A microfluidic device with valves

Abstract

There is provided a microfluidic device comprising a microfluidic device comprising a middle layer (2), a first layer (1) and a second layer (3), a cell trapping region (4) with an inlet (41), media inlet ports (5) each being fluidly connected to the cell trapping region (4) by an individual media channel (6), and a plurality of pressure controlled valves (7). The cell trapping region (4), the individual media channels (6), and the valves (7) each are formed by cavities in the layers. A fluid flow accessibility of each individual media channel (6) is controllable by operation of a valve (7). The device facilitates analysis of the effects of a plurality of reagents on cells in a sample comprising a low number of cells. Two types of methods to be performed using such a device is provided.

Description

[0001] A microfluidic device with valves

[0002] Technical Field

[0003] The invention relates to a microfluidic device for cell capturing and analysis as well as a method for capturing cells and a method for analysing cells using such a device.

[0004] Background

[0005] Microfluidic devices are well known in the art and 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] Summary

[0010] It is an object of the invention to obviate at least some of the problems in the prior art and provide a microfluidic device as well as a method of capturing and of analysing cells, the device and method in particular providing the possibility use the same rare cells for several analytical tests.

[0011] In a first aspect there is provided a microfluidic device comprising

[0012] - 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,

[0013] - a cell trapping region 4 having at least one inlet 41,

[0014] - a plurality of media inlet ports 5 each being fluidly connected to the cell trapping region 4 by an individual media channel 6,

[0015] - a plurality of pressure controlled valves 7, wherein the cell trapping region 4, the individual media channels 6, and the valves 7 each are 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, and wherein a fluid flow accessibility of each individual media channel 6 is controllable by operation of at least one valve 7 configured to control the fluid flow accessibility through that individual media channel 6.

[0016] The device facilitates analysis of the effects of a plurality of reagents on cells in a desired temporal order. The device is further easy to manufacture. All the features may for example be provided by casting in one middle layer located between two outer layers. Providing all such fine features in one layer reduces the need for accurate alignment at assembly.

[0017] In a second aspect, there is provided 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 such 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 said first reagent 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 or to a 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.

[0018] In a third aspect, these is provided a method of characterizing genotypes of strains of cells in a sample, comprising : al) providing a microfluidic device such 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 ) .

[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.

[0025] 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. 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 identifies it as E. coli .

[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 identifies E. coli or Klebsiella.

[0029] Detailed description

[0030] The following detailed description discloses by way of examples details and embodiments by which the invention may be practised.

[0031] 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.

[0032] 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.

[0033] The term "valve" is used to denote 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.

[0034] As used herein, the phrase "at least one" means that there may be one or more of that object. Throughout the text, characteristics disclosed to describe that object may be applied to all such objects present in the device, even if it is described in relation to "an / the" object, if not clearly specified that it regards just one specific such object or certain such objects.

[0035] 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. In a first aspect , there is provided 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 ( 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 .

[0036] 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 .

[0037] 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 .

[0038] 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 .

[0039] 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 .

[0040] 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 .

[0041] 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 .

[0042] 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 .

[0043] 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 . 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 .

[0044] 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 .

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 .

[0050] 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 .

[0051] 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 . 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 .

[0052] 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 .

[0053] 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 . Figures 5 and 6 do not show any valve pressure control channel 9 that may lead to the cavity forming valve 7 .

[0054] 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 ) , 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 .

[0055] 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 .

[0056] 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.

[0057] 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.

[0058] 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.

[0059] In a second aspect, there is provided 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: e) providing a microfluidic device as the device disclosed herein, f) 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, g) 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, h) performing at least one of observing, measuring and collecting data regarding the cells at different time points.

[0060] 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.

[0061] 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 .

[0062] 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.

[0063] In one embodiment, the collected data represents phenotypic traits of the cells, such as growth rate, 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, time-course analysis, and machine learning.

[0064] 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. 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.

[0065] The microfluidic device used in the method may be any of the embodiments of the microfluidic device disclosed herein .

[0066] In a third aspect, these is provided 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 ) .

[0067] In one embodiment, a step of observing or measuring a phenotype of the living cells is performed between step bl ) and dl ) .

[0068] 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 ) .

[0069] 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.

[0070] In one embodiment, step dl) comprises in situ sequencing by sequential introduction of reagents in at least on cell trap. Other methods for in si tu 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 .

[0071] In one embodiment , the cells are bacteria, and the fluorescent oligo probes are se l ected to bind to ribosomal RNA associated with a certain bacterial species to identi fy the species or class of bacteria .

[0072] 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. If the growth rate is not impacted by the antibiotic the bacterium may be resistant.

Claims

Claims1. A microfluidic device comprising- 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) ,- a cell trapping region (4) having at least one inlet (41) ,- a plurality of media inlet ports (5) each being fluidly connected to the cell trapping region (4) by an individual media channel (6) ,- a plurality of pressure controlled valves (7) , wherein the cell trapping region (4) , the individual media channels (6) , and the valves (7) each are 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) , wherein all cavities are formed by recesses in the middle layer (2) , and wherein a fluid flow accessibility of each individual media channel (6) is controllable by operation of at least one valve (7) configured to control the fluid flow accessibility through that individual media channel ( 6 ) .

2. The microfluidic device according to claim 1, wherein the individual media channels (6) are formed at least partly by at least one cavity between the middle layer(2) and the first layer (1) , and wherein the valves are formed at least partly by cavities between the middle layer (2) and the second layer (3) .

3. The microfluidic device according to any of the preceding claims, wherein a section of the middle layer (2) between a cavity partly forming a valve (7) and cavity forming an individual media channel (6) is movable based on a pressure in the valve cavity.

4. The microfluidic device according to any of the preceding claims, wherein at least two individual media channels (6) fluidly connect in a common channel (8) upstream of the cell trapping region inlet (41) .

5. The microfluidic device according to any of the preceding claims, wherein at least one of the cavities forming a valve (7) is in fluid connection with a valve pressure control channel (9) .

6. The microfluidic device according to any of the preceding claims, wherein at least a part of the microfluidic device is transparent.

7. The microfluidic device according to any of the preceding claims, further comprising a plurality of media reservoirs (51) each fluidly connected to a media inlet port (5) , 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) .

8. The microfluidic device according to any of the preceding claims, wherein the volume of the cell trapping region (4) is variable.

9. The microfluidic device according to any of the preceding claims, wherein the cell trapping region (4) comprises an outlet (42) and one or more constrictions (10) configured to hinder cells in the cell trapping region (4) from exiting through the outlet (42) .

10. The microfluidic device according to any of the preceding claims, wherein 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.

11. 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: i) providing a microfluidic device comprising- 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) ,- a cell trapping region (4) having at least one inlet (41) ,- a plurality of media inlet ports (5) each being fluidly connected to the cell trapping region (4) by an individual media channel (6) ,- a plurality of pressure controlled valves (7) , wherein the cell trapping region (4) , the individual media channels (6) , and 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 ) ,wherein all cavities are formed 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) . j) introducing a sample comprising less than 20 cells into the cell trapping region (4) , k) 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 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 said first 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 andconcentrations of reagents that are to be analysed at different time points,1) performing at least one of observing, measuring and collecting data regarding the cells at different time points.

12. The method according to claim 11, comprising 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) .

13. The method according to any of claims 11 - 12, wherein the collected data represents phenotypic traits of the cells, such as growth rate, fluorescence, morphology or lysis rate.

14. The method according to any of claims 11 - 13, wherein collecting data involves a microscope providing imaging data of the cells in the cell trapping region (4) .

15. The method according to any of claims 11 - 14, wherein 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, time-course analysis, and machine learning.

16. The method according to any of claims 11 - 15, wherein the reagents are antibiotics.

17. The method according to claim 16, wherein antibiotic susceptibility of the cells is determinedbased on comparing phenotypic traits observed or measured at different time points.

18. The method according to any of claims 16-17, wherein a minimum inhibitory concentration (MIC) is determined .19 The method according to any of claims 11-18, wherein the sample is a sample provided from a patient with suspected sepsis.

20. The method according to any of claims 11-19, wherein the microfluidic device comprises a first layer (1) , a middle layer (2) and a second layer (3) , wherein the cell trapping region, the individual media channels (6) , and 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 second layer (3) , such as by recesses in the middle layer (2) .

21. A method of characterizing genotypes of strains of cells in a sample, of: al) providing a microfluidic device comprising- 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) ,- a cell trapping region (4) having at least one inlet (41) ,- a plurality of media inlet ports (5) each being fluidly connected to the cell trapping region (4) by an individual media channel (6) ,- a plurality of pressure controlled valves (7) ,wherein the cell trapping region (4) , the individual media channels (6) , and 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 ) , wherein all cavities are formed 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) , bl) introducing a sample of cells into the cell trapping region (4) , cl) optionally 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 ) .

22. The method of characterizing genotypes of strains of cells in a sample according to claim 21, further comprising, between step bl) and dl) , a step of observing or measuring a phenotype of the living cells.

23. The method of characterizing genotypes of strains of cells in a sample according to claim 21 or 22, wherein the step dl) comprises fluorescence in situ hybridization by sequential introduction of reagents in at least on cell trap.

24. The method of characterizing genotypes of strains of cells in a sample according to claim 23, wherein the cells are bacteria, and wherein the fluorescent oligonucleotide probes used that are selected to bind to ribosomal RNA associated with certain bacterial species or classes.

25. The method of characterizing genotypes of strains of cells in a sample according to claim 21 or 22, wherein the step dl) comprises in situ sequencing by sequential introduction of reagents in at least on cell trap.

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