Method for studying cells

Abstract

There is provided a method for evaluating the effect of a plurality of reagent compositions on living cells, comprising the sequential steps of introducing a sample with a living cell, sequentially exposing it to at least two different reagent compositions, and collecting data from at least one cell or descendants in its lineage of cells at different points in time corresponding to exposure to the different reagent compositions using an optical microscope, and determining a property of the sample by comparing data regarding cells in the same lineage of cells cell during exposure to the different reagent compositions.

Description

[0001] Method for studying cells

[0002] Technical Field

[0003] The invention relates to a method for studying individual cells or a cell lineage over time during influence of different reagents and to determine the impact of one reagent in relation to that of another.

[0004] Background

[0005] Microfluidic devices as well as methods performed in such devices are well known in the art. Their use include diagnosis as well as research. The microfluidic devices may comprise cell traps, in which cells may be captured, cultured and analysed.

[0006] In samples of cells to be analysed, it is not uncommon to encounter a low number of cells, rendering it impractical to divide the sample into numerous compartments for analysis of their reactions to different reagents. This limitation arises particularly in situations where the quantity of cells is scarce or precious, such as in clinical samples or rare cell populations.

[0007] Time may also be a crucial factor when analysing samples, especially in time-sensitive applications such as clinical diagnostics. Delayed analysis can lead to degradation of samples or inaccurate results, underscoring the importance of rapid and efficient analysis methods. Furthermore, in clinical settings, time is of the essence as patients may be awaiting the results for timely treatment decisions. Therefore, the need for expedited analysis methods is paramount to ensure swift and accurate diagnoses, ultimately improving patient outcomes.

[0008] Although the microfluidic devices according to the prior art are successfully used for studying cells, there is still room for an improvement regarding the function and manufacture of microfluidic devices.

[0009] EP3601588B1 discloses method for phenotyping cells, said method comprising: a) loading a biological sample comprising biological material including target cells of a cell type and non-target cells of another cell type into a microfluidic device comprising spatially defined and separated cell compartments to capture biological material in said spatially defined and separated cell compartments , b) monitoring biological material in said spatially defined and separated cell compartments prior to exposing biological material in said spatially defined and separated cell compartments to a test agent, c) identifying a subset of said spatially defined and separated cell compartments as comprising target cells exhibiting at least one target phenotype characteristic as determined based on said monitoring of biological material in said spatially defined and separated cell compartments in b) , identifying remaining spatially defined and separated cell compartments as comprising non-target cells and / or non-cell material not exhibiting said at least one target phenotype characteristic as determined based on said monitoring of biological material in said spatially defined and separated cell compartments (20, 120) in b) , d) exposing biological material in said spatially defined and separated cell compartments to said test agent , e) monitoring target cells in said identified subset of said spatially defined and separated cell compartments by: taking at least one image of said spatially defined and separated cell compartments, processing said at least one image for detection of said identified subset of said spatially defined and separated cell compartments and target cells in said identified subset of said spatially defined and separated cell compartments while disregarding said remaining spatially defined and separated cell compartments and non-target cells and / or non-cell material in said remaining spatially defined and separated cell compartments, f) determining a phenotypic response of said target cells to said test agent based on said monitoring of target cells in said identified subset of said spatially defined and separated cell compartments in e) .

[0010] In EP3601588B1, temporal analysis of cells is used to discriminate compartments, including living cells, from compartments, including dead cells or debris. The phenotypic response is monitored for one test agent.

[0011] In general, it is important to determine the relative response to different test agents. In prior art, US 10, 913, 969 B2, cells in different spatial regions are exposed to different test agents, and for example, antibiotic susceptibility is determined by the response to an antibiotic for cells in one spatial region as compared to that of cells in another spatial region the cells were treated with no antibiotic.

[0012] In the prior art there is still room for an improvement regarding the study of individual cells and cells in the same cell lineage with over time.

[0013] Summary

[0014] It is an object of the invention to obviate at least some of the problems in the prior art and provide a method of capturing and of analysing cells.

[0015] In a first aspect there is provided a method for evaluating the effect of a plurality of reagent compositions on living cells, comprising the sequential steps of: a. providing a microfluidic device comprising at least one cell trapping region (4) , at least one media port (5) , and at least one channel (6) connecting said cell trapping region (4) and said at least one media port ( 5 ) , b. introducing a sample comprising at least one living cell into the cell trapping region (4) via the at least one media port (5) , wherein the at least one cell consists of one selected from of : i. one cell, that can be the origin of one linage of cell ii. a plurality of cells, that can be the origins of different lineages of cells, c. sequentially exposing the at least one cell in the cell trapping region (4) to different reagent compositions, by sequentially introducing the different reagent compositions into the cell trapping region (4) via the at least one media port (5) and at least one channel (6) , and collecting data from at least one cell or descendants in its lineage of cells at different points in time corresponding to exposure to the different reagent compositions using an optical microscope, d. determining a property of the at least one cell by using data regarding cells in the same lineage collected in step c) during exposure to at least two different reagent compositions

[0016] It is an advantage that it is possible to study one single cell or a cell lineage over time when it is exposed to different reagents, such as a reagent comprising different concentrations of an antibiotic.

[0017] It is particularly important to get out as much information as possible from a single or a few cells when there are only this many cells in the sample, such as in a bloodstream infection sample, which may include as few as one bacteria per millilitre. Given the current invention, it is possible to sequentially apply different antibiotics to the single cell until one is found for which the bacterium is not resistant.

[0018] The present invention makes it possible to (i) use one individual cell as a non-treated reference to itself instead of comparing different cells in different spatial regions, and (ii) sequentially test different treatments on the same cell or lineage of cells until one is effective. Getting out the same information from different cells exposed to different reagents at different spatial positions would require many more cells to reach statistically significant data in each position .

[0019] Brief description of the drawings

[0020] Figure 1 illustrates a microfluidic device according to the invention in four stages of performance of an analysis .

[0021] Figure 2 illustrates a similar device, wherein the volume of the cell trapping region 4 is variable.

[0022] Figure 3 illustrates a similar device, wherein the cell trapping region 4 comprises a plurality of spatially separated cell traps 43.

[0023] Figure 4 illustrates a microfluidic device from a side angle (to the left) and a front angle (to the right) , with a first layer 1, middle layer 2 and second layer 3. A individual channel 6 is formed by a cavity between the first 1 and middle 2 layer. A valve 7 is formed by a cavity between the middle 2 and second 3 layer.

[0024] Figure 5 illustrates a microfluidic device from a side angle, with an individual channel 6 in opened (to the left) and closed (to the right) state. Figure 6 illustrates a microfluidic device from a front angle , with an individual channel 6 in an opened ( to the left ) and closed ( to the right ) state .

[0025] Figure 7 illustrates a middle layer 2 of a microfluidic device , in a transparent version to the left and a nontransparent version to the right .

[0026] Figure 8 illustrates a microfluidic device similarly to the one in figure 7 . An cross-section along an imaginary line B-B is shown, illustrating the relative thickness of the cavities forming the valve pressure control channels 9 and the cavities forming the valves 7 .

[0027] Figure 9 illustrates growth of bacterial cells (E . coli ) in a single cell trapping region 4 when its sequentially exposed to media without antibiotic and 5 increasingly high concentrations of antibiotic ( ciprofloxacin) . Each exposure is for 30 min . Finally the cells are fixed and exposed to a fluorescent probe that identi fies it as E . coli . The lengths of a single cell and the cells in its lineage are indicated with a length bar between each media condition . The relative length increase during a media condition is indicated by a percentage .

[0028] Fig 10 sequential phenotyping of growth in a microfluidic cell trapping region 4 followed by in situ FISH genotyping of the species ID with probes that identi fies E . coli or Klebsiella .

[0029] Figure 11 illustrates two examples ( top and bottom) of the growth of bacterial cells (E . coli ) in a single lineage originating from the single cell in the bottom of the trapping region 4 (formed as a channel) . The cells are sequentially exposed to (i)no antibiotic, (ii) chloramphenicol, (iii)no antibiotic, ( iv) tetracycline, (v)no antibiotic. Each sub-panel shows the same cell trapping region 4 photographed in time-lapse (4 min in between each image) , from the left to the right. The individual grey rods are the single cells and when they are split by a white line, the cell divides into two daughter cells. The dashed markings indicate when the cells are exposed to the antibiotic. The cells in the cell linage in the top panel are susceptible to both antibiotics, and the cells' length increase stops when exposed to the antibiotics. The cells in the cell lineage in the bottom panel are resistant to chloramphenicol and keep growing exponentially while exposed to chloramphenicol but then stop growing when later exposed to tetracycline. The effects of the antibiotics on bacterial growth (in length or area) can be seen in both individual cells and in the sum of lengths (or areas) of cells descending from the same mother cell.

[0030] Detailed description

[0031] The following detailed description discloses by way of examples details and embodiments by which the invention may be practised.

[0032] It is to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention is limited by the appended claims. If nothing else is defined, any terms and scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains.

[0033] In the following, each of the described methods, devices, apparatuses, examples and aspects, which do not fully correspond to the invention as defined in the claims is thus not according to the invention and is, as well as the whole following description, present for illustration purposes only or to highlight specific aspects or features of the claims.

[0034] As used herein the term "reagent composition" refers to the specific formulation or mixture of chemical substances used in the method. The reagent composition can differ with regard to its chemical components, i.e. the substances that make up the reagent, the concentration or proportions, i.e. the relative amounts or concentrations of each component, the physical state, i.e. whether the reagent is in solid, liquid, or gaseous form and with regard to additional additives, i.e. stabilizers, solvents, or catalysts that enhance or control the reagent's behavior. In one embodiment the reagent composition can be only cell culture media to allow comparative experiments with a reagent composition comprising for instance another additive in addition to cell culture media.

[0035] As used herein the term "living cells" refers to structural and functional units of life that exhibit key characteristics such as growth, metabolism, reproduction, and response to stimuli. They comprise biomolecules, including proteins, nucleic acids, lipids, and carbohydrates, and are enclosed by a membrane that regulates the exchange of substances with their environment. The term living cells include prokaryotic cells, which are simple, unicellular organisms without a nucleus (e.g., bacteria and archaea) and eukaryotic cells, which are more complex cells with a nucleus and membrane-bound organelles (e.g., plant, animal, fungal, and protist cells) .

[0036] As used herein the term "microfluidic device" refers to a miniaturized system that precisely manipulates small volumes of fluids (typically in the microliter to nanoliter range) within microscale channels. These devices are designed using microfabrication techniques.

[0037] As used herein the term "cell trapping region" denotes a designated microscale area within a microfluidic device designed to capture, isolate, and hold individual or groups of living cells for imaging, analysis, manipulation, and / or experimentation. It can use features including but not limited to channels, chambers, or wells to physically confine cells. It can rely on techniques including but not limited to size-based filtration, hydrodynamic forces, optical trapping or biochemical interactions .

[0038] As used herein the term "media port" denotes an inlet or outlet in a microfluidic device, that allows the controlled introduction, exchange, or removal of fluids such as growth media, reagents, or waste. The media port has a fluid exchange to facilitate the supply of nutrients, removal of waste, or addition of reagents. The media port is often designed to connect with tubing, syringes, or pumps for precise fluid control and may include filters or sealing mechanisms to prevent contamination .

[0039] As used herein the term "channel" is a narrow, confined passage that directs and controls the flow of fluids , cells , or particles .

[0040] As used herein the term " lineage of cells" refers to a population of cells derived from a single original cell , which can continuously grow and divide under controlled conditions and that was present at the start of the experiment .

[0041] As used herein the term "daughter cell" refers to a cell produced as a result of cell division, speci fically from the division of a parent cell . These cells inherit genetic material from the parent cell and may undergo further di f ferentiation or division themselves . A parent cell and a daughter cell belong to the same lineage of cells .

[0042] As used herein the term "phenotypic trait" refers to any observable characteristic or feature of an organism that results from the interaction of its genetic makeup ( genotype ) and environmental factors .

[0043] As used herein the term "minimum inhibitory concentration" (MIC ) is the lowest concentration of an antimicrobial agent ( such as an antibiotic or anti fungal ) that prevents visible growth of a microorganism in a culture medium .

[0044] As used herein the term "wild type MIC" refers to the minimum inhibitory concentration (MIC ) of an antimicrobial agent for a wild-type strain of a microorganism . A wild-type strain is the naturally occurring, non-mutated form of the organism, as opposed to strains that have acquired mutations , especially those that may confer resistance to drugs . It represents the MIC for the organism in its most common, unaltered form, without any genetic modi fications or resistance traits . The MIC value of a strain or isolated is determined by a standardi zed method as defined by EUCAST or CLS I . MIC values are reported in a log2 scale , and the accuracy of the reference methods are commonly accepted to be one log2 unit .

[0045] As used herein the term "near zero concentration" of an antibiotic refers to a concentration level of the antibiotic that is extremely low and so close to zero that it has negligible biological activity . This concentration may still be capable of having an extremely slight ef fect on microbial growth, but it is insuf ficient to cause anything else but a negligible biological activity . The near zero concentration does not have any measurable impact on the growth or survival of bacteria, fungi , or other pathogens .

[0046] As used herein the term "valve" denotes a mechanical device by which a flow of fluid may be controlled by a movable section . It may be a device for controlling the movement of fluid in a space or passage of fluid through a channel , pipe , duct , etc .

[0047] As used herein, the phrase "at least one" means that there may be one or more of that obj ect . Throughout the text , characteristics disclosed to describe that obj ect may be applied to all such obj ects present in the device , even i f it is described in relation to "an / the" obj ect , if not clearly specified that it regards just one specific such object or certain such objects.

[0048] As used herein, "media" encompasses any reagent, cell culture media or other fluid relevant for use in the device or method, if not otherwise specified. In other words, a media inlet port is not restricted to introduction of a certain media.

[0049] In the first aspect there is provided a method for evaluating the effect of a plurality of reagent compositions on living cells, comprising the sequential steps of: a. providing a microfluidic device comprising at least one cell trapping region (4) , at least one media port (5) , and at least one channel (6) connecting said cell trapping region (4) and said at least one media port ( 5 ) , b. introducing a sample comprising at least one living cell into the cell trapping region (4) via the at least one media port (5) , wherein the at least one cell consists of one selected from of : i. one cell, that can be the origin of one linage of cell ii. a plurality of cells, that can be the origins of different lineages of cells, c. sequentially exposing the at least one cell in the cell trapping region (4) to different reagent compositions, by sequentially introducing the different reagent compositions into the cell trapping region (4) via the at least one media port (5) and at least one channel (6) , and collecting data from at least one cell or descendants in its lineage of cells at different points in time corresponding to exposure to the different reagent compositions using an optical microscope, d. determining a property of the at least one cell by using data regarding cells in the same lineage collected in step c) during exposure to at least two different reagent compositions

[0050] One cell or a number of cells are introduced into the microfluidic device. The cells may divide to give rise to one cell lineage. Various reagent compositions are added so that the cell (s) is / are exposed to different reagent compositions. The reagent compositions can differ in for instance concentration and / or composition. The cell (s) is / are observed and data is collected over time and in particular data is collected for the same individual cell and / or the same cell lineage. The data is collected for at least two different reagent compositions. This offers a systematic approach to evaluate the effects of various reagent compositions on living cells, allowing for precise control and observation through a microfluidic device. This enhances the ability to study cellular responses in a controlled environment.

[0051] By working with cells in the same lineage over time, it is possible to overcome the biological cell-to-cell variation that is otherwise always present in a population of cells, without analyzing so many cells that it's possible to make accurate population averages .

[0052] In one embodiment, fewer than 20 living cells are introduced in step b) . This is often as many bacterial cells that can be obtained from a sample from blood stream infection or meningitis.

[0053] In one embodiment, in step c) , the length of one cell or the sum of lengths of daughter-cells descending from it is determined by optical microscopy and / or image analysis. This enables the determination of cell or daughter-cell lengths through optical microscopy, providing valuable data on cell growth and morphology changes in response to reagents. In a confined channel or tube the length of a cell can easily be defined and determined in the direction of the channel or tube.

[0054] In one embodiment, in step c) , the size of one cell or the sum of sizes of daughter-cells descending from it is determined by optical microscopy and / or image analysis. The size differs from the length. The size is in one embodiment the two dimensional area of the cell (s) as determined from optical microscopy and / or image analysis. Alternatively the size is the maximum size in any direction of the cell.

[0055] In one embodiment, in step d) the property determined is the difference in percentage growth between different time points corresponding to different reagents compositions. This facilitates the calculation of percentage growth differences, which can be critical for understanding the effects of reagents on cell proliferation over time.

[0056] In one embodiment, in step c) , at least two of the different reagent compositions are reagent compositions comprising different concentrations of the same reagent. This allows for the assessment of different concentrations of the same reagent, providing insights into dose-dependent cellular responses. By increasing the concentration stepwise, it is possible to determine the MIC value as the concentration where growth is inhibited. This is also possible for a single cell. For example, as in Fig 9.

[0057] In one embodiment, in step c) , at least two of the different reagent compositions are reagent compositions comprising different reagents or different combinations of reagents. This supports testing of different reagents or combinations, broadening the scope of experimental conditions and allowing for comprehensive analysis of synergistic or antagonistic effects.

[0058] In one embodiment, in step c) , at least two of the different reagent compositions are reagent compositions comprising different reagents of different concentration. Combining different reagents at varying concentrations, enables a thorough exploration of complex reagent interactions on cellular behaviour.

[0059] In one embodiment, the media composition in the next step can be determined by the response to the reagents in the previous steps, such that, for example, combinations of antibiotics are tested if none of the individual antibiotics has given an impact.

[0060] In one embodiment, step d) involves analyzing the change in a phenotypic trait due to a change in reagent composition for several individual cell lineages originating in different single cells by calculating a property from changes in response for the several individual cell lineages. This analyzes phenotypic changes across different individual cell lineages, such that the similarity or differences in response between different lines can be evaluated.

[0061] In one embodiment, the method comprises a step of exposing the cells in the cell trapping region (4) to cell culturing media. This includes a step for cell culturing media exposure, which is vital for maintaining cell viability and simulating physiological conditions.

[0062] In one embodiment, the method comprises a step of exposing the cells in the cell trapping region (4) to cell culturing media in order to establish a reference for the cells' distribution of growth rates, division times, or cell size, such that deviations from this reference distribution can be monitored when exposing the cell or cell linage to another reagent.

[0063] In one embodiment, the collected data represents phenotypic traits of the cells, such as growth rate, generation time, fluorescence, morphology or lysis rate, where morphology can include for example, cell size at cell division. This focuses on phenotypic traits like growth rate and morphology, providing a comprehensive understanding of cellular responses beyond molecular changes .

[0064] In one embodiment , the optical microscope in step c ) provides imaging data of the at least one cell in the cell trapping region ( 4 ) . The utili zation of imaging data from optical microscopes , allows for non-invasive , realtime observation of cellular processes .

[0065] In one embodiment , the collected data is analysed using a computer software performing at least one selected from image analysis , automated cell counting, feature extraction and quanti fication, statistical analysis , dose-response analysis , time-course analysis , and machine learning . This allows for incorporation of advanced data analysis techniques such as image analysis and machine learning, enhancing the accuracy and depth of experimental insights .

[0066] In one embodiment , at least one reagent is an antibiotics . This facilitates the study of antibiotics , crucial for determining bacterial resistance and developing new antimicrobial strategies .

[0067] In one embodiment , the next reagent composition is decided in real-time based on the detected phenotypic response to a previous media composition . This allows for a flexible and time saving experiment where the result for one response determines a future addition of reagent composition . This is preferably controlled by a programmable computer . In one embodiment , at least one reagent is a plurality of di f ferent antibiotics and wherein the at least one cell is at least one bacterial cell . In this way the influence of di f ferent antibiotics on one bacterial cell or a bacterial cell lineage can be tested rapidly . It is possible to quickly find a suitable antibiotics for a very low number of bacterial cells . The same principle applies to other cells as well , for instance cancer cells and suitable drugs for treatment .

[0068] In one embodiment , a minimum inhibitory concentration (MIC ) is determined by gradually increasing the concentration until the growth inhibiting concentration is reached . Determining the minimum inhibitory concentration (MIC ) , is key step for evaluating the ef ficacy of antimicrobial agents .

[0069] In one embodiment , the relative growth increase per time for a cell near the wild type MIC concentration of an antibiotic is compared to that of a cell in the same lineage at an earlier timepoint corresponding to near zero concentration of the antibiotics . The wording "near" the wild type MIC concentration is interpreted as the wild type MIC concentration ± one log2 unit , since the MIC scale is logarithmic . The MIC is determined experimentally according to known and accepted methods as outlined above .

[0070] In one embodiment , the cells are bacterial cells . This of fers targeted insights into microbial behaviour and treatment responses . In one embodiment, the sample is provided from a patient with suspected sepsis, meningitis, gonorrhoea, urinary tract infection or tuberculosis. Since this applies to samples from patients with various infections, it is relevant for clinical diagnostics and personalized medicine. In one embodiment, the sample is prepared from a body fluid from a patient with sepsis, meningitis, gonorrhoea, urinary tract infection, or tuberculosis.

[0071] In one embodiment, the at least one reagent comprises a phage. Including phages as reagents, expands the method's applicability to phage therapy research and viral interactions .

[0072] In one embodiment, the at least one reagent is a cancer drug and the cell is a cancer cell. Incorporating cancer drugs, allows for applications within real time resistance development during cancer treatment, oncology research and drug development.

[0073] In one embodiment, at least one reagent is a genotyping reagent, such as a set of FISH probes or a reagent for in situ sequencing.

[0074] In one embodiment, the microfluidic device comprises at least two media ports (5) each connected to the cell trapping region (4) by an individual media channel (6) . Using a microfluidic device with multiple ports, enhances the flexibility and throughput of experimental setups.

[0075] In one embodiment, the microfluidic device comprises a first outer layer (1) , a middle layer (2) and a second outer layer (3) , wherein the cell trapping region (4) and the media channel (s) (6) , are formed at least partly by cavities between the middle layer (2) and at least one of the first layer (1) and second layer (3) , such as by recesses in the middle layer (2) . A layered microfluidic device structure, optimizes the fluid dynamics and reagent delivery.

[0076] In one embodiment, sequential exposure of reagents in step c) is controlled by aid of external structures that involve valve functions and / or pump functions. Utilizing external structures for reagent exposure control, improves precision and reproducibility in experiments.

[0077] In one embodiment, the microfluidic device comprises a plurality of media inlet ports (5) each being fluidly connected to the cell trapping region (4) by an individual media channel (6) , and step c) comprises: i. introducing a plurality of reagents through at least one media inlet port (5) , ii. at a first time point, controlling fluid flow accessibility through the individual media channels (6) , such that a certain flow rate of at least one reagent, and optionally of a dilution media, is allowed, such that the cells in the cell trapping region (4) are exposed to a certain concentration of, and optionally combination of, reagent (s) , iii. at a second time point, adapting the fluid flow accessibility through the individual media channels (6) , such that the cells in the cell trapping region (4) are exposed to a different concentration of at least one reagent present in step ii) and / or at least one different reagent, and iv. optionally repeating the last step a number of times, such that the cells in the cell trapping region (4) are exposed to all concentrations and / or combinations of reagent (s) that are to be analysed at different time points.

[0078] This system for reagent introduction and concentration control, allows for dynamic and complex experimental designs .

[0079] In one embodiment, the microfluidic device comprises a plurality of pressure controlled valves (7) , wherein the valves (7) are formed at least partly by cavities between the middle layer (2) and at least one of the first layer (1) and the second layer (3) , such as by recesses in the middle layer (2) , and wherein a fluid flow accessibility from each media inlet port (5) to the cell trapping region (4) is controllable by operation of at least one valve (7) configured to control the fluid flow accessibility through the individual media channel (6) fluidly connecting that media inlet port (5) to the cell trapping region (4) . The pressure-controlled valves, provide precise fluid flow control for complex microfluidic experiments.

[0080] In order to facilitate the implementation of the above described method there is disclosed the following microfluidic device, in which it is possible to perform the method. There is disclosed a microfluidic device comprising three layers being a middle layer 2, a first layer 1 on a first side of the middle layer 2, and a second layer 3 on a second side of the middle layer 2

[0081] ( see Figs . 4-5 ) . The microfluidic device comprises a cell trapping region 4 having at least one inlet 41 , and optionally an outlet 42 , and a plurality of media inlet ports 5 each being fluidly connected to the cell trapping region 4 each by an individual media channel 6 , through which media may be introduced into the cell trapping region 4 . The microfluidic device comprises a plurality of pressure controlled valves 7 that control the fluid flow accessibility through the individual media channels 6 .

[0082] The cell trapping region 4 , the individual media channels 6 , and the valves 7 are each formed at least partly by at least one cavity between the middle layer 2 and at least one of the first layer 1 and the second layer 3 . In one embodiment , all three features are formed by recesses in the middle layer 2 . These may be cast structures in the middle layer 2 . The individual media channels 6 and the at least one cell trapping region 4 may alternatively, or additionally, be formed at least partly by cavities in the first layer 1 , between the first layer and the middle layer . The valves 7 may alternatively, or additionally, be formed by cavities in the second layer 3 , being between the second layer 3 and the middle layer .

[0083] The fluid flow accessibility of each individual media channel 6 is controllable by operation of at least one of the valves 7 , which is configured to control the fluid flow accessibility through that individual media channel 6 . As a valve acts upon an individual channel 6 , the cross-section of a segment of that channel is af fected . A decreased cross-section leads to a restricted fluid flow accessibility through the channel . Each individual media channel may be closed or opened by action of a valve 7 controlling its fluid flow accessibility . An individual media channel may also be partly closed by a valve 7 , such that fluid flow is reduced but not fully hindered . This way, the fluid flow from each media inlet port 5 ( and indirectly from a media source ) to the cell trapping region is controllable .

[0084] For this purpose , the each valve 7 comprises a movable part which acts on an individual channel 6 . A valve 7 being formed by a cavity means that the cavity constitutes a functional part of that valve 7 , in that the cavity enables movement of such a movable part , for example by a varied fluidic pressure in the valve cavity . Mechanical pressure by an obj ect , or a magnetic force , may also be used to control a movable valve part . Such a movable part may for example be a section of the middle layer 2 adj acent to a valve cavity and an individual channel 6 , such as between them .

[0085] In one embodiment , the individual media channels 6 are formed by cavities between the middle layer 2 and the first layer 1 , and the valves are formed by cavities between the middle layer 2 and the second layer 3 . The channel cavities may then be located ( at least partly) opposite to the valve cavities , such that they have overlapping sections on a line perpendicular to both the first layer and the second layer . A thin section of the middle layer is thus formed between the cavities that is relatively thin in comparison to at least one adj acent section of the middle layer 2 . The thin section may be <100pm . This thin section is adj acent to an individual channel 6 and may be movable in relation to that individual channel 6 , such that it moves towards and away from the individual channel based on pressure , and in doing so af fects the cross-section of the individual channel at a channel segment , which af fects the fluid flow accessibility through that individual channel 6 . The thin section may be movable towards the channel 6 such that fluid flow may be partly or completely cut of f . The thin section thereby forms a movable valve part of the valve 7 .

[0086] The valves 7 may be configured such that they are open in a relaxed state , and closed when pressure is increased in the valve cavities . The valves 7 may be configured such that they are closed in a relaxed state , and opened when pressure is actively reduced in the valve cavities .

[0087] The setup enables several media sources to be connected to one cell trapping region 4 at the same time , while only one or a certain combination of media is allowed to reach the cell trapping region at a certain time point , based on the status of the valves at that time point . The device may thereby be used for sequentially exposing a cell sample present in the cell trapping region to di f ferent media ( reagents ) by stepwise or gradual adaptation of the status of the valves , without having to connect or disconnect any media sources during the performance . At one time point , the valves may close of f all individual channels except one , such that only one reagent reaches the cell trapping region . It is also possible that two or more channels are open at the same time , providing a combination of reagents , that can be mixed before reaching the cell trapping region, for example using a staggered herringbone mixer ( Fig 3 ) . The ratio of the flow from di f ferent media sources may thereby be combined such that a desired concentration of a certain reagent is achieved .

[0088] When analysing a sample comprising a low number of cells , and time is of the essence such that cell number ampli fication by culturing is not an option, there is a need to be able to analyse the ef fect on the cells by di f ferent reagents without splitting the cells up into several sub-samples in separate devices or compartments , as each sub-sample may be too small for a reliable result . This device enables a quick and easy way of performing such an analysis . The device is also easy to manufacture . All the features may be provided by casting in the middle layer, which suitably is located between two outer layers , and without fine alignment between layers . Such fine alignment would be needed i f the features are cast in di f ferent layers .

[0089] In one embodiment , at least two individual media channels 6 fluidly connect in a common channel 8 upstream ( in the intended fluid flow direction) of the cell trapping region inlet 41 , as is seen in figures 1-3 , 7 , and 8 . The common channel 8 may form a mixing channel , in which media simultaneously being introduced is mixed before reaching the cell trapping region 4 . This may be used to mix a reagent such as an antibiotic with cell culture media, in order to dilute the reagent to a certain concentration .

[0090] In one embodiment , at least one of the valve forming cavities is in fluid connection with a valve pressure control channel 9 , as is seen in figures 1-4 , 7 , and 8 . Such a control channel 9 may be considered to act upon, or constitute , one or more valve forming cavities . When the pressure in a control channel 9 is increased, the pressure is increased in the valve cavities it is connected to , which in turn act on movable parts . In figures 1-3 , 7 , and 8 it is illustrated that each control channel 9 is in fluid contact with a valve cavity adj acent to an individual channel 6 . The control channel 9 may thereby be used to control that individual channel . In an embodiment not shown in the figures , each control channel is configured to control all except one individual channel 6 , such that it may be used to close all individual channels except that channel .

[0091] In one embodiment , at least a part of the microfluidic device is transparent . The microfluidic device may be made of materials such as silicon, glass , or polymers . Transparent materials may be suitable , at least in some areas of the device adj acent to the trap, for optical analysis purposes . For example , a silicone rubber substrate (middle layer ) may be sandwiched between two flat substrates ( outer layers ) and the thickness of the silicone rubber substrate at the position of the valves ( the thin section) may be <100pm . The silicone rubber substrate may be bonded to the other substrates , for example using a chemical process .

[0092] In one embodiment , the microfluidic device comprises a plurality of media reservoirs 51 each fluidly connected to a media inlet port 5 . They may be configured such that when pressure is applied on the media in a media reservoir 51 , media is introduced from that reservoir into an individual media channel 6. Such reservoirs are shown in figures 7-8. The volume of the reservoirs 51 may be variable by control of a valve, such that media may be pushed from a reservoir into an individual channel 6 by use of such a valve. Alternatively, a pump may be used. In another embodiment, an external source of media is connected to each inlet port 5 without any reservoir 51 being part of the device.

[0093] In one embodiment, the volume of the cell trapping region 4 is variable (Fig. 2) . By increasing the volume of the region, media may be drawn in from an individual channel through the inlet. By decreasing the volume, media may be pushed out from the region, through the inlet. In this case, the inlet acts as both inlet and outlet port. Obstacles may be present that are configured to keep cells in the region.

[0094] An outlet 42 from the cell trapping region, separate from the inlet, may be present. In one embodiment, the cell trapping region comprises an outlet 42 (Figs. 1, 3, 7, 8) . There may be one or more constrictions 10 configured to hinder cells in the cell trapping region 4 from exiting through the outlet 42. This way media, but not cells, may exit. Such a constriction may be a filter.

[0095] In one embodiment, each cell trapping region 4 is divided into a plurality of spatially separated cell traps 43, each having an inlet in fluid connection with the inlet 41 of the cell trapping region 4, each configured to receive media and cells from the inlet 41 of the cell trapping region and to accommodate cells, and optionally each having an outlet in fluid connection with an outlet 42 of the cell trapping region for exit of media. Such cell traps 43 are illustrated in figure 3, 7, 8. The cell traps 42 may comprise constrictions 10 that hinder cells, but not media, from leaving the cell traps 43.

[0096] Figure 1 illustrates a microfluidic device in four stages of performance of an analysis. The device has three media ports 5, each in fluid contact with an individual channel 6, leading to a cell trapping region 4. The three individual channels join in a common channel 41 before the cell trapping region inlet 41. The cell trapping region has an outlet 42. There are three pressure controlled valves 7 each adjacent to an individual channel, such that the fluid flow accessibility of each individual channel is adaptable by use of a valve 7. Each valve forming cavity is fluidly connected to a valve pressure control channel 9. In figure la) , a first individual channel 6 is open, and the other two are each closed by a valve 7, such that a first reagent is allowed to flow through first individual channel 6 and enter the cell trapping region 4. (In this figure cells are already present in the cell trapping region 4. la) could otherwise represent introduction of cells into the cell trapping region 4 from the first media inlet port 5) . lb) illustrates a later time point, where under continuous flow of that reagent, the cells have grown (growth here symbolizing any type of effect on the cells, including multiplication (division into more cells) or phenotypic change (alteration in appearance or characteristics) ) . In 1c) , a second reagent is instead allowed to enter the cell trapping region through a second individual channel 6, by opening this channel while closing the first and third individual channel, using the valves 7. Id) illustrates a later time point , where under continuous flow of the second reagent , the cells have shrunk ( also symboli zing any type of ef fect ) . A natural continuation would be to similarly analyse the ef fect of a third reagent from the third media inlet port . It is also possible that the third media port is used to provide a media used for dilution of the reagents , such as a cell culture media, which may be introduced simultaneously as the first or the third reagent , to analyse the ef fects of di f ferent concentrations of a reagent . The common channel may provide a mixing zone .

[0097] Figure 2 illustrates a similar device , but without a separate cell trapping region outlet 42 . Flow into and out from the cell trapping region 4 may be controlled by increasing and decreasing the volume of the cell trapping region, represented by the arrow .

[0098] Figure 3 illustrates a similar device , having two media inlet ports 5 and two individual channels 6 . The cell trapping region 4 comprises a plurality of spatially separated cell traps 43 , each configured for receiving cells that enter through the inlet 41 . Each cell trap 43 has an outlet leading to a cell trapping region outlet 42 , and each cell trap 43 has a constriction 10 hindering cells from exiting the cell trap . The common channel 8 comprises a mixing zone , for example comprising a staggered herringbone mixer .

[0099] Figure 4 illustrates a microfluidic device from a side angle ( to the left ) and a front angle ( to the right ) . There is a first layer 1 , middle layer 2 and second layer 3 . A individual channel 6 is formed by a cavity between the first 1 and middle 2 layer . A valve 7 is formed by a cavity between the middle 2 and second 3 layer, said cavity defining a thin section (membrane ) of the middle layer 2 , which is thinner than the surrounding middle layer 2 , which may be pushed towards the individual channel 6 as the cavity expands , which may occur due to an increased pressure in the valve cavity optionally in combination with the middle layer 2 being formed by an elastic material . The cavities vertically overlap, on a line perpendicular to both the first layer and the second layer . The cavity forming the valve 7 is partly expanded by a pressure in the figure , and partly closes the individual channel 6 .

[0100] Figure 5 illustrates a microfluidic device from a side angle , with an individual channel 6 in opened ( to the left ) and closed ( to the right ) state . To the right , the channel is closed as the cavity of the valve 7 is expanded and pushes a thin section of the middle layer 2 towards and all the way into the channel 6 , fully reducing the cross-section of a segment of the channel 6 . The valve cavity is expanded as the pressure within it is increased .

[0101] Figure 6 illustrates a microfluidic device from a front angle , with an individual channel 6 in an opened ( to the left ) and closed ( to the right ) state .

[0102] Figures 5 and 6 do not show any valve pressure control channel 9 that may lead to the cavity forming valve 7 .

[0103] Figure 7 illustrates a middle layer 2 of a microfluidic device , in a transparent version ( to the left ) and a non- transparent version ( to the right ) , both viewed obliquely from above . There is shown three media inlet ports 5 , three media reservoirs 51 , three individual channels 6 , a common channel 8 , three valves 7 each being formed by a cavity adj acent to an individual channel 6 , three valve pressure control channels 9 , each being fluidly connected to one of the valve cavities , a cell trapping region 4 with an inlet 41 , an outlet 42 with a constriction 10 , and with a plurality of cell traps 43 also having constrictions 10 by their outlet .

[0104] Figure 8 illustrates a microfluidic device similarly to that in figure 7 , from the side (upper drawing) and from above ( lower drawing) . An cross-section along an imaginary line B-B is shown (upper drawing) , illustrating the relative thickness of the cavities of the valve pressure control channels 9 and the cavities of the valves 7 .

[0105] Figure 9 illustrates growth of bacterial cells (E . coli ) in a single cell trapping region 4 ( formed as a channel ) when its sequentially exposed to no antibiotic and five increasingly high concentrations of antibiotic ( ciprofloxacin) for 30 min each and finally its fixed and exposed to a fluorescent probe that identi fies it as E . coli . Each of the six sub- figures show the same cell trapping region 4 photographed in time-lapse over the course of 30 minutes , from the left to the right . The markings on the left and right side of each of the six figures shows the length of the cell at the bottom of the channel or the sum of lengths of the daughter-cells descending from it . The percentage growth over 30 min is calculated and indicated beneath each sub- figure . Fig 10 illustrates sequential phenotyping of growth in a microfluidic cell trapping region 4 followed by in situ FISH genotyping of the species ID with probes that identifies E. coli or Klebsiella.

[0106] The device is suitable for performance of methods where a sample of cells, such as a sample comprising a relatively small number of cells, are to be sequentially exposed to different reagents, or concentrations of reagents, in order to determine their reaction to those reagents / concentrations . The following methods are thereby suitable for performance using the device.

[0107] In addition there is disclosed a method of evaluating the effect of a plurality of reagents on living cells in a sample comprising less than 20 cells, comprising the steps of: a) providing a microfluidic device as the device disclosed herein, b) introducing a sample comprising less than 20 cells into the cell trapping region 4, for example by introducing the sample through a media inlet port 5 in the same manner as the other reagents are introduced, c) sequentially exposing the cells in the cell trapping region 4 to different reagent compositions by: i. introducing a plurality of reagents through separate media inlet ports 5, ii. by use of the valves 7, controlling fluid flow accessibility through the individual media channels 6, such that flow of at least one first reagent is allowed, such that the cells in the cell trapping region 4 are exposed to a certain concentration of reagent (s) at a first time point , iii. by the valves 7, adapting the fluid flow accessibility through the individual media channels 6, such that the cells in the cell trapping region 4 are exposed to a different concentration of said first reagent (s) or to at least one second reagent at a second time point, iv. optionally repeating the last step until the cells in the cell trapping region 4 have been exposed to all reagents and concentrations of reagents that are to be analysed at different time points, d) performing at least one of observing, measuring and collecting data regarding the cells at different time points.

[0108] In other words, media sources are connected to the media inlet ports 5 or media reservoirs 51 are filled with media (such as reagents to be analysed, media comprising cells, cell culture media) . The cell sample is introduced into the cell trapping region 4 before or after connecting the other media, suitably through a media inlet port 5 and an individual channel 6. The fluid flow accessibility of the individual channels 6 are then controlled by the valves 7, such as by adding / relieving pressure in cavities forming the valves 7, such that at a first time point, a desired composition of first reagent (s) is allowed to flow to the cell trapping region 4, while the individual channels 6 that are in fluid contact with other reagents are closed by their respective valves 7. The first reagent (s) may be only one reagent to be analysed, or a reagent to be analysed and a cell culture media for dilution of the first reagent to a certain concentration. The second reagent (s) may similarly be one reagent, several reagents, or reagent plus dilution media.

[0109] The method may be performed on a sample comprising more than 20 cells, such as 20-1000 cells, but the device and method is specifically adapted for the purpose of being able to analyse a small number of cells. In one embodiment, the sample comprises less than 10 cells .

[0110] In one embodiment, the method further comprises a step of culturing the cells in cell culturing media in the cell trapping region 4 for a time period before exposing the cells to the first reagent (s) . The number of cells that will be exposed to reagents may thereby be increased.

[0111] In one embodiment, the collected data represents phenotypic traits of the cells, such as growth rate, generation time, morphology, fluorescence, or lysis rate. The data may be collected using a microscope providing imaging data of the cells in the cell trapping region 4. In one embodiment, the collected data is analysed using a computer software performing at least one of image analysis, automated cell counting, feature extraction and quantification, statistical analysis, dose-response analysis, timecourse analysis, and machine learning.

[0112] In one embodiment, at least one of the reagents that the cells are exposed to are antibiotics. In one embodiment, antibiotic susceptibility of the cells is determined based on comparing phenotypic traits observed or measured at different time points. In one embodiment, a minimum inhibitory concentration (MIC) is determined. The MIC is the lowest concentration of a substance (such as an antibiotic) that inhibits growth of a microorganism.

[0113] In one embodiment, the sample is provided from a patient with suspected sepsis. This is an example of a case where time is of the essence, and where a sample from a patient may be provided that comprise a low number of cells to be analysed.

[0114] The microfluidic device used in the method may be any of the embodiments of the microfluidic device disclosed herein .

[0115] There is further disclosed a method of characterizing genotypes of strains of cells in a sample, comprising: al) providing a microfluidic device as the device disclosed herein, bl) introducing a sample of cells into the cell trapping region 4, cl) culturing the cells in the cell trapping region 4 in the presence of a cell culturing media, dl) performing in situ identification of at least one RNA or DNA sequence of the cells by introducing reagents needed for performance of such identification into the cell trapping region (4) in a suitable order, the order being regulated by controlling the flow accessibility through the individual media channels (6) using the valves ( 7 ) .

[0116] In one embodiment, a step of observing or measuring a phenotype of the living cells is performed between step bl ) and dl ) .

[0117] The method may be combined with step g) and h) from the method of evaluating the effect of a plurality of reagents on living cells, being performed between step bl ) and dl ) .

[0118] In one embodiment, the step dl) of performing in situ identification of RNA or DNA comprises fluorescence in situ hybridization by sequential introduction of reagents in at least one cell trap. The cells may be bacteria, and fluorescent oligonucleotide probes may be used that are selected to bind to ribosomal RNA associated with certain bacterial species or classes.

[0119] In one embodiment, step dl) comprises in situ sequencing by sequential introduction of reagents in at least on cell trap.

[0120] Other methods for in situ genotyping that require that the cells exposed to a sequence of reagents may be performed in the microfluidic device ; these can be isothermal such as LAMP or depend on temperature cycling .

[0121] In one embodiment , the cells are bacteria, and the fluorescent oligo probes are s el ected to bind to ribosomal RNA associated with a certain bacterial species to identi fy the species or class of bacteria .

[0122] While the microfluidic device as disclosed herein is suitable for performance of the methods disclosed herein, the methods may also be performed by use of a di f fering microfluidic device comprising a cell trapping region and at least one channel and at least two media ports . Such a device may be without any integrated pressure controlled valves 7 formed by cavities . The sequential introduction of reagents may be provided by aid of external structures that may involve valve functions or pump functions controlling sequential introduction of reagents into the device . In other words it is possible to grow a small number (<20 ) of bacteria in a microfluidic device and exposing them to di f ferent concentration of antibiotics , or di f ferent antibiotics , by supplying these in a controlled sequence to the microfluidic device from an external source not controlled by integrated microfluidic valves . Also in this case antibiotic susceptibility of the cells can be determined based on comparing phenotypic traits observed or measured at di f ferent time points . For example , the relative growth increase per time for a cell at the wild type MIC concentration of antibiotic can be compared to that of a cell in the same lineage at an earlier timepoint corresponding to zero concentration of the antibiotics . I f the growth rate is not impacted by the antibiotic the bacterium may be resistant . The wild- type MIC concentration should here be interpreted to be within the commonly accepted measurement accuracy of one log2 from the MIC concentrations reported by EUCAST or CLS I . Zero concentration should be interpreted as a concentration that is so low that it has the same phenotypic response as i f there was no antibiotic molecule in the growth media .

Claims

Claims1. A method for evaluating the effect of a plurality of reagent compositions on living cells, comprising the sequential steps of: a. providing a microfluidic device comprising at least one cell trapping region (4) , at least one media port (5) , and at least one channel (6) connecting said cell trapping region (4) and said at least one media port ( 5 ) , b. introducing a sample comprising at least one living cell into the cell trapping region (4) via the at least one media port (5) , wherein the at least one cell consists of one selected from of: i. one cell, that can be the origin of one linage of cell ii. a plurality of cells, that can be the origins of different lineages of cells, c. sequentially exposing the at least one cell in the cell trapping region (4) to different reagent compositions, by sequentially introducing the different reagent compositions into the cell trapping region (4) via the at least one media port (5) and at least one channel (6) , and collecting data from at least one cell or descendants in its lineage of cells at different points in time corresponding to exposure to the different reagent compositions using an optical microscope, d. determining a property of the at least one cell by using data regarding cells in the same lineagecollected in step c) during exposure to at least two different reagent compositions.

2. The method, according to claim 1, wherein fewer than 20 living cells are introduced in step b) .

3. The method, according to any one of claims 1-2, wherein in step c) , the length of one cell or the sum of lengths of daughter-cells descending from it is determined by optical microscopy and / or image analysis .

4. The method, according to any one of claims 1-3, wherein in step c) , the size of one cell or the sum of sizes of daughter-cells descending from it is determined by optical microscopy and / or image analysis .

5. The method according to any one of claims 1-4, wherein in step d) the property determined is the difference in percentage growth between different time points corresponding to different reagents compositions.

6. The method according to any one of claims 1-5, wherein in step c) , at least two of the different reagent compositions are reagent compositions comprising different concentrations of the same reagent.

7. The method according to any one of claims 1-6, wherein in step c) , at least two of the different reagent compositions are reagent compositions comprising different reagents or different combinations of reagents .

8. The method according to any one of claims 1-7, wherein in step c) , at least two of the different reagent compositions are reagent compositions comprising different reagents of different concentration.

9. The method according to any one of claims 1-8, wherein step d) involves analyzing the change in a phenotypic trait due to a change in reagent composition for several individual cell lineages originating in different single cells by calculating a property from changes in response for the several individual cell lineages.

10. The method according to any one of claims 1-9, comprising a step of exposing a cell or a cell lineage in the cell trapping region (4) to cell culturing media and measuring properties of the cells to determine a reference distribution for the cells' growth rates, generation times, or cell sizes, and using this reference distribution to monitor deviation from the distribution when exposing the cells another reagent composition.

11. The method according to any one of claims 1-10 wherein the collected data represents phenotypic traits of the cells, such as growth rate, generation time, fluorescence, morphology or lysis rate, where morphology can include cell size at division.

12. The method according to any one of claims 1-11, wherein the optical microscope in step c) provides imaging data of the at least one cell in the cell trapping region (4) .

13. The method according to any one of claims 1-12, wherein the collected data is analysed using a computer software performing at least one selected from image analysis, automated cell counting, feature extraction and quantification, statistical analysis, dose-response analysis, time-course analysis, and machine learning.

14. The method according to any one of claims 1-13, wherein the next reagent composition is decided in real-time based on the detected phenotypic response to a previous media composition.

15. The method according to any one of claims 1-14, wherein at least one reagent is an antibiotics.

16. The method according to any of claims 13-15, wherein a minimum inhibitory concentration (MIC) is determined .

17. The method according to any one of claims 1-16, wherein at least one reagent is a plurality of different antibiotics and wherein the at least one cell is at least one bacterial cell.

18. The method according to any of claims 13-17, wherein the relative growth increase per time for a cell near the wild type MIC concentration of an antibiotic is compared to that of a cell in the same lineage at an earlier timepoint corresponding to near zero concentration of the antibiotics.

19. The method according to any one of claims 1-18, wherein the cells are bacterial cells.

20. The method according to any one of claims 1-19, wherein the sample is prepared from a body fluid from a patient with sepsis, meningitis, gonorrhoea, urinary tract infection, or tuberculosis.

21. The method according to any one of claims 1-20, wherein at least one reagent comprises a phage.

22. The method according to any one of claims 1-21, wherein at least one reagent is a cancer drug.

23. The method according to any one of claims 1-22, wherein the microfluidic device comprises at least two media ports (5) each connected to the cell trapping region (4) by an individual media channel (6) .

24. The method according to any one of claims 1-23, wherein the microfluidic device comprises a first outer layer (1) , a middle layer (2) and a second outer layer (3) , wherein the cell trapping region (4) and the media channel (s) (6) , are formed at least partly by cavities between the middle layer (2) and at least one of the first layer (1) and second layer (3) , such as by recesses in the middle layer (2) .

25. The method according to any one of claims 1-24, wherein sequential exposure of reagents in step c) is controlled by aid of external structures that involve valve functions and / or pump functions.

26. The method according to any one of claims 1-25, wherein the microfluidic device comprises a plurality of media inlet ports (5) each being fluidly connected to the cell trapping region (4) by an individual media channel (6) , and step c) comprises: i. introducing a plurality of reagents through at least one media inlet port (5) , ii. at a first time point, controlling fluid flow accessibility through the individual media channels (6) , such that a certain flow rate of at least one reagent, and optionally of a dilution media, is allowed, such that the cells in the cell trapping region (4) are exposed to a certain concentration of, and optionally combination of, reagent (s) , iii. at a second time point, adapting the fluid flow accessibility through the individual media channels (6) , such that the cells in the cell trapping region (4) are exposed to a different concentration of at least one reagent present in step ii) and / or at least one different reagent, and iv. optionally repeating the last step a number of times, such that the cells in the cell trapping region (4) are exposed to all concentrations and / or combinations of reagent (s) that are to be analysed at different time points.

27. The method according to any one of claim 24 or claims 25-26, when dependent on claim 24, wherein the microfluidic device comprises a plurality of pressurecontrolled valves (7) , wherein the valves (7) are formed at least partly by cavities between the middle layer (2) and at least one of the first layer (1) and the second layer (3) , such as by recesses in the middle layer (2) , and wherein a fluid flow accessibility from each media inlet port (5) to the cell trapping region (4) is controllable by operation of at least one valve (7) configured to control the fluid flow accessibility through the individual media channel (6) fluidly connecting that media inlet port (5) to the cell trapping region (4) .

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