A DEVICE AND METHOD FOR VASCULARISING A CELL AGGREGATE

Abstract

There is provided a device for vascularising a cell aggregate, the device comprising: a matrix region configured to contain a gel-like matrix and cells that form a vasculature network within th ematrix region. The matrix region having at least one opening for positioning the cell aggregate therein based on a desired three-dimensional spatial location; and one or more fluidic regions configured to contain a supporting fluid that is capable of supporting vascularisation of the cell aggregate, the one or more fluidic regions being in fluid communication with the matrix region, wherein a flow passage from the one or more fluidic regions to a gel-like matrix disposed in the matrix region is configured to allow three-dimensional vascularisation around the cell aggregate and perfusion of the vasculature once formed. There is also provided a chip comprising a plurality of the device as disclosed herein and a method for vascularising a cell aggregate.

Claims

1. A device for vascularising a cell aggregate, the device comprising: a matrix region configured to contain a gel-like matrix and the matrix region having at least one opening for positioning the cell aggregate therein based on a desired three-dimensional spatial location; and one or more fluidic regions configured to contain a supporting fluid that is capable of supporting vascularisation of the cell aggregate, the one or more fluidic regions being in fluid communication with the matrix region, wherein a flow passage from the one or more fluidic regions to a gel-like matrix disposed in the matrix region is configured to allow three-dimensional vascularisation around the cell aggregate and perfusion of the vasculature once formed.

2. The device as claimed in claim 1, wherein one or more fluidic regions comprise at least two fluidic regions.

3. The device as claimed in claim 2, wherein one fluidic region is disposed lateral to the matrix region on one side and another fluidic region is disposed lateral to the matrix region on the opposite side.

4. The device as claimed in claim 1, wherein the flow passage is substantially free from intervening structural obstacles disposed between the one or more fluidic regions and the matrix region.

5. The device as claimed in claim 1, wherein the at least one opening is positioned substantially central to the matrix region.

6. The device as claimed in claim 1, wherein each fluidic region comprises at least two openings for facilitating introduction of the supporting fluid in each of the fluidic region.

7. The device as claimed in claim 1, wherein the matrix region is substantially symmetrical in shape along its longitudinal length.

8. The device as claimed in claim 2, wherein the at least two fluidic regions are symmetrically disposed about the matrix region.

9. The device as claimed in claim 1, further comprising a gel-like matrix disposed within the matrix region.

10. The device as claimed in claim 1, wherein at least part of walls defining the matrix region and the one or more fluidic regions comprises an elastomer.

11. The device as claimed in claim 1, wherein the device comprises an elastomer disposed on a substrate, wherein the elastomer comprises patterns formed on an open surface of the elastomer, the patterns corresponding to a layout of the matrix region and the one or more fluidic regions, and wherein the substrate substantially fluidically seals the patterns at the open surface of the elastomer to form the matrix region and the one or more fluidic regions of the device.

12. The device as claimed in claim 2, wherein the at least two fluidic regions are separated from one another by the matrix region.

13. A chip comprising a plurality of the device of claim 1.

14. A method for vascularising a cell aggregate, the method comprising: providing a device comprising a matrix region configured to contain a gel-like matrix and the matrix region having at least one opening for positioning the cell aggregate therein based on a desired three-dimensional spatial location; and one or more fluidic regions configured to contain a supporting fluid that is capable of supporting vascularisation of the cell aggregate, the one or more fluidic regions being in fluid communication with the matrix region, wherein a flow passage from the one or more fluidic regions to a gel-like matrix disposed in the matrix region is configured to allow three-dimensional vascularisation around the cell aggregate and perfusion of the vasculature once formed; introducing the gel-like matrix into the matrix region; positioning a cell aggregate into the gel-like matrix via the at least one opening; and introducing the supporting fluid into the one or more fluidic regions.

15. The method as claimed claim 14, wherein the gel-like matrix is substantially maintained in the matrix region by surface tension.

16. The method as claimed in claim 14, wherein the cell aggregate is positioned in the gel-like matrix such that the gel-like matrix fully surrounds the cell aggregate.

17. The method as claimed in claim 14, wherein the method further comprises introducing cells capable of supporting vascularisation of the cell aggregate into the one or more fluidic regions.

18. The method as claimed in claim 14, wherein the method further comprises introducing one or more cell types into the gel-like matrix capable of supporting vascularisation of the cell aggregate and/or interacting with the cell aggregate.

19. The method as claimed in claim 14, wherein the method further comprises vascularising the cell aggregate to obtain a three-dimensional vascularisation around the cell aggregate.

20. The method as claimed in claim 14, further comprising introducing one or more test agents into the one or more fluid regions and/or the matrix region; and analysing the effect of the one or more test agents on the cell aggregate.

Description

BRIEF DESCRIPTION OF FIGURES

[0087] FIG. 1A is a three-dimensional (3D) rendering of a chip in accordance with various embodiments disclosed herein. FIG. 1B is a schematic top view drawing of a chip, in accordance with various embodiments disclosed herein. FIG. 1C is a detail view drawing of portion X of FIG. 1B (4:1), showing a device on a chip, in accordance with various embodiments disclosed herein. FIG. 1D is a schematic drawing illustrating an isometric view of a device on a chip, in accordance with various embodiments disclosed herein. FIG. 1 E is a cross-sectional view diagram of a device taken along the line Y-Y of FIG. 1D, in accordance with various embodiments disclosed herein. FIG. 1F is a photograph of a chip with devices fabricated with PDMS (polydimethylsiloxane) bonded to a microscope glass slide in accordance with various embodiments disclosed herein. FIG. 1G is a zoom photo of a circular opening located substantially in the middle of a matrix region of a PDMS device, in accordance with various embodiments disclosed herein.

[0088] FIG. 2 is a schematic drawing of a mold for fabricating PDMS devices, in accordance with various embodiments disclosed herein.

[0089] FIG. 3 is a schematic drawing of another chip, in accordance with various embodiments disclosed herein.

[0090] FIGS. 4A and 4B are schematic cross-sectional diagrams illustrating a process of introducing an organoid into a device, in accordance with various embodiments disclosed herein.

[0091] FIG. 5 is a diagram showing an exemplary workflow for a 3D in vitro vascularised tumour model, in accordance with various embodiments disclosed herein.

[0092] FIG. 6A shows images of HepG2 (GFP, green) and Hep3b (GFP, green) liver tumour cell lines that were used to form monoculture (only liver cancer cells) or triculture spheroids (liver cancer cell co-cultured with stellate cells (LX2) and endothelial cells (RFP-HLSEC)), which, after 5 days, were seeded into devices in an ECM containing GFP-HUVEC and NHLF. Images of the control (No Spheroid) show GFP-HUVEC and NHLF that were seeded into devices. The scale bars represent 200 m. FIG. 6B shows a graph showing vasculature coverage (%) against days post-seeding for each condition. Mean and SEM are shown. **P<0.01.

[0093] FIG. 7A is a graph showing spheroid growth over time with and without vasculature. FIG. 7B shows representative fluorescence images, as well as expanded images of the boxed portions of the respective fluorescence images, from spheroids and DRAQ7 quantification. FIG. 7C is a graph showing quantification of DRAQ7 in the spheroids. Mean and SEM from 5 samples are shown. FIG. 7D is a graph showing expression of the proliferation marker Ki-67 in vascularised spheroids relative to spheroids grown in ECM without vasculature. FIGS. 7E and 7F are graphs showing expression of proliferation marker Ki-67 in vascularised spheroids and spheroids grown in ECM without vasculature, in the core and outer regions of the spheroids respectively. FIG. 7G is a representative fluorescence image showing the distribution of the proliferation marker Ki-67 in a HepG2 monoculture spheroid with vasculature. FIG. 7H is a representative fluorescence image showing the distribution of the proliferation marker Ki-67 in a HepG2 triculture spheroid with vasculature. FIG. 7I is a graph showing the extent of apoptosis in the vascularised spheroids relative to non-vascularised spheroids, detected by immunofluorescence using Caspase-3 antibody. FIGS. 7J and 7K are graphs showing the extent of apoptosis in the vascularised spheroids relative to non-vascularised spheroids, in the core and outer regions of the spheroids respectively. FIG. 7L is a representative fluorescence image showing the extent of apoptosis in a HepG2 triculture spheroid without vasculature. FIG. 7M is a representative fluorescence image showing the extent of apoptosis in a Hep3b triculture spheroid with vasculature. FIG. 7N is a graph showing the extent of hypoxia in the vascularised spheroids relative to non-vascularised spheroids, measured using anti-Hif1a (Hypoxia-inducible factor-1 alpha) antibody. FIGS. 7P and 7Q are graphs showing the extent of hypoxia in the vascularised spheroids relative to non-vascularised spheroids, in the core and outer regions of the spheroids respectively. FIG. 7R is a representative fluorescence image showing the extent of hypoxia detected in a HepG2 triculture spheroid without vasculature. FIG. 7S is a representative fluorescence image showing the extent of hypoxia detected in a HepG2 triculture spheroid with vasculature. Fluorescence images were acquired using the same acquisition parameters in all the conditions and representative images to show the significant changes are shown. Data are represented as meanSEM. The scale bars represent 100 m, *P<0.05, **P<0.01, *** P<0.001, ****P<0.0001.

[0094] FIG. 8 is a graph showing vasculature coverage close to a spheroid (or adjacent to a spheroid, wherein adjacent means within a distance of 350 m from the spheroid) and distant from the spheroid (wherein distant means more than 350 m from the spheroid) across different conditions. Data are represented as meanSEM. *P<0.05, **P<0.01.

[0095] FIG. 9A shows representative fluorescence images of vascularised and non-vascularised Hep3b monoculture and triculture spheroids subjected to Sorafenib (against a control), taken on Day 0 (starting of drug treatment) and Day 5 post-treatment. FIG. 9B is a graph showing the GFP signal detected for vascularised and non-vascularised Hep3b monoculture spheroids and Hep3b triculture spheroids over time subjected to Sorafenib treatment, based on 3 independent replicates. The scale bars represent 200 m.

[0096] FIG. 10A is a graph showing DRAQ7 signals in HepG2 monoculture and triculture spheroids seeded into devices with and without vasculature and which were treated with TCR T cells or Mock T cells. FIG. 10B shows representative fluorescence images of HepG2 monoculture and triculture spheroids seeded into devices with and without vasculature and which were treated with TCR T cells or Mock T cells used for the DRAQ7 quantification. The scale bars represent 200 m. FIGS. 10C and 10D are graphs showing DRAQ7 signals in Hep3b monoculture and triculture spheroids seeded into devices with and without vasculature which were treated with CD133 CAR-T cells. FIG. 10E is a graph showing CD133 CAR-T cell count in devices seeded with Hep3b monoculture and triculture spheroids with and without vasculature. FIG. 10F shows representative fluorescence images of vascularised and non-vascularised Hep3b monoculture and triculture spheroids treated with CD133 CAR-T cells or mock cells.

[0097] FIGS. 11A and 11B show images taken on day 3 of a vascularised patient biopsy (the biopsy obtained from an ovarian cancer patient) which was cultured in a device with endothelial cells (HUVEC) and fibroblasts (NHLF) in accordance with various embodiments disclosed herein.

EXAMPLES

[0098] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural and biological changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0099] The following examples describe a device (e.g., a microfluidic device) to culture and vascularise a cell aggregate (or an organoid or a patient biopsy) in a controlled location within a gel-like matrix (e.g., a 3D hydrogel matrix). A plurality of such devices can be arranged in a chip in the form of an array.

[0100] In the following examples, each microfluidic device is designed and prototyped with three different regions (or compartments), two regions (referred to as fluidic regions) for containing a supporting fluid (or liquid) and one region (referred to as a matrix region) for containing a gel-like matrix such as an extracellular matrix (ECM)-like hydrogel. The matrix region presents a circular opening (or openings with other shapes) to precisely control the position of a cell aggregate inside the matrix and to consistently form a vascular network within the gel-like matrix such as an ECM-like hydrogel.

[0101] Device

[0102] FIG. 1A shows a three-dimensional (3D) rendering of a chip 100 with a microscope glass slide 102 being its base. The chip 100 comprises a plurality of devices 104 (see FIG. 1B) which are arranged in a 5 by 1 array on the microscope glass slide 102.

[0103] FIG. 1C shows a detail view drawing of portion X of FIG. 1B, showing one of the devices 104. As shown in FIG. 1C, the device 104 (e.g., a microfluidic device) is designed with three different regions (or compartments), two regions (fluidic regions 106 and 108) for containing a supporting fluid (or liquid) and one region (matrix region 110) for containing a gel-like matrix such as an extracellular matrix (ECM)-like hydrogel. See also FIG. 1D which shows an isometric view of the device 104 and FIG. 1E which shows a cross-sectional view diagram of the device 104 taken along line Y-Y of FIG. 1D.

[0104] As shown in FIGS. 1C, 1D, and 1E, the matrix region 110 comprises a circular opening 112 which is accessible from the top surface of the device 104. The circular opening 112 allows precise control the position of a cell aggregate 114 inside the gel-like matrix (see FIG. 1E), e.g., by hand or by automation (such as with the use of a liquid handler). That is, the cell aggregate 114 can be seeded in a controlled position of the gel-like matrix, as shown in FIG. 1E.

[0105] The fluidic regions 106 and 108 on the other hand each comprise two openings, i.e., openings 116 and 118 for fluidic region 106 and openings 120 and 122 for fluidic region 108. The openings 116, 118, 120, 122 are provided as annexures to the main portion (i.e., the part adjacent to the matrix region that allows the supporting fluid to directly perfuse the gel-like matrix of the matrix region) of the fluidic regions 106 and 108. These annexures are in the form of wells that have a tapered profile (i.e., tapered side profile) tapering from a larger/broader opening at the top to a smaller/narrower bottom.

[0106] These openings of the fluidic regions 106 and 108 allow the supporting fluid (for example, cell culture media) to be introduced into and/or be removed from the fluidic regions 106 and 108. These openings can also act as a connection point to the external environment or external components, for example, tubes, pumps, or additional devices.

[0107] In this example, the device 104 has the following dimensions (see FIG. 1C): the width of the matrix region 110 (see A) is 2.4 mm; the diameter of the circular opening 112 (see B) is 1.5 mm; the length of the fluidic regions 106, 108 parallel to the matrix region (see C) is 5 mm; the direct distance between the centre of one opening of the fluidic region to the centre of the other opening of the same fluidic region (e.g., the distance between opening 116 to opening 118, and the distance between opening 120 to opening 122) (see D) is 6 mm; the direct horizontal distance between the centre of one opening of one fluidic region to the centre of an opening of the other fluidic region (e.g., the distance between opening 116 to opening 120, and the distance between opening 118 to opening 122) (see E) is 10 mm; the width of the regions (see F) is 0.8 mm, while the width of the complete fluidic channel (see G) is 1.8 mm; and the diameter of the openings (or inlet/outlet ports) of the whole fluidic channel (e.g., openings 116, 118, 120, 122) (see H) is 4.0 mm. In the above example, while the device 104 may have the dimensions described above, it will be appreciated that the dimensions are not limited as such and can be changed e.g., depending on the application.

[0108] The device 104 as shown in FIGS. 1C, 1D, and 1E is configured to maintain or keep the gel-like matrix, such as an ECM-like matrix, in the matrix region by surface tension. Further, as shown in FIG. 1E, the device 104 is configured such that the flow passage from the fluidic regions 106, 108 to the matrix region 110 is substantially free from intervening obstacles (e.g., pillars).

[0109] In the device 104 as shown in FIGS. 1C and 1D, the circular opening 112 is substantially in the middle of the matrix region 110 along the longitudinal length of the device 104. Positioning the circular opening 112 substantially in the middle of the matrix region 110 allows a precise control the cell aggregate seeding position in the device 104.

[0110] The device 104 can allow cells, such as endothelial cells, to enter the fluidic regions 106 and 108 (e.g., when present in the supporting fluid introduced into the fluidic regions 106 and 108) and/or the matrix region 110 (e.g., when the endothelial cells are seeded in the gel-like matrix directly). In so doing, the vasculature as well as cell aggregate layout can have different spatial distributions. Namely, vasculature can form by self-assembling of endothelial cells already inside the gel-like matrix containing the cell aggregate to completely surround the cell aggregate in all directions; and/or vasculature can form by sprouting of endothelial cells from the fluidic regions into the gel-like matrix (e.g., ECM-like matrix) to mimic angiogenesis towards the cell aggregate (e.g., to mimic newly vascularised tumours). For the latter, see vasculature 124 of FIG. 1E.

[0111] The central circular opening 112 of the device 104 further allows insertion of a cell aggregate from the top of the device 104 directly into the matrix region 110 without the need for them to be exposed to physico-mechanical changes that could potentially occur when cells are made to flow through a fluidic channel. The central circular opening 112 of the device 104 also allows the extraction of the sample (e.g., cell aggregate, an organoid, or a patient biopsy) if needed, to conduct further investigations such as histology, flow cytometry, and omics studies.

[0112] Fabricating the Device

[0113] The devices 104 described with reference to FIGS. 1C and 1D for example can be fabricated with polydimethylsiloxane (PDMS) through a soft-lithography process and specifically by replica moulding. FIG. 1F shows a photograph of PDMS devices bonded to a microscope glass slide (compare microscope glass slide 102 described with reference to FIG. 1A), FIG. 1G shows a zoom photo of a circular opening located substantially in the middle of a matrix region of a PDMS device (compare central circular opening 112 described with reference to FIG. 1C), and FIG. 2 shows a schematic drawing of a mold for fabricating PDMS devices.

[0114] An exemplary protocol for device fabrication can comprise the steps of: (i) casting PDMS into a mould (e.g., by first mixing the PDMS elastomer with a PDMS curing agent in a plastic cup, then using a vacuum machine to remove all air bubbles before pouring the PDMS mix into the mould); (ii) using the vacuum machine to remove all air bubbles thoroughly; (iii) placing the mould in an 80 C. oven for about 1 hour 45 minutes; (iv) using a pair of tweezers, removing the cured PDMS from the mould; (v) placing the cured PDMS into a petri dish, and placing in the 80 C. oven overnight; (vi) cutting out the PDMS replicas and punching holes accordingly; (vii) using tape to remove any dirt or other particles from both sides of the PDMS chip; and (viii) autoclaving the PDMS replicas in a pipette box.

[0115] After the PDMS replica is prepared, the PDMS replica can be bonded to a glass coverslip. Bonding of the PDMS replica to a glass coverslip can comprise the steps of: cleaning glass coverslips with acetone, followed by isopropanol, and then 70% ethanol; using tape to clean dust off surface of PDMS and glass slide; cleaning the inside of a vacuum chamber with ethanol and making sure it is dry; performing plasma treatment on PDMS replicas and glass cover slips; bonding glass to PDMS replica; and placing the bonded chip into an oven at 70 C.

[0116] Chip Modification

[0117] In the examples described above, a chip comprises one array of devices. See FIG. 1B for example.

[0118] The chip can be modified to increase throughput for example. One example of a modified chip 300 is shown in FIG. 3 which shows the chip 300 comprising three (i.e., a plurality of) array of devices 302, 304, and 306. In the chip 300, each array 302, 304, and 306 comprises a 5 by 1 array of devices (similar to the 5 by 1 array of devices described with reference to FIG. 1B) and each device of the arrays 302, 304, and 306 is substantially similar to the device 104 described with reference to FIGS. 1C and 1D for example. The array 302, 304, and 306 are each bonded onto a microscope glass slide 308 to form the chip 300. The chip 300 with the microscope glass slide 308 as its base can be placed onto a plate holder for example for downstream analytics.

[0119] Introducing Cell Aggregates and/or Organoids into Devices

[0120] In the examples described above, a matrix region of a device comprises an opening which is substantially in the middle of the matrix region along its longitudinal length. See circular opening 112 of FIG. 1C for example.

[0121] The opening mentioned above allows cell aggregates to be introduced into a device by a number of ways. For example, the cell aggregates can be introduced into the device via the opening by one of: microinjection, pipetting and/or gravity.

[0122] FIGS. 4A and 4B are schematic cross-sectional diagrams illustrating one example of a process of introducing an organoid into a device by pipette injection. The device 400 shown in FIGS. 4A and 4B comprises a matrix region 402 (compare matrix region 110 described with reference to FIG. 1C) and two fluidic regions 404 and 406 (compare fluidic regions 106 and 108 described with reference to FIG. 1C). The matrix region 402 provides an opening 408 (compare circular opening 112 described with reference to FIG. 1C) through which an organoid can be introduced.

[0123] In this example, a gel-like matrix 410 is present in the matrix region 402 and a supporting fluid 412 is present in the fluidic regions 404 and 406. An organoid 414 with a size of about 300 m to about 500 m is introduced into the device 400 via the opening 408 (see FIG. 4A), and thereafter, additional gel-like matrix 416 (e.g., collagen or other types of hydrogel such as fibrin solution and/or a mix of collagen solution, fibrin solution or the like) is introduced into the device, also through the opening 408 (see FIG. 4B). As shown in FIG. 4B, the introduction of the additional gel-like matrix 416 through the opening 408 causes the organoid 414 to sink into the gel-like matrix 410 present in the matrix region 402 (see arrow 418). In this way, the opening 408 allows the organoid 414 to be embedded in the gel-like matrix 410 with a substantially precise control of its position.

[0124] Exemplary Workflow for 3D In Vitro Vascularised Tumour Model

[0125] FIG. 5 is a diagram showing an exemplary workflow for a 3D in vitro vascularised tumour model. The workflow 500 shown in FIG. 5 comprises step 502, step 504, step 506, and step 508.

[0126] At step 502, tumour spheroids derived from HepG2 and Hep3b cell lines are formed using the hanging drop technique.

[0127] After 5 days, at step 504, spheroids (or cell aggregates) 510 are formed. Separately, a mixture containing endothelial cells (e.g., HUVEC (Human Umbilical Vein Endothelial Cells) and human induced pluripotent stem cell-derived endothelial cell (hiPSC-EC)) 512 and fibroblasts (e.g., NHLF (Normal Human Lung Fibroblasts)) 514 are prepared.

[0128] At step 506, the spheroids 510 are seeded into a middle channel 516 of a device (compare matrix region 110 described with reference to FIG. 1C) in an ECM (extracellular matrix) containing endothelial cells (e.g., HUVEC, hiPSC-EC) 512 and fibroblasts (e.g., NHLF) 514. See arrow 518 showing that a spheroid 510 is seeded into a middle channel via an opening 520 (compare circular opening 112 described with reference to FIG. 1C).

[0129] After 7 days, at step 508, vasculature 522 forms spontaneously around a tumour 524 (formed from spheroids 510 seeded into the device at step 506).

[0130] The prepared 3D in vitro vascularised tumour model can subsequently be used for various applications. Such applications can include analysing the effect of one or more test agents (e.g., through drug treatment or cell therapy) on the tumour 524 formed. Other applications can include analysing the tumour 524 formed via immunofluorescence (live/fixed) 526, histology characterisation 528 (e.g., to analyse cellular spatial organisation in the tumour 524), scRNA sequencing and digital spatial proteomics/transcriptomics 530.

[0131] Vasculature Formation over Time

[0132] Based on the above exemplary workflow, vasculature formation in the device was investigated.

[0133] In this example, HepG2 and Hep3b liver tumour cell lines were used to form monoculture (only liver cancer cells) or triculture spheroids (liver cancer cell co-cultured with stellate cells (LX2) and endothelial cells). After 5 days of allowing the cells to organise and grow in an aggregate format outside the device, the spheroids formed were seeded into devices in an ECM containing GFP-HUVEC and NHLF. As a control, GFP-HUVEC and NHLF were seeded into the devices to monitor the natural formation of vessels. Representative images of the different conditions are shown in FIG. 6A.

[0134] Based on the data shown in FIG. 6A, vasculature quantification was performed using Fiji and the results are shown in FIG. 6B. For vasculature quantification, GFP-signal was quantified in the device excluding the spheroid and represented as a percentage of the total surface.

[0135] Effect of the Vasculature on Spheroid Viability

[0136] The effect of vasculature on spheroids cultured on the devices was also investigated.

[0137] First, the effect of vasculature on spheroid growth was investigated. FIG. 7A is a graph showing spheroid growth over time measured with and without vasculature. As shown in FIG. 7A, the various spheroids showed growth with time (relative to day 1), both with and without vasculature.

[0138] Next, the effect of vasculature on the spheroid viability was quantified with DRAQ7. FIG. 7B shows representative fluorescence images, as well as expanded images of the boxed portions of the respective fluorescence images, from spheroids and DRAQ7 quantification. FIG. 7C is a graph showing quantification of DRAQ7 in the spheroids. As shown in FIG. 7C, spheroid viability was significantly improved when grown with vasculature, particularly for HepG2 monoculture spheroids, HepG2 triculture spheroids, and Hep3b monoculture spheroids.

[0139] The effect of vasculature on the expression of Ki-67 was also investigated. FIGS. 7D, 7E, 7F, 7G, and 7H show the distribution and expression of the proliferation marker Ki-67 in vascularised spheroids detected by immunofluorescence and compared to the proliferation in the spheroids grown in ECM without vasculature. As shown in FIGS. 7D, 7E, and 7F, a significant difference in expression of the proliferation marker Ki-67 was found particularly in Hep3b monoculture spheroids grown with vasculature, both in its core and outer regions of the spheroids. Overall the data show a decreased Ki-67 expression in cell aggregates grown with vasculature.

[0140] The effect of vasculature on apoptosis was also investigated. FIGS. 7I, 7J, 7K, 7L, and 7M show the extent of apoptosis in the spheroids detected by immunofluorescence using Caspase-3 antibody. As shown in these figures, vascularised spheroids mostly showed less apoptosis compared to spheroids in ECM without vasculature, suggesting a positive effect of the vasculature on the spheroids' viability. The difference was particularly significant for vascularised HepG2 monoculture spheroids (see FIG. 7I) and for vascularised Hep3b triculture spheroids, both in its core and outer regions of the spheroids (see FIGS. 7I and 7K).

[0141] The effect of vasculature on reducing hypoxia was also investigated. This was detected and measured using anti-Hif1 a (Hypoxia-inducible factor-1 alpha) antibody. As shown in FIGS. 7N, 7P, and 7Q (see also FIGS. 7R and 7S as examples), vasculature reduced hypoxia in most of the conditions tested. As shown in FIG. 7N, the presence of vasculature significantly reduced hypoxia, particularly for vascularised HepG2 triculture spheroids, vascularised Hep3b monoculture spheroids (especially in its core region, as shown in FIG. 7Q), and vascularised Hep3b triculture spheroids (also especially in its core region, as shown in FIG. 7Q).

[0142] Effect of Spheroid Type on Vasculature Formation

[0143] The effect of spheroid type on vasculature formation was investigated. For this, quantification of vasculature coverage close to the spheroid (or adjacent to the spheroid, wherein adjacent means within a distance of 350 m from the spheroid) and distant from the spheroid (wherein distant means more than 350 m from the spheroid) was performed using concentric analysis in Fiji and the results are shown in FIG. 8. As shown in FIG. 8, Hep3b spheroids were more angiogenic (vasculature coverage was close to the tumour) and monoculture HepG2 spheroids decreased the vasculature around the tumour compared to the region far/distant from the spheroid.

[0144] Effect of Drug Treatment on Tumour Spheroids and Vasculature

[0145] Next, the effect of Sorafenib treatment on the tumour spheroids and the vasculature was investigated. In this example, Hep3b monoculture and triculture spheroids were seeded in the devices with and without vasculature. At day 5, 10 M of Sorafenib was used to treat the tumours for 7 days. GFP signal (representing the live cells) was quantified along the time.

[0146] In FIG. 9A, representative images from all the conditions are shown at Day 0 (starting of the drug treatment) and Day 5 post-treatment. FIG. 9B is a graph showing the GFP signal over time represented from 3 independent replicates. As can be seen in the graph of FIG. 9B, Sorafenib transport is affected by vasculature. That is, in the Sorafenib treated samples the viability of the vascularised spheroids is lower compared to the non-vascularised spheroids, suggesting an improvement in drug delivery into the tumour due to the vasculature.

[0147] Use of Device to Test Cell Therapy

[0148] The use of the devices to test cell therapy was also investigated.

[0149] In the following first example, TCR-engineered T cells were used to test the efficiency in tumour killing of engineered T cells in 3D in vitro. HepG2 spheroids (TCR T cells targets) were seeded into the devices with and without vasculature. Vasculature was developed for 7 days. At day 7, 100 k TCR T cells or Mock T cells were added into the devices (to the side channels; compare fluidic regions 106 and 108 described with reference to FIG. 1C) and killing of the tumour was detected with DRAQ7 18 hours after the treatment.

[0150] As shown in FIGS. 10A and 10B, TCR-engineered T cells showed an increase in killing HepG2 spheroids compared to Mock T cells in vascularised spheroids and no difference was detected in non-vascularised spheroids, suggesting than vascularisation of spheroids in the device can be used as a model to test cell therapies in vitro.

[0151] In the following second example, CD133 CAR-T cells were used instead to perform a killing assay in 3D in vitro in devices. Hep3b spheroids (CD133 CAR-T cells targets) were seeded into the devices with and without vasculature. 5 days post-seeding, 100 k CD133 CAR-T cells were added to the side channels of the devices and tumour killing was monitored using DRAQ7 at day 2, 4 and 6 post treatment and compared to the non-treated tumours (control).

[0152] As shown in FIGS. 10C and 10D, CD133 CAR T-cells were able to target and perform their cytotoxic activity on the spheroids in the devices in both vascularised and non-vascularised spheroids. For Hep3b monoculture spheroids in particular, a significant difference was observed at day 4 in the capability of the CD133 CAR T-cells to perform a higher cytotoxic activity on vascularised spheroids over the non-vascularised spheroids.

[0153] FIG. 10E shows that it's possible to quantify the amount of T cells reaching the tumor aggregate and that the CD133 CAR T-cell count grew from day 2 to day 6 in the devices with or without vasculature.

[0154] FIG. 10F shows representative fluorescence images of vascularised and non-vascularised Hep3b monoculture and triculture spheroids treated with CD133 CAR-T cells or mock cells.

[0155] Use of Device for Culturing Biopsies

[0156] The devices can be used to culture tumor biopsies. As an example, FIGS. 11A and 11B show images taken on day 3 of a vascularised patient biopsy (the biopsy was obtained from an ovarian cancer patient) which was cultured in a device with endothelial cells (HUVEC) and fibroblasts (NHLF).

APPLICATIONS

[0157] Various embodiments of the devices and methods disclosed herein provide possible uses/applications in translational research, including drug screening and drug development; immunotherapy and cell therapy testing and QC (Quality Control); and personalised medicine strategies.

[0158] Various embodiments of the devices and methods disclosed herein also provide possible uses/applications in basic research, including: investigation of cancer development mechanisms, cancer vascularisation, and cancer metastasis; discovery of molecular pathways in cancer-endothelium-stroma interactions; and investigation on organoid differentiation and development.

[0159] Various embodiments of the devices and methods disclosed herein may be further utilised to develop an organ-on-a-chip platform that is ready for adoption in industry (i.e., develop from lab prototype version 1.0 to manufacturing-ready prototype version 2.0).

[0160] Various embodiments of the devices and methods disclosed herein may also be utilised to validate and optimise protocols for a human 3D complex vascularised cell aggregate in a device.

[0161] Various embodiments of the devices and methods disclosed herein may further be utilised to validate downstream analytics to analyse cellular spatial organisation in a 3D complex model, including histology characterisation.

[0162] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.