WO2021009201A1

WO2021009201A1 - Microfluidic device

Info

Publication number: WO2021009201A1
Application number: PCT/EP2020/069954
Authority: WO
Inventors: Dirk Cornelis VAN GENT; Roland Kanaar; Maayke Maria Petronella KUIJTEN
Original assignee: Erasmus University Medical Center Rotterdam
Priority date: 2019-07-15
Filing date: 2020-07-15
Publication date: 2021-01-21

Abstract

A microfluidic device for holding three dimensional cell structures (2), with a top cover plate (3), a bottom cover plate (4), and an insert (5) having a membrane (6) provided in an aperture in the insert (5) and extending in a plane of the insert (5). In operation the insert (5) is positioned between the top cover plate (3) and bottom cover plate (4) to form two flow channels (3a; 4a) on either side of the insert (5). The membrane (6) is provided with a plurality of pores (6a) for holding three dimensional cell structures, with a pore size of at least 50pm. The microfluidic device (1) may be applied for a real-time imaging application.

Description

Microfluidic device Field of the invention

The present invention relates to a microfluidic device, an insert for such a microfluidic device and a method of using such a microfluidic device.

Background art

Microfluidic devices are well known, and are used in many diagnostic and research applications.

Summary of the invention

The present invention seeks to provide an improved microfluidic device compatible with real-time microscopic imaging and especially suitable for use with three dimensional cell structures such as organoids and spheroids.

According to the present invention, a microfluidic device is provided, in accordance with the claims as appended. Furthermore, in a further aspect, the present invention relates to methods of using such a microfluidic device, e.g. wherein the microfluidic device is applied for a real-time imaging application.

Short description of drawings

The present invention will be discussed in more detail below, with reference to the attached drawings, in which

Fig. 1 shows a cross sectional view of a microfluidic device according to an embodiment of the present invention;

Fig. 2 shows a perspective view of an insert for use in a microfluidic device in accordance with a further embodiment of the present invention;

Fig. 3A and 3B shows a cross sectional view of two embodiments of the insert as shown in Fig. 2;

Fig. 4 shows a partial cross sectional view of a microfluidic device according to a further embodiment of the present invention; and

Fig. 5 shows a perspective view of an optical window of an exemplary embodiment of the microfluidic device of the present invention.

Description of embodiments

The present invention embodiments are related to a microfluidic system, especially suited for three dimensional cell structures such as organoids, organotypic (tissue) slices, biopsies, etc. compatible with confocal imaging. E.g. a microfluidic device is provided as well as methods suitable to culture primary tumor material for extended culture periods (days to months) ex vivo, enabling real-time (live) imaging and functional pathology. The technology of (tumor) organoid cultures has great potential for especially drug screening purposes. Organoids may be less suitable for prognostic purposes due to time between biopsy and required clinical decision, and selective advantage of faster growing cells in the material used to derive the organoid. Viable organotypic slices obtained from (primary) tumors from cancer patients, or spheroids/organoids derived from such tumor slices, can provide a suitable alternative approach. The present invention embodiments, as described in more detail below with reference to a number of exemplary embodiments, relate to techniques to culture thin tumor slices ex vivo. It has been shown that these materials can be used in "functional" pathology, because a functional response to therapies can be measured. This will become of great importance in precision (personalized) medicine approaches. The next frontier in this development is to include various aspects of the tumor microenvironment. This can be achieved by e.g. incubating the thin tumor slices ex vivo in microfluidic devices. It is also possible to build tumor like structures by co-culture of various cell types (immune cells, blood vessel endothelial cells, microbiome) in microfluidic devices.

In one aspect, a microfluidic chip device is provided that can accommodate long-term culture of tumor slices ex vivo, suitable for diagnostic procedures and integration into clinical practice. The microfluidic device embodiments as described herein have great potential to be applied in clinical practice for diagnostic purposes, not only for breast cancer, but virtually any (solid) tumor type. The present invention embodiments are e.g. provided as a Cancer-On-Chip (CoC) facility, which will not only allow extended culturing of tumor tissue ex vivo, but also development of models for immunotherapy, as (immune) cells can be introduced in a controlled fashion. Furthermore, CoC systems can also be imaged microscopically in real-time, which will be of great value to follow various processes in real-time (for example, DNA damage response and apoptosis). Generically, in a further aspect the present invention relates to methods of using such a microfluidic device, e.g. wherein the microfluidic device is applied for a real-time imaging application.

Fig. 1 shows a cross sectional view of a microfluidic device 1 according to an embodiment of the present invention. The microfluidic device 1 is arranged for holding three dimensional cell structures 2, such as organoids, spheroids, tissue samples, organotypic slices, etc. The microfluidic device 1 comprises a top cover plate 3 (with thickness tt), a bottom cover plate 4 (with thickness tb), and an insert 5 having a membrane 6 provided in an aperture in the insert 5 and extending in a plane of the insert 5. In operation the insert is positioned between the top cover plate 3 and bottom cover plate 4 to form two flow channels 3a; 4a on either side of the insert 5. This arrangement allows to obtain a laminar flow of fluids in the two flow channels 3a, 4a having respective channel heights hi , ti2 of e.g. 250pm. It is noted that by using different heights hi , h_å in the flow channels 3a, 4a, the fluid volume above and below the insert 5 and membrane 6 can be made different. The fluids can be entered into the flow channels 3a, 4a, using flow channel inputs 3b, 4b, and can be extracted at the other ends of the flow channels 3a, 4a, e.g. using flow channel outputs 3c, 4c. This can be accomplished e.g. using pump and control set-ups of microfluidic arrangements which are known as such. In the microfluidic device 1 , the flow channels 3a, 4a are sealed using separate seal elements 8. It is noted that in the microfluidic device 1 , the three dimensional cell structures 2 positioned in pores 6a of the membrane 6 are embedded in a hydrogel 7, allowing interaction with the fluid in the two flow channels 3a, 4a.

In accordance with a first group of embodiments of the present invention, the membrane 6 is provided with a plurality of pores 6a for holding three dimensional cell structures, with a pore size of at least 50pm. The pore size is larger than pore sizes in prior art microfluidic devices having an arrangement with an insert having a membrane, allowing to adequately position and hold the three dimensional cell structures 2 as mentioned above. In a further aspect of the present invention, an insert 5 is provided for use in a microfluidic device 1 according to any one of the microfluidic device embodiments described herein. The features described below with reference to exemplary microfluidic device embodiments can, where applicable, also be included in insert 5 embodiments. Fig. 2 shows a perspective view of such an insert 5 for use in a microfluidic device 1 , the arrows indicating the general direction of flow of a fluid in the (upper) flow channel 3a.

In yet a further aspect of the present invention, a method is provided of using a microfluidic device 1 according to any one of the present invention embodiments, comprising feeding a first fluid in a first flow channel 3a between the insert 5 and the top cover plate 3, and feeding a second fluid in a second flow channel 4a between the insert 5 and the bottom cover plate 4. It is noted that the first fluid can be the same as the second fluid, but alternatively, the first fluid is different from the second fluid. E.g. in an exemplary embodiment, the method may further comprise adding an imaging agent to the second fluid, e.g. for influencing a refractive index. This imaging agent can be advantageous when using the microfluidic device 1 for (confocal) imaging, such as agents to increase refractive index of fluids such as iodixanol.

In further embodiments of the present invention, the pore size of the individual pores 6a is at least 100pm, e.g. at least 250pm. This allows to accommodate even bigger three dimensional cell structures 2 in operation. The pore size of the pores 6a can even be as high as 400pm.

The pores 6a in the membrane 6 can be provided in a specific pattern, e.g. in a specific embodiment the plurality of pores 6a comprises an array of at least two by at least two pores (6a). To allow more cell structures 2 to be kept ex vivo and to be subjected to diagnostics or imaging, the array of cells can alternatively be a 3x3 array, 3x4 array, 4x4 array, etc., etc. The array configuration and pore size can e.g. be adapted to the specific three dimensional cell structures 2 for which the microfluidic device 1 is being used, as the requirements in this respect can be different for organoids, spheroids and organotypic slices. Advantageous combinations are e.g. a 2x2 array of 400pm pores 6a, or a 3x4 array of 250pm pores 6a.

In the exemplary embodiments described herein, the membrane 6 is positioned symmetrical in the insert 5. This allows to have a symmetrical contact of the three dimensional cell structures 2 with the fluids in the flow channels 3a, 4a via the respective hydrogel 7 layers.

In a particular advantageous embodiment, the membrane 6 is an integral part of the insert 5, and can be manufactured as an integrated body, a single piece body, and can then be used as a one piece disposable item, greatly enhancing usability of the insert 5 and/or microfluidic device 1 (low cost and easy to handle). Alternatively, the membrane 6 can be a separate item, affixed in an aperture in the insert 5, e.g. using glue or other fixation means. To allow use of the insert 5 and/or the microfluidic device 1 in existing set-ups with pumps etc., the membrane 6 has a dimension of 10x10mm in a further embodiment, e.g. in combination with insert 5 dimensions of 45 x 15 mm. For symmetry and laminar flow reasons, it is advantageous if the membrane 6 is a sunken part of the insert 5, e.g. with symmetric depths on both sides of the membrane 6 (of e.g. 0.8mm).

Fig. 3A shows a cross sectional view of the insert embodiment of Fig. 2 with some exemplary detail dimensions. As shown in this exemplary embodiment (not to scale), the membrane

6 has a thickness t_m which is less than a thickness t, of the insert 5, and a hydrogel material 7 is provided on (both sides of) the membrane 6. The membrane thickness t_m is e.g. 10pm, and the insert thickness t, is e.g. 0.8mm. The material of the membrane 6 is advantageously a biocompatible material, having as little as possible or no interference with (multiphoton or confocal) imaging of the three dimensional cell structures 2 in the microfluidic device 1 .

It is noted that the material of the hydrogel 7 is e.g. a polyethylene glycol (PEG) based hydrogel, or a polyethylene glycol diacrylate (PEGDA) based hydrogel, which has the advantage that no cell adhesion occurs from the three dimensional cell structures 2. Furthermore, the hydrogel

7 will prevent the three dimensional cell structures 2 to transform is a (pseudo-) two dimensional structure in/on the membrane 6.

In an even further embodiment, shown in the cross sectional view of Fig. 3B, the insert 5 further comprises a secondary membrane layer 6b (below or above the‘primary’ membrane 6) having a plurality of secondary pores with a smaller dimension than the pores 6a of the primary membrane 6, e.g. less than 20pm (e.g. about 8pm). This secondary membrane 6b may in an even further embodiment be provided with a coating layer on both sides, of the same or different coating material.

The combination of the primary membrane 6 and secondary membrane 6b in the insert 5 in this group of embodiments would allow to form special 3D cell culture models such as spheroids with a cell monolayer (e.g. endothelial cells to mimic a blood vessel). As an example, it would be possible to use the secondary membrane 6b with small pore sizes (around 8pm) to form a cell monolayer, and use the primary membrane 6 on top of the secondary membrane 6b to form the spheroids directly in this insert. As an advantage, no intermediate transfer is necessary to form such special 3D cell culture models. This embodiment can be further enhanced using a spatial patterning of cell-adherent and cell-repellent materials to guide formation of spheres 2 on specific positions of the primary membrane 6 and/or secondary membrane 6b. In a further alternative embodiment, two membranes of different structure (such as the primary membrane 6 and secondary membrane 6b) are combined in a single insert 5.

All components of the microfluidic device 1 embodiments described herein are made of biocompatible materials. Furthermore, the structure and materials used are compatible with X-ray exposure, i.e. there will be no issues with radiation absorption, and also compatible with use of a (confocal) microscope.

To allow imaging access to the three dimensional cell structures 2, in a further group of embodiments, the bottom cover plate 4 comprises a transparent material. The transparent material e.g. comprises glass, or an optically transparent polymer, such as a cyclic olefin copolymer (COC). To further enhance the optical relevant characteristics of a microfluidic set-up, the bottom cover plate 4 has a thickness (tb) of less than 0.3mm, e.g. less than 0.2mm, e.g. 0.17mm, in a further embodiment.

Fig. 4 shows a partial cross sectional view of a microfluidic device 1 according to an even further group of exemplary embodiments of the present invention. As shown in this set-up, the bottom cover plate 4 comprises an optical window 9. The optical window 9 may comprise the same material as the bottom cover plate 4, but alternatively comprises an optically special material. E.g. the bottom cover plate 4 may be of a glass material specifically arranged to provide sufficient strength to prevent breaking of the microfluidic device 1 , while the optical window 9 comprises an optically optimized material. In an even further embodiment, the optical window 9 has a thickness of less than 0.3mm, e.g. less than 0.2mm, e.g. 0.17mm, whereas the bottom cover plate 4 can then have a larger thickness. In this group of exemplary embodiments, the thickness of the optical window 9 may be smaller than the thickness tb of the bottom plate 4, and the optical window 9 may be positioned flush with the bottom side of the bottom plate 4, flush with the bottom channel 4a, or at an intermediate position.

In a further embodiment, an exemplary embodiment of which is shown in perspective view in Fig. 5, the optical window 9 is provided as an optical waveguide assembly, comprising cladding material 9a with a first refractive index, and one or more cores 9b with a material having a second refractive index. The one or more cores 9b can then be aligned with the pores 6a of the membrane 6 above the optical window 9. As an alternative, the optical waveguide assembly with cladding material 9a and cores 9b is positioned on top of optical window 9, i.e. within the bottom channel 4a of the microfluidic device 1 .

E.g. in case of one core 9b, the size and position can be aligned with the circumference of the plurality of pores 6a of the membrane 6. As an alternative (shown in Fig. 5), the number of cores 9b is larger than the number of pores 6a, allowing the pores 6a in the (primary) membrane to be aligned with multiple cores 9b.

(Confocal) imaging of cell models on the membrane 6 through the bottom flow in flow channel 4a is difficult due to signal intensity loss as a result of scattering. The cause of scattering is the difference in refractive index (Rl) of the flow in the bottom flow channel 4a and the cell models 2 on top of the membrane 6. The optical window 9 as described in the previous paragraph will solve this problem, as light is able to travel through the one or more cores 9b more efficiently. As an example, the optical window 9 is provided as a plate or block of e.g. 1 cm x 1 cm (depending on the membrane 6 size) and 0.25mm height, made of agarose (first refractive index of about 1 .34) as cladding material 9a and PEG-based hydrogel as core material (second refractive index of about 1 .47). Such an optical window 9 can be prepared using a mold to pore the agarose material 9a to make the optical window 9 with slots for the smaller cores 9b, and when cooled the hydrogel material can be poured in these slots, and allowed to solidify.

In an exemplary embodiment, the insert 5 and membrane 6 have the following dimensions. Number of pores = 16; membrane dimensions 10x10 mm (square), thickness 10pm, radius of pores 6a = 150 pm; length of insert = 45 mm; width of insert = 15 mm. The further components have e.g. the following dimensions: bottom cover plater thickness = 0.7 mm; top cover plate 3 thickness = 1 .1 mm.

The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims.

Claims

1 . A microfluidic device for holding three dimensional cell structures (2), the microfluidic device (1) comprising

a top cover plate (3),

a bottom cover plate (4), and

an insert (5) having a membrane (6) provided in an aperture in the insert (5) and extending in a plane of the insert (5),

the insert (5) in operation being positioned between the top cover plate (3) and bottom cover plate (4) to form two flow channels (3a; 4a) on either side of the insert (5),

wherein the membrane (6) is provided with a plurality of pores (6a) for holding three dimensional cell structures, with a pore size of at least 50pm.

2. The microfluidic device according to claim 1 , wherein the membrane (6) has a thickness (tm) which is less than a thickness (t) of the insert (5), and a hydrogel material (7) is provided on the membrane (6).

3. The microfluidic device according to claim 1 or 2, wherein the pore size is at least 100pm, e.g. at least 250pm.

4. The microfluidic device according to any one of claims 1 -3, wherein the plurality of pores (6a) comprises an array of at least two by at least two pores (6a).

5. The microfluidic device according to any one of claims 1 -4, wherein the membrane (6) is positioned symmetrical in the insert (5).

6. The microfluidic device according to any one of claims 1 -5, wherein the membrane (6) is an integral part of the insert (5).

7. The microfluidic device according to any one of claims 1 -6, wherein the membrane (6) has a dimension of 10x10mm.

8. The microfluidic device according to any one of claims 1 -7, wherein the insert (5) further comprises a secondary membrane layer (6b) having a plurality of secondary pores with a smaller dimension than the pores (6a) of the primary membrane (6).

9. The microfluidic device according to any one of claims 1 -8, wherein the bottom cover plate (4) comprises a transparent material.

10. The microfluidic device according to claim 9, wherein the transparent material comprises glass, or an optically transparent polymer.

1 1 . The microfluidic device according to any one of claims 1 -10, wherein the bottom cover plate (4) has a thickness (tb) of less than 0.3mm, e.g. less than 0.2mm, e.g. 0.17mm.

12. The microfluidic device according to any one of claims 1 -1 1 , wherein the bottom cover plate (4) comprises an optical window (9).

13. The microfluidic device according to claim 12, wherein the optical window (9) has a thickness of less than 0.3mm, e.g. less than 0.2mm, e.g. 0.17mm.

14. The microfluidic device according to claim 12 or 13, wherein the optical window (9) comprises an optical waveguide assembly having cladding material (9a) with a first refractive index, and one or more cores (9b) with a material having a second refractive index, the one or more cores (9b) being aligned with the plurality of pores (6a) of the membrane (6).

15. An insert for use in a microfluidic device (1) according to any one of claims 1 -14.

16. A method of using a microfluidic device (1) according to any one of claims 1 -14, comprising feeding a first fluid in a first flow channel (3a) between the insert (5) and the top cover plate (3), feeding a second fluid in a second flow channel (4a) between the insert (5) and the bottom cover plate (4).

17. The method according to claim 16, further comprising adding an imaging agent to the second fluid.

18. The method according to claim 16 or 17, wherein the microfluidic device (1) is applied for a real-time imaging application.

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