PL242812B1 - Magnetic microfluidic device for rapid screening tests and a method of conducting tests in a magnetic microfluidic device - Google Patents

Abstract

The subject of the invention is a magnetic microfluidic device for rapid screening consisting of a lid, a base, tank covers and a functional layer of the chip, and the lid of the chip contains supply tanks and a millimeter scale, and the functional layer of the chip contains culture chambers, characterized in that it consists of a microfluidic chip (1) transparent to visible light and UV, equipped with at least three linear culture chambers, each of which connects at the ends to tanks supplying a flexible base, and a magnetic microfield generator (1) consisting of a platform containing a chip socket, spacer supports, a generator base assembly consisting of a top and a bottom, containing a magnet socket, a permanent magnet and a series of ferromagnetic pins, the system of culture chambers in the microfluidic chip (2) and the system of ferromagnetic pins of the magnetic microfield generator (1) being mutually correlated in such a way that the pins ferromagnetic in the platform run parallel to each other from the surface of the chip culture chambers to the upper surface of the permanent magnet. The subject of the invention is also a method of conducting research in a magnetic microfluidic device.

Description

Description of the invention

The subject of the invention is a magnetic microfluidic device for rapid screening tests, constituting a set of replaceable single-use microfluidic chip for conducting three-dimensional, island cell cultures in a stationary system, in a continuous gradient of the active substance, and a device generating a magnetic microfield (the so-called magnetic microfield generator) with preset properties, and in in particular the given distribution of the magnetic field strength.

The magnetic microfluidic device is used as a tool for rapid screening of drugs and bioactive substances (screening tests), which makes it applicable at various stages of research into new drugs. The device allows you to run tests simultaneously for an infinite number of dilutions (in a continuous gradient) of the active substance. In addition, the device is used to conduct basic research in neurobiology and cell biology, in research on the potential protective effect of active substances preventing neurodegenerative diseases (neuroprotection) and in research on the effect of active substances on cell regeneration (neuroregeneration).

The method of conducting research with the use of a magnetic microfluidic device and commonly available biocompatible, immunoreactive, surface-modified with biotylated antibodies polymer microcapsules containing nanoparticles with ferromagnetic properties in the core allows for three-dimensional, island cell cultures and research using them in a controlled, continuous concentration gradient of the active substance. This enables screening tests to be carried out in a simple, fast, reproducible and efficient way using advanced cellular 3D models.

In the body, concentration gradients of biomolecules are regulated and control many basic cell functions, including cell differentiation and migration. In vivo, cells in tissues are exposed to gradient concentrations of biologically active compounds that pass from the blood to the cell environment. Depending on the distance of individual cells from the site of absorption of the biologically active compound, the cells will be exposed to different concentrations of this compound - the cells will respond with different intensity of the metabolic and/or phenotypic effect. They will also release into the environment (cellular milleu) different amounts of signaling compounds for neighboring cells in their environment (e.g. prostanoids). Thus, it is a complex system in which the total effect of an extracellular compound acting on a group of cells in a tissue is dependent on the distance of the cells through the concentration gradient of that compound and local differences in the concentration of cell-released signaling compounds acting on neighboring cells in the tissue. Signaling compounds secreted from cells exposed to extracellular factor affect the modification of the function of neighboring cells. Conducting research using three-dimensional cell cultures in a microfluidic device makes it possible to take into account the mutual interaction of cells, and not only the effect of the tested active substance, as in classical solutions, on cell metabolism. Further advantages of the microfluidic device are higher accuracy and better control of the generated gradient, compared to classic solutions, thanks to the dimensions adapted to the cellular scale. In addition, microfluidic systems operate on small fluid volumes, in the order of microliters, which significantly reduces research costs (Mahdi Karimi et al., Microfluidic systems for stem cell-based neural tissue engineering, Lab on a Chip 2016, 16: 2551-2571).

Currently, screening of bioactive substances for their potential use as therapeutic substances and the phenomena of neuroprotection and neuroregeneration in vitro is carried out using classic multi-well culture plates and cell suspension or adherent cultures, which do not allow for conducting research in a continuous gradient of the test substance or they do not allow testing using three-dimensional cell cultures.

From the patent application US20180141047 A1, a multi-well microfluidic device for conducting three-dimensional cell cultures in stationary conditions, using support materials for cell immobilization, is known. Microchannels supplying substances stimulating cell growth and proliferation were led to the centrally located breeding chambers. Patent application US20130143230 A1 describes a microfluidic platform for studying the influence of physicochemical and biological factors on cell cultures and cocultures. Cell cultures were carried out in stationary and flow-through systems on synthetic scaffolds. From the patent application US20080233607 A1, a microfluidic flow device is known in the form of a simple channel with a number of partitions for conducting cell cultures in a laminar flow of the culture medium. In turn, the patent application US20090023608 A1 describes a microfluidic device having a matrix system of culture chambers for conducting a number of simultaneous tests and cell cultures. The system allowed observation of the culture under an optical microscope. Similarly, patent application US20120003732 A1 presents a system provided with a matrix system of culture chambers for simultaneous research using a number of cell cultures under an optical microscope. The US patent application US20070084706 A1 describes a microfluidic device and a method for cultivating cell cultures on a porous membrane.

None of the known design solutions and methods of screening bioactive substances and drugs, in particular in terms of usefulness in regenerative and preventive medicine, uses a magnetic microfluidic device to conduct three-dimensional cell cultures in a continuous gradient of the active substance in stationary (non-flow) conditions, and through it does not allow the formation of cell islands of a given diameter and height and a given distance along the culture chambers due to the formation and control of a low-intensity magnetic microfield and discrete distribution and the use of commonly available biocompatible, immunoreactive, surface-modified with biotylated antibodies polymer microcapsules containing nanoparticles with ferromagnetic properties in the core , previously combined into microcapsule-chamber agglomerates in a selective manner permanently or separably with the cell membrane of specific types of cells due to the formation of antigen-antibody immune complexes, and thus does not allow screening tests of potential medicinal substances for an infinite number of dilutions (in a controlled continuous gradient) of substances in in one experiment and using complex island 3D cell cultures, and therefore does not allow for screening in cell models with intercellular signaling present in living tissues.

The aim of the invention was to develop a new design solution in the form of a magnetic microfluidic device, in particular for the needs of regenerative and preventive medicine, which would solve the above-mentioned problems.

Magnetic microfluidic device for rapid screening consisting of a lid, base, tank covers and a functional layer of the chip, and the lid of the chip contains supply tanks and a millimeter scale, and the functional layer of the chip contains culture chambers, it is characterized in that it consists of a light-transparent visible and UV microfluidic chip equipped with at least three linear culture chambers with axes of 5 to 25 mm apart, each connected at the ends to supply tanks, a flexible base and a magnetic microfield generator consisting of a platform containing a chip socket , which is adapted in size to the size of the microfluidic chip, spacers, generator base assembly consisting of a top and bottom, containing a magnet socket, a permanent magnet and a series of ferromagnetic pins, which are positioned perpendicularly to the surface of the microfluidic chip culture chambers, the arrangement of the culture chambers in microfluidic chip and the system of ferromagnetic pins of the magnetic microfield generator are mutually correlated in such a way that the ferromagnetic pins in the platform run parallel to each other from the surface of the chip's culture chambers to the upper surface of the permanent magnet.

Preferably, the base of the microfluidic chip is made of a polycarbonate film, polyethylene terephthalate or COC cyclic polyolefin copolymer with a thickness of 100 to 300 microns.

Preferably, the tank covers are made of a self-adhesive polyvinylidene fluoride (PVDF) film with a thickness of 200 to 500 microns.

Preferably, the surface of the magnet receptacle in the base of the generator covers the area of the microfluidic chip culture chambers.

Preferably, plate permanent magnets from the N35 to N52 group are used.

Preferably, the dimensions of the permanent magnet match the dimensions of the magnet socket in the base of the generator.

Preferably, the ferromagnetic pins are made of ferromagnetic steel, most preferably of the SS 430 type.

Preferably, the ferromagnetic pins have a diameter of 100 to 300 microns.

Preferably, the ferromagnetic pins have a length of 30 to 100 millimeters.

Preferably, the ferromagnetic mandrels are distributed under each of the culture chambers of the microfluidic chip along their longer sides at mutual distances not smaller than twice the diameter of the ferromagnetic mandrel.

Preferably, the ferromagnetic pins are located on at least % of the length of the breeding chamber.

Preferably, the ferromagnetic pins are arranged in at least one row.

Preferably, the ends of the ferromagnetic pins are flush with the upper surface of the chip socket and with the upper surface of the magnet socket.

Preferably, the spacer supports are equipped with an inspection opening enabling access to the ferromagnetic pins.

Advantageously, the height of the spacer supports is correlated with the length of the ferromagnetic pins in such a way that it ensures their ends flush with the upper surface of the chip socket and the upper surface of the magnet socket.

Preferably, the height of the magnet socket is equal to the height of the permanent magnet.

Preferably, the top and bottom of the generator base are permanently connected.

Preferably, the spacer supports are permanently connected to the platform and the base of the generator.

Preferably, the ferromagnetic pins are inserted into the through holes of the ferromagnetic pins and permanently connected to the generator platform and base.

Preferably, the microfluidic chip comprises magnetic microcapsules.

Most preferably, the concentration of the magnetic microcapsules incorporated into the microfluidic chip is in the range of 1x106 to 1x107 agglomerates per milliliter.

The magnetic microfluidic device for rapid screening according to the invention enables screening of biologically active substances with potential healing, regenerative or protective properties in a continuous gradient of this substance, in a wide range of concentrations, using three-dimensional, island cell cultures in a stationary (non-flow) system, which are possible to achieve without the use of support structures, in particular, it does not require the use of hydrogels, semi-permeable membranes and scaffolds, and is possible thanks to the use of ferromagnetic polymer microcapsules with modified surface properties, thus adhering to the cells that enable the microcapsule-chamber agglomerates to be held in specific places within the chambers of the microfluidic chip by means of a magnetic microfield generated by the field generator, and this is possible while maintaining undisturbed concentration profiles of biochemical factors that are subject to natural secretion from cells. The construction of the device enables simultaneous control culture (the so-called blank test) and at least one proper culture, as well as indirect detection of the concentration profile of the test substance and its changes over time thanks to spectrophotometric measurements of the color indicator introduced into the indicator chamber. In addition, the design of the device ensures the possibility of introducing any active substances into the culture environment and conducting microscopic observations as well as research and analysis using standard research techniques used in biology, medicine, pharmacy and biomedical engineering, in particular optical epifluorescence microscopy and spectrofluorimetry.

The subject of the invention is presented in detail in the examples and in the drawing, where: Fig. 1 shows a magnetic microfluidic device for rapid screening, Fig. 2 shows an exploded drawing of a microfluidic chip with three linear culture chambers, Fig. 3 shows an exploded drawing of a discrete microfield generator magnetic. Example 1

The magnetic microfluidic device for rapid screening tests is shown in Fig. 1. The magnetic microfluidic device is a detachable assembly of the magnetic microfield generator 1 and the gradient microfluidic chip 2. The microfluidic chip 2 has been positioned on the magnetic microfield generator 1 by inserting the microfluidic chip 17 into the socket, in such a way that the base of the chip 6 adheres closely to the surface of the socket of the microfluidic chip 2.

A three-chamber microfluidic chip 2 with dimensions of 25/75/3.43 mm (width/length/height) for conducting research in a continuous gradient of a bioactive substance is shown in Fig. 2. The microfluidic chip 2 consists of three layers and two tank covers 3, the arrangement of which is shown in Fig. 2. The lid of the chip 4 is made of PMMA transparent to visible light and UV, 3 mm thick. At the age 4, at the height of the inlets and outlets from the breeding chambers 9 of the device, six cylindrical tanks 8 with a diameter of 5 mm were made, matching the width of the breeding chambers 9 and located at both ends of each chamber 9. Along the working area of each chamber 9, centrally between the chambers 9 there is a millimeter scale 7. The functional layer 5 is made of a commercially available adhesive film prepared on the basis of acrylic adhesive with a thickness of 0.13 mm. In the functional layer 5, three elongated, symmetrically arranged, mutually parallel culture chambers 9 were made, each 5 mm wide and 70 mm long, with the working area of each chamber 9 being 50 mm long. The base of the chip 6 is made of a polycarbonate foil permeable to visible light and UV, 300 micrometers thick and with dimensions adapted to the dimensions of the microfluidic chip 2. The functional layer 5 connects the lid 4 with the base 6 in an inseparable and hydraulically tight manner. The inner surface of each of the chambers 9 was hydrophilized with low-temperature air plasma according to a generally known procedure. The upper surfaces of the supply tanks 8 are protected by tank covers 3 made of 0.5 mm thick, 25/20 (width/length) self-adhesive PVDF film, located at each end of the device. The covers of the tanks 3 are connected to the lid of the chip 4 in a detachable and hydraulically tight manner.

The magnetic microfield generator 1 is shown in Fig. 3. The generator 1 consists of a generator platform 10 with dimensions of 50/100/3.6 mm (width/length/height), 300 cylindrical ferromagnetic pins 11 with a diameter of 0.3 mm and 30 mm long, made of SS430 steel, two spacers 12 with dimensions of 26.4/50/3 (height/length/thickness) equipped with a centrally located inspection opening with dimensions of 15/25 (height/length), neodymium rectangular permanent magnet 14 25/55/20 mm (width/length/height) of the N35 type positioned in the magnet socket 20 in the generator base assembly consisting of a top 13 and a bottom 15 layer, each 50/100/11.8 mm (width/length /height) and magnet 14. In the base of the generator, a centrally and symmetrically placed magnet socket 20 with dimensions of 25/55/20 mm (width/length/height). In the generator platform, through holes 16, 18 ferromagnetic pins 11 with a diameter of 0.3 mm were made. The holes 16, 18 are located in the area of the microfluidic chip socket 17 under each of the culture chambers 9 of the microfluidic chip 2, within their working areas, in two rows running parallel and symmetrically along the longer axes of the culture chambers 9, each at a distance of 1.5 mm from axis and spaced 1 mm apart over a length of 50 mm. The chip socket 17 in the magnetic microfield generator platform 10 has a depth of 1.8 mm. Positioning holes 19 have been made in the corners of the microfluidic chip 2 and in the corresponding places of the chip socket 17 and the generator base assembly, enabling precise positioning of the chip 2 in the socket 17. The diameter of each positioning hole 19 is 0.5 mm. All elements of the magnetic microfield generator, apart from the ferromagnetic pins 11 and the magnet 14, were made of PMMA and joined using a commercially available photo-curable PMMA adhesive. The ferromagnetic pins 11 were inserted into the holes of the ferromagnetic pins 16 and 18 in the platform and base of the generator, so that their ends were flush with the planes of the chip socket 17 and the magnet socket 20, and permanently joined with a photo-curing adhesive for PMMA.

Example^

The magnetic microfluidic device for rapid screening was made according to the procedure presented in embodiment 1 with the difference that:

A four-chamber microfluidic chip 2 with dimensions of 25/75/3.23 mm (width/length/height) for conducting two cell cultures in a bioactive substance gradient was placed in the microfluidic chip socket 17 of the magnetic microfield generator 1. Eight cylindrical 8 tanks with a diameter of 3 mm. In the functional layer 5, four symmetrically arranged cultivation chambers 9 were made, each 3 mm wide and 60 mm long, with the working area of each chamber 9 being 40 mm long. The base of the chip 6 is made of 100 micrometer thick COC foil. Tank covers 3 are made of self-adhesive PVDF foil with a thickness of 0.2 mm.

Magnetic microfield generator 1 includes a generator platform with dimensions of 50/100/4.8 mm (width/length/height), 800 cylindrical ferromagnetic pins 11 with a diameter of 0.1 mm and a length of 100 mm, two spacers 12 with dimensions of 94/ 50/3 (height/length/thickness) equipped with a centrally located inspection opening with dimensions of 60/25 (height/length), permanent magnet 14 with dimensions of 25/40/10 mm (width/length/height) of type N52 positioned in the socket magnet 20 in the assembly of the generator base consisting of a top layer 13 and a bottom layer 15, each with dimensions of 50/100/8 mm (width/length/height) and a permanent magnet 14. In the base of the generator, a centrally and symmetrically placed magnet socket 20 with dimensions of 25 /40/10 mm (width/length/height). In the generator platform, through holes 19 ferromagnetic pins 11 with a diameter of 0.1 mm were made. The openings 19 are arranged symmetrically in one row under each of the working areas of the cultivation chambers 9 of the chip and run in their axis at 0.2 mm intervals over a length of 40 mm.

Example 3 ''

A method of screening a factor with a potential neuroprotective effect in order to determine its optimal concentration.

The tests were carried out in a stable concentration gradient of the active substance in a three-chamber magnetic microfluidic device with the use of magnetic microcapsules modified with surface biotylated monoclonal antibodies against the A2B5 surface antigen and with the use of the following substances: wogonin - a bioactive substance, a suspension rat cell line PC12, type I collagen - a substance to modify cell surfaces, methylene blue as an indicator substance, nerve growth factor NGF as a substance inducing cell differentiation, amyloid beta as an active agent causing cell damage, and commercial solutions of propidium iodide for the determination of apoptosis of nerve cells. The method of conducting research consists in the following:

Reagents before testing: RPMI culture medium, distilled water, 0.1% CTAB solution in glucose-free culture medium, 0.1% methylene blue indicator dye solution in 0.1% CTAB culture medium without glucose, and wogonin solution with 100 micromoles/liter culture medium and a 5 micromoles/liter beta amyloid solution in culture medium are placed in an incubator and thermostated at 37°C for 1 hour.

1. In the first step, neuronal cells of rat PC12 lines are separated and concentrated in a test tube with 1 ml of their suspension at a concentration of 1x10 ⁵ cells in a reference culture medium using commercially available magnetic microcapsules with a diameter of one micrometer, surface modified with monoclonal antibodies against the surface antigen A2B5 according to the microcapsules manufacturer's reference procedure. The microcapsule-cell aggregates are then resuspended in 0.1 ml of culture medium, thus obtaining a 10-fold concentration of the original cell suspension.

2. In a second step, 20 microliters of a 0.1mg/ml type I collagen solution is introduced into each of the supply reservoirs 8 located on the same side of the microfluidic chip 2, and the device is tilted 30 degrees towards the empty reservoirs to force the collagen solution to flow through the chambers 9 of the microfluidic chip 2. The supply reservoirs 8 of the culture chambers 9 are then emptied by means of an adjustable automatic pipette set to 20 microliters, the liquid remaining within the chambers 9. Each of the reservoirs 8 of the microfluidic chip is then filled 2 with 30 μl collagen solution. Finally, the tanks 8 of the microfluidic chip 2 are protected with tank covers 3 and the device prepared in this way is placed in a refrigerator at 4-8°C for 12 hours. The tanks 8 on either side of the culture chambers 9 are then emptied by an automatic adjustable pipette, after which the chambers 9 of the microfluidic chip are flushed with PBS by dispensing a measured volume of PBS into the supply tanks 8 and tilting the chip 2 at an angle of 60 degrees towards the empty tanks 8 to forcing the liquid through the chambers 9 and emptying the tanks 8 by means of an automatic pipette. The operation is repeated five times, each time 30 microliters of PBS are poured into each of the chambers 9. Then the device is irradiated with UVC rays for 30 minutes for sterilization.

3. In the third step, 20 microliters of culture medium are introduced into the two supply tanks 8 on one side of the microfluidic chip 2 (blank and proper culture), and 20 microliters of 0.1% CTAB solution in culture medium without glucose are introduced into the third tank 8 of the chamber 9 (indicator) and the microfluidic chip 2 is tilted at 30 degrees towards the empty reservoirs 8 to force flow through the chambers 9 of the microfluidic chip 2. The reservoirs 8 of the culture chambers 9 are then emptied by an adjustable automatic pipette with a setting of 20 microliters, liquid remains within chambers 9.

4. In the fourth step, 20 microliters of the suspension of cell-microcapsule agglomerates in the culture medium are introduced into two supply tanks 8 on one side of the microfluidic chip 2 (blank and proper culture), and 20 microliters of 0.1% CTAB solution in the culture medium without glucose into the third reservoir 8 of chamber 9 (indicator). The microfluidic chip 2 is then tilted 30 degrees towards the empty supply tanks 8, which forces the slurry and CTAB solution from the supply tanks 8 through the chambers 9 to the tanks 8 located on the opposite side. The downstream tanks 8 are then emptied. The process is repeated twice. Then, all reservoirs 8 of the microfluidic chip 2 are emptied and reservoirs adjacent to chambers 9 containing the suspension of cell-microcapsule agglomerates and 0.1% CTAB solution in culture medium without glucose are filled with culture medium, reservoirs 8 adjacent to chamber 9 (indicator) respectively. Then the tanks are secured with covers 3.

5. In the fifth stage, the microfluidic chip 2 is placed in the socket 17 of the magnetic microfield generator, thanks to which the microcapsules and agglomerates are positioned inside the cultivation chambers 9 of the microfluidic chip 2 in the areas determined by the placement of the ferromagnetic pins 11.

6. In the sixth step, the cells are cultured for 24 hours. at 37°C in 5% CO2 in an incubator for their adhesion to the surface of the cultivation chambers 9, and then the microfluidic chip 2 is disconnected from the magnetic microfield generator 1.

7. In the seventh step, the supply tanks 8 on both sides of the culture chambers 9 are emptied by an automatic adjustable pipette, while the chambers 9 of the microfluidic chip 2 remain filled with liquid, and 20 microliters of NGF solution in culture medium at a concentration of 100 nanogram/ml are introduced. to the supply tank 8 of the first and second chambers 9 (blank, proper culture) and 20 microliters of a 0.1% CTAB solution in culture medium without glucose to the supply tank 8 of the third chamber 9 (indicator). The microfluidic chip 2 tilts at 30 degrees towards the empty reservoirs 8, forcing fluid through the chambers 9 of the microfluidic chip 2 until the supply reservoirs 8 previously filled with liquid are completely emptied, but without emptying the contents of the chambers 9, after which the liquid from the reservoirs 8 is removed on the opposite side of the chamber 9 of the microfluidic chip 2 by means of an automatic adjustable pipette. The process is repeated three times. Tanks 8 are secured with covers 3, and then culture is carried out in an incubator at 37°C in 5% CO2 for 72 hours. for cell differentiation under the influence of NGF.

8. In the eighth step, the supply tanks 8 on both sides of the cultivation chambers 9 are emptied by an automatic adjustable pipette, while the chambers 9 of the microfluidic chip 2 remain filled with liquid, and 14 microlitres of culture medium are introduced into the supply tank 8 of the first chamber 9 (blank ), a solution of the active substance in the culture medium to the supply tank 8 of the second chamber 9 (culture proper) and a solution of the dye in 0.1% CTAB in the culture medium without glucose to the supply tank 8 of the third chamber 9 (indicator). The microfluidic chip 2 is then tilted at an angle of 60 degrees towards the empty reservoirs 8, forcing the fluid to flow through the chambers 9 of the microfluidic chip 2 until the supply reservoirs 8 previously filled with fluid are completely emptied, but without emptying the contents of the chambers 9, after which the liquid from the reservoirs is removed 8 of the microfluidic chip 2 using an automatic adjustable pipette.

9. In the ninth stage, 5 microliters of culture medium are introduced to the first and second tank 8 (blank sample, proper culture) on the outflow side, and to the third tank 8 of chamber 9 (indicator) 0.1% CTAB solution in culture medium without glucose and the microfluidic chip 2 is tilted 45 degrees towards the empty reservoirs 8 of the microfluidic chip 2 to force the fluid flow in the chambers 9 of the microfluidic chip 2 to completely empty the supply reservoirs 8. The microfluidic chip 2 is then tilted 45 degrees in the opposite direction and forces the fluid to flow back in the chambers 9. The alternating deflections are repeated fifteen times until a linear distribution of the concentration of the color substance in the chamber 9 (indicator) is obtained, which is facilitated by the reciprocating movement of the fluid in the culture chambers 9, after which the liquid is removed from the reservoirs 8 of the microfluidic chip 2 using an automatic adjustable pipette.

10. In the tenth stage, the tanks 8 of the microfluidic chip 2 are filled with 30 microliters of liquid each time, according to the composition of the solution at the outlets from the chambers 9 adjacent to them - culture medium, 0.1% CTAB solution in culture medium without glucose, dye solution and 0. 1% CTAB in glucose-free culture medium or active substance solution. The reservoirs 8 of the microfluidic chip 2 are then protected by the covers 3 of the supply reservoirs to ensure stabilization of the concentration gradients.

11. In the eleventh step, the absorbance of light with a wavelength of 650 nm is measured in chamber 9 (indicator) using a plate spectrophotometer.

12. In the twelfth step, the cells are incubated in a microfluidic chip 2 in an incubator at 37°C in 5% CO2 for 24 hours.

13. In the thirteenth step, he again measures the absorbance in chamber 9 (indicator) using a plate spectrophotometer.

14. In the fourteenth step, the feed tanks 8 on either side of the cultivation chambers 9 are emptied by an automatic adjustable pipette, while the chambers 9 of the microfluidic chip 2 remain filled with liquid. The microfluidic chip 2 is tilted 10 degrees so that the supply reservoirs 8 are above the reservoirs 8 on the opposite side of the microfluidic chip 2, and a measured volume of the amyloid beta solution in culture medium is introduced into the supply reservoirs 8 of the first and second chambers 9 (blind sample, proper culture), and 0.1% CTAB solution in medium without glucose to the supply tank 8 of the last chamber 9 (indicator). The inclination of the device forces the fluid to flow through the chambers 9 of the microfluidic chip 2 until the supply tanks 8 previously filled with liquid are completely emptied, but without emptying the contents of the chambers 9, after which the liquid is removed from the tanks 8 on the opposite side of the chamber 9 by means of an automatic adjustable pipette. The action is repeated three times. Then, all reservoirs 8 of the first and second chambers 9 (blank, proper culture) are filled with a solution of amyloid beta in culture medium, and reservoirs 8 of the third chamber 9 (indicator) with 0.1% CTAB solution in glucose-free medium. Then, the reservoirs 8 of the microfluidic chip 2 are secured with covers 3 and the cells are incubated in the microfluidic chip 2 placed in an incubator for 24 hours. at 37°C in 5%CO2.

15. In the fifteenth step, the culture of cells in the first and second chambers 9 of the microfluidic chip 2 (blank, proper culture) is evaluated microscopically.

16. In the sixteenth step, tracer dyes in the form of commercially available propidium iodide cellular apoptosis solutions are added to chambers 9 (blank, proper culture). Then incubation is carried out in the dark for 20 min at room temperature.

17. In the seventeenth step, the culture of cells in the first and second chambers 9 of the microfluidic chip 2 (blank, proper culture) is evaluated under a fluorescence microscope. Example 4

The method of conducting cytostatic screening tests in order to determine the range of therapeutic concentrations.

The tests were carried out in a stable concentration gradient of the active substance in a four-chamber magnetic microfluidic device using magnetic microcapsules modified with surface biotylated monoclonal antibodies BerEP4 against the EpCAM human antigen and the following substances: doxorubicin - active substance (cytostatic) at a concentration of 1000 ppm w/w in the culture medium, MDA-MB-231 tumor cell suspension of the human breast cancer adherent line, L-15 reference culture medium, 0.01% solution of the indicator dye - fluorescein in distilled water and a commercially available live/dead test for the fluorescence determination of live and dead cells.

The method of conducting research consists in the following:

Prior to testing, the reference culture medium, distilled water, indicator dye solution and doxorubicin solution are placed in an incubator and thermostated at 37°C for half an hour.

1. In the first stage, separation and concentration are carried out in a test tube MDA-MB-231 of the human breast cancer adherent line with 1 ml of their suspension with a concentration of 5x105 cells in a reference culture medium using commercially available magnetic microcapsules with a diameter of three micrometers, surface-modified with antibodies against the EpCam surface antigen according to the microcapsule manufacturer's reference procedure. The microcapsule-cell aggregates are then resuspended in 0.5 ml of culture medium, thus obtaining a 10-fold concentration of the original cell suspension.

2. In the second stage, to each of the supply tanks 8 located on the same side of the microfluidic chip 2, 30 microliters are introduced: culture medium to the first, second and third chamber 9 (blank sample and two proper cultures), and distilled water to the fourth chamber chamber 9 (indicator), then the microfluidic chip 2 is tilted at 30 degrees towards the empty reservoirs 8 to force fluids to flow through chambers 9 of the microfluidic chip 2. All reservoirs 8 are then emptied using an adjustable automatic pipette set to 30 microliters, the liquid remaining within the chambers 9, filling them completely due to surface tension forces.

3. In the third stage, 30 microliters of the suspension of cell-microcapsule agglomerates are introduced into three tanks 8 of liquids supplying the culture chambers 9 on one side of the microfluidic chip 2 (two cultures and a blank sample), and 30 microliters of distilled water are introduced into the fourth tank 8 of chamber 9 (indicator) and the device is tilted towards the empty tanks 8 at an angle of 60 degrees, so as to force the flow of fluids from the supply tanks 8 through the chambers 9 of the microfluidic chip 2 to the tanks 8 located on the opposite side, until the level of fluids in the tanks 8 of the chip is equalized microfluidic 2 and the tanks 8 are secured with covers 3.

4. In the fourth stage, the microfluidic chip 2 is placed in the socket of the magnetic microfield generator 17, thanks to which the agglomerates are positioned inside the cultivation chambers 9 of the microfluidic chip 2 in the areas determined by the location of the ferromagnetic pins 11.

5. In the fifth stage, the cells are cultured for 6 hours at 37°C in 5% CO2 in a microfluidic device in an incubator in order to adhere them to the surface of the cultivation chambers 9, and then the microfluidic chip 2 is disconnected from the magnetic microfield generator 1.

6. In the sixth step, the tanks 8 on both sides of the culture chambers 9 are emptied by an automatic adjustable pipette, while the chambers 9 of the microfluidic chip 2 remain filled with liquid, and 10 microliters of culture medium are introduced into the supply tank 8 of the first chamber 9 (blank) the active substance solution to the supply tank 8 of the second and third chambers 9 (cultivation proper) and the colored indicator solution to the supply tank of the fourth chamber 9 (indicator). Then the device is tilted at an angle of 60 degrees towards the empty tanks 8, forcing the fluid to flow through the chambers 9 of the microfluidic chip 2, until the supply tanks 8 previously filled with liquid are completely empty, but without emptying the contents of the chambers 9, after which the liquid from the tanks 8 is removed microfluidic chip 2 using an automatic adjustable pipette.

7. In the seventh stage, 10 microliters of liquid are introduced into each of the tanks 8 on the outflow side: culture medium to the tank 8 of the first, second and third chambers 9, and distilled water to the tank 8 of the fourth chamber 9 and the microfluidic chip 2 is tilted at an angle 60 degrees towards the empty reservoirs 8 in order to force fluid flow in the channels of the microfluidic chip 2 and to completely empty the reservoirs 8 previously filled with fluid. Then, the microfluidic chip 2 tilts at an angle of 60 degrees in the opposite direction and forces the fluid to flow back in the chambers 9. The activities of alternating tilts are repeated 5 times until the desired distribution of the concentration of the color substance in the chamber 9 (indicator) is obtained, which is facilitated by the reciprocating movement of the fluid in the chambers 9 of the microfluidic chip 2, after which the liquid is removed from the reservoirs 8 by means of an automatic adjustable pipette.

8. In the eighth stage, the tanks of the microfluidic chip 2 are filled with 30 microliters of liquid each time, according to the composition of the solution, at the outlets of the chambers 9 adjacent to them: both tanks 8 of the first chamber 9 of the culture medium (blank sample), tanks 8 (inflow) of the second and third chamber 9 (two proper cultures) with an active substance solution, and tanks 8 (outflow) of the second and third chambers 9 with culture medium, tank 8 (inflow) of the fourth chamber 9 (indicator) with a dye solution, and tank 8 (outflow) of the fourth chamber 9 with distilled water and tanks 8 are protected with covers 3.

9. In the ninth step, the fluorescence measurement is performed at the incident wavelength of 480 nm and the emitted wavelength of 530 nm in chamber 9 (indicator) using a plate spectrophotometer.

10. In the tenth step, the cells are incubated in a microfluidic chip 2 in an incubator at 37°C in 5% CO2 for 3 hours.

11. In the eleventh step, he again measures the absorbance in chamber 9 (indicator) using a plate spectrophotometer.

12. In the twelfth step, the supply tanks 8 on either side of the cultivation chambers 9 are emptied by an automatic adjustable pipette, while the chambers 9 of the microfluidic chip 2 remain filled with liquid. The microfluidic chip 2 is tilted 10 degrees so that the supply tanks 8 are above the tanks 8 on the opposite side of the microfluidic chip 2, and then a measured volume of the live/dead test solution is introduced into the supply tanks 8 of the first, second and third chambers 9 ( blank and proper cultures), and distilled water to the feed tank 8 of the last chamber 9 (indicator). The inclination of the microfluidic chip 2 forces the fluid to flow through the chambers 9 until the supply tanks 8 previously filled with liquid are completely emptied, but without emptying the contents of the chambers 9, after which liquid is removed from the tanks 8 on the opposite side of the chamber 9 by means of an automatic adjustable pipette. The action is repeated twice. Then, all the tanks 8 of the first, second and third chambers 9 (blank, true cultures) are filled with the live/dead test solution and the tanks 8 of the third chamber 9 (indicator) with distilled water. Then, the reservoirs 8 of the microfluidic chip 2 are secured with covers 3 and the cells are incubated in the microfluidic chip 2 placed in an incubator for 3 hours at 37°C.

13. In the thirteenth step, the culture of cells in the first and second chambers 9 of the microfluidic chip 2 (blank, proper culture) is evaluated microscopically.

Claims (21)

1. Magnetic microfluidic device for quick screening consisting of a lid (4), a base (6), tank covers (3) and a functional layer (5) of the chip, and the chip lid contains the supply tanks (8) and a millimeter scale (7) , and the functional layer of the chip includes cultivation chambers (9), characterized in that it consists of a microfluidic chip (1) transparent to visible and UV light, equipped with at least three linear cultivation chambers (9), the axes of which are spaced at distances from 5 up to 25 mm, each of which connects at the ends to the supply tanks (8), a flexible base (6) and a magnetic microfield generator (1) consisting of a platform (10) containing a socket (17) of the chip, which is adapted in size to the size a microfluidic chip (2), spacers (12), a generator base assembly consisting of a top (13) and bottom (15), containing a magnet socket (20), a permanent magnet (14) and a series of ferromagnetic pins (11), which are positioned perpendicularly to the surface of the culture chambers (9) of the microfluidic chip (2), the system of culture chambers (9) in the microfluidic chip (2) and the system of ferromagnetic pins (11) of the magnetic microfield generator (1) are mutually correlated in such a way that the pins ferromagnetic (11) in the platform (10) run parallel to each other from the surface of the culture chambers (9) of the chip to the upper surface of the permanent magnet (14). 2. A magnetic microfluidic device as claimed in claim 1. 1, characterized in that the base of the microfluidic chip (6) is made of polycarbonate foil, polyethylene terephthalate or cyclic polyolefin copolymer COC with a thickness of 100 to 300 microns. 3. A magnetic microfluidic device as claimed in claim 1. 1, characterized in that the tank covers (3) are made of a self-adhesive polyvinylidene fluoride (PVDF) film with a thickness of 200 to 500 microns. 4. A magnetic microfluidic device as claimed in claim 1. 1, characterized in that the surface of the magnet seat (20) in the generator base assembly covers the area of the culture chambers (9) of the microfluidic chip (2). 5. A magnetic microfluidic device as claimed in claim 1. 1, characterized in that permanent plate magnets from the group from N35 to N52 are used. 6. A magnetic microfluidic device as claimed in claim 1. 1, characterized in that the dimensions of the permanent magnet (14) match the dimensions of the magnet socket (20) in the generator base assembly. 7. A magnetic microfluidic device as claimed in claim 1. 1, characterized in that the ferromagnetic pins (11) are made of ferromagnetic steel type SS 430. 8. The magnetic microfluidic device of claim 1. 1, characterized in that the ferromagnetic pins (11) have a diameter of 100 to 300 micrometers. 9. A magnetic microfluidic device as claimed in claim 1. 10, characterized in that the ferromagnetic pins (11) have a length of 30 to 100 millimeters. 10. The magnetic microfluidic device of claim 1. 1, characterized in that the ferromagnetic pins (11) are distributed under each of the culture chambers (9) of the microfluidic chip (2) along their long sides at mutual distances not less than twice the diameter of the ferromagnetic pin (11). 11. The magnetic microfluidic device of claim 1. 1, characterized in that the ferromagnetic pins (11) are located on at least % of the length of the breeding chamber (9). 12. The magnetic microfluidic device of claim 1. 1, characterized in that the ferromagnetic pins (11) are arranged in at least one row. 13. The magnetic microfluidic device of claim 1. 1, characterized in that the ends of the ferromagnetic pins (11) are flush with the upper surface of the chip socket (17) and with the upper surface of the magnet socket (20). 14. The magnetic microfluidic device of claim 1. 1, characterized in that the spacer supports (12) are equipped with an inspection opening enabling access to the ferromagnetic pins (11). 15. The magnetic microfluidic device of claim 1. 1, characterized in that the height of the spacers (12) is correlated with the length of the ferromagnetic pins (11) in such a way that their ends are aligned with the upper surface of the chip socket (17) and with the upper surface of the magnet socket (20). 16. The magnetic microfluidic device of claim 1. 1, characterized in that the height of the magnet socket (20) is equal to the height of the permanent magnet (14). 17. The magnetic microfluidic device of claim 1. 1, characterized in that the top (13) and bottom (15) of the generator base assembly are permanently connected. 18. The magnetic microfluidic device of claim 1. 1, characterized in that the spacer supports (12) are permanently connected to the platform and the base of the generator. 19. The magnetic microfluidic device of claim 1. 1, characterized in that the ferromagnetic pins (11) are inserted into the through holes (16), (18) of the ferromagnetic pins and permanently connected to the platform (10) and the generator base assembly. 20. The magnetic microfluidic device of claim 1. 1, characterized in that the microfluidic chip (2) comprises magnetic microcapsules. 21. The magnetic microfluidic device of claim characterized in that the concentration of the magnetic microcapsules introduced into the microfluidic chip (2) is in the range of ^1x106 to ^1x107 agglomerates per milliliter.