Patent Description:
Antibiotic-resistant bacteria represent a growing global problem, as these bacteria cannot be killed or made to stop dividing by antibiotics. The generation time of bacteria can in many cases be very fast (around <NUM> minutes), and due to the short generation time and relative genetic instability of bacteria, the bacteria may quickly acquire resistance towards antibiotics. There is an increasing prevalence of antibiotic-resistant bacterial infections in the human population, and some of these bacteria have even become multi-resistant, sometimes meaning that there are no efficient antibiotics available to halt their growth. These multi-resistant bacteria are a serious public health problem as patients infected with such bacteria may die since their bacterial infections cannot be treated.

The traditional approaches for the identification and study of microorganisms, including but not limited to bacteria, fungi, parasites and viruses, and their resistance to test agents, such as antibiotics, which kill or inhibit the growth of the microorganism, have mainly, in the example of bacteria, been limited to broth micro dilution where varying concentrations of the antibiotics and the bacteria to be tested are added to different wells of a micro titer plate. After the allotted time, typically <NUM> to <NUM> hours, of incubation the wells are checked for bacterial growth by measuring the optical turbidity in the different wells.

The Kirby-Bauer test, which is basically an agar diffusion method, is also commonly used. Here wafers, or discs, that contain bactericidal or bacteriostatic antibiotics of defined concentrations are placed on agar plates where bacteria have been spread. The agar plates are left to incubate and if an antibiotic stops the bacteria from growing or kills the bacteria, a zone of inhibition will become visible after incubation.

Another prior art approach is the so-called E-test. The E-test uses a rectangular strip impregnated with different concentrations of a test agents to be evaluated for its killing or growth inhibiting effect. In a typical approach, bacteria are spread and grown in a 2D culture on an agar plate, where after the E-test strip is placed on top of the agar plate. The E-test strip releases the test agent by diffusion and the growth inhibitory effects of the released test agent are typically inspected after <NUM> hours of incubation.

The Kirby-Bauer test and the E-test as two-dimensional (2D) culture methods for antibiotic susceptibility testing (AST) and evaluation of the effects of antibiotics or other test agents on microorganisms have several limitations. For instance, these setups normally require that the microorganisms, e.g., bacteria, are cultured over night to allow for a clear result readout showing if a particular bacteria strain is resistant or not to a given antibiotic. A further limitation is that readouts of the inhibitory concentration of the test substance is only possible in distinct digital steps and in the selected concentrations used in the E-test strip. The shortcomings for the broth microdilution approach for traditional AST are basically the same.

In order to reduce the AST time, microfluidic channel systems for rapid AST (RAST) have been developed. Such RAST approaches include droplet-based microfluidic channel systems, in which bacteria are captured in a droplet that includes an antibiotic. A limitation with the droplet-based system is that only a single antibiotic concentration can be tested. Other RAST approaches include using polydimethylsiloxane (PDMS) microchannels, dielectrophoretic capturing of bacteria in microfluidic electrode structures, preloaded PDMS layers with antibiotics, covalently binding bacteria to microfluidic channels and subjecting them to mechanical shear stress, using asynchronous magnetic bead rotation (AMBR) biosensors or tracking single cell growth in a microfluidic agarose channel system. A major limitation of these various PAST approaches is that they can only test a single antibiotic concentration or a set of a few selected antibiotic concentrations.

It has further been proposed to use a microfluidic system for analysis of antibiotic susceptibility of bacterial biofilms. Such a microfluidic system, however, requires <NUM> hours of incubation and the bacteria to be tested must contain a plasmid able to express green fluorescent protein (GFP).

Hence, there is still a need for systems for response testing of microorganism that do not have the disadvantages of the prior art. There is a particular need for a cassette assembly that can be used in such systems.

<CIT> discloses a device having a first surface having a plurality of first areas and a second surface having a plurality of second areas. The first surface and the second surface are opposed to one another and can move relative to each other from at least a first position where none of the plurality of first areas, having a first substance, are exposed to plurality of second areas, having a second substance, to a second position. When in the second position, the plurality of first and second areas, and therefore the first and second substances, are exposed to one another. The device may further include a series of ducts in communication with a plurality of first second areas to allow for a substance to be disposed in, or upon, the plurality of second areas when in the first position.

<CIT> discloses an apparatus that integrates one dimensional separation to another dimensional separation and automates the operation of the two dimensional separation. The first dimensional separation is performed in one column while the second dimensional separation is performed in multiple separation columns. The integration is achieved using a one-piece, a two-piece, or a three-piece interface.

<CIT> discloses an off-column sample injection scheme for introducing samples into micro-reaction channels in microfabricated devices. The off-column sample injection is effected by introducing sample from a sample reservoir provided on the substrate of the microfabricated device into a reaction channel via a constricted channel or opening interface. Alternatively, the off-column sample injection is effected by introducing sample from a sample reservoir that is provided outside the substrate of the microfabricated device. A through-hole is provided in the substrate to facilitate sample introduction into the reaction channel. The free-end of a capillary tube connected to the sample-channel is moved alternatively to a sample and an auxiliary solution to bring multiple samples in series to the vicinity of a reaction channel for convenient sample introduction and high-throughput assays. Fixed volumes of samples are metered into the reaction channel using one or more slidable blocks having at least one fixed-length sample metering channel. A sample injection scheme based on injection time is implemented using relatively sliding blocks of separation channels and sample channels. Separation channels are configured in relation to the slidable block in a manner that enables separations to be conducted continuously for high-throughput assays.

<CIT> discloses devices, systems, methods, and kits for performing separation, immobilization, blotting, and/or detection of analytes from biological samples. The devices are constructed from two solid substrates with surfaces in contact. The devices include a plurality of channels formed from indentations in these surfaces. The indentations can be aligned with each other across the interface between the substrates, and realigned by shifting or sliding one substrate relative to the other. The devices are constructed from three layers of a solid substrate. A separation channel in the middle layer of the device is first used for analyte separation. The middle layer can then be slid relative the top and/or bottom layer, thereby aligning the separation channel with a blotting membrane. Analytes can then be transferred to the membrane using electrodes in the top and bottom layers.

It is a general objective to provide a cassette assembly that can be used in systems for response testing of microorganisms.

This and other objectives are met by embodiments as disclosed herein.

The present invention is defined in the independent claim. Further embodiments of the invention are defined in the dependent claims.

An aspect of the invention relates to a cassette assembly comprising a cover, a first cassette half and a second cassette half and a slider. The cover is configured to be positioned onto the first and second cassette halves and keep the first and second cassette halves together with the slider sandwiched between the first and second cassette halves. The slider comprises N≥<NUM> test chambers in the form of through holes through the slider. Each of the first and second cassette halves comprises N waste tanks connected in series and separated by respective walls and an excessive liquid tank in fluid connection with the waste tanks by a vertical outlet channel and by an air valve. An inlet port of the first cassette half is configured to receive a liquid caused to sequentially fill each waste tank and a head space defined above the waste tanks. Opening of the air valve causes drainage of liquid in the head spaces into the excessive liquid tanks through the vertical outlet channels and a separation of equal volumes of liquid in the waste tanks.

In an embodiment, the slider comprises a first through hole in connection with a first end of the slider and a second through hole in connection with a second end of the slider. In this embodiment, the first and second cassette halves each comprises a circumferential channel having a first opening configured to be aligned with the first through hole and a second opening configured to be aligned with the second through hole. Each of the first and second cassette halves also comprises a serpentine channel interconnecting the circumferential channel and the waste tanks. In this embodiment, the inlet port of the first cassette half is in fluid connection with the circumferential channel. The serpentine channels present a higher flow resistance to the liquid as compared to the circumferential channel to enable filling of the circumferential channels prior to entering of the liquid into the waste tanks through the serpentine channels.

The cassette assembly enables an accurate metering of predefined volumes of liquid in reservoir tanks to achieve accurate concentrations of test agents and controlled establishment of concentration gradients over 3D culture matrices in the test chambers. As a consequence, microorganisms present in a biological sample can be exposed to well-defined concentration gradients for the purpose of determining the response of the microorganisms to the test agents, including determination of minimum inhibitory concentration (MIC) values.

Throughout the drawings, the same reference numbers are used for similar or corresponding elements.

The embodiments generally relate to cassette assemblies for fluidic systems, and in particular to such cassette assemblies that can be used to determine a response of microorganisms to test substances or agents.

It has been concluded that fluidic systems, such as microfluidic systems, can be designed to be particularly suitable to monitor or determine the response of microorganisms to various test agents. In more detail, such fluidic systems, having a test chamber with a three-dimensional (3D) culture matrix containing a culture of the relevant microorganisms, provide an efficient tool to quickly determine the response of the microorganisms to the test agent or indeed to any combination of multiple test agents.

Such fluidic systems have key features that make them a very efficient tool. Firstly, a steady-state gradient of the relevant test agent can quickly and accurately be established over at least a portion of the 3D culture matrix. This means that a continuous range of test agent concentrations is established from a high concentration at one of the end portions or sides of the 3D culture matrix down to a low or zero concentration at another end portion or side of the 3D culture matrix. Hence, a continuous range of concentrations of the test agent can be tested. This is in clear contrast to prior art techniques where one or at most a few predefined concentrations but not any continuous range of concentrations can be tested. Accordingly, a more exact determination of, for instance, minimum inhibitory concentration (MIC) of an antibiotic can be made.

Secondly, the microorganisms are cultured in a 3D culture matrix. Accordingly, the microorganisms are allowed to grow in three dimensions. This in turn provides a significant difference between areas of the 3D culture matrix where viable and growing microorganisms are present and areas with cell death or low growth. Accordingly, the embodiments provide an enhanced signal-to-noise ratio. Hence, it is much easier to differentiate between different areas or zones in the 3D culture matrix as compared to growing microorganisms as a biofilm on a two-dimensional (2D) surface where fewer microorganisms can be grown and, thus, lower detection signals are generated.

A gradient of the test agent can quickly be established over at least a portion of the 3D culture matrix. This together with the possibility of microorganism growth in three dimensions enables reading the response of the microorganisms to the test agent in a very short time, generally within one or at most a few hours. This should be compared to several of the prior art techniques, typically requiring incubation overnight.

The present invention relates to a cassette assembly for a fluidic system and that is designed to house 3D culture matrices. The cassette assembly can therefore be used in the fluidic system to monitor and determine responses of microorganisms to test agents. A typical application of the cassette assembly is to analyze the susceptibility of a biological sample, such as a blood sample, from a patient suffering from a bacterial infection, such as sepsis, to different antibiotics and to determine the MIC of each of the antibiotics for the bacteria in the biological sample in a short period of time, typically within <NUM>-<NUM> hours.

The cassette assembly is preferably in the form of a disposable cassette assembly preloaded with test agents, such as antibiotics, into which the biological sample is loaded. The fluidic system in addition comprises an analysis instrument that is used to monitor and analyze, preferably optically monitor and analyze, bacterial growth while the biological sample is exposed to gradients of antibiotics.

The cassette assembly <NUM>, see <FIG>, is composed of four main components, two cassette halves 200A, 200B, a slider <NUM> and a cover <NUM>. The cassette halves 200A, 200B comprise inlet ports <NUM>, <NUM> for the biological sample and a liquid and connection ports <NUM> for a pressure interface. The slider <NUM> comprises test chambers <NUM> in the form of through holes through the thickness of the slider <NUM>. The 3D culture matrices will be formed in these test chambers <NUM> and microorganism growth and response to test agents will be monitored and analyzed. The cover <NUM> is employed to keep the main components together and has a supporting function.

The cover <NUM> has a lid <NUM> and four side walls <NUM>, <NUM>, <NUM>, <NUM> extending from the lid <NUM> and comprising two end walls <NUM>, <NUM> and two longitudinal walls <NUM>, <NUM>. The two end walls <NUM>, <NUM> preferably comprise a respective window <NUM> that is configured to receive end walls <NUM>, <NUM> of the two cassette halves 200A, 200B as shown in <FIG>.

The lid <NUM> of the cover <NUM> comprises openings <NUM>, <NUM>, <NUM>, <NUM> positioned to be aligned with inlet and connection ports <NUM>, <NUM>, <NUM> in the two cassette halves 200A, 200B when the main components are assembled together as shown in <FIG>. The lid <NUM> also comprises a central opening or window <NUM> providing visual access through the cover <NUM> to the slider <NUM> and the test chambers <NUM> provided therein. In a preferred embodiment, supporting structures <NUM> extend from the bottom surface of the lid <NUM> and supports a slider cover <NUM> configured to be aligned with and covering the slider <NUM> in the cassette assembly <NUM>. The supporting structures <NUM> are arranged at the lid <NUM> with spaces between adjacent supporting structures <NUM> to form windows <NUM> allowing visual access to reservoirs <NUM> in the cassette halves 200A, 200B in the cassette assembly <NUM>. The slider cover <NUM> comprises openings or windows <NUM> that are aligned with the test chambers <NUM> in the slider <NUM> to provide visual access through the slider cover <NUM> and into the test chambers <NUM>.

In an embodiment, snap-fit connectors may be used to interconnect the cover <NUM> with the cassette halves 200A, 200B with the slider <NUM> sandwiched between the first and second cassette halves 200A, 200B. In such a case, the cover <NUM> may comprise at least one snap-in area or through hole, such as in each of its end walls <NUM>, <NUM> and/or longitudinal walls <NUM>, <NUM>. The first and second cassette halves 200A, 200B may then comprise a protruding structure or lip configured to enter the snap-in area or through hole in the cover <NUM> to achieve a snap-fit lock, preferably a cantilever snap-fit lock.

In an embodiment, the cover <NUM> may comprise two snap-in areas or through holes positioned one on top of the other. In such an embodiment, the cover <NUM> can be locked to the first and second cassette halves 200A, 200B in two different positions, preferably corresponding to a transportation position, see <FIG>, and a loading position, see <FIG> and <FIG>. In such a case, the cassette assembly <NUM> may be delivered in the transportation position with the protruding structures or lids of the first and second cassette halves 200A, 200B in the lowest of the snap-in areas or through holes in the cover <NUM>. This means that the components of the cassette assembly <NUM> are preferably interlocked also during transport to thereby prevent the components from falling apart. In the loading position, the cover <NUM> is pressed downwards thereby causing the protruding structures or lids of the first and second cassette halves 200A, 200B to enter the uppermost of the snap-in areas or through holes in the cover <NUM> to thereby keep the components interlocked in this loading position.

In the embodiments above, the snap-fit connector has been described with the snap-in area in the cover <NUM> and the protruding structures or lids in the first and second cassette halves 200A, 200B. In another embodiment, the snap-in areas are instead in the first and second cassette halves 200A, 200B with the matching protruding structure or lids in the cover <NUM>.

Also other solutions of interlocking the components of the cassette assembly <NUM>, preferably in two different positions corresponding to the transportation and loading positions, are possible and can be used instead of snap-fit connectors.

The slider <NUM> comprises two through holes <NUM>, each provided in connection with a respective end of the slider <NUM>. These through holes <NUM> form part of a fluidic system to enable liquid to flow between the cassette halves 200A, 200B and through the slider <NUM>. These two through holes <NUM> are arranged to be aligned with respective openings <NUM> in front walls <NUM> of the cassette halves 200A, 200B as is more clearly shown in <FIG>. The end sides of the slider <NUM> preferably comprise a projection <NUM> that can be slid into a matching indentation in a side wall <NUM> of the cassette halves 200A, 200B to align the slider <NUM> relative to the cassette halves 200A, 200B. The slider <NUM> can preferably be vertically moved relative to the cassette halves 200A, 200B between a sample filling position (<FIG>) and a flow position (<FIG>) as is further described herein.

The two cassette halves 200A, 200B are preferably identical with the exception that one of the cassette halves 200A is preferably preloaded with test agents, such as antibiotics, in reservoirs <NUM>, whereas the other cassette half 200B preferably lacks such test agents. In an embodiment, one of the two cassette halves 200A, 200B also comprises a plug, membrane or tape in the liquid inlet to prevent leakage through the circumferential channel during filling as is further described herein. In the following, a cassette half <NUM> is described in more detail and this description applies to both cassette halves 200A, 200B unless otherwise indicated.

A cassette half <NUM> comprises end sides or walls <NUM>, <NUM>, of which one is preferably longer than the other one. One of the end walls <NUM> comprises a projection <NUM> configured to be slid into a matching groove <NUM> in an end wall <NUM> of the other cassette half. The cassette half <NUM> also comprises a back wall <NUM> and a front wall <NUM> configured to face the slider <NUM>. The front wall <NUM> comprises openings or connections <NUM> that are to be aligned with through holes <NUM> in the slider <NUM> to provide a circumferential liquid flow in a fluidic system <NUM>. The front wall <NUM> also comprises multiple pairs of openings or interfaces <NUM>, <NUM>, preferably one pair per test chamber <NUM> in the slider <NUM>. Each such pair comprises an upper opening or interface <NUM> to be used for a liquid flow and a lower opening or interface <NUM> to be used for a sample flow. Each pair of openings or interfaces <NUM>, <NUM> and the opening or connection <NUM> and liquid enclosing interface <NUM> in the front wall <NUM> preferably comprises a respective gasket or seal <NUM> circumferentially enclosing the openings, interfaces or connections <NUM>, <NUM>, <NUM>, <NUM>. The gaskets or seals <NUM> are configured to contact the main surfaces of the slider <NUM> and thereby be pressed between the slider <NUM> and the front wall <NUM> to form a liquid tight connection.

The cassette half <NUM> also comprises the previously mentioned inlet ports <NUM>, <NUM> for the biological sample and a liquid and connection ports <NUM> for a pressure interface of the analysis instrument forming part of the fluidic system. The cassette half <NUM> further comprises a number of waste tanks <NUM> separated by a respective wall <NUM>. There is a head space <NUM> above the waste tanks <NUM> and the walls <NUM> as more clearly shown in <FIG> and <FIG>. The cassette half <NUM> preferably comprises one waste tank <NUM> per test chamber <NUM> in the slider <NUM>. The waste tanks <NUM> are in fluid connection with the inlet port <NUM> in at least one of the cassette halves 200A, 200B through a fluidic system <NUM> shown in <FIG> running along the end walls <NUM>, <NUM> and the back wall <NUM> of the cassette half <NUM>. As a consequence, the fluidic system <NUM> forms a circumferentially continuous channel in the cassette assembly <NUM> formed by a first channel part in one of the cassette halves 200A, 200B, the through holes <NUM> in the slider <NUM> and a second channel part in the other of the cassette halves 200A, 200B. The fluidic system <NUM> also comprises a serpentine channel <NUM> per cassette half <NUM> interconnecting the circumferential continuous channel with the waste tanks <NUM>.

The waste tanks <NUM> are in fluid connection with an excessive liquid tank <NUM> through a vertical outlet channel <NUM>. There is also a fluid connection between the waste tanks <NUM> and the excessive liquid tank <NUM> in the form of a narrow channel <NUM> acting as an air valve between the waste tanks <NUM> and the excessive liquid tank <NUM>. In an embodiment, the excessive liquid tank <NUM> comprises markings <NUM> in its bottom to identify the respective sets of waste tanks <NUM>, reservoirs <NUM> and test chambers <NUM>.

The cassette half <NUM> further comprises a number of reservoirs <NUM>, preferably one such reservoir <NUM> per waste tank <NUM>. Each waste tank <NUM> is then in fluid connection with a respective reservoir <NUM> through a channel <NUM> extending from the waste tank <NUM>, preferably passing a restrictive filter <NUM>, turning at the slider <NUM> and then continuing to the reservoir <NUM> as shown in <FIG>. The reservoirs <NUM> in one of the cassette halves 200A, 200B are preferably prefilled with a respective test agent, such as freeze dried antibiotic, whereas the reservoirs <NUM> in the other of the cassette halves 200A, 200B preferably lack the test agents.

<FIG> illustrate the four main components of the cassette assembly <NUM> as separate components prior to interconnecting the components. <FIG> illustrates the cassette assembly <NUM> with the cassette halves 200A, 200B interconnected and with the slider <NUM> sandwiched between the front walls <NUM> of the cassette halves 200A, 200B. <FIG> illustrates the interconnected cassette halves 200A, 200B and slider <NUM> as seen from above. <FIG> illustrates the cassette assembly <NUM> with the cover <NUM> attached to the cassette halves 200A, 200B in a loading position, i.e., with the cover <NUM> pushed down towards the cassette halves 200A, 200B so that the end walls <NUM>, <NUM> of the cassette halves 200A, 200B enter the window <NUM> in the end wall <NUM> of the cover <NUM>.

<FIG> and <FIG> illustrate a cassette half <NUM> together with various tapes, membranes and covers according to an embodiment. <FIG> illustrates the tapes, membranes and covers attached to the cassette half <NUM> as seen in a bottom view (top panel), a side view (middle panel) and a top view (bottom panel). In this embodiment, two bottom tapes <NUM>, <NUM> are attached to the underside of the cassette half <NUM> to enclose the fluidic components and channels in the cassette half <NUM>, see top panel of <FIG>. A first top tape <NUM> covers the waste tanks <NUM> and excessive liquid tank <NUM>. A membrane <NUM>, such as a polyethersulfone (PES) membrane, is interposed between a shoe tape <NUM> and a restriction bar <NUM>. In another embodiment, the membrane <NUM> is omitted as is further described herein. Furthermore, also the shoe tape <NUM> and the restriction bar <NUM> may be omitted, which is further discussed herein. A membrane <NUM>, such as a polytetrafluoroethylene (PTFE) membrane, is used to cover the reservoirs <NUM>. A second top tape <NUM> is designed to cover the fluidic components and channels in the cassette half <NUM>.

In an embodiment, the cassette assembly <NUM> is shipped with cover <NUM> in a transport position as shown in <FIG>, where the cover <NUM> keeps the main components together but prevents compression of the gaskets or seals <NUM> between the cassette halves 200A, 200B and the slider <NUM>. In an embodiment, the cassette assembly <NUM> is also positioned in a cassette tray <NUM> during transportation as shown in the figure. The cassette tray <NUM> then comprises an opening <NUM> in its top surface <NUM> into which the cassette assembly <NUM> inserted.

The user then preferably pushes the cover <NUM> down into the loading position shown in <FIG>. This causes a compression of the gaskets or seals <NUM> between the front walls <NUM> of the cassette halves 200A, 200B and the slider <NUM> to thereby keep the cassette assembly <NUM> tight to avoid leaks in the points of contact between the cassette halves 200A, 200B and the slider <NUM> when the sample and the liquid are injected.

<FIG> illustrates the cassette assembly <NUM> when liquid, such as water, an aqueous solution, a buffer solution, a culture medium, etc., is loaded through an opening <NUM> in the cover <NUM> positioned to be aligned with an inlet port <NUM> in one of the cassette halves 200A, 200B. The corresponding inlet port <NUM> in the other cassette half is preferably plugged with a plug <NUM> as shown in <FIG> or could be sealed with a membrane or tape. The injection of liquid through the opening <NUM> allows filling of the waste tanks <NUM> in both cassette halves 200A, 200B in a first step followed by transferring metered volumes of the liquid from the waste tanks <NUM> into the reservoirs <NUM>.

As is more clearly shown in <FIG>, the liquid first flows through a circumferential channel <NUM> connecting the two cassette halves 200A, 200B. The liquid then flows through two serpentine and narrow channels <NUM>, which divide the injected liquid into two equal volumes, one for each cassette half 200A, 200B. The serpentine channels <NUM> present a significantly higher flow resistance as compared to the circumferential channel <NUM>. Accordingly, once the liquid is injected through the opening <NUM> and the inlet port <NUM>, the liquid first fills up the circumferential channel <NUM> before entering the serpentine channel <NUM>. This design of the serpentine channels <NUM> and the circumferential channel <NUM> achieves a division of the injected liquid into two equal volumes, one for each cassette half 200A, 200B. In addition, the serpentine channels <NUM> enable a substantially timed filling of the two series of waste tanks <NUM> in the two cassette halves 200A, 200B with substantially the same flow rates in both cassette halves 200A, 200B.

From the respective serpentine channel <NUM>, the liquid fills up, via a vertical inlet channel <NUM>, a series of waste tanks <NUM> connected via a head space <NUM> as shown in <FIG>. Thus, the vertical inlet channel <NUM> is connected to the serpentine channel <NUM> in connection with its lower end and the vertical inlet channel <NUM> is connected to the series of waste tanks <NUM> in connection with its upper end.

The waste tanks <NUM> are separated by walls <NUM> to form a series of waste tanks <NUM> that are in fluid connection with each other through the head space or volume <NUM> present between the walls <NUM> and the top tape <NUM>. The waste tanks <NUM> become filled one after each other. Hence, liquid coming from the serpentine channel <NUM> enters a first waste tank <NUM> and starts filling this first waste tank <NUM>. Once the first waste tank <NUM> is full liquid flows over the wall <NUM> separating the first waste tank <NUM> and a second waste tank through the head space <NUM> and starts filling the second waste tank <NUM>. This process continues until all waste tanks <NUM> are full with liquid as shown in <FIG>. Any excessive liquid escapes into an excessive liquid tank <NUM> via a vertical outlet channel <NUM>.

The head space <NUM> of the waste tanks <NUM> is also connected to the excessive liquid tank <NUM> via an air valve <NUM> in the form of a narrow channel. The air valve <NUM> presents a higher flow resistance as compared to the vertical outlet channel <NUM>. This means that during filling, liquid will not penetrate through the air valve <NUM> but rather flow between waste tanks <NUM> and then, when the waste tanks <NUM> are full, enter the excessive liquid tank <NUM> via the vertical outlet channel <NUM> with the air valve <NUM> still remaining closed, i.e., no liquid passing there through.

Liquid may also flow from the waste tanks <NUM> into a portion of the liquid path or channel <NUM> interconnecting the waste tanks <NUM> and the reservoirs <NUM>, see <FIG>. However, any such escaping liquid at most goes to the restrictive filter <NUM> or slit. This restrictive filter <NUM> or slit has an opening pressure that is higher than any pressure that the escaping liquid may exert on the restrictive filter <NUM> or slit during the liquid filling procedure.

The excessive liquid tank <NUM> may comprise an absorbent material configured to absorb any excessive liquid entering the excessive liquid tank <NUM> from the waste tanks <NUM> and through the vertical outlet channel <NUM>. The absorbent material may be in form of a sponge, film or other device made of an absorbent material, preferably a so-called superabsorbent material or superabsorbent polymer (SAP). The absorbent material may then be arranged in the excessive liquid tank <NUM> to have a length substantially corresponding to the length of the excessive liquid tank <NUM> or a portion of the length of the excessive liquid tank <NUM>. The height of the absorbent material in the excessive liquid tank <NUM> is preferably selected so that the absorbent material does not reach the first top tape <NUM> covering the waste tanks <NUM> and excessive liquid tank <NUM> even when absorbing any liquid entering the excessive liquid tank <NUM>. Hence, there is preferably an air gap between the top of the absorbent material and the first top tape <NUM> to enable opening of the air valve <NUM>. This means that the height of the absorbent material is preferably less than the height of the excessive liquid tank <NUM>.

The absorbent material can be made of any material capable of absorbing liquid entering the excessive liquid tank <NUM>. In an embodiment, the absorbent material is made of SAP including, but not limited to, poly-acrylic acid sodium salt (sodium polyacrylate), polyacrylamide copolymer, ethylene maleic anhydride copolymer, cross-linked carboxymethylcellulose, polyvinyl alcohol copolymer, cross-linked polyethylene oxide, starch grafted copolymer of polyacrylonitrile. Also other absorbent materials could be used, including polyurethane (PUR).

The absorbent material in the excessive liquid tank <NUM> reduces the risk of any liquid in the excessive liquid tank <NUM> blocking the air valve <NUM>, for instance if a user would tilt the cassette assembly <NUM> to cause any liquid in the excessive liquid tank <NUM> to contact the air valve <NUM> and remain in the air valve <NUM> once the cassette assembly <NUM> is once more levelled. Any such liquid captured in the air valve <NUM> might prevent or at least obstruct opening of the air valve <NUM>.

Another advantage with the absorbent material is that it will keep the vertical outlet channel <NUM> interconnecting the waste tanks <NUM> and the excessive liquid tank <NUM> open, i.e., not full or at least partly full with liquid. Having an open vertical outlet channel <NUM> enables an even flow of liquid during operation (analysis) even if the air valve <NUM> would be unintentionally closed by a water droplet since air can then instead flow into the head space <NUM> through the vertical outlet channel <NUM> rather than through the air valve <NUM>.

In very specific situations, such as dry ambient air, and depending on the material(s) of the components of the cassette assembly <NUM> and the type of liquid used, electrostatic forces may affect the transport of the liquid in the cassette assembly <NUM>, and in particular the filling of the waste tanks <NUM>. For instance, if such electrostatic forces are built up in the cover <NUM> and/or the first top tape <NUM>, such forces may attract the liquid during filling causing at least some of the liquid to be transported along the underside of the first top tape <NUM> rather than flowing from waste tank <NUM> to waste tank <NUM>. This may cause an incorrect filling of at least some of the waste tanks <NUM> in the cassette halves 200A, 200B.

The build-up of electrostatic forces in such very specific conditions can be avoided or at least significantly suppressed or inhibited according to various embodiments. For instance, the bottom side of the cover <NUM> could be sprayed with an electrically conductive composition to form a thin electrically conductive layer or film on at least the part of the cover <NUM> facing the waste tanks <NUM>. Alternatively, or in addition, the cover <NUM> could be made of an electrically conductive material, such an electrically conductive plastic or polymer, or an electrically conductive additive could be added to the material of the cover <NUM> to get an electrically conductive cover <NUM>. A further alternative is to provide a thin electrically conductive tape or film, such as an aluminum tape or film, between the waste tanks <NUM> and the first top tape <NUM>. Yet other alternatives include providing a laminated first top tape <NUM> having an electrically conductive film attached to at least the surface of the first top tape <NUM> facing the waste tanks <NUM> or providing a coated first top tape <NUM> having an electrically conductive material coated or deposited onto at least the surface of the first top tape <NUM> facing the waste tanks <NUM>. It would also be possible to use an electrically conductive first top tape <NUM>. The above described solutions to prevent or at least inhibit build-up of electrostatic forces can be combined.

At this point either the sample is injected into the cassette assembly <NUM> or the head space <NUM> of the waste tanks <NUM> is cleared from liquid, which is further described herein.

The sample is injected into the cassette assembly <NUM> through an opening <NUM> in the cover <NUM> aligned with an inlet port <NUM> in one of the cassette halves as shown in <FIG>. The sample is preferably a biological sample comprising a gel suspension that can be polymerized into a culture matrix in the respective test chambers <NUM> in the slider <NUM>. The sample may optionally also comprise a culture medium allowing growth of any microorganisms, such as bacteria, in the culture matrix. The present invention can be used in connection with any type of culture matrix material known in the art and that can be injected into cassette assembly <NUM> and polymerized to form a solid 3D culture matrix. Additionally, the culture matrix once formed should preferably be transparent to allow visual inspection and visual access to the biological sample included therein.

Examples of suitable matrix material include agarose materials. An illustrative example of such an agarose material is ultra-low-gelling-temperature (ULGT) agarose. Other suitable materials include collagen materials, such as collagen I. Collagen I is well documented to support 3D cultures. Other gels that can be used include Engelbreth-Holm-Swarm (ECM) gels, such as Matrigel (BD Bioscience, Bedford, MA, USA) or hydrogels, including a mixture of phenylalanine (Phe) dipeptide formed by solid-phase synthesis with a fluorenylmethoxycarbonyl (Fmoc) protector group on the N-terminus, and Fmoc-protected lysine (Lys) or solely phenylalanine. However, any type of biocompatible matrix could be used as long as the matrix can be applied in soluble form and cast or polymerized to form a solid culture matrix.

The sample flows, see <FIG>, from the inlet port <NUM> in one of the cassette halves 200A, 200B through a channel system <NUM> and the test chambers <NUM> in the slider <NUM> in a meander pattern to fill respective test chamber <NUM> with the sample. The corresponding port <NUM> in the other cassette half 200B is preferably plugged with a filter, such as a filter plug, allowing air but not liquid to escape through the filter. This filter prevents any bacteria in the sample from escaping through the port <NUM> and thereby contaminating the outside of the cassette assembly <NUM>.

Such a filter may, as an illustrative, but non-limiting, example, be a PFTE membrane allowing air to escape through the filter when dry. However, when the sample reaches the filter and wets it, the filter no longer lets air, or liquid, to escape through the filter. This can be used as a feedback signal indicating that no more sample should be injected into the inlet port <NUM>.

The cassette assembly <NUM> is then preferably placed inside a refrigerator to initiate and finish the gel reaction of the sample and thereby formation of the solid 3D culture matrices in the test chambers <NUM> of the slider <NUM>. The cassette assembly <NUM> is then brought out from the refrigerator and is now ready for running an analysis of the response of microorganisms, such as bacteria, present in the biological sample to test agents, such as antibiotics, preloaded, such as in freeze-dried form, in reservoirs <NUM> of one of the cassette halves 200A, 200B. At this point, the cassette assembly <NUM> can therefore be inserted into an analysis instrument of the fluidic system.

Pressure interfaces, such as air pressure interfaces, are connected to openings <NUM>, <NUM> in the cover <NUM> as shown in <FIG> and thereby to aligned connection ports <NUM> in the cassette halves 200A, 200B. A short over pressure pulse, typically a duration of one or few tens of a second, is introduced via the pressure interface to clear the head space <NUM> of the waste tanks <NUM> from liquid. This over pressure pulse opens up the air valve <NUM> connecting the head space <NUM> and the excessive liquid tank <NUM>. The opening of the air valve <NUM> by the over pressure pulse causes drainage of the liquid in the head space <NUM> through the vertical outlet channel <NUM> and into the excessive liquid tank <NUM>, see <FIG> and <FIG>. This operation divides the liquid in the waste tanks <NUM> into a number of separate and equal liquid volumes, one for each waste tank <NUM>.

The air valve <NUM> is preferably in the form of a narrow channel interconnecting the head space <NUM> of the waste tanks <NUM> and the excessive liquid tank <NUM>. The narrow channel presents a significantly higher flow resistance as compared to the vertical outlet channel <NUM>. This means that during the serial filling of the waste tanks <NUM>, any excess liquid will flow from the head space <NUM> and into the vertical outlet channel <NUM> rather than out through the air valve <NUM>. The dimensions, in particular the cross-sectional dimensions, of the vertical outlet channel <NUM> relative to the dimensions of the narrow channel in the air valve <NUM> are preferably designed to create an under pressure by gravitation and capillary force that is sufficiently large to draw the excess liquid into the excessive liquid tank <NUM> but not that large to open the air valve <NUM> during the liquid filling stage.

As previously mentioned herein, the head space <NUM> could be cleared from liquid prior to sample filling. In such a case, gravitational forces acts on the liquid in the vertical outlet channel <NUM> causing a suction of air from the excessive liquid tank <NUM> through the air valve <NUM> and into the head space <NUM>, i.e., opens the air valve <NUM>. This causes drainage of the liquid in the head space <NUM> into the excessive liquid tank <NUM> through the vertical outlet channel <NUM> and thereby a separation of the liquid into equal volumes in the waste tanks <NUM>.

Once the separate and equal volumes of liquid have been obtained in the waste tanks <NUM>, a longer in time over pressure pulse, typically one or a few seconds, is applied to the waste tanks <NUM> via the excessive liquid tank <NUM> and the air valve <NUM>. This pressure pulse presses the liquid out from the waste tanks <NUM> and into the reservoirs <NUM> via a liquid path or channel <NUM> as shown in <FIG> (showing liquid in only one of the waste tanks <NUM>, reservoirs <NUM> and liquid paths or channels <NUM>). The pressure pulse is sufficiently strong or high to open the restrictive filter <NUM> or slit arranged in the liquid path or channel <NUM> and thereby allow an emptying of liquid in the waste tanks <NUM> into the reservoirs <NUM>.

The restrictive filter <NUM> or slit thereby has the function of preventing, during filling of the waste tanks <NUM>, liquid from passing the restrictive filter <NUM> or slit in the liquid path or channel <NUM> but enable the liquid to flow through the liquid path or channel <NUM> past the restrictive filter <NUM> or slit once the opening pressure of the restrictive filter <NUM> or slit has been overcome by application of the pressure pulse.

In an embodiment, the restrictive filter <NUM> is formed by the membrane <NUM>, such as PES membrane, and the restriction bar <NUM> shown in <FIG>, which is closed by the shoe tape <NUM>. In the case of a slit instead of the restrictive filter <NUM>, the membrane <NUM> may be omitted. In such a case, the restriction bar <NUM> comprises the slits or the slits can be formed in the liquid path or channel <NUM>. In this latter case, no restriction bar <NUM> is needed and can therefore be omitted together with the membrane <NUM> and the restrictive filter <NUM>.

The reservoirs <NUM> are filled until the liquid reaches the membrane <NUM>, preferably PTFE membrane, attached to the top of the reservoirs <NUM>. The membrane <NUM> arranged on top of the reservoirs <NUM> is preferably designed to let out air but not an aqueous liquid. As a consequence, the membrane <NUM> guarantees that all reservoirs <NUM> are filled with the same volume of liquid.

<FIG> illustrates the reservoirs <NUM> filled with the same defined volumes of liquid. In a preferred embodiment, the reservoirs <NUM> in one cassette half 200A are preloaded with test agents, such as freeze dried antibiotics, which become dissolved into the liquid in the reservoirs <NUM>. The reservoirs <NUM> in the other cassette half 200B preferably do not contain these test agents and will therefore only comprise the liquid at this point.

A short in time, typically in the range of one to a few seconds, under pressure pulse is applied to open up the membrane <NUM> for air flow. Thus, the under pressure pulse lets in a small volume of air <NUM> at the top of the reservoirs <NUM> as shown in <FIG>. At this point, the slider <NUM> is moved upwards relative to the cassette halves 200A, 200B, from a sample filling position shown in <FIG> into a flow position shown in <FIG>, to bring the 3D culture matrices present in the test chambers <NUM> in contact with the liquid paths or channels <NUM>.

The relative movement between the slider <NUM> and the cassette halves 200A, 200B can be achieved either by pressing the cassette halves 200A, 200B and the cover <NUM> downwards with the bottom of the slider <NUM> resting on a surface or by pushing the slider <NUM> upwards relative to the cassette halves 200A, 200B and the cover <NUM>.

The relative movement between the slider <NUM> and the cassette halves 200A, 200B additionally forms well defined ends or sides of the 3D culture matrices in the test chambers <NUM>. Hence, the relative movement achieves a cutting of the gelled sample to form the 3D culture matrices. The newly cut ends or sides of the 3D culture matrices are further moved to be aligned with the liquid openings or interfaces <NUM> in the front walls <NUM> of the cassette halves 200A, 200B to thereby be exposed to the respective liquids filled in the two cassette halves 200A, 200B.

As is more clearly shown in <FIG>, the cassette halves 200A, 200B have front walls <NUM> with two rows of openings or interfaces <NUM>, <NUM>, an upper sample filling row and a lower liquid flow row. The front wall <NUM> also has two cross flow connections or openings <NUM> coupling together the circumferential flow channel <NUM>. The front wall <NUM> further comprises liquid enclosing interfaces <NUM> arranged to capture and enclose the minute liquid volume contained in the through holes <NUM> in the slider <NUM> when the slider <NUM> is moved relative to the cassette halves 200A, 200B to the position shown in <FIG>. An optional center reaction or culture chamber act as the reference chamber <NUM> and has therefore no connection to the flow system. The figure also illustrates the gaskets or seals <NUM> around the interfaces and flow connections <NUM>, <NUM>, <NUM>, <NUM>.

In an embodiment, the gasket or seal <NUM> configured to be aligned with and enclose the reference chamber <NUM> preferably has uniform height, i.e., forms a solid seal rather than a circumferential seal around the reference chamber <NUM> as is preferably used for the test chambers <NUM>. The reason being that such a circumferential seal may trap a minute air volume when moving the slider <NUM> relative to the cassette halves 200A, 200B and where this air volume will then flank the gelled sample (3D culture matrix) in the reference chamber <NUM>. The trapped air may then dry the gelled sampled in the reference chamber <NUM> and cause a deformation of the gelled sample during such a drying process. However, the preferred solid seal <NUM> prevent or at least significantly reduces the risk of entrapment of air next to the gelled samples and thereby avoids or at least suppresses any undesired drying of the gelled sample in the reference chamber <NUM>.

Application of a constant under pressure starts a constant flow, such as about <NUM>-<NUM>µl/minute, through the liquid interfaces in connection with the test chambers <NUM>. This liquid flow goes from the reservoirs <NUM> and into the waste tanks <NUM>, i.e., basically opposite to the flow direction shown in <FIG> during filling of the reservoirs <NUM>. As a consequence, one side of the 3D culture matrix <NUM> in the test chamber <NUM> is exposed to the liquid <NUM> comprising a test agent, such as an antibiotic, whereas the other side of the 3D culture matrix <NUM> is exposed to the liquid <NUM> lacking the test agent. Hence, there is a constant concentration of the test agent in the liquid <NUM> flowing past one side the 3D culture matrix <NUM> and preferably a zero concentration of the test agent in the liquid <NUM> flowing past the opposite side of the 3D culture matrix. As a consequence, a linear concentration gradient of the test agent will be established and maintained over the 3D culture matrix <NUM>.

The linear concentration gradient of the test agent is preferably formed due to diffusion of the test agent through the 3D culture matrix. Hence, the diffusion is from a so-called source side, which has a higher concentration of the test agent in the liquid relative the other side, denoted sink side. In a preferred embodiment, the flow rates of the liquids on either side of the 3D culture matrix are preferably kept substantially similar since then no flow of the liquid is present through the 3D culture matrix <NUM> in the test chamber <NUM>. Substantially similar indicates that the two flow rates are preferably identical but can differ slightly due to inherent variations in the flow rate of the pumping systems. Thus, the difference in flow rate is preferably less than <NUM> %, more preferably less than <NUM> %, such as less than <NUM> % and most preferably less than <NUM> %.

It is also possible, in at least some of the test chambers <NUM>, to use a combination of multiple, such as two, test agents. In such a case, concentration gradients may be established over the 3D culture matrix <NUM> in the test chamber <NUM> for all or both test agents. Alternatively, a concentration gradient is established over the 3D culture matrix <NUM> for one of the test agents, whereas the other test agent should be present in a substantially uniform concentration over the 3D culture matrix <NUM>. This is possible by including the first test agent only in one of reservoirs <NUM> connected to a test chamber <NUM> whereas the second test agent is present, preferably at a same amount and concentration, in both reservoirs <NUM> connected to the test chamber <NUM>. Non-limiting, but illustrative, examples of such combinations of test agents include piperacillin/tazobactam, ceftazidime/avibactam and ceftolozane/tazobactam.

As is shown in the figures, the cassette assembly <NUM> preferably comprises multiple test chambers <NUM>. This means that different test agents can be provided in the different reservoirs <NUM> in one of the cassette halves 200A to thereby, in a single run of the cassette assembly <NUM>, monitor and analyze the responses of microorganisms, such as bacteria, in the biological sample to the different test agents. For instance, the MIC of different antibiotics can be determined for bacteria in a biological sample to thereby select a suitable antibiotic or suitable antibiotics that can be used to combat or treat a bacterial infection in a subject.

The design of the cassette assembly <NUM> enables accurate formation of equal volumes of liquid in the reservoirs <NUM> and therefore, once the test agents provided therein in one of the cassette halves 200A, 200B become dissolved in the liquid, well-defined concentrations of test agents in the reservoirs <NUM> in one of the cassette halves 200A, 200B. This further means that well-defined concentration gradients of test agents can be established over the 3D culture matrices <NUM> in the test chambers <NUM> and an accurate determination of responses, such as MICs, of any microorganisms present in the sample and the 3D culture matrices <NUM>.

In particular, the shedding or draining of liquid in the head space <NUM> above the waste tanks <NUM> by opening of the air valve <NUM> guarantees that each waste tank <NUM> in both cassette halves 200A, 200B contain the same volume of liquid that can be transferred to the respective reservoirs <NUM>.

An aspect of the invention relates to a cassette assembly <NUM> comprising a cover <NUM>, a first cassette half 200A, a second cassette half 200B and a slider <NUM>. The cover <NUM> is configured to be positioned onto the first cassette half 200A and the second cassette half 200B and keep the first cassette half 200A and second cassette half 200B together with the slider <NUM> sandwiched between the first cassette half 200A and the second cassette half 200B. In this aspect, the slider <NUM> comprises N≥<NUM> test chambers <NUM> in the form of through holes through the slider <NUM>. In this aspect, each of the first cassette half 200A and the second cassette half 200B comprises N waste tanks <NUM> connected in series and separated by respective walls <NUM>, and an excessive liquid tank <NUM> in fluid connection with the waste tanks <NUM> by a vertical outlet channel <NUM> and by an air valve <NUM>. In this aspect, an inlet port <NUM> of the first cassette half 200A is configured to receive a liquid to sequentially fill each waste tank <NUM> and a head space <NUM> defined above the waste tanks <NUM>. According to this aspect, the air valves <NUM> are configured to be opened to drain liquid in the head spaces <NUM> into the excessive liquid tanks <NUM> through the vertical outlet channels <NUM> and to form a separation of equal volumes of liquid in the waste tanks <NUM>.

In an embodiment, the air valves <NUM> are configured to present a higher flow resistance to the liquid as compared to the vertical outlet channels <NUM> to prevent liquid from entering the excessive liquid tanks <NUM> through the air valves <NUM> during filling of the waste tanks <NUM>.

In an embodiment, each of the first cassette half 200A and the second cassette half 200B comprises a connection port <NUM> configured to be aligned with respective openings <NUM>, <NUM> in the cover <NUM>. In this embodiment, the air valves <NUM> are configured to be opened by a first overpressure pulse applied through the openings <NUM>, <NUM> in the cover <NUM>.

In another embodiment, the air valves <NUM> are configured to be opened by a suction of air from the excessive liquid tanks <NUM> through the air valves <NUM> and into the head spaces <NUM>. In this embodiment, the suction of air is caused by gravitational forces acting on liquid in the vertical outlet channels <NUM> or by absorption of liquid in the vertical outlet channels <NUM> by absorbent material comprised in the excessive liquid tanks <NUM>.

In an embodiment, the air valves <NUM> are in the form of channels interconnecting the head spaces <NUM> and the excessive liquid tanks <NUM>. In this embodiment, the cross-sectional dimensions of the vertical outlet channels <NUM> relative to the channels of the air valves <NUM> are configured to create an under pressure by gravitation and capillary force that is sufficiently large to draw excess liquid into the excessive liquid tank <NUM> but not sufficiently large to open the air valves <NUM> during filling of the waste tanks <NUM>.

In an embodiment, each of the first cassette half 200A and the second cassette half 200B comprises N reservoirs <NUM>. In this embodiment, each reservoir <NUM> in one of the first cassette half 200A and the second cassette half 200B comprises a respective test agent and each corresponding reservoir <NUM> in the other of the first cassette half 200A and the second cassette half 200B lacks the respective test agent. Furthermore, each waste tank <NUM> is in fluid connection with a respective reservoir <NUM> through a liquid channel <NUM> extending from the waste tank <NUM> turning at the slider <NUM> and continuing to the reservoir <NUM>.

Corresponding reservoir <NUM> in the other of the first and second cassette halves 200A, 200B as used herein relates to a reservoir <NUM> that is in fluid connection with a same test chamber <NUM> in the slider <NUM> as a reservoir <NUM> in one of the first and second cassette halves 200A, 200B. This means that liquid present in the reservoir <NUM> comprising a particular test agent is transported through the liquid channel <NUM> and the upper opening <NUM> in the front wall <NUM> of the cassette half 200A, 200B to present the liquid flow with the particular test agent to one side of the 3D culture matrix <NUM> in the test chamber <NUM>. Correspondingly, liquid present in the corresponding reservoir <NUM> lacking the particular test agent is transported through the liquid channel <NUM> and the upper opening <NUM> in the front wall <NUM> of the other cassette half 200A, 200B to present the liquid flow lacking the particular test agent to the other side of the 3D culture matrix <NUM> in the test chamber <NUM>.

In an embodiment, each of the first cassette half 200A and the second cassette half 200B comprises a connection port <NUM> configured to be aligned with respective openings <NUM>, <NUM> in the cover <NUM>. In this embodiment, the equal volumes of liquid in the waste tanks <NUM> are configured to be pressed out from the waste tanks <NUM> and into the reservoirs <NUM> via the liquid channels <NUM> by application of a second over pressure pulse through the openings <NUM>, <NUM> in the cover <NUM>.

In an embodiment, each liquid channel <NUM> comprises a restrictive filter or slit <NUM>. In this embodiment, the second over pressure pulse is configured to open the restrictive filters or slits <NUM> to allow emptying of the equal volumes of liquid in the waste tanks <NUM> into the reservoirs <NUM>.

In an embodiment, the restrictive filters or slits <NUM> are configured to prevent liquid from passing the restrictive filters or slits <NUM> during filling of the waste tanks <NUM> but enable liquid to flow through the liquid channels <NUM> past the restrictive filters or slits <NUM> once an opening pressure of the restrictive filters or slits <NUM> has been overcome by application of the second over pressure pulse through the openings <NUM>, <NUM> in the cover <NUM>.

In an embodiment, each of the first cassette half 200A and the second cassette half 200B comprises a connection port <NUM> configured to be aligned with respective openings <NUM>, <NUM> in the cover <NUM>. In this embodiment, membranes <NUM> are arranged on top of the reservoirs <NUM> and configured to allow air but not liquid to pass through the membranes <NUM>. Furthermore, the membranes <NUM> are configured to be opened by application of an under pressure pulse at the openings <NUM>, <NUM> in the cover <NUM> to introduce a volume of air <NUM> between the liquid in the reservoirs <NUM> and the membranes <NUM>.

In an embodiment, the slider <NUM> is movable relative to the first cassette half 200A and the second cassette half 200B between a sample filling position and a flow position. In this embodiment, the liquid channels <NUM> are in fluid connection with the test chambers <NUM> in the slider <NUM> in the flow position but not in the sample filling position.

In an embodiment, each of the first cassette half 200A and the second cassette half 200B comprises a connection port <NUM> configured to be aligned with respective openings <NUM>, <NUM> in the cover <NUM>. In this embodiment, liquid in the reservoirs <NUM> are configured to flow through the liquid channels <NUM> into the waste tanks <NUM> by application of an under pressure at the openings <NUM>, <NUM> in the cover <NUM> when the slider <NUM> is in the flow position. Furthermore, one side of a respective three dimensional (3D) culture matrix <NUM> in the test chambers <NUM> is exposed to liquid <NUM> comprising a test agent and the other side of the respective 3D culture matrix <NUM> is exposed to liquid <NUM> lacking the test agent to establish a linear concentration gradient of the test agent over the respective 3D culture matrix <NUM>.

In an embodiment, the slider <NUM> comprises a first through hole <NUM> in connection with a first end of the slider <NUM> and a second through hole <NUM> in connection with a second end of the slider <NUM>. In this embodiment, each of the first cassette half 200A and the second cassette half 200B comprises a circumferential channel <NUM> having a first opening <NUM> configured to be aligned with the first through hole <NUM> and a second opening <NUM> configured to be aligned with the second through hole <NUM>. Each of the first cassette half 200A and the second cassette half 200B also comprises a serpentine channel <NUM> interconnecting the circumferential channel <NUM> and the waste tanks <NUM>. The inlet port <NUM> of the first cassette half 200A is in fluid connection with the circumferential channel <NUM>, and the serpentine channels <NUM> are configured to present a higher flow resistance to the liquid as compared to the circumferential channels <NUM> to enable filling of the circumferential channels <NUM> prior to entering of the liquid into the waste tanks <NUM> through the serpentine channels <NUM>.

In an embodiment, each of the first cassette half 200A and the second cassette half 200B comprises a vertical inlet channel <NUM> in fluid connection at its lower end with the serpentine channel <NUM> and in fluid connection with the waste tanks <NUM> in its upper end.

In an embodiment, the slider <NUM> is movable relative to the first cassette half 200A and the second cassette half 200B between a sample filling position and a flow position. In this embodiment, one of the first cassette half 200A and the second cassette half 200B comprises an inlet port <NUM> configured to receive a biological sample comprising a gel suspension that can be polymerized into three dimensional (3D) culture matrices <NUM> in the test chambers <NUM> in the slider <NUM> when the slider <NUM> is in the sample filling position. Furthermore, each of the first cassette half 200A and the second cassette half 200B comprises a channel system <NUM> forming, together with the test chambers <NUM> in the slider <NUM>, a meander pattern and configured to sequentially fill the test chambers <NUM> with the biological sample received at the inlet port <NUM>.

In an embodiment, the slider <NUM> is configured to be moved relative to the first cassette half 200A and the second cassette half 200B from the sample filling position into the flow position once the gel suspension has been polymerized into the 3D culture matrices <NUM> in the test chambers <NUM>. In this embodiment, the relative movement between the slider <NUM> and the first cassette half 200A and the second cassette half 200B is configured to cut the polymerized biological sample to form well defined sides of the 3D culture matrices <NUM> and to align the sides of the 3D culture matrices <NUM> with liquid openings <NUM> in front walls <NUM> of the first cassette half 200A and the second cassette half 200B.

In an embodiment, each of the first cassette half 200A and the second cassette half 200B comprises a front wall <NUM> configured to face the slider <NUM>. In this embodiment, the front walls comprises N pairs of openings <NUM>, <NUM> comprising a respective upper opening <NUM> in fluid connection with a respective liquid channel <NUM> and a respective lower opening <NUM> forming part of the channel system <NUM>. Furthermore, optional but preferred gaskets or seals <NUM> are arranged in the front walls <NUM> to circumferentially enclose the openings <NUM>, <NUM>.

In an embodiment, the cover <NUM> comprises a lid <NUM> and supporting structures extending from the lid <NUM> and supporting a slider cover <NUM> comprising windows <NUM> configured to be aligned with the test chambers <NUM> in the slider <NUM> to provide visual access through the slider cover <NUM> into the test chambers <NUM>.

Another aspect of the invention relates to a cassette assembly <NUM> comprising a cover <NUM>, a first cassette half 200A, a second cassette half 200B and a slider <NUM>. The cover <NUM> is configured to be positioned onto the first cassette half 200A and the second cassette half 200B and keep the first cassette half 200A and second cassette half 200B together with the slider <NUM> sandwiched between the first cassette half 200A and the second cassette half 200B. In this aspect, the slider <NUM> comprises a first through hole <NUM> in connection with a first end of the slider <NUM> and a second through hole <NUM> in connection with a second end of the slider <NUM>. In this aspect, each of the first cassette half 200A and the second cassette half 200B comprises a circumferential channel <NUM> having a first opening <NUM> configured to be aligned with the first through hole <NUM> and a second opening <NUM> configured to be aligned with the second through hole <NUM>. Each of the first cassette half 200A and the second cassette half 200B further comprises N≥<NUM> waste tanks <NUM> connected in series and separated by respective walls <NUM>. Each of the first cassette half 200A and the second cassette half 200B also comprises a serpentine channel <NUM> interconnecting the circumferential channel <NUM> and the waste tanks <NUM>. The first cassette half 200A comprises, in this aspect, an inlet port <NUM> in fluid connection with the circumferential channel <NUM>. According to this aspect, the serpentine channels <NUM> are configured to present a higher flow resistance to the liquid as compared to the circumferential channels <NUM> to enable filling of the circumferential channels <NUM> prior to entering of the liquid into the waste tanks <NUM> through the serpentine channels <NUM>.

A further aspect of the invention relates to a cassette assembly <NUM> comprising a cover <NUM>, a first cassette half 200A, a second cassette half 200B and a slider <NUM>. The cover <NUM> is configured to be positioned onto the first cassette half 200A and the second cassette half 200B and keep the first cassette half 200A and second cassette half 200B together with the slider <NUM> sandwiched between the first cassette half 200A and the second cassette half 200B. In this aspect, the slider <NUM> comprises N≥<NUM> test chambers <NUM> in the form of through holes through the slider <NUM>. In this aspect, the slider <NUM> is movable relative to the first cassette half 200A and the second cassette half 200B between a sample filling position and a flow position. One of the first cassette half 200A and the second cassette half 200B comprises an inlet port <NUM> configured to receive a biological sample comprising a gel suspension that can be polymerized into three dimensional (3D) culture matrices <NUM> in the test chambers <NUM> in the slider <NUM> when the slider <NUM> is in the sample filling position. According to this aspect, each of the first cassette half 200A and the second cassette half 200B comprises a channel system <NUM> forming, together with the test chambers <NUM> in the slider <NUM>, a meander pattern and configured to sequentially fill the test chambers <NUM> with the biological sample received at the inlet pot <NUM>.

In an embodiment, each of the first cassette half 200A and the second cassette half 200B comprises a front wall <NUM> configured to face the slider <NUM>. In this embodiment, the front walls comprises N pairs of openings <NUM>, <NUM> comprising a respective upper opening <NUM> in fluid connection with a respective liquid channel <NUM> and a respective lower opening <NUM> forming part of the channel system <NUM>. Optional gaskets or seals <NUM> are arranged in the front walls <NUM> to circumferentially enclose the openings <NUM>, <NUM>.

The cassette assembly <NUM> can be made by polymer materials, such as plastics, using, for instance, injection molding. In more detail, the cover <NUM> can be made of any polymer or plastic material having sufficient stiffness to keep the main components of the cassette assembly <NUM> together. An illustrative, but non-limiting, example of a suitable material for the cover <NUM> is polycarbonate (PC). The two cassette halves 200A, 200B can be made of any polymer or plastic material that is inert to the test agents preloaded into the reservoirs <NUM>. This means that the material of the cassette halves 200A, 200B should not react with or decompose the test agents. In an embodiment, the polymer or plastic material of the cassette halves 200A, 200B is a hydrophobic polymer or plastic material. In such a case, the hydrophobicity of the material can be used to produce a (back) pressure in the meander channels. In addition, or alternatively, the polymer or plastic material is preferably optically transparent to enable monitoring the liquid level in the reservoirs <NUM> through the windows <NUM> in the cover <NUM>. An illustrative, but non-limiting, example of a suitable material for the cassette halves 200A, 200B is polypropylene (PP). The slider <NUM> can be made of any polymer or plastic material that is inert to the sample loaded into the test chambers <NUM>. The material preferably also presents sufficient adhesion to the 3D culture matrices <NUM> in the test chambers <NUM> to prevent the 3D culture matrices <NUM> from being pushed out of the test chambers <NUM> and into the fluidic system of the cassette halves 200A, 200B. The slider <NUM> is preferably also made of an optically transparent material to enable monitoring of the 3D culture matrices <NUM> in the test chambers <NUM> with a suitable optical, preferably microscopy-based system. Illustrative, but non-limiting, examples of suitable materials for the slider <NUM> include polyester, such as Eastar™ copolyester MN211; styrene methyl methacrylate (SMMA), such as NAS® <NUM>; styrene-butadiene copolymer (SBC), such as Styrolux® T.

The response of microorganisms, such as bacteria, to the established concentration gradients of test agents, such as antibiotics, can be determined by monitoring the 3D culture matrices <NUM> present in the test chambers <NUM> either from above, i.e., through the windows <NUM> in the slider cover <NUM> of the cover <NUM> that are aligned with the test chambers <NUM>, or from below. In either case, light is preferably provided from the other side of the slider <NUM>, i.e., from above if the monitoring is done from below and from below if the monitoring is done from above of the slider <NUM>.

The monitoring or analysis of the responses of microorganisms to the test agents can be performed as disclosed in <CIT>. Briefly, a response of the microorganisms to the test agent can be determined based on a position, relative to one of the sides of the 3D culture matrix <NUM>, of any border zone in the 3D culture matrix between a first response zone, in which the microorganism shows a first response to the test agent, and a second response zone, in which the microorganism shows a second, different response to the test agent.

In a particular embodiment, the response of the microorganism to the test agent can be determined based on the position, relative to one of the sides of the 3D culture matrix <NUM>, of the any border zone in the 3D culture matrix and based on a width of the border zone. In another particular embodiment, the response of the microorganism to the test agent is determined based on the position, relative to one of the sides of the 3D culture matrix <NUM>, of the any border zone in the 3D culture matrix <NUM> and based on a shape of the border zone and optionally based on the width of the border zone.

For instance, if the test agent is an antibiotic or another bactericide or bacteriostat the second response zone, in which the microorganisms, here represented by bacteria, are exposed to a comparatively low concentration of the test agent, could be the portion of the 3D culture matrix <NUM> with viable and growing bacteria. The first response zone is then preferably the portion of the 3D culture matrix <NUM> where there is substantially no viable or growing bacteria, hence the first response zone is characterized by a relative lack of bacteria (due to cell death or no cell growth). The border zone then constitutes the border or portion between the growing/viable portion and the non-growing/cell death portion.

The position of the border zone thereby provides information of the particular concentration of the test agent at which the response of the microorganisms to the test agents changes from the response occurring in the first response zone to the response occurring in the second response zone. A particular example of such a concentration of a test agent that can be determined based on the position of the border zone is MIC.

The width of the border zone provides additional information of the microorganisms and their response to the test agent. For instance, if the width of the border zone is substantially zero, i.e. basically a boundary or border between the first and second response zones, the change in response of the microorganisms occurs at a specific concentration of the test agent. However, if the border zone has a substantially non-zero width the change in response of the microorganisms occurs at a concentration range corresponding to the respective ends of the border zone.

A border zone with a non-zero width may further provide information with regard to any resistance of the microorganisms to the test agent. Hence, an extended border zone may imply that a resistance to the test agent is present in some of the microorganisms, for instance since they are able to grow at concentrations of the test agent which otherwise kill or prevent growth to non-resistant microorganisms. This means that the width of the border zone can be used in order to determine or detect any resistance at a given time of the microorganisms to the test agent.

In fact, it is actually possible with the cassette assembly <NUM> of the embodiments to detect any mutation in the microorganisms that induced resistance to the test agent or indeed caused loss of resistance to the test agent. Thus, the width of the border zone over time could be monitored when running the cassette assembly <NUM> with the culture of microorganisms in the 3D culture matrix <NUM>. An increase in the width of the border zone over time then typically implies gain of resistance to the test agent among at least some of the microorganisms. Correspondingly, a decrease in the width of the border zone typically implies loss of resistance to the test agent among microorganisms that previously showed resistance to the test agent.

In an embodiment, see <FIG>, an analysis instrument <NUM> of a system <NUM> can be used to take at least one image of the 3D culture matrix <NUM> and process the at least one image in order to identify the border zone in the image, including its position and optionally width and/or shape. Alternatively, the processing of the at least one image is performed by a processor <NUM> of or connected to the analysis instrument <NUM>. The analysis instrument <NUM> may take the at least one image of the 3D culture matrix <NUM> using, for instance, a dark-field microscopy, a bright-field microscope or a phase-contrast microscope and then process the at least one image by a computer or processor <NUM> configured to identify the border zone based on detected light intensity in the at least one image.

In a particular embodiment, the analysis instrument <NUM> comprises a microscope and camera that are used to take images of the 3D culture matrices <NUM>. In an embodiment, the analysis instrument <NUM> moves the microscope and camera step-by-step between the 3D culture matrices <NUM> in the slider <NUM>. The slider <NUM> may then preferably comprise a respective marking or identifier in connection with each test chamber <NUM> to thereby clearly identify the particular 3D culture matrix <NUM> in an image taken by the camera at a given position relative to the cassette assembly <NUM>. In another embodiment, the analysis instrument <NUM> comprises a camera configured to take an image of multiple, such as all, 3D culture matrices <NUM>. This embodiment thereby relaxes the need for moving the camera between the 3D culture matrices <NUM>.

Yet another aspect of the invention relates to a system <NUM> for determining a response of microorganisms to test agents, see <FIG>. The system <NUM> comprises a cassette assembly <NUM> according to any of the embodiments. The system <NUM> also comprises an analysis instrument <NUM> configured to take at least one image of three dimensional (3D) culture matrices <NUM> in the test chamber <NUM>. The 3D culture matrices <NUM> are formed by polymerization of a biological sample comprising the microorganisms and a gel suspension. The system <NUM> also comprises a processor <NUM> configured to determine a response of the microorganisms to test agents based on the at least one image of the 3D culture matrices <NUM>.

In <FIG>, the processor <NUM> has been illustrated as a separate unit connected, by wired or wirelessly, to the analysis instrument <NUM>. For instance, the processor <NUM> could represent or form part of a computer connected to, and optionally controlling, the analysis instrument <NUM>. In another embodiment, the analysis instrument <NUM> comprises the processor <NUM>.

The cassette assembly <NUM> of the invention can be used to determine the identity of and to monitor various microorganisms, test agents and responses. Hence, the invention finds many different uses within hospital clinics, laboratories, diagnostic laboratories, healthcare facilities, etc..

For instance, the cassette assembly <NUM> can be used to identify MIC, or other concentration thresholds that have an effect or no effect, of a bioactive compound in order to identify the minimum concentration of the bioactive compound that has an effect on any given microorganism with regard to growth, proliferation, death or survival of the microorganism.

Furthermore, by establishing MIC for a set of different test agents, the phenotypic identity of a tested microorganism can be established in, for example, a diagnostic test. The analysis of phenotype could result in either full identification of the microorganism strain or result in a general classification of the microorganism with regard to responses to various test agents.

The cassette assembly <NUM> can also be used to follow the change of MIC for a test agent on the growth, proliferation, viability or other behavioural aspect of the microorganism over time. This approach enables monitoring of development or loss of resistance, such as antibiotic resistance, in the microorganism over time through several generations, thereby altering the ability of the microorganism to metabolize or resist the effects of the test agent. This type of experiment can be used to provide an indication of suitable clinical dose of the test agent and/or frequency of administration. Hence, the cassette assembly <NUM> can be used to study both the pharmacodynamics and the pharmacokinetics of any test agent or combination of test agents in any given type of microorganism.

The cassette assembly <NUM> can further be used to evaluate and identify combinations of test agents, such as antibiotics, which together are more efficient than the individual agents, or to evaluate if the test agents have synergistic effect, or if they together have no effects on, for example, the growth, proliferation, viability or other behavioural aspect of the microorganism.

The cassette assembly <NUM> can be used to screen novel drugs or chemical compounds for their effects on microorganism growth, proliferation, viability, death, ability to spread, or other behavioural aspects of the microorganism.

The cassette assembly <NUM> can also be used to quickly get a first profile of response of any given microorganism to agents used in clinical practice. The aim could then be to identify agents that are suitable for anti-microorganism treatment of patients suffering from microbial infections. This can be used even in cases where the life of the patient depends on a fast identification of the microorganism causing the infection in order to design an appropriate treatment.

The cassette assembly <NUM> can further be used to study the effects of drugs or test agents on host cells that have been infected with viruses, with effects on cell behaviour, cell survival, cell proliferation, cell death and/or cell differentiation used as readouts for the amount and effect of the viruses and test agents on the host cells. In this application, the viruses are preferably placed together with the cells that they are known to be able to infect. Thus, both direct effects on the virus particles as well as on the infected host cells, such as cytopathic effects, can be used to study the effects of test agents at different concentrations.

As an example of the study of the effects of drugs on parasites, Malaria parasites could be studied using human or mouse red blood cells infected with the parasite and cultured in the 3D culture matrix <NUM> of the cassette assembly <NUM>. The response of the parasites and red blood cells to one or more gradients of drug(s) can then be tested.

Culturing microorganisms in a 3D culture matrix <NUM> in test chambers <NUM> of a cassette assembly <NUM> enables keeping the microorganisms inside the closed test chambers <NUM>. This provides protection for the personnel against potentially harmful microorganisms but also protects against contamination of the sample in the 3D culture matrices <NUM>.

Claim 1:
A cassette assembly (<NUM>) comprising:
a cover (<NUM>);
a first cassette half (200A);
a second cassette half (200B); and
a slider (<NUM>), wherein
the cover (<NUM>) is configured to be positioned onto the first cassette half (200A) and the second cassette half (200B) and keep the first cassette half (200A) and second cassette half (200B) together with the slider (<NUM>) sandwiched between the first cassette half (200A) and the second cassette half (200B);
the slider (<NUM>) comprises N≥<NUM> test chambers (<NUM>) in the form of through holes through the slider (<NUM>);
characterized in that each of the first cassette half (200A) and the second cassette half (200B) comprises:
N waste tanks (<NUM>) connected in series and separated by respective walls (<NUM>); and
an excessive liquid tank (<NUM>) in fluid connection with the waste tanks (<NUM>) by a vertical outlet channel (<NUM>) and by an air valve (<NUM>);
an inlet port (<NUM>) of the first cassette half (200A) is configured to receive a liquid to sequentially fill each waste tank (<NUM>) and a head space (<NUM>) defined above the waste tanks (<NUM>); and
the air valves (<NUM>) are configured to be opened to drain liquid in the head spaces (<NUM>) into the excessive liquid tanks (<NUM>) through the vertical outlet channels (<NUM>) and to form a separation of equal volumes of liquid in the waste tanks (<NUM>).