IT201700004589A1 - Kit and method for placing one or more fluids in a microfluidic device - Google Patents

Description

Title: "Kit and method for placing one or more fluids in a microfluidic device"

Description

The subject of the present invention is a system for discharging one or more fluids into a microfluidic device. A discharge region (70) comprised in a microfluidic device (1) is described which comprises: a discharge container (12) in fluidic connection with said microfluidic device (1) via at least one discharge channel (22) and a discharge port exit (10).

State of the art

The handling of fluids in microfluidic devices typically uses pumps, vacuum or thrust, and / or valves. The combination of pumps and valves allows fine control of fluid movements in a circuit.

As an example, Byun et al. (Pumps for Microfluidic Cell Culture, Electrophoresis 2014, 35: 245–257) describe microfluidic devices for cell culture and associated pumping systems. Also in Au et al. (Microvalves and Micropumps for BioMEMS, Micromachines 2011, 2: 179-220) describes a wide variety of valves and pumps, to be used in specific combinations, each with unique characteristics that make it applicable in certain contexts and not in others.

From the available literature, it emerges that there are no standard parameters on which to base the choice of microvalves and micropumps, making it necessary to carry out a specific study for each specific system.

A strongly felt problem is that of efficiently managing the bidirectional movement of fluids in a microcircuit, without necessarily having to rely on bulky and demanding pumps and valves from the point of view of purchase and management costs.

A further problem associated with valve-based microfluidic devices is that, if the integration of valves in the microsystem is envisaged to obtain high parallelism and / or reduced dimensions, the technological complexity required is high, given for example the need to integrate elastomers in addition to rigid materials.

The present invention offers a simple and advantageous solution to the problem, allowing the use of a common liquid handling tool for loading, pumping and possibly the high-precision discharge of fluids into a microfluidic device. Description of the invention

Description of the figures

Figure 18: scheme of the kit according to the present invention which includes a tip and a microfluidic device. Figure 19: schematic representation of an embodiment of the kit according to the present invention. (A) tip, (B) entrance region, (C) tip inserted into the entrance region.

Figure 20: schematic representation of an embodiment of the kit according to the present invention, tip inserted in the inlet region.

Figure 21: schematic representation of a further embodiment of the kit according to the present invention. (A) tip, (B) entrance region, (C) tip inserted into the entrance region.

Figure 22: schematic representation of a further embodiment of the kit according to the present invention, tip inserted in the inlet region which comprises a vertical channel and a connector.

Figure 23: schematic representation of a further embodiment of the kit according to the present invention. (A) tip, (B) entrance region, (C) tip inserted in the entrance region, (D) tip inserted in the entrance region in an alternative embodiment.

Figure 24: schematic representation of a further embodiment of the kit according to the present invention. (A) tip, (B) entrance region, (C) tip inserted in the entrance region, (D) tip inserted in the entrance region in an alternative embodiment.

Figure 25: inverted open microwell system according to the present invention, seen from above.

Figure 26: inverted open microwell system according to a further embodiment, seen from above.

Figure 27: three embodiments, in panels A, B and C, of the unloading region (70) of a microfluidic device (1).

Figure 1: exemplary diagram of an inverted open microwell system used in the method of the present invention, perspective view (a), vertical section (b) and top view (c).

Figure 2: diagram of an inverted open microwell system used in a further embodiment of the method of the present invention, perspective view (a), vertical section (b) and top view (c).

Figure 3: diagram of an inverted open microwell system used in a preferred embodiment of the method of the present invention, perspective view (a), vertical section (b) and top view (c).

Figure 4: diagram of an inverted open microwell system used in a further preferred embodiment of the method of the present invention, perspective view (a), vertical section (b) and top view (c).

Figure 5: sectional view of a portion of an inverted open microwell system used in the method of the present invention.

Figure 6: Example of images acquired in the visible (A), in the DAPI fluorescent channel (B), in the FITC fluorescent channel (C) and in the CY5 fluorescent channel (D). (E) is the result of superimposing the images acquired in the FITC channel and the CY5 channel.

Figure 7: classification of the cell population of Figure 6 according to the different parameters evaluated.

Figure 8: Classification of tumor cells in a bone marrow sample extracted from a patient with acute myeloid leukemia (AML). In (A) and (B) results obtained at FACS, in (C) and (D) results obtained according to the method described in the present application. In (A) and (C) the basal reading, carried out on unlabeled control cells, which allows to define a threshold value, indicated by the arrow; in (B) and (D) the cell count positive for the anti CD34 antibody, or events above the defined threshold value.

Figure 9: data obtained on a mix of cells extracted from a healthy donor and a cell line of Burkitt's lymphoma (Raji) according to the method described in the present application. Classification of tumor cells within a mix of cells extracted from healthy donor and tumor cells (A), analysis of cell death in the tumor and healthy population (B) and dose / response curve after drug exposure (C).

Figure 10: schematic sectional representation of an inverted open microwell system used in an alternative form to the method according to the present description, which also includes electrodes in the microwell and on the lower surface of the channel. Figure 11: assay on SU-DHL-4 tumor cells treated (RTX) and untreated (CTRL) with the monoclonal antibody Rituximab. (A) observation of live cells (light gray) and dead cells (indicated by the arrow) at time zero (T0), and after 30 (T1) and 60 (T2) minutes of treatment using the method according to the present invention; (B) live cell count at T0, T1 and T2 obtained by flow cytometry; (C) comparison between the cell count data obtained after 60 minutes of treatment by flow cytometry or using the method according to the present invention.

Figure 12: Cell count indicated as percentage of SU-DHL-4 tumor cells surviving following treatment, obtained at FACS (squares) and using the method according to the present invention (circle) in control cells (light gray) and treated with Rituximab (dark gray).

Figure 13: classification of Raji tumor cells within a mix of cells extracted from a healthy donor and called Raji tumor cells using the method according to the present invention. (A) phase contrast and fluorescence observation; (B) cell-specific morphological data; (C) classification of cells considering morphological (diameter) and functional (CD38 expression) parameters; (D) Statistical analysis of morphological (diameter) and functional (CD38 expression) data from a population of healthy (peripheral blood mononuclear cells, PBMC) and tumor (Raji) cells.

Figure 14: Variation of the fluorescence signal over time. Data obtained in time-lapse in cell-specific mode (A) or as a total observation of the cell population (B). In figure (A) columns of the histogram are highlighted that correspond to the subpopulation that has suffered the least cell damage, or the least relative variation of fluorescent signal. In figure (B) the columns corresponding to subpopulations containing the same cells identified in graph (A) are highlighted.

Figure 15: Automated marking process. In (A) the background signal is reported and in (B) the variation of said signal over time, as measured in a microwell operating with a flow rate of 16 µl / minute. The dashed line in (A) represents the background signal that is measured not by operating in timelapse, but by alternating washing with measurements. In (C) the signal / background ratio is reported expressed as the average signal measured on the cells divided by the average background signal. In (D) there are representative photos of the collection of time-lapse images during the washes.

Figure 16: Temporal analysis at the flow cytometer of the variation of the signal emitted at the wavelength of 480 nm (FITC) from Jurkat cells labeled with Fluo-4 following stimulation with (a) Ionomycin and (b) purified OKT3 antibody.

Figure 17: Temporal analysis in the open microwell system of the variation of the signal emitted at the wavelength of 480nm (FITC) from Jurkat cells labeled with Fluo-4 following stimulation with (a) Ionomycin and (b) purified OKT3 antibody. The dashed curves represent the trend of the signal obtained for single cells inside the microwells. The solid curve represents the average value of the signal.

Detailed description of the invention:

A microfluidic device (1), a kit comprising a tip (20) and an inlet region (18) of a microfluidic device (1), an unloading region (70) of said microfluidic device (1) are described here. Also part of this description is a method for the introduction and / or discharge of one or more fluids from said microfluidic device (1) and a method for high-content analysis in said microfluidic device (1).

Definitions:

By interference coupling we mean here a cooperation between two elements, such that said two elements can be considered interlocked. When said two elements, in this case a tip and a vertical channel, are interference coupled, a fluid loaded in said tip and released in said vertical channel is forced to move inside the channel, said interference coupling being such as to prevent fluid passage, i.e. said interference coupling, is such as to seal the two elements together.

By connector we mean here any tubular element, cylindrical in shape, more or less tapered, converging or diverging, functional to put two compartments in fluid communication.

With half-opening of the truncated cone we mean the angle formed by the generating line of said truncated cone with the line that constitutes the axis of rotation of the same.

Fluid: any substance in liquid or gaseous form. Biological sample: sample comprising cells obtained from a microorganism, an animal and / or a man, preferably from a man, where said sample is preferably selected from the group comprising biological fluids or biopsies. Said sample comprises cells in suspension, or is a tissue. In a preferred embodiment, it is a blood sample or a bone marrow aspirate. Alternatively, said biological sample consists of cells in culture, such as for example a cell line, or of a composition which includes cells in culture and patient cells.

High-content assay: phenotypic assay conducted on cells.

Time-lapse: image acquisition technique that involves a series of shots of the same field collected in temporal succession.

Ex-vivo: experimentation carried out on a tissue obtained from an organism in an environment external to the organism itself, with minimal alteration of the natural conditions.

Kit and method for injecting one or more fluids into a microfluidic device

The subject of the present invention is a kit which comprises a tip (20) and a microfluidic device (1) which comprises at least one microchannel (3) and an inlet region (8) which includes at least one vertical channel (18), said tip ( 20) and said vertical channel (18) being sized so as to produce an interference fit therebetween.

Said tip is selected from one of the commercially available tips which comprise at least a proximal portion intended to cooperate with a fluid dispensing system and an open tapered distal portion.

With reference to Figure 18, said tip (20) comprises a proximal portion (22) intended to cooperate with a fluid dispensing system and a distal portion (21), said proximal portion (22) of generally tubular configuration and said distal portion (21) tapered open where the terminal base (25) of said distal portion (21) has an outer diameter of size d3, and the upper base (24) of said distal portion (21) has an outer diameter of size d4, where said inlet region (8) comprises a vertical channel (18) which optionally introduces, through one or more connectors, into said at least one microchannel (3), the upward opening of said vertical channel (18) having a diameter measuring d2, where d3 <d2, said tip (20) and said vertical channel (18) being sized so as to produce an interference fit between them. Said interference coupling typically takes place according to one of the modes schematised in Figures 19 and 20. With reference to Figure 19, said tip (20) and said channel (18) have the upper base (9) of said channel as a sealing contact point. vertical (18). With reference to Figure 20, the sealed contact point is inside the vertical channel (18), being in this version the terminal base (25) of the distal portion (21) of the tip (20) to come into contact with the internal wall of said vertical channel (18). In both cases, tight contact occurs. Preferably, said end portion (21) of said tip (20) and said vertical channel (18) are made of plastic material and make the system elastic enough to guarantee the seal, avoiding gaskets. In a particularly preferred form, the geometries of the system described below ensure that the contact between said vertical channel (18) and said tip (20) does not take place in a single point but is distributed over a portion of the surface, further guaranteeing an effective seal. . In particular, this condition is advantageously verified where the half-opening angle of said end portion (21) of said tip and of said vertical channel (18) are slightly different from each other, preferably they differ from each other by less than 10 °. Even more preferably, said vertical channel (18) is a cylinder, optionally slightly tapered downwards.

With reference to Figure 19A, the length of said distal portion (21) of said tip (20) is h2, the half-opening of the truncated cone formed by said distal portion (21) measures (90 ° - �) and the length of said proximal portion (22) measures h3. Please refer to the same figure 19A for an even more exhaustive description of said tip, which indicates the angle (26) of measurement � and the longitudinal axis x which allows the definition of said half-opening of the truncated cone. Figures 20 to 24 and the description that follows are intended to represent some particularly preferred embodiments of the invention, do not in any way want to limit the scope of protection of the same which extends to what is claimed in claim 1.

In a preferred embodiment, the reference is to Figure 19, where said vertical channel (18) is a downwardly tapered channel which has an upper base (9) and a lower base (12), said lower base (12) having a diameter of dimensions d1 and said upper base (9) having a diameter of dimensions d2, said vertical channel (18) having a height h1 and the half-opening of the truncated cone formed by said tapered channel measures (90 ° - �1). In a preferred form, �1 is 90 ° and said vertical channel (18) is a cylinder.

Preferably, said measurements � and �1 differ from each other by a maximum of 15 °, preferably by 10 °, even more preferably by a value between 4 and 5 °.

In a preferred embodiment, with reference to Figures 19A, 19C, said distal portion (21) of said tip (20) has a diameter section of dimensions d2 at a point (28) positioned along said distal portion (21) to a height h_x with respect to the terminal base (25) of said distal portion, said height h_x being lower than the distance between said upper base (9) of said vertical channel (18) and the entry point in said microchannel (3), where said distance is h1 in the absence of any connector, where d3 <d2 <d4 e (90 ° - �1����� (90 ° - ���� preferably �1 is between 80 ° and 90 °, even more preferably it is equal to 90 °. In particular, said tip (20) is inserted in said inlet region (8) which is said vertical channel (18) for a portion (27) of length h_x, that is, said tip is inserted in said inlet region (8) reaching the interference coupling point before reaching the microchannel (3), i.e. said tip and and said vertical channel (18) included in said inlet region (8) reach the sealing position when the portion of said tip inserted in said vertical channel (18) is not such as to make said tip reach said microchannel (3). In this embodiment, (90 ° -�1) <(90 ° -�) and h_x = ((d2-d3) / 2) * tg�, preferably �1 is between 80 ° and 90 °, even more preferably it is 90 °.

Alternatively, with reference to Figure 22, said tip (20) is inserted in said vertical channel (18) included in said inlet region (8) for a portion (27) of length h_x, where d1 <d3 <d2, (90 ° -�1����� (90 ° - ����. Again with reference to the same figure 22, optionally said inlet region (8) comprises a connector (60), having an upper base (12) which coincides with the lower base (12) of said vertical channel (18).

In an alternative embodiment, with reference to Figure 21, said inlet region (8) also comprises a defined portion of a hollow truncated conical shaped flare (30), having a height h4 and an upper base (33) and a lower base (9) which coincides with the upper base (9) of said vertical channel (18), said upper base (33) having a diameter d5 greater than the diameter d2 of said lower base (9), where the half-opening of the cone forming said portion of flare (30) measures (90 ° -�2), where (90 ° -��)> (90 ° -�).

Preferably, with reference to Figure 23, said inlet region (8) also comprises, above said flare portion (30), a storage region (40) which comprises at least two portions: an upper portion (42) and a lower portion (41), where said upper portion (42) has a generally tubular shape having an upper base (43) and a lower base (44) of diameter measuring d6 and said lower portion (41) is tapered downwards and has an upper base (44) which coincides with said lower base (44) of said upper portion (42) and a lower base (45) of measuring diameter d5, said storage region (40) has a height h5 and the half-opening of the truncated cone that forms called the lower portion (41) measures (90 ° - �3), where �3 is less than or equal to 90 °, preferably �3 is 0 °.

Where said inlet region also comprises said storage region (40), said tip (20) is inserted in said inlet region (8) for a length greater than the length of said distal portion (21) of said tip (20), the distance between said terminal base (25) of said distal portion (21) of said tip (20) and the upper base (43) of said storage region (40) measures h_tot, being (h_tot> h2) and the distance between said upper base (24) of said distal portion (21) of said tip (20) and the lower base (45) of said storage region (40) measures h_y, being 2 * h_y * cot�3 d5> d4.

Alternatively, and with reference to Figure 23D, said tip (20) is inserted in said inlet region (8) for a length less than the length of said distal portion (21) of said tip (20), the distance between said terminal base (25) of said distal portion (21) of said tip (20) and the upper base (43) of said storage region (40) measures h_tot, and the distance between said terminal base (25) of said distal portion (21 ) of said tip (20) and the upper base (9) of said vertical channel (18) measures h_x being h_tot = h_x h4 h5 and the distance between said upper base (24) of said distal portion (21) of said tip ( 20) and the lower base (45) of said storage region (40) measures h_y, being h_y = h2 - h_x –h4.

In a further embodiment, with reference to Figure 24, said inlet region (8) also comprises a defined storage portion (50) which comprises at least two portions: an upper portion (52) and a lower portion (51) , where said upper portion (52) has a generally tubular shape having an upper base (53) and a lower base (54), of diameter d6 and said lower portion (51) is tapered downwards and has an upper base (54 ) which coincides with said lower base (54) of said upper portion (52) and a lower base (55) of diameter d2, said storage region (50) has a height h5 and the half-opening of the truncated cone which forms said storage portion (50) measures (90 ° - �3), where (90 ° - �3)> (90 ° - �).

Where said inlet region comprises said storage region (50), said tip (20) is inserted into said inlet region (8) for a length less than the total length of said tip (20), the distance between said terminal base ( 25) of said distal portion (21) of said tip (20) and the upper base (53) of said storage region (50) measures h_tot, being h_tot less than or equal to h2 and (2 * h_tot * cot � d3) <d6.

Alternatively, and the reference is to Figure 24D, said tip (20) is inserted in said inlet region (8) for a length less than the total length of said tip (20), the distance between said terminal base (25) of said distal portion (21) of said tip (20) and the upper base (53) of said storage region (50) measures h_tot and the distance between said terminal base (25) of said distal portion (21) of said tip (20 ) and the upper base (9) of said vertical channel (18) measures h_x, being h_tot> h2 and h_tot = h_x h5.

The expert in the sector understands that further forms of realization are possible.

The embodiment that provides for the presence of a storage tank in said inlet region (8) finds advantageous use whenever it is necessary to control evaporation in the microfluidic device. In fact, said storage region can advantageously be left full of the fluid loaded into the microfluidic device so that, even where long incubation times are required, unwanted evaporation effects do not occur.

The optional presence of the storage region allows to have a vertical channel (18) and, optionally, connectors (60) whose volumes are reduced to a minimum, so as to avoid wasting fluids, being able to have one or more storage tanks to be to be used only when necessary.

The possibility of having a flare portion (30) offers the advantage of providing a sufficiently wide opening to allow a tip (20) that is inserted in said inlet region (8) even in a way that is not centered along the central axis of the inlet channel (18) to enter said flare portion (30) and, subsequently, during the downward descent of said tip (20), into the inlet channel (18), by sliding the same tip along the internal walls of the flared portion and of the inlet channel, even with a possible bending / curvature of the tip (20).

Optionally, said microfluidic device (1) also comprises an impedance calibration plate and said tip (20) is connected to a dispensing system equipped with an impedance detection system.

Optionally, said microfluidic device comprises a closure element of said inlet region (8), by way of example said closure element is a cap or a protective film. Said protective film is, in one embodiment, of elastomeric material, by way of example a silicone.

The surprising solution highlighted by the authors of the present invention and described in claim 1 allows, with the method described below, to manage the introduction of one or more fluids into a microfluidic device only by using the interference coupling that is achieved between said tip and said vertical channel, without having to resort to pumps or valves.

In particular, the method of loading / unloading fluids in the microfluidic device (1) included in the described and claimed kit comprises the following steps: a) Providing a kit according to one of claims 1 to 8;

b) Optionally, load a fluid into said tip (20);

c) Position said tip (20) above said inlet region (8) and insert it until reaching a position of interference coupling between said distal region (21) of said tip (20) and said vertical channel (18) in said inlet region (8);

d) Releasing the fluid contained in said tip (20) in said microfluidic device (1) through said inlet region (8) or, alternatively, sucking with said tip (20) fluid already contained in said microfluidic device.

When the method is applied to the suction of fluid, and not to the release, and said fluid is a liquid, conveniently said vertical channel (18) and said possible connectors (60) also contain said liquid, i.e. said liquid is not only contained in the microfluidic device. The presence of liquid in the portion in contact with the tip (20), by lowering the surface tension, facilitates the suction process which would be more difficult if said tip initially sucked air before being able to suck the fluid contained in said microfluidic device.

Microfluidic device

Also disclosed is a microfluidic device (1) which is an inverted open microwell system comprising a matrix of open microwells 2, at least one microchannel 3, at least one inlet port 8 for reagents and / or for one or more biological samples and at least an outlet port 10 for the same, said inlet and outlet ports being in microfluidic communication with one or more of said microchannels 3, wherein said microchannel 3 has a cross-sectional area of micrometric dimensions and supplies fluid to said microwells 2 , wherein said inverted open microwell system is in one embodiment inserted in an automated management system which includes the following functionalities: incubator at controlled temperature, humidity and CO2, fluid dispensing system, acquisition of images in phase contrast and in fluorescence.

In a particularly preferred embodiment, an input door 8 and an output door 10 are associated with each of the microchannels 3.

In a preferred form, said microfluidic device (1) also comprises reservoirs 6, 7, where said reservoirs are at least one reservoir 6 for reagents and at least one reservoir 7 for one or more biological samples. Said reservoirs are selected in the group which includes: plates, one or more multi-well plates, for example 96-well plates, eppendorf tubes. Said reservoirs 6 and 7 can be 2, or 4, 8, 16, 24, 48, 96, 384.

In a preferred embodiment, schematized in Figure 3, 26 said reservoirs 6, 7 are integrated in said inverted open microwell system 1.

In a further embodiment, schematized in Figure 4, said at least one inlet port 8 for reagents also comprises a storage area 11. In this embodiment, said reagents and / or said biological sample are stationed in said storage area before to pass through said entrance door 8. Also said exit door 11 can optionally comprise a storage area 11. Said storage area is preferably positioned above said entrance door and, in a preferred embodiment, consists advantageously of two portions: an upper portion 13 and a lower portion 14. Said upper portion 13 has a cylindrical shape and said lower portion 14 has a funnel shape, where said upper portion 13 is a cylinder with a base having a diameter greater than diameter of said inlet port 8 and said lower portion 14 is a funnel which connects said upper portion with said inlet port entrance.

Region of discharge

In a further embodiment, with reference to Figure 27, said discharge region (70) dedicated to the discharge of fluids from said microfluidic device (1) which comprises at least one inlet region (8), comprises a discharge container ( 12) in fluidic connection with said microfluidic device (1) through at least one discharge channel (22) and an outlet port (10). In one embodiment, said fluid, pushed by a pressure applied in said inlet region (8) in said microfluidic device (1), unidirectionally reaches said discharge container (12) where the volume V of said fluid is smaller. or equal to the volume of said discharge container (12). Preferably, with reference to Figure 27A, said discharge channel (22) emerges from at least one microchannel (3) included in said microfluidic device (1) and is a siphon.

Preferably, the diameter of said discharge channel (22) is such that the siphon exerts a capillary force on the fluid contained in said microchannel (3).

In a further embodiment, schematized in figure 27C, said discharge channel (22) is placed on the bottom of at least one micro-channel (3) and is almost orthogonal to it and connects said at least one micro-channel (3) with said waste container (12) which is positioned below the same micro-channel (3).

In a further embodiment, with reference to Figure 27B, said outlet door (10) is placed on the bottom of said at least one microchannel (3) and leads into a first discharge container (12a), positioned below the to said microchannel (3) and from said first discharge container (12a) emerges said discharge channel (22) which enters into said discharge container (12). Preferably, with reference to Figure 27B, the discharge channel (22) is a siphon.

Said discharge channel (22) preferably has a cross-sectional area of micrometric dimensions, said dimensions being between 100 �m and 5mm, preferably between 500 �m and 2 mm.

In a particularly preferred embodiment, the kit according to the present invention comprises a microfluidic device as described above.

Even more preferably, the kit according to the present invention comprises the microfluidic device as described above, also characterized by the fluid discharge region described above.

In a preferred embodiment, shown in Figure 25, the microfluidic device (1) comprises an inlet region (8) suitable for being included in the kit according to one of claims 1 to 8 and an unloading region (70) according to claim 10.

Therefore, the subject of the present invention is a method for loading / unloading fluids from a microfluidic device where said method comprises:

a) Provide a microfluidic device (1) which comprises an inlet region (8) and an unloading region (70) according to one of claims 1 to 6, wherein said microfluidic device (1) is loaded with at least a first fluid;

b) Optionally, exert a pressure on said at least one first fluid entering said microfluidic device (1);

c) Alternatively, having a discharge region (70) where said discharge channel (22) has a diameter such as to allow said fluid to pass from said microfluidic device to said discharge channel by capillarity;

characterized in that, where the volume V of said at least one first fluid is less than or equal to the volume of said discharge container (12), said at least one fluid reaches said discharge container (12) in a unidirectional manner.

In a further embodiment, said method also includes:

-injecting a second or further fluid through said inlet region (8) into said microfluidic device (1), wherein said first, second and / or further fluids are independently the same or different from each other and are selected in the group comprising liquids and gases ;

- optionally, said second and / or further fluid completely replaces in said microchannel (3) or in said microchannel (3) and in said inverted open microwells (2), where present in said microfluidic device (1), the fluid previously introduced ; characterized in that said first, second and / or further fluid do not mix with each other.

In a further embodiment, shown in figure 26, the microfluidic device (1) is an inverted open microwell microfluidic device (2) and also comprises reservoirs (6) for reagents and reservoirs (7) for one or more biological samples.

In a particularly advantageous embodiment, said device comprises 16 microchannels (3), each having an inlet region (8) and an outlet region (10), where each of said microchannels (3) faces 1200 inverted open microwells .

In addition to the advantages highlighted above, it should be noted that the microfluidic device (1) which comprises the inlet region (8) according to the present invention is particularly advantageous because:

i) allows direct interfacing, ie without the aid of seals, of a microfluidic device with systems commonly used in the sector for the management and transfer of liquids and gases; ii) allows to withdraw fluids from plates or containers by inserting them under pressure into microchannels of the microfluidic device, where the volumes involved in such plates or containers are greater or much greater (for example 50 �l or more, 300 �l, 1 ml or more ) compared to those characteristics of the microchannel (40 �l or less) and where the transfer takes place using standard tools already commonly used in combination with the aforementioned plates or containers,

iii) allows you to insert sequences of fluids, even of different types, into the microfluidic device, without the use of additional equipment, such as pumps or valves.

Method of loading / unloading fluids from a microfluidic device

A further aspect of the present invention is a method for managing a microfluidic device (1) with inverted open microwells (2) which comprises at least one inlet region (8), at least one outlet region (10) and at least one microchannel (3 ), where said method includes:

- introducing at least a first fluid through said inlet region (8) into said microfluidic device (1);

- optionally, introducing a second or further fluid through said inlet region (8) into said microfluidic device (1), wherein said first, second and further fluids are independently the same or different from each other and are selected in the group comprising liquids and gases ; - optionally, said second or further fluid completely replaces the previously introduced fluid in said microchannel (3) or said microchannel (3) and in said inverted open microwells (2);

- optionally, aspirating from said inlet region (8) the volume of fluid contained in the microchannel (3), leaving said fluid in said microwells (2).

In one embodiment, said method is implemented in a microfluidic device (1) which comprises an inlet region (8) according to one of claims 1 to 8 and an unloading region (70) according to claim 10 and is characterized by fact that where the overall volume of said first and, optionally, second and further fluids introduced into said microfluidic device (1) is less than the volume of said discharge container (12), said fluids do not mix with each other.

Method for the high-content analysis of biological samples The method described below surprisingly allows not only a time-lapse analysis but also a processing during said time-lapse analysis. That is, thanks to the method of the present invention it is possible not only to monitor the same sample at different times (classic time-lapse analysis), but also to manipulate the same sample at different times, varying the conditions over time and in a controlled and automated manner. to outline. By way of example, the method described here allows to evaluate, for each individual cell, the variation of morphological and functional parameters following a controlled exposure to a pharmacological agent, where said agent is added during said monitoring. Alternatively, a dynamic analysis of the sample is possible with the same method, where dynamic analysis here means an analysis of the sample carried out at different times after exposure to treatments of interest. Furthermore, thanks to the dynamic analysis carried out according to the present invention, it is possible to identify cells in a sample that are insensitive to a treatment so as to expose them to a different treatment. The method claimed here allows the implementation of a large-scale high-content assay with time-lapse processing, independent operator, having equipment that can be installed in any analysis laboratory.

The method of the present invention is implemented in a system with inverted open microwells, the structure of which is exemplified in Figure 1. Said system with inverted open microwells 1 comprises a series of microwells 2, open at both ends, arranged according to a matrix scheme. Said system with inverted open microwells also comprises at least one microchannel 3, where said at least one microchannel 3 has a cross-sectional area of micrometric dimensions and supplies fluid to said microwells 2. Said system also comprises at least one inlet port 8 for reagents and / or for one or more biological samples and at least one output port 10 for the same, said inlet and outlet ports being in microfluidic communication with one or more of said microchannels 3.

In a particularly preferred embodiment, an input door 8 and an output door 10 are associated with each of the microchannels 3.

In a preferred form, said reagents are contained in reservoirs 6, 7, where said reservoirs are at least one reservoir 6 for reagents and at least one reservoir 7 for one or more biological samples. Said reservoirs are selected in the group which includes: plates, one or more multi-well plates, for example 96-well plates, eppendorf tubes. Said reservoirs 6 and 7 can be 2, or 4, 8, 16, 24, 48, 96, 384.

In an embodiment, represented in Figure 2, said reservoirs 6, 7 are external to said inverted open microwell system 1. In a preferred embodiment, schematized in Figure 3, said reservoirs 6, 7 are integrated in said system with inverted open microwells 1.

In a further embodiment, schematized in Figure 4, said at least one inlet port 8 for reagents also comprises a storage area 11. In this embodiment, said reagents and / or said biological sample are stationed in said storage area before to pass through said entrance door 8. Also said exit door 11 can optionally comprise a storage area 11. Said storage area is preferably positioned above said entrance door and, in a preferred embodiment, consists advantageously of two portions: an upper portion 13 and a lower portion 14. Said upper portion 13 has a cylindrical shape and said lower portion 14 has a funnel shape, where said upper portion 13 is a cylinder with a base having a diameter greater than diameter of said inlet port 8 and said lower portion 14 is a funnel which connects said upper portion with said inlet port entrance.

The subject of the present invention is a method for the high-content analysis of large-scale biological samples, where said biological samples are defined as above, which includes the following steps, not necessarily in the order indicated:

a) Provide an inverted open microwell system comprising a matrix of open microwells 2, at least one microchannel 3, at least one inlet port 8 for reagents and / or for one or more biological samples and at least one outlet port 10 for the same, said inlet and outlet ports being in microfluidic communication with one or more of said microchannels 3, wherein said microchannel 3 has a cross-sectional area of micrometric dimensions and supplies fluid to said microwells 2;

b) Provide an automated management system for said inverted open microwell system which includes the following functions: temperature, humidity and CO2 controlled incubator, fluid dispensing system, acquisition of phase contrast and fluorescence images;

c) Insertion of said system with inverted open microwells 1 in said automated system; d) Loading of the reagents through one or more of said inlet ports for reagents 8, where said reagents include: filling buffer and / or washing solution and / or one or more drugs and / or one or more tracers and / or one or multiple labeled antibodies and / or one or more cell viability markers;

e) Loading of said one or more biological samples through one or more of said inlet ports 8;

i) Optionally, staining of said cells with one or more tracers, and / or one or more labeled antibodies and / or one or more cell viability markers;

j) Acquisition of images from one or more of said microwells 2, where said images are defined as T0 images;

p) Classification of the displayed cells, where said classification is carried out with morphological and / or functional parameters detected by the T0 images.

In one embodiment, said inverted open microwell system is the microfluidic device according to claim 9; in a further embodiment it is the microfluidic device according to claim 11, or it is a microfluidic device (19 which also includes the kit according to one of claims 1 to 8.

In one embodiment, where said reagents and biological sample are contained in 6, 7, said reservoirs 6, 7 are external to said inverted open microwell system 1, as shown schematically in Figure 2, and said loading of the reagents and of the at least a biological sample takes place manually, or by means of automated systems for dispensing fluids, taking from the reservoirs and loading into the inlet ports 8. Preferably, said reservoirs 6, 7 are inserted into the automated system with the inverted open microwell system.

In an even more preferred form, schematized in Figure 3, said reservoirs 6, 7 are integrated in said inverted open microwell system 1, and said loading of the reagents and of the at least one biological sample takes place manually, or by means of automated systems for the dispensing of fluids, taking from the reservoirs and loading into the inlet ports 8. Alternatively, said reservoirs 6, 7, integrated in said inverted open microwell system 1, are in fluid communication with said at least one inlet port 8.

Even where said inlet door 8 comprises said storage area 11, said reservoirs 6, 7 are located as described above, ie they are external or integrated in the inverted open microwell system.

In a preferred embodiment, some of said reservoirs 6 are preloaded with said reagents prior to carrying out the method, even days or months before carrying out the same, so as to be able to have specific reservoirs 6 ready for use. In this embodiment, the only manual step required by the operator is that of preloading the biological sample in said one or more reservoirs 7.

In a further preferred embodiment, in addition to said biological sample loading step, a further manual step performed by the operator is the loading of one or more drugs in one or more of said reservoirs 6.

In a preferred embodiment, said reservoirs 6, 7, integrated or external to said inverted open microwell system, pre-loaded with reagents and biological sample, are introduced into said automated system. In this embodiment, said loading passages d) and e) through said inlet gates, also said inlet regions (8) occur after said step c) of insertion into the automated system of said inverted open microwell system in which said reservoirs 6, 7 or of said inverted open microwell system and of said reservoirs 6, 7 external to it are integrated, preferably by automated sampling of the biological sample from said reservoirs (7) with consequent arrangement of the cells contained therein in one or more more than said microwells (2) and, optionally, subsequent insertion of drugs in one or more of said microwells and / or staining of said cells with one or more tracers, and / or one or more labeled antibodies and / or one or more markers cell viability, where said tracers, and / or one or more labeled antibodies and / or one or more cell viability markers are fed to said cell viability gates entrance (8) from said reservoirs (6).

Said morphological / functional classification derives from the fluorescence analysis with resolution of a single cell, a single cell aggregate or the entire cell population that is contained in a single microwell.

These parameters are acquired and analyzed automatically, through the use of systems known to the expert in the sector.

By morphological parameters we mean measures relating to the size and shape of the cell. By functional parameters we mean those characteristics observed thanks to the markers, such as the expression of specific antigens.

In a preferred embodiment, said method also comprises, after the insertion of said inverted open microwell system in said automated system:

f) Filling at least one said microchannel 3 with filling buffer;

g) Acquisition of images from one or more of said microwells 2, individually or by subgroups, where said images are defined as baseline images (Tbaseline);

and, in said classification step p), said classification is carried out with morphological and / or functional parameters detected by comparing the images T0 with said baseline images.

Even more preferably, said method also includes:

k) Dispensing of one or more of said drugs, individually or in combination, where said drugs are preferably withdrawn in an automated manner from said reservoirs 6 and dispensed into one or more of said microwells 2;

l) Incubation.

In said embodiment, said method allows, by way of example, the analysis of the ex vivo drug activity, where the classification of said cells present in said biological sample carried out in said step p) allows a direct comparison between, a as an example, the responsiveness to treatment of healthy cells and tumor cells present in the same biological sample, or a comparison between the response of the same cell type to different treatments.

In an even more preferred embodiment, said method also comprises, after said step l):

m) Acquisition of at least two images from said one or more microwells 2, at successive times during said incubation, where said images are defined as T1, T2, Tn images, where n is any number equal to or greater than 2, preferably 1,000, or 100, even more preferably 25, in a preferred embodiment is 9;

n) Optionally, between said acquisitions of said T1, T2, Tn images, further staining of said cells with one or more tracers, and / or one or more labeled antibodies and / or one or more cell viability markers;

o) Optionally, between said acquisitions of said images T1, T2, Tn, further dispensing of one or more of said drugs into a microwell 2 or into one or more subgroups of microwells 2, individually or in combination;

and said classification referred to in step p) comprises the comparison of morphological and / or functional parameters detected by images T0, T1, Tn and, optionally, Tbaseline.

Even more preferably, said method also comprises, after said classification step p):

q) Analysis of cell viability, where said analysis is specific cell, or is carried out at the cellular aggregate level, or even takes into consideration the entire cell population contained in one of said microwells 2.

In a preferred embodiment, in said step k) said drugs are dispensed in at least two different concentrations and said cell viability analysis, where present, leads to obtaining a specific dose / cell response curve for the one or more drugs tested.

Optionally, dilutions of each drug are prepared by the liquid dispensing system by mixing the drug with a diluent.

In a further embodiment, the method provides, after said acquisition of images referred to in step j), to count the number of cells on average present in each of the microwells 2 and the subsequent dilution of the sample, by means of said dispensing system of the fluids, so as to reach an objective concentration, i.e. a concentration such as to guarantee the desired number of cells for each microwell, and the subsequent loading of the sample thus diluted into one or more microchannels of said inverted open microwell system, microchannels other than one or multiple channels used in step d) above.

In said step k), where the combined administration of at least two drugs is required, said two or more drugs are optionally combined by a liquid dispensing system, mixing before loading the contents of at least two reservoirs 6 containing said drugs.

Said method is characterized by using a biological sample whose volume is between 1 µl and 1 ml, preferably between 10 µl and 100 µl, with a cell concentration between 5,000 cells per ml and 5,000,000 cells per ml.

In a preferred form, said filling buffer comprises RPMI medium added with 10% Fetal Bovine Serum (FBS) and, preferably, a cell death marker, typically 5 µM propidium iodide (PI).

Said system with inverted open microwells 1, of which Figure 1 shows a perspective view (a), in vertical section (b) and a top view (c) and in Figure 5 a perspective view in section, in a preferred embodiment comprises 1200 microwells 2 for each microchannel 3 and 16 microchannels 3; in a further embodiment, it comprises 500 microwells 2 and 5 microchannels 3.

In a preferred embodiment, said method analyzes a biological sample containing only about 10-20 cells and uses the inverted open microwell system of the type described in WO2012 / 072822. In a preferred embodiment, said biological sample is obtained from an animal and / or a human being affected by a tumor pathology and said biological sample comprises healthy cells and tumor cells.

The totality of the cells is highlighted in the images acquired in phase contrast and / or, where these cells are stained with a tracer, in a fluorescence channel, for example in the DAPI if the tracer is CMAC (7-amino-4-chloromethylcoumarin) . Other tracers usable in the method can be selected from the group which includes: Calcein-AM, carbocyanins such as DiD, DiO, DAPI (4 ', 6-diamidino-2-phenylindole).

Other tracers that can be used for the purposes of this method are cell death markers such as PI, Calcein-AM, JC1, Caspase 3/7, Annexin V. In a further embodiment, labeled antibodies are used as tracers.

In a particularly preferred embodiment, said method comprises a repetition of the treatment, where the cells that survive a first exposure to one or more of said drugs referred to in step k), are re-exposed to a further pharmacological treatment, where said further treatment involves exposure to one or more of said drugs already used in said step k) at a higher concentration, or where in said further step one or more drugs other than those used in the previous step are used.

The reiteration of the method takes place, in one embodiment, on the cells that remain alive in said one or more microwells 2, where this further treatment is made possible by the fact that, in said inverted open microwell system, said microchannel 3 is emptied of said fluid which comprises said one or more drugs and subsequently filled with a second fluid which comprises said further one or more drugs. The cells, remaining positioned on the meniscus at the air / fluid interface, are not altered by the change of fluid in said microchannel. These cells positioned on the meniscus, even if they are not cells that grow in adhesion, from a fluidic point of view behave like cells that grow in adhesion on the bottom of a closed well, where typically it is possible to replace the culture medium without affecting said cells in accession. The advantage linked to the present embodiment is to be found in its use also with cells that grow in suspension, where said meniscus allows to mimic the bottom of a well, without however imposing an obligatory adhesion to said cells, for example by using substrates that stimulate cell adhesion known to those skilled in the art. This is particularly advantageous since it allows to impact as little as possible on the biological sample, avoiding imposing external conditions extraneous to the physiological context.

In a preferred embodiment, said method includes the following steps, in the order indicated:

a) Provide an inverted open microwell system (1) that includes a matrix of open microwells (2), at least one microchannel (3), at least one inlet port (8) for reagents and / or for one or more samples and at least one outlet port (10) for the same, said inlet and outlet ports being in microfluidic communication with one or more of said microchannels (3), where said microchannel (3) has a cross-sectional area of micrometric dimensions and supplies fluid to said microwells (2);

b) Provide an automated management system for said inverted open microwell system which includes the following functions: temperature, humidity and CO2 controlled incubator, fluid dispensing system, acquisition of phase contrast and fluorescence images;

c) Insertion of said inverted open microwell system (1) in said automated system;

f) Optionally, filling of at least one said microchannel (3) with filling buffer;

g) Optionally, acquisition of images from one or more of said microwells (2), individually or by subgroups, where said images are defined as baseline images;

d) Loading of the reagents through one or more of said inlet ports (8), where said reagents include: filling buffer and / or washing solution and / or one or more drugs and / or one or more tracers, and / or one or more antibodies labeled and / or one or more cell viability markers and where said reagents are contained in reservoirs (6) for reagents;

e) Loading of said one or more biological samples through one or more of said inlet gates (8), where said one or more biological sample is contained in reservoirs (7) per biological sample;

i) Optionally, staining of said cells with one or more tracers, and / or one or more labeled antibodies and / or one or more cell viability markers;

j) Acquisition of images from one or more of said microwells (2), where said images are defined T0 images;

k) Dispensing of one or more of said drugs, through said inlet doors (8), into one or more of said microwells (2);

l) Incubation;

m) Acquisition of at least two images from said one or more microwells (2), at successive times during said incubation, where said images are defined as T1, T2, Tn images, where n is any number equal to or greater than 2, preferably 1,000 , or 100, even more preferably 25, in a preferred embodiment it is 9;

n) Optionally, between said acquisitions of said T1, T2, Tn images, further staining of said cells with one or more tracers, and / or one or more labeled antibodies and / or one or more cell viability markers;

o) Optionally, between said acquisitions of said images T1, T2, Tn, further dispensing of one or more of said drugs in a microwell (2) or in one or more subgroups of microwells (2), individually or in combination;

p) Classification of the displayed cells, where said classification is carried out with morphological and / or functional parameters detected by images T0, T1, Tn and, optionally, Tbaseline.

Tumor cells are identified by size and shape parameters, for example membrane roughness, and by binding to specific labeled antibodies for tumor antigens, where the presence or absence of a specific signal emitted by one or more specific labeled antibodies determines identification of the cell type. By way of example, where the tumor in question is a lymphoma, an anti-CD38 antibody is used for the differential analysis of tumor cells; where the tumor in question is acute myeloid leukemia (AML), an anti CD34, or anti CD117, or anti HLA-DR, or anti CD33 / CD14 antibody is used.

In a preferred embodiment, the tested drugs are selected from monoclonal antibodies, by way of example Alemtuzumab, Bevacizumab, Cetuximab, Ibritumomab, Ofatumumab, Panitumumab, Rituximab, Tositumomab, Trastuzumab, chemotherapeutic agents, by way of example cytarabine, idarudarabine, idarudarabine, idarudarabine, decitabine, 5-azacitidine and small molecules, by way of example ibrutinib, idasanutlin, venetoclax, whereby the method of the present invention measures complement-mediated cytolytic activity, in the case of monoclonal antibodies, or direct cytotoxic, in the case of chemotherapeutic agents and small molecules.

The automated analysis of the images obtained according to the method of the present invention makes it possible to extract the information related to each single cell, or to an aggregate of cells, captured in the acquired images.

At the end of this method, the cells contained in the microwells can be collected and used for subsequent analyzes. In particular, the method described here makes it possible to select one or more microwells containing cells of particular interest, for example cells resistant to the drug injected into a specific microchannel, to recharge said cells in the reservoir (7) of a further open microwell system. inverted, dispensing the same at a higher dilution, so as to obtain microwells containing only one cell. Once the desired cell has been identified, it can be isolated. Assays known to those skilled in the art, for example RT-PCR, are conducted on said one or more isolated cells.

The biological sample can be loaded as taken / explanted, or it can be previously processed so as to make it usable in the method according to the present invention. In particular, where it is a blood or bone marrow sample, said processing preferably involves a step of separation of the red blood cells with techniques known to the skilled in the art, preferably on a density gradient, or through the use of a buffer. of lysis. Where said sample comprises cells in suspension, said cells are counted and diluted to the dispensing concentration. Alternatively, said counting and dilution step is carried out automatically by the system. Where the sample is a biopsy, said processing typically includes the isolation of a fragment with dimensions between 10 µm and 1000 µm, dimensions compatible with the microfluidics used. In an alternative embodiment, said biological sample is used as such as isolated from animals and / or humans.

Figure 6 shows an example of a series of images acquired at time T1 in a biological sample consisting of leukemia cells from a cell line mixed with blood obtained from a healthy donor. The biological sample therefore contains healthy PBMCs and cancer cells. The image is obtained in the visible (A), in the DAPI fluorescent channel with a tracer (B), in the FITC fluorescent channel for the anti-CD45 labeled antibody signal (C) and in the CY5 fluorescent channel for the antibody marked anti CD34 (D).

Finally, (E) shows the superimposition of said images (C) and (D). The method has been shown to have a high sensitivity accompanied by a high specificity when applied to the classification of the cells, as evidenced by the image (E) in which all the cells present in the sample are found, i.e. those of the image in (B ), where all cells are CD45 positive (C) and only a portion are also CD34 positive (D). By analyzing the cells using morphological (cell diameter) and functional parameters (expression of CD45, CD34), a correct classification is obtained for almost all of these tumor cells, as shown in Figure 7, a figure that clearly shows that the CD34 marker is the parameter which allows to identify tumor cells with high sensitivity and specificity, as evident from the ROC curve for which an area under the curve equal to 99.8% of the cells that actually are (CD34 ROC curve) is obtained. The CD45 parameter, on the other hand, produces an area under the curve equal to 85.2%, thus demonstrating lower sensitivity and specificity. This experiment shows how, for the tumor type being analyzed, CD34 is the marker of choice and allows an extremely accurate analysis of the cell population.

Figure 8 shows the graphs relating to the analysis of the acquired data, in relation to the classification of cancer cells. In (C) the distribution of cells stained with the CMAC tracer and not stained with anti-CD34 / CD45 antibodies is reported. From this distribution it is possible to determine the maximum signal identified in this negative control and consequently set a minimum threshold for classification. Subsequently, in (D) the count carried out after anti-CD34 / CD45 labeling was carried out by maintaining the threshold identified in point (C) and by classifying the cells characterized by a signal emitted by the antibody above the threshold as tumors. In particular, tumor cells can be classified as such because they are positive for the anti-CD34 (CD34 +) antibody and because they are positive for both anti-CD34 and anti-CD45 (CD34 + / CD45 +) antibodies. As a control of the method, panels (A) and (B) report the results obtained by flow cytometry on the same samples. These results show that the data obtained with the method of the present invention are absolutely comparable with those obtained for the same sample by cytofluorimetry, which is currently the technique of choice for this type of analysis and cell count.

Desiring to monitor cell viability, in said step i) said cells are stained with cell viability markers. By way of example, Propidium Iodide (PI) or Annexin V are commonly used markers in this regard: if the membrane is damaged, PI enters the cells by emitting fluorescence after binding to nucleic acids. The labeled cells therefore correspond to those with the compromised or destroyed membrane and are to be considered as dead. Another commonly used cell viability marker is calcein - acetoxymethyl (calcein-AM) (Lichtenfels R et al., CARE-LASS calceinrelease-assay, an improved fluorescence-based test system to measure cytotoxic T lymphocyte activity. J Immunol. Methods 1994, 172: 227-239). Calcein acetoxymethyl ester passively crosses the cell membrane and, once inside, is converted by an intracellular esterase into calcein, which is a polar fluorescent product that is believed to be inside viable cells but released by cells whose membrane is damaged and therefore to be considered death. Figure 9A-9B shows an example of an analysis carried out on a biological sample that includes Raji cells, lymphoma cell line, and leukocytes. In the samples under examination, a treatment with a drug, specifically with Rituximab, was carried out according to the method of the present invention. Panel (A) highlights the ability of the method according to the invention presence to distinguish between tumor cells (circle) and healthy cells (square), simultaneously using morphological parameters and the CD38 marker. In (B) the cell viability data is represented, obtained by quantifying the presence of Calcein-AM in the cell, in particular by comparing the signal emitted after treatment with the drug with the signal emitted initially, where the signal reduction is associated with the loss of vitality. In this case the reference threshold is determined using a control consisting of a channel in which the sample is not exposed to the drug. Alternatively, known references in the literature regarding physiological signal loss are used (Neri S et al., Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyzes on Recovered Effector Cells and Supernatants. Clin. Diagn. Lab. Immunol. 2001 , 8: 1131–1135).

Figure 9C shows the dose / response curves obtained for a different model consisting of leukemic cell lines (KG-1, Kasumi-1 and HL-60) for which a response analysis to the chemotherapy drug idarubicin was performed. The results show 3 different levels of response, observable, for example, by the different measurable IC50 values indicated with the vertical dashed lines: sensitive (IC50 = 0.0049 µM), medium sensitive (IC50 = 0.0218 µM) and resistant (IC50 = 0.2127 µM) .

By way of example, a biological sample deriving from explantation of a liver tumor can be used in the present method, after partial non-enzymatic disintegration. The biological sample is then labeled and loaded into said reservoirs. In less than an hour from the explant, you have an inverted open microwell system ready for analysis, which includes cell clusters and isolated cells. The possibility of evaluating the effect of drugs also on cell clusters is extremely advantageous, since it also allows for the evaluation of any changes in the volume of the cluster itself, as well as different marking areas in the same cluster. It is also possible to acquire images on different focal planes, thus being able to reconstruct a 3D image of the cluster.

Figure 10 shows the diagram of a system with inverted open microwells which also comprises electrodes 5 arranged around said microwells 2 and on the base of said microchannel 3. Said electrodes 5 have the function of conveying inside the microwell 2 at the times and in the the desired ways the cells 4 present in the microchannel 3. The electrodes therefore favor the formation of cell clusters inside the microwells, thus allowing an analysis that also takes into consideration the specific cell / cell interaction.

BENEFITS

The method described here facilitates the personalized medicine approach, which is strongly necessary especially when it is necessary to intervene in tumoral pathologies. In fact, the method described here surprisingly makes it possible to test on an ex vivo biological sample, which includes even only a few cells, for example even only about 10-20 cells where the inverted open microwell system used in said method is of the type described in WO2012 / 072822, the effect of different drugs so as to allow the choice of the most effective drug for the specific patient and / or the optimal dose for the specific patient and / or the determination of the resistance of the patient sample to one or more drug therapies . The analysis is carried out immediately after the collection of the biological sample and is completed in a period of about 24 hours. The fast timing allows the cells within the biological sample not to undergo significant changes in their functionality, vitality and gene expression, with reference to the characteristics they possess in-vivo. The speed with which the data are obtained allows the use of the same method in clinical situations for which it is necessary to make decisions quickly, for example in acute leukemia.

Furthermore, given the scarcity of biological sample required, this method is applied without having a substantial impact on the operational routine, as it does not require dedicated sampling. For example, in the case of using blood, a volume of less than 1 ml is typically sufficient. In the case of the spinal cord, volumes equal to 1 ml are typically sufficient for the execution of multiple tests, or 0.1 ml or less for the execution of a single test. A needle aspiration sample may offer enough material.

The method developed here allows the use of complex samples, including multicellular samples and serum, with enormous advantages from the point of view of conditions as close to physiological conditions, to improve the predictivity of the assay.

The method described here allows a high content analysis, with the resolution up to a single cell, performing both a dynamic analysis (timelapse), and a dynamic processing of the sample (through programmable replacement over time of the fluids surrounding the sample) these characteristics are highly sought-after since they allow to monitor and compare the evolution over time of a given cell having specific characteristics, to make sample preparation and drug administration automatic and to create complex protocols that provide, for example, the insertion of drug sequences or labeling with certain reagents, for example Annexin V, at the end of the incubation period with the drug to determine the outcome of the experiment in terms of viability, induction of apoptosis or evaluation of the activation of specific associated pathways the activity of the drug. Among the numerous applications for which the method described here is particularly functional, we mention here the possibility of comparing, in a rapid and automated way, the responsiveness of a patient's tumor cells to a specific treatment and the average responsiveness of the tumor cells of a population of patients affected by the same type of tumor. Furthermore, there is the possibility of comparing the responsiveness of a patient's tumor cells to a specific treatment with the average responsiveness of cell lines relating to the same type of tumor and / or to different types of tumor, for example by referring to literature data.

The process is completely automated, the operator's intervention is reduced to a minimum, that is to say only the loading of biological samples and reagents and drugs in the reservoirs. In some embodiments, the intervention is exclusively limited to loading the biological sample, where the inverted open microwell system includes pre-loaded reservoirs of reagents and drugs, minimizing errors, reducing the risk of cross-contamination and increasing the reproducibility of the data obtained is considerable. It is also possible to load the biological sample as it is, without any preparation: the system itself is able to detect the number of cells contained in it, dilute them to the optimal dilution and dispense them into the microwells of interest.

In the embodiment that includes electrodes in the microwells, it is possible to obtain specific groupings of cells in single microwells, so as to evaluate cell-dependent mechanisms of action.

As a further advantage, it is highlighted here that working on a micrometric scale also the consumption of reagents and drugs is absolutely contained, for example drugs are diluted in volumes of only 20-50 microliters, with a considerable saving compared to what is required by having to work on volumes. larger, an advantage that is particularly evident where the drugs used are extremely expensive drugs, or in the drug development phase, where the availability of the drug may be limited, especially in the case, for example, of biological drugs.

In a particularly preferred embodiment, a method for the high-content analysis of large-scale biological samples is described which includes the following steps, not necessarily in the order indicated:

a) Providing a kit according to claim 9 or 10;

b) Provide an automated management system for said inverted open microwell system which includes the following functions: temperature, humidity and CO2 controlled incubator, fluid dispensing system, acquisition of phase contrast and fluorescence images;

c) Insertion of said inverted open microwell system (1) in said automated system;

d) Loading of the reagents through one or more of said inlet ports (8), where said reagents include: filling buffer and / or washing solution and / or one or more drugs and / or one or more tracers, and / or one or more labeled antibodies and / or one or more cell viability markers;

e) Loading of said one or more biological samples through one or more of said inlet gates (8);

i) Optionally, staining of said cells with one or more tracers, and / or one or more labeled antibodies and / or one or more cell viability markers;

j) Acquisition of images from one or more of said microwells (2), where said images are defined T0 images;

p) Classification of the displayed cells, where said classification is carried out with morphological and / or functional parameters detected by the T0 images.

Example 1: Evaluation of the efficacy of Rituximab on non-Hodgkin's lymphoma cells

Cells: SU-DHL-4 lymphoma cells and Raji cells (ATCC CCL-86) were used. 3 10 ^ 5 cells / ml were grown in RPMI medium supplemented with 10% FBS, 1% puromycin / streptomycin at 37 ° C, 95% humidity, 5% CO2. The cells were amplified, dividing them when at confluence. PBMCs were isolated from blood by gradient centrifugation. Whole blood, from healthy donors, was collected in tubes treated with anticoagulants in accordance with the directive "ethical principles and guidelines for the protection of human subjects in research". Isolated PBMCs were washed twice with 10 mL of cold HBSS after red blood cell lysis. Cell concentration and viability were determined by Trypan blue on a hemocytometer. The concentration was adjusted to 1 million cells / mL. Several batches of SU-DHL-4 or Raji cells were harvested at peak growth, washed with PBS and counted in a hemocytometer with Trypan Blue. The cell concentration was adjusted to 1 million cells / ml and the cells were labeled with Calcein AM (CAM) 5 nM for the flow cytometry and 250 nM for the assay according to the present invention. The labeled cells were washed once in PBS and resuspended in RPMI. The same protocol was used to label freshly isolated PBMC CAMs. Cell viability was finally assessed with PI and flow cytometry.

Flow cytometry (comparative)

10 ^ 5 CAM-labeled cells for each lot were seeded in duplicate on a 96-well plate. The control group (CTRL) was treated with RPMI spiked with PI at a final concentration of 1.5 µM. The treatment group (RTX) was treated with Rituximab at a final concentration of 10 µg / ml in RPMI supplemented with PI at a final concentration of 1.5 µM and incubated at room temperature for 5 min. Immediately thereafter, human serum (hS) was added to a 1% final concentration. The final volume of each well was 200 µl. Samples of 50 µl were taken at time 0, 30 and 60 min after the hS addition, and analyzed at FACS (FACSAria BD, USA).

Assay in open microwells

Several batches of CAM-labeled cells were resuspended in RPMI at a final concentration of 2 · 10 ^ 6 / ml. Similar to the procedure followed for flow cytometry, the RTX group was treated with Rituximab at a final concentration of 10 µg / ml off chip and incubated at room temperature for 5 min. CTRL and RTX cells were seeded in the inverted open microwell system as described, resulting in an optimal number of 7 cells or less per microwell. The channels were then rinsed with PBS added with 20% FBS. A first series of images had been acquired to set the initial conditions (Tbaseline). After loading, the CTRL channel was treated with RPMI supplemented with hS at a final concentration of 1% and PI at a concentration of 5 μM. The RTX channel was treated with RPMI supplemented with Rituximab 10 µg / ml, HS 1% and PI 5 μM.

Time-lapse images T1, T2, T3, T4 were acquired every 15 'and up to one hour after treatment.

Microscopy

The images were acquired in bright field (BF) to evaluate the morphological characteristics and then in fluorescence to evaluate the signal in the DAPI bands (ex: 360/40 nm, em: 460/50 nm), FITC (ex: 480/30 nm, em: 535 / 40nm), TRITC (ex: 540/25 nm, em: 605/55 nm). All images were captured with the 10x lens with a Nikon camera. During imaging the microscope worked in a controlled atmosphere at 37 ° C, 95% humidity, 5% CO2.

Visualization

SU-DHL-4 tumor cells were labeled with CAM and subsequently loaded into the open microwell system, treated with drug in the case of the RTX group and observed.

The results are shown in Figure 11. Panel A shows a sequence of time-lapse images of CTRL and RTX treated samples at the times T05-10 min, T1 30 min and T3 60 min. The cells indicated by arrows refer to the staining acquired in the TRITC (PI) channel and represent dead cells. They are present only at T2 and T3 in the RTX group. In greater contrast, the cells acquired in the FITC channel are seen, ie the living cells marked with Calcein-AM. Panel B shows the data obtained with the FACS. Panel C reports the histograms of the analysis carried out at FACS and according to the method of the present invention at time T2 60 minutes. The percentages indicated refer to the population of Calcein-AM positive cells, therefore alive. As can be seen, the data obtained with the two methods are absolutely comparable. To confirm, the data are reported linearly in Figure 12, leading to the conclusion that the system according to the present invention can be used to quantify live and dead cells over time, providing data equivalent to what is currently the preferred technique. , that is flow cytometry, with all the advantages linked to the possibility of performing high-content analyzes, not possible with flow cytometry.

The method according to the present invention is also shown to be capable of providing important results, which cannot be obtained with flow cytometry. At first, it was observed that from the mixture of Raji tumor cells and PMBC it was possible, thanks to the CD38 labeling, to stain and selectively observe tumor cells. To this end, Raji and PBMC tumor cells were labeled with CAM and an anti-CD38 Alexa Fluor 488 antibody 1:10, separately. A portion of the cells was then mixed in a 1: 1 ratio. Raji cells, PMBC, and the mixture were then plated in three different channels of the inverted open microwell system and then observed. As shown in Figure 13, tumor cells, highlighted in A for selective staining with anti-CD38 antibody compared to total cells stained with CAM, are easily highlighted by the method of the present invention not only for the fluorescence associated with the specific marker ( panel C) but also for morphological parameters (panel B and D)

The acquisition of time-lapse images has the advantage of providing a precise analysis of each cellular response to stimuli represented, for example, by pharmacological treatment, where the precise analysis is obtained through the evaluation of the relative variation of the fluorescence which, in case of staining with a vital dye, it provides a means of determining the cellular response. Even where the initial staining is not uniform, the signal emitted by each cell is thus normalized to its initial value. In comparison, the analysis of absolute fluorescence after application of a treatment, which is typically provided by flow cytometry, gives a less accurate result in this case.

For example, Figure 14 compares the distribution obtained by dynamic analysis or relative fluorescence variation, in A, towards the distribution of the data obtained in absolute terms, in B. The first distribution in Figure 14A was obtained according to the method described here on Raji cells after exposure to Rituximab. Cells showing less signal variation represent a sub-population of cells that are possibly resistant, or that have suffered less damage. Using this distribution, those cells for which the variation was less than a 45% decrease, indicated with a star (*) in the figure, were selected and analyzed within an analysis that reports the variation in terms absolute (Figure 14B). In particular, in this figure the columns marked with a star indicate the presence of at least one cell identified as resistant by the previous analysis. As shown in Figure 14B, absolute analysis does not provide a means of identifying cells with low damage with the same accuracy as the method reported in Figure 14A, as evidenced by the fact that there is a column on the left side of the histogram showing a subgroup of cells having minimum absolute intensity but which are not classified as resistant according to the relative analysis, in fact the column is not marked with any star. Similarly, on the right side of the graph, there are cells that despite having a high signal intensity have shown an important signal loss and which, therefore, must be considered sensitive to therapy according to the relative analysis.

Example 2: automated marking process, time-lapse analysis

Cells are loaded into 2 microwells of an inverted open microwell system. The medium in which said cells were contained is then eliminated with a series of repeated washes, where said washes occur by making a washing fluid, for example HBSS, pass through said microchannels 3. Images are acquired at successive times during said washing operations and panel D of Figure 15 shows images acquired at time 0 and after 3, 5, 7, 8, 10 and 12 minutes from washing. In the graphs shown in panels B and C of the same Figure 15, the curves indicate the reduction of the background noise obtained thanks to these washes. In panel A, the procedure according to the method described here is compared with a standard method, which requires successive centrifugations. In particular, the continuous line marked by the diamond indicates the result over time with the method according to the present invention, where a 79.1% decrease in the background noise can be observed after 5 minutes of continuous infusion of the washing solution at a rate of 16 µl / minute. The dotted line represents the result obtained by the standard centrifugation process, where the result obtained after 4 wash cycles separated by 5 minutes of centrifuge at 1000 rcf is reported. The solid line marked by the triangle shows the result obtained after 2 washing cycles separated by 5 minutes of spinning at 1000 rcf. The method according to the present invention allows to obtain comparable results, with the obvious advantage of not requiring laborious centrifugations which inevitably involve a loss of biological material, a loss which in some situations can be significant to the point of compromising the analysis itself. , as well as exposure of the biological sample to additional stresses.

Example 3: Evaluation of the efficacy of OKT3 on T lymphoblasts

Cells of the Jurkat cell line (human leukemic T cells) were obtained from ATCC and cultured in RPMI 1640 medium supplemented with 10% inactivated FBS, 10 mM Hepes, 1 mM Sodium pyruvate, 2 mM Ultraglutamine and 1% Penicillin / Streptomycin, in flasks of 25 cm <2> at 37 ° C and 5% CO2.

OKT3 hybridomas were grown in bioreactors (CellLine, Integra Biosciences) in RPMI 1640 complete medium containing 3% FBS and the supernatant was collected twice a week. Purification of monoclonal antibodies was performed by double precipitation in 30% and 50% saturated ammonium sulfate, followed by dialysis in PBS. The purity of OKT3 was evaluated by SDS-PAGE gel and the amount of antibody was determined by NanoDrop (Thermo Scientific). The antibody was then frozen at -80 ° C before use.

For comparative purposes, the measurement of the calcium signal on entire populations of Jurkat cells was performed following the Fluo-4 protocol (Life Technologies). 1x10 <6> Jurkat cells were washed once in HBSS and incubated with Fluo-4 (4 ng / µl) for 30 minutes at 37 ° C. After washing and resuspension in HBSS, the cells are treated with the purified OKT3 antibody at concentrations of 1, 5 and 10 µg / ml or, alternatively, using 50 μl of supernatant taken from the hybridoma culture. As a positive calcium flow control, Ionomycin (Sigma-Aldrich) 2 μg / ml was added. The cells thus treated were then analyzed on a flow cytometer.

For the open microwell assay according to the present invention, Jurkat cells were labeled with Fluo-4 20µM in HBSS, centrifuged and resuspended in serum-free medium (RPMI 1640 with Hepes 25 mM or HBSS) at a concentration of 1.6 x 10 < 6> cells / mL in 1.5 mL Eppendorf tubes used as inlet reservoirs for the inverted open microwell system. After depositing the Jurkat cells in the microwells (approximately 1-10 cells per microwell) and washing the channels with HBSS, the solutions containing Ionomicin (2 µg / ml) or OKT3 (10 µg / ml), are made to flow in specific microchannels of the open microwell system by peristaltic pump. The images were acquired by a camera connected to the microscope at a distance of 1 minute and processed by software developed in LabView language, quantifying the dynamic trend of the signal itself on each cell of the sample.

Results on the flow cytometer: the trend of the average fluorescence of the sample following the injection of Ionomycin (a) or OKT3 (b) into the sample was quantified by the flow cytometer, obtaining the results shown in Figure 16. The graphs show the variation of the average of the fluorescence intensity and confirm the functioning of the model used.

Background signal change in open microwells: The change in signal emitted by Fluo-4-labeled single cells in the absence of stimulation was quantified after loading the cells into the open microwell system. The analysis reported a signal increase of 38%.

Stimulation with Ionomycin in open microwells: stimulation with Ionomycin showed an average increase in signal, compared to the baseline value, equal to 193% / - 139% in a series of 10 experiments. The typical trend observed is that shown in Figure 17 (a). As observed also with the flow cytometer, in case of stimulation with ionicin, following an initial increase in fluorescence, the signal then settles on stable high values.

Pacing with OKT3 reported a peak signal increase from baseline averaging 178% / - 90% in a series of 3 experiments. The typical trend observed is that shown in Figure 17 (b). As also observed with the flow cytometer, in case of stimulation with OKT3, following an initial increase in fluorescence, the signal reached the peak value within a few minutes.

The comparative experiment demonstrates how the method according to the present invention allows to have data comparable to those obtained with the preferred method which is flow cytometry.

Claims (8)

CLAIMS 1. A discharge region (70) comprised in a microfluidic device (1) which comprises: a discharge container (12) in fluidic connection with said microfluidic device (1) via at least one discharge channel (22) and a discharge port exit (10). The discharge region (70) according to claim 1, wherein said microfluidic device (1) comprises at least one microchannel (3) and said discharge channel (22) emerges from said at least one microchannel (3) and is a siphon. The discharge region (70) according to claim 1 or 2, characterized in that the diameter of said discharge channel (22) is such that the siphon exerts a capillary force on the fluid contained in said micro-channel (3). 4. The unloading region (70) according to one of claims 1 to 3, where said unloading channel (22) is placed on the bottom of said at least one microchannel (3) and is practically orthogonal to it and connects said at least a microchannel (3) with said discharge container (12) which is positioned below the same microchannel (3). The discharge region (70) according to one of claims 1 to 3, where said outlet door (10) is placed on the bottom of said at least one microchannel (3) and enters a first discharge container (12a), positioned below said microchannel (3) and from said first discharge container (12a) emerges said discharge channel (22) which leads into said discharge container (12). The unloading region (70) according to one of claims 1 to 5, where said unloading channel (22) has a cross-sectional area of micrometric dimensions, said dimensions being between 100 µm and 5mm, preferably between 500 �m and 2 mm. 7. A method for loading / unloading fluids from a microfluidic device where said method comprises: a) Provide a microfluidic device (1) which comprises an inlet region (8) and an unloading region (70) according to one of claims 1 to 6, wherein said microfluidic device (1) is loaded with at least a first fluid; b) Optionally, exert a pressure on said at least one first fluid entering said microfluidic device (1); c) Alternatively, having a discharge region (70) where said discharge channel (22) has a diameter such as to allow said fluid to pass from said microfluidic device to said discharge channel by capillarity; characterized in that, where the volume V of said at least one first fluid is less than or equal to the volume of said discharge container (12), said at least one fluid reaches said discharge container (12) in a unidirectional manner. 8. The method for loading / unloading fluids according to claim 7, where said method also comprises: -injecting a second or further fluid through said inlet region (8) into said microfluidic device (1), wherein said first, second and / or further fluids are independently the same or different from each other and are selected in the group comprising liquids and gases ; - optionally, said second and / or further fluid completely replaces in said microchannel (3) or said microchannel (3) and in said inverted open microwells (2), where present in said microfluidic device (1), the fluid previously introduced ; characterized in that said first, second and / or further fluid do not mix with each other.