Device and method for cultivating and testing tissue sections

Abstract

The invention relates to a fluidic, in particular microfluidic device (100) for cultivating and testing tissue sections (2), the device comprising a large number of fluidically interconnected branches (10), wherein each branch (10) is formed by a large number of cultivation elements (50) fluidically interconnected in series, and wherein each cultivation element (50) comprises at least one supply channel (4a, 4a') and at least one discharge channel (4b, 4b'), and also comprises a cultivation chamber (1) into which a tissue section (2) can be introduced, and wherein all cultivation elements (50) of a branch (10) can be uniformly fluidically addressed or wherein each cultivation element (50) can be individually fluidically addressed.

Description

[0001] Description

[0002] title

[0003] Device and method for culturing and testing tissue sections

[0004] The present invention relates to a fluidic, in particular microfluidic, device, a method for operating the same and a cartridge comprising the microfluidic device, according to the preamble of the independent claims.

[0005] State of the art

[0006] In cancer, the exact tumor type or cell type is determined, among other things, by biopsy or by pathology examination of the surgically removed tumor. Then, based on these results and in combination with previous experience, for example from studies, further treatment, particularly chemotherapy, is decided upon. There are various approaches to making cancer therapy more successful for patients. The one examined here deals with personalized medicine. The aim is to find the most suitable treatment method for each individual patient. This is possible, for example, through drug tests on tissue taken from the patient. In order to carry out (drug) tests on the taken tissue, it is first further cultivated, for example. For this purpose, the tissue can be cut into tissue sections and these can be kept alive in a culture.

[0007] If tissue sections are used, they are placed in so-called culture inserts and cultured statically in a culture medium in an incubator for several days. The culture medium must be changed manually on a regular basis. Different tissue sections can be tested with different active substances, although only a subsequent, retrospective analysis of the effects is possible. In addition to traditional cultivation in culture inserts, cultivation options in microfluidics are increasingly being investigated.

[0008] So-called lab-on-a-chip systems, or LoC systems for short, are microfluidic systems that integrate the functionalities of a macroscopic laboratory onto a plastic substrate for automated processing. Such systems enable biochemical processes to be largely or completely automated.

[0009] Lab-on-a-chip systems typically comprise a test carrier, for example in the form of a cartridge, which includes structures and mechanisms for manipulating a sample.

[0010] US 2018 / 0274020 describes a method and apparatus for rapidly assessing whether a microorganism present in a sample is susceptible or resistant to treatment.

[0011] US 2020 / 0080050 discloses a system for culturing cells, comprising a bioreactor chamber, a delivery system that supplies a perfusion solution to the bioreactor chamber, a dialysis system, and a filter for reducing the ammonia content in the dialysate.

[0012] Disclosure of the invention

[0013] Tissue removed from the body, especially tumor tissue, reflects the heterogeneity of the tumor even in tissue sections. Therefore, testing substances, especially active ingredients, on the entire section is advisable. To prepare the tissue sections, the tissue is usually divided into 100–500 μm thick, and preferably 150–300 μm thick, sections using a vibratome.

[0014] According to the invention, a fluidic, in particular microfluidic, device for cultivating and testing tissue sections, a method for operating the same, and a cartridge comprising the fluidic device are provided, having the features of the independent patent claims. This is based, in particular, on the fact that the fluidic, in particular microfluidic, device comprises a plurality of fluidically interconnected strands, wherein each strand is formed by a plurality of fluidically interconnected cultivation elements in series. For example, the fluidic device comprises four strands, wherein each strand, for example, in turn has three fluidically interconnected, sequentially arranged cultivation elements.Each cultivation element comprises at least one supply 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 inserted. For this purpose, the cultivation elements have, for example, a holder for the tissue sections so that they are held securely during cultivation and testing, ensuring good fluid contact without damaging the tissue sections. The holder for the tissue sections can be implemented using frames, nets, grids, filters, embedding in hydrogels, gluing to a carrier, or other means.

[0015] All culture elements of a strand are uniformly fluidically addressable, meaning that all culture elements of a strand receive the same fluidic supply, allowing multiple determinations. Alternatively or additionally, each culture element can be individually fluidically addressed.

[0016] The integration of valves allows for targeted control of the desired fluid channels. For example, valves are integrated into the fluidic supply and / or discharge channels of the cultivation elements and / or valves are integrated into a main supply and / or discharge channel. The main supply channel is understood to be a fluidic network located upstream of the plurality of strands of the fluidic device. The main supply channel comprises, for example, fluidic, in particular microfluidic, channels that lead from at least one fluidic inlet or a reservoir to the respective strands.

[0017] The main drainage channel is understood to be a fluidic network located downstream of the plurality of strands of the fluidic device. The main drainage channel comprises fluidic, in particular microfluidic, channels that lead from the respective strands, for example, to a fluidic outlet, a reservoir, a waste container, and / or a collection container. The terms "downstream" and "downstream" refer to two opposite directions. Downstream refers to the direction coming from a fluidic inlet or a reservoir, via the main supply channel to the strands and through them, further to a main drainage channel and from there to a fluidic outlet, a reservoir, a waste container, and / or a collection container; "upstream" refers to the opposite direction.This is intended to describe the arrangement of the various components and not necessarily the flow of a fluid.

[0018] For example, each strand upstream of the cultivation elements comprises a pump, in particular a diaphragm pump. Alternatively, a central pump can also transport the liquids, for example, from the at least one reservoir, into and through the cultivation elements via the fluidic network, which is controlled in particular by valves.

[0019] The advantage of the fluidic, particularly microfluidic, device is that it enables the cultivation and analysis of still-living, removed tissue in the form of tissue sections, for example, tumor tissue sections. This allows for direct, so-to-speak "live" recording of the reaction of the tumor cells in the tissue section to different substances, such as drugs, for example, in different concentrations and combinations of potential active ingredients.

[0020] Another advantage is that the cultivation and analysis of tissue sections is automated using the device according to the invention, making cultivation and analysis reproducible and comparatively convenient. This also prevents, for example, contamination that can occur through manual human laboratory work.

[0021] Further advantageous embodiments of the microfluidic device emerge from the subclaims.

[0022] In an advantageous embodiment, the cultivation elements each comprise two supply channels and two discharge channels. A first supply channel forms a direct fluidic connection from the upstream adjacent element in the strand, for example, from the previous cultivation element or from a supply channel of the main supply channel, to the respective cultivation 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 downstream adjacent cultivation element or a channel of the main discharge channel.

[0023] In addition, a second supply channel forms an indirect fluidic connection from the upstream main supply channel of the device to the respective cultivation element.

[0024] A second drainage channel forms an indirect fluidic connection from the cultivation element to a main drainage channel located downstream.

[0025] A direct connection refers to a connection that directly connects adjacent elements of the fluidic device. An indirect connection refers to a connection that connects elements that are not necessarily adjacent to one another and that may not be directly connected to one another.

[0026] Alternatively, in a particularly advantageous embodiment, only one supply channel 4a is provided for the first cultivation element of each strand. The second supply channel can be omitted, since it would supply the same liquids as the first supply channel. Likewise, only one discharge channel can be provided for the last cultivation element of each strand. The second discharge channel can be omitted, since it would discharge the same liquids as the first discharge channel.

[0027] A majority of the cultivation elements thus have at least two supply channels and / or at least two discharge channels. The cultivation elements located centrally in the strand each have two supply channels and two discharge channels.

[0028] The advantage here is that each cultivation element can be fluidically addressed uniformly with the other cultivation elements in the strand, as well as individually and independently of the other cultivation elements in the strand. This allows for a highly defined and flexible analysis and characterization of the tissue sections. In an alternative embodiment, the cultivation elements comprise only a first fluidic supply channel and a first fluidic discharge channel, so that all cultivation elements in a strand can be fluidically addressed uniformly. The advantage here is that this device is less complex.

[0029] In a particularly advantageous embodiment, the fluidic device further comprises at least one sensor located downstream of 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.

[0030] 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.

[0031] Further alternatively, the fluidic device comprises a sensor located after each cultivation element which is designed to measure a parameter in a discharged liquid of the respective cultivation element.

[0032] The advantage of this approach is that the effects of different substances on tissue sections can be measured and analyzed. For example, the oxygen content in the culture medium allows conclusions to be drawn about cell viability and cell activity. Furthermore, measuring glucose levels, especially in combination with oxygen levels, can detect changes in cell metabolism and, for example, differentiate between aerobic and anaerobic combustion. Measuring lactate levels, for example, can be used to differentiate between normal cells and tumor cells.

[0033] Comparing these parameters, particularly in a time-resolved manner, in parallel in the various fluidic strands, each with multiple tissue sections exposed to different medications, allows valuable conclusions to be drawn about the sensitivity of tumor cells to the various treatment options. This advantage naturally also applies to comparing parameters in the drained fluid from each culture element. In all the embodiments mentioned here, the integrated sensors allow monitoring of the influence of the various substances even during their application (in situ) at close intervals.

[0034] Furthermore, this enables low-risk research and evaluation of new active ingredients in direct comparison with established procedures.

[0035] 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 ingredient solutions and / or staining reagents. Furthermore, the device comprises, for example, a waste container for waste products. The reservoir or waste container serves in particular for receiving, temporarily storing, and / or mixing liquids. The advantage here is that the required liquids can be pre-stored, thus enabling rapid, efficient, and low-contamination addition of these liquids.

[0036] In a further advantageous embodiment, the fluidic device has at least one side channel, which leads from the main supply channel to a main discharge channel and serves to flush the fluidic channels and / or the at least one sensor and / or to provide a calibration fluid for the at least one sensor. In this case, the side channel does not pass through the cultivation elements.

[0037] The advantage here is that the at least one sensor can be calibrated with reference solutions in a simple and time-saving manner at the beginning of the measurement and, if necessary, in between, and / or the fluidic channels and / or the at least one sensor can be rinsed at the beginning or if necessary.

[0038] In an advantageous embodiment, the fluidic device has a collecting container through which a defined volume of a drained liquid can be extracted for further analysis. This allows the measurement results to be comparable and reproducible.

[0039] Advantageously, the collection container is coupled, in particular via valves, to a waste container into which the liquid not drained via the collection container is discharged. Furthermore, in a further advantageous embodiment of the fluidic device, a withdrawal point for a liquid is arranged before and after the cultivation chamber, in particular of each cultivation element. The advantage here is that the supplying or draining liquid of the cultivation element can be examined and further analyzed before and after a substance, in particular an active ingredient, comes into contact with the tissue section in the cultivation element. In this way, direct and very precise conclusions about the effect of the respective substance are possible.

[0040] The invention further relates to a method for cultivating and testing tissue sections, comprising the following steps: a) Inserting tissue sections into the cultivation elements. Preferably, one tissue section is inserted into each cultivation element. Alternatively, for example, two, three or more tissue sections are inserted into each cultivation element. b) Applying a different substance to each of the strands, in particular at least one active ingredient in a defined concentration and / or combination and / or a staining reagent. c) Measuring at least one parameter, in particular the oxygen content and / or the glucose content and / or the lactate content and / or the pH

[0041] value in the drained liquid of a culture element, a strand and / or all strands and / or measuring fluorescence of staining reagents bound to specific markers of the tissue sections.

[0042] The term "application" in step b) refers to the fact that the respective fluid is delivered or applied in a defined amount and with an adjustable flow profile, i.e., the flow rate, from a reservoir or an external fluidic connection via the main fluidic supply channel into the respective strands. The process for culturing and testing or examining the tissue sections in the device according to the invention takes place, for example, for several hours or even several days, depending on the question.

[0043] In this case, washing steps can be performed in between, for example, with phosphate-buffered saline (PBS). The substance applied in step b) can also be a nutrient medium, such as Dulbecco's Modified Eagle Medium (DMEM), especially temporarily. The exact composition of the nutrient medium can be adapted in each individual case to the tumor type being examined.

[0044] In the method according to the invention, for example, culture elements of different strains are subjected to different treatment patterns. A treatment pattern can be understood, for example, as the application of a specific concentration of a drug over a defined period of time. Various active ingredients, combinations of active ingredients, or temporally alternating active ingredients or concentrations are conceivable as treatment patterns.

[0045] The effect of the different treatment patterns on the tissue sections can be observed “live” and automatically using the measurements described below.

[0046] By measuring the oxygen content, for example in the nutrient medium, changes in the oxygen content per unit of time allow conclusions to be drawn about the viability of the cells in the tissue section and their cell activities.

[0047] By measuring the glucose content, especially in conjunction with the oxygen content, changes in cell metabolism can be detected and, for example, aerobic and anaerobic combustion can be distinguished.

[0048] The measurement of lactate content can be used to distinguish the metabolism of normal cells (aerobic glycolysis with citric acid cycle in the mitochondria) from that of tumor cells, which often involve glycolysis followed by excretion of lactate (“Warburg effect”).

[0049] Comparing these parameters, particularly in a time-resolved manner, in parallel in the various fluidic strands, each with multiple tissue sections exposed to different medications, allows valuable conclusions to be drawn about the sensitivity of the cells, particularly tumor cells, in the tissue section to the various treatment options. Another advantage is that the integrated sensors (measurement of oxygen, carbon dioxide, glucose, lactate, and / or pH levels) allow monitoring of the influence of the various treatment patterns even during their application (in situ) at closely spaced intervals. Another advantage is that the method according to the invention can also be used for the low-risk research and evaluation of new active ingredients in direct comparison with established methods.

[0050] The staining of biomarkers in tissue sections using staining reagents that bind to them can already occur during the method according to the invention. For example, proliferation markers and / or live and dead dyes are used here. Furthermore, cell nuclei, for example, can be stained to enable the unambiguous assignment of additional signals and the cell position in the tissue section. This is particularly useful when the same, fixed markers are always present. To introduce the staining reagents, the fluidic path is adapted, for example, to allow the addition of small amounts of staining reagents. These can either bind directly to markers, particularly tumor-specific ones, or be administered via coupling to antibodies. To ensure their analysis, optical and / or sensory evaluation is carried out, for example.Traditionally, the evaluation is carried out via optical observation with a fluorescence microscope, so that the image of the tissue sections of the culture elements is at least partially optically transparent in this case.

[0051] Alternatively, the biomarkers of the tissue sections are stained only after they have been removed from the fluidic device.

[0052] 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 to it as a reference.

[0053] The advantage here is that in this way a comparison point of the cultivation is set with the other strands which are exposed to a substance.

[0054] The negative control also provides early information about viability and aging effects under the established conditions, or a possible or necessary need for changes to the established cultivation conditions. Furthermore, in a particularly advantageous embodiment, step a' is performed after step a): a') Uniform temperature control of all strands and supply of all strands with nutrient medium for a predefined time.

[0055] After inserting the tissue sections into the cartridge, a stabilization phase is advantageously performed, during which all tissue sections are maintained at a uniform temperature and supplied with culture medium to establish defined and uniform initial conditions. This step typically lasts 1–3 hours, but can also take up to 12 hours. During this phase, the metabolism of the tissue sections is monitored and compared using, for example, the built-in oxygen sensor. Optionally, glucose consumption and / or lactate concentration can also be monitored.

[0056] In a further advantageous embodiment, the nutrient medium is enriched with oxygen and / or carbon dioxide, as the oxygen and carbon dioxide supply to the tissue sections can thus be provided via liquids. In this way, optimal conditions can be set with regard to the amount of oxygen or carbon dioxide available in the tissue sections. The concentrations to be set for this purpose depend heavily on the tissue type whose tumor is being examined. For example, the oxygen requirement of liver cells is comparatively high, so that an additional oxygen storage device (hemoglobin analogue) can sometimes be used for them. In the fluidic arrangement, the oxygen requirement can advantageously be adjusted and ensured by increasing the supply of nutrient medium.For this purpose, the nutrient medium is enriched with oxygen, for example to over 80% of the respective solubility limit of the gas for the nutrient medium used at the set cultivation temperature.

[0057] A further advantageous embodiment provides for a gas to be introduced into the strands of the fluidic device, allowing an exchange with a gas atmosphere. This allows the cells of the tissue section to absorb the required amount of oxygen and carbon dioxide particularly well. Furthermore, a further advantageous embodiment provides for an evaluation of the measured parameters in a further step d) of the method and, if necessary, an adjustment of the application of the substance, in particular the active ingredient, to at least one strand or to at least one cultivation element.

[0058] By measuring the oxygen, glucose and / or lactate content and, if necessary, the pH value, the influence of different treatment patterns on the cells of the tissue section can be monitored over short periods of time.

[0059] This allows, for example, variations in treatment patterns over a longer period: if, for example, no measurable change occurs under any of the treatment patterns, the active ingredient concentrations can be increased. If the sensor signals between the strands with different treatment patterns show significant deviations, individual tissue sections can be removed from the culture chamber for more detailed analysis.

[0060] In a further advantageous embodiment, in a step e) following step b) and / or c) and / or d), a liquid is taken for analysis via the respective sampling point before and / or after a cultivation element.

[0061] The advantage here is that before and after a substance, particularly an active ingredient, comes into contact with the tissue section in the culture element, the fluid introduced into or removed from it can be examined and further analyzed. This allows direct and very precise conclusions to be drawn about the effect of the respective substance on the tissue section.

[0062] After the end of the fluidic, especially microfluidic, cultivation and testing of the tissue sections in the device, these can be removed and, as with previous static cultures, classically stained, embedded and retrospectively examined using all desired methods.

[0063] Furthermore, following the procedure, individual tissue sections can be divided into smaller fragments or individual cells, which can then be prepared for further analysis. Depending on the intended analysis, this can involve either initially dissolving the tissue into individual cells or performing complete cell lysis, which then allows for the analysis of cell components, deoxyribonucleic acid (DNA), mRNA (messenger ribonucleic acid), and enzymes.

[0064] Another analytical option is the integration of established assays. For example, assays that only analyze the culture medium used are conceivable. One example is the lactate dehydrogenase (LDH) assay. This can be used to measure the amount of lactate dehydrogenase released from destroyed cells.

[0065] 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.

[0066] A further subject matter of the invention is 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.

[0067] The pumps and valves of the device according to the invention are implemented, for example, by deflecting an elastic membrane using compressed air, and the media reservoirs can be represented by so-called reagent bars, as described in patents EP2322277 B1 and DE102011078770 B4. The introduction of samples by the user can also be implemented, for example, as described therein.

[0068] If the pumps are implemented as small diaphragm pumps for each strand, a subsequent strand can be connected modularly with cultivation elements. This offers the advantage that the number of strands can be varied, especially when more tissue sections are available and a larger number of treatment scenarios are to be tested.

[0069] Short description of the drawing

[0070] Embodiments 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: a schematic representation of a cultivation element in a first embodiment with two supply channels and two discharge channels in a cross-section,

[0071] Fig. 2: the schematic representation 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,

[0072] Fig. 3: the schematic representation of a fluidic, in particular microfluidic, device according to the invention in a second embodiment in a cross section,

[0073] Fig. 4: the schematic representation of an inventive

[0074] Cartridge comprising the microfluidic device according to Figure 2, and

[0075] Fig. 5: the schematic representation of an embodiment of the method according to the invention.

[0076] Embodiments of the invention

[0077] Figure 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 latter is held securely during cultivation and testing and good fluid contact with the tissue section 2 is ensured.

[0078] The cultivation element 50 further comprises a first supply channel 4a and a first discharge channel 4b. These run, for example, vertically on opposite sides of the cultivation chamber 1 and are connected to it in such a way that a direct fluidic connection runs from the first supply channel 4a through the tissue incision 2 to the first discharge channel 4b. Furthermore, the cultivation element 50 comprises a second supply channel 4a' and a second discharge channel 4b', which are also arranged on opposite sides of the cultivation chamber 1, in particular at a defined angle to the first supply and discharge channels 4a, 4b. A valve 5 is incorporated into each of the supply and discharge channels 4a, 4a', 4b, 4b', so that these channels can be opened and closed individually.

[0079] Figure 2 shows a fluidic, in particular microfluidic, device 100 according to the invention for cultivating and testing tissue sections 2 in a first embodiment with cultivation elements 50 according to Figure 1. Three successively arranged cultivation elements 50 form a strand 10. The fluidic device 100 comprises four such strands 10, which are fluidically connected to one another via a main supply channel 4c and a main discharge channel 4d.

[0080] The main supply channel 4c is formed from a fluidic network located upstream of the strands 10. The main supply channel 4c comprises fluidic, in particular microfluidic, channels, which in Figure 2 lead from various reservoirs 7a, 7b, 7c, 7d, 7e to the various strands 10 and comprise valves 5, via which the individual channels of the main supply channel 4c can be individually opened and closed.

[0081] In the reservoirs 7a, 7b, 7c, 7d, 7e, in particular liquids are stored 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.

[0082] Furthermore, a pump 9, in particular a diaphragm pump, is arranged upstream of each strand 10, which pumps a liquid from at least one reservoir 7a, 7b, 7c, 7d, 7e via the fluidic network of the main supply channel 4c into and through the cultivation elements 50. Alternatively, and not shown in Figure 2, a central pump can also transport the liquids for each strand 10 instead of the pumps 9.

[0083] The main drainage channel 4d is formed from a fluidic network located downstream of the strands 10. The main drainage channel 4d comprises fluidic, in particular microfluidic, channels that lead from the respective strands 10 to a fluidic outlet (not shown), a waste container 13, and / or a collection container 15. The individual channels of the main drainage channel 4d comprise valves 5, via which the individual channels of the main drainage channel 4d can be individually opened and closed.

[0084] Furthermore, sensors 11a, 11b, 11c, 11d are arranged in the channels of the main drainage channel 4d. A first sensor 11a is located directly downstream of each branch 10, which is designed to measure a parameter, in particular an oxygen content in a drained liquid of the respective branch 10. Alternatively or additionally, the sensor 11a is designed to measure a glucose content and / or a lactate content and / or a pH value in the drained liquid of the respective branch 10. Furthermore, in Figure 1, further sensors 11b, 11c, 11d are arranged in a channel of the main drainage channel 4d, through which the drained liquid of all branches 10 can pass. These sensors 11 b, 11 c, 11 d are designed, for example, to measure an oxygen content, a carbon dioxide content, a glucose content, a lactate content and / or a pH value.Each strand 10 further comprises a side channel 4e, which leads from the main supply channel 4c to the main discharge channel 4d and serves to flush the main fluidic channels 4c, 4d and the sensors 11a, 11b, 11c, 11d between measurements and / or to provide calibration fluids for the sensors 11a, 11b, 11c, 11d. Fluid conducted via one of the side channels 4e does not pass through the cultivation elements 50.

[0085] Liquids that are no longer needed can be collected in at least one waste container 13. The waste containers 13 shown in Figure 2 can also be combined into one container and have only two alternative inlets.

[0086] In particular, liquids such as the nutrient medium are collected in the collecting container 15, which can then be made available for further analyses outside the fluidic device 100.

[0087] The cultivation elements 50 each comprise two supply channels 4a, 4a' and two discharge channels 4b, 4b'.

[0088] The first supply channel 4a forms a direct fluidic connection from the main supply channel 4c to the first cultivation element 50 of each strand 10. The first supply channel 4a of the cultivation elements 50 arranged after the first cultivation element 50 in the strand 10 leads from the previous cultivation element 50 to this.

[0089] The first discharge channel 4b of 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 4d.

[0090] All cultivation elements 50 of each strand 10 can be uniformly fluidically addressed via the first supply channel 4a and the first discharge channel 4b. Thus, all cultivation elements 50 of a strand 10 receive the same fluidic supply, enabling multiple determinations.

[0091] Furthermore, the cultivation elements 50 comprise a second supply channel 4a', which forms an indirect fluidic connection from the upstream main supply channel 4c of the device 100 to the respective cultivation element 50. Furthermore, 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. Each cultivation element 50 of a strand 10 can be individually fluidically addressed via the second supply channel 4a' and the second discharge channel 4b'.

[0092] In an alternative advantageous embodiment, not shown in Figure 2, only one supply channel 4a is provided for each first cultivation element 50 of each strand 10. The second supply channel 4a' is omitted, since it supplies the same liquids as the first supply channel 4a. Furthermore, only one discharge channel 4b is provided for each last cultivation element 50 of each strand 10. The second discharge channel 4b' is omitted, since it discharges the same liquids as the first discharge channel 4b.

[0093] For example, a withdrawal point for a liquid (not shown in Figure 2) is arranged before and after the cultivation chamber 1 of the cultivation elements 50.

[0094] First, in a first method step a), a tissue section 2 is introduced into a cultivation element 50, so that the device 100 comprises twelve tissue sections 2 for cultivation and testing.

[0095] Optionally, a stabilization phase follows in a process step a'), in which all tissue sections 2 are uniformly tempered and supplied with nutrient medium for 1-3 hours, for example, in order to create identical initial conditions. For this purpose, a nutrient medium is pumped, for example by means of the pump 9, from at least one of the reservoirs 7a, 7b, 7c, 7d, 7e into the strands 10, so that the tissue sections 2 in the cultivation elements 50 are supplied with it. In this case, the oxygen content and thus the metabolism of the tissue sections 2 can be monitored and compared via a sensor 11a, for example. Optionally, the glucose consumption and / or the lactate concentration in the medium removed from the cultivation elements 50 can also be measured. Subsequently, in a process 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 medication is applied over a defined period of time into each strand 10, in particular from at least one of the reservoirs 7a, 7b, 7c, 7d, via the main supply channel 4c and the first supply channel 4a of the cultivation elements 50. Various active ingredients or combinations of active ingredients, or temporally alternating active ingredients or concentrations, are conceivable as treatment patterns. One strand 10 is preferably further cultivated as a reference under the initial cultivation conditions. This provides information about viability and aging under the prevailing conditions. Thus, in this example, three different active ingredients, three combinations of active ingredients, or three concentrations of an active ingredient can be tested.

[0096] First, the liquid containing the substance is introduced into the strands 10 containing the tissue sections 2, and the strands are allowed to act for a defined time, or a very low flow rate is set to continue supplying the liquid containing the substance. A nutrient medium is then introduced into the strands 10 and finally flushed out of the cultivation chambers 50, so that, in a process step c), the oxygen content, glucose content, lactate content, and / or pH value, for example, can be measured in the discharged nutrient medium via at least one sensor 11a downstream of each strand 10 and / or downstream of all strands 10. During the supply of nutrient medium, the aforementioned parameters can be measured repeatedly.In this case, either the nutrient solutions discharged from 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 supply channel 4a' and the second discharge channel 4b', the liquid is exchanged and measured by means of the sensors.

[0097] In addition, a staining reagent can be applied into the strands 10 and the fluorescence of staining reagents bound to specific markers of the tissue sections 2 can be measured.

[0098] In process steps a') and / or b), the nutrient medium can be enriched with oxygen and / or carbon dioxide, for example. Furthermore, in step b), an atmospheric gas can also be applied to the strands 10 of the fluidic device 100, so that an exchange with the gas atmosphere can take place.

[0099] In addition, in a further optional step d), for example, an evaluation of the measured parameters and, if necessary, an adjustment of the application of the substance, in particular the active ingredient, into at least one strand 10 or into at least one cultivation element 50 takes place.

[0100] Figure 3 shows a second, simpler embodiment of a fluidic, in particular microfluidic, device 100 according to the invention for cultivating and testing tissue sections 2. In contrast to the first embodiment shown in Figure 2, the device 100 comprises only one central pump 9 for each strand 10, instead of the pumps 9, which transports the fluids through the fluidic device 100. Alternatively, and not shown, each strand 10 can also have a pump 9.

[0101] The main difference from the first embodiment shown in Figure 2 is that the cultivation elements 50 comprise only a first supply channel 4a and a first discharge channel 4b. 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.

[0102] Furthermore, the device 100 shown in Figure 3 includes a separate side channel 4e, which leads from the main supply channel 4c to the main discharge channel 4d or discharges into a waste container 13, depending on which valves 5 are open or closed. The separate side channel 4e has the same function as the side channel 4e described in Figure 2.

[0103] Figure 4 shows a cartridge 1000 according to the invention, which, as an example for all embodiments of the microfluidic device 100 according to the invention, comprises a microfluidic device 100 in the first embodiment according to Figure 2.

[0104] Figure 5 shows a flowchart of an embodiment of the method 800 according to the invention for culturing and testing tissue sections 2, in particular tumor tissue sections. In particular, the method steps a), a'), b), c), and d) are carried out, which are illustrated and described in / for Figures 2 and 3.

Claims

Claims 1. A fluidic, in particular microfluidic, device (100) for cultivating and testing tissue sections (2), comprising a plurality of fluidically interconnected strands (10), wherein each strand (10) is formed by a plurality of cultivation elements (50) fluidically interconnected in series, and wherein each cultivation element (50) comprises at least one supply channel (4a, 4a') and at least one discharge channel (4b, 4b'), as well as a cultivation chamber (3) into which at least one tissue section (2) can be introduced, and wherein all cultivation elements (50) of a strand (10) are uniformly fluidically addressable and / or wherein each cultivation element (50) is individually fluidically addressable.

2. Fluidic device (100) according to claim 1, wherein at least a plurality of the cultivation elements (50) each comprise two supply channels (4a, 4a') and / or two discharge channels (4b, 4b'), of which a first supply channel (4a) forms a direct fluidic connection from the upstream adjacent element in the strand (10) to the cultivation element (50), and of which at least a first discharge channel (4b) forms a direct fluidic connection to the downstream adjacent element in the strand (10), and of which a second supply channel (4a') forms an indirect fluidic connection from the upstream main supply 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 downstream main drainage channel (4d) forms 3. 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 (11 a, 11 b, 11 c, 11 d) 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 cultivation element (50), a strand (10) or all strands (10).

4. 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 ingredient solutions and / or coloring reagents, and wherein the at least one reservoir (7a, 7b, 7c, 7d, 7e) serves for receiving, temporarily storing and / or mixing liquids.

5. 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 supply 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 fluids for the at least one sensor (11a, 11 b, 11 c, 11 d), whereby the cultivation elements (50) are not passed.

6. Fluidic device (100) according to one of the preceding claims, wherein the device has a collecting container (15) via which a defined 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 a removal point for a liquid is located before and after the cultivation chamber (1) of the cultivation elements (50), by means of which removal point a defined volume of liquid can be removed.

7. Fluidic, in particular microfluidic method (800) for cultivating and testing tissue sections (2) by means of a device (100) according to one of claims 1-6, comprising the following steps: a) introducing tissue sections (2) into the cultivation elements (50); b) applying a different substance to each of the strands (10), in particular at least one active ingredient in a defined concentration and / or combination, a nutrient medium and / or a staining 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 pH value in the drained liquid of a culture 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).

8. Method (800) according to claim 7, wherein after step a) a step a') takes place: a') Uniform tempering of all strands (10) and supply of all strands (10) with nutrient medium for a predefined time.

9. The method (800) according to any 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 set cultivation temperature.

10. The method (800) according to any one of claims 7-9, wherein in step b) an atmospheric gas is introduced into the strands (10) of the fluidic device (100).

11. Method (800) according to one of claims 7-10, wherein in a further step d) an evaluation of the measured parameters is carried out and, if necessary, an adjustment of the application of the substance, in particular of the active ingredient, into at least one strand (10) or into at least one cultivation element (50) is carried out.

12. Cartridge (1000) comprising a microfluidic device (100) according to any one of claims 1-6.

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