Microfluidic assay devices for supporting multi-layer cellular matrices

Abstract

Microfluidic assay devices that support multi-layer cellular matrices (e.g., ex-vivo skin cell matrices) and associated methods of fabrication and use are provided. A holder including a microfluidic chamber includes multiple sidewall ports disposed at different levels, multiple inlet ports in communication with different sidewall ports, and a liquid-permeable membrane bounding a chamber bottom. Stratified layers of cell-containing material may be formed in the chamber by sequential steps of introducing cell-hydrogel precursor material through a sidewall port and crosslinking the same, then repeating the steps for each sidewall port, to form cell-containing material layers in contact with one another. A cover with distribution channel(s) is provided below the liquid-permeable membrane. A continuous upper extension of the chamber may be open from above to provide interface(s) for receiving analytes and / or for environmental interaction. A holder may removably receive multiple assay devices.

Description

MICROFLUIDIC ASSAY DEVICES FOR SUPPORTING MULTI-LAYER CELLULAR MATRICESCross-Reference to Related Application^)

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 688,480 filed on August 29, 2024, wherein the entire disclosure of the foregoing application is hereby incorporated by reference herein.Technical Field

[0002] This disclosure relates to microfluidic assay devices, including devices configured to support multi-layer cellular matrices (optionally embodying ex-vivo skin cell matrices), and apparatuses incorporating plural microfluidic assay devices, as well as associated methods of fabricating and using such devices.Background

[0003] Dermatology and beauty product development research / testing has traditionally been limited to animal testing. Due to increasing controversy with the use of animal testing, there is an increasing need for ex-vivo models to perform skin research and testing. There is a further need for performing skin sensitization studies (or chemical skin irritation studies) for assessing safety of various chemicals, such as for publishing information in Material Safety Data Sheets.

[0004] Recent advances in micro- and nano-scale (e.g., micropatterning and microfluidics) technologies have enabled the development of platforms to study interactions with cells within well-defined microenvironments. Cells have been encapsulated in hydrogel matrices to allow them to move in various directions, as well as to increase their contact with their surroundings, making these models highly biomimetic. These models have been incorporated into microfluidic devices to provide a more lifelike ex-vivo environment as compared to larger-scale platforms such as petri dishes, etc. Various prior devices have permitted tissue types such as skin, liver, epithelial, endothelial, and other tissues to be studied and / or assayed.

[0005] Prior testing platforms incorporating cellular materials have suffered from limitations that have restricted their utility. For example, certain platforms have exhibited limited capability to incorporate stratified layers of cellular material, limited flexibility in supplying media and analytes (or samples) to interact with cellular material, limited ability to simultaneously analyze multiple cellular layers in a single microfluidic device, and limitedability to simultaneously image and analyze multiple microfluidic assay devices (e.g., for multi-sample and / or multi-condition studies).

[0006] Need therefore exists in the art for microfluidic assay devices that address limitations associated with conventional devices, as well as methods for fabricating and using the same, and related apparatuses incorporating microfluidic assay devices.Summary

[0007] This disclosure relates to a microfluidic assay devices, including devices configured to support multi-layer cellular matrices (optionally embodying ex-vivo skin cell matrices), and apparatuses incorporating plural microfluidic assay devices, as well as associated methods of fabricating and using such devices. A microfluidic assay device includes a holder that defines a first microfluidic chamber laterally bounded by a sidewall with multiple sidewall ports disposed at different levels, multiple inlet ports in fluid communication with different sidewall ports, and a liquid-permeable membrane bounding a bottom of the microfluidic chamber. The different sidewall ports permit formation of stratified layers of cell-containing material (e.g., cells with hydrogel) in the first microfluidic chamber (e.g., by supplying hydrogel precursor and first cells to the chamber through a first sidewall port and crosslinking the hydrogel precursor, followed by supplying hydrogel precursor and second cells to the first microfluidic chamber through a second sidewall port and crosslinking the hydrogel precursor, etc.). In certain embodiments, additional sidewall ports may be provided at one or more additional levels to permit formation of additional layers of cell-containing material. Each layer of cellcontaining material is in contact with at least one other layer of cell-containing material. A cover having at least one distribution channel may be arranged below the liquid-permeable membrane, with the membrane being configured to support the cell-containing material from below and configured to permit passage of liquid media to contact the cell-containing material. A second microfluidic chamber may serve as a continuous upper extension of the first microfluidic chamber, and may be open from above to provide an interface for receiving analytes (or samples) and / or for providing an interface (e.g., air / liquid interface) for interacting with a surrounding environment. Multiple inlet ports and multiple media ports may be accessible along the top of the body structure. A microfluidic apparatus may include a holder having multiple primary recesses and device retaining features, with the holder being configured to receive and removably retain multiple microfluidic assay devices in the primary recesses (i.e., with one microfluidic assay device per primary recess). In certain embodiments,the holder may be compatible in size and shape with a conventional microplate, to permit multiple microfluidic assay devices to be interfaced with microplate handling equipment, automated pipettors, imaging equipment, and the like.

[0008] In one aspect, a microfluidic assay device configured to support a multi-layer cellular matrix comprises a body structure having a top, having a bottom, and defining a first microfluidic chamber laterally bounded by a sidewall, wherein the first microfluidic chamber comprises a chamber bottom, at least one first sidewall port arranged at a first level, and at least one second sidewall port arranged at a second level, the chamber bottom being closer to the first level than to the second level. At least one first inlet port and at least one second inlet port are accessible from the top of the body structure, with the at least one first inlet port being arranged in fluid communication with the at least one first sidewall port, with and the at least one second inlet port being arranged in fluid communication with the at least one second sidewall port. A liquid-permeable membrane bounds the chamber bottom.

[0009] In certain embodiments, the body structure further comprises at least one first channel arranged in fluid communication with the at least one first sidewall port, at least one first via extending from the at least one first inlet port to the at least one first channel, at least one second channel arranged in fluid communication with the at least second first sidewall port, at least one second via extending from the at least one first inlet port to the at least one first channel.

[0010] In certain embodiments, the body structure comprises a plurality of liquid media ports accessible from the top of the body structure; and a plurality of liquid media vias between the plurality of liquid media ports and at least one distribution channel defined in a cover underlying the liquid-permeable membrane.

[0011] In certain embodiments, the at least one first sidewall port includes a pair of horizontally opposed first sidewall ports, the at least one first inlet port includes a pair of first inlet ports, the at least one second sidewall port includes a pair of horizontally opposed second sidewall ports, the at least one second inlet port includes a pair of second inlet ports.

[0012] In certain embodiments, the first microfluidic chamber contains a first layer of first cells within hydrogel material, and a second layer of second cells within hydrogel material; the first layer is arranged on the liquid-permeable membrane and is substantially aligned with the first level; the second layer is arranged on the first layer and is substantially aligned with the second level; and the second cells differ in cell type from the first cells.

[0013] In certain embodiments, the first microfluidic chamber further comprises at least one third sidewall port arranged at a third level, the chamber bottom being closer to the second level than to the third level; and the microfluidic assay device further comprises least one thirdinlet port accessible from the top of the body structure, the at least one third inlet port being arranged in fluid communication with the at least one third inlet port. In a related embodiment, the first microfluidic chamber may further contain a third layer of third cells within hydrogel material, the third layer being arranged on the second layer and being substantially aligned with the third level; and the third cells differing in cell type from the first cells and from the second cells.

[0014] In certain embodiments, the liquid-permeable membrane defines a plurality of membrane vias aligned with the plurality of liquid media vias. In certain embodiments, the liquid-permeable membrane defines a plurality of membrane vias aligned with the plurality of liquid media vias.

[0015] In certain embodiments, the first microfluidic chamber comprises a tapered sidewall, wherein a width of the first microfluidic chamber decreases with distance away from the chamber bottom. In certain embodiments, the first microfluidic chamber comprises a frustoconical shape. In certain embodiments, the body structure comprises a second microfluidic chamber (optionally having a constant width) that embodies an upper extension of the first microfluidic chamber, wherein the upper microfluidic chamber is open along an upper boundary thereof.

[0016] In certain embodiments, a lower portion of the body structure comprises a plurality of protrusion receiving features configured to receive upwardly extending protrusions of a microfluidic device holder, wherein such features may be received by corresponding protrusions associated with a primary recess of a holder of a microfluidic apparatus, with the holder including multiple primary recesses and configured to receive multiple microfluidic assay devices.

[0017] In one aspect, the disclosure relates to a microfluidic apparatus comprising a holder and one or more microfluidic assay devices as described herein received within one or more primary recesses of the holder. The holder comprises a holder body structure defining an upper holder surface, a lower holder surface, a plurality of primary recesses extending downward relative to the upper holder surface, and a plurality of device retaining features. One or more microfluidic assay devices are received within one or more corresponding primary recesses of the plurality of primary recesses, wherein each microfluidic assay device is removably retained by one or more device retaining features of the plurality of device retaining features. In certain embodiments, at least some device retaining features of the plurality of device retaining features comprise upwardly-extending protrusions; optionally, the upwardly-extendingprotrusions each comprise a generally cylindrical shape, and extend upward from floor portions proximate to corner areas of primary recesses of the plurality of primary recesses.

[0018] In certain embodiments, the holder body structure further comprises at least one well that opens to the upper holder surface, and that is configured to hold a liquid, wherein the at least one well is non-coincident with the plurality of primary recesses.

[0019] In certain embodiments, each primary recess comprises a window opening extending through the lower holder surface, and the window opening is sized and shaped to receive at least a portion of the cover of a corresponding microfluidic assay device of the plurality of microfluidic assay devices.

[0020] In another aspect, the disclosure relates to a method for fabricating a multi-layer cellular matrix in a microfluidic assay device, the method comprising multiple steps. A first step includes providing a microfluidic assay device comprising: (i) a body structure having a top, having a bottom, and defining a first microfluidic chamber laterally bounded by a sidewall, wherein the first microfluidic chamber comprises a chamber bottom, at least one first sidewall port arranged at a first level, and at least one second sidewall port arranged at a second level, the chamber bottom being closer to the first level than to the second level; (ii) at least one first inlet port and at least one second inlet port accessible from the top of the body structure, the at least one first inlet port being arranged in fluid communication with the at least one first sidewall port, and the at least one second inlet port being arranged in fluid communication with the at least one second sidewall port, and (iii) a liquid-permeable membrane bounding the chamber bottom. A second step includes supplying a first mixture of hydrogel precursor and first cells through the at least one first inlet port and the at least one first sidewall port into the first microfluidic chamber. A third step includes crosslinking the hydrogel precursor of the first mixture to produce, on the liquid-permeable membrane and in the first microfluidic chamber, a first layer of first cells within crosslinked hydrogel, the first layer being substantially aligned within the first level. A fourth step includes supplying a second mixture of hydrogel precursor and second cells through the at least one second inlet port and the at least one second sidewall port into the first microfluidic chamber, the second cells differing in type from the first cells. A fifth step includes crosslinking the hydrogel precursor of the second mixture to produce, on the first layer and in the first microfluidic chamber, a second layer of second cells within crosslinked hydrogel, the second layer being substantially aligned within the second level.

[0021] In certain embodiments, the first microfluidic chamber further comprises at least one third sidewall port arranged at a third level, the chamber bottom being closer to the second level than to the third level, and the microfluidic assay device further comprises least one thirdinlet port accessible from the top of the body structure, the at least one third inlet port being arranged in fluid communication with the at least one third inlet port, wherein the method further comprises additional steps. One additional step includes supplying a third mixture of hydrogel precursor and third cells through the at least one third inlet port and the at least one third sidewall port into the first microfluidic chamber, the third cells differing in type from the first cells and the second cells. Another additional step includes crosslinking the hydrogel precursor of the third mixture to produce, on the second layer and in the first microfluidic chamber, a third layer of third cells within crosslinked hydrogel, the third layer being substantially aligned within the third level. In certain embodiments, the first cells comprise adipocytes, the second cells comprise fibroblasts, and the third cells comprise keratinocytes.Brief Description of Drawings

[0022] FIG. 1A is an exploded lower perspective view of a microfluidic assay device configured to support a multi-layer cellular matrix, the microfluidic assay device including a body structure, a liquid-permeable membrane, adhesive material, and a cover.

[0023] FIG. IB is an exploded upper perspective view of the microfluidic assay device of FIG. 1A.

[0024] FIG. 2A is a top plan view of the body structure of the microfluidic assay device of FIGS. 1A-1B.

[0025] FIG. 2B is a bottom plan view of the body structure of the microfluidic assay device ofFIGS. 1A-1B.

[0026] FIG. 2C is an upper perspective view of the body structure of the microfluidic assay device of FIGS. 1A-1B.

[0027] FIG. 3 is a perspective view of the cover of the microfluidic assay device of FIGS. 1 A-1B, showing one or more distribution channels formed therein.

[0028] FIG. 4A is a top plan view of the body structure of the microfluidic assay device of FIGS. 1A-1B, with addition of section lines 4B-4B, 4C-4C, and 4D-4D.

[0029] FIG. 4B is a cross-sectional view of the body structure of FIG. 4A taken along section line 4B-4B.

[0030] FIG. 4C is a cross-sectional view of the body structure of FIG. 4A taken along section line 4C-4C.

[0031] FIG. 4D is a cross-sectional view of a partially assembled microfluidic assay device including a cover according to FIGS. 1A-1B affixed to the body structure of FIG. 4A taken along section line 4D-4D.

[0032] FIG. 4E is a cross-sectional view including excerpts from FIG. 4D, namely: the first and second microfluidic chambers of the body structure, a lower recess defined in the body structure for receiving a porous membrane, and a distribution channel defined in the cover.

[0033] FIG. 5 is a cross-sectional view of an assembled microfluidic assay device including the items of FIG. 4E with addition of a porous membrane between the body structure and the cover, and following formation of a multi-layer cellular matrix in the first microfluidic chamber.

[0034] FIG. 6 is a top plan view of an assembled microfluidic assay device according to FIGS. 1A-1B, with addition of section lines A-A, B-B, and C-C, respectively.

[0035] FIG. 7A is a cross-sectional view of the assembled microfluidic assay device taken along section line A-A of FIG. 6, after supplying a first mixture of hydrogel precursor and first cells through one first inlet port, one first via, one first channel, and one first sidewall port into the first microfluidic chamber to contact an underlying membrane.

[0036] FIG. 7B is a cross-sectional view of the assembled microfluidic assay device taken along section line B-B of FIG. 6, after supplying a second mixture of hydrogel precursor and second cells through one second inlet port, one second via, one second channel, and one second sidewall port into the first microfluidic chamber, to contact an underlying first layer of crosslinked hydrogel and first cells.

[0037] FIG. 7C is a cross-sectional view of the assembled microfluidic assay device taken along section line C-C of FIG. 6, after supplying a third mixture of hydrogel precursor and third cells through one third inlet port, one third via, and one third sidewall port into the first microfluidic chamber, to contact an underlying second layer of crosslinked hydrogel and second cells.

[0038] FIG. 8A is a cross-sectional view of the assembled microfluidic assay device taken along section line A-A of FIG. 6, after supplying a first mixture of hydrogel precursor and first cells through multiple first inlet ports, multiple first vias, multiple first channels, and multiple first sidewall ports into the first microfluidic chamber to contact an underlying membrane.

[0039] FIG. 8B is a cross-sectional view of the assembled microfluidic assay device taken along section line B-B of FIG. 6, after supplying a second mixture of hydrogel precursor and second cells through multiple second inlet ports, multiple second vias, multiple secondchannels, and multiple second sidewall ports into the first microfluidic chamber, to contact an underlying first layer of crosslinked hydrogel and first cells.

[0040] FIG. 8C is a cross-sectional view of the assembled microfluidic assay device taken along section line C-C of FIG. 6, after supplying a third mixture of hydrogel precursor and third cells through multiple third inlet ports, multiple third vias, multiple third channels, and multiple third sidewall port into the first microfluidic chamber, to contact an underlying second layer of crosslinked hydrogel and second cells.

[0041] FIG. 9A is a cross-sectional view of the assembled microfluidic assay device taken along section line A- A of FIG. 6, after supplying a first mixture of hydrogel precursor and first cells through one first inlet port, one first via, and one first sidewall port into the first microfluidic chamber to contact an underlying membrane, with hydrogel precursor (but without cells) being present in another first channel, another first via, and another first inlet port.

[0042] FIG. 9B is a cross-sectional view of the assembled microfluidic assay device taken along section line B-B of FIG. 6, after supplying a second mixture of hydrogel precursor and second cells through one second inlet port and one second sidewall port into the first microfluidic chamber, to contact an underlying first layer of crosslinked hydrogel and first cells, with hydrogel precursor (but without cells) being present in another second channel, another second via, and another second inlet port.

[0043] FIG. 9C is a cross-sectional view of the assembled microfluidic assay device taken along section line C-C of FIG. 6, after supplying a third mixture of hydrogel precursor and third cells through one third inlet port and one third sidewall port into the first microfluidic chamber, to contact an underlying second layer of crosslinked hydrogel and second cells, with hydrogel precursor (but without cells) being present in another third channel, another third via, and another third inlet port.

[0044] FIG. 10A is a cross-sectional view of the assembled microfluidic assay device taken along section line A- A of FIG. 6, after supplying a first mixture of hydrogel precursor and first cells through one first inlet port and first sidewall port into the first microfluidic chamber to contact an underlying membrane, without hydrogel precursor or first cells being present in another first channel, another first via, and another first inlet port.

[0045] FIG. 10B is a cross-sectional view of the assembled microfluidic assay device taken along section line B-B of FIG. 6, after supplying a second mixture of hydrogel precursor and second cells through one second inlet port and one second sidewall port into the first microfluidic chamber, to contact an underlying first layer of crosslinked hydrogel and firstcells, without hydrogel precursor or second cells being present in another second channel, another second via, and another second inlet port.

[0046] FIG. 11A is an upper perspective view of a holder including nine primary recesses with device retaining features embodying upwardly-facing protrusions and configured to receive multiple microfluidic assay device as disclosed herein.

[0047] FIG. 1 IB is a bottom plan view of the holder of FIG. 11 A.

[0048] FIG. 11C is a top plan view of the holder of FIG. 11 A.

[0049] FIG. 1 ID is a cross-sectional view of the holder of FIG. 11 A.

[0050] FIG. 1 IE is a magnified cross-sectional view of a single upwardly-facing protrusion of FIG. 11D.

[0051] FIG. 12A is an exploded perspective view of a microfluidic apparatus according to one embodiment, showing an assembled microfluidic assay device as disclosed herein positioned above a primary recess of the holder of FIGS. 11 A-l ID.

[0052] FIG. 12B is an assembled perspective view of the microfluidic apparatus of FIG. 12A, with the microfluidic assay device received within one primary recess of the holder of FIGS.11 A-l ID.Detailed Description

[0053] This disclosure relates to a microfluidic assay devices, including devices configured to support multi-layer cellular matrices (optionally embodying ex-vivo skin cell matrices), and apparatuses incorporating plural microfluidic assay devices, as well as associated methods of fabricating and using such devices. A microfluidic assay device includes a holder that defines a first microfluidic chamber laterally bounded by a sidewall with multiple sidewall ports disposed at different levels, multiple inlet ports in fluid communication with different sidewall ports, and a liquid-permeable membrane bounding a bottom of the microfluidic chamber. The different sidewall ports permit formation of stratified layers of cell-containing material (e.g., cells with hydrogel) in the first microfluidic chamber (e.g., by supplying hydrogel precursor and first cells to the chamber through a first sidewall port and crosslinking the hydrogel precursor, followed by supplying hydrogel precursor and second cells to the first microfluidic chamber through a second sidewall port and crosslinking the hydrogel precursor, etc.). In certain embodiments, additional sidewall ports may be provided at one or more additional levels to permit formation of additional layers of cell-containing material. Each layer of cellcontaining material is in contact with at least one other layer of cell-containing material. Acover having at least one distribution channel may be arranged below the liquid-permeable membrane, with the membrane being configured to support the cell-containing material from below and configured to permit passage of liquid media to contact the cell-containing material. A second microfluidic chamber may serve as a continuous upper extension of the first microfluidic chamber, and may be open from above to provide an interface for receiving analytes (or samples) and / or for providing an interface (e.g., air / liquid interface) for interacting with a surrounding environment. Multiple inlet ports and multiple media ports may be accessible along the top of the body structure. A microfluidic apparatus may include a holder having multiple primary recesses and device retaining features, with the holder being configured to receive and removably retain multiple microfluidic assay devices in the primary recesses (i.e., with one microfluidic assay device per primary recess). In certain embodiments, the holder may be compatible in size and shape with a conventional microplate, to permit multiple microfluidic assay devices to be interfaced with microplate handling equipment, automated pipettors, imaging equipment, and the like.

[0054] FIGS. 1 A and IB provide exploded perspective views of a microfluidic assay device 10 configured to support a multi-layer cellular matrix according to one embodiment, with the device 10 including a body structure 20, a liquid-permeable membrane 60, adhesive material 65, and a cover 70. FIG. 1A shows a lower surface 22 (including peripheral lower surface portions 22A and central lower surface portion 22B) of the body structure 20, and FIG. IB shows an upper surface 21 of the body structure 20. The body structure 20 has a generally rectangular shape with opposing first (e.g., shorter) sidewalls 23 and opposing second (e.g., longer) sidewalls 24. The body structure 20 includes a central portion 25 arranged between two peripheral portions 26, wherein a thickness T2 of the central portion 25 is greater than a thickness Ti of each peripheral portion 26. A bottom of the body structure 20 is coincident with the central lower surface portion 22B of the central portion 25. Each peripheral portion 26 of the body structure 20 defines two peripheral recesses 29 with protrusion receiving features 29’ (which project inwardly into the peripheral recesses 29) that are configured to receive upwardly extending protrusions of a microfluidic device holder (e.g., protrusions 171 of microfluidic device holder 155 in FIG. 11 A).

[0055] The central portion 25 of the body structure 20 has a substantially rectangular shape and defines a lower recess 28 that is laterally bounded by the central power surface portion 22B. A first microfluidic chamber 30 is centrally arranged in the lower recess 28 and is bounded by a sidewall 31 A, with the first microfluidic chamber 30 having a tapered width that is reduced toward a top of the first microfluidic chamber, causing the first microfluidic chamber30 to have a frustoconical (truncated conical) shape. The lower recess 28 includes four liquid media vias 36’ (leading to liquid media ports 36 along the top surface 21) arranged proximate to comers thereof, and three pairs of vias (including paired first vias 34-1’, paired second vias 34-2’, paired third vias 34-3’ shown in FIG. 2B) surrounding the first microfluidic chamber 30. Three pairs of sidewall ports (including a pair of first sidewall ports 41, a pair of second sidewall ports 42, and a pair of third sidewall ports 43) are defined at different levels in the sidewall 31 A. The first pair of sidewall ports 41 is arranged at a first level (closer than the other pairs of sidewall ports 42, 43 to a bottom of the first microfluidic chamber 30), the third pair of sidewall ports 43 is arranged at a third level (closer than the other pairs of sidewall ports 41, 42 to a top of the first microfluidic chamber 30), and the second pair of sidewall ports 42 is arranged at a second level that is intermediately arranged between the first level and the second level. Each sidewall port 41, 42, 43 leads to a corresponding laterally extending microfluidic channel 51, 52, 53 (51 being shown in FIGS. 1 A, 7A, 8A, and 9A; 52 being shown in FIGS. 7B, 8B, and 9B; and 53 being shown in FIGS. 7C, 8C, and 9C) that itself is in fluid communication with a corresponding vertically extending via (34-1’, 34-2’, 34-3’ shown in FIG. 2B) that leads to a corresponding inlet port (34-1, 34-2, 34-3 shown in FIGS. IB and 2B) arranged substantially coplanar with a top surface 21 of the body structure 20. Specifically, each first sidewall port 41 leads to a first microfluidic channel (51 in FIGS. 1A, 7A, 8A, and 9A) that is in fluid communication with a first via 34-1’ and a first inlet port 34-1, each second sidewall port 42 leads to a second microfluidic channel (52 in FIGS. 4C, 7B, 8B, and 9B) that is in fluid communication with a second via 34-2’ and a second inlet port 34-2, and each third sidewall port 43 leads to a third microfluidic channel (53 in FIGS. 7C, 8C, and 9C) that is in fluid communication with a third via 34-3’ and a third inlet port 34-3. A second microfluidic chamber 32 having a generally cylindrical shape is arranged above (i.e., as a vertical extension of) the first microfluidic chamber 30.

[0056] With continued reference to FIG. 1A, the liquid-permeable membrane 60 is configured to be received in the lower recess 28, with the liquid-permeable membrane 60 including four membrane vias 63 proximate to comers thereof, and the membrane vias 63 being registered (i.e., aligned) with liquid media vias 36’ defined in the body structure 20 and extending in a vertical direction. The liquid permeable membrane 60 may comprise a suitable liquid-permeable material (e.g., a porous polyolefin material such as polypropylene, optionally surface treated by etching, plasma exposure, etc.) that prohibits passage of cells and hydrogel. The liquid-permeable membrane 60 includes an upper surface (61 in FIG. IB) configured to serve as a lower boundary of the first microfluidic chamber 30, and includes an opposing lowersurface 62 configured to be arranged proximate to the cover 70 with the membrane 60 serving as an upper boundary of a distribution network 74 defined in the cover 70. FIG. 1 A also shows an adhesive layer 65 having a generally rectangular shape with a large aperture 66 defined therein, causing the adhesive layer 65 to resemble an open rectangular picture frame in shape. Either a wet or dry adhesive material may be used in certain embodiments to form the adhesive layer 65.

[0057] With reference to FIG. IB and FIG. 3, the cover 70 includes a peripheral upper surface 71 A and a central upper surface 71B, wherein the adhesive layer 65 is configured to be received by the peripheral upper surface 71 A to adhere the cover 70 to the central lower surface portion 22B of the body structure 20. A crossing (e.g., X-shaped) distribution network 74, including a central distribution channel region 75 and four peripheral distribution channels 76, is defined in the central upper surface 7 IB of the cover 70, wherein ends of the peripheral distribution channels 76 are aligned with the membrane vias 63 defined in the liquid-permeable membrane 60. FIG. IB additionally shows an upper surface recess 27 bordered by the upper surface 21 of the body structure 20, with six smaller diameter tubes 35 and four larger diameter tubes 37 extending vertically into the upper surface recess 27. Terminal ends of the larger diameter tubes 37 form media ports 36 that permit fluid communication with the liquid media vias 36’ (e.g., shown in FIG. 2B), and terminal ends of the smaller diameter tubes 35 form inlet ports 34-1, 34-2, 34-3 that permit fluid communication with the vias 34-1’, 34-2’, 34-3’ (e.g., shown in FIG. 2B).

[0058] FIGS. 2A, 2B, and 2C provide top plan, bottom plan, and upper perspective views, respectively, of the body structure 20 of the microfluidic assay device 10 of FIGS. 1A-1B. As shown in FIG. 2B, the central portion 25 is arranged between the two peripheral portions 26 of the body structure 20, with the central portion 25 including all fluid handling features and defining the lower recess 28 (for receiving the liquid permeable membrane 60 shown in FIGS. 1A-1B), while features defined by the peripheral portions 26 are useable to permit the body structure 20 to be removably received by a multi-device holder (e.g., 155 in FIGS. HAto 12B). FIGS. 2 A and 2C show the upper surface recess 27 bordered by the upper surface 21 of the body structure 20, with six smaller diameter tubes 35 and four larger diameter tubes 37 extending vertically into the upper surface recess 27. Terminal ends of the larger diameter tubes 37 form media ports 36 that permit fluid communication with the liquid media vias 36’ shown in FIG. 2B, and terminal ends of the smaller diameter tubes 35 form inlet ports 34-1, 34-2, 34-3 that permit fluid communication with the vias 34-1’, 34-2’, 34-3’ shown in FIG. 2B). As shown in FIGS. 2A and 2C, the second microfluidic chamber 32 is accessible fromabove and is centrally located between the inlet ports 34-1, 34-2, 34-3. FIG. 2B also shows that each peripheral portion 26 of the body structure 20 defines two peripheral recesses 29 with protrusion receiving features 29’ that project inwardly into the peripheral recesses 29 and that are configured to receive upwardly extending protrusions of a microfluidic device holder (e.g., protrusions 171 of microfluidic device holder 155 in FIG. 11 A).

[0059] In certain embodiments, the body structure 20 has a width of 19 mm and a length of 25 mm, the central portion 25 has a thickness T2 of 7 mm and a length of 11.6 mm, each peripheral portion 26 has a thickness Ti of 5 mm, the first microfluidic chamber 30 has height of 1 mm, the lower recess 28 has depth of 1 mm, the second microfluidic chamber 32 has a diameter of 3 mm, and each sidewall port 41-43 has a width of 1 mm.

[0060] FIG. 4A is a top plan view of the body structure of the microfluidic assay device of FIGS. 1 A-1B, showing the three pairs of inlet ports 34-1, 34-2, 34-3 and the media ports 36, with addition of section lines 4B-4B, 4C-4C, and 4D-4D defining the cross-sectional views shown in FIGS. 4B, 4C, and 4D, respectively. FIG. 4B shows first, second, and third sidewall ports 41, 42, 43 (distributed sixty degrees apart from one another) opening into the frustoconical-shaped first microfluidic chamber 30, with a second microfluidic chamber 32 (bounded by second sidewall 3 IB and having a substantially constant width) extending continuously upward from the first microfluidic chamber 30. An open volume 33 is arranged above the second microfluidic chamber 32. Larger diameter tubes 35 (containing vias 36’) are shown to either side of the second microfluidic chamber 32. The lower recess 28 is arranged along a lower boundary of the first microfluidic chamber 30. FIG. 4C shows the third vias 34- 3’ in fluid communication with third microfluidic channels 53 that lead to third sidewall ports 43 defined in the sidewall 31 A of the first microfluidic chamber 30.

[0061] FIG. 4D is a cross-sectional view of a partially assembled microfluidic assay device including the body structure 20 of FIGS. 4A-4C having a cover 70 (according to FIG. 3) mated thereto, with the peripheral upper surface 71A of the cover 70 being proximate to the central lower surface portion 22B of the body structure 20. Although adhesive material is not shown, it is to be understood that adhesive material 65 of FIGS. 1A-1B would be arranged between the peripheral upper surface 71 A and the central lower surface portion 22B. In an operative microfluidic assay device, a liquid-permeable porous membrane (60 in FIGS. 1A-1B) would be arranged in the lower recess 28 defined in the body structure 20, but such membrane is omitted in FIG. 4D. Such figure also shows the distribution network 74 defined in the cover 70. The liquid media vias 36’ extend through larger tubes 37 defined in the body structure 20A and extend to the lower recess 28. FIG. 4E is a cross-sectional view including excerpts fromFIG. 4D, namely: the first and second microfluidic chambers 30, 32 (bounded by sidewalls31 A, 3 IB, respectively) of the body structure 20, the lower recess 28 defined in the body structure 20 for receiving a porous membrane (60 in FIGS. 1 A-1B), and a distribution network 74 (including central distribution channel region 75 and peripheral distribution channels 76) defined in the cover 70.

[0062] FIG. 5 shows the same cross-sectional view of the microfluidic assay device as FIG. 4D, following addition of a porous membrane layer 60 to the lower recess 78, and following formation of a multi-layer cellular matrix in the first microfluidic chamber 30, including a first cellular matrix layer 101 (i.e., a subdermal layer of adipocytes 92 in hydrogel), a second cellular matrix layer 102 (i.e., a dermal layer of fibroblasts 94 in hydrogel), and a third cellular matrix layer 103 (i.e., an epidermal layer of keratinocytes 96 in hydrogel). FIG. 5 additional shows presence of liquid media 99 in liquid media vias 36’ and in at least one channel of the distribution network 74 defined in the cover 70, and shows the second microfluidic chamber32 as being devoid of cellular matrix material. In certain embodiments, the epidermal layer (third cellular matrix layer 103) is substantially thinner than the dermal layer (second cellular matrix layer 102), while the epidermal layer (third cellular matrix layer 103) may have any suitable thickness relative to the overlying layers. The layers of cell -containing material (cellular matrix layers 101-103) in the first microfluidic chamber 30 may be formed by supplying hydrogel precursor and first cells (e.g., 92) to the first microfluidic chamber 30 through a first sidewall port (41 in FIG. 4D) and crosslinking the hydrogel precursor to form a first cellular matrix layer 101 on the porous membrane 60, then supplying hydrogel precursor and second cells (e.g., 94) to the first microfluidic chamber 30 through a second sidewall port (42 in FIG. 4D) and crosslinking the hydrogel precursor to form a second cellular matrix layer 102 contacting the first cellular matrix layer 101, and then supplying hydrogel precursor and third cells (e.g., 96) to the first microfluidic chamber 30 through a third sidewall port (43 in FIG. 4D) and crosslinking the hydrogel precursor to form a third cellular matrix layer 103 contacting the second cellular matrix layer 102. Such crosslinking of hydrogel may be accomplished by at least one of thermal, chemical, or photonic polymerization. In certain embodiments, a hydrogel precursor solution may be cured by placing a microfluidic device in an incubator (e.g., for 30 minutes or more at 37°C in a 5% CO2 atmosphere in certain embodiments. Examples of hydrogels that may be used include collagen type I hydrogel and / or fibrin hydrogel.

[0063] The multiple cellular matrix layers 101-103 in the first microfluidic chamber 30 of the microfluidic assay device 10 of FIG. 5 are in direct contact with one another, with the firstcellular matrix layer 101 being in direct contact with the underlying fluid-permeable membrane 60. The fluid-permeable membrane 60 is arranged to permit passage of liquid media 99 from the distribution channel network 74 defined in the cover 70, to permit perfusion of liquid media 99 from the bottom to the top of the cellular matrix layers 101-103 contained in the first microfluidic chamber 30. The liquid media vias 36’ may be coupled to one or more flow apparatuses (e.g., incorporating pump(s), regulator(s), flow control! erf s), etc.) to control flow of media through the microfluidic assay device 10.

[0064] FIG. 6 is a top plan view of an assembled microfluidic assay device 10 according to FIGS. 1 A-1B, and illustrating the same items shown in FIG. 4A, with addition of section lines A-A, B-B, and C-C. Section lines A-A define the cross-sectional views shown in FIGS. 7A, 8A, 9A, and 10A; section lines B-B define the cross-sectional views shown in FIGS. 7B, 8B, 9B, and 10B; and section lines C-C define the cross-sectional views shown in FIGS. 7C, 8C, and 9C.

[0065] FIGS. 7A-7C illustrate steps in forming a multi-layer cellular matrix in the first microfluidic chamber 30 of the microfluidic assay device 10 of FIGS. 1A-1B and 5 according to a fabrication method involving supply of hydrogel precursor material and cells through one of each paired inlet port 34-1 to 34-3 and through one of each paired via 34-1’ to 34-3’, respectively, according to one embodiment. FIG. 7A illustrates the microfluidic assay device 10 after supplying a first mixture of hydrogel precursor 90 and first cells (e.g., adipocytes 92) through one first inlet port 34-1 (at left), one first via 34-1’ (at left), one first microfluidic channel 51, and one first sidewall port 41 into the first microfluidic chamber 30 to contact an underlying membrane 60, without hydrogel precursor or cells being present in another first via 34-1’ (at right), another first channel 51 (at right), and another first inlet port 34-1 (at right). The hydrogel precursor 90 in the first microfluidic chamber 30 is then crosslinked to form a first cellular matrix layer 101. FIG. 7B illustrates the microfluidic assay device 10 of FIG. 7 A after supplying a second mixture of hydrogel precursor 90 and second cells (e.g., fibroblasts 94) through one second inlet port 34-2 (at left), one second via 34-2’ (at left), one second microfluidic channel 52 (at left), and one second sidewall port 42 into the first microfluidic chamber 30, to contact an underlying first cellular matrix layer 101, without hydrogel precursor or cells being present in another second via 34-2’ (at right), another second microfluidic channel 52 (at right), and another second inlet port 34-2 (at right). The hydrogel precursor 90 in the first microfluidic chamber 30 is then crosslinked to form a second cellular matrix layer 102. FIG. 7C illustrates the microfluidic assay device 10 after supplying a third mixture of hydrogel precursor 90 and third cells (e.g., keratinocytes 96) through one third inlet port 34-3,one third via 34-3’ (at left), one third microfluidic channel 53 (at left), and one third sidewall port 42 (at left) into the first microfluidic chamber 30, to contact the underlying second cellular matrix layer 102, without hydrogel precursor or cells being present in another third via 34-3’ (at right), another third microfluidic channel 53 (at right), and another third inlet port 34-3 (at right). The hydrogel precursor 90 in the first microfluidic chamber 30 is then crosslinked to form a third cellular matrix layer 103.

[0066] FIGS. 8A-8C illustrate steps in forming a multi-layer cellular matrix in the first microfluidic chamber 30 of the microfluidic assay device 10 of FIGS. 1A-1B and 5 according to a fabrication method involving supply of hydrogel precursor material and cells through each paired inlet port 34-1 to 34-3 and each paired via 34-1’ to 34-3’, respectively, according to one embodiment. FIG. 8 A illustrates the microfluidic assay device 10 after supplying a first mixture of hydrogel precursor 90 and first cells (e.g., adipocytes 92) through both first inlet ports 34-1, both first vias 34-1’, both first microfluidic channels 51, and both first sidewall ports 41 into the first microfluidic chamber 30 to contact an underlying membrane 60, followed by crosslinking of hydrogel precursor 90 in the first microfluidic chamber 30 to form a first cellular matrix layer 101. FIG. 8B illustrates the microfluidic assay device 10 of FIG. 8A after supplying a second mixture of hydrogel precursor 90 and second cells (e.g., fibroblasts 94) through both second inlet ports 34-2, both second vias 34-2’, both second microfluidic channels 52, and both second sidewall ports 42 into the first microfluidic chamber 30, to contact an underlying first cellular matrix layer 101, followed by crosslinking of hydrogel precursor 90 in the first microfluidic chamber 30 to form a second cellular matrix layer 102. FIG. 8C illustrates the microfluidic assay device 10 after supplying a third mixture of hydrogel precursor 90 and third cells (e.g., keratinocytes 96) through both third inlet ports 34-3, both third vias 34-3’, both third microfluidic channels 53, and both third sidewall ports 42 into the first microfluidic chamber 30, to contact the underlying second cellular matrix layer 102, followed by crosslinking of hydrogel precursor 90 in the first microfluidic chamber 30 to form a third cellular matrix layer 103.

[0067] FIGS. 9A-9C illustrate steps in forming a multi-layer cellular matrix in the first microfluidic chamber 30 of the microfluidic assay device 10 of FIGS. 1A-1B and 5 according to a fabrication method involving supply of hydrogel precursor material 90 and cells through one of each paired inlet port 34-1 to 34-3 and through one of each paired via 34-1’ to 34-3’, respectively, and supplying hydrogel precursor material 90 without cells through the other of each paired inlet port 34-1 to 34-3 and through the other of each paired via 34-1’ to 34-3’, respectively, according to one embodiment. FIG. 9A illustrates the microfluidic assay device10 after supplying a first mixture of hydrogel precursor 90 and first cells (e.g., adipocytes 92) through one first inlet port 34-1 (at left), one first via 34-1’ (at left), one first microfluidic channel 51, and one first sidewall port 41 into the first microfluidic chamber 30 to contact an underlying membrane 60, with hydrogel precursor 90 (but without cells) already being present in another first via 34-1 ’ (at right), another first channel 51 (at right), and another first inlet port 34-1 (at right). The hydrogel precursor 90 in the first microfluidic chamber 30 is then crosslinked to form a first cellular matrix layer 101. FIG. 9B illustrates the microfluidic assay device 10 of FIG. 9 A after supplying a second mixture of hydrogel precursor 90 and second cells (e.g., fibroblasts 94) through one second inlet port 34-2 (at left), one second via 34-2’ (at left), one second microfluidic channel 52 (at left), and one second sidewall port 42 into the first microfluidic chamber 30, to contact an underlying first cellular matrix layer 101, with hydrogel precursor 90 (but without cells) being present in another second via 34-2’ (at right), another second microfluidic channel 52 (at right), and another second inlet port 34-2 (at right). The hydrogel precursor 90 in the first microfluidic chamber 30 is then crosslinked to form a second cellular matrix layer 102. FIG. 9C illustrates the microfluidic assay device 10 after supplying a third mixture of hydrogel precursor 90 and third cells (e.g., keratinocytes 96) through one third inlet port 34-3, one third via 34-3’ (at left), one third microfluidic channel 53 (at left), and one third sidewall port 42 (at left) into the first microfluidic chamber 30, to contact the underlying second cellular matrix layer 102, with hydrogel precursor 90 (but without cells) being present in another third via 34-3’ (at right), another third microfluidic channel 53 (at right), and another third inlet port 34-3 (at right). The hydrogel precursor 90 in the first microfluidic chamber 30 is then crosslinked to form a third cellular matrix layer 103.

[0068] Although various embodiments shown in FIGS. 5, 7C, 8C, and 9C each show formation of three cellular matrix layers 101-103, the present disclosure is not so limited. In certain embodiments, additional cellular matrix layers may be formed atop the third cellular matrix layer 103 with appropriate modification to the microfluidic assay device 10. In certain embodiments, the microfluidic assay device 10 may be used to form only one cellular material layer 101 or only two cellular matrix layers 101, 102. For example, FIGS. 10A-10B illustrate formation of first and second cellular material layers 101, 102 in the microfluidic assay device 10.

[0069] FIG. 10A is a cross-sectional view of the assembled microfluidic assay device 10 taken along section line A-A of FIG. 6, after supplying a first mixture of hydrogel precursor and first cells through one first inlet port and first sidewall port into the first microfluidicchamber to contact an underlying membrane, without hydrogel precursor or first cells being present in another first channel, another first via, and another first inlet port.

[0070]

[0071] FIG. 10A is a cross-sectional view of the assembled microfluidic assay device 10 taken along section line A-A of FIG. 6, after supplying a first mixture of hydrogel precursor 90 and first cells (e.g., adipocytes 92) through one first inlet port 34-1 (at left), one first via 34- 1’ (at left), one first microfluidic channel 51, and one first sidewall port 41 into the first microfluidic chamber 30 to contact an underlying membrane 60, without hydrogel precursor or cells being present in another first via 34- 1 ’ (at right), another first channel 51 (at right), and another first inlet port 34-1 (at right). The hydrogel precursor 90 in the first microfluidic chamber 30 is then crosslinked to form a first cellular matrix layer 101. FIG. 10B illustrates the microfluidic assay device 10 of FIG. 10A after supplying a second mixture of hydrogel precursor 90 and second cells (e.g., fibroblasts 94) through one second inlet port 34-2 (at left), one second via 34-2’ (at left), one second microfluidic channel 52 (at left), and one second sidewall port 42 into the first microfluidic chamber 30, to contact an underlying first cellular matrix layer 101, without hydrogel precursor or cells being present in another second via 34- 2’ (at right), another second microfluidic channel 52 (at right), and another second inlet port 34-2 (at right). Further cellular matrix layers be omitted, with the microfluidic assay device 10 being suitable for use.

[0072] In certain embodiments, multiple microfluidic assay devices as disclosed herein may be removably received by a holder to form a microfluidic apparatus by which multiple assays may be performed simultaneously. In certain embodiments, a holder may be compatible in size and shape with a conventional microplate, to permit multiple microfluidic assay devices to be interfaced with microplate handling equipment, automated pipettors, imaging equipment, and the like.

[0073] FIGS. 11 A-l ID illustrate a holder 155 according to one embodiment, the holder 155 including a plurality of primary recesses 165A-165I with device retaining features embodying upwardly-facing protrusions 171 and being configured to receive multiple microfluidic assay devices as disclosed herein. FIG. 11A is an upper perspective view of the holder 155, while FIGS. 11B-11D provide bottom plan, top plan, and cross-sectional views of the holder 155, and FIG. 1 IE provides a magnified cross-sectional view of a portion thereof (including a single upwardly-facing protrusion 171 having a height of 1.6 mm relative to a floor portion 274).

[0074] Referring to FIGS. 11 A-l ID, the holder 155 comprises a holder body structure 160 that defines an upper holder surface 161, a lower holder surface 162, a lateral boundary wall164, and nine primary recesses 165A-165I (arranged in a three by three array) that extending downward relative to the upper holder surface 161. Each primary recess 165A-165I comprises a window opening 166A-166I extending through the lower holder surface 162. Each primary recess 165A-165I further includes floor portions 174 along comer portions thereof, with an upwardly-facing protrusion 171 extending upward from each floor portion 174. Pillars 168 defining wells 169 are provided at intersections between adjacent primary recesses 165A-165I, and secondary recesses 167 extend downward from the upper holder surface 161, with the secondary recesses 167 being useful for handling (e.g., inserting and / or extracting) microfluidic assay devices. Indents 171’ corresponding to the device retaining features (upwardly facing protrusions 171) may be visible along a lower surface 162 of the holder 155, as shown in FIG. 1 IB. The holder 155 may be sized and shaped to correspond to a conventional microplate (e.g., a 40-well plate, or any other suitable conventional microplate), as may be advantageous to utilize conventional plate handling and / or optical imaging equipment therewith. Each primary recess 165A-165I is configured to receive and removably retain a single microfluidic assay device as disclosed herein, such that up to nine microfluidic assay devices 10 may be received and retained by the holder 155.

[0075] In use, a microfluidic assay device (e.g., as described previously herein) may be inserted into a primary recess (e.g., 165 A) of the holder 155 and pressed downward to cause upwardly-facing protrusions 171 of the holder 155 to be received and removably engaged by protrusion receiving features 116 of the microfluidic assay device. After use, one or more microfluidic assay devices 10 may be removed from the holder 155 (e.g., by pulling the microfluidic assay device upward).

[0076] FIG. 12A is an exploded perspective view of a microfluidic apparatus according to one embodiment, showing an assembled microfluidic assay device 10 as disclosed herein positioned above a primary recess (e.g., recess 165E) of the holder 155 of FIGS. 11A-11D. FIG. 12B is an assembled perspective view of the microfluidic apparatus of FIG. 12A, with the microfluidic assay device 10 received within one primary recess 165E of the holder 155 of FIGS. 11A-11D.

[0077] In certain embodiments, a multi-layer stratified matrix of cellular materials (e.g., cellular matrix layers 101-103 within the first microfluidic chamber 30 of a microfluidic assay device 10 as disclosed herein) comprises a skin-on-a chip construct, and may include items such as hair follicles, immune cells, melanocytes, langerhan cells, vasculature, nerves, dendrites, and any other cell type associated with human skin.

[0078] In certain embodiments, a biopsy of skin may be inserted (e.g., through open volume 33 and second microfluidic chamber 32) into the first microfluidic chamber 30 of a microfluidic assay device 10 as disclosed herein. Alternatively, or additionally, organoids and spheroids can be inserted into and / or cultured in the first microfluidic chamber 39 to recapitulate a skin model. With the overlying second microfluidic chamber 32 being open, thereby providing an air-liquid interface, the recapitulated skin layer(s) may be biopsied for histological analysis, RNA analysis, and / or other testing for instances in which there is a need for a tissue sample.

[0079] In certain embodiments, analytes or other compounds to be tested may be supplied to cellular material (e.g., cellular matrix layers 101-103) in the first microfluidic chamber 30, whether by direct application through the air-liquid interface provided by the overlying second microfluidic chamber 32 and / or by injection through one or more of the first, second, and / or third inlet ports 34-1, 34-2, 34-3. In certain embodiments, an analyte or compound to be tested (e.g., a drug, cosmetic, or other solution) may be directly applied (to simulate topical application / transdermal analyte testing) from the top of the assay device through the open volume 33 and the second microfluidic chamber 32 to contact cellular material (e.g., cellular matrix layers 101-103) in the first microfluidic chamber (e.g., to achieve highest concentration on an uppermost cell-containing material within the first microfluidic chamber, such as a second cellular matrix layer 102, a third cellular matrix layer 103, or a subsequent cellular matrix layer or other layer of cells). Optionally, a same or different analyte or compound to be tested (e.g., a drug, cosmetic, or other solution) may also be supplied through one or more of the first, second, and third inlet ports 34-1, 34-2, 34-2 (e.g., to achieve highest concentration in the first, second, or third layers of cell -containing material (e.g., cellular matrix layers 101- 103) proximate to sidewall ports 41-43 of the first microfluidic chamber 30), such as to simulate non-transdermal interaction.

[0080] In certain embodiments, one or more microfluidic assay devices 10 (optionally incorporated into a holder 155 as disclosed herein) may be used to simulate various skin conditions, such as: acne, acne scars, atopic dermatitis, cellulite, contact dermatitis, eczema, keloid scars, Lyme disease, melanoma, melasma, Merkel cell carcinoma, psoriasis, rash, rosacea, scleroderma, sebaceous carcinoma, seborrheic dermatitis, seborrheic keratoses, shingles, squamous cell carcinoma, vitiligo, warts, sarcoidosis, neurodermatitis, nickel allergy, moles, lupus, bacterial skin infection, alopecia, genital herpes, genital warts, folliculitis, diabetic skin condition, cold sores, stretch marks, wrinkles, athlete’s foot, actinic keratosis, and surgical scars.

[0081] As noted previously, the first microfluidic chamber 30 of a microfluidic assay device 10 may have a tapered width, optionally embodied in a frustoconical shape. Such configuration may facilitate imaging of all layers of material (e.g., cellular matrix layers 101-103) within the first microfluidic chamber 30.

[0082] In certain embodiments, the body structure 20 and / or cover 70 of a microfluidic assay device 10 disclosed herein comprises a thermoplastic or thermoset material, permitting the microfluidic assay device 10 to be produced by three-dimensional (3D) printing, injection molding, casting, or hot embossing. In certain embodiments, the body structure 20 is a unitary (one-piece) structure that is substantially rigid in character (thereby excluding soft materials such as silicone, PDMS, and PMMA) to maintain a high degree of dimensional integrity, by avoiding the need for a separate rigid substrate that would otherwise be required if compliant materials were used. A rigid body structure 20 is also preferably substantially non-ab sorptive of, and non-reactive with, drugs, reagents, and cellular material, wherein such material is adequate or well-suited for culturing living cells. In certain embodiments, a rigid body structure 20 comprises a material that is transparent or at least highly optically transmissive (e.g., at least 90% light transmissive, at least 95% light transmissive, or at least 98% light transmissive), or to facilitate viewing, imaging, and / or optical analysis (including but not limited to microscopy) of contents of a first microfluidic chamber 30. In certain embodiments, a rigid body structure 20 comprises a polyolefin material, such as (but not limited to) a cyclic olefin polymer or a cyclic olefin copolymer. In certain embodiments, a rigid body structure 20 comprises polystyrene, polypropylene, polytetrafluoroethylene, a modified fluoropolymer, polymethylpentene, or combinations thereof.

[0083] Microfluidic assay devices 10 as disclosed herein, optionally in combination with a holder 155, may be used to support multi-layer structures of living cells (e.g., within cellular matrix layers 101-103) that may be useful for various purposes, including preclinical testing (especially irritation / biocompatibility testing) for cosmetic, pharmaceutical, medical device, and chemical manufacturers (e.g., as an alternative to performing tests on animals). In certain embodiments, cellular matrix layers 101-13 may provide a multi-layer skin-on-a-chip model including live cells may be used for high-throughput testing instead of using mouse, rat, rabbit, or porcine skin.

[0084] The terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting of the disclosure. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the term "and / or" includes any and all combinations of one or moreof the associated listed items. It will be further understood that the terms "comprises," "comprising," "includes," and / or "including" when used herein specify the presence of stated features, integers, actions, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, actions, steps, operations, elements, components, and / or groups thereof.

[0085] It will be understood that, although the terms first, second, etc., may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first element could be termed a second element, and, similarly, a second element could be termed a first element without departing from the scope of the present disclosure.

[0086] It will be understood that when an element is referred to as being "connected" or "coupled" to another element, it can be directly connected or coupled to the other element, or intervening elements may be present. In contrast, when an element is referred to as being "directly connected" or "directly coupled" to another element, there are no intervening elements present.

[0087] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms used herein should be interpreted as having a meaning consistent with their meaning in the context of this specification and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0088] It is to be understood that the present disclosure is not limited to the aspects described above and illustrated in the drawings; rather, the skilled person will recognize that many changes and modifications may be made within the scope of the present disclosure and appended claims. In the drawings and specification, there have been disclosed aspects for purposes of illustration only and not for purposes of limitation, the scope of the disclosure being set forth in the following claims.

[0089] Those skilled in the art will recognize improvements and modifications to the preferred embodiments of the present disclosure. All such improvements and modifications are considered within the scope of the concepts disclosed herein and the claims that follow

Claims

ClaimsWhat is claimed is:

1. A microfluidic assay device configured to support a multi-layer cellular matrix, the microfluidic assay device comprising: a body structure having a top, having a bottom, and defining a first microfluidic chamber laterally bounded by a sidewall, wherein the first microfluidic chamber comprises a chamber bottom, at least one first sidewall port arranged at a first level, and at least one second sidewall port arranged at a second level, the chamber bottom being closer to the first level than to the second level; at least one first inlet port and at least one second inlet port accessible from the top of the body structure, the at least one first inlet port being arranged in fluid communication with the at least one first sidewall port, and the at least one second inlet port being arranged in fluid communication with the at least one second sidewall port; and a liquid-permeable membrane bounding the chamber bottom.

2. The microfluidic assay device of claim 1, further comprising at least one first channel arranged in fluid communication with the at least one first sidewall port, at least one first via extending from the at least one first inlet port to the at least one first channel, at least one second channel arranged in fluid communication with the at least second first sidewall port, at least one second via extending from the at least one first inlet port to the at least one first channel.

3. The microfluidic assay device of any one of claims 1 or 2, wherein: the at least one first sidewall port includes a pair of horizontally opposed first sidewall ports, the at least one first inlet port includes a pair of first inlet ports, the at least one second sidewall port includes a pair of horizontally opposed second sidewall ports, and the at least one second inlet port includes a pair of second inlet ports.

4. The microfluidic assay device of any one of claims 1 to 3, further comprising a cover arranged below the body structure, the cover comprising at least one distribution channel in a top surface thereof, wherein the liquid-permeable membrane is arranged between the chamber bottom and the at least one distribution channel.

5. The microfluidic assay device of claim 4, further comprising an adhesive material coupling the cover to the body structure, with openings in the adhesive material being aligned with liquid media vias defined in the body structure.

6. The microfluidic assay device of claim 1, wherein the body structure further comprises: a plurality of liquid media ports accessible from the top of the body structure; and a plurality of liquid media vias between the plurality of liquid media ports and the at least one distribution channel.

7. The microfluidic assay device of claim 6, wherein the liquid-permeable membrane defines a plurality of membrane vias aligned with the plurality of liquid media vias.

8. The microfluidic assay device of any one of claims 1 to 7, wherein the body structure comprises a lower recess, wherein the liquid-permeable membrane is arranged in the lower recess.

9. The microfluidic assay device of any one of claims 1 to 7, wherein the first microfluidic chamber comprises a tapered sidewall, wherein a width of the first microfluidic chamber decreases with distance away from the chamber bottom.

10. The microfluidic assay device of claim 9, wherein the first microfluidic chamber comprises a frustoconical shape.

11. The microfluidic assay device of any one of claims 1 to 7, further comprising a second microfluidic chamber that embodies an upper extension of the first microfluidic chamber, wherein the upper microfluidic chamber is open along an upper boundary thereof.

12. The microfluidic assay device of claim 11, wherein the second microfluidic chamber comprises a constant width, and wherein a width of the first microfluidic chamber decreases with distance away from the chamber bottom.

13. The microfluidic assay device of any one of claims 1 to 7, wherein:the first microfluidic chamber contains a first layer of first cells within hydrogel material, and a second layer of second cells within hydrogel material; the first layer is arranged on the liquid-permeable membrane and is substantially aligned with the first level; the second layer is arranged on the first layer and is substantially aligned with the second level; and the second cells differ in cell type from the first cells.

14. The microfluidic assay device of any one of claims 1 to 7, wherein: the first microfluidic chamber further comprises at least one third sidewall port arranged at a third level, the chamber bottom being closer to the second level than to the third level; and the microfluidic assay device further comprises at least one third inlet port accessible from the top of the body structure, the at least one third inlet port being arranged in fluid communication with the at least one third inlet port.

15. The microfluidic assay device of claim 14, wherein: the first microfluidic chamber further contains a third layer of third cells within hydrogel material, the third layer being arranged on the second layer and being substantially aligned with the third level; and the third cells differ in cell type from the first cells and from the second cells.

16. The microfluidic assay device of claim 15, wherein the first cells comprise adipocytes, the second cells comprise fibroblasts, and the third cells comprise keratinocytes.

17. The microfluidic assay device of any one of claims 1 to 7, wherein a lower portion of the body structure comprises a plurality of protrusion receiving features configured to receive upwardly extending protrusions of a microfluidic device holder.

18. A microfluidic apparatus comprising: a holder comprising a holder body structure defining an upper holder surface, a lower holder surface, a plurality of primary recesses extending downward relative to the upper holder surface, and a plurality of device retaining features; andone or more microfluidic assay devices according to any one of claims 1 to 7 received within one or more corresponding primary recesses of the plurality of primary recesses, wherein each microfluidic assay device is removably retained by one or more device retaining features of the plurality of device retaining features.

19. The microfluidic apparatus of claim 18, wherein the holder body structure further comprises at least one well that opens to the upper holder surface, and that is configured to hold a liquid, wherein the at least one well is non-coincident with the plurality of primary recesses.

20. The microfluidic apparatus of claim 18, wherein each primary recess comprises a window opening extending through the lower holder surface, and the window opening is sized and shaped to receive at least a portion of the cover of a corresponding microfluidic assay device of the plurality of microfluidic assay devices.

21. The microfluidic apparatus of claim 18, wherein at least some device retaining features of the plurality of device retaining features comprise upwardly-extending protrusions.

22. The microfluidic apparatus of claim 21, wherein the upwardly-extending protrusions each comprise a generally cylindrical shape, and extend upward from floor portions proximate to corner areas of primary recesses of the plurality of primary recesses.

23. A method for fabricating a multi-layer cellular matrix in a microfluidic assay device, the method comprising: providing a microfluidic assay device comprising: (i) a body structure having a top, having a bottom, and defining a first microfluidic chamber laterally bounded by a sidewall, wherein the first microfluidic chamber comprises a chamber bottom, at least one first sidewall port arranged at a first level, and at least one second sidewall port arranged at a second level, the chamber bottom being closer to the first level than to the second level; (ii) at least one first inlet port and at least one second inlet port accessible from the top of the body structure, the at least one first inlet port being arranged in fluid communication with the at least one first sidewall port, and the at least one second inlet port being arranged in fluidcommunication with the at least one second sidewall port, and (iii) a liquid-permeable membrane bounding the chamber bottom; supplying a first mixture of hydrogel precursor and first cells through the at least one first inlet port and the at least one first sidewall port into the first microfluidic chamber; crosslinking the hydrogel precursor of the first mixture to produce, on the liquid- permeable membrane and in the first microfluidic chamber, a first layer of first cells within crosslinked hydrogel, the first layer being substantially aligned within the first level; supplying a second mixture of hydrogel precursor and second cells through the at least one second inlet port and the at least one second sidewall port into the first microfluidic chamber, the second cells differing in type from the first cells; and crosslinking the hydrogel precursor of the second mixture to produce, on the first layer and in the first microfluidic chamber, a second layer of second cells within crosslinked hydrogel, the second layer being substantially aligned within the second level.

24. The method of claim 23, wherein: the first microfluidic chamber further comprises at least one third sidewall port arranged at a third level, the chamber bottom being closer to the second level than to the third level; the microfluidic assay device further comprises least one third inlet port accessible from the top of the body structure, the at least one third inlet port being arranged in fluid communication with the at least one third inlet port; and the method further comprises: supplying a third mixture of hydrogel precursor and third cells through the at least one third inlet port and the at least one third sidewall port into the first microfluidic chamber, the third cells differing in type from the first cells and the second cells; and crosslinking the hydrogel precursor of the third mixture to produce, on the second layer and in the first microfluidic chamber, a third layer of third cells within crosslinked hydrogel, the third layer being substantially aligned within the third level.

25. The method of claim 24, wherein the first cells comprise adipocytes, the second cells comprise fibroblasts, and the third cells comprise keratinocytes. 1

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