CZ308789B6 - Microfluidic chip for flow-through cultivation of biological objects - Google Patents

Abstract

The invention relates to a microfluidic chip for flow-through cultivation of biological objects and testing the effects of chemicals on these objects, comprising a chip (1) provided with a culture space divided into an upper channel and a lower channel which are separated from each other by an immobilization barrier with immobilization holes. Furthermore, the chip (1) is provided with flushing channels with inlet openings and an inlet port, an outlet port and an ejection 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 region of the immobilization openings is made of a transparent material for an optical method for observing objects located in the region of the immobilization openings in the immobilization barrier.

Description

Microfluidic chip for flow-through cultivation of biological objects

Field of technology

The invention relates to a microfluidic chip for flow-through cultivation of biological objects.

Prior art

The presented technical solution is a microfluidic device designed mainly for in vitro and in vivo cultivation of biological objects in the flow regime, with the possibility of use for testing the biological effects of chemicals on these objects. Testing the biological effect of chemicals is important, for example, for determining the toxicity of substances to the environment, for living organisms, for human health, but it can also be important for determining the properties of pharmacologically active substances in the field of biomedical applications.

By biological object in this case is meant, 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 multicellular organism, for example fish eggs - embryos. The size of biological objects can range from several tens of micrometers to several units of millimeters.

For cultivation tests of biological objects and tests of the effect of biologically active substances on biological objects, traditional technologies and procedures using standardized multi-well plates, ie 6, 12, 24, 96, 128, 384, 1536 wells, or for example approaches with cultures in microstructured wells or in a "hanging drop", which are, however, 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 tumor spheroids, is that their behavior and manifestations are very similar to tumor tissues in vivo, and are therefore very suitable for testing, for example, tumor development dynamics, drug distribution and effect in tumor tissues, etc. 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 requirements. Therefore, suitable alternatives to other multicellular 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 tests of medicines. One suitable model is a fish of the genus 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 stage of development. 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 work with 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%. Static conditions are usually used for different variants of cultures and tests of the mentioned biological objects, ie without continuous medium change.

One of the main disadvantages of experiments with biological 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 acquiring 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. In addition, when making records is in addition to complex manipulation

-1 CZ 308789 B6 samples need to be fixed with other materials, such as agarose, or. it is necessary to use the immobilization of objects with the use of anesthetics.

New technologies that enable the development and production of multifunctional micro-devices for automated sample handling and analysis have great potential for the elimination of the described negatives in tests with such small biological objects. In the field of bioapplications, these are microfluidic systems, for which the already established terms are used, 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 micro-devices for working with biological objects. During the development of these devices, the problems related to the reliable placement of a sufficient number of objects in the chip, the correct flow of objects by liquids distributed inside microfluidic channels, the possibility of manipulating objects during experiments and related possibilities of parallelization and automation of the whole experiment and high-quality image recording are solved.

The essence of the invention

The above drawbacks are largely eliminated by a microfluidic chip for the flow-through cultivation of biological objects and the testing of the effects of chemicals on these objects according to the invention. Its essence is that it contains a chip which is provided with a culture space divided into an upper channel and a lower channel, which are separated from each other by an immobilization partition with immobilization holes. Furthermore, the chip is provided with flushing channels with inlet openings and an inlet port, an outlet port and an outlet 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 region of the immobilization openings of the transparent material. for an optical method for observing objects located in the region of the immobilization holes in the immobilization barrier.

The number of immobilization openings is preferably equal to the number of flushing channels and at the same time the mouth of the flushing channels is located below the immobilization openings. The inlet port is divided into two inlet channels and a capture barrier is located at the outlet port.

The upper channel preferably has a shape selected from the group consisting of a rectangle, a semicircle and a semi-ellipse, the top and / or bottom edges being straight and / or rounded and the mouth of the ejection port leading to the upper channel and being located in front of the chip immobilization holes. In a preferred embodiment, the cross section of the lower channel has the shape of a horizontal letter L, the edges of the lower channel being sharp and / or rounded.

The mouth of the washing channels into the lower channel is at the lowest point of the lower channel, whereby the bottoms of the lower channel and the washing channels are in one plane.

The present invention relates to a microfluidic chip intended in particular for the flow-through cultivation of biological objects with the possibility of testing the biological effects of chemical substances on these objects. It is possible to plant a large number of objects in the chip and their number is limited by the length of the cultivation area of the chip and the size of the immobilization well.

The chip is characterized in that its cultivation area is oriented horizontally in the working position and also in that the cultivation area is divided into an upper and a lower channel, the two channels being separated by an immobilization barrier with immobilization openings into which biological objects are immobilized. The placement of objects uses a combination of gravitational force and the entrainment force of the liquid in the upper channel. These 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 immobilization wells of the chip.

- 2 CZ 308789 B6

The proposed design of the lower channel is characterized by a lateral extension so as to avoid unwanted hydrodynamic phenomena, such as an increased risk of leaching of immobilized objects in front of the chip.

Furthermore, the lower channel is also characterized in that it provides an even flow of liquid around the objects and ensures a rapid metabolism in close proximity to the objects in almost the entire area of their contact area with the newly flowing liquid and at the same time guarantees fast flushing of substances excreted by the objects.

The designed shape of the chip allows fast fluid 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.

The proposed chip design is characterized in that the immobilized objects can be selectively washed out of the immobilization wells at any time, even during the experiment, by means of flushing channels opening into the lower part of the lower channel in the culture area of the chip.

The chip is also characterized in that when using the drop-off port, no objects are jammed below the drop-off port, since 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 ° with the upper channel.

The presented technical solution can be realized either by 3D printing technology, while the complex internal structure of the chip is made as a whole, or by other technologies enabling layering of individual microstructured sheets of materials on each other by various joining techniques, such as gluing, pressing, etc., into one whole so that the required shape of the inner channels was made. The condition is the use of biocompatible materials. The individual layers of the chip can also be produced by etching techniques, pressing or also by casting.

The advantage of the present solution is that the chip can be made by composing several layers of different materials, while the lower layer of the lower channel or the 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 immobilized objects. .

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 possibility of cheap and fast production of fully functional systems with the possibility of quick adaptation of system parameters, such as the size of wells, channels and other microchip elements, according to the type and parameters of the selected biological object.

The advantage of the solution is the possibility of reusing the chip, because the immobilized objects can be easily washed out of the chip.

Another advantage is the possible adjustment of the hydrodynamic properties of the fluid flow in the upper and lower channels by changing the size of the lateral extension, which allows controlled control of the parameters of the flow around the biological object in the well by the test substances.

The advantage of the described solution over other known solutions is also the possibility of selective removal of a selected biological object by means of a washing channel without interrupting the culture experiment and thus the possibility of its subsequent analysis by other methods, eg genetic analysis, enzyme activity analysis, chemical composition analysis, etc. cultivation chip. Thus, this procedure allows to perform long-term dynamic experiments with continuous image recording of biological object development, eg using inverted microscope, confocal microscope, so-called light sheet microscopy, etc., and correlate them with results obtained by other analytical techniques requiring advance of biological object. that

-3GB 308789 B6 system, eg for extraction, homogenization, isolation of nucleic acids, proteins or other cellular components.

Individual biological objects exposed in the same chip, ie exposed to the same test substance, can be removed from the well at different time intervals during a long-term experiment and subsequently analyzed by other techniques, see above. In one culture experiment, a complex time frame of the effect of the studied substance on a biological 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.

Explanation of drawings

The microfluidic chip for flow-through cultivation of biological 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:

Giant. 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 foil in an exploded view.

Giant. 2 shows the complete microfluidic chip in the folded state.

Giant. 3a shows a top view and FIG. 3b a bottom view of a microfluidic chip.

Giant. Fig. 4a shows a side view of the chip - section in the plane AA in Fig. 3, Fig. 4b rear view of the chip - section in the plane BB in Fig. 3 and Fig. 4c detail of the arrangement and shape of both channels in the immobilization hole area C section in the BB plane.

Giant. 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.

Giant. 6 shows a detail of the front area of the channels with the main and the discharge port - area D in FIG. 5.

Giant. 7 shows a detail of the rear area of the channels with the outlet port - area E in FIG. 5.

Giant. 8 shows a detail of the arrangement of the upper and lower channels in the region of the immobilization opening - section in the plane FF in FIG. 5.

Giant. 9 shows a detail of the arrangement of the upper and lower channels in the region of the immobilization partition - section in the plane GG in FIG. 5.

Giant. 10 shows a view of the total volume of the upper, lower and all flushing channels of the microfluidic chip.

Giant. 11 shows a detail of the front area of the channels with biological objects planted into the immobilization wells, i.e. area I in FIG. 10.

Giant. 12 shows a view of the total volume of all flushing channels with a precisely given position and representation of planted biological objects in the immobilization openings.

-4GB 308789 B6

Examples of embodiments of the invention

Experiments with the designed chip can be performed 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 capturing a large number of high-resolution image recordings.

To observe cultured biological objects with a microscope or binocular magnifying glass, it is advisable to fix the chip in a suitable holder. Tubes are connected to the microfluidic chip ports. The chip is filled with liquid. Biological objects with a suitable spacing are sucked into the external settling tube. The external hose is connected to the drop-off port. The defined fluid flow to the chip inlet port is started and the biological fluid flow from the external tubing introduces biological objects from the discharge tube into the chip's upper channel. The fluid flow in the upper channel and gravity cause the biological objects to move in the direction of the fluid flow in the area, and the objects will be immobilized in the immobilization well above the immobilization well. By repeating this process, we achieve the filling of all immobilization wells with biological objects. After immobilizing the objects, a cultivation experiment can take place. The purpose of a culture experiment may be, for example, to test the effects of chemicals on biological objects. By triggering a 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 immobilization well, the immobilized object is washed out of the immobilization well. The subsequent short-term increase in the flow of liquid in the upper channel will wash away the object from the chip. This washout can be performed at any time during the experiment.

An example of a technical solution can be a microfluidic chip designed for culturing fish embryos of the genus Danio rerio 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 1 itself - the body of the chip, ii) the module for connecting the hoses to the flushing channels 18 of the chip, iii) the biocompatible transfer laminating adhesive 7, iv) the polymer foil 9.

The chip 1 is characterized in that a laminating adhesive 7 is applied to its lower part, to which a polymeric film 9 with precisely given dimensions is adhered. The foil 9 forms the lower part of the lower channel 17 and the flushing channels 18 and allows optical observation with, for example, an inverted microscope and image recording of planted and immobilized embryos 25 in chip 1. The hose connection module 18 is attached to chip 1 by eight M3 screws to upper part of the chip 1. The chip 1 has on its outer upper side one inlet port 10. one discharge port 14 and one outlet port 12. inlet openings to the flushing channels 3 and threaded holes 2 for attaching the module for connecting hoses to the flushing holes by means of eight screws. On the lower outer side, the chip 1 has openings which are part of the lower channel 17 and the flushing channels 18. The inner part of the chip 1 is characterized in that it has an upper channel 16 and a lower channel 17, the two channels being separated from each other by an immobilization partition 20 of thickness. 0.6 mm. The immobilization baffle 20 is characterized in that it has twenty-four immobilization openings 21, these openings 21 being located between the discharge port 14 and the outlet port 12. The liquid inlet to the upper channel 16 and the lower channel 17 is provided by an inlet port 10 which is in chip 1 is divided into two supply channels 17.2, with the same cross-sectional area, the supply channels 17.2 having a width of 1.0 mm and a height of 0.7 mm. The supply channels 17,2 open into the upper channel 16 and the lower channel 17 of the chip E. The drainage of liquid from the chip 1 is ensured by a connection of the upper channel 16 and the lower channel 17 in the rear part of the chip 1 below the outlet port 12. which prevents the flushed embryo from falling from the chip to the bottom of the lower channel 17 in this joint. This design of the connection ensures a reliable removal of embryos from the chip 1 through the outlet port 12. In front of the immobilization openings 21, in the upper channel 16, there is the mouth of the insertion port 14, which has a chamfer of 45 °. The side channels 17.1 of the lower channel 17 are located perpendicularly into the lower channel 17 on one side and on the other side. The lower channel 17 with its side extension 17.1 has an L-shaped cross-section. The lower channel 17 along the immobilization channel the partition 20 is 0.65 mm high and in the area of the lateral extension

-5GB 308789 B6

17.1 is 2.15 mm high. The upper edges of the upper channel 16 and the lateral extensions of the lower channel 17.1 have a rounding at their edges.

Industrial applicability

The proposed chip is intended for use in areas where parallel cultivation of biological objects is required or, for example, a large number of toxicity tests or other types of effects of biologically active substances on biological objects are required. These tests are performed by companies and their 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 parallelization of a large number of experiments with automated collection of image documentation.

Claims (7)

Microfluidic chip for flow-through cultivation of biological objects and testing the effects of chemical substances on these objects, characterized in that it comprises a chip (1) which is provided with a cultivation space divided into an upper channel (16) and a lower channel (17). which are separated from each other by an immobilization partition (20) with immobilization openings (21) and further the chip (1) is provided with flushing channels (18) with inlet openings (3) and inlet port (10), outlet port (12) and drop-off port (14) which are connected to the upper channel (16), wherein at least one of the boundaries in the region above the upper channel (16) and / or below the lower channel (17) in the region of the immobilization openings (21) is made of a transparent material for the optical method. for observing objects located in the area of the immobilization openings (21) in the immobilization partition (20). Microfluidic chip according to Claim 1, characterized in that the number of immobilization 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 immobilization openings (21). Microfluidic chip according to Claim 1 or 2, characterized in that the input port (10) is divided into two supply channels (17.2). Microfluidic chip according to any one of claims 1 to 3, characterized in that a catch baffle (23) is arranged at the outlet port (12). Microfluidic chip according to any one of claims 1 to 4, characterized in that the upper channel (16) has a shape selected from the group consisting of a rectangle, a semicircle and a semi-ellipse, the upper and / or lower edges being straight and / or rounded and the mouth (15) ) of the discharging port (14) is led into the upper channel (16) and at the same time is located in front of the immobilization holes (21) of the chip (1). Microfluidic chip according to any one of claims 1 to 5, characterized in that the cross section of the lower channel (17) has the shape of a lying letter L, the edges of the lower channel (17) being sharp and / or rounded. Microfluidic chip according to any one of claims 1 to 6, characterized in that the mouth (19) of the flushing channels (18) into the lower channel (17) is at the lowest point of the lower channel (17), whereby the bottoms of the lower channel (17) are ) and flushing channels (18) in one plane.