Device and method for culturing and testing tissue sections

Abstract

A fluidic, in particular microfluidic, device (100) for cultivating and testing tissue sections (2) is described, comprising a plurality of fluidically interconnected strands (10), wherein each strand (10) is formed by a plurality of cultivating elements (50) fluidically interconnected in series, and wherein each cultivating element (50) comprises at least one supply channel (4a, 4a') and at least one discharge channel (4b, 4b'), as well as a cultivating chamber (1) into which a tissue section (2) can be introduced, and wherein all cultivating elements (50) of a strand (10) are uniformly fluidically addressable or wherein each cultivating element (50) is individually fluidically addressable.

Description

The present invention relates to a fluidic, in particular microfluidic device, a method for operating the same and to a cartridge comprising the microfluidic device, according to the preamble of the independent claims.Prior ArtIn the case of cancers, the exact tumor type or cell type is determined, inter alia, by biopsy or by examination of the operably removed tumor in pathology and then, on the basis of these results, in combination with earlier experiences, for example by studies, it is decided about the further, in particular chemotherapeutic, treatment. In order to make the cancer therapy more successful for the patients, there are various approaches. The subject here deals with personalized medicine. The most suitable treatment method is to be found individually for the patient. This is possible, for example, by drug tests on tissue removed from the patient. In order to be able to carry out (medicament) tests on the removed tissue, this is, for example, first cultivated further. For this purpose, the tissue can be broken down into tissue sections and these can be kept alive in a culture.If tissue sections are used, these are placed, for example, in so-called cultivation inserts and are cultivated statically in nutrient medium in an incubator for a few days. In this case, the nutrient medium must regularly be exchanged manually. Different tissue sections can be tested with different active ingredients, wherein only a subsequent retrospective analysis of the effect can be made.In addition to the classic cultivation in cultivation inserts, cultivation options in microfluidics are also increasingly being studied.So-called lab-on-a-chip systems, LoC systems for short, are microfluidic systems which accommodate functionalities of a macroscopic laboratory on a plastic substrate for automated processing. Such systems make it possible to process biochemical processes in a largely or completely automated manner.Lab-on-a-chip systems typically comprise a test carrier, for example in the form of a cartridge, which comprises structures and mechanisms for the manipulation of a picked sample.US 2018 / 0274020 describes a method and apparatus for rapidly assessing whether a microorganism present in a sample is susceptible or resistant to treatment.US 2020 / 0080050 discloses a system for growing cells comprising a bioreactor chamber, a delivery system which supplies a perfusion solution to the bioreactor chamber, a dialysis system and a filter for reducing the ammonia content in the dialysate.Disclosure of the InventionTissue taken from a body, in particular tumor tissue, also maps the heterogeneity of the tumor as a tissue section. Testing of substances, in particular active substances, over the entire section is therefore expedient. To produce the tissue sections, the tissue is usually divided by means of a vibratom into tissue sections 100-500 μm thick, and preferably 150-300 μm thick.According to the invention, a fluidic, in particular microfluidic device for culturing and testing tissue sections, a method for operating the same and a cartridge comprising the fluidic device having the features of the independent claims are provided.This is based in particular on the fact that the fluidic, in particular microfluidic, device comprises a plurality of strands connected fluidically to one another, wherein each strand is formed by a plurality of culturing elements connected fluidically in series to one another. For example, the fluidic device comprises four strands, wherein each strand in turn has, for example, three culturing elements which are fluidically connected to one another and are arranged one after the other. Each cultivation element comprises at least one feed channel and at least one discharge channel, which can be opened and closed in particular via valves, as well as a cultivation chamber into which at least one tissue section can be introduced. For this purpose, the culture elements have, for example, a receptacle for the tissue sections, so that they are held firmly during the culture and testing and good fluid contact is ensured without the tissue sections being damaged. For this purpose, the acquisition for tissue sections can be realized via frames, nets, grids, filters, embedding in hydrogels, adhering to a carrier or otherwise.All culture elements of a strand are uniformly fluidically addressable, i.e. all culture elements of a strand experience the same fluidic supply, whereby multiple determination is possible. Alternatively or additionally, each cultivation element is individually fluidically addressable.By integrating valves, a targeted control of the desired fluid channels can be implemented. Here, for example, valves in the fluidic inlet and / or outlet channels of the cultivation elements and / or valves are integrated into a main inlet and / or main outlet channel. The main feed channel is understood to mean a fluidic network which is located upstream of the plurality of strands of the fluidic device. The main feed channel comprises, for example, fluidic, in particular microfluidic channels, which lead from at least one fluidic inlet or a reservoir to the respective strands.The main discharge channel is understood to mean a fluidic network which is located downstream of the plurality of strands of the fluidic device. The main discharge channel comprises fluidic, in particular microfluidic channels, which lead from the respective strands, for example, to a fluidic outlet, a reservoir, a waste container and / or a collecting container.Here, the terms "downstream" and "downstream" mean two opposite directions. In this case, downstream means the direction coming from a fluidic inlet or a reservoir via the main feed duct to the strands and leading through them, further to a main discharge duct and from this to a fluidic outlet, a reservoir, a waste container and / or a collecting container, "upstream" means the opposite direction. This is intended to describe the arrangement of the various components and not necessarily the flow of a fluid.For example, each strand comprises a pump, in particular a membrane pump, upstream of the cultivation elements. Alternatively, a central pump can also transport the liquids, for example from the at least one reservoir, via the fluidic network controlled in particular by valves into and through the cultivation elements.An advantage of the fluidic, in particular microfluidic, device is that it allows culturing and analysis of still living removed tissue in the form of tissue sections, for example tumor tissue sections. This allows direct, so-called "live" detection of the reaction of the tumor cells in the tissue section to different substances, such as, for example, medicaments, for example in different concentrations and combinations of potential active substances. It is furthermore advantageous that the culture and analysis of the tissue sections is automated by means of the device according to the invention, whereby the culture and analysis is reproducible and comparatively comfortable. This also makes it possible, for example, to prevent contamination which can arise as a result of manual human laboratory work.Further advantageous embodiments of the microfluidic device are evident from the dependent claims.In an advantageous embodiment, the cultivation elements each comprise two feed channels and two discharge channels. In this case, a first feed channel forms a direct fluidic connection from the adjacent element in the strand located upstream, for example from the preceding culturing element or from a feeding channel of the main feed channel, to the respective culturing element.Furthermore, at least one first discharge channel forms a direct fluidic connection from the cultivation element to the next element in the strand, for example to the adjacent cultivation element located downstream or a channel of the main discharge channel.In addition, a second feed channel forms an indirect fluidic connection from the upstream main feed channel of the device to the respective culturing element.A second discharge channel forms an indirect fluidic connection from the culturing element to a downstream main discharge channel.In this context, a direct connection means a connection which connects elements of the fluidic device located directly adjacent to one another in a direct way. By an indirect connection is meant such a connection that connects elements to one another that are not necessarily located adjacent to one another and that may not be connected to one another directly.Alternatively, in a particularly advantageous embodiment, only one feed channel 4 ais implemented for each first culturing element of each strand. The second feed channel can be omitted, since it would feed the same liquids as the first feed channel. Likewise, for the last culturing element of each strand, only one discharge channel can be realized. The second discharge channel can be omitted, since this would discharge the same liquids again the first discharge channel.A plurality of the cultivation elements thus has at least two feed channels and / or at least two discharge channels. The cultivation elements located centrally in the strand each have two feed channels and two discharge channels.It is advantageous here that each cultivation element is fluidically addressable uniformly both with the other cultivation elements in the strand and is fluidically addressable individually independently of the other cultivation elements in the strand. This allows a very defined and flexible analysis and characterization of the tissue sections.In an alternative embodiment, the cultivation elements comprise only a first fluidic feed channel and a first fluidic discharge channel, so that all cultivation elements of a strand are uniformly fluidically addressable. It is advantageous here that this device is of less complex design.In a particularly advantageous embodiment, the fluidic device further comprises at least one sensor located downstream at least each strand, which is designed to measure a parameter, in particular an oxygen content and / or a carbon dioxide content and / or a glucose content and / or a lactate content and / or a pH value, in a discharged liquid of each strand.Alternatively, the fluidic device comprises at least one sensor located downstream of all strands, which is designed to measure a parameter in a discharged liquid of all strands.Furthermore alternatively, the fluidic device comprises a sensor located after each culturing element, which is designed to measure a parameter in a discharged liquid of the respective culturing element.It is advantageous here that the effect of different substances on the tissue sections can be measured and analyzed in this way. For example, the oxygen content in the nutrient medium allows conclusions to be drawn about the viability of the cells and their cell activity. Moreover, changes in the metabolism of the pulp can be detected by measuring the glucose content, in particular in combination with the measurement of the oxygen content, and a distinction can be made, for example, between aerobic and anaerobic combustion. A measurement of the lactate content serves, for example, to distinguish between normal cells and tumor cells.If these parameters are compared, in particular in a time-resolved manner, in parallel in the different fluidic strands with a respective plurality of tissue sections which are exposed to different medications, this allows valuable conclusions to be drawn about the sensitivity of the tumor cells to the different treatment options. This advantage naturally also applies to the comparison of parameters in the discharged liquid of each cultivation element.In all embodiments mentioned here, the integrated sensor system allows monitoring of the influence of the various substances already during their application (in situ) in narrow-mesh time segments.Furthermore, low-risk research or evaluation of new active ingredients is possible in this way in direct comparison with established methods.A further advantageous embodiment provides that the fluidic device comprises at least one reservoir for liquids, in particular for at least one rinsing liquid and / or at least one nutrient medium, and / or a calibration liquid and / or active substance solutions and / or coloring reagents. Furthermore, the apparatus comprises, for example, a waste container for waste products. The reservoir or waste container serves in particular for receiving, buffering and / or mixing liquids. It is advantageous here that the required liquids can already be stored in front and thus a rapid, efficient and low-contamination addition of these liquids is possible.In a further advantageous embodiment, the fluidic device has at least one side channel which leads from the main feed channel to a main discharge channel and serves for flushing the fluidic channels and / or the at least one sensor and / or for providing a calibration liquid for the at least one sensor. In this case, the side channel does not pass through the cultivation elements.It is advantageous here that the at least one sensor can thus be calibrated in a simple and time-saving manner at the beginning of the measurement and, if required, also intermittently with reference solutions and / or the fluidic channels and / or the at least one sensor can be rinsed at the beginning or if required.In an advantageous embodiment, the fluidic device has a collecting container, via which a defined liquid volume of a discharged liquid can be removed for further analyses. In this way, the measurement results are comparable and reproducible.Advantageously, the collecting container is coupled, in particular via valves, to a waste container into which the liquid not discharged via the collecting container is discharged.In addition, in a further advantageous embodiment of the fluidic device, a respective removal point for a liquid is arranged before and after the culturing chamber, in particular of each culturing element. It is advantageous here that the supplying or discharging liquid of the culturing element can thus be examined and further analyzed before and after a substance, in particular an active substance, comes into contact with the tissue section in the culturing element. In this way, direct and very precise conclusions can be drawn about the effect of the respective substance.The invention furthermore relates to a method for culturing and testing tissue sections, comprising the following steps: a) introducing tissue sections into the culturing elements. In this case, preference is given to eacha tissue section is introduced into each culture element. Alternatively,for example, two, three or more tissue sections each into a culture elementintroduced.b) Application of a different substance in each of the strands, in particular of at least one active ingredient in a defined concentration and / or combination and / or of a coloring reagent. c) Measurement of at least one parameter, in particular of the oxygen contentand / or the glucose content and / or the lactate content and / or the pH value,in the discharged liquid of a culturing element, a strand and / or all strands and / or measuring a fluorescence of staining reagents bound to specific markers of the tissue sections.The term application in step b) is understood here to mean that the respective liquid is guided or applied in a defined quantity and with an adjustable flow profile, i.e. the flow rate, from a reservoir or an external fluidic connection via the fluidic main feed channel into the respective strands.The method for culturing and testing or examining the tissue sections in the device according to the invention is carried out, for example, for several hours or even several days, depending on the question.In the meantime, washing steps can be carried out, for example, using phosphate-buffered saline solution (PBS). The substance applied in step b) can also be, for example, in particular temporarily, a nutrient medium, such as, for example, Dulbecco's Modified Eagle Medium (DMEM). The exact composition of the nutrient medium can be adapted in each case to the tumour type to be investigated.In the method according to the invention, for example, culture elements of different strands are subjected to different treatment patterns. A treatment pattern can be understood here to mean, for example, the administration of a specific concentration of a medicament over a defined time. In this context, different active ingredients, active ingredient combinations, active ingredients or concentrations alternating over time are conceivable as treatment patterns.The effect of the different treatment patterns on the tissue sections can be observed "live" and automatically with the aid of measurements described below.By measuring the oxygen content, for example in the nutrient medium, changes in the oxygen content per unit time allow conclusions to be drawn about the viability of the cells in the tissue section and their cell activities.By means of the measurement of the glucose content, in particular in conjunction with the measurement of the oxygen content, changes in the metabolism of the pulp can be detected and, for example, aerobic and anaerobic combustion can be differentiated.The measurement of the lactate content can be used to distinguish the metabolism of normal cells (aerobic glycolysis with citrate acid cycle in the mitochondria) from that of tumor cells, in which glycolysis frequently takes place with subsequent excretion of lactate ("Warburg effect").If these parameters are compared, in particular in a time-resolved manner, in parallel in the various fluidic strands with a respective plurality of tissue sections which are exposed to different medications, this allows valuable conclusions to be drawn about the sensitivity of the cells, in particular of the tumor cells, of the tissue section to the different treatment options.It is also advantageous that the integrated sensor system (measurement of the content of oxygen, carbon dioxide, glucose, lactate and / or the pH) allows monitoring of the influence of the various treatment patterns already during their application (in situ) in narrow-mesh time segments. It is also advantageous that the method according to the invention can also be used for the low-risk investigation or evaluation of new active ingredients in direct comparison with established methods.Staining of biomarkers of the tissue sections by means of staining reagents binding to the tissue sections can already take place during the method according to the invention. In this case, proliferation markers and / or live and dead dyes are used, for example. Furthermore, the nuclei can be stained, for example, in order to enable a clear assignment of further signals and the cell position in the tissue section. This is particularly appropriate with always identical, fixed markings. For the input of the coloring reagents, the fluidic path is adapted, for example, in such a way that the addition of small amounts of coloring reagents is possible. These can either bind directly to, in particular tumor-specific, markers or are administered via a coupling to antibodies. In order to ensure their analysis, optical and / or sensory evaluation is carried out, for example. Conventionally, the evaluation takes place via an optical observation with a fluorescence microscope, so that the acquisition of the tissue sections of the culture elements is designed at least partially optically transmissive in this case.Alternatively, the staining of the biomarkers of the tissue sections takes place only after their removal from the fluidic device.In a particularly advantageous embodiment of the method, one of the strands of the fluidic device serves as a negative control, so that only a nutrient medium is applied therein as a reference.It is advantageous here that in this way a comparison point of the cultivation with the other strands to which a substance is applied is set.The negative control additionally provides early information about viability and aging effects under the conditions set or a possible or necessary change requirement in the culture conditions set.Furthermore, in a particularly advantageous embodiment, step a') takes place after step a):a') uniform temperature control of all strands and supply of all strands with nutrient medium for a predefined time.After the tissue sections have been introduced into the cartridge, a stabilization phase advantageously follows first, in which all tissue sections are uniformly tempered and supplied with nutrient medium in order to produce defined and uniform initial conditions. This step extends for example over 1-3 hours, but can also take up to 12 hours. In this case, the metabolism of the tissue sections is monitored and compared, for example, via the built-in oxygen sensor system. Optionally, glucose consumption and / or lactate concentration is additionally monitored.In a further advantageous embodiment, the nutrient medium is enriched with oxygen and / or carbon dioxide, since the oxygen and carbon dioxide supply of the tissue sections can thus take place via liquids. In this way, optimum conditions can be set with respect to the available oxygen or carbon dioxide quantity in the tissue sections. The concentrations to be adjusted for this purpose are strongly dependent on the tissue type of which the tumor is examined. For example, the oxygen demand of liver cells is comparatively high, so that an additional oxygen storage (hemoglobin analog) can also be used for these in part. In the fluidic arrangement, the demand for oxygen can be advantageously adapted and ensured by increased supply of nutrient medium. For this purpose, the nutrient medium is enriched in particular with oxygen, for example up to more than 80% of the respective solubility limit of the gas for the nutrient medium used at the established cultivation temperature.A further advantageous embodiment provides that a gas is conducted into the strands of the fluidic device, so that an exchange with a gas atmosphere can take place. Thus, the cells of the tissue section can absorb the required amount of oxygen and carbon dioxide particularly well.In addition, in a further advantageous embodiment, it is provided that in a further step d) of the method, the measured parameters are evaluated and, if appropriate, the application of the substance, in particular of the active substance, into at least one strand or into at least one cultivation element is adapted.By measuring the oxygen, glucose and / or lactate content and optionally the pH, the influence of different treatment patterns on the cells of the tissue section can be monitored in short time periods.This allows for example variations of the treatment patterns during a longer phase: if, for example, no measurable change occurs among any of the treatment patterns, the active ingredient concentrations can be increased, for example. If the sensor signals show strong deviations between the strands with different treatment patterns, individual tissue sections can be taken from the receptacle of the cultivation chamber for a more detailed analysis, for example.In a further advantageous embodiment, in a step e) following step b) and / or c) and / or d), a liquid is removed via the respective removal site for analysis before and / or after a culturing element.It is advantageous here that, thus before and after a substance, in particular an active substance, comes into contact with the tissue section in the cultivation element, the liquid supplied to it or the liquid discharged from it can be examined and analyzed further. In this way, direct and very precise conclusions can be drawn about the effect of the respective substance on the tissue section.After the end of the fluidic, in particular microfluidic, cultivation and testing of the tissue sections in the device, these can be removed and, as in the case of previous static cultures, classically colored, embedded and retrospectively examined using all desired methods.Furthermore, individual tissue sections can be divided into smaller fragments or individual cells following the method, in order to then provide these in a targeted manner for further analyses. Depending on the intended analysis, it is possible to dissolve the tissue in individual cells or even completely lyse the cells, which then allows the analysis of cell constituents, deoxyribonucleic acid (DNA), mRNA (messenger ribonucleic acid) and enzymes.A further possibility for analysis is possible via integration of already established assays. Here, for example, those are conceivable which only analyze the culture medium used. An example of this is the lactate dehydrogenase (LDH) assay. This can be used to measure the amount of lactate dehydrogenase released from destroyed cells.Furthermore, the fluidic, in particular microfluidic, method according to the invention is controlled, for example, by means of a control unit, for example by an electrical actuation of at least one valve and / or at least one pump.The invention further relates to a cartridge, in particular a cartridge, as described for example in DE102016222072A1 or DE102016222075A1 comprising the fluidic, in particular microfluidic device according to the invention.The pumps and valves of the device according to the invention are realized, for example, by deflecting an elastic membrane by means of compressed air, and the media reservoirs can be imaged via so-called reagent bars, as described in patents EP2322277 B1 and DE102011078770 B4. The introduction of samples by the user can also be realized, for example, as described therein.If the pumps are realized as small membrane pumps for one line each, a respectively following line with cultivation elements can be connected in modular fashion. This offers the advantage that the number of strands can be varied, in particular increased, if more tissue cuts are present and a larger number of treatment scenarios are to be tested.BRIEF DESCRIPTION OF THE DRAWINGEmbodiments of the present invention are illustrated in the drawing and explained in more detail in the following description of the figures. It shows: FIG. 1 : shows the schematic illustration of a cultivation element in a first embodiment with two feed channels and two discharge channels in a cross section, FIG. 2 : shows the schematic illustration of a fluidic, in particular microfluidic, device according to the invention in a first embodiment with cultivation elements according to FIG. 1 in a cross section, FIG. 3 : shows the schematic illustration of a fluidic, in particular microfluidic, device according to the invention in a second embodiment in a cross section, FIG. 4 : shows the schematic illustration of a cartridge according to the invention comprising the microfluidic device according to FIG. 2, and FIG. 5 : shows the schematic illustration of an exemplary embodiment of the method according to the invention.Embodiments of the InventionFIG. 1 shows a cultivation element 50 with a cultivation chamber 1. the cultivation chamber 1 comprises a receptacle 3 for a tissue section 2, for example for a tumor tissue section, so that the tissue section is held firmly during the cultivation and testing and good fluid contact with the tissue section 2 is ensured.The culture element 50 further includes a first supply channel 4 aand a first discharge channel 4 b. These extend, for example, vertically on opposite sides of the cultivation chamber 1 and are connected thereto in such a way that a direct fluidic connection runs from the first feed channel 4 athrough the tissue section 2 to the first discharge channel 4 b. Furthermore, the cultivation element 50 comprises a second feed channel 4a' and a second discharge channel 4b', which are likewise arranged on opposite sides of the cultivation chamber 1, in particular at a defined angle to the first feed and discharge channel 4a, 4b. A valve 5 is introduced into each of the inlet and outlet ducts 4a, 4a', 4b, 4b', so that these ducts can be individually opened and closed.FIG. 2 shows a fluidic, in particular microfluidic, device 100 according to the invention for culturing and testing tissue sections 2 in a first embodiment with culturing elements 50 according to FIG. 1. Three successive cultivation elements 50 form one strand 10 in each case. the fluidic device 100 comprises four such strands 10, which are fluidically connected to one another via a main feed channel 4 cand a main discharge channel 4 d.The main feed channel 4 cis formed from a fluidic network which is located upstream of the strands 10. The main feed channel 4 ccomprises fluidic, in particular microfluidic, channels which, in FIG. 2, lead from different reservoirs 7 a, 7 b, 7 c, 7 d, 7 eto the different strands 10 and comprise valves 5, via which the individual channels of the main feed channel 4 cmay be individually opened and closed.In the reservoirs 7 a, 7 b, 7 c, 7 d, 7 e, in particular, liquids are arranged upstream, for example a rinsing liquid and / or at least one nutrient medium, and / or a calibration liquid and / or active substance solutions and / or coloring reagents.Furthermore, a pump 9, in particular a membrane pump, is arranged upstream of each strand 10, which pump conveys a liquid from at least one reservoir 7 a, 7 b, 7 c, 7 d, 7 evia the fluidic network of the main feed channel 4 cin and through the cultivation elements 50. Alternatively and not shown in FIG. 2, instead of the pumps 9, a central pump can also convey the liquids for each strand 10.The main discharge channel 4 dis formed from a fluidic network which is located downstream of the strands 10. The main discharge channel 4 dcomprises fluidic, in particular microfluidic, channels which lead from the respective strands 10 to a fluidic outlet, a waste container 13 and / or a collecting container 15, not shown. The individual channels of the main discharge channel 4 dcomprise valves 5, by means of which the individual channels of the main discharge channel 4 dmay be individually opened and closed.In the channels of the main discharge channel 4d, sensors 11a, 11b, 11c, 11d are furthermore arranged. Here, in each case a first sensor 11 ais located directly downstream of each strand 10, which is designed to measure a parameter, in particular an oxygen content, in a discharged liquid of the respective strand 10. Alternatively or additionally, the sensor 11 ais designed to measure a glucose content and / or a lactate content and / or a pH value in the discharged liquid of the respective strand 10. Furthermore, in FIG. 1, further sensors 11 b, 11 c, 11 dare arranged in a channel of the main discharge channel 4 d, which can be passed by the discharged liquid of all strands 10. These sensors 11 b, 11 c, 11 dare designed, for example, to measure an oxygen content, a carbon dioxide content, a glucose content, a lactate content and / or a pH value. Each line 10 further comprises a side channel 4 e, which leads from the main feed channel 4 cto the main discharge channel 4 dand serves for flushing the fluidic main channels 4 c, 4 dand the sensors 11 a, 11 b, 11 c, 11 dbetween the measurements and / or for providing calibration fluids for the sensors 11 a, 11 b, 11 c, 11 d. Liquid that is guided via one of the side channels 4 edoes not pass through the cultivation elements 50.Liquids that are no longer required can be collected in at least one waste container 13. The waste containers 13 shown in FIG. 2 can also be combined in one container and have only two alternative feeds.In particular, liquids such as, for example, the nutrient medium are collected in the collecting container 15, which can then be provided outside the fluidic device 100 for further analyses.The culture elements 50 each comprise two feed channels 4a, 4a' and two discharge channels 4b, 4b'.The first feed channel 4 aconstitutes a direct fluidic connection from the main feed channel 4 cto the first culturing element 50 of each strand 10.The first discharge channel 4 bof the cultivation elements 50 forms a direct fluidic connection to the adjacent cultivation element 50 located downstream or, in the case of the last cultivation element 50, in the strand 10 to the main discharge channel 4 d.Via the first feed channel 4 aand the first discharge channel 4 b, all culture elements 50 of each strand 10 are uniformly fluidically addressable. Thus, all culture elements 50 of a strand 10 are supplied with the same fluidic supply, whereby multiple determination is possible.Furthermore, the cultivation elements 50 comprise a second feed channel 4a', which forms an indirect fluidic connection from the upstream main feed channel 4c of the device 100 to the respective cultivation element 50. Moreover, the cultivation elements 50 comprise a second discharge channel 4b' which forms an indirect fluidic connection from the respective cultivation element 50 to a downstream main discharge channel 4d. Above the second feed channel 4a' and the second discharge channel 4b', each cultivation element 50 of a strand 10 is individually fluidically addressable.In an alternative advantageous embodiment, not shown in FIG. 2, only one feed channel 4 ais realized for each of the first cultivation elements 50 of each strand 10. The second feed channel 4a' is omitted, since it feeds the same liquids as the first feed channel 4a. Furthermore, for the respective last cultivation element 50 of each strand 10, only one discharge channel 4 bis realized. The second discharge channel 4b' is omitted in each case, since it discharges the same liquids as the first discharge channel 4b.Before and after the culture chamber 1 of the culture elements 50, for example, a liquid withdrawal site not shown in FIG. 2 is arranged.First, in a first method step a), one tissue section 2 each is introduced into one culture element 50 each, so that the device 100 comprises twelve tissue sections 2 for culture and testing.Optionally, in a method step a'), a stabilization phase follows subsequently, in which all tissue sections 2 are uniformly tempered for 1-3 hours, for example, and supplied with nutrient medium in order to produce the same starting conditions. For this purpose, a nutrient medium is conveyed into the strands 10 for example by means of the pump 9 from at least one of the reservoirs 7 a, 7 b, 7 c, 7 d, 7 e, such that the tissue sections 2 in the cultivation elements 50 are supplied with the latter. In this case, for example, the oxygen content and thus the metabolism of the tissue sections 2 can be monitored and compared via a sensor 11 a. Optionally, the glucose consumption and / or the lactate concentration in the medium discharged from the cultivation elements 50 can also be measured additionally.Subsequently, in a method step b), the different strands 10 and thus three tissue sections 2 each are subjected to different treatment patterns. For this purpose, a specific concentration of a substance or of a medicament is applied over a defined time in a respective strand 10, in particular from at least one of the reservoirs 7 a, 7 b, 7 c, 7 d, via the main feed channel 4 cand the first feed channel 4 aof the cultivation elements 50. In this context, different active ingredients or active ingredient combinations, active ingredients or concentrations alternating over time, are conceivable as treatment patterns. A strand 10 is preferably further cultivated as a reference with the initial cultivation conditions. Thus, information is obtained about viability and aging under the prevailing conditions. Thus, in this example, three different active ingredients, three active ingredient combinations or three concentrations of an active ingredient can be tested.First, the liquid with the substance is conducted into the strands 10 with the tissue sections 2 and an action takes place for a defined time or a very low flow rate is set for further supply with the liquid containing the substance. Subsequently, a nutrient medium is fed into the strands 10 and finally flushed out of the cultivation chambers 50 again, so that in the discharged nutrient medium, in a method step c), for example, the oxygen content, the glucose content, the lactate content and / or the pH value can be measured via at least one sensor 11 adownstream of each strand 10 and / or downstream of all strands 10. During the supply of nutrient medium, the above-mentioned parameters can be measured repeatedly. In this case, either the discharged nutrient solutions of a strand 10 can be measured by means of the sensors, or individual cultivation elements 50 of a strand 10 are individually fluidically addressed via the second feed channel 4a' and the second discharge channel 4b', the liquid is exchanged and measured by means of the sensors.In addition, a staining reagent can be applied to strands 10 and the fluorescence of staining reagents bound to specific markers of tissue sections 2 measured.In process steps a') and / or b), the nutrient medium can be enriched, for example, with oxygen and / or carbon dioxide. In addition, in step b), an atmospheric gas can also be applied into the strands 10 of the fluidic device 100, so that an exchange with the gas atmosphere can take place.In addition, in a further optional step d), for example, the measured parameters are evaluated and, if appropriate, the application of the substance, in particular of the active substance, into at least one strand 10 or into at least one cultivation element 50 is adapted.FIG. 3 shows a fluidic, in particular microfluidic, device 100 according to the invention for culturing and testing tissue sections 2 in a second, simpler embodiment. In contrast to the first embodiment shown in FIG. 2, the device 100 comprises, instead of the pumps 9, for each strand 10, only one central pump 9 which conveys the liquids through the fluidic device 100. Alternatively and not shown, however, each line 10 can also have a pump 9.The main difference from the first embodiment shown in FIG. 2 is that the culture elements 50 comprise only a first supply channel 4 aand a first discharge channel 4 b. As a result, separate fluidic addressing of individual cultivation elements 50 is not possible in this embodiment, so that all cultivation elements 50 of a strand 10 always receive the same fluidic supply of supplied substances.Furthermore, the device 100 comprises in FIG. 3 a separate side channel 4 ewhich leads from the main feed channel 4 cto the main discharge channel 4 dor opens into a waste container 13, depending on which valves 5 are opened or closed. The separate side channel 4 ehas the same function as the side channel 4 edescribed with reference to FIG. 2.FIG. 4 shows a cartridge 1000 according to the invention, which comprises, by way of example, for all embodiments according to the invention of the microfluidic device 100, a microfluidic device 100 in the first embodiment according to FIG. 2.FIG. 5 shows a flow diagram of an exemplary embodiment of the method 800 according to the invention for culturing and testing tissue sections 2, in particular tumor tissue sections. In this case, the method steps a), a'), b), c) and d) in particular proceed, which are illustrated and described in / with respect to FIGS. 2 and 3.References included in the specificationThis list of documents cited by the applicant has been produced in an automated manner and is only included for the better information of the reader. The list is not part of the German patent application or utility model application. The DPMA does not take any adhesion for any faults or omissions.Patent Literature citedUS 2018 / 0274020

[0007] US 2020 / 0080050

[0008] DE 102016222072A1

[0070] DE 102016222075A1

[0070] EP 2322277 B1

[0071] DE 102011078770 B4

[0071]

Claims

Fluidic, in particular microfluidic device (100) for culturing and testing tissue sections (2) comprising a plurality of strands (10) which are fluidically connected to one another, wherein each strand (10) is formed by a plurality of culturing elements (50) which are fluidically connected to one another in series, and wherein each culturing element (50) comprises at least one feed channel (4a, 4a') and at least one discharge channel (4b, 4b'), and a culturing chamber (3) into which at least one tissue section (2) can be introduced, and wherein all culturing elements (50) of a strand (10) are uniformly fluidically addressable and / or wherein each culturing element (50) is individually fluidically addressable.Fluidic device (100) according to claim 1, wherein at least a plurality of the cultivation elements (50) each comprise two feed channels (4a, 4a') and / or two discharge channels (4b, 4b'), of which a first feed channel (4a) each forms a direct fluidic connection from the upstream adjacent element in the strand (10) to the cultivation element (50), and of which at least one first discharge channel (4b) each forms a direct fluidic connection to the downstream adjacent element in the strand (10), and of which a second feed channel (4a') each forms an indirect fluidic connection from the upstream main feed channel (4c) of the device (100) to the cultivation element (50), and of which a second discharge channel (4b') forms an indirect fluidic connection from the cultivation element (50) to a main discharge channel (4d) located downstream.Fluidic device (100) according to one of the preceding claims, wherein the fluidic device (100) comprises at least one sensor (11a, 11b, 11c, 11d) located downstream of at least each strand (10) and / or wherein the fluidic device (100) comprises at least one sensor (11a, 11b, 11c, 11d) located downstream of all strands (10), which is designed to measure a parameter, in particular an oxygen content and / or a carbon dioxide content and / or a glucose content and / or a lactate content and / or a pH value, in a discharged liquid of a respective culturing element (50), a strand (10) or all strands (10).Fluidic device (100) according to one of the preceding claims, wherein the fluidic device (100) comprises at least one reservoir (7a, 7b, 7c, 7d, 7e) for liquids, in particular for at least one rinsing liquid and / or at least one nutrient medium, and / or a calibration liquid and / or active substance solutions and / or coloring reagents, and wherein the at least one reservoir (7a, 7b, 7c, 7d, 7e) serves for receiving, buffering and / or mixing liquids.Fluidic device (100) according to one of the preceding claims, wherein the fluidic device (100) has at least one side channel (4e), which leads from the main feed channel (4c) to a main discharge channel (4d), for flushing the fluidic main channels (4c, 4d) and / or the at least one sensor (11a, 11b, 11c, 11d) and / or for providing calibration liquids for the at least one sensor (11a, 11b, 11c, 11d), wherein the cultivation elements (50) are not passed.Fluidic device (100) according to one of the preceding claims, wherein the device has a collecting container (15), via which a defined liquid volume of a discharged liquid can be removed, in particular wherein the collecting container (15) is coupled to a waste container (13), into which the liquid not discharged via the collecting container (15) is discharged and / or wherein before and after the cultivation chamber (1) of the cultivation elements (50) a removal point for a liquid is located in each case, by means of which a defined liquid volume can be removed.Fluidic, in particular microfluidic, method (800) for culturing and testing tissue sections (2) by means of a device (100) according to one of Claims 1-6, having the following steps: a) introducing tissue sections (2) into the culturing elements (50) b) applying a different substance in each case to each of the strands (10), in particular at least one active substance in a defined concentration and / or combination, of a nutrient medium and / or a coloring reagent. c) measuring at least one parameter, in particular the oxygen content and / or the carbon dioxide content and / or the glucose content and / or the lactate content and / or the pH, in the discharged liquid of a culturing element (50), a strand (10) and / or all strands (10) and / or measuring a fluorescence of staining reagents bound to specific markers of the tissue sections (2).Method (800) according to claim 7, wherein after step a) a step a') takes place: a') uniform temperature control of all strands (10) and supply of all strands (10) with nutrient medium for a predefined time.Method (800) according to one of claims 7 or 8, wherein the nutrient medium is enriched with oxygen and / or carbon dioxide above 80% of the respective solubility limit of the respective gas for the nutrient medium used at the established culture temperature.The method (800) according to any one of claims 7 - 9, wherein in step b) an atmospheric gas is directed into the strands (10) of the fluidic device (100).Method (800) according to one of claims 7 - 10, wherein in a further step d) an evaluation of the measured parameters takes place and optionally an adaptation of the application of the substance, in particular of the active substance, into at least one strand (10) or into at least one cultivation element (50) takes place.A cartridge (1000) comprising a microfluidic device (100) according to any of claims 1-6.

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