) having wash conduits (142) with internal funnel surfaces (144), and a wash-filter array (150) having washing conduits (152) that continue as underside extensions (154) with an internal filter (155). Bidirectional imaging can be performed by overlaying a coverslip (160) over selected culture cavities (102) or by introducing an immersion medium (162) into the underside cavity (104) at the step (105). Retrieval may be performed directly by a micropipette (182) and optionally by on-substrate embedding (170) followed by lift-out with forceps (180). Referring to Fig.1, the upper face of the device (100) defines a plurality of culture cavities (102) each having a concave bottom region (103) configured to promote aggregation during hanging-drop culture and to assist gravity-based seating of the formed 3D tissue upon re-inversion. The cavities (102) may be arranged in rows and columns on the central platform (109) to realize array formats for example, 24-well, 48-well, 96-well or 384- well embodiment without limitation to any particular density or pitch. In certain embodiments, coverslip positioning guides (L-brackets) (110) facilitate stable placement of a coverslip (160), while a triangular lift-off recess (112) assists removal. A peripheral groove (106) is provided for cover engagement, and handling features such as support studs (107) and finger grips (108) may assist placement and manipulation of the device (100). Referring to Fig.2 and 3, the underside of the device (100) includes one or more underside imaging cavities (104) disposed beneath respective culture sites. In section, each underside cavity (104) incorporates an inner step (105) dimensioned to receive and retain an immersion medium (162), thereby providing a stable optical interface and working distance compatible with high magnification. Herein, immersion medium (162) encompasses immersion oil and functionally equivalent media. During working, referring to Fig. 13, the underside configuration enables bidirectional observation—top-side coverslip-based imaging can be performed independently or in combination with underside immersion imaging—without removing the 3D tissue from its culture site. The geometries of (104) and (105) (e.g., diameter, depth, and step width) may be selected to suit objective front-lens dimensions, focal constraints, and stage clearance, while maintaining compatibility with standard microscope hardware. Referring to Fig.4 and 5, a detachable cover (120) is provided to cooperate with the slide- like device (100) and establish a closed arrangement suitable for incubation. The cover (120) includes finger grips (122) for handling and a retaining collar (125) arranged to engage the peripheral groove (106) of the slide to provide positional stability and reduce inadvertent separation during inversion and transfer. In certain embodiments, the cover incorporates hydration provisions (124, 126), wherein an inward step (124) and a hydration chamber (126) are configured to hold a hydrating medium to mitigate droplet evaporation during hanging-drop culture. Fig.5 illustrates the cover (120) in an inverted orientation to show the spatial relationship of the finger grips (122), retaining collar (125), and inward step (124). The cover geometry may be selected to achieve reliable seating and sealing with the groove (106) while permitting repeated assembly or disassembly, and maintaining compatibility with standard incubator and microscope workflows. Referring to Fig.6, the slide-like device (100) is shown engaged with the detachable cover (120) to form an assembled, closed arrangement for incubation and handling. In the illustrated orientation, the assembly is presented bottom-side up to reveal the underside imaging cavities (104) with inner steps (105) on the slide (100), as well as the slide finger grips (108). The slide (100) is seated on and retained by the cover’s collar (125), which interfaces with the slide peripheral groove (106) as illustrated in Figs. 1 and 4, while the cover’s finger grips (122) facilitate secure manipulation of the assembled unit during inversion and transfer. This arrangement permits formation and maintenance of hanging- drops within the closed environment and preserves alignment of the slide relative to the cover throughout use. In use, a cell suspension is dispensed into selected culture cavities (102) on the slide (100) to define droplets when the slide is inverted. The slide is then engaged with the cover (120) to establish the closed arrangement, and the assembly is inverted to orient the droplets downward for hanging-drop culture. During incubation, the cover’s hydration provisions (124, 126) may hold a hydrating medium to mitigate evaporation. Following aggregation and tissue formation, the assembly is re-inverted, whereupon the formed 3D tissue (134) settles by gravity and is seated by the concave bottom region (103) of the corresponding cavity (102), as illustrated in Fig. 3. This seating facilitates repeatable positioning for subsequent handling and observation, including on-slide washing and imaging. Referring to Fig. 9, a wash accessory (140) is configured to interface with the underside of the slide-like device (100) to deliver fluids across selected culture cavities (102). The wash accessory (140) includes one or more wash conduits (142) that, in certain embodiments, incorporate internal funnel surfaces (144) arranged to direct flow toward the culture site while discouraging displacement or egress of a formed 3D tissue (134) during washing or permeabilization. As illustrated in Fig.11, the wash accessory (140) is shown in an assembled relation with the slide (100) (and, where employed, with the wash- filter array described below), such that the wash conduits (142) are aligned with corresponding culture sites to provide controlled, localized fluid delivery. Referring to Fig. 10, a wash-filter accessory (150) includes washing conduits (152) on the top side that continue to the underside as extensions (154). As illustrated in Fig. 11, the extensions (154) are arranged to interface with the top side of the wash array (140) when the assemblies are mated to the slide (100), thereby establishing a coupled flow path aligned to the culture sites (102). As illustrated in Fig. 12, the wash-filter accessory (150) incorporates an internal filter element (155) positioned within the conduit path - within or downstream of an extension (154) to retain fragments or small constructs during washing or permeabilization while permitting through-flow of the processing solution. The placement and characteristics of the filter (155) may be chosen to balance retention against flow resistance, and can be adapted for different tissue sizes and fluid properties without departing from the disclosed arrangement. Referring to Fig. 7, coverslip positioning guides (L-brackets) (110) on the upper face of the slide-like device (100) facilitate accurate placement of a coverslip (160) over selected culture cavities (102) for top-side observation. As illustrated in Fig.8, a triangular lift-off recess (112) assists controlled removal of the coverslip after imaging. As illustrated in Fig. 13, underside observation is enabled by introducing an immersion medium (162) (e.g., immersion oil) into the inner step (105) of the underside imaging cavity (104), thereby providing a stable optical interface compatible with high-magnification, high numerical aperture (NA) objectives. These arrangements support bidirectional observation—top-side coverslip imaging and underside immersion imaging without transferring the 3D tissue from its culture site. Referring to Fig.14, on-substrate embedding (170) may be employed to stabilize a formed 3D tissue (134) within a solidifiable medium, after which the embedded construct is oriented outward for lift-out with forceps (180) to facilitate downstream histological sectioning. As illustrated in Fig.15, an alternative retrieval approach allows the 3D tissue (134) to be manually aspirated by suction using a micropipette system (182) directly from the culture cavity (102). These retrieval options enable collection for analysis without centrifugation and are compatible with the bidirectional imaging arrangements described above. Fig. 16, illustrates a method for 3D tissue formation, including dispensing a cell suspension into selected culture cavities (102), engaging the cover (120) to establish a closed arrangement, inverting the assembly to form hanging-drops, incubating with hydration provided by (124, 126), and thereafter re-inverting so that the formed 3D tissue (134) is seated by the concave bottom region (103). As illustrated in Fig.17, downstream use-workflow variants may include top-side coverslip imaging and, in some implementations, underside immersion imaging (using coverslip (160) and immersion medium (162) at the underside cavity (104) and inner step (105)), optional live imaging or drug-treatment sequences conducted on the slide (100), temporary sample storage, and re-inversion for embedding (170) followed by sectioning. Steps may be performed in different orders or repeated as appropriate, and parameters may be selected according to the application and tissue type without departing from the disclosed arrangements. The slide-like device (100) and cover (120) may be formed from optically transparent, biocompatible materials selected to balance manufacturability, imaging performance, and sterilization compatibility. In certain embodiments, the slide body is fabricated from glass to provide low autofluorescence and high scratch resistance, while alternative embodiments employ polymeric substrates to enable high-volume molding. As illustrated in Figs.1 and 4, the peripheral groove (106) and retaining collar (125) may be dimensioned for an interference fit or a clearance fit, chosen in view of material selection and anticipated temperature and humidity conditions, to maintain positional stability while allowing repeated assembly and disassembly. Surface treatments may be applied locally or globally to tune wetting characteristics for hanging-drop formation at the culture cavities (102) and concave bottoms (103), and to promote retention of an immersion medium (162) at the inner step (105) within the underside imaging cavity (104). The wash array (140) and wash-filter array (150) may be produced using similar processes; in some embodiments, a filter element (155) is provided as a replaceable cartridge or insert, and the extensions (154) and conduits (152, 142) are molded or machined with tapers consistent with the funnel surface (144) geometry to guide flow while minimizing shear on a seated tissue (134). Materials and finishes may be selected to be compatible with common sterilization methods (e.g., ethanol rinse, gamma, or autoclave where suitable for the material), and standard laboratory consumables (e.g., coverslip (160) dimensions) can be accommodated by appropriate selection of L-brackets (110) and recess (112) geometry. Referring to Fig. 1 to 17, the culture cavities (102) and concave bottom regions (103) are dimensioned to support stable hanging-drop formation, aggregation, and subsequent gravity-based seating upon re-inversion; the underside imaging cavities (104) and inner steps (105) are sized to accommodate an immersion medium (162) while preserving objective working distance and providing a stable optical interface; the array layout on the central platform (109) is chosen to realize the illustrated formats (e.g., 24-well / 48- well / 96-well / 384-well styles) while maintaining adequate pitch for dispensing, imaging, and accessory alignment; the groove-to-collar engagement (106 125) provides reliable seating and repeatable assembly and disassembly; the L-brackets (110) and lift-off recess (112) are proportioned to accept standard coverslips and common handling tools; and the wash conduits (142) with funnel surfaces (144), together with the wash-filter conduits (152, 154) and filter (155), are proportioned to deliver processing solutions while discouraging displacement of a seated 3D tissue (134) and retaining fragments as required. Manufacturing tolerances are selected according to the chosen materials and processes (e.g., machined glass or molded polymers) so that mating features assemble reliably and optical interfaces remain sufficiently flat and smooth for microscopy. Quantitative dimensions may be defined for implementations without departing from the scope of the present disclosure. Referring to Fig.1 and 6, the array of culture cavities (102) on the central platform (109) may be realized in alternative densities and layouts to suit experimental requirements, including but not limited to 24-well, 48-well, 96-well or 384-well embodiments. The layouts may be rectangular or circular while maintaining compatibility with dispensing, incubation, washing, and imaging workflows. As illustrated in Fig. 17, the use workflow variants are supported by the same core architecture. Optional features such as coverslip guides (110), lift-off recess (112), support studs (107), and finger grips (108) may be included or omitted in different implementations. The device may be provided alone or together with the cover (120) and one or both processing accessories—wash array (140) and wash-filter array (150)—as a kit adapted for the foregoing workflows. Where fluid communication between sites is desired, controlled delivery through wash conduits (142) and through conduits (152) with extensions (154) can be arranged while discouraging tissue egress via funnel surfaces (144) and, where appropriate, retention by the filter element (155), without departing from the disclosed arrangements. The disclosed hanging-drop cell culture device is applicable in pharmaceutical discovery, toxicology, oncology, regenerative medicine, and academic research settings. By enabling stable formation of three-dimensional tissues, direct on-substrate imaging (including high-magnification observation), on-slide washing or permeabilization, and simplified retrieval or embedding, the device supports end-to-end workflows encompassing assay development, drug-response profiling, phenotypic screening, and histology. The architecture is compatible with standard laboratory equipment and can be manufactured from glass or polymeric materials using established processes, allowing scalable production and integration into existing laboratory pipelines.

Claims

I claim:

1. A hanging-drop cell culture device (100) comprising: a) a slide-like body defining a plurality of culture cavities (102) on an upper face, each culture cavity having a concave floor (103) configured to promote cell aggregation in a hanging-drop condition and to seat a formed three-dimensional tissue upon re-inversion; and b) an underside imaging cavity (104) aligned with the corresponding culture cavities and including an inner step (105) configured to retain an immersion medium (162) for optical observation from below.

2. The device of claim 1, further comprising a detachable cover (120) configured to cooperate with the slide-like body (100) to form a closed arrangement suitable for incubation.

3. The device of claim 2, wherein the slide-like body (100) comprises a peripheral groove (106) and the cover (120) comprises a collar (125) configured to engage the peripheral groove (106).

4. The device of any of claims 1 to 3, wherein the cover (120) comprises a hydration feature (124, 126) configured to hold a hydrating medium to reduce droplet evaporation during incubation.

5. The device of any of claims 1 to 4, further comprising a coverslip guide (110) on the upper face and a lift-off recess (112) arranged to facilitate placement and removal of a coverslip (160) for top-side imaging.

6. The device of any of claims 1 to 5, further comprising at least one handling feature selected from a support stud (107) and a finger grip (108).

7. The device of any of claims 1 to 6, wherein the concave floor (103) is arranged to gravity-seat the formed three-dimensional tissue for repeatable positioning after re-inversion.

8. The device of any of claims 1 to 7, wherein the underside imaging cavity (104) and the inner step (105) are dimensioned to accommodate an immersion medium (162) compatible with observation using an immersion objective.

9. The device of any of claims 1 to 8, wherein the culture cavities (102) are arranged in an array on the slide-like body (100) corresponding to a multi-well format selected from 24-well, 48-well, 96-well, and 384-well layouts.

10. The device of any of claims 1 to 9, wherein at least a portion of the slide-like body (100) comprises an optically transparent material selected from glass and polymeric substrates.

11. An on-slide processing assembly comprising: a) a wash array (140) configured to couple to a hanging-drop cell culture device (100) and having at least one conduit (142) arranged to deliver a processing fluid toward a culture cavity (102); and b) a wash-filter array (150) configured to couple to the wash array (140) and comprising a conduit path that continues as an underside extension (154) and a filter element (155) positioned in the conduit path to retain fragments during processing while permitting through-flow of the processing fluid.

12. The assembly of claim 11, wherein at least one conduit (142) of the wash array (140) includes a funnel surface (144) configured to guide flow and discourage egress of a three-dimensional tissue (134) from the culture cavity (102).

13. The assembly of claim 11 or 12, wherein, when the wash array (140) and the wash-filter array (150) are mated to the device (100), the conduit path (152, 154) is aligned with a culture cavity (102) of the device.

14. A method for forming and processing a three-dimensional tissue (134) on a slide-like hanging-drop cell culture device (100), the method comprising: a) dispensing a cell suspension into a culture cavity (102) to define a droplet;b) establishing a closed arrangement with a cover (120) and inverting the device (100) and the cover (120) to provide a hanging-drop condition; c) incubating to form the three-dimensional tissue (134); d) re-inverting the device (100) such that the formed tissue (134) is seated by a concave floor (103) of the culture cavity (102); e) delivering a processing fluid toward the culture cavity (102) using a wash array (140) and, optionally, retaining fragments using a wash-filter array (150) including a filter element (155); f) imaging the formed tissue (134) from a top side using a coverslip (160); and, in some implementations, from an underside by retaining an immersion medium (162) at an inner step (105) of an underside imaging cavity (104); and g) retrieving the formed tissue (134) by aspiration using a micropipette (182); 15. The method of claim 14, wherein the processing comprises washing or permeabilization performed while the formed tissue (134) remains seated within the culture cavity (102).

16. The method of claim 14 or 15, wherein underside imaging is performed using an immersion objective while the immersion medium (162) is retained at the inner step (105) of the underside imaging cavity (104).

17. The method of any of claims 14 to 16, further comprising placing and removing a coverslip (160) using a coverslip guide (110) and a lift-off recess (112) on the device (100).

18. A kit comprising: the device of any of claims 1 to 10; at least one detachable cover (120) configured to engage the device (100); and at least one accessory selected from a wash array (140) and a wash-filter array (150).

19. The kit of claim 18, further comprising one or more coverslips (160) and one or more consumables selected from an embedding medium (170) and a filter (155).

20. The kit of claim 18 or 19, wherein the components are packaged for workflows comprising hanging-drop formation, on-slide processing, bidirectional imaging, and retrieval or embedding of three-dimensional tissues (134).

Images