PL71591Y1 - Multifunctional flow-through micro-system - Google Patents

Description

Pattern description The subject of the utility model is a multifunctional flow microsystem for the cultivation of cell lines in a monolayer, controlled by microfluidic conditions for the formation of a co-culture, and migration and testing of cytotoxic phototoxic activity of drugs. This type of microsystem can be found in application in cell / tissue engineering as well as material sciences where screening for newly designed drug delivery systems.

A microcircuit for assessing the effectiveness of photodynamic therapy procedures is known from the Polish patent specification PL 214 546, consisting of: made of a hydrophilic glass plate and a polymer plate made of a material with hydrophobic properties, transparent, gas-permeable, biocompatible and capable of forming microstructures therein. Both the glass and polymer tiles are with you? permanently connected using oxygen plasma. In a microchip, in a glass plate, there is matrix of at least 2 × 2 culture microcells, while in the polymer plate there are si? microchannels, where microchannels connect microcells along the The microchip, and in addition, the microsystem includes inlet holes drilled in the polymer plate in an amount corresponding to the number of microchannels, and one inlet-outlet port. In the described microcircuit, cells can creates? aggregates, which is influenced by the low width? microchannels and cause their clogging in the case of introducing high-density cell suspension.

From the publications of Tomecka E., Tokarska K., Jastrz? Bska E., Chudy M., Brzózka Z. - Cell engineering in lab-on-a-chip systems - Chemical News, 2015 [Z] 69, 9 -10, 909-929 known s? microcircuits containing in their geometry places intended for cell docking and growth (culture microcells), microchannels supplying cells and a network microstructures allowing the continuous supply of nutrients and test compounds. This publication discloses microcircuits for testing the cytotoxicity of compounds, studying chemotaxis and cell migration, disclosing the so-called concentration gradient generators (Concentration Gradient Generator, CGG), in which it is possible to generate, in one stage of the analysis, several solutions introduced into the microcircuit. The simplest generators include T- or Y-shaped microchannels [19]. The principle of operation of CGG is based on the fact that solutions flowing in two channels introducing part of si? in one, where they are mixed by diffusion. The gradient control can be done? si? through a properly designed Aug? channels, the number of inlets and outlets of which determines the number of obtained concentrations? used for research factor. Also described? a microsystem with 576 culture microcells, which were used to test the toxic effects of compounds such as: digitonin, saponin, CoCl2, NiCl2 and acrolein [21]. The microsystem made in PDMS includes? microchannels in their geometry with you? perpendicular. The structures are also described. of a microsystem that uses three microchannels arranged in parallel, connected to each other microchannels.

From the publication of Roman Szafran - Methodology of designing lab-on-a-chip micro-cameras for research? The survival in the capillary of neoplastic tumors is described in the vector map? channels in Autodesk AutoCAD that then supplemented with August? channels for supplying and draining fluid from the structure of the vessel network hairline. The incoming and outgoing channels were connected with ports placed in the chip cover plate, allowing for the connection of microchip supplying capillaries.

From the patent description US2008254997 there is known an analytical kit containing: i) an analytical device containing a allowing flow through it? liquid, formed by joining together the first element having a groove, a second element covering the groove, and the first nucleic acid (N1) having any sequence bases and immobilized in the capture zone on the first and / or second members before bonding the first and second members together; ii) Reagent A comprising a conjugate (N2-L1) consisting of a second nucleic acid (N2) with a sequence at least complementary to the base sequence of the first nucleic acid and (2) the first ligand (L1) capable of specific binding zania from research? substance biological (ABOUT); iii) reagent B containing a conjugate (L2-M) resulting from the binding of the marker (M) to a second ligand (L2) capable of specific binding to from research? substance biological (ABOUT).

The solutions known from the state of the art do not allow for culturing in a monolayer of any three, both cancerous and normal, cell lines in various configurations and observing cell migration, which is extremely important in the process of neoplasia. It is also important? the need for the possibility of observation intercellular cells in co-culture (e.g., a normal tumor cell), where cells can with you? affect? by way of e.g. sending death signals and controlling the flow rates of the introduced streams and obtaining co-cultures composed of two and three cell lines in different proportions.

According to the utility formula, the microsystem consists of from two permanently joined layers: a glass plate and a poly (dimethylsiloxane) (PDMS) layer. Matter? this one stayed selected for its favorable properties in relation to the conduct of research biological. It is transparent in a wide range of electromagnetic waves, non-toxic, gas-permeable and enables the mapping of microstructures with large dimensions. accuracy. Glass as a material hydrophilic provides the basics? for cell adhesion and proliferation. On the other hand, hydrophobic PDMS contains in its structure microchannels and culture microcells obtained using the replication method. In the poly (dimethylsiloxane) layer there is Aug? three, not connected together main microchannels. Every microchannel? main has an inlet at one end and contains six culture microcells for cell culture in monoculture. At the other end of each main microchannel there is a microchannel? with two additional culture microchamber each for culturing a particular cell line, the microchannels going downstream each. si? at one point, and furthermore, these microchannels are with you? connected by two transverse microchannels of different lengths running through additional culture microcells for the formation of cell co-culture and migration observation. The microchannels run from the confluence point of the microchannels? the outlet ends with an outlet opening and along the length of this canal there are si? the last three growing microcells, where are mixing the three cell lines to a varying degree in the step of introducing cell suspensions.

Preferably wide ?? and high ?? According to the utility pattern, the microchannels are 150 µm and 100 µm, respectively, and the diameter of the round microcells is 1 mm.

The three main microchannels with inlet holes are to introduce cell suspensions and fresh culture medium to provide the cells with the necessary nutritional properties. At the end of the micro-pattern there is kana? an outlet with an opening enabling the flushing fluids to be drained from the microcircuit.

Moreover, the geometry of the microchannel network in the developed microsystem was adapted to the measuring system of the multi-well plate reader and corresponds to the arrangement of the wells on a commercially available 384-well plate. Therefore, with the use of a special coin holder, it is possible to direct spectrofluorimetric measurements in a multi-well plate reader, which significantly increases the efficiency of conducted research? Thanks to the use of microfluidic conditions, the physiological environment of cell growth is much better mapped compared to cell cultures carried out in the classical laboratory scale (macroscale).

Beings? the microchip according to the pattern is possible culturing in a monolayer of any three, both cancerous and normal, cell lines in various configurations. This allows you to observe cellular migration, which is extremely important in the process of neoplasm. The microchip according to the utility formula also gives possibility ?? of observation affect? intercellular cells in co-culture (e.g., a normal tumor cell), where cells can with you? affect? on the road, eg sending death signals. The creation of such a co-culture is possible due to the phenomenon of hydrodynamic focusing in a microscale. Beings? The formula is that by controlling the flow rates of the injected streams, it is possible to obtain co-cultures composed of two and three cell lines in different proportions. According to the formula, the flow microsystem may have application at the research stage? preclinical drug screening.

The microsystem has been shown in the drawing, in which Fig. 1 shows the microsystem in an assembled perspective view, Fig. 2 shows the circuit board Fig. 3 shows the microsystem in a section along the bottom section. channel 8b.

According to the utility formula, the microsystem consists of with two permanently connected layers: a glass plate 1 with dimensions of 80 × 50 mm and a layer of poly (dimethylsiloxane) (PDMS) 2. Material? this one stayed selected for its favorable properties in relation to the conduct of research biological. It is transparent in a wide range of electromagnetic waves, non-toxic, gas-permeable and enables the mapping of microstructures with large dimensions. accuracy. Glass as a material hydrophilic provides the basics? for cell adhesion and proliferation. On the other hand, hydrophobic PDMS contains in its structure microchannels and culture microcells obtained using the replication method. The polymer layer contains Aug? three, not connected together main microchannels 8a, 8b and 8c. Every microchannel? the main one has an inlet 3a, 3b and 3c at one end and includes six lines respectively. culture microcells 5a, 5b and 5c, respectively, for culturing cells in a monoculture. At the other end of each main microchannel 8 there is a microchannel? 8 'with two additional culturing microcells 6a, 6b and 6c, respectively, each for culturing a particular cell line, the microchannels going downstream. si? at one point, and furthermore, these microchannels are with you? connected by two transverse microchannels 9 of different lengths running through additional growth microcells 6 for cell co-culture formation and migration observation. In the microcells 6b it is possible to obtain a co-culture by mixing three cell lines to varying degrees, while in the microcells 6a and 6c two lines, at the stage of introducing cell suspensions. A microchannel runs from the confluence of microchannels 8 '' an outlet 10 ended with an outlet 4 and along the length of this channel 10 there are si? the last three growing microchamber 7, where are mixing the three cell lines at the stage of introducing cell suspensions. Widely?? and high ?? all microchannels are 150 µm and 100 µm, respectively, and the diameter of all circular microcells is 1 mm.

The three main microchannels 8 together with 3 inlet openings serve to to introduce cell suspensions and fresh culture medium to provide the cells with the necessary nutritional properties. At the end of the micro-pattern there is kana? an outlet 10 together with an opening 4 for draining flush fluids from the microcircuit.

Creating a microsystem according to a utility pattern consists in designing a microsystem in the program, e.g. SolidWorks, with the accuracy of dimension mapping of? 1 µm. Then, this model is translated into machine language using the CAM module - SolidCAM. The microsystem was prepared in poly (dimethylsiloxane) (PDMS) by methods replication. The method consists in cross-linking the PDMS prepolymer (mixture of monomer with a cross-linking agent) in a suitable form that is a negative reflection of the final element. There are many methods of making molds, among which the predominant ones are photolithography based methods when? assure they are very big? resolution, and also allow for fabrication of structures smaller than 1 µm. Undoubtedly? disadvantages? of these methods, however, is their multi-stage, and also difficult? in obtaining structures of different heights.

The form of the microsystem is made by the method? mechanical with the help of micro-milling. Form? prepared in polymethyl methacrylate (PMMA). As matter? base form used? made of PMMA with dimensions of 100 mm × 100 mm and a thickness of 5 mm. In the first step, a rectangular square was fabricated in the center of the base plate. pocket? with dimensions (80 × 50 mm) and a depth of 3.9 mm. Pocket? was made with a rotating end mill with speed? 10,000 rpm and a moving speed along the longitudinal axis with speed? 500 mm / min. Then, directly at the bottom of the previously made pocket, the appropriate shape was milled. stamp by removing the 100 µm layer of material. Stamp t? was made with a 500 mm diameter cutter, spinning at a speed of 12,000 rpm and a moving speed in a plane parallel to the milled surface with a linear speed? 500 mm / min. The cutter overlap on adjacent passes was set to 70%. As a result of these actions? arose convex model of microchannels and chambers 100 µm high. So made? After removing the chips, washing and drying, the prepolymer was poured and allowed to cross-link at 70 ° C for 1 h. from a PDMS block, getting a ready element with faithfully mapped microchannels. After the PDMS layer was frozen in liquid nitrogen, inlet and outlet openings were drilled. Then, after cleaning and drying, the PDMS plate was connected to the PDMS plate. glass using the low-temperature oxygen plasma technique.

After placing the tubing in the inlet and outlet openings, the microcircuit was subjected to sterilization by UV radiation and the action of 70% ethanol. Three separate cell suspensions were introduced through the inlet holes into the microcircuit prepared in this way: A375, HaCaT and MeWo. After 24 hours, a photosensitizer - a drug used in anti-cancer photodynamic therapy - was introduced through the inlet holes, and its cytotoxic and phototoxic effects were examined. For this purpose, the fluorescent dye AlamarBlue was introduced into the microcircuit and a fluorimetric measurement was performed using a multi-well plate reader.

Claims (1)

Multifunctional flow microsystem consisting of from two permanently connected layers: a glass plate and a poly (dimethylsiloxane) layer, one of the plates containing microchannels with culture microcells and inlet and outlet openings, characterized in that in the poly (dimethylsiloxane) layer (2) there is Aug? three, not connected together main microchannels (8a, 8b and 8c) having an inlet port (3a, 3b and 3c) at one end, respectively, and including six lines culture microcells (5a, 5b and 5c) respectively, and at the other end of each main microchannel (8a, 8b and 8c) is microchannel? (8 ') with two additional growth microchamber (6a, 6b and 6c) respectively, with the microchannels (8') coming down from the si? at one point, and furthermore, these microchannels are with you? connected by two transverse microchannels (9) of different lengths running through additional breeding microchamber (6), and a microchannel extending from the junction point of the microchannels (8 ') outlet (10) ended with an outlet opening (4) and along the length of this channel (10) there are si? the last three breeding microchamber (7). The microsystem according to claims According to claim 1, characterized in that the width and high ?? all microchannels are 150 µm and 100 µm, respectively, and the diameter of all circular microcells is 1 mm.