Standard Multiwell Plates Research Articles

Robust Surfactant-Assisted One-Pot Sample Preparation for Label-Free Single-Cell and Nanoscale Proteomics.

With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk cells. However, such bulk measurement obscures cell-to-cell heterogeneity, precluding proteome profiling of single cells and small numbers of cells of interest. To address this issue, in the recent 5years, there has been a surge of small sample preparation methods developed for robust and effective collection and processing of single cells and small numbers of cells for in-depth MS-based proteome profiling. Based on their broad accessibility, they can be categorized into two types: methods based on specific devices and those based on standard PCR tubes or multi-well plates. In this chapter, we describe the detailed protocol of our recently developed, easily adoptable, Surfactant-assisted One-Pot (SOP) sample preparation coupled with MS method termed SOP-MS for label-free single-cell and nanoscale proteomics. SOP-MS capitalizes on the combination of an MS-compatible surfactant, n-dodecyl-β-D-maltoside (DDM), and standard low-bind PCR tube or multi-well plate for "all-in-one" one-pot sample preparation without sample transfer. With its robust and convenient features, SOP-MS can be readily implemented in any MS laboratory for single-cell and nanoscale proteomics. With further improvements in MS detection sensitivity and sample throughput, we believe that SOP-MS could open an avenue for single-cell proteomics with broad applicability in biological and biomedical research.

Utilizing primary human airway mucociliary tissue cultures to model ramifications of chronic E-cigarette usage

Electronic cigarettes are battery powered devices that use a vape-liquid to produce a vapor that is inhaled. A consequence of the rise in e-cigarette usage was the 2019 emergence of a vaping-induced respiratory disease denoted as ‘e-cigarette or vaping use-associated lung injury’ (EVALI). One of the suspected causes of EVALI is Vitamin E Acetate (VEA), which was found to be a diluent in certain illicit vape-pens, whereas nicotine is commonly diluted in equal parts propylene glycol and vegetable glycerin (PG:VG). The prevalent use of e-cigarettes and the emergence of a novel illness has made understanding how e-cigarette vapors affect our respiratory tissues a public health concern. We have designed and produced a simple device that can operate e-cigarettes and deliver the vapor to a chamber containing a standard cell culture multi-well plate. Here we utilize our device to model the response of human airway mucociliary tissue after chronic exposure to vapors produced from either PG:VG or VEA. We note several differences between how PG:VG and VEA vapors interact with and alter airway tissue cultures and suggest potential mechanisms for how VEA-vapors can exacerbate EVALI symptoms. Our device combined with primary human airway tissue cultures make an economical and compact model system that allows for animal-free investigations into the acute and chronic consequences of e-cigarette vapors on primary respiratory cells.

Combinatorial drug screening on 3D Ewing sarcoma spheroids using droplet-based microfluidics

Culturing and screening cells in microfluidics, particularly in three-dimensional formats, has the potential to impact diverse areas from fundamental biology to cancer precision medicine. Here, we use a platform based on anchored droplets for drug screening. The response of spheroids of Ewing sarcoma (EwS) A673 cells to simultaneous or sequential combinations of etoposide and cisplatin was evaluated. This was done by culturing spheroids of EwS cells inside 500nL droplets then merging them with secondary droplets containing fluorescent-barcoded drugs at different concentrations. Differences in EwS spheroid growth and viability were measured by microscopy. After drug exposure such measurements enabled estimation of their IC50 values, which were in agreement with values obtained in standard multiwell plates. Then, synergistic drug combination was evaluated. Sequential combination treatment of EwS with etoposide applied 24h before cisplatin resulted in amplified synergistic effect. As such, droplet-based microfluidics offers the modularity required for evaluation of drug combinations.

Abstract 155: Towards the development of an in vitro human vascularized immunocompetent metastatic melanoma-on-chip model

Abstract Despite recent extraordinary clinical success of immune checkpoint inhibitors (ICIs) in treating people affected by melanoma, a relevant number of patients still develops an adaptive resistance resulting in poor prognosis. To accelerate access to new therapies, there is a strong need for an in vitro human melanoma model mimicking the complexity of the tumor's cellular and physical microenvironment. Such model should recapitulate 3 critical features: (1) a reconstructed human melanoma-in-skin (Mel-RhS) to model melanoma cell invasion, (2) an endothelium to mimic the vascular system, and (3) circulation of, and ultimately, infiltration by, immune cells. Here, we present the development of these 3 individual biological features separately in dynamic conditions. These were constructed in standard multi-well plates (MW) by means of our microenviromentally controlled microphysiological system (MPS) (CubiX). This is a critical step towards the creation of a fully differentiated vascularized immunocompetent metastatic melanoma-on-chip model to recapitulate human pathophysiology, suitable for testing immunotherapies and studying the onset of possible resistance mechanisms. (1) Mel-RhS were constructed on transwells (8 μm pore size) and consisted of a human fibroblasts-populated collagen-fibrin dermal compartment, on top of which A375 melanoma cells and human keratinocytes were seeded. Mel-RhS were air-exposed for > 14 days to form of a fully differentiated and stratified epidermis. Melanoma nests developed and expanded into the dermal compartment in the 3D model, mimicking the initial stages of invasive melanoma. Moreover, Mel-RhS were viable under flow conditions for up to 3 days and their histology was comparable to that of the static controls. (2) The endothelial layer consisted of a monolayer of CD31+ endothelial cells on the bottom side of the transwell membrane. The shear stress provided by medium perfusion was able to induce alignment (75-80%) of cells within 24 hours (3 times faster than published protocols) as well as Von Willebrand Factor production. Real-time pH, lactate, glucose, and oxygen consumption were measured by using in-line sensors during the culture time. (3) Circulation of immune cells (MUTZ-3 progenitor cells) was achieved up to 24 hours in the presence of a healthy reconstructed human skin (RhS) model in a proof-of-concept study. The specific design of the developed MPS allowed for both immune cell flow and collection for downstream phenotypic analysis. In conclusion, we have individually recapitulated these major biological features of melanoma in vitro, in standard MW under dynamic culture conditions. In the future, the integration of these features into a single model would provide a unique in vitro vascularized immunocompetent human melanoma-on-chip model as a novel tool to investigate and test new therapies targeting adaptive resistance to ICIs. Citation Format: Elisabetta Michielon, Tanja de GruIijl, Taco Waaijman, Matteo Boninsegna, Divyasree Prabhakaran, Hadhemi Mejri, Antoni Homs Corbera, Pierre Gaudriault, Susan Gibbs, Dario Fassini. Towards the development of an in vitro human vascularized immunocompetent metastatic melanoma-on-chip model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 155.

Abstract 193: A high throughput vascularized immunocompetent tumoroids model in a standard multiwell plate for precision oncology

Abstract Recent advances in immuno-oncology have significantly increased the therapeutic arsenal available for clinicians. However, being able to identify the responder to a given treatment remains difficult and time consuming. This is due to the lack of translational preclinical models that recapitulate the complete cellular and physical tumor-immune micro-environment (TME). We report for the first time a high throughput vascularized immunocompetent breast tumoroids model in standard multiwell culture plate (MW). The model uses micro physiological system (MPS) and microfabrication to recapitulate and precisely control the TME. The tumor model includes fibroblasts, tumor cells (MDA-MB-231), immune cells (CD81+, CD64+), endothelial cells (CD31+), and a collagen extra cellular matrix. These are essential for the TME and are often lacking in preclinical models, potentially biasing the observed therapeutic response. The physical microenvironment was recapitulated using Cherry Biotech’s MPS, CubiX, and 4DCell SmartSphero Plates (SSoP). A combined system, which to the best of our knowledge is the only one able to recreate the complete TME in a 24 MW with multiplexed and uniformized tumoroid sizes. The SSoP technology is based on microstructured hydrogels, where microwells with an anchoring point at the bottom, allow the formation of the tumoroids, and to keep them in place, making it easy to trace them. The non-adherent properties of the gels help maintain the tumoroids shape. The CubiX system allowed controlling the cell culture conditions: temperature (37°C) and medium perfusion (150 µL/min). The presence of an enriched gas mix with 5% CO2 and O2, was also provided to the tumoroids, and pH, lactate, glucose, O2 consumption were monitored along all culture periods. We focused on optimizing the recapitulation of the physiopathology of breast cancer in 3 aspects: growth rate; tumor cell migration; and oxygen gradient within the MW plate to mimic different depths of the tumor (normoxic to the hypoxic core). Those features are essential for accurate drug efficacy testing. We were able to grow and maintain up to 91 tumoroids per well in a 24MW. After tumoroids formation, we obtained a fully vascularized and immuno-competent model in 48h, the fastest to the best of our knowledge. The system enabled monitoring of the tumoroids growth rate, the differentiation of the endothelium cells (Kfl 2/4, Van Willebrand Factor, eNOS, -% actin fiber alignment), differentiation of CD81+ and CD64+ cells into Macrophage type 1 or 2. All cell types were kept viable for 7 days, and endothelial cells alignment was found physiological (80%). Furthermore, we were able to induce on demand the invasiveness phenotype of the tumoroids. We envision that this model will evolve into a vascularized immunocompetent patient derived tumor model that can be used routinely in precision oncology to predict the drug response of a given patient. Citation Format: Stijn Robben, Anais Peyron, Ana Rita Ribeiro, Divyasree Prabhakaran, Antoni Homs Corbera, Pierre Gaudriault, Patricia Davidson, Dario Fassini. A high throughput vascularized immunocompetent tumoroids model in a standard multiwell plate for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 193.

Patient-Specific Microfluidic Cancer Spheroid Cultures for Testing Cancer Therapies.

The field of oncology increasingly focuses on strategies to predict effectiveness of a given therapy on a patient-by-patient basis. Such precision or personalized oncology has the potential of significantly extending patient survival time. Patient-derived organoids are seen as the main source of patient tumor tissue that may be used for therapy testing in personalized oncology. The gold standard approach for culturing cancer organoids is in standard multi-well plates coated with Matrigel. Despite their effectiveness, these standard organoid cultures have drawbacks, namely, requirement of a large starting cell population and polydispersity of cancer organoid sizes. The latter drawback makes it challenging to monitor and quantify changes in organoid size in response to therapy. Microfluidic devices with integrated arrays of microwells may be used to both decrease the amount of starting cellular material required to form organoids and to standardize organoid size to make therapy assessment easier. Herein, we describe methodology for making microfluidic device as well as for seeding patient-derived cancer cells, culturing organoids, and testing therapies using these devices.

Open-Source Culture Platform for Multi-Cell Type Study with Integrated Pneumatic Stimulation

Mechanical forces can influence the structure and development of healthy and cancerous cells and tissue microenvironments, acting on their physical shape and promoting non-genetic alterations during growth. For this reason, it is interesting to investigate the role of dynamic hydrostatic compression on such cultures, to assess the role of such stimuli on key parameters, such as cell differentiation, cell stiffness and cytoskeleton rearrangements. In this work, we present a versatile Arduino-based pneumatic system for the stimulation of a cell culture performed in a standard multi-well plate, designed to work inside a CO2 incubator. The system is capable of modifying the hydrostatic pressure inside a dedicated culture chamber following the desired pattern, and, thus, providing a mechanical hydrostatic stimulus to a cell culture growing inside it. In the present work, a human respiration-like compression pattern was used, to mimic the mechanical stress conditions inside the human lung alveoli, and make the platform compatible with the development of lung tissues and organoids.

An open-source LED array illumination system for automated multiwell plate cell culture photodynamic therapy experiments

Photodynamic therapy (PDT) research would benefit from an automated, low-cost, and easy-to-use cell culture light treatment setup capable of illuminating multiple well replicates within standard multiwell plate formats. We present an LED-array suitable for performing high-throughput cell culture PDT experiments. The setup features a water-cooling loop to keep the LED-array temperature nearly constant, thus stabilizing the output power and spectrum. The setup also features two custom-made actuator arms, in combination with a pulse-width-modulation (PWM) technique, to achieve programmable and automatic light exposures for PDT. The setup operates at ~ 690 nm (676–702 nm, spectral output full-width half-maximum) and the array module can be readily adapted to other LED wavelengths. This system provides an illumination field with adjustable irradiance up to 400 mW/cm2 with relatively high spectral and power stability comparing with previously reported LED-based setups. The light doses provided by the LED array were validated with comparison to traditional laser PDT. This open-source illumination platform (including the detailed technical description, fabrication protocols, and parts list provided here) helps to make custom light sources more accessible and of practical use for photomedicine research.

A programmable culture platform for hydrostatic stimulation and in situ pH sensing of lung cancer cells with organic electrochemical transistors

In this work, we present a programmable dynamic cell culture stimulation platform designed to work in a CO 2 incubator, compatible with a standard multi-well plate. An epithelial lung cell culture can be mechanically stimulated with a cyclic increase of the hydrostatic pressure inside the culture chamber in the range 1–12 kPa, trying to mimic the human respiration rate. An in situ live pH-monitoring system based on PEDOT:PSS Organic ElectroChemical Transistors was also designed, fabricated and characterized, in order to merge both the stimulated cell culture and the pH sensing in a compact platform. • A culture chamber for the stimulation of lung cells by means of the cyclic increaseof its internal pressure was fabricated. • The tightness of the chamber and its reliability have been tested. • PEDOT:PSS based Organic ElectroChemical Transistors were fabricated by using photolithographic steps. • OECTs were tested in NaCl 0.1 M solution and in cell culture medium, showing good electrical response. • Cell proliferation tests were carried out inside the chamber, showing identical proliferation with respect to control. • pH tests have been carried out with OECTs, showing high sensitivity below pH 4.

An automated do-it-yourself system for dynamic stem cell and organoid culture in standard multi-well plates

SummaryWe present a low-cost, do-it-yourself system for complex mammalian cell culture under dynamically changing medium formulations by integrating conventional multi-well tissue culture plates with simple microfluidic control and system automation. We demonstrate the generation of complex concentration profiles, enabling the investigation of sophisticated input-response relations. We further apply our automated cell-culturing platform to the dynamic stimulation of two widely employed stem-cell-based in vitro models for early mammalian development: the conversion of naive mouse embryonic stem cells into epiblast-like cells and mouse 3D gastruloids. Performing automated medium-switch experiments, we systematically investigate cell fate commitment along the developmental trajectory toward mouse epiblast fate and examine symmetry-breaking, germ layer formation, and cardiac differentiation in mouse 3D gastruloids as a function of time-varying Wnt pathway activation. With these proof-of-principle examples, we demonstrate a highly versatile and scalable tool that can be adapted to specific research questions, experimental demands, and model systems.

Multi-Layered Human Blood Vessels-on-Chip Design Using Double Viscous Finger Patterning.

Blood vessel-on-a-chip models aim at reproducing vascular functions. However, very few efficient methods have been designed to address the need for biological replicates in medium- to high-throughput screenings. Here, vessels-on-chip were designed in polydimethylsiloxane-glass chips using the viscous finger patterning technique which was adapted to create channels with various internal diameters inside a collagen solution and to simultaneously seed cells. This method was refined to create blood vessels composed of two concentric, distinct, and closely appositioned layers of human endothelial and perivascular cells arranged around a hollow lumen. These approaches allowed the formation of structurally correct blood vessels-on-chips which were constituted of either only endothelial cells or of both cell types in order to distinguish the vascular barrier reactivity to drugs in the presence or not of perivascular cells. The established vessels showed a tight vascular barrier, as assessed by immunostaining of the adherens junctions, and were reactive to the natural vasopermeant thrombin and to inflammatory cytokines. The presence of perivascular cells markedly increased the tightness of the vascular barrier and lowered its response to thrombin. The design allowed us to simultaneously challenge in real-time several tens of 3D-reconstituted, multicellular blood vessels in a standard multiwell plate format suitable for high-throughput drug screening.

Megapixel camera arrays enable high-resolution animal tracking in multiwell plates

Tracking small laboratory animals such as flies, fish, and worms is used for phenotyping in neuroscience, genetics, disease modelling, and drug discovery. An imaging system with sufficient throughput and spatiotemporal resolution would be capable of imaging a large number of animals, estimating their pose, and quantifying detailed behavioural differences at a scale where hundreds of treatments could be tested simultaneously. Here we report an array of six 12-megapixel cameras that record all the wells of a 96-well plate with sufficient resolution to estimate the pose of C. elegans worms and to extract high-dimensional phenotypic fingerprints. We use the system to study behavioural variability across wild isolates, the sensitisation of worms to repeated blue light stimulation, the phenotypes of worm disease models, and worms’ behavioural responses to drug treatment. Because the system is compatible with standard multiwell plates, it makes computational ethological approaches accessible in existing high-throughput pipelines.

Using Microfluidics to Generate Human Naïve and Primed Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) are generated from somatic cells by the expression of a cocktail of transcription factors, and iPSCs have the capacity to generate in vitro all cell types of the human body. In addition to primed (conventional) iPSCs, several groups recently reported the generation of human naïve iPSCs, which are in a more primitive developmental state and have a broader developmental potential, as shown by their ability to form cells of the placenta. Human iPSCs have broad medical potential but their generation is often time-consuming, not scalable and requires viral vectors or stable genetic manipulations. To overcome such limitations, we developed protocols for high-efficiency generation of either conventional or naïve iPSCs by delivery of messenger RNAs (mRNAs) using a microfluidic system. In this protocol we describe how to produce microfluidic devices, and how to reprogram human somatic cells into naïve and primed iPSCs using these devices. We also describe how to transfer the iPSC colonies from the microfluidic devices over to standard multiwell plates for subsequent expansion of the cultures. Our approach does not require stable genetic modifications, is reproducible and cost-effective, allowing to produce patient-specific iPSCs for cell therapy, disease modeling, and in vitro developmental studies.

A programmable culture platform for stimulation and <em>in situ</em> sensing of lung epithelial cells

A programmable dynamic cell culture chamber compatible with a standard multi-well plate was designed and characterized. The system is integrated with an array of OECT biosensors, in view of an in-situ monitoring of the dynamic cultures.

A programmable culture platform for stimulation and <em>in situ</em> sensing of lung epithelial cells

A programmable dynamic cell culture chamber compatible with a standard multi-well plate was designed and characterized. The system is integrated with an array of OECT biosensors, in view of an <i>in situ</i> monitoring of the dynamic cultures.

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

Lab-on-a-Chip System for Developing and Fluorescence Imaging a Three-Dimensional Model of Pancreatic Islets Under Flow Conditions

Lab-on-a-Chip System for Developing and Fluorescence Imaging a Three-Dimensional Model of Pancreatic Islets Under Flow Conditions

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Automated dendritic cell generation in a closed system

Background & Aim Dendritic cells (DC) are an attractive vehicle for therapeutic manipulation and are used in antigen-pulsed autologous DC therapies, DC-stimulated autologous T cell therapies, and a variety of in vitro cellular immunology assays. Given the low abundance of DC in blood, they are typically generated ex vivo from monocytes or stem cell precursors in a manual culture process. The standard multiwell plate or flask culture used for DC generation requires several solution exchanges including cytokine refresh and wash steps. When DCs must be generated in a patient-specific manner, the multistep nature of the protocol presents significant logistical challenges for consistent results. To this end, the Corning MicroDEN® system relies upon medium perfusion to supply fresh cytokines throughout the duration of monocyte-derived DC generation. This study demonstrates performance of the MicroDEN system for immature DC (iDC) generation from a source of enriched monocytes. Methods, Results & Conclusion Monocytes were isolated from whole blood via Ficoll separation and CD14 magnetic bead purification. Monocyte differentiation to iDCs was achieved by a 6 day culture with CellGenix DC medium containing IL4 (350 U/mL;CellGenix), GM-CSF (350 U/mL;CellGenix), and 1% penicillin-streptomycin in both 6-well tissue culture-treated (TCT) multiwell plates (Corning) and the MicroDEN system. Flow cytometry data indicated that the iDCs from MicroDEN and 6-well plates were phenotypically comparable with regard to expression of CD209, CD80, CD83, CD86 and CD14. Allogeneic T cell proliferation assays and an antigen-specific assay demonstrated that iDCs generated via MicroDEN are functionally competent and can successfully generate a T cell response. A DOE-based approach was employed to characterize the relationship between perfusion flow rate, monocyte seeding density, and yield of dendritic cells. DCs generated by MicroDEN are functionally equivalent to DCs generated by the standard well plate culture. The larger surface area of MicroDEN cartridge allows generation of a higher number of dendritic cells in single run when compared to the standard 6-well plate, thereby reducing time and labor involved in harvesting cells from multiple plates. Higher perfusion flow rates enable the processing of larger numbers of seeded monocytes, upto a seeding density level of 1.3 million monocytes/cm2. MicroDEN is an effective tool to generate dendritic cells for applications ranging from cell-based assays to cell-based immunotherapies.

Lab-on-a-Chip System for Developing and Fluorescence Imaging a Three-Dimensional Model of Pancreatic Islets Under Flow Conditions

Introduction Diabetes mellitus is a group of metabolic diseases characterized by elevated blood sugar resulting from a defect in the production of insulin by pancreatic β-cells or tissue insensitivity for this peptide hormone [1]. The most common form of the disease is diabetes mellitus type 2 (insulin-dependent), which is diagnosed in about 80% of all patients [2]. In patients with this type of diabetes, both the efficiency and the secretion of insulin are disturbed [3]. For this reason, it is very important to thoroughly understand the kinetics of insulin release in response to various conditions, which can help develop effective therapy. The most important cells for this disease are insulin secreting β-cells and their antagonist - glucagon-secreting α-cells [4]. In our research, we focused on imitate the pancreatic islet structure, which can be a universal model for testing the impact of various environmental factors on disease development. The idea of using a Lab-on-a-chip system for three-dimensional islet cells culture came from the possibility of mimicking in vivo conditions. This can give a wider and more accurate view of the pancreatic islets function than the cell culture in static conditions. Lab-on-a-chip system In our research we present a Lab-on-a-chip system in which a pancreatic “pseudoislet” model will be developed. This system is composed of biocompatible, non-toxic and transparent materials, such as: poly(dimethylsiloxane) (PDMS) and thin glass. Thanks to this, it is possible to conduct cell culture and observe the results in various types of microscopes. The geometry of Lab-on-a-chip system consist of two elliptical cell culture chambers, one for the test sample and second for the reference sample. In in each of the chambers there are 15 round microtraps. Each of the microtraps is made of 6 micropillars. This solution forces the dense packing of cells and their aggregations. The geometry of the developed system is consistent with the culture wells on a standard multi-well plate, which allows measurements in a multi-well plate reader. Methods All experiments were performed using two commercially purchased pancreatic islets cell lines: β-cells (INS-1E) and α-cells (α-TC1-6). Appropriate α-cells and β-cells ratios were selected to reflect the morphology and composition of the pancreatic islet found in the body. The cells were introduced through the inlet to the Lab-on-a-chip system and placed for 24h in an incubator to form spherical aggregates (“pseudoislets”). In the next stage immunostaining protocol using primary and secondary antibody solutions was developed. Due to the use this method and confocal microscope it was possible to confirm the correct distribution of cells in the obtained aggregate. To confirm cells viability previously prepared solution of Calcein AM and Propidium Iodide were introduced through the microsystem inlet with a flow rate 5µL/min for 10 min. After 10 min incubation (37oC, 5% CO2) the viability of the cells was determined using fluorescence microscope. Proliferation of the ells was measured using spectrofluorimetric multi-well plate reader after incubation with AlamarBlue solution. The “pseudoislets” were then stimulated with various glucose concentrations (imitation of health and disease state). After this, the standard ELISA test procedure was adapted to the microfluidic conditions to examine the insulin secretion profile from the obtained “pseudoislets”. Results and Conclusions It was confirmed that 15 spherical α and β-cell aggregates with diameters of 160-180 µm were obtained in one culture chamber using the method described earlier. As was expected the participation of β-cells in the core and α-cells on the periphery of the aggregate was confirmed by the use of immunostaining method. Moreover, the compatibility of the designed microsystem with the multi-well plate reader allowed to monitor cell proliferation and viability in situ during the culture. A high viability after 24 and 48 hours of culture in the microchip was confirmed and was 97% and 95% respectively. The cells maintained their morphology, function and high level of proliferation for up to 48 hours of culture. Moreover, in the developed model health and disease conditions were simulated and the level of insulin secreted from this model was determined using the ELISA test. At this stage, a research model corresponding to the model in vivo conditions was obtained. The next step will be to develop a method for analyzing insulin secretion in real time (such as Surface Plasmon Resonance). This study presents basic research and in the future, this model could be utilized to simulate diabetes, testing new drugs and therapy in diabetes mellitus treatment.