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We present the development and application of a microfluidic device that can efficiently and reproducibly isolate a programmable number of cells into microwells. The chip consists of a 3x4 array of 100 µm microwells drilled through a flex-PCB substrate along microchannels. Negative DEP focusing of particles is used to axially centre particles within the channel and also to shield the microwell entrance for flowing particles. An optical detection routine analyses and selects the cells to be delivered with single cell resolution. Cell viability results based on a calcein release assay performed on single cells are within the range of physiological loss of fluorescence intensity.

We present a novel method for the isolation, functional analysis and sorting of single cells or small cellular aggregates. In- verted open microwells featuring a microchannel on top, an open air-fluid interface on bottom side and embedded electrodes were implemented on flexible-PCB technology. Microwell arrays in pitch with standard microtiter plates were implemented. K562 cells were delivered to the microwells and trapped at the air-flow interface. Cell delivery and aggregate formation were controlled by dielectrophoresis. Continuous fluid replacement around the cells during the experiment was demonstrated. After analysis, live cells were easily recovered to standard plates where cell growth was demonstrated.

We present here an innovative architecture based on negative-dielectrophoresis (nDEP) to perform the controlled isolation and patterning of a programmable number of cells inside microwells. The high efficiency solution we propose features electrodes placed on the floor and sidewalls of the microfluidic channels where microwells are realized and allows for the successful isolation, patterning and live-cell imaging of both single cells and cell clusters, with single cell resolution. Cell viability was assessed after delivery performing a calcein release assay in the microwells.

In this work we present a method for delivering small fluid volumes possibly containing cells and particles over a flexible large area substrate. Particles are deposited by a dispensing device that performs serial delivery of liquid volumes in the range of 200 pL. Due to the required precision, the alignment and the calibration of the spotting system are crucial for the proper performance of the delivery process. The system provides automated motion of mechanical parts with micrometric precision. To address the problem of the device deformation caused by the flexibility of the substrate, the 3D coordinates of each site need to be known, but the serial acquisition of all the coordinates would be highly time consuming for high-throughput arrays and large devices. We solved this problem by sampling the coordinates of a small subset and interpolating the positions of the remaining sites by means of a custom algorithm. System calibration for%2

In this article we present an impedance-based measurement technique for single-cell detection within open microwells. It is used for continuous monitoring of cells delivered in open-microwell arrays which enable simplified cell recovery procedures.

In this work we present a novel concept of active microwells based on cylindrical wells able to vertically trap and control single particles by means of negative dielectrophoresis. The device is fabricated by drilling through holes on a polyimide substrate with copper-gold or aluminum metals, forming three annular electrodes within the well. A channel under the device provides a fluid flow filling the microwell by capillarity. Particles are delivered from the top by a microdispenser and applying sinusoidal signals to the electrodes at frequencies ranging from 100kHz to 1.5MHz and amplitudes between 2V and 7V they are successfully trapped and levitated at the level of the central electrode in the middle of microwells with a diameter of 125mum. By changing signal phases, other configurations are also enabled to load particles in the well or eject them from the bottom. The extension to an array of microwells is presented and design rules are described for routing electrode connections and setting signal parameters. K562 cells cultured with Ara-C 1000nM were successfully trapped and controlled in physiological media. Polystyrene beads were also levitated in water and were used for experimental measurements on minimum amplitudes and phase differences in the signals required to levitate beads, confirming the results obtained by simulation.

There has been a fast progress in clinical use of antibody-based immunotherapy, given the superior efficacy commonly achieved in clinic and the limited toxicity. However, personalization of treatment remains of major importance, both to achieve better clinical performance for monotherapy and to identify the best combinations on a patient-by-patient basis. Predicting patient's response is complex due to the need to characterize both tumor response and immunologic mechanisms possibly activated by the therapy, including antibody dependent cellular cytotoxicity (ADCC). We present the Inter-Cell Networking Profiling (ICNP), a novel analytical method enabling a comprehensive and precise characterization of the modulatory effect of immunotherapies on immune-tumor cell interactions. ICNP works on the Open Microwell (OMW) microfluidic system which recreates 20,000 unique cell clusters from ex-vivo patient samples and exposes them to anticancer drugs. We validated the ICNP using multiple myeloma (MM) patient samples to characterize the efficacy of Daratumumab, an anti-CD38 antibody (Ab). Bone marrow samples in EDTA were collected from 11 MM patients. 8 samples were processed by Ficoll-Paque, preserving the original composition of effector (E) and target (T) cells, i.e. NK and plasma cells respectively. 3 samples were processed to obtain co-cultures of WBC depleted of plasma cells (which include NK cells) and U-266 MM cell line. NK and plasma cells were stained with anti-CD16/CD56 and anti-CD138 fluorescently-labeled Abs, respectively. Propidium Iodide (PI) was used as death marker. A statistical model was created to project the optimal experimental setup (E:T ratio, cell concentration) to maximize the co-localization of E/T cells in the 20,000 microwells of the OMW platform. After seeding, cells were incubated with Daratumumab 10µg/mL or no drug and analyzed through fluorescence time lapse microscopy for up to 12 hours. ICNP analysis first separates microwells with both E/T cells in close proximity from those not featuring both cell types (Fig 1A). Then, anti-CD38 efficacy is evaluated in microwells with E/T co-localization, thus implementing a miniaturized ADCC assay (Fig 1B). At the same time, spontaneous NK activity is measured in microwells with E/T co-localization and no drug, while direct effect of the drug on target cells can be measured in microwells with T but not E cells. We first validated our statistical model of co-localization in microwells against the actual number of wells with E/T co-localization (correlation R2: 0.79-0.97, n=5). Then, we characterized the immune composition of 8 MM patients samples with the OMW, finding that E/T cell fractions were in the ranges 5-21% (E) and 1-28% (T). Interestingly, according to our statistical model, co-localization occurs in at least 1% of microwells for all the 8 samples, making ICNP applicable with good statistical significance in the OMW system on ex-vivo clinical samples without any pre-enrichment. Finally, ICNP was evaluated on 4 cases (3 obtained by mixing patient's WBC with U-266 MM cell line and 1 complete MM patient sample). We measured the effect of anti-CD38 Ab using i) the standard approach, considering all microwells regardless of the co-presence of E and T; ii) ICNP approach, considering only microwells featuring E/T co-localization. Results show that drug effect is much evident with ICNP in all the 4 cases with an average increase in target cell death of 40%, indicating a higher sensitivity of this approach than the averaged analysis (Fig 1C, right). Importantly, in one case, the standard analysis did not identify significant differences between anti-CD38 treated and control cells, that could instead be observed with ICNP (p ICNP proved to enable a comprehensive profiling of the immune system by evaluating in one test the immune composition and the fitness of immune cells, both native and drug-treated. These results open the opportunity to develop functional precision medicine approaches for predicting patient's response to drugs with immune-mediated mechanisms of action. AB and RR equally contributed Figure 1 Disclosures Bettelli: CellPly.S.r.l.: Employment. Ruggiano:Cellply S.r.l.: Employment. Bocchi:CellPly S.r.l.: Employment. Rocchi:CellPly.S.r.l.: Employment. Faenza:CellPly S.r.l.: Employment. Zamagni:Janssen: Honoraria, Other: Advisory board, Speakers Bureau; Amgen: Honoraria, Other: Advisory board, Speakers Bureau; BMS: Honoraria, Other: Advisory Board, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Sanofi: Honoraria, Other: Advisory Board, Speakers Bureau; Celgene Corporation: Honoraria, Other: Advisory board, Speakers Bureau. Bettelli:CellPly S.r.l.: Employment. Rambelli:CellPly S.r.l.: Employment. Guadagnuolo:CellPly S.r.l.: Employment. Pecorari:CellPly S.r.l.: Employment. Giulianelli:CellPly S.r.l.: Employment. Pisani:CellPly S.r.l.: Employment. Biscarini:CellPly S.r.l.: Employment. Cavo:amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Guerrieri:CellPly S.r.l.: Equity Ownership. Bocchi:CellPly S.r.l.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Background: Patient stratification to match individual patients with the most effective drug treatment is still a major open challenge in cancer care. For instance, cytarabine is the main drug used for AML treatment but 30% of patients fail to respond to this agent. Laboratory developed tests determining ex-vivo cellular response to cytotoxic anticancer drugs have demonstrated good correlations with clinical response, sometimes surpassing the predictive power of molecular and genetic profiling. Standardizing sample processing to remove operator-dependent biases and maintaining live cells in a functional status that closely resembles in-vivo function are major challenges affecting these tests. Here we present the open microwell (OMW) platform, a microfluidic-based system that integrates the entire process of ex-vivo testing of anticancer drug efficacy and enables drug testing in the clinical setting prior to therapy administration. The concept was validated for the first time on 13 AML patients at Sant'Orsola hospital, Italy, showing the possibility to initiate the analysis readily after sample collection, thus minimizing drifts in cell function that typically start occurring within hours from sampling, and provide results in about 24 hours, with a fully-automated system. Methods: 13 refractory, relapsed or newly diagnosed AML patients were enrolled in the study. 2 ml of fresh bone marrow in EDTA and 1 ml of serum blood were collected from each patient. White blood cells (WBC) were separated from bone marrow by standard Ficoll-Paque, suspended in medium additioned with 2% autologous serum and loaded in microfluidic chips featuring 16 microchannels and 1200 microwells/channel (70 μm diameter), open at the bottom end (Fig. 1A-B). After settling down in microwells, cells were stained with CMAC cell tracker, anti-human-CD34/CD45 fluorescently-labeled antibodies and Propidium Iodide (PI) by injecting the reagents in the microchannels. Cytarabine or combination therapies (FLAI-3, FLAI-5, FLA, MEC-4) were then injected in the microchannels and cells were incubated for 24 hours in the system (Fig. 1C). Four channels were used per therapeutic condition, including reference (ref), high (ref x 10) and low (ref/10) dosages plus a non-treated channel as reaction control. Finally, a custom software was used to detect cells in images, classify AML blasts and analyze cell death (Fig. 1D). Drug efficacy was determined by evaluation of both cell depletion and apoptosis induction. Results: We first validated the OMW platform against gold standard (FacsAria flow cytometer) for the detection of tumor cells and measurement of cell viability and apoptosis. Count of blast frequency was carried out on 5 patient samples using anti-CD34/CD45 staining. Results (Fig. 2B) show a correlation between the gold standard and OMW (n=5, R2=0.99, p Conclusion: The OMW platform was able to count AML blasts and analyze viability and apoptosis showing high concordance with conventional diagnostics represented by flow cytometry. The assay requires 30 μl of bone marrow sample and simple manual pre-processing. Profiling of patient response to specific drugs or combination is provided within 24h. These features make the OMW platform suitable for bedside analysis, to evaluate drug activity on tumor cells within a heterogeneous sample and promptly guide personalized treatment in hematologic malignancies, with a first proof achieved on AML patients. LRo and AF equally contributed. Disclosures Rocchi: CellPly S.r.l.: Employment. Faenza:CellPly S.r.l.: Employment. Rambelli:CellPly S.r.l.: Employment. Guadagnuolo:CellPly S.r.l.: Employment. Pecorari:CellPly S.r.l.: Employment. Giulianelli:CellPly S.r.l.: Employment. Biscarini:CellPly S.r.l.: Employment. Martinelli:Novartis: Speakers Bureau; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Celgene: Consultancy, Speakers Bureau; Genentech: Consultancy. Guerrieri:CellPly S.r.l.: Equity Ownership. Bocchi:CellPly S.r.l.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.