WO2024261063A1

WO2024261063A1 - Ex vivo tissue sampling system

Info

Publication number: WO2024261063A1
Application number: PCT/EP2024/067104
Authority: WO
Inventors: Mario Modena; Martin Baumgartner; Jana PETR
Original assignee: Eth Zurich; Universität Zürich
Priority date: 2023-06-23
Filing date: 2024-06-19
Publication date: 2024-12-26

Abstract

Ex vivo tissue culturing and sampling system (2) comprising a microfluidic cartridge (4) and a microfluidic sampling apparatus (3) that receives the microfluidic cartridge and a tissue support substrate (5) on which a tissue sample (1) to be cultured and analysed is mounted, the tissue support substrate separable from the microfluidic cartridge and comprising a porous hydrophilic membrane (19), the microfluidic cartridge comprising - a body (10), - a reaction chamber (20) formed within the body and configured to be sealingly closed by a seal (24) around the tissue sample (1) on the tissue support substrate (5), and - a transparent observation window (16) bounding one side of the reaction chamber configured to allow microscope imaging of the tissue sample, the microfluidic sampling apparatus comprising - a cartridge holder (6) in which the microfluidic cartridge and biological sample support substrate is removably mounted, - a reagent fluid flow system (7) configured to pump liquid through the microfluidic cartridge reaction chamber, and - a gas to liquid diffusion device (8) connected to the reagent fluid flow system upstream of the microfluidic cartridge reaction chamber.

Description

EX VIVO TISSUE SAMPLING SYSTEM

The present invention relates to a system for culture and analysis of tissue samples ex vivo.

Organotypic tissue slices, i.e. dissections of primary tissue of either murine or human origin, are becoming an increasingly popular model for cancer research. The culturing of such tissue slices enables to preserve ex vivo the original heterogeneity and organization of the cells. Slice maintenance relies on static culturing on porous membranes by placing the tissues at the air-medium interface, or on periodic exposure of the slices to medium and environmental air (roller-tube method) to support long-term tissue viability. While these methods have been proven effective for maintaining a large variety of tissue slices of healthy and disease tissues, they do not allow for continuous optical access to the tissue section or the controlled delivery of temporally defined chemical stimuli (e.g., drugs or compounds) to investigate dynamic processes. This problem is particularly evident in, e.g., cancer-invasion studies, where cancer cells are loaded onto tissue slices to investigate their invasion into the target tissues. Currently, implanted slices are fixed at the end of the assay to then proceed with the evaluation of cancer invasion. However, information on how tumor cells could respond to drug treatment, or how drug treatment affects the invasiveness of the tumor cannot be monitored in real time, due to the poor optical properties of the supporting porous membrane or the necessary continuous rotation of the slices in the roller-tube culturinq method.

A known method for organotypic cultures of nervous tissue is described in Stoppini L., Buchs P.-A. and Muller D. “A Simple Method for Organotypic Cultures of Nervous Tissue", Journal of Neuroscience Methods 37,2,173-182 (1991_. This journal article, published in 1991 , introduces the air-liquid-interface culturing method. This is the current state of the art in organotypic culturing of tissue slices. In this method, freshly cut slices are placed on a porous membrane, with one side exposed to air and the other side in contact with the medium solution. This method simplified culturing, with respect to the previously used rollertube approach. However, as stated in the manuscript itself, the membrane inserts, which are used to culture the slices, are not compatible with rapid exchange of solutions for, e.g., drug studies and limit live microscopy resolution due to the optical properties of the porous membrane. A similar culturing method is further described in W02010020875A3. It is also known to perform multiplexed drug testing of tumor slices using a microfluidic platform as described in Horowitz, L.F., Rodriguez, A.D., Dereli-Korkut, Z. et al. “Multiplexed drug testing of tumor slices using a microfluidic platform." npj Precis. One. 4, 12 (2020). In this paper, the authors introduced a platform for the parallelized culturing of tissue slices on a porous membrane, similarly to the standard culturing method, as introduced in the previous paper (here above). However, in contrast to the standard static culturing method, the medium chamber below the porous membrane is connected to perfusable channels to provide rapid exchange of fluids. Furthermore, multiple channels are placed below each membrane, so that the same slice is exposed to different conditions. However, image quality is still highly limited by the porous membrane in the optical path so that monitoring of dynamic processes and immunofluorescence staining with subcellular resolution cannot be carried out in the system.

Another proposed approach to enable the handling and transferring of organoids is reported in Govindan S., Batti L., Osterop S. F. et al. “Mass Generation, Neuron Labeling, and 3D Imaging of Minibrains” Front. Bioeng. Biotechnol. 8 (2021). This document describes a culturing method for brain organoids, in which the organoids are grown on a porous membrane section, which is then placed on a standard slice-culturing porous-membrane insert. This approach enables to maintain a large number of organoids in static culturing, while individual organoids can be transferred with their supporting porous membranes to perform electrophysiological experiments or to perfuse and image them. However, the small membrane on which the organoids are grown is not a hard support and the handling of the tissues is difficult.

A general object of the invention is to provide a system that enables monitoring of dynamic processes with high image resolution of ex vivo tissue samples, for instance ex vivo tissue slices.

It is advantageous to provide an ex vivo tissue culturing and sampling system that allows immunofluorescence staining and imaging.

It is advantageous to provide an ex vivo tissue culturing and sampling system that is cost- effective to operate and maintain.

It is advantageous to provide an ex vivo tissue culturing and sampling system that allows automated analysis. It is advantageous to provide an ex vivo tissue culturing and sampling system that allows imaging with subcellular resolution.

For certain applications, it is advantageous to provide a system that enables culturing and analysis of the ex vivo tissue sample over an extended duration, from a few hours to up to 2 to 4 weeks (depending on the specific application).

Objects of the invention have been achieved by providing a system according to claim 1. Dependent claims set out various advantageous features of embodiments of the invention.

Disclosed herein is an ex vivo tissue culturing and sampling system comprising a microfluidic cartridge and a microfluidic sampling apparatus that receives the microfluidic cartridge and a tissue support substrate on which a tissue sample to be cultured and analysed is mounted, the tissue support substrate separable from the microfluidic cartridge and comprising a porous hydrophilic membrane, the microfluidic cartridge comprising a body, a reaction chamber formed within the body and configured to be sealingly closed by a seal around the tissue sample on the tissue support substrate, and a transparent observation window bounding one side of the reaction chamber configured to allow microscope imaging of the tissue sample, the microfluidic sampling apparatus comprising a cartridge holder in which the microfluidic cartridge and biological sample support substrate is removably mounted, a reagent fluid flow system configured to pump liquid through the microfluidic cartridge reaction chamber, and a gas to liquid diffusion device connected to the reagent fluid flow system upstream of the microfluidic cartridge reaction chamber.

In an advantageous embodiment, the porous hydrophilic membrane extends over a zone bounded by a porous hydrophilic membrane perimeter arranged within an outer perimeter of the tissue support substrate.

In an advantageous embodiment, the porous hydrophilic membrane perimeter is arranged within a perimeter of said seal.

In an advantageous embodiment, a support substrate portion surrounding the porous hydrophilic membrane perimeter is non-porous, or hydrophobic, or non-porous and hydrophobic.

In an advantageous embodiment, the porous hydrophilic membrane is integrally formed with the surrounding support substrate portion and formed of a same base material, whereby the substrate base material is hydrophilic and a hydrophobic coating is applied to the surrounding support substrate portion, or the base material is hydrophobic and a hydrophilic coating is applied to the zone bounded by a porous hydrophilic membrane perimeter forming the porous hydrophilic membrane.

In an advantageous embodiment, the gas to liquid diffusion device comprises a diffusion unit and a gas source connected to the diffusion unit, optionally via one or more valves to control the supply of gas to the diffusion unit.

In an advantageous embodiment, the gas source comprises at least one gas being a source of oxygen for the liquid medium.

In an advantageous embodiment, the gas source comprises a plurality of gases.

In an advantageous embodiment, the diffusion unit comprises a gas chamber body comprising at least one gas chamber having an inlet fluidically connected to the gas source, and at least one outlet, a liquid chamber body comprising at least one liquid chamber having an inlet connected upstream to the reagent source and an outlet connected downstream to the reaction chamber of the microfluidic chip, and a gas-permeable membrane between the liquid chamber and gas chamber configured to allow gas to diffuse from the gas chamber to the liquid chamber.

In an advantageous embodiment, the gas-permeable membrane comprises or consists of a hydrophobic porous PTFE (Polytetrafluoroethylene) membrane.

In an advantageous embodiment, the diffusion unit comprises a seal that surrounds the liquid chamber and that is sandwiched between the liquid chamber body and the gas- permeable membrane.

In an advantageous embodiment, a height of the liquid chamber is less than 500 microns and a surface area of the liquid chamber is greater than 10mm². In an advantageous embodiment, the diffusion unit comprises more than one pair of overlying liquid and gas chambers.

In an advantageous embodiment, the reaction chamber of the microfluidic cartridge has a substantially rectangular shape defined by an inlet edge, an outlet edge and lateral edges, a height H of the reaction chamber between a top side and a bottom side that engages the tissue support substrate being in a range from 0.02 mm to 1 mm, and a surface area LxW defined by a perimeter of the inlet, outlet and lateral edges being in a range of 100mm² to 600mm² .

In an advantageous embodiment, the cartridge holder includes a base part and a cover part movable with respect to the base part, the microfluidic cartridge and substrate being removably mountable between the base part and the cover part.

In an advantageous embodiment, the microfluidic sampling apparatus comprises an image capture and processing system comprising an imaging device for capturing images of the tissue sample, and the microfluidic cartridge and cartridge holder comprise a viewing window configured to allow optical access to the reaction chamber for the imaging device .

In an advantageous embodiment, the reagent fluid flow system comprises reagent sources, a fluidic circuit connected to an inlet conduit and an outlet conduit of the cartridge holder, and a pump for circulating reagents through the microfluidic cartridge via the inlet conduit and the outlet conduit.

In an advantageous embodiment, the ex vivo tissue culturing and sampling system is configured for multiplex cycling of reagents through the reaction chamber.

Further advantageous features of the invention will be apparent from the following detailed description of embodiments of the invention and the accompanying illustrations.

Brief description of the figures

Figure 1a is a schematic simplified diagram of an ex vivo tissue culturing and sampling system according to an embodiment of the invention;

Figures 1 b and 1c are illustrations of a cartridge holder and microfluidic cartridge of an ex vivo tissue culturing and sampling system according to an embodiment of the invention; Figure 2 is a perspective exploded view of a diffusion unit of a gas to liquid diffusion device, in particular for oxygenation of the liquid medium pumped into a microfluidic cartridge of an ex vivo tissue culturing and sampling system according to an embodiment of the invention;

Figure 3 is a schematic chart illustrating steps of an experimental procedure to study cancer invasion in brain slices using an ex vivo tissue culturing and sampling system according to an embodiment of the invention for static and dynamic culturing of tissue slices for up to 24 days in vitro (DIV);

Figure 4: illustrate confocal microscope images of live cells in a microfluidic cartridge of an ex vivo tissue culturing and sampling system according to an embodiment of the invention, whereby: in A, the brain tissue slices were perfused with the live-cell dyes Hoechst and SiR Actin and stained on-chip; to assess the viability of the tissues slices on chip, the apoptotic cell dye Caspase-3/7, a nuclear stain, was used; the brain tissue slices were perfused with Hoechst and Caspase-3/7 after DIV 4 of perfusion culture as visualized in B and C; B shows an edge section of the brain tissue slice, where the higher apoptotic signal at the edge stems from suboptimal handling while transferring and placing several brain slices on the tissue culture substrate;

Referring to figures, starting with figures 1a to 1c and 2, an ex vivo tissue culturing and sampling system 2 according to embodiments of the invention comprises a microfluidic sampling apparatus 3 that receives a microfluidic cartridge 4 assembled to a tissue support substrate 5 on which a tissue sample 1 to be analysed is mounted.

The microfluidic sampling apparatus 3 comprises:

- a cartridge holder 6 including a base part 6a and a cover part 6b between which the microfluidic cartridge and substrate is removably mounted, the cartridge holder configured to securely clamp the cartridge 4 on the base 6a, the cover part 6b pressing against a top face 14 of the microfluidic cartridge 4,

- a reagent fluid flow system comprising one or more reagent sources, at least one said reagent source comprising a liquid culture medium for culturing of the ex vivo tissue, a fluidic circuit connected to an inlet conduit 17i and an outlet conduit 17o of the cartridge holder, and a pump P and one or more valves (not shown) for circulating reagents through the microfluidic cartridge via the inlet conduit 17i and an outlet conduit 17o,

- a gas to liquid diffusion device 8 connected to the fluidic circuit configured to diffuse gas into the liquid culture medium flowing in the fluidic circuit, - an image capture and processing system comprising an imaging device 13 for capturing images of the tissue sample,

- a user interface 11 for inputting commands, displaying operation information, and accessing data and images captured by the imaging device, and

- a control system 9 for controlling operation of the ex vivo tissue culturing and sampling system 2 including software 21 , 23 for control inter alia of the reagent fluid flow system and of the image capture and processing system.

The microfluidic cartridge 4 comprises a cartridge body 10 having a top face 14 and a mounting face 12. The microfluid cartridge 4 comprises a seal 24 forming a closed circuit on the mounting face 12 surrounding a reaction chamber 20, the seal configured to engage the tissue support substrate 5 and provide sealing of the reaction chamber 20 formed between the tissue support substrate 5 and the cartridge body 10.

An observation or viewing window 16 may be provided in the top face 14, or from the chamber bottom in case of a transparent apparatus, for observation of the tissue sample 1 mounted on the tissue support substrate 5 with a microscope 13 of the image capture and processing system.

The reaction chamber 20 is connected to an inlet 18i of the microfluidic cartridge 4 via a fluid flow network 22 formed within the microfluidic cartridge 4 on the one side of the reaction chamber, and on the other side of the reaction chamber is connected to an outlet 18o of the microfluidic cartridge 4 via a fluid flow network 22 formed within the microfluidic cartridge 4. The reaction chamber 20 has an inlet edge 26 with a plurality of chamber inlets 22i of the fluid flow network 22 spanning across the entire width of the reaction chamber, and similarly the reaction chamber has an outlet edge 30 on an opposed side of the chamber from the inlet edge 36 with a plurality of chamber outlets 22o spanning across the width of the chamber. The chamber is bounded on its sides by lateral edges 22 and on a top side 32 by the cartridge body and observation window 16. An open bottom side 34 of the reaction chamber 20 is covered and closed by the tissue support substrate 5 that engages the seal 24.

The reaction chamber 20 has a substantially rectangular shape defined by the inlet and outlet edges 26, 30 and the lateral edges 28. The height H of the chamber between the top side 32 and the open bottom side 34 that engages the tissue support substrate 5 is typically in the range from 0.02 mm to 1mm and the surface area LxW defined by the perimeter of the inlet, outlet and lateral edges may typically be in a range of 100mm² to 600mm² These dimensions however are not absolute limits and smaller dimensions or larger dimensions may be provided without departing from the scope of the invention.

The ratio of the chamber height H to the length L of the lateral edge H I L is typically in a range of 1/5 to 1/100. The reaction chamber 20 thus has a thin flat aspect and in conventional systems the reagents react with the tissue sample 1 to the flow of liquid from the inlet to the outlet.

The tissue support substrate 5 comprises a porous hydrophilic membrane 19 positioned facing the reaction chamber when the open bottom side 34 of the reaction chamber 20 is covered and closed by the tissue support substrate 5 that engages the seal 24. In other words, the porous hydrophilic membrane 19 forms at least part of the side of the reaction chamber covered by the tissue support substrate. The ex vivo tissue sample is intended to be positioned on the porous hydrophilic membrane 19 to receive liquid culture medium through the porous hydrophilic membrane.

In an advantageous embodiment, the porous hydrophilic membrane 19 extends overa zone bounded by a porous hydrophilic membrane perimeter 25 arranged within the outer perimeter 27 of the tissue support substrate, in particular arranged within the perimeter of the seal 24. The latter configuration ensures good sealing by avoiding the perfusion of liquid outside of the reaction chamber through the tissue support substrate.

In preferred embodiments, the support substrate portion surrounding the porous hydrophilic membrane perimeter 25 is non-porous, or hydrophobic, or non-porous and hydrophobic. In an embodiment the porous hydrophilic membrane 19 may be integrally formed with the surrounding support substrate portion and formed of the same base material. The substrate base material may be hydrophilic, in which case a hydrophobic coating is applied to the surrounding support substrate portion, or alternatively if the base material is hydrophobic, a hydrophilic coating is applied to the zone bounded by a porous hydrophilic membrane perimeter 25 forming the porous hydrophilic membrane 19.

In embodiments, the tissue support substrate 5 may comprise a mechanical support layer on which the porous hydrophilic membrane 19, and/or the support substrate portion surrounding the porous hydrophilic membrane perimeter 25, is mechanically supported. Alternatively, the porous hydrophilic membrane 19 and the support substrate portion surrounding the porous hydrophilic membrane perimeter 25 may comprise a base material of sufficient strength and rigidity to be without an additional mechanical support layer. Different types of porous membranes can be employed, with different pore sizes, membrane base materials and coatings, depending on the type of tissue to be cultured, the liquid medium properties, and the operating parameters such as pressure, temperature and duration.

The tissue support substrate may, in an advantageous embodiment, be provided in a rectangular shape. The dimensions of the rectangular shape (i.e. outer perimeter 27) may advantageously correspond to a conventional microscope coverslip, for instance of rectangular dimensions in a range from 20mm to 30mm wide and 50mm to 100mm long, for instance 25mm wide and 75mm long. This configuration is well adapted for microscope imaging and allows convenient coupling with the microfluidic cartridge, as well as easy handling of the tissue sample prior to coupling with the microfluidic cartridge. The handling of the tissue sample prior to coupling allows various culture steps, for instance static monoculture and static co-culture steps as illustrated in figure 3.

In embodiments, dimensions of the tissue support substrate may be smaller, or larger, than those indicated above, depending on the applications and the size of the microfluidic cartridge. Larger dimensions may in particular accommodate larger tissue sample surface areas and larger imaging surface areas.

The gas to liquid diffusion device 8 serves to diffuse a gas into the liquid medium that is pumped by the pump P through the reaction chamber 20. In particular, the gas, or at least one of the gases, diffused into the liquid culture medium, contains and supplies oxygen for culture of the ex vivo tissue sample 1 . The oxygen supplying gas may for instance comprise or consist of a carbogen gas. Other gases, such as nitrogen may also be diffused into the liquid medium connected to the fluidic circuit.

The gas to liquid diffusion device 8 comprises a diffusion unit 8a and a gas source 8b connected to the diffusion unit, optionally via one or more valves V to control the supply of gas to the diffusion unit. The gas source may comprise one gas being a source of oxygen for the liquid medium, or may comprise a plurality of gases, at least one thereof being a source of oxygen for the liquid medium, connected via corresponding valves to the diffusion unit to supply the required gas at the required process time. In a variant, the gas to liquid diffusion device 8 may comprise a plurality of diffusion units. In the latter variant, each diffusion unit may be supplied with a different gas from one or more gas sources. An embodiment of a diffusion unit 8a is illustrated in figure 2. In this example embodiment, the diffusion unit 8a comprises a gas chamber body 36 comprising at least one gas chamber 37 having an inlet 37i fluidically connected to the gas source 8b, and at least one outlet 37o that may feed into the atmosphere, or return to the gas source, or be fed into another gas containing system for processing (e.g recycling). The diffusion unit 8a further comprises a liquid chamber body 40 comprising at least one liquid chamber 42 having an inlet 42i connected upstream to the reagent source 7 and an outlet 42o connected downstream via the fluidic circuit to the the inlet 18i of the reaction chamber 20 of the microfluidic chip.

The at least one gas chamber 36 overlies and is separated from the at least one liquid chamber 42 by a gas-permeable membrane 38. The gas-permeable membrane may for instance comprise or consist of a hydrophobic PTFE (Polytetrafluoroethylene) membrane, for instance with an approximately 0,2 micron pore size .

The diffusion unit may further comprise a seal 41 that surrounds the liquid chamber 42 and that is sandwiched between the liquid chamber body 40 and the gas-permeable membrane 38. The height of the liquid chamber 42 may advantageously be less than 500 microns, for instance between 100 microns and 400 microns, and the surface area of the liquid chamber 42 may advantageously be greater than 10mm², for instance in a range of 10mm² to 100mm². The high surface area to height ratio of the liquid chamber in contact with the gas- permeable membrane 38 promotes a highly efficient gas diffusion from the gas chamber to the liquid medium.

The diffusion unit may comprise more than one pair of overlying liquid and gas chambers 42, 36. The illustrated embodiment in figure 2 has two liquid chambers 42 interfacing through the gas-permeable membrane 38 with two aligned gas chambers 36. Such configuration provides a large gas diffusion surface area while providing mechanical support to the gas-permeable membrane, taking into account the pressure differences between the gas circuit and liquid flow circuit. In effect, the liquid medium in the liquid chamber may be at a pressure of 0.1 or more bars greater than the pressure of the gas in the gas chamber.

Embodiments of the invention are configured for the ex vivo culturing of organotypic tissue slices with continuous optical access to the slices for high-resolution microscopy, for instance confocal or fluorescence microscopy, and the controlled delivery of drugs and reagents for drug testing and automated staining. Tissue-slice culturing can be performed in : - a static manner on the tissue support substrate, so as to for instance accommodate for tissue maturation or recovering from lesions related to the tissue preparation, and

- under perfusion in the microfluidic cartridge chamber enabling high-resolution microscopy and the controlled delivery of reagents and drugs.

The configuration of an open-sided microfluidic chip for coupling to the tissue support substrate enables simple transfer of the tissue slices onto the porous membrane using standard assays on organotypic-slice inserts. This allows for static culturing in medium reservoirs, to allow for tissue recovery and tissue culturing, if necessary.

The microfluidic cartridge may be made of plastic materials, to enable high-throughput, low- cost production and to ensure biocompatibility and no absorption of small hydrophobic molecules, which are typical issues of materials commonly employed in microfluidic prototyping.

The overall thickness of the microfluidic cartridge may be kept below 2mm, preferably below 1 mm, to ensure compatibility with high magnification and high numerical aperture (NA) microscope objectives.

The reagent fluid flow system provides nutrients and reagents to the tissue sample slices in culture during perfusion in the reaction chamber of the microfluidic cartridge.

In conventional methods, organotypic tissue slices are typically cultured at the air-liquid interface to ensure high levels of oxygenation to the tissues. However, to enable high- resolution microscopy as in the present invention, no air bubbles in the liquid layer above the sample imaged by the microscope should be present, as the bubbles would drastically degrade image quality. The gas to liquid diffusion device provides a bubble free solution to this problem, because it allows to saturate the liquid culture medium with oxygen without creating bubbles, prior to the delivery of the liquid culture medium to the tissue sample slice in the microfluidic cartridge reaction chamber.

The gas to liquid diffusion device integrated in the reagent fluid flow circuit upstream of the microfluidic cartridge enables the rapid exchange of gas in the liquid culture medium without bubble creation. Carbogen gas (for instance composed of 95% oxygen, 5% carbon dioxide) may be used to ensure high oxygen content in the liquid culture medium and to provide a stable pH of the liquid culture medium. The use of a small-volume, high surface area, liquid chamber for rapid gas exchange also enables to vary the gas composition in the liquid medium to realize different experimental microenvironments, for instance to investigate anoxia by removing oxygen from the medium by saturating the liquid medium with nitrogen gas.

Examples of live imaging of the organotypic slices using live, confocal microscopy are shown in Fig. 4.

In summary, some of the advantages of the invention include the ability to perform static (as in the current state-of-the-art procedure) as well as dynamic (under flow) culturing of ex vivo tissue slices; continuous, high-resolution microscopy on ex vivo cultured tissue slices, in which it is also possible to perform real-time monitoring of cellular responses and behaviors using fluorescent probes and sensors; high level of control over the microenvironment by varying the oxygen and nutrient concentration in medium in a dynamic way; sampling of cell culture supernatant for off-substrate analysis; perfusion-based fixation and staining of tissues on a flat substrate, which can then be used for automated staining protocols with coupling to a microfluidic cartridge.

The combination of tissue support substrate and microfluidic cartridge features a low fabrication complexity, as the microfluidic cartridge may be manufactured with high- throughput fabrication technologies (e.g. injection moulding) and the tissue support substrate may be based on commercially available porous membranes, which may be modified to ensure sealing against the microfluidic cartridge.

Embodiments of the invention offer a complete culturing environment to maintain the slices for multiple days under perfusion and to perform in situ live high-resolution imaging, perfusion, supernatant sampling, staining and immunofluorescence image acquisition.

The possibility of performing both static and dynamic culturing on the same sample further expands the application range of ex vivo tissue slices, as it enables to operate the chip under perfusion according to the scientific questions, e.g., after waiting fortissue maturation or cellular reorganization. Furthermore, the possibility of continuously perfusing the tissue slices enables to further automate the experimental procedure by performing in situ fixation, staining, and imaging of the tissue slices.

Applications Cancer invasion studies, maintenance of tissue slices/biopsies requiring perfusion to improve tissue viability, drug discovery , personalized medicine and personalized drug testing

Example of an experimental method

Fig. 3 shows an example of an experimental protocol. After slicing of the tissue, the tissue slices are transferred onto the porous hydrophilic membrane 19 supported on a holder, and the holder is placed on top of a culture medium reservoir. The culture medium contacts the lower part of the porous hydrophilic membrane 19, while the tissue slices 1 are cultured on the top surface, so as to realize an air-liquid-interface culture method, which is known to support tissue viability in static culturing. Organotypic slices can be maintained for multiple days, and during this period the slices adhere to the porous membrane and reorganize their cell layers to recover from the slicing lesions, replicating the gold standard ex vivo culturing method routinely performed for, e.g., organotypic brain slices. This static culturing time was previously reported in literature as a critical parameter for different assays (see Neve, A., Santhana Kumar, K., Tripolitsioti, D. et al. Investigation of brain tissue infiltration by medulloblastoma cells in an ex vivo model. Sci Rep 7, 5297 (2017), and Organotypic brain slice cultures: A review, C. Humpel, Laboratory of Psychiatry and Experimental Alzheimer’s Research, Department of Psychiatry and Psychotherapy, Medical University of Innsbruck).

During static culturing, multiple experimental interventions can be performed on the tissue slices, such as viral transduction and/or the implantation of disease tissue models (e.g., cancer). These interventions can then be used to, e.g., introduce foreign DNA into specific sub-cell populations to monitor protein expression or to study cancer invasion on the tissue slice, respectively. After static culturing and to be able to perform live, high-resolution and long-term imaging, the tissue support substrate is then sealingly coupled to a microfluidic cartridge in a cartridge holder.

The flow of oxygenated liquid medium through the reaction chamber provides oxygen to support the viability of the tissue slices in the reaction chamber. The small distance between the microscope viewing window and the tissue slices enable unhindered optical access to the samples for high-resolution microscopy. Live-cell, long-term imaging can, e.g., be used to monitor the activity of neurons on the brain slices, and to follow the migration and invasion of cells onto the host tissue slices. Fluidic access can also be used to provide external stimuli to the sample with high temporal resolution, e.g., by flowing drugs and compounds. At the end of the assay, the samples can be fixed and stained on the tissue support substrate within the reaction chamber, without the need of removing the samples from the holder and the microscope, so as to provide high spatial correlation between live and fixed imaging.

Figure 4 illustrates confocal microscope images of live cells in a microfluidic cartridge of an ex vivo tissue culturing and sampling system according to an embodiment of the invention, whereby: in A, the brain tissue slices were perfused with the live-cell dyes Hoechst and SiR Actin and stained on-chip; to assess the viability of the tissues slices on chip, the apoptotic cell dye Caspase-3/7, a nuclear stain, was used; the brain tissue slices were perfused with Hoechst and Caspase-3/7 after DIV 4 of perfusion culture as visualized in B and C; B shows an edge section of the brain tissue slice, where the higher apoptotic signal at the edge stems from suboptimal handling while transferring and placing several brain slices on the tissue culture substrate; The tissue health in the center is not affected by the perfusion culture as seen in panel C.

List of references

Tissue sample 1

Ex vivo tissue culturing and sampling system 2

Microfluidic sampling apparatus 3

Cartridge holder 6

Base 6a

Cover clamp 6b viewing window reagent fluid flow system 7 reagent sources pump P inlet conduit 17i outlet conduit 17o gas to liquid diffusion device 8 diffusion unit 8a gas chamber body 36 gas chamber 37 inlet 37i outlet 37o gas-permeable membrane 38 e.g. hydrophobic PTFE membrane liquid chamber body 40 seal 41 liquid chamber 42 inlet 42i outlet 42o gas source 8b control system 9 image capture and processing module 21 reagent pump and valve control module 23 user interface 11 imaging device (e.g. microscope) 13

Microfluidic cartridge 4

Cartridge body 10

Mounting face 12

Top face 14

Observation Window 16

Inlet 18i

Outlet 18o

Reaction chamber 20

Inlet edge 26

Lateral edges 28

Outlet edge 30

Top side 32

Open bottom side 34

Fluid flow network 22

Chamber inlets 22i

Chamber outlets 22o

Seal 24 Tissue support substrate 5

Outer perimeter 27 porous hydrophilic membrane 19 porous hydrophilic membrane perimeter 25 non-hydrophilic or non-porous part 29

Claims

1. Ex vivo tissue culturing and sampling system (2) comprising a microfluidic cartridge (4) and a microfluidic sampling apparatus (3) that receives the microfluidic cartridge and a tissue support substrate (5) on which a tissue sample (1) to be cultured and analysed is mounted, the tissue support substrate separable from the microfluidic cartridge and comprising a porous hydrophilic membrane (19), the microfluidic cartridge comprising

- a body (10),

- a reaction chamber (20) formed within the body and configured to be sealingly closed by a seal (24) around the tissue sample (1) on the tissue support substrate (5), and

- a transparent observation window (16) bounding one side of the reaction chamber configured to allow microscope imaging of the tissue sample, the microfluidic sampling apparatus comprising

- a cartridge holder (6) in which the microfluidic cartridge and biological sample support substrate is removably mounted,

- a reagent fluid flow system (7) configured to pump liquid through the microfluidic cartridge reaction chamber, and

- a gas to liquid diffusion device (8) connected to the reagent fluid flow system upstream of the microfluidic cartridge reaction chamber.

2. The ex vivo tissue culturing and sampling system according to claim 1 wherein the porous hydrophilic membrane (19) extends over a zone bounded by a porous hydrophilic membrane perimeter (25) arranged within an outer perimeter (27) of the tissue support substrate (5).

3. The ex vivo tissue culturing and sampling system according to the preceding claim wherein the porous hydrophilic membrane perimeter (25) is arranged within a perimeter of said seal (24).

4. The ex vivo tissue culturing and sampling system according to either of the two directly preceding claims wherein a support substrate portion surrounding the porous hydrophilic membrane perimeter (25) is non-porous, or hydrophobic, or non-porous and hydrophobic.

5. The ex vivo tissue culturing and sampling system according to the preceding claim wherein the porous hydrophilic membrane is integrally formed with the surrounding support substrate portion and formed of a same base material, whereby the substrate base material is hydrophilic and a hydrophobic coating is applied to the surrounding support substrate portion, or the base material is hydrophobic and a hydrophilic coating is applied to the zone bounded by a porous hydrophilic membrane perimeter forming the porous hydrophilic membrane.

6. The ex vivo tissue culturing and sampling system according to any preceding claim wherein the gas to liquid diffusion device (8) comprises a diffusion unit (8a) and a gas source (8b) connected to the diffusion unit, optionally via one or more valves V to control the supply of gas to the diffusion unit.

7. The ex vivo tissue culturing and sampling system according to the preceding claim wherein the gas source comprises at least one gas being a source of oxygen for the liquid medium.

8. The ex vivo tissue culturing and sampling system according to either of the two directly preceding claims wherein the gas source comprises a plurality of gases.

9. The ex vivo tissue culturing and sampling system according to any of the three directly preceding claims wherein the diffusion unit (8a) comprises

- a gas chamber body (36) comprising at least one gas chamber (37) having an inlet (37i) fluidically connected to the gas source, and at least one outlet (37o),

- a liquid chamber body (40) comprising at least one liquid chamber (42) having an inlet (42i) connected upstream to the reagent source (7) and an outlet (42o) connected downstream to the reaction chamber (20) of the microfluidic chip, and

- a gas-permeable membrane (38) between the liquid chamber and gas chamber configured to allow gas to diffuse from the gas chamber to the liquid chamber.

10. The ex vivo tissue culturing and sampling system according to any of the four directly preceding claims wherein the gas-permeable membrane comprises or consists of a hydrophobic porous PTFE (Polytetrafluoroethylene) membrane.

11. The ex vivo tissue culturing and sampling system according to either of the two directly preceding claims wherein the diffusion unit comprises a seal (41) that surrounds the liquid chamber (42) and that is sandwiched between the liquid chamber body (40) and the gas-permeable membrane (38).

12. The ex vivo tissue culturing and sampling system according to any of the three directly preceding claims wherein a height of the liquid chamber (42) is less than 500 microns and a surface area of the liquid chamber is greater than 10mm².

13. The ex vivo tissue culturing and sampling system according to any of the four directly preceding claims wherein the diffusion unit comprises more than one pair of overlying liquid and gas chambers (42, 36).

14. The ex vivo tissue culturing and sampling system according to any preceding claim wherein the reaction chamber (20) of the microfluidic cartridge has a substantially rectangular shape defined by an inlet edge (26), an outlet edge (30) and lateral edges (28), a height H of the reaction chamber between a top side (32) and a bottom side (34) that engages the tissue support substrate (5) being in a range from 0.02 mm to 1 mm, and a surface area LxW defined by a perimeter of the inlet, outlet and lateral edges being in a range of 100mm² to 600mm² .

15. The ex vivo tissue culturing and sampling system according to any preceding claim wherein the cartridge holder (6) includes a base part (6a) and a cover part (6b) movable with respect to the base part, the microfluidic cartridge and substrate being removably mountable between the base part (6a) and the cover part (6b).

16. The ex vivo tissue culturing and sampling system according to any preceding claim wherein the microfluidic sampling apparatus comprises an image capture and processing system comprising an imaging device (13) for capturing images of the tissue sample, and the microfluidic cartridge and cartridge holder comprise a viewing window configured to allow optical access to the reaction chamber (20) for the imaging device (13).

17. The ex vivo tissue culturing and sampling system according to any preceding claim wherein the reagent fluid flow system comprises reagent sources, a fluidic circuit connected to an inlet conduit (17i) and an outlet conduit (17o) of the cartridge holder, and a pump (P) for circulating reagents through the microfluidic cartridge via the inlet conduit (17i) and the outlet conduit (17o).

18. The ex vivo tissue culturing and sampling system according to any preceding claim wherein the ex vivo tissue culturing and sampling system is configured for multiplex cycling of reagents through the reaction chamber.