WO2021181337A1

WO2021181337A1 - Microfluidic chip for flow-through culturing of bio-objects

Info

Publication number: WO2021181337A1
Application number: PCT/IB2021/052053
Authority: WO
Inventors: Michaela Liegertová; Jan Maly; Regina Herma; Jiri SMEJKAL; Petr Panuska; Zuzana NEJEDLA; Petr Aubrecht; Marcel STOFIK; Jaromir HAVLICA
Original assignee: Univerzita Jana Evangelisty Purkyne v Ústí nad Labem
Priority date: 2020-03-12
Filing date: 2021-03-11
Publication date: 2021-09-16
Also published as: CZ308789B6; CZ2020134A3

Abstract

The technical solution relates to a microfluidic chip for flow-through culturing of bio-objects and testing the effects of chemical substances on these objects, comprising a chip (1) with a culture space divided into an upper channel and a lower channel separated from each other by an immobilization partition with immobilization openings. Furthermore, the chip (1) is provided with flushing channels with inlet openings and an inlet port, an outlet port and a planting port, which are connected to the upper channel. At least one of the boundaries in the area above the upper channel and/or below the lower channel in the area of immobilization openings is made of a transparent material for an optical method for observing objects located in die area of the immobilization openings in the immobilization partition.

Description

Microfluidic chip for flow-through culturing of bio-objects Field of the Invention

[001] The invention relates to a microfluidic chip for flow-through culturing of bio objects State of the Art

[002] The presented technical solution is a microfluidic device designed primarily for in vitro and in vivo culturing of bio-objects in the flow-through regime, with the possibility of being used for testing biological effects of chemicals on these objects. Testing biological effects of chemicals is essential, for example, for determining the toxicity of substances to the environment, living organisms or human health, but may also be necessary for determining the properties of pharmacologically active substances in the field of biomedical applications.

[003] In this case, a bio-object means, for example, a three-dimensional (3D) organized cluster of cells, it can be, for example, a 3D cell culture in the form of an organoid, spheroid, etc., or the germ stage of a multi-cellular organism, such as fish egg - embryo. The size of bio-objects can range from several tens of micrometres to several units of millimetres.

[004] For culture tests of bio-objects and tests of the effect of biologically active substances on bio-objects, traditional technologies and procedures are employed using standardized multi-well plates, i.e. with 6, 12, 24, 96, 128, 384, 1536 wells, or, for example, approaches with cultures in micro-structured wells or in a ‘hanging drop’, but which are part of a culture system with a static culture regime. These culture methods are used for in vitro experiments with 2D and 3D cell cultures. The advantage of 3D cultures, for example, tumour spheroids, is that their behaviour and manifestations are very similar to in vivo tumour tissues and are therefore very suitable for testing, for example, the dynamics of tumour development, distribution and effect of drugs in tumour tissues, etc. In the case of in vivo tests of biologically active substances, their effect is verified mainly in rodents - mammalian models. Testing on mammalian models is very costly and is also technologically demanding, including high administrative demands. Therefore, suitable alternatives to other multi cellular organisms are currently being sought for testing purposes. In recent years, model fish, for example, have been increasingly used for various genetic studies, for studies in the field of chemical biology, for monitoring the effect of chemicals or for preclinical drug trials. One suitable model is a zebrafish ( Danio rerio). The advantage of testing on this fish model is that the embryos of this fish are available in large quantities, with minimal financial costs, and at the same developmental stage. Their development is relatively fast; within 72 hours, the larvae have well-developed internal organs, which allows monitoring of developmental changes and defects in a very short time. Embryos are small, and we work with them in an aquatic environment. Their advantage is transparency, which allows observation of all internal organs and recording their development by non-invasive microscopic techniques. The great advantage of this model is a very high genetic similarity to humans, about 70 per cent. Static conditions are usually used for different variants of cultures and tests of the mentioned bio-objects, i.e. without continuous medium exchange.

[005] One of the main disadvantages of experiments with bio-objects under static conditions is the need to perform many time-consuming steps, which must be performed manually. These include, for example, sorting objects into individual wells, regularly exchanging solutions in wells for fresh ones, finding the position of an object in wells when creating image documentation, etc. It is not always possible to use technologies providing high-resolution image recording. There is also a limitation in reducing the total volume of test substances, which can often be very expensive or available only in small quantities. When recording, in addition to the complex handling of samples, it is also necessary to fix them using other materials such as agarose or immobilise objects with the use of anaesthetics.

[006] New technologies that enable the development and production of multi- functional microdevices for automated sample handling and analysis have great potential for the elimination of the described negatives in tests with such small bio objects. In the field of bio-applications, these are microfluidic systems, for which the already established terms are used, such as the biological micro-electromechanical systems (from the English abbreviation BioMEMS) or also laboratories on a chip, the so-called Lab-On-Chips (LOC). These technologies are currently beginning to be used for the development of microdevices for working with bio-objects. During the development of these devices, the problems related to the reliable placement of a sufficient number of objects into the chip, correct flow around the objects by liquids distributed inside microfluidic channels, the possibility of handling with objects during experiments and related possibilities of parallelization and automation of the whole experiment and high-quality image recording.

Summary of the Invention [007] The above drawbacks are largely eliminated by a microfluidic chip for flow through culturing of bio-objects and testing the effects of chemicals on these objects according to the invention. The chip features a culture space divided into an upper channel and a lower channel; they are separated from each other by an immobilisation partition with immobilisation openings. The chip also has flushing channels with inlet openings and an inlet port, an outlet port and a planting port, which are connected to the upper channel; wherein at least one of the boundaries in the region above the upper channel and/or below the lower channel in the area of the immobilisation openings is made of a transparent material for an optical method for observing objects located in the region of the immobilisation openings in the immobilisation partition. [008] The number of immobilisation openings is preferably equal to the number of flushing channels. At the same time, the mouth of the flushing channels is located below the immobilisation openings. The inlet port is divided into two feeding channels, and a baffle plate is located at the outlet port.

[009] The upper channel preferably has a shape selected from the group consisting of a rectangle, a semi-circle and a semi-ellipse, the top and/or bottom edges being straight or rounded. The mouth of the planting port leads to the upper channel. Simultaneously it is located in front of the immobilisation openings of the chip. In a preferred embodiment, the cross-section of the lower channel shape is that of a horizontal letter L, wherein the edges of the lower channel are sharp or rounded. [010] The mouth of the flushing channels into the lower channel is at the lowest point of the lower channel, whereby the bottoms of the lower channel and the flushing channels are levelled.

[011] The present invention relates to a microfluidic chip intended in particular for the flow-through culturing of bio-objects with the possibility of being used for testing the biological effects of chemicals on these objects. A large number of objects can be planted in the chip, which number is limited by the length of the culture area of the chip and the size of the immobilisation well.

[012] The chip is characterised in that its culture area is oriented horizontally in the working position and also in that the cultivation area is divided into upper and lower channels. An immobilisation partition with immobilisation openings into which bio objects are immobilised, separates the two channels. The planting of objects uses a combination of gravitational force and the carrying force of the liquid in the upper channel. These forces act on the planted objects in the upper channel of the culture space so that it is possible to perform automated planting of objects into the immobilisation wells of the chip.

[013] The proposed design of the lower channel is characterised by a lateral extension so as to avoid unwanted hydrodynamic phenomena, such as an increased risk of washing out immobilised objects in front part of the chip.

[014] Furthermore, the lower channel is also characterised in that it provides an even flow of liquid around the objects and ensures rapid metabolism in close proximity to the objects in almost the entire area of their contact with the newly inflowing liquid. At the same time, it guarantees fast flushing of substances excreted by objects into the surroundings.

[015] The designed shape of the chip allows quick liquid exchange in the entire volume of the chip even at low flow rates in a relatively short time. This property is necessary, for example, when the medium needs to be replaced with the test substance.

[016] The proposed chip design is characterised in that the immobilised objects can be selectively, i.e. any immobilised object separately, washed out of the immobilisation wells at any time, even during the experiment, via flushing channels opening into the lower part of the lower channel in the culture area of the chip.

[017] The chip is also characterised in that when using the planting port, there is no jamming of objects below the planting port, as this port is not connected perpendicular to the upper channel, but the area of its mouth into the upper channel forms an angle of less than 90 degrees with the upper channel. [018] The presented technical solution can be realized either by 3D printing technology, wherein the complex internal structure of the chip is made as a whole or by other technologies enabling layering of individual micro-structured sheets of materials on each other by various joining techniques, such as glueing, pressing, etc., into one unit to produce the desired shape of the inner channels. The condition is the use of biocompatible materials. The individual layers of the chip can also be made by etching techniques, pressing or casting.

[019] The advantage of the present solution is that the chip can be made by putting together several layers of different materials, wherein the lower layer of the lower channel or upper layer of the upper channel can be made of both polymeric material and special glass suitable for high-resolution microscopy, or other material suitable for optical observation of immobilised objects.

[020] The advantage of the proposed solution is the possibility of its production using 3D printing, or by combining 3D printing with other production processes, which brings the opportunity of cheap and fast production of fully functional systems that can quickly adapt the system parameters, such as the size of wells, channels and other microchip elements, according to the type and parameters of the selected bio-object.

[021] The advantage of the solution is the possibility of reusing the chip because the immobilised objects can be easily washed out of the chip.

[022] Another advantage is the possible adjustment of the hydrodynamic properties of the liquid flow in the upper and lower channels by changing the size of the lateral extension, which allows influencing in a controlled way the parameters of flowing around the bio-object by the test substances in the well.

[023] The advantage of the described solution over other known solutions is also the possibility of selective removal of a selected bio-object via the flushing channel without interrupting the culture experiment and thus the possibility of its subsequent analysis by other methods, for example genetic analysis, enzyme activity analysis, chemical composition analysis, etc., outside the culture chip itself. Thus, this procedure allows the carrying out of long-term dynamic experiments with continuous image recording of bio-object development, e.g. using an inverted microscope, confocal microscope, so-called light-sheet microscopy, etc., and correlate them with the results obtained by other analytical techniques, for which it is necessary to remove the bio-object from the system in advance, e.g. for extraction, homogenization, isolation of nucleic acids, proteins or other cellular components. [024] Individual bio-objects in the same chip exposed to the same test substance can be removed from the well at different time intervals during the long-term experiment and subsequently analysed by other techniques as seen above. In one culture experiment, a comprehensive time frame of the effect of the studied substance on a bio-object can be obtained by a combination of imaging techniques and other analytical techniques. At the same time, compared to known solutions, this can significantly speed up the testing of the effects of substances in biological models using a wider range of complementary techniques.

Brief Description of Drawings [025] The microfluidic chip for flow-through culturing of bio-objects according to the invention will be described in more detail on a specific exemplary embodiment with the aid of the accompanying drawings, in which:

[026] Figure 1 shows a complete microfluidic chip with a module for connecting hoses to the flushing channels of the chip, with a laminating adhesive and a transparent film in an exploded view.

[027] Figure 2 shows the complete microfluidic chip in the folded state.

[028] Figure 3a shows a top view and Figure 3b bottom view of the microfluidic chip.

[029] Figure 4a shows a side view of the chip - section in AA plane in Fig. 3; Figure 4b shows a rear view of the chip - section in BB plane in Fig. 3; and Figure 4c shows a detail of the arrangement and shape of the two channels in the area of the immobilisation opening - area C of the section in BB plane BB.

[030] Figure 5 shows a view of the total volume of the upper and lower channels of the microfluidic chip and a top view of the upper and lower channels. [031] Figure 6 shows a detail of the front area of the channels with the main and the planting ports - area D in Fig. 5.

[032] Figure 7 shows a detail of the rear area of the channels with the outlet port - area E in Fig. 5.

[033] Figure 8 shows a detail of the arrangement of the upper and lower channels in the area of the immobilisation opening - section in FF plane in Fig. 5. [034] Figure 9 shows a detail of the arrangement of the upper and lower channels in the area of the immobilisation partition - section in FF plane in Fig. 5.

[035] Figure 10 shows a view of the total volume of the upper, lower and all flushing channels of the microfluidic chip. [036] Figure 11 shows a detail of the front area of the channels with bio-objects planted into the immobilisation wells, i.e. area I in Fig. 10.

[037] Figure 12 shows a view of the total volume of all flushing channels with a precisely given position and display of planted bio-objects in the immobilization openings. Examples of Embodiments of the Invention

[038] Experiments with the designed chip can be carried out in the mode of continuous or sequential timed flow of liquid around the object. The designed chip enables parallelized experiments with automated real-time recording and with the possibility of making a large number of high-resolution image recordings. [039] For observing the cultured bio-objects with a microscope or binocular magnifying glass, it is advisable to fix the chip in a suitable holder. Hoses connect to the ports of the microfluidic chip. The chip fills with liquid. Then the bio-objects are sucked into the external planting hose with a suitable spacing. The external hose connects to the planting port. A defined flow of liquid to the chip inlet port starts, and the defined flow of liquid in the external hose introduces bio-objects from the planting hose into the upper channel of the chip. The liquid flow in the upper channel and the gravity cause the bio-objects to move in the direction of the liquid flow of the area, and the objects above the immobilisation well are immobilised into the immobilisation well. By repeating this process, we achieve that all immobilisation wells are filled with bio-objects. After immobilising the objects, a culture experiment can take place.

The purpose of the culture experiment may be, for example, to test the effects of chemicals on bio-objects. By starting the defined flow of liquid in the direction from a particular flushing channel to the lower channel of the chip in a particular mouth of the flushing channel below the immobilisation well, the immobilised object is washed out of the immobilisation well. The subsequent short-term increase in the flow of liquid in the upper channel will wash out the object from the chip. This washout can be carried out at any time during the experiment.

[040] An example of the technical solution can be a microfluidic chip for culturing Danio rerio embryos, produced by 3D printing technology. The microfluidic chip 1 is manufactured using DLP 3D printing technology. The complete culture chip has 4 parts: i) the chip \ itself - the body of the chip; ii) the module for connecting the hoses to the flushing channels J_8 of the chip; iii) the biocompatible transfer laminating adhesive 7; iv) the polymeric film 9.

[041] The chip \ is characterised in that the laminating adhesive 7 is applied to its lower part, to which a polymeric film 9 with precisely given dimensions adheres. The film 9 forms the lower part of the lower channel T7 and flushing channels 18 and allows optical observation, for example with an inverted microscope, and making image recordings of the planted and immobilised embryos 25 in the chip T The module for connecting hoses to the flushing channels 18 is attached to the upper part of the chip 1 with 8 M3 screws. On its outer upper side, the chip 1 has one inlet port 10 one planting port 14, and one outlet port 12, inlet openings to flushing channels 3 and threaded openings 2 for attaching the module for connecting hoses to the flushing holes with 8 screws. On its outer lower side, the chip 1 has openings which are part of the lower channel G7 and flushing channels 18. The inner part of the chip 1 is characterised in that it has an upper channel 16 and a lower channel G7 both channels are separated from each other by an immobilisation partition 20 with a thickness of 0.6 mm. The immobilisation partition 20 is characterised in that it has 24 immobilisation openings 21; these openings 21 are located between the planting port 14 and the outlet port 12. The entry of liquid into the upper channel 16 and the lower channel T7 is ensured via an inlet port K), which is divided in the chip 1 into two feeding channels 172 with the same cross-sectional area, the feeding channels 172 having a width of 1.0 mm and a height of 0.7 mm. The feeding channels 172 open into the upper channel 16 and the lower channel T7 of the chip 1. The drainage of liquid from the chip 1 is ensured by connection of the upper channel 16 and the lower channel T7 in the rear part of the chip 1 below the outlet port 12. In this connection, there is a baffle plate 23, which prevents the washed out embryo from falling out of the chip to the bottom of the lower channel G7 in this connection. This design of the connection ensures a reliable removal of embryos from the chip 1 via the outlet port 12. In front of the immobilisation openings 21 in the upper channel 16, there is the mouth of the planting port 14, which has a bevel of 45 degrees. The flushing channels 18 open perpendicularly into the lower channel G7 on one side, and on the other side there is a lateral extension 17 1 of the lower channel T7, wherein the lower channel T7 with its lateral extension 17 1 has a horizontal L-shaped cross-section. The lower channel 17 along the immobilisation partition 20 is 0.65 mm high and 2.15 mm high in the area of the lateral extension 17 1. The upper edges of the upper channel 16 and the lateral extension of the lower channel 17 1 have rounded edges.

Industrial Applicability [042] The proposed chip is intended for use in areas where carrying out of parallelised culturing of bio-objects or, for example, a large number of tests of toxicity or other types of effects of biologically active substances on bio-objects are required. These tests are performed by companies and workplaces that focus on environmental and health protection or the development of pharmacologically active substances or other workplaces with an experimental focus. The advantage of the proposed solution is the possibility of automation and parallelisation of a large number of experiments with an automated collection of image documentation.

Reference signs list:

1 - Microfluidic chip 2 - Threaded openings

3 - Inlet openings to flushing channels

4 - Module for connecting hoses to the flushing channels of the chip

5 - Inlet ports of the module for connecting hoses to the flushing channels of the chip

6 - Holes for attaching the module for connecting hoses to the chip with screws 7 - Laminating adhesive

8 - Holes in the laminating adhesive

9 - Polymeric film

10 - Inlet port 11 - Mouth of the inlet port to the channels

12 - Outlet port

13 - Mouth of the outlet port to the channels

14 - Planting port 15 - Mouth of the planting port to the upper channel

16 - Upper channel

17 - Lower channel

17.1 - Lateral extension of the lower channel 17.2 - Feeding channel 18 - Flushing channel

19 - Mouth of the flushing channel to the lower channel

20 - Immobilisation partition

21 - Immobilisation opening

22 - Dividing inlet to the upper and lower channels 23 - Baffle plate

24 - Rear connection of upper and lower channels

25 - Bio-object (for example, a fish embryo)

Claims

C L A I M S

1. A microfluidic chip for flow-through culturing of bio-objects and testing the effects of chemical substances on these objects, characterized in that it comprises a chip (1) which has a culture space divided into an upper channel (16) and a lower channel (17) separated from each other by an immobilisation partition (20) with immobilisation openings (21); further the chip (1) is provided with flushing channels (18) with inlet openings (3) and an inlet port (10), an outlet port (12) and a planting port (14) which are connected to the upper channel (16), wherein at least one of the boundaries in the area above the upper channel (16) and/or below the lower channel (17) in the area of the immobilisation openings (21) is made of a transparent material for the optical method for observation of objects located in the area of immobilisation openings (21) in the immobilisation partition (20).

2. The microfluidic chip according to claim 1, wherein the number of immobilisation openings (21) is equal to the number of flushing channels (18) and at the same time the mouth of the flushing channels (19) is located below the immobilisation openings (21).

3. The microfluidic chip according to claim 1 or 2, wherein the inlet port (10) is divided into two feeding channels (17.2).

4. The microfluidic chip according to any one of claims 1 to 3, wherein a baffle plate (23) is arranged at the outlet port (12).

5. The microfluidic chip according to any one of claims 1 to 4, wherein the upper channel (16) has a shape selected from the group consisting of a rectangle, a semi circle and a semi-ellipse, wherein the upper and/or lower edges are straight or rounded and the mouth (15) of the planting port (14) leads to the upper channel (16) and at the same time it is located in front of the immobilisation openings (21) of the chip (1).

6. The microfluidic chip according to any one of claims 1 to 5, wherein the cross-section of the lower channel (17) has the shape of a horizontal letter L, wherein the edges of the lower channel (17) are sharp or rounded.

7. The microfluidic chip according to any one of claims 1 to 6, wherein the mouth (19) of the flushing channels (18) into the lower channel (16) is at the lowest point of the lower channel (16), whereby the bottoms of the lower channel (17) and the flushing channels (18) are levelled.