IT202300008451A1 - Method for characterization of cell therapy products - Google Patents

Description

DESCRIPTION

Of the Patent Application for Industrial Invention Titled:

?Method for the characterization of cell therapy products?

State of the art

Assays commonly used to characterize and understand the mode of action of cell therapies, to identify optimal effector cells, or to monitor the potency of cell therapies during process development or product release are cytotoxicity and cytokine release assays that reveal interactions between immune cells and target cells. These tests can typically be performed for 4-72 hours.

Cytotoxicity assays used in cell therapy rely on the ability to maintain the viability of target cells over the long term. It is therefore essential to maintain ideal conditions and minimize measurement noise due to unwanted and unrelated death of target cells, while at the same time ensuring that effector cells are maintained in an environment where they can exert their cytotoxic activity against target cells, with minimal disruption of the surrounding environment.

Currently available methodologies do not allow for the verification of functional heterogeneity of cell therapy products. In fact, bulk assays are used, from which information is derived that is given by the average of the cell population being analyzed, without the possibility of tracing the cytotoxicity of each individual effector cell. Flow cytometry assays define subpopulations on the basis of phenotypic markers, not on the basis of functionality.

Microfluidic devices are available as cutting-edge tools that enable high-content analyses at scale. Working with nanoscale volumes, while offering unique advantages such as the ability to colocalize single effector cells with multiple target cells and measure cytokines secreted by individual cells or to successively perform multiple assays on the same cells over extended periods, requires the highest level of control of the local environment to minimize target cell death due to factors unrelated to effector-target interactions.

There remains a strong need for a method that can characterize a cell therapy product with specific single-cell information.

Description

The present invention relates to a method suitable for cell-mediated cytotoxicity tests.

In one embodiment, the method is performed in an open microwell microfluidic device, which allows for rapid automated exchange of liquid in the wells without displacement of the loaded suspension cells. This capability allows for automation of the preparation and execution of the assay in the previously loaded sample wells and for feeding the microwells with nutrients at desired time intervals, thus achieving periodic perfusion of the medium.

Description of the drawings

Figure 1: Exemplary schematic 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: Workflow showing (A) seeding of cells in microwells, (B) integrated staining of cells, (C) perfusion of channels with culture medium supplemented with propidium iodide (PI), (D) incubation without perfusion. Steps (C) and (D) can be repeated to generate periodic perfusions multiple times and at specific times.

Figure 3: Cell viability of KG-1 (A) and K562 (B) at 72 hours. While for K562 cells the data do not show statistically significant differences between conditions, for KG-1 the 24-hour periodic perfusion preserves cell viability by limiting changes to the environment surrounding the cells. (C) Kinetics of KG-1 and K562 cell viability and cell count in the assay at 72 hours. Data refer to the 24-hour perfusion condition.

Figure 4: Image sequence of a KG-1 cell line showing that cell viability is maintained as well as the persistence of the CMAC marker, with an increase in cell number over time due to proliferation. The PI marker is not visible as no cell death was observed in this microwell.

Figure 5: Cell viability measured after 72 hours in culture, measured by varying reperfusion frequency. Solid line: target cells only. Dashed line: target cells co-cultured with effector cells.

Figure 6: (A) Representative image of a set of microwells visualized by fluorescence and phase contrast microscopy. Arrows indicate the effector cell, highlighted by Calcein AM staining and CD16/CD56 marker. Wells containing a single effector cell, framed in the figure, are selected for analysis whose results are reported in the graph. (B) Graph showing the % of effector cells that are able to kill the indicated number of target cells.

Figure 7: Representative image of a set of microwells containing (A) a non-active effector cell or (B) an active effector cell. The effector cell is highlighted by the arrow.

Figure 8: Quantification of effector cell potency obtained from D1-D6 donors. (A) effector cells killing 1 or 2 target cells; (B) effector cells killing 3 or more target cells; (C) effector cell potency derived from the results in graphs (A) and (B).

Figure 9: Potency of effector cells from D1-D3 donors.

Figure 10: Potency on two different phenotypes of effector cells (A) obtained from DA donor; B) obtained from DC donor.

Detailed description

Definitions

Interference coupling here means a cooperation between two elements, whereby said two elements can be considered as joined. When said two elements, in this case a tip and a vertical channel, are coupled by interference, a fluid loaded into said tip and released into said vertical channel is forced to move inside the channel, said interference coupling being such as to prevent the passage of fluid, or said interference coupling is such as to seal the two elements together.

By connector we mean here any tubular, cylindrical, more or less tapered, convergent or divergent element capable of connecting two compartments in a fluidic manner.

Re-perfusion here means replacing the medium in which the culture is located with a fresh medium. Said fresh medium is either the same or different from the medium in which said culture is already located. In one embodiment, said medium is culture medium. In one embodiment, it is culture medium that comprises one or more drugs and/or one or more dyes, and/or one or more labeled antibodies and/or one or more cell viability markers.

Fluids: Any substance in liquid or gaseous form.

Biological sample: A sample comprising cells obtained from a microorganism, an animal and/or a human being, preferably human, wherein said sample is preferably selected from the group comprising biological fluids or biopsies. Said sample comprises suspended cells or is a tissue. In a preferred embodiment, it is a blood sample or a bone marrow aspirate. Alternatively, said biological sample is composed of cultured cells, such as a cell line, or a composition comprising cultured cells and cells from a patient.

High content assay: phenotypic assay conducted on cells.

Time-lapse: An imaging technique that involves a series of shots of the same field taken in a time sequence.

Ex vivo: test performed on tissue obtained from an organism in an environment external to the organism itself, with minimal alteration of the natural conditions. The object of the present invention is a method suitable for long-term cell-mediated cytotoxicity tests, i.e. culture longer than 24 h, or 48 h, preferably 72 h.

In one embodiment, the method is performed in the open-well microfluidic device described in WO2017/216739.

In one embodiment, the open microwells are approximately 0.5 nL open microwells.

Advantageously, the open microwell interface provides a source of gas exchange that contributes to long-term viability.

In one embodiment, the method claimed herein is performed by having a kit comprising a tip and a microfluidic device (1), Figure 1, which comprises at least one microchannel (3) and an inlet region (8) comprising at least one vertical channel (18), said tip and said vertical channel (18) being sized to produce an interference fit between them.

Said tip is selected from among commercially available tips that comprise at least a proximal portion intended to cooperate with a fluid delivery system and an open tapered distal portion.

Preferably, said distal portion of said tip and said vertical channel (18) are made of plastic and make the system sufficiently elastic to guarantee the seal, avoiding gaskets. In a particularly preferred embodiment, the geometries of the system described below ensure that the contact between said vertical channel (18) and said tip does not occur at a single point but is distributed over a surface portion, also ensuring an effective seal. This condition is advantageously verified where the semi-opening angle of said terminal portion of said tip and of said vertical channel (18) are slightly different, preferably less than 10?. Even more preferably, said vertical channel (18) is a cylinder, possibly slightly tapered towards the bottom.

The loading/unloading mode of fluids in the microfluidic device (1) included in the kit described includes the following phases:

a) Provide a kit as described herein;

b) optionally, load a fluid into said tip;

c) Position said tip above said inlet region (8) and insert it until it reaches an interference fit position between said distal region of said tip and said vertical channel (18) in said inlet region (8);

d) Release the fluid contained in said tip into said microfluidic device (1) through said inlet region (8) or, alternatively, with the same tip, aspirate fluid already contained in said microfluidic device.

Also disclosed is a microfluidic device, which is an inverted open microwell system comprising an open microwell array (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) therefor, 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 into an automated management system comprising the following features: a temperature, humidity and CO2 controlled incubator, fluid delivery system, phase contrast and fluorescence image acquisition.

Said automated management system is obtained by assembling elements known in the art such as incubators for the control of temperature, humidity and CO2, pipetting systems for microplates, lenses for fluorescence and phase contrast microscopy connected to an image acquisition camera, such as a CMOS or CCD camera, where said elements are managed in whole or in part by software known to those skilled in the art via hardware connected to them. In a particularly preferred embodiment, each microchannel (3) is associated with an input port (8) and an output port (10).

In a preferred embodiment, the microfluidic device (1) also comprises reservoirs, wherein said reservoirs are at least one reservoir for reagents and at least one reservoir for one or more biological samples. Said reservoirs are selected from the group comprising: plates, one or more multiwell plates, such as 96-well plates, Eppendorf tubes. Said reservoirs may be 2, or 4, 8, 16, 24, 48, 96, 384.

In one embodiment, the method according to the present invention comprises:

- Make available at least two cell populations, where at least a first cell population comprises effector cells growing in suspension, at least a second cell population comprises target cells;

- Co-cultivate said cells for at least 24h, for example 48h or 72h, periodically re-perfusing the culture, where said reperfusion occurs once in a period of time between 1 and 30 hours, for example once every two hours, or once every 12 hours or once every 24 hours;

- Evaluation of cell viability.

In one embodiment, said method is conducted in a microfluidic system.

In one embodiment, said method is performed in an inverted open microwell system (1) comprising an array 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).

In one embodiment, said microfluidic device comprises 16 microchannels (3).

In one embodiment, 1,200 open microwells (2) are connected to each of said microchannels (3).

In one embodiment, said inverted open microwell system is managed by an automated management system that includes the following features: temperature, humidity and CO2 controlled incubator, fluid delivery system, phase contrast and fluorescence imaging acquisition.

In one embodiment, said method comprises:

- Providing an inverted open microwell system (1) comprising an array 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) therefor, 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 micrometer dimensions and supplies fluid to said microwells (2);

- Provide an automated management system for said inverted open microwell system that includes the following features: temperature, humidity and CO2 controlled incubator, fluid delivery system, phase contrast and fluorescence imaging acquisition.

- Place said inverted open microwell system (1) in said automated system;

- Load reagents through one or more of said input ports (8), wherein said reagents comprise: filling buffer and/or washing solution and/or one or more drugs and/or one or more dyes, and/or one or more labeled antibodies and/or one or more cell viability markers;

- Load said at least two cell populations,

- Staining of said cells with one or more dyes and/or one or more labeled antibodies and/or one or more cell viability markers (Figure 2B);

- Acquire images from one or more of said microwells (2), at a time point T0;

- Periodically re-perfuse the culture with fresh media, optionally including one or more drugs and/or one or more dyes, and/or one or more labeled antibodies and/or one or more cell viability markers (Figure 2C-D);

- Acquire images from one or more of said microwells (2), at a time point T1;

in which the aforementioned re-perfusion and image acquisition phases are repeated over time.

In one embodiment, said at least two cell populations are loaded at a concentration of approximately 2x10<6 >cells/mL (Figure 2A).

In one embodiment, one of said at least two cell populations is an effector cell population.

In one embodiment, at least 400 of the 1200 microwells (2) connected to each microchannel (3) are loaded with a single effector cell.

In one embodiment, one of said at least two cell populations is KG-1, a human macrophage cell line.

In one embodiment, one of said at least two cell populations is K562, a human lymphoblast cell line.

In one embodiment, said effector cells are NK lymphocytes.

In one embodiment, said effector cells are T lymphocytes.

In one embodiment, said T or NK cells are genetically modified.

These effector cells are co-cultured in the presence of target cells. These target cells are primary cells obtained from a subject, or are a cell line.

In one embodiment, said target cells express the antigen recognized by said effector cells.

In one embodiment, the test and time lapse are performed for up to 72 hours.

In one embodiment, said marker is used for cellular tracking and is 7-amino-4-chloromethylcoumarin (CellTracker? Blue CMAC).

In one embodiment, said marker is used to assess cell death, and it is propidium iodide (PI).

In one embodiment, said marker is Calcein AM.

In one embodiment, homogeneous co-cultures are selected, that is, those microwells containing a single effector cell and target cells in a number within a defined range are selected. For example, with reference to Figure 6A, microwells containing a single NK effector cell are selected, highlighted thanks to Calcein AM staining. In these microwells, dead target cells are counted, stained with PI. This allows to define the % of effector cells capable of killing 0 target cells, 1, 2, 3, 4 or more than 5 target cells (Figure 6B). From this profile, the so-called potency score is calculated, consisting of the weighted average of the number of target cells killed by each effector cell, having first subtracted from the count the number of average dead target cells in microwells that do not contain any effector cells.

Where ikj indicates the number of target cells killed by the effector cell /, normalized to the spontaneous death of the same target cells in the absence of effector cells and N indicates the total number of effector cells.

A further object of the present invention is therefore a method for determining the efficacy of a cell therapy (or immunotherapy), where said method comprises;

a. Prepare a first set of microwells each containing effector cells and target cells;

b. Prepare a second set of microwells, each containing target cells;

c. Select those microwells that include a single effector cell and n target cells, where n is between 1 and 50, preferably between 1 and 20;

d. Maintain the cells in culture for a time t;

e. Measure the percentage of dead target cells in each of the microwells selected to contain a single effector cell;

f. Calculate a potency score consisting of the weighted average of the number of target cells killed by each effector cell, having first subtracted from the count the number of target cells on average killed in the second set of microwells.

In one embodiment, said microwells are inverted open microwells of a microfluidic device as described in the preceding paragraphs.

In one embodiment, effector cells are selected only if positive, or only if negative, for a surface marker which, for example, is selected from the group comprising CD3, CD4, CD4, CD56, CD45RA, CCR7.

In one embodiment, dead target cells are counted only if positive, or only if negative, to a surface marker which, for example, is selected from the group that includes CD38, HLA-DR, CD34. This potency score advantageously allows to represent in an absolute way the efficacy of the therapy.

The potency score defined here is the average number of target cells that a single effector cell is able to eliminate, having tested this activity using a well-controlled replicate of the test conditions, i.e., a known number of cells in a known volume.

An advantage of this approach is that it allows comparisons between different variants of a cell therapy, e.g. different sources of immune cells (donors or patients), different engineering methods, different manufacturing processes. From the data available so far, it is observed for example that a CAR-NK therapy can achieve a score >1 for t=12h or t=24. In another series of experiments it is observed that non-engineered NK cells can typically achieve a score of <1.

Advantageously, the method according to the present invention allows a combined functional and phenotypic characterization.

For example, by being able to observe 4 different colors, up to 2 surface markers can be evaluated in combination with cytotoxicity. In one embodiment, the cytoplasm of the target cell is stained with CMAC (in the blue DAPI channel), and PI is used as a cell death marker (in the yellow TRITC channel). It is therefore possible to use two surface markers, in the green and red channels. In this embodiment, for example, it is possible to select the cell population positive for antibody 1, and in this to identify serial killer cells, killer cells and inactive cells. This allowed us to observe that the presence of a surface marker correlates with an increase in the potency score (in other words, there are subpopulations defined based on the immunophenotype that have potency scores higher than the mean potency score of the parent population). According to the method described here, the viability of the target cells can be be maintained for 72 hours outside the presence of immune effector cells even in the prolonged presence of specific dyes used for cell detection, such as 7-amino-4-chloromethylcoumarin (CellTracker? Blue CMAC) and time-lapse death assessment, e.g. propidium iodide (PI).

The entire setup has been optimized in terms of imaging, fluidics and staining methods to achieve a reagent concentration high enough to allow cells to be detected and classified correctly, without significantly affecting their viability.

The method described here allows these stains to be applied throughout the test to support time-lapse analysis.

In conclusion, the conditions identified here provide a model consisting of target cell lines, a set of dyes, and cell processing methods that warrant downstream use for cell death-based potency assays.

Examples

Example 1: Feasibility analysis conducted on KG-1, a human macrophage cell line, and K562, human lymphoblast cell lines, loaded, processed, and imaged over a time frame of up to 72 hours.

K562 and KG-1 cell lines were loaded separately into multiple microchannels of a microdevice and the experimental workflow steps included:

? cell seeding in microwells at a concentration of 2x10<6 >cells/m L; ? automated preparation/dilution of the cytoplasmic marker, CMAC, at a concentration of 7??;

? automated cell staining by infusing CMAC into microfluidic channels;

? automated immunophenotyping, with automated dilution, preparation and infusion of reagents;

? automated preparation and dilution of the cell death marker, PI, at 5 mM concentration with complete medium (RPMI 10% FBS 1%L-Glu 1% Penstrep);

? automated cell staining by infusion of complete medium supplemented with PI;

? time-lapse imaging in brightfield/phase contrast and 4 fluorescent channels repeated every 12 hours for 72 hours.

Periodic preparation and perfusion of culture media with PI was repeated at specific time points to refresh nutrients and restore the fluorescence intensity of the PI signal. For each reinfusion, 100 µl were injected into each microchannel at a flow rate of 10 ??/s (rinse time: 10 seconds). Repeatability of the assay was assessed by replicating the assay with independent runs on three different days. Cell viability was measured over a time span by AI-based single-cell detection and quantification of the percentage of cells with PI uptake.

The K562 cell line showed a mean viability at the 72-hour endpoint, after normalization to T0, of 90.58% ± 6.8% for a perfusion every 12 hours, 86.24% ± 7.3% for a perfusion every 24 hours and 87% ± 0.44% for the no-rinse condition.

There was no significant difference between conditions (Figure 3B).

The results for the KG-1 cell line, Figure 3A, with perfusion of the culture medium every 12 h or every 24 h showed a mean viability at the endpoint of 72 h, after normalization to T0, of 80.96% ± 8.19% and 87.54% ± 3.36%, respectively. For the same cell line, the viability at 24 h without perfusion of the medium was 96.71% ± 2.36% demonstrating an optimal state of health. However, cell viability decreased over time to reach a viability of 61.11% ± 14.16% at 72 h, thus demonstrating the significant benefit brought by periodic perfusion of the medium.

Between the two perfusion strategies we tested, there was no statistical difference; therefore, the preferred approach is to rinse every 24 hours to minimize changes in the cellular environment.

In comparison, other microtechnologies (Halldorsson et al., Biosensors and Bioelectronics, 2015, 63: 218-231) commonly require continuous perfusion to maintain ideal conditions while the method according to the present invention preserves cell viability by applying short and fast periodic perfusions. As reported in Figure 3C, these conditions also support cell growth inside the microwells and, as visible in Figure 4, the CMAC cell tracker propagates to daughter cells and remains visible for the 72-hour period.

Figure 4 shows the results of a co-culture experiment, compared with a single culture experiment.

The target cells were co-cultured with effector cells (according to the invention) or by themselves.

Cell viability was assessed after 72 hours of incubation.

Different microwells were exposed to different re-perfusion frequency where the frequency ranged from once every hour to once every 30 hours.

The solid line in the graph represents target cells in the absence of effector cells. The dashed line, target cells co-cultured with effector cells.

In the absence of re-perfusion, a high % of cell death is observed in both experimental settings, showing the need to change the medium to maintain a healthy cell population for up to 72 hours.

By increasing the re-perfusion frequency, the cells are healthy. At the extreme, re-perfusion makes the effect of co-culture questionable.

Surprisingly, a "working range" is demonstrated here, i.e. an optimal re-perfusion frequency to maximize the possibility of measuring the effect of effector cells on target cells, without it being influenced by suboptimal culture conditions. This "working range" is the frequency at which the maximum difference between the two experimental settings is observed. Effector cells cultured alone or in the presence of target cells show a viability pattern comparable to that observed when target cells are cultured alone.

Example 2: characterization of 6 cell therapy lots, lots D1-D6.

Three microfluidic devices, I, II and III, respectively, each comprising 16 microchannels, were provided. Each of said microfluidic devices was loaded with effector cells obtained from donors, said cells being CAR-NK. In particular, 7 microchannels of each device were loaded with effector cells from one donor, 7 with effector cells from a different donor. 6 of said channels loaded with effector cells were also loaded with target cells, while the seventh was kept as a control channel containing only effector cells. The target cells used were Raji cells, a cell line obtained from a human lymphoma. 12 remaining channels on each device were loaded only with target cells, as a negative control.

In the co-culture microwells, effector cells were loaded in a 1:10 ratio with respect to target cells. For cell tracking, target cells were stained with the CMAC marker. Effector cells were instead visualized in bright field, after evaluating that the CMAC and Calcein AM markers, which do not impact the cell viability of Raji cells, instead induced a share of cell death in CAR-NK.

Through an automated process, the effector cells contained in each well were counted, thus arriving at selecting a total of 1079 wells containing a single effector cell each. These 1079 microwells were selected for the subsequent steps. The target cells contained in the same wells were then counted.

Note that these results were achieved in terms of plating by seeding in each microwell 90 µl of medium where CAR-NK cells were suspended at a concentration of 3 * 10<6 >cells/ml and 330 µl of medium where Raji cells were suspended at the same concentration of 3 * 10<6 >cells/ml. In the control microwells, 300 µl of medium where CAR-NK cells were suspended at a concentration of 1.2 * 10<5 >cells/ml or 1200 µl of medium where Raji cells were suspended at a concentration of 3 * 10<6 >cells/ml were seeded.

By staining with PI, cell death was then measured after 24 hours of incubation of the co-culture.

For illustrative purposes, images of some microwells are shown in Figure 7. In panel A, microwells are shown where the CAR-NK cell present is not active, as evidenced by the absence of dead target cells. Conversely, panel B shows microwells exemplifying the presence of serial killing CAR-NK cells, where, proceeding from left to right, one goes from 1 dead target cell per well to over 5.

Advantageously, the rapid automated liquid exchange system in the wells allows re-labeling with PI every 12 hours, thus compensating for the natural loss of labeling.

The results obtained were reported in a graph, shown in Figure 8. For each donor, it is reported how many NK cells kill 1 or 2 target cells (A), how many NK cells kill 3 or more target cells (B). Finally, panel C shows, for each donor, the potency score, defined according to the present invention. The Potency score identifies the donor D4 as the most potent, being the one with the highest Potency score.

Example 3: characterization of 3 allogeneic cell therapy lots, lots D1-D3.

NK cells from 3 different donors and target cells were loaded onto the same 16-channel microfluidic device. The target cells used were the K562 cell line.

After 24 hours of co-culture incubation, the % of NK cells able to kill 1 target cell, defined as killer NK cells, and the % of NK cells able to kill 2 or more target cells, defined as serial killer NK cells, were assessed.

The results obtained are reported in Figure 9 and show the greater potency of donor D1 compared to the other two donors tested in the same assay.

Example 4: Phenotypic/functional multiparametric analysis

NK cells from donor A and donor C were provisioned and plated in a microfluidic device as described in the examples above.

This NK cell population was co-cultured with a heterogeneous population of target cells, expressing either phenotype A or phenotype B. The potency of the effector cells was then calculated according to the method of the present invention.

As shown in Figure 10, for donor C, panel B, an increase in killing potency on cells having the B phenotype is evident.

Claims (9)

1. A method for determining the efficacy of a cell therapy or immunotherapy, where said method comprises; a. Prepare a first set of microwells each containing effector cells and target cells; b. optionally, prepare a second set of microwells each containing target cells; c. Select those microwells that include a single effector cell and n target cells, where n is between 1 and 50, preferably between 1 and 20; d. Maintain the cells in culture for a time t; e. Measure the percentage of dead target cells in each of the selected microwells containing a single effector cell; f. Calculate a potency score consisting of the weighted average of the number of target cells killed by each effector cell. 2. The method according to claim 1, where to calculate said potency score the number of average dead target cells in the second set of microwells is subtracted from the weighted average of the number of target cells killed by each effector cell. 3. The method according to claim 1, where t is between 0 and 120h, or about 72h, or about 24h. 4. The method according to one of claims 1-3 which is conducted in a microfluidic system, where said method comprises: - Providing an inverted open microwell system (1) comprising an array 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) supplies fluid to said microwells (2); - Provide an automated management system for said inverted open microwell system that includes the following features: temperature, humidity and CO2 controlled incubator, fluid delivery system, phase contrast and fluorescence imaging acquisition. - Place said inverted open microwell system (1) in said automated system; - Load reagents through one or more of said input ports (8), wherein said reagents comprise: filling buffer and/or washing solution and/or one or more drugs and/or one or more dyes, and/or one or more labeled antibodies and/or one or more cell viability markers; - load said at least two cell populations; - staining of said cells with one or more dyes and/or one or more labeled antibodies and/or one or more cell viability markers; - periodically re-perfuse the culture with fresh media, possibly including one or more drugs and/or one or more dyes, and/or one or more labeled antibodies and/or one or more cell viability markers. 5. The method according to one of claims 1-4, where in each microwell of the co-culture are seeded 90 µl of medium where effector cells are in suspension at a concentration of 3 * 10<6 >cells/ml and 330 µl of medium where target cells are in suspension at a concentration of 3 * 10<6 >cells/ml. 6. The method according to one of claims 1-5, where said effector cells are NK lymphocytes and/or T lymphocytes, possibly genetically modified. 7. The method according to claim 1, wherein said cells are cultured for at least 24h, for example 48h or 72h, periodically reperfusing the culture, wherein said reperfusion occurs once in a period of time between 1 and 30 hours, for example once every two hours, or once every 12 hours or once every 24 hours. 8. The method according to claim 4, wherein said microfluidic system comprises 16 microchannels (3). 9. The method according to claim 4, wherein said microfluidic system comprises 1,200 open microwells (2).