WO2018143831A1

WO2018143831A1 - Method of manufacturing a multilayer microfluidic device for dynamic tissue culture

Info

Publication number: WO2018143831A1
Application number: PCT/PL2018/000010
Authority: WO
Inventors: Roman SZAFRAN; Agnieszka PAWŁOWSKA; Jakub FICHNA; Urszula LEWANDOWSKA
Original assignee: Politechnika Wrocławska; Uniwersytet Medyczny W Lodzi
Priority date: 2017-01-31
Filing date: 2018-01-26
Publication date: 2018-08-09
Also published as: PL420395A1; PL238703B1

Abstract

The method of manufacturing a multilayer microfluidic device for the conduct of tissue culture under dynamic conditions is presented, which consists of at least six layers (these are: bottom and lid, adhesive layer, at least one auxiliary glass layer, at least one elastomeric functional layer and at least one hydrogel layer). The method is characterized by the fact that in the first stage, on the basis of pre-designed maps of each layer of the device, its elements (vascular and connecting channels and microchannels, inspection, positioning and through holes, culture chambers, partitions and tanks) are produced. For this purpose, elastomeric layers are prepared in such a way that the medium is coated with a layer of elastomer and fixed. Then the focused laser beam does the engraving and cuts the structures. The auxiliary glass layers, the adhesive layer and the bottom and lid of the microdevices are engraved and the structure are cut by means of a focused laser beam operating on material sheets. In the second stage, non-adhesive auxiliary glass layers and elastomeric functional layers are combined into sets. Each set consists of one glass layer and one elastomeric layer on a carrier which forms the temporary basis of the set. In the third stage, the previously prepared liquid hydrogel is introduced through inspection holes into the culture chambers of the sets where it is fixed and then disconnected from the temporary base of the unit. In this way, in the exposed hydrogel layer, vascular microchannels are engraved, whereas connecting channels are engraved in partitions, by means of a focused laser beam. In the fourth stage, without the use of glue, one or more sets are joined together or to the bottom or to the lid by means of an adhesive layer. In the fifth stage, the microsystem is combined with liquid supply and outlet hoses using an adhesive. In the sixth stage, the microsystem is cleaned and washed. In the seventh stage, suspension of cells of any type is introduced into the microsystem, once or several times, at intervals allowing their immobilization on the surface of microchannels. In the eighth stage, cell culture is carried out under flow conditions in order to form a network of capillary vessels within the artificial tissue.

Description

Method of manufacturing a multilayer microfluidic device for dynamic tissue culture

The subject of the invention is the method of making microfluidic device (biochip) for conducting tissue culture in the flow system and conducting research into biological processes occurring within artificial tissues. Microdevices of this type are used in biology, medicine, pharmacy, biotechnology, biomedical engineering and tissue engineering to conduct research into cell differentiation mechanisms and methods of intercellular signalling, research of tissue response to physical, chemical and biological damage and growth of cancerous tissue in the presence of bioactive agents and cytostatics. These devices enable creating in silico dynamic conditions resulting from the flow of fluid in blood vessels and lymphatic system as well as in the area of extracellular matrix, imitating the phenomena present in living organisms, and impossible to be imitated in vitro while conducting classical tissue culture: suspension cultures, on solid media and scaffolding. The production method of microfluidic devices for conducting tissue culture enables the direct production of complex three-dimensional hydrogel structures with a pre-set, repeatable geometry of microchannels within the culture chambers, without the need for semipermeable membranes, which makes it suitable for mass production of microdevices. In addition, the use of hydrogel materials enables spontaneous formation of new capillary vessels within the cultured tissues in the presence of proangiogenic factors, thus better imitating the conditions of cell growth in vivo. By using materials permeable to visible light and ultraviolet (UV), the method enables the fabrication of microchips for observation and detection with the use of fluorescent microscopy - a classic technique commonly used for examining biological materials.

Tissue engineering is a promising area for the future, hoping to solve the problems of patients suffering from irreversible organ damage caused by diseases, injuries and degeneration {Robert Longer, Joseph P. Vacanti, (1993) Tissue Engineering, Science, New Series, Vol. 260, No. 5110: 920-926). Currently, three-dimensional tissue structures are manufactured on the basis of porous scaffolding, in which cells are closed inside. These scaffolds serve as synthetic extracellular matrices and enable the spatial organization of cells and are responsible for providing physico-chemical stimuli stimulating cell division and growth within the cells. (Gaharwar AK, Peppas NA, Khademhosseini A (2014) Nanocomposite hydrogels for biomedical applications. Biotechnol Bioeng 111:441-453). This technique was used to create a narrow group of synthetic tissues that do not require complex vascular structure: cartilage and skin. Production of tissues with complex vascular structure, e. g. heart or liver is confronted with serious difficulties due to the impossibility of forming a functional network of blood vessels within their area, capable of proper nutrition and oxygenation. Therefore, the main challenge of tissue engineering nowadays is to develop a method for blood vessel formation inside the porous hydrogel structure, capable of maintaining proper activity of the cells within the matrix. Two approaches can be distinguished in this area. The first one consists in supplying to the hydrogel matrix pro- angiogenic growth factors, stem cells and endothelial cells and thus forcing spontaneous formation of the vascular network within artificial tissue (Naito Y, Shinoka T, Duncan D, Hibino N, Solomon D, Cleary Met al (2011) Vascular tissue engineering: towards the next generation vascular grafts. Adv Drug Deliv Rev 63:312-323). The second approach is to use microfabrication techniques to form the microchannel structure in the hydrogel before implementing cell culture inside. (Zorlutuna P, AnnabiN, Camci-Unal G, NikkhahM, Cha JM, NicholJW et al (2012) Microfabricated biomaterials for engineering 3D tissues. Adv Mater 24:1782-1804). The first method is ineffective in the production of stable, branched vascular high level structures, while the second one poses problems in the fabrication of small capillaries within tissues.

Although conventional macroscopic cell culture methods have been known for more than 100 years and remain basic methods of research in biology, medicine and biotechnology, the high demands placed by emerging fields of medicine, including tissue engineering, require the use of new precision techniques for cell culture. Micro scale culture provides better, precise control of cell development conditions and their interactions, and above all, it enables breeding in flow conditions, in a dynamic system that closely imitates the in vivo phenomena. Microfluidic tissue culture systems enable limiting the use of animal models in research on new drugs and acceleration pre-clinical tests due to high repeatability of results and their compatibility of artificial tissues with human genetic material. These microsystems not only better imitate the three-dimensional micro architecture of tissues than classical cultures, but also reflect the tissue-specific physical conditions. The most complex microfluidic systems are able - to a limited extent - to reproduce the specific properties of lung, liver, kidney and digestive system tissues and even of several organs simultaneously, which enables pharmacokinetic and pharmacodynamic studies of new medicaments in silico. (F. Sonntag, et al, (2010), Design and prototyping of a chip-based multi-micro-organoid culture system for substance testing, predictive to human (substance) exposure, J. Biotechnol. 148 (1), 70 -75). American patent no. US7960166 (B2) describes the flow microdevice and its manufacturing method, consisting of two parallel straight channels separated by a porous membrane, designed to carry out cell co-culture. The device enables parallel - made up of two different cell types - cell culture under flowing conditions, while nutrients and metabolites are transported through a porous membrane between the channels, making one of the cells act as a blood vessel and the other as a culture chamber. Similarly, German patent no. DE102011112638 (B4) describes a microdevice consisting of at least one flowing channel and one culture chamber separated by a porous membrane for the simulation of biological phenomena occurring in blood vessels. The method of producing both microsystems requires the integration of a porous membrane inside the device, which is technically difficult to implement and does not enable the formation of complex vascular structures of any configuration.

American patent no. US8647861 (B2) refers to the microsystem for tissue culture, its mode of operation and the method of microchip production. The device consists of a central microchannel divided by porous membranes into several parallel compartments inside which fluids can flow. The surface of the membranes has been modified with substances that facilitate the adhesion of cells to their surface, which enables tissue formation on the outer surfaces of the vessels (flow channels) inside the non-flowing culture chambers. The microsystem is connected to the system of supply tanks and pumps, and the system is controlled via a computer. The method of microsystem implementation, similarly to the previously discussed patents, requires the placement of porous, semipermeable membranes separating the channels and microsystem chambers, which is technically difficult to implement and does not allow for forming complex vascular structures of desired configurations and does not allow for self-organization of vessels within the cultivated tissues. This prevents the internal structure of any tissue from being accurately reproduced. Another American patent no. US7622298 (B2) reveals the in vitro method of producing permeable blood vessel networks, which are integrated with the microfluidic device. Micro vessels were produced by endothelial cell culture on straight, cylindrical, elongated cores. After the vascular walls were formed, cores surrounded by a cell wall were submerged in a microfluidic chamber filled with hydrogel, then the core was removed and further cell culture was carried out under flowing conditions, stimulating the spontaneous blood vessel formation process inside the chamber. This method is multi-stage and tedious; it requires initial cell culture on the cores and precise integration of micro vascular walls with the chamber of microchip, which is technically very difficult to implement, and the process of removing the cores from the vessels may cause their damage. This method does not allow for the formation of a blood vessel network with a designed, repeatable configuration and does not allow for effective serial production of microsystems with a repeatable vessel geometry for tissue culture. On the other hand, American patent no. US9081003 (B2) describes the microsystem and system for simulation of dynamic in vivo phenomena occurring in tissues, organs and three-dimensional cellular structures and resulting from the flow of fluid inside them. The device is used for testing new medicines and for studying the phenomena of transport accompanying drug distribution in tumours, especially in targeted therapies. The microsystem consists of a culture chamber equipped with partitions and separated by a porous membrane from the channel serving as a blood vessel. The semi-adhesive membrane supported cell culture and constituted a barrier separating the feed channel from the culture chamber and lymphatic channels. The method of manufacturing the multi-layer microsystem included the production of microdevice components in a multi-stage photolithographic method in poly(dimethylsiloxane) (PDMS) and their combination with a porous semipermeable membrane, which was one of the layers of the microsystem. The described method of microsystem production is multi-stage and tedious. The construction and method of its manufacturing do not enable the formation of complex vascular structures of any configuration and does not allow the self-organization of vessels inside the cultured tissues. American patent application no. WO2015153451 (Al) describes a microsystem capable of producing biologically active substances, enabling tissue cultures under flow-through conditions. The system consists of several parallel channels separated by a partition with holes enabling mass transfer between channels. The microsystem production was based on a multistage method of soft photolithography and included the preparation of replication matrices using silicon wafers, photoresist coating, photomask preparation, UV radiation, developing photoresist, casting of functional layers in PDMS and combining elements, surface coating with substances modifying the surface properties of microchannels. The method is multi-stage, labour- intensive and time-consuming. The way of preparing multilayer cellular co-cultures and conducting tests of obtained tissue cultures in a microfluidic device is known from American patent application no. US20060141617 (Al). The device was equipped with rectilinear culture chambers, inside of which the partitions were placed made of hydrogel matrix and cultured cells. The device was made using the classic photolithography method in PDMS, and the surface of the channels was modified with organo-silicon compounds. Similarly, American patent no. US8119394 (B2) describes a device for 3-dimensional cell culture on the surface of porous membranes.

Polish patent no. PL219444 (Bl) describes the method of making a microvascular device, and patent applications P409877 (Al) and P415009 (Al) describe the method of making a microfluidic device for encapsulating active substances and the method of making a microfluidic device for conducting nerve cell culture. In the documents referred to above, a direct method based on laser engraving of thin PDMS layers and their bonding with the layers of glass and plastic lid was used to manufacture the microfluidic device. However, the method of production of microfluidic devices for tissue culture, in particular the method of production of hydrogel layers and the method of production of channels in these layers, which is the basic element of microsystems for tissue culture, has not been revealed. Neither was the method of forming cell walls on the surfaces of the hydrogel layer channels described.

None of the known methods of making microfluidic tissue culture devices under dynamic conditions enables the direct production of complex three-dimensional, stable and branched high-level vascular structures with a given, reproducible diameter and spatial distribution in hydrogel layers and without the need for semi-adhesive membranes, while allowing spontaneous self-forming of capillaries in the cultured tissues in the presence of pro-angiogenic substances.

As a result, there was a need to develop a new way of producing microsystems for tissue culture, which solves these technical problems.

The method of manufacturing a multilayer microfluidic device for dynamic tissue culture consists of at least six layers. These are: bottom and lid, adhesive layer, at least one auxiliary glass layer, at least one elastomeric functional layer and at least one hydrogel layer. This is done in eight stages. In the first step, the device elements are created based on previously designed maps of each layer. These include channels and vascular microchannels or and connecting channels, inspection and positioning holes as well as culture chambers, partitions and tanks. Elastomer layers are prepared in such a way that the carrier is coated with a layer of elastomer and cured. Then the structures are engraved and cut with a focused laser beam. The auxiliary glass layers, the adhesive layer and the bottom and lid of the microdevice are engraved, while the structure of the sheets of the material is cut with a focused laser beam. In the second stage, the glass auxiliary layers and elastomeric functional layers are joined without the use of glue into sets, each consisting of one glass layer and one elastomeric layer on a carrier, which is a temporary base of the assembly. In the third stage, the prepared liquid hydrogel is introduced through inspection holes into the culture chambers of the units and solidified, next the temporary base of the unit is disconnected. In this way, by means of focused laser beam irradiation, vascular microchannels are engraved in the exposed hydrogel layer and connecting channels are engraved in the partitions. In the fourth stage, one or more sets are combined with each other or with their bottoms without adhesive and with a lid using glue. In the fifth stage, the microsystem is combined with hoses supplying and draining liquid, using adhesive. In the sixth stage, the microsystem is cleaned and washed. In the seventh stage, a suspension of cells of any type is introduced into the microsystem, once or several times at intervals allowing their immobilisation on the surface of microchannels. In the eighth stage, cell culture is carried out under flow conditions in order to form a network of capillary vessels in the artificial tissue.

Advantageously, the hydrogel is prepared on the basis of acrylic acid or anhydride solution, polyvinyl alcohol, poly(lactic acid), polyethylene glycol, collagen, hyaluronic acid, alginate, chitosan or their mixtures or mixtures of compounds forming the extracellular matrix in water or culture medium.

Advantageously, the hydrogel is sterilized by filtration on a hydrophilic membrane filter with a pore diameter no greater than 0.5 micrometre.

Advantageously, to the hydrogel are added substances limiting the growth of microorganisms, preferably antibiotics.

Advantageously, pro-angiogenic substances are added to the hydrogel; VEGF vascular endothelial growth factor is the most favourable.

Advantageously, fibroblasts and stem cells are added to the hydrogel.

Advantageously, maps of vascular microchannels are prepared on the basis of pictures of the physical structure of the blood vessel system in the tissue, mathematical model or according to design assumptions in the CAD graphic environment.

Advantageously, coating of the supports with an elastomer layer is carried out in a spin coater.

Advantageously, elastomeric layers are prepared by applying polydimethylsiloxane (PDMS) onto polyester film (PET).

Advantageously, the lid and bottom of the device are made using glass sheet, PMMA poly (methyl methacrylate) or polyester film (PET).

Advantageously, commercially available sheets of acrylic adhesive film, doubly covered with paper are used for the adhesive layer.

Advantageously, curing of PDMS layers is carried out in a cabinet drier or on a heating plate at temperatures between 60 - 120 °C for 30 minutes and 12 hours respectively, or at room temperature for at least 24 hours.

Advantageously, engraving and cutting out structures is done by CO₂ laser beam with a wavelength of 10.6 μιη.

Advantageously, laser beam and laser head movement parameters are selected in such a way as to minimize thermal impact of the laser beam on the material outside the cutting or engraving area.

Advantageously, the mutual positioning of the microdevice layers is carried out using positioning holes arranged in the same location on each layer and templates equipped with positioning pins, adjusted to the positioning holes.

Advantageously, the elastomeric layers are favourably bonded to the glass auxiliary layers in the sets after prior exposure of the elastomer surface to ultraviolet (UV) radiation.

Advantageously, the elastomeric layers are favourably bonded to the glass auxiliary layers in sets after preheating the glass layer at a temperature of 300 °C for at least 30 minutes.

Advantageously, the layers are tightly bonded hydraulically.

Advantageously, the microdevice is hydraulically tightly bonded and inseparable with feed and drain hoses by means of a photoset adhesive and UV radiation for not less than 30 seconds.

Advantageously, the microchannel network is favourably cleaned and washed with deionised water, buffered saline (PBS) or aqueous EDTA solution and surfactant of a temperature of not more than 60 °C.

Advantageously, in order to immobilise the cells inside the microchannels, a cell suspension is introduced into the microdevice in the culture medium and left for a period of at least 30 minutes.

Advantageously, cell culture under flow conditions is carried out at a flow rate of not less than 1 ml/24h of culture medium.

Most advantageously, pro-angiogenic substances are added to the culture medium.

Most advantageously, the growth factor of VEGF vascular endothelial endothelium is added to the culture medium.

The invention provides for an eight-stage method of producing a multilayer microfluidic device for conducting tissue culture under dynamic conditions, thanks to which the process is simple, cheap and fast, and enables a direct production of complex three-dimensional hydrogel structures of a defined, repeatable geometry without the need to use semipermeable membranes. This enables the production of a basic, branched, stable vascular network, as well as secondary, spontaneous formation of capillary vessels inside the cultured tissues. In addition, thanks to the use of visible light transmitting materials and ultraviolet (UV) radiation, this method enables the manufacture of microchips for observation and detection using fluorescent microscopy.

The subject of the invention is presented in more detail in examples of its implementation. Example 1.

The method of producing a six-layer, single-chamber flow microfluidic device for managing connective tissue culture with the use of hydrogel structure based on collagen, (a) Hydrogel preparation

Before starting to manufacture the microdevice, liquid hydrogel was prepared. For this purpose, commercially available lyophilized type IV collagen was prepared in 0.2% v/v aqueous acetic acid solution, yielding a 5 mg/ml collagen solution. All reagents and dishes were cooled in the refrigeration chamber to 4°C before preparation of the solution and, after preparation, the solution was stored in liquid form in a refrigeration chamber or in an ice bath at a temperature of no more than 4°C. Subsequently, a culture medium with the following composition was prepared: 89% of high glucose DMEM culture medium with L-glutamine, 10% of foetal bovine serum, 1% of solution 100X penicillin/streptomycin, which was stored in a refrigeration chamber at 4°C. Immediately before the hydrogel layer was manufactured, both solutions were combined in 1:5 volume proportions (collagen:medium) and cold-filtered on a syringe filter with a pore diameter of 0.5 micrometre. The mixture was then heated to 37°C and combined in a ratio of 10:1 (hydrogel: cell concentrate) with a concentrated suspension of endothelial cells of L929 mouse line (1x107 cells/mL). All operations were carried out in sterile conditions in the laminar chamber,

b) Device design

The microfluidic device design with dimensions 24/60/3.6 mm (width/length/height) has been prepared in the CAD graphical environment for spatial modelling. The chip structure elements are arranged on six layers. The inlet and outlet openings were connected to the culture chamber by means of intake and outlet channels and through flow-through holes. Flow-through holes ensure vertical fluid flow between the individual layers while channels allow for planar fluid flow within a single layer. The culture chamber together with the feed and discharge channels were placed on a functional layer made of PDMS, each channel was separated hydraulically from the chamber by a transverse partition, in which microchannels connecting the spaces of feed and outlet channels with the culture chamber are made. The functional layer is limited from the bottom by the base and from the top by a supporting glass layer with through holes and inspection holes. The glass layer is in turn connected to the lid by means of an adhesive layer, in which through holes are designed. On the basis of a microscopic picture of neoplastic tissue in the hydrogel layer located in the culture chamber, a distribution of vascular microchannels has been designed, which faithfully imitates the distribution of fine veins and arterioles in the tissue. The lid on the side walls has connection ports for micro hoses that feed or drain the liquids to and from the device. The lid also has tanks connected by connection ports with micro hoses and with channels that feed or drain the liquids to and from the culture chamber. Positioning holes are located at four corners of each layer, excluding the hydrogel layer. The three-dimensional project of the microdevice was transferred to 6 two-dimensional drawings, i.e. maps depicting each layer of the microsystem. Different colours have been used to distinguish between edges of different cutting depths and widths. Drawings of the designed microdevice were sequentially and directly exported from the CAD graphic environment to the engraving station operating environment for each layer. This was done in such a way that the drawing layers of one microdevice layer were exported simultaneously while the other drawing layers were excluded.

c) First stage: manufacture of microdevice components

The elastomeric functional layer with a thickness of 100 micrometres was produced by spin coating in a spin coater. The layer was made in such a way that the mixture of PDMS prepolymer and polymerization activator with a weight ratio of 10: 1 was de-aerated in vacuum and then fed in the amount of 4 cm³ on a disk with a diameter of 80 mm rotating at a speed of 500 revolutions per minute in the coating chamber. The disc was made of transparent PET polyester film with a thickness of 150 micrometres. The material was rotated for 1.5 minutes and then the rotational speed of the disk was gradually reduced within 1 minute to zero. During the coating, an excess pressure was generated in the coating chamber by means of compressed, dust-free, dry air. Then, the polyester foil with an elastomer layer was placed on a Petri dish in a cabinet drier, where polymerization tool- place at 80°C for 6 hours. Then, a sheet of adjacent protective paper was applied to the polymer surface to protect it from engraving dust. The finished substrate was stored in a desiccator over 4A molecular sieves in a dry air atmosphere with a relative humidity of 1%. Subsequently, the PDMS substrate was engraved with a focused CO₂ laser beam with a wavelength of 10.6 μηι, a rated power of 30 watts, a spot diameter of 25 micrometres and a maximum head travel speed of 0.25 m/s in places where the elastomer layer was designed to be cut to the media surface. For this purpose, the vector mode of the engraving station is assigned to each line of a specific colour and the laser beam power is set at 0.3% of the rated power. The pulse frequency was set at 1000 DPI, laser head travel speed at 0.9% of the nominal maximum value, and working surface distance from laser beam focus point at 0.2 mm. These settings made it possible to cut the PDMS layer to the surface of the polyester medium while achieving a basic channel width of 45 micrometres and at the same time reducing the contamination of the PDMS surface. To obtain channels with a width of more than 100 micrometres, in vector mode, an elastomer layer on the perimeter of the channel was cut into the media surface, and PDMS was mechanically removed from the inside of the channel with a sharp spike under a stereoscopic microscope. The total intersection of the PDMS layer and polyester medium required for positioning holes was possible for the following settings of the engraving machine: power 1.5%, head speed 1.7%, impulse frequency 1000 DPI, focusing distance 0.2 mm.

The auxiliary glass layer was made of borosilicate glass sheet with a thickness of 150 micrometres and dimensions of 24/60 mm. In order to make through holes, positioning holes and inspection holes, the glass surface was engraved three times with a focused laser beam driven around the circle in places where, according to the design, the glass layer was to be cut. For this purpose, the following operating parameters of the engraving machine have been assigned to particular colours of drawing lines: power 0.4%, head speed 0.3%, impulse frequency 1000 DPI, focusing distance 0.0 mm.

The adhesive layer was made of a commercially available sheet of adhesive film with an acrylic glue layer 100 micrometres thick, covered on both sides with protective paper. In order to make through holes and cut out the geometry of the layer, the surface of the sheet was engraved with a focused laser beam in places where the glue film was to be cut. For this purpose, the following operating parameters of the engraving machine were assigned to particular colours of the drawing lines: power 30%, head speed 40%, impulse frequency 1000 DPI, focusing distance 0.3 mm. The total cut of the adhesive film and two layers of the protective paper, required for making positioning holes, was possible for the following settings of the engraving device: power 100%, head speed 40%, impulse frequency 1000 DPI, focusing distance 0.3 mm.

The lid of the microdevice was made of PMMA in such a way that the sheet of material was cut with a concentrated 30 watt CO2 laser beam, obtaining rectangular sheets of 24/60 mm and 3 mm thick. In the side walls of the base, holes were made for attaching feed and discharge capillaries in such a way that the designated areas were treated with a focused laser beam, led around the circle. The laser settings were as follows: power 10%, head speed 1%, impulse frequency 1000 DPI, focusing distance 27 mm. The second time: power 18%), head speed 1%, impulse frequency 1000 DPI, focusing distance 27 mm. As a result side connection ports for micro hose were obtained. The tanks of the channels that feed and discharge fluids to/from the functional layer group of the chip were made in the microdevice lid in such a way that the designated places were treated with a laser beam led around the circle. The settings of the laser were: power 100%, head speed 4.5%, impulse frequency 1000 DPI, focusing distance 3.0 mm.

The microdevice base was made of PET foil in such a way that the sheet of material was cut and then positioning holes were made with the use of focused CO₂ laser beam of 30 watts. The laser's settings were: power 5%, head speed 20%, impulse frequencylOOO DPI, focusing distance 0.1 mm.

d) The second stage: connecting the layers into units

In order to connect the elastomer layer with the glass auxiliary layer into a complex, the protective paper sheet was removed from the elastomer surface. The functional layer on the polyester foil medium was then washed with KOH solution in ethanol at a concentration of 50 g/dm3, then three times washed with deionized water and dried in a stream of clean compressed air. The glass sheet ( microscope cover slip) was then heated at 300°C in the oven, in the air atmosphere for 30 minutes. Simultaneously, the functional layer was treated with 185 nm and 253.7 nm wavelength radiation for 30 seconds in the UV chamber. Then both elements were positioned using a template equipped with positioning bolts: the positioning bolts were inserted into the positioning holes of subsequent layers and the elements were pressed. After removing the template, the elements were pressed against each other using a rubber roller to remove air bubbles from the space between layers. Finally, the unit was placed on a hot plate thermostatically heated at a temperature of 120 °C for 30 minutes. In this way, a culture chamber unit was obtained, consisting of a glass and elastomeric layer, protected from the bottom by a polyester support, as its temporary base.

e) Third stage: preparation of the hydrogel layer

In order to prepare the hydrogel layer, the previously prepared hydrogel was introduced by a disposable syringe equipped with a needle with a diameter matched to the diameter of inspection holes, through inspection holes of the culture chamber, which was completely filled. Subsequently, the unit was placed in an incubator at 37 °C for 1 hour to solidify the hydrogel. After this period, the temporary polyester base of the unit was removed and the exposed hydrogel layer was engraved with a focused laser beam to produce 50 micrometre wide microchannels. The laser settings were as follows: power 0.1%, head speed 10%, impulse frequencyl 000 DPI, focussing distance 0.3 mm. The laser operating parameters have been selected so as to minimize thermal effects in the hydrogel area outside the microchannel space and not to damage the glass layer below the hydrogel layer. In addition, in the partitions separating the culture chamber from the liquid supply and discharge channels, connecting microchannels were made in such a way that the surface of partitions was treated with a focused laser beam in places where according to the design there were supposed to be connecting microchannels. In this case, the laser settings were as follows: power 1.1%, head speed 1.6%, impulse frequency 1000 DPI, focusing distance 0.3 mm.

c) Fourth stage: connecting the unit of the culture chamber with the bottom and lid of the microdevice

In order to connect the assembly with the lid of the microdevice, elements are put together using a template equipped with positioning bolts in such a way that the bolts of the template are inserted into the positioning holes of the subsequent layers, i.e. the culture chamber unit, adhesive layer and lid, after which the elements are pressed. Before the unit was combined with the bottom, the bottom was treated with 185nm and 253.7nm radiation for 30 seconds in a UV chamber, and then combined with the unit using a template and pressed.

d) Fifth stage: connecting the microdevice to micro hoses

In order to connect the micro hoses to the device, their ends were inserted into the side connection ports after applying a light cure adhesive at their ends. Then they were exposed to UV light for 30 seconds. The surface of the chip above the hydrogel chamber was covered with a UV-impermeable foil to prevent the negative impact of UV radiation on the hydrogel structure.

e) Sixth stage: cleaning of the microsystem In order to remove contaminants after the engraving of the hydrogel layer, the microsystem is three times washed with buffered PBS physiological saline solution, each time with a volume of 1 cm³ and a temperature of 37°C.

f) Seventh stage: cell immobilisation

In order to immobilize endothelial cells of B 129 mouse line on the internal surface of the vascular microchannels, a concentrated cell suspension (1x107 cells/mL) in the reference culture medium Ml 168, which contains, inter alia, growth factors of the mouse endothelium VEGF and EGF, was introduced into the device's microchannels and incubated for 4 hours at 37°C under stationary conditions, which led to their adhesion to the surface of the microchannels.

g) Eighth stage: cell culture under flowing conditions

In order to stimulate spontaneous formation of capillary blood vessels inside artificial tissue, cell culture under flowing conditions was performed for 5 days after placing the microsystem in an incubator at 37°C, 5% CO₂ and 95% humidity. A syringe pump with a 50 ml syringe filled with the reference culture medium Ml 168 was placed outside the incubator and connected to inlet of the microsystem in the incubator via micro hoses. Microsystem outlets were connected to the waste tank. The culture was carried out at the medium flow rate of 5 ml/24h.

Example 2.

The method of production of a six-layer, single-chamber fluid-flow microfluidic device for the culture of human breast cancer tissue using a hydrogel structure based on alginate. The device has been manufactured in a similar way as shown in Example 1 , except that:

a) Hydrogel preparation

Before starting to manufacture the microdevice, liquid hydrogel was prepared. In order to obtain 10 cm³ of sodium alginate solution with a concentration of 5 % w/v of in PBS, PBS and sodium alginate were intense stirred on a magnetic stirrer in a closed vessel in a water bath at 80°C until complete dissolution. The solution was cooled and stored in a refrigeration chamber at 4°C.

Subsequently, a culture medium composed of 89% of high glucose L-15 culture medium with L-glutamine, 10% foetal bovine serum, 1% solution of 100X penicillin/streptomycin was prepared and stored in a refrigeration chamber at 4°C. Immediately before the hydrogel layer was fabricated, both solutions were combined in 1:5 volume proportions (alginate:medium) and cold-filtered on a syringe filter with a pore diameter of 0.2 micrometre. The mixture was then heated to 37°C and combined in a ratio of 10:1 (hydrogel: cell concentrate) with a concentrated suspension of human breast cancer cells of the MB-231 line (1x107 cells/mL). All operations were carried out in sterile conditions in the laminar chamber.

e) Third stage: preparation of the hydrogel layer

In order to prepare the hydrogel layer, the previously prepared liquid hydrogel was introduced into the chamber using a disposable syringe through the inspection holes of the culture chamber with a diameter matching the syringe outlet diameter, filling the chamber up completely. Then, the hydrogel was removed from the inspection holes and the holes were filled with a 5% aqueous calcium chloride solution, after which, in order to solidify the hydrogel, the unit was placed in an incubator at 37°C for 6 hours. Next, the temporary polyester base of the unit was removed and an exposed hydrogel layer was treated with a focused laser beam to produce 80 micrometre wide microchannels. The laser settings were as follows: power 0.2%, head speed 10%, impulse frequencyl 000 DPI, focussing distance 0.5 mm. The laser operating parameters were selected so as to minimize thermal effects in the hydrogel area outside the microchannel space and not to damage the glass layer below the hydrogel layer. In addition, in the partitions separating the culture chamber from the liquid supply and discharge channels, connecting microchannels were made in such a way that the surface of the partitions was treated with a focused laser beam in places where, according to the design, connecting microchannels were to be located. In this case, the laser settings were as follows: power 0.3%, head speed 0.9%, impulse frequency 1000 DPI, focusing distance 0.2 mm.

f) Seventh stage: cell immobilisation

In order to immobilize HUVEC endothelial cells on the internal surface of the microchannels, a cell suspension (1x106 cells/mL) concentrated in the reference culture medium 211-500 and supplemented by 211-GS, which contained the endothelial growth factor VEGF, was introduced into the device's microchannels and incubated for 2 hours at 37°C under stationary conditions. In this way their adhesion to the surface of microchannels was obtained.

g) Eighth stage: cell culture under flow conditions

In order to stimulate the spontaneous formation of capillary blood vessels in artificial tissue, cell culture under flowing conditions was performed for a period of 3 days after placing the microsystem in an incubator at 37°C and 95% humidity. For this purpose, a 50-ml syringe pump filled with 89% reference culture medium L-15 with L-glutamine, 10% foetal bovine serum, 1% solution 100X penicillin/streptomycin solution and human VEGF medium were placed outside the incubator and connected to the inlet of the microsystem in the incubator with micro hoses. Microsystem outlets are connected to the waste tank. The culture was carried out at the medium flow rate of 5 ml/24h.

Claims

1. The method of manufacturing a multilayer microfluidic device for the conduct of tissue culture under dynamic conditions, which consists of at least six layers, including: bottom and lid, adhesive layer, at least one auxiliary glass layer, at least one elastomeric functional layer and at least one hydrogel layer, wherein in the first stage, on the basis of pre-designed maps of each layer of the device, its elements - vascular and connecting channels and microchannels, inspection, positioning and through holes, culture chambers, partitions and tanks - are produced; for this purpose, elastomeric layers are prepared in such a way that the medium is coated with a layer of elastomer and fixed, then the focused laser beam does the engraving and cuts the structures; the auxiliary glass layers, the adhesive layer and the bottom and lid of the microdevices are engraved and the structure are cut by means of a focused laser beam operating on material sheets; in the second stage, non-adhesive auxiliary glass layers and elastomeric functional layers are combined into sets, each consisting of one glass layer and one elastomeric layer on a carrier which forms the temporary basis of the set; in the third stage, the previously prepared liquid hydrogel is introduced through inspection holes into the culture chambers of the sets where it is cured and then disconnected from the temporary base of the unit and in this way, in the exposed hydrogel layer, vascular microchannels are engraved, whereas connecting channels are engraved in partitions, by means of a focused laser beam; in the fourth stage, without the use of glue, one or more sets are joined together or to the bottom or to the lid by means of an adhesive layer; in the fifth stage, the microsystem is combined with liquid supply and outlet hoses using an adhesive; in the sixth stage, the microsystem is cleaned and washed; in the seventh stage, suspension of cells of any type is introduced into the microsystem, once or several times, at intervals allowing their immobilization on the surface of microchannels; in the eighth stage, cell culture is carried out under flow conditions in order to form a network of capillary vessels within the artificial tissue.

2. The method in accordance to claim 1, wherein the hydrogel is prepared based on a solution of acrylic acid or anhydride, polyvinyl alcohol, poly(lactic acid), polyethylene glycol, collagen, hyaluronic acid, alginate, chitosan or their mixtures, or mixtures of compounds forming part of the extracellular matrix, in water or culture medium.

3. The method in accordance to claim 2, wherein the hydrogel is sterilized by filtration on a hydrophilic membrane filter with a pore diameter no greater than 0.5 micrometre.

4. The method in accordance to claim 2, wherein substances limiting microbial growth are added to the hydrogel.

5. The method in accordance to claim 4, wherein antibiotics are added to the hydrogel.

6. The method in accordance to claim 2, wherein pro-angiogenic substances are added to the hydrogel.

7. The method in accordance to claim 6, wherein vascular endothelial growth factor VEGF is added to the hydrogel.

8. The method in accordance to claim 2, wherein fibroblasts and stem cells are added to the hydrogel.

9. The method in accordance to claim 1, wherein maps of microchannels of vascular microchannels are prepared based on photographs of the physical structure of the blood vessel system in the tissue, mathematical model or according to design assumptions in the CAD graphic environment.

10. The method in accordance to claim 1, wherein coating of the support with a layer of elastomer is carried out in a spin coater.

11. The method in accordance to claim 1 , wherein the elastomeric layers are prepared by applying polydimethylsiloxane (PDMS) to polyester foil (PET).

12. The method in accordance to claim 1 , wherein glass sheets, poly(methyl methacrylate) PMMA or polyester foil (PET) are used for the lid and bottom of the equipment.

13. The method in accordance to claim 1 , wherein commercially available acrylic adhesive film sheets based on acrylic glue are used for the adhesive layer, protected on both sides with cover paper.

14. The method in accordance to claim 1, wherein PDMS layer curying is carried out in a cabinet drier or on a hot plate at a temperature between 60 - 120°C for 30 minutes and 12 hours respectively, or in room conditions for at least 24 hours.

15. The method in accordance to claim 1, wherein engraving and cutting out structures is done with a concentrated CO2 laser beam with a wavelength of 10.6 μπι.

16. The method in accordance to claim 1, wherein the laser beam and laser head travel parameters are selected so as to minimize thermal interaction between the laser beam and the material outside the cutting or engraving area.

17. The method in accordance to claim 1, wherein mutual positioning of the microdevice layers is carried out with the use of positioning holes located in the same places on each layer and templates equipped with positioning pins, adjusted to the positioning holes.

18. The method in accordance to claim 1, wherein the bonding of elastomeric layers with glass auxiliary layers in sets is carried out after prior exposure of the elastomer surface to ultraviolet (UV) radiation.

19. The method in accordance to claim 1, wherein bonding of the elastomeric layers with the glass auxiliary layers into sets takes place after the glass layer has been warmed up at a temperature of 300°C for at least 30 minutes.

20. The method in accordance to claim 1, wherein the layers are bonded hydraulically tight.

21. The method in accordance to claim 1, wherein the microdevice is connected hydraulically tight and inseparable with the liquid supply and discharge hoses by means of a light cure adhesive and exposure to UV radiation for not less than 30 seconds.

22. The method in accordance to claim 1, wherein the microchannel network is cleaned and washed with deionised water, buffered saline (PBS) or aqueous EDTA and surfactant solution with a temperature of not more than 60°C.

23. The method in accordance to claim 1, wherein in order to immobilise the cells inside the microchannels, a cell suspension in the culture medium is inserted into the microdevice and left for at least 30 minutes.

24. The method in accordance to claim 1 , wherein cell culture under flow conditions is carried out at a flow rate of not less than 1 ml/24h of culture medium.

25. The method in accordance to claim 24, wherein pro-angiogenic substances are added to the culture medium.

26. The method in accordance to claim 25, wherein vascular endothelial growth factor VEGF is added to the culture medium.