WO2018106132A1

WO2018106132A1 - Microfluidic device for cell culture in gradient of bioactive substance

Info

Publication number: WO2018106132A1
Application number: PCT/PL2017/000122
Authority: WO
Inventors: Roman SZAFRAN; Kazimierz GĄSIOROWSKI; Katarzyna GĘBCZAK; Benita WIATRAK
Original assignee: Politechnika Wrocławska; Uniwersytet Medyczny Im. Piastów Śląskich
Priority date: 2016-12-09
Filing date: 2017-12-08
Publication date: 2018-06-14
Also published as: PL237365B1; PL419754A1

Abstract

A multilayer microfluidic device for cell culture in the gradient of a bioactive substance characterised by three layers and two protective cups (3) of tanks, the top layer being a cover (6), which contains tanks of fluids feeding the culture chambers (1), reference points (4) and a millimetre graduation (5); in the middle functional layer (7), made of adhesive film, there is a set of channels serving as culture and indicator chambers (2) with semi-circular ends (8), at least one of which is intended for cell culture in concentration gradient of the active substance (culture chamber), one for cell culture without an active agent (blind test) and one to develop a concentration profile of the indicator substance (indicator chamber); and the bottom layer is the base (9).

Description

Microfluidic device for cell culture

in gradient of bioactive substance

The subject of the invention is a microfluidic device (biochip) for conducting cell culture in a stationary system, in a continuous gradient of the active substance and conducting research on biological processes occurring in its gradient. The device is used in biology, medicine, pharmacy, biotechnology and biomedical engineering. The construction of the biochip enables simultaneous control cultivation (so-called blind test) and cultivation in the stable gradient of the active substance with a very low concentration and continuous indirect detection of this concentration due to the use of the indicator chamber. In particular, the device enables the creation of microenvironment suitable for basic examinations: proliferation, growth and differentiation of cells, immune response, damage treatment, embryogenesis and neoplastic metastasis, i.e. phenomena dependent on molecular gradients, necessary for the mapping and regulation of cell signalling routes characteristic for these processes. These gradients in the device are created in a simple, quick and reproducible way thanks to the use of the phenomenon of the advective transport of mass, and cultures and research are conducted in stationary (non-flow) conditions, so that the natural secretion of biochemical factors by the cells remains undisturbed.

In the human body, gradients of biomolecular concentrations are regulated and perform control tasks for many basic cell functions. Biomolecules include, among others, lipids, glycolipids, sterols, vitamins, hormones, neurotransmitters and metabolites. In order to understand the effect of chemical stimuli on cellular signal pathways on cellular biology, methods of mapping the microenvironmental conditions in living organisms under artificial conditions are being sought. For this purpose, the Boyden, Dunn or Zigmond chambers are used, and recently cultures are also carried out on agarose mediums on Petri dishes, where gradients of concentration are produced by microaspirators. Due to their size, standard solutions for cell culture in gradient concentrations do not allow for the examination of phenomena occurring at the cellular level and in their immediate surroundings. For example, the diameters of most cells are in the range of 1-100 micrometres, and the chemical intercellular signals emitted by them (e.g. cytokines and chemokines) cover distances of 250 micrometres. Compared to the sizes of typical culture chambers, which range from a few millimetres to several centimetres, microfluidic systems allow for greater accuracy and better control of the generated gradient, thanks to cellular scale dimensions (e.g. height/width of the channel), ranging from a few to several hundred micrometres. This in turn allows us to carry out research into phenomena occurring at the cellular level. Additionally, the microfluidic systems operate on small volumes of liquid, of microliter magnitude, which significantly reduces testing costs. (Alicia G. G. Toh, Z.Nam-Trung Nguyen, P. Wang Chun Yang; Engineering microfluidic concentration gradient generators for biological applications, Microfluid Nanofluid, 2014, 16:1-18). Microfluidic gradient generators have already been successfully used to conduct research into the development of new drugs, chemotaxis, the influence of biochemical factors on cell survival and stem cell differentiation.

There are two basic types of microfluidic gradient generators: static and dynamic. Dynamic systems are characterized by a continuous fluid flow within the culture chamber and the concentration gradient is produced by an advective mixing of fluids at different concentrations. Devices of this type are used for investigating phenomena in the presence of shear stress and are used for cells that are natively exposed to such strain and require the use of cells from adherent lines. In addition, they require devices forcing a continuous and stable fluid flow, which interferes with the concentration profiles of the substances released by the cells. American patent application no. US20020113095 Al describes a microchip consisting of a system of channels to generate dilutions of the desired concentration and concentration gradients. The flow device consisted of a number of connected channels and mixers, thanks to which it was possible to obtain a number of streams of a given concentration. Their parallel flow through the outlet channel generated a discontinuous transverse gradient of concentration in the culture chamber. In another American patent no. US8216526 (B2) on the other hand, a microfluidic device with a centrally positioned round culture chamber, tangential on the edges with the flowing channel system, was described. The active substance flowing through the chamber, in its short section, produced a gradient of concentration inside the chamber, which was the effect of diffusional-advective mass transport. The diffusion-advective mechanism of mass transport allowed for quick formation and control of continuous gradients of concentrations; however, the construction of the device required very precise control of pressures on inlets and outlets from flow channels.

Static microfluidic gradient generators utilize a diffusive mass transport mechanism, which is a few magnitudes slower mechanism than the advective mechanism. Therefore, the time of establishing a gradient is long and frequently requires the introduction of cells only after the gradient is determined, which is technically difficult to implement. Static generators make it possible to conduct tests with non-adherent cells and without shear stress affecting the body of the cell, as well as to preserve the concentration profiles of naturally released substances. However, these solutions often require membranes and hydrogels to be used to stabilise the concentration profile and, due to the long time of concentration establishment, the culture chambers are short and generate proportionally large concentration gradients, resulting in low resolution of the test results. Patent no. US8377685 (B2) describes a microfluidic device for the examination of chemotaxis in a stable gradient of active substance. The device consisted of two reservoirs connected with a channel in which cell migration to the chemotaxis activator took place. The gradient of the active agent in the channel connecting the tanks was obtained by the process of diffusion of the active substance towards the cell tray. On the other hand, patent application no. US20110003372 (Al) describes a microfluidic device for testing chemical and biological processes in a concentration gradient of the active substance, which consists of containers connected by channels running parallel in the working section, which are crosswise connected by connectors. The concentration gradients are generated in short transverse connectors due to active substance diffusion occurring between parallel channels. Similarly, patent application no. US20070253868 (Al) reveals a microfluidic device to generate a gradient of particle concentration, which is constructed of a single channel with inlet and outlet, and porous diaphragms at both ends of the channel, permeable to particles that stabilized the gradient concentration. European patent application no. EP1741487 (Al) describes a microfluidic device with an observation field between at least two chambers and a diffusive gradient of the active substance is generated within the field of observation. Another American patent no. US8449837 (B2) describes a technically complex microfluidic device for researching phenomena in biological systems in concentration gradients of active substance. The device is equipped with a number of channels and supply tanks and two parallel flow- through channels connected with transverse connection channels, and diffusional gradients of active substance are produced within the connection channels in various dilution ranges. German patent no. DE102014109468 (B3) describes a multi-layer microfluidic device for cell culture in a concentration gradient of the active substance, which consists of three channels arranged one above the other, separated by a porous membrane through which the active component of the mixture diffuses. In the middle channel, which is a culture chamber, a transverse diffusion gradient of the active substance concentration is produced. International patent application no. WO2015032900 (Al) describes a microfluidic device equipped with a single polygon-shaped culture chamber. Inside the culture chamber, a concentration gradient of active substance was created by conducting channels parallel to the sides of the polygon through which the active substance was flowing, and which only came into contact with the chamber through a semi-permeable membrane, so that a diffusion gradient of active substance was created inside the chamber. On the other hand, patent application no. US20130171682 (Al) concerns a multiple-inlet microfluidic device allowing for parallel cultures, their analysis and observation of growth and cell migration. Polish patent applications no. P415008 (Al) and P415010 (Al) describe flow equipment for nerve cell culture, which enables the generation of damage to neurons network. However, these devices do not enable testing in the concentration gradient of active substance.

None of the known design solutions of microchips for cell culture in the concentration gradient of active substance allows for fast creation of concentration gradient of active substance as a result of fluid advective movements in the culture chamber and conducting culture in stationary conditions, without shear stress and without disturbing the profile of biochemical agents subject to natural secretion from cells. The solutions known from the scientific literature do not enable conducting many simultaneous cell cultures in concentration gradients of factors and without their participation (so-called blind test) in one device, nor the continuous, indirect detection of gradient of a medium of ultra-low concentration, whose direct measurement is not possible, and without direct contact of the indicator substance with cell culture.

As a result, there was a need for a new construction to address these problems.

The microfluidic device for cell culture in the gradient of a bioactive substance according to the invention consists of three layers and two protective cups of feed tanks, with the top layer consisting of a cover containing tanks of fluids feeding the culture chambers, reference points and a millimetre graduation; in the middle functional layer made of adhesive membrane, there is a set of channels serving as culture and indicator chambers, at least one of which is intended for cell culture in the concentration gradient of active substance (proper culture), one for conducting culture without active agent (so- called blind test) and one to develop the concentration profile of the indicator substance (indicator chamber); and the bottom layer is the base. Advantageously, the cover is made of PMMA methyl poly(methacrylate).

Advantageously, the functional layer is made of acrylic adhesive film.

Advantageously, the height of the functional layer is not smaller than the diameter of the cells planned for culture and testing.

Advantageously, the microchip chambers are elongated in shape and run parallel to the longer edges of the device.

Advantageously, microchip's chambers have semi-circular ends, matching in size to the diameter of the tanks.

Advantageously, each chamber at its ends is connected to the feed tank.

Advantageously, all chambers are exactly the same shape and dimensions.

Advantageously, all feed tanks are cylindrical in shape and have a diameter that is adapted to the diameter of the tip of the disposable syringe.

Advantageously, all feed tanks are of exactly the same shape and volume.

Advantageously, the volume of each container is at least equal to that of the chamber.

Advantageously, tanks are protected from above by means of protective covers.

Advantageously, the tank protective cups are connected to the lid in a separable and hydraulically tight way.

Advantageously, tank protective cups protrude beyond the edge of the micro equipment from 10 to 50% of their length.

Advantageously, the millimetre graduation runs along each chamber along the entire length of their working space, outside the chamber boundaries.

Advantageously, the reference points are in the form of equally distant circles with a diameter of not more than 0.1 mm set in one or more rows symmetrically along the axis of each chamber.

Advantageously, the base is made of sodium glass, borosilicate or methyl poly (methacrylate) PMMA.

Advantageously, the adhesive functional layer connects the cover to the base in an inseparable, durable and hydraulically tight way.

Advantageously, the surface properties of the culture chambers have been modified to ensure better adhesion of the cells to their surface and reduce the adsorption of active substances on their internal surface.

Advantageously, all layers are made of low auto-luminescence materials.

Advantageously, all layers are made of transparent materials for visible and ultraviolet lighting.

Advantageously, the layers are made of biocompatible materials.

The microfluidic device for cell culture in gradient of bioactive substance according to the invention enables stationary (non-flow) cell culture to be carried out without exposure of cells to stress associated with the occurrence of shear stress induced by flowing fluids and with the preservation of undamaged concentration profiles, subject to natural secretion of biochemical agents from cells; enables culture in the continuous gradient of the active substance and conducting research into biological processes occurring in its gradient, while the structure of biochip allows simultaneous control culture (so-called blind test) and at least one proper culture in a stable gradient of the active substance at a low concentration that cannot be detected directly and a continuous indirect detection of the indicator concentration by the spectrophotometric method without exposing the cells to the indicator substance.

The subject of the invention is presented more comprehensively in the following examples and in drawings:

Fig. 1 represents a three-chamber microfluidic device,

Fig. 2 represents the arrangement of layers in a three-chamber microfluidic device, Fig. 3 represents a four-chamber microfluidic device,

Fig. 4 shows the arrangement of layers in a four-chamber microfluidic device. Example 1.

Figure 1 and Figure 2 shows a three-chamber open microfluidic device with dimensions 25/75/5.13 mm (width/length/high) for single cell culture in the gradient of a bioactive substance. Fig. 1 shows the device in a general view with fluid reservoirs (1) feeding a system of three parallel chambers (2), two protective caps (3) of the tanks, with reference points (4), and a millimetre graduation (5). The microfluidic device consists of three layers and two protective covers for the tanks, the arrangement of which is shown in Fig. 2.

The covering layer (6) of the microchip was made of PMMA 3 mm thick. In this layer, six cylindrical tanks (1) were made at the level of inlets and outlets from microchip chambers, with a diameter of 4.3 mm matched to the diameter of microchip chambers and located at both ends of each chamber. In the axis of each chamber at equal distances of 1 mm, on the underside of the cover there are points of reference (4) in the form of 0.05 mm diameter dots, arranged in one row. A millimetre graduation scale (5) is placed along each chamber along the length of their working area, outside the working space of the chambers. The functional layer (7) is made of commercially available adhesive film based on an acrylic adhesive 0.13 mm thick. On the surface of this layer three longitudinal, symmetrically distributed, parallel chambers (2) were constructed, each 4.3 mm wide and 68.6 mm long, with the working area of each chamber being 50 mm long. Each chamber had a semi-circular end (8) of 4.3 mm in diameter, arranged so as to coincide with the edges of the tank openings placed concentrically with the centres of the semi-circular ends. The base (9) was made of a 2mm thick basic microscopic slide. The adhesive layer connects the lid to the base in an inseparable and hydraulically tight way. The internal surface of each chamber has modified surface properties that limit the adsorption of active substances on their internal surfaces and facilitate the immobilization of cells in the microdevice chambers. The upper surfaces of the feed tanks are protected by a rectangular 1 mm thick sections of self-adhesive polypropylene film with dimensions 25/20 (width/length) at each end of the device, forming covers (3) for feed tanks. The edge of the tank cover is 10 mm away from the edge of the microdevice and extends beyond its perimeter. Tank covers are connected to the lid in a separable and hydraulically tight way.

Example 2.

Fig. 3 and Fig. 4. present a four-chamber open-fluid microfiuidic device measuring 45/100/10.06 mm (width/length/height) for two parallel cell cultures in the gradient of the bioactive substance. Fig. 3 shows the device in a general view with fluid tanks (1) feeding the system of four parallelly running chambers (2), two protective caps (3) of tanks, reference points (4) and a millimetre graduation (5). The microfiuidic device consists of three layers and two casings of tanks, the arrangement of which is shown in Fig. 2.

The microchip was constructed in a similar way as shown in Example 1 , except that the top layer of the cover (6) was made of PMMA 5 mm thick. In this layer, eight cylindrical feed tanks with a diameter of 4.3 mm were made. The underside of the lid is equipped with reference points (4) in the form of dots 0.05 mm in diameter, arranged in two parallel rows 2 mm apart, symmetrically to the axis of each chamber at equal distances of 1 mm. The functional layer (7) was made of a commercially available 0.06 mm thick adhesive film. Four chambers, each 4.3 mm wide and 80 mm long, were constructed in this layer, with the working area of each chamber being 60 mm long. The base (9) is made of PMMA 5 mm thick. The upper surfaces of the feed tanks are protected by a rectangular section of 0.5 mm thick polypropylene self-adhesive foil with dimensions 45/25 (width/length). The edge of the tank cover is 5 mm away from the edge of the microchip and extends beyond its perimeter.

Claims

1. A multilayer microfluidic device for cell culture in the gradient of a bioactive substance wherein it consists of three layers and two protective caps (3) of tanks, the top layer being a cover (6), which contains fluid tanks (1) supplying the culture chambers, landmarks (4) and a millimetre graduation (5); in the middle functional layer (7) of the adhesive film there is a set of channels (2) acting as breeding and indicator chambers, with at least one of the channels (2) intended for cell culture in concentration gradients of the active substance (so-called culture chamber), one for cell culture without an active agent (blind test) and one for developing the concentration profile of the indicator substance (indicator chamber); and the bottom layer is the basis (9).

2. The device in accordance with claim 1, wherein the cover (6) is made of PMMA- (poly)methacrylate.

3. The device in accordance with claim 1 , wherein the functional layer (7) is made of an acrylic adhesive film.

4. The device in accordance with claim 1, wherein the height of the functional layer (7) is not smaller than the diameter of the cells planned for culture and testing.

5. The device in accordance with claim 1, wherein the channels (2) are elongated in shape and run parallel to the longer edges of the device.

6. The device in accordance with claim 1 and 5, wherein the channels (2) have semicircular ends (8) matching the diameter of the fluid tanks (1).

7. The device in accordance with claim 1, wherein each channel (2) at its ends is connected to the fluid tank ( 1 ) .

8. The device in accordance with claim 1 and 5 wherein all channels (2) are of exactly the same shape and size.

9. The device in accordance with claim 1, wherein all fluid tanks (1) have cylindrical shapes and diameters adapted to the diameter of the tip of the disposable syringe.

10. The device in accordance with claim 1, wherein all fluid tanks (1) are of exactly the same shape and volume.

11. The device in accordance with claim 1, wherein the volume of each fluid reservoir (1) is at least equal to the volume of the channel (2).

12. The device in accordance with claim 1, wherein the fluid tanks (1) are protected from above by protective cups (3).

13. The device in accordance with claim 1, wherein tanks protective cups (3) protrude beyond the edge of the microdevice from 10 to 50% of their length.

14. The device in accordance with claim 1, wherein the protective cups (3) of the feed tanks are connected to the cover (6) in a separable and hydraulically tight manner.

15. The device in accordance with claim 1, wherein a millimetre graduation (5) runs along each chamber along the entire length of their working spaces, except within the channel area (2).

16. The device in accordance with claim 1, wherein reference points (4) are in the form of equally distant circles with a diameter of not more than 0.1 mm arranged in at least one row symmetrically along the axis of each chamber.

17. The device in accordance with claim 1, wherein base (9) is made of sodium or borosilicate glass, and poly (methacrylate) or PMMA methyl.

18. The device in accordance with claim 1, wherein the adhesive functional layer (7) connects the lid (6) to the base (9) in an inseparable, durable and hydraulically tight way.

19. The device in accordance with claim 1, wherein surface properties of the channels (2) have been modified to ensure better adhesion of the cells to their surface and reduce the adsorption of active substances on their internal surface.

20. The device in accordance with claim 1, wherein all layers of the device are made of low auto-luminescence materials.

21. The device in accordance with claim 1, wherein all layers are made of transparent materials in the visible and ultraviolet range.

22. The device in accordance with claim 1, wherein the layers are made of biocompatible materials.