1.&#160;Chip design for adherent and floating cells 2D co-cultures
NOTE: The 2D co-culture layout (Figure 1A-C) is characterized by three chambers (100 &#181;m high) interconnected by two sets of microchannel arrays (500 x 12 x 10 &#181;m3, L&#215;W&#215;H). The intermediate chamber forms two closed dead-end compartments which block floating immune cells overflowing into the tum...

The authors have nothing to disclose. AS is supported by the Fondazione Italiana per la Ricerca sul Cancro (AIRC, Start-Up 2016 #18418) and Ministero Italiano della Salute (RF_GR-2013-02357273). GS and FM are supported by the Italian Association for Cancer Research (AIRC) no. 21366 to G.S.).

The described methods try to design a general approach to recapitulate, with modulable degree of complexity, two significant aspects in the field of onco-immunology, which can benefit from the adoption of more relevant in vitro models. The first one involves the tumor cell population side, where tackling single cell characteristics may lead to a better description of heterogeneity and correlated biological and clinical significance including resistance to therapy, propension to metastasis, stem cell and differentiation g...

The evolution of different branches of medicine as experimental disciplines has depended on the ability to manipulate cell population and organ functions under controlled conditions1. Such ability has its roots in the availability of measurable models able to recapitulate processes happening in our body.
In the age of immunotherapy and single-cell genomic profiling2, cancer biology needs to take advantage of emerging in vitro and computational models for investigating the tumor-immune interface in a proper spatiotemporal context2,3.
The tumor microenvironment4 (TME) is a complex tissue where cancer cells continuously interact and dynamically co-evolve with the other cellular (immune, stromal, and endothelial cells) and non-cellular (the extracellular matrix, ECM) components. The dynamic nature of this complex landscape dictates whether immune cells play as friends or foes of malignant cells, thus strongly affecting both disease progression and response to therapy. Nowadays, great efforts from onco-immunologists, bioinformaticians, and systems biology experts are converging to address the clinical significance of cancer heterogeneity5,6, either in the space (i.e., in distinct tumoral regions) and time (i.e., at distinct tumor progression stages)5,6, and to characterize cancer and immune cell phenotype and function at a single-cell level. As an example of this synergy, advanced computer-vision techniques are now routinely used for spatial mapping of immune infiltrate in histological samples7,8.
On the front of experimental models, bridging animal studies and traditional in vitro methods, advances in microfluidics and co-culturing techniques give access to different classes of micro-engineered cellular models such as organoids, micro-physiological systems9,10,11 (MPS), and organs-on-chip12,13,14 (OOC). They share the common trait to zoom in the &#39;big picture&#39; view of the cellular ecosystems and expanding the in vitro potential to control microenvironmental factors while exploiting high-content microscopy15 and image processing approaches.
Nowadays, state-of-the-art- MPS and OOC systems have begun to include immunological aspects , incorporating different subtypes of immune cells in existing tissues- and co-cultures, so to explore and measure a variety of processes like inflammatory diseases, wound healing, mucosal immunity, and response to toxins or daily food products16. TME-on-a-chip models10,11,12,13,14,15,16,17, also integrated with perfusable microvessels18,19,20,21, have been developed to investigate cell-type-dependent interactions, physical and chemical perturbations, and the cytotoxic activity of infiltrating lymphocytes22, as well as clinically relevant immunomodulatory agents23.
Here, we provide versatile protocols, spanning from loading cells in chips to image processing tools, to exploit advanced tumor-immune microfluidic co-cultures in 2D (section 1) and 3D (section 2) settings16, compatible with dynamic, multiparametric24 monitoring and visualization of cellular functions. This is achieved maintaining easiness of use and flexibility both in sample management and data analysis, taking advantage of Fiji freeware software and its toolboxes25,26.
The microfluidic device, described in section 1, is designed to perform 2D co-cultures of adherent cancer and floating immune cells. This platform was validated for the in vitro measurement of immune cell behavior in the presence of genetic mutations27 and/or immunodeficiencies28. Here, we illustrate steps for tracking immune cells in time-lapse bright-field images, by exploiting a semi-automatic method based on Trackmate (a plugin implemented in Fiji software). This procedure enables the extraction of kinematic descriptors of immune migration 29 and response (i.e., interaction times) to target cancer cells, treated or not with immunogenic cell death inducers27.
Importantly these parameters, extracted from time-series images, can be processed with advanced mathematical machinery. As an example of the potentiality of this approach, our groups recently published an analysis based on mathematical methods from stochastic processes and statistical mechanics to model cellular network properties and provide a parametrized description of immune cell behavior (i.e., biased or uncorrelated random walk, highly or not coordinated motion30,31).
The 3D setting, provided in the second section, is based on a co-culture protocol to recreate more complex immunocompetent TMEs embedded in two gel regions with different combinations of cell types and drugs in a competitive fashion. Here, image processing steps are described to measure, at different timepoints, the infiltration of stained immune cells in human A375M melanoma cells cultivated within Matrigel, to evaluate antitumor agent combinations32. A375M line, an A375P derived cell line characterized by a highly metastatic phenotype was chosen to evaluate their metastatic capability in the presence of immune cells32.
The described models can be fully compliant with different cell sources (murine and human immortalized or primary cell lines, organoids, xenografts, among the others). In recent studies of our lab, by combining high-content video microscopy with image analysis, the competitive 3D layout was applied to investigate: i) an anti-tumoral (antibody-dependent cell-mediated cytotoxicity, ADCC) immune response and dissect the role of fibroblasts in resistance to trastuzumab therapy in HER2+ breast cancer on-chip models33; ii) the action of myeloid cells (i.e., cancer-associated macrophages) in mechanisms of tumor evasion and recruitment of T cells34; iii) the efficacy of immunotherapeutic regimes, specifically based on Interferon-&#945;-conditioned dendritic cells (IFN-DCs), cultivated with drug-treated colon cancer cells in collagen matrices, and to evaluate efficient motion and the succeeding phagocytosis events35; iv) the chemotactic migration of bone marrow-derived eosinophils towards IL-33 treated or untreated melanoma cells36.
These advanced models could serve as observation windows for understanding the role of immune contexture in cancer metastasis and resistance mechanisms, but efforts are required to translate findings into the clinics, closing the gap with basic research37.
As an emerging scenario, harnessing the power of automated high-content microscopy coupled to the use of more physiologically-relevant microsystems is opening novel potential challenges for the handling, processing, and interpretation of hundreds, and even thousands, of Gigabytes of multiparametric data, which can be generated from a single experimental campaign. This implies a direct link of OOC experiments with artificial intelligence38,39,40,41,42 (AI)-based algorithms both for advanced automated analysis, and generation of features which can feed in turn in silico models of cancer-immune interplay43, with exciting new applications at the horizon, such as the development of predictive drug screening assays44.
An ever-expanding flow of efforts is focused on the design of disease models jointly with the optimization of strategies to implement the large-scale perturbation screens with single-cell multi-omics readouts. This will undoubtedly help the development and, hopefully, the clinical implementation, accompanied by an appropriate degree of method standardization, of a systematic onco-immunology-on-a-chip approach to gain novel insights into immune disorders and cancer dissemination mechanisms.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture materials&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Corning-Sigma Aldrich, St. Louis, MO</td>
			<td>CLS430828</td>
			<td>centrifuge tubes</td>
		</tr>
		<tr>
			<td>5-aza-2&#39;-deoxycytidine DAC</td>
			<td>Millipore-Sigma; St. Louis, MO</td>
			<td>A3656</td>
			<td>DNA-hypomethylating agent</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning-Sigma Aldrich, St. Louis, MO</td>
			<td>CLS3506</td>
			<td>culture dishes</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flask</td>
			<td>Corning, New York, NY</td>
			<td>430641U</td>
			<td>culture flasks</td>
		</tr>
		<tr>
			<td>A365M</td>
			<td>American Type Culture Collection (ATCC), Manassas, VA</td>
			<td> 
			CVCL_B222</td>
			<td>human melanoma cell line</td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Millipore-Sigma; St. Louis, MO</td>
			<td>D1515</td>
			<td>anthracycline antibiotic&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium DMEM</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECM0728L</td>
			<td>Culture medium for SK-MEL-28&#160; cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffer Saline w/o Calcium w/o Magnesium</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECB4004L</td>
			<td>saline buffer solution</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECS0180L</td>
			<td>ancillary for cell culture</td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>GE-Heathcare</td>
			<td>17-1440-02</td>
			<td>separation of mononuclear cells from human blood.&#160;</td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Neubauer</td>
			<td></td>
			<td>Cell counter</td>
		</tr>
		<tr>
			<td>Heparinized vials</td>
			<td>Thermo Fisher Scientific Inc., Waltham, MA</td>
			<td></td>
			<td>Vials for venous blood collection</td>
		</tr>
		<tr>
			<td>interferon alpha-2b</td>
			<td>Millipore-Sigma; St. Louis, MO</td>
			<td>SRP4595</td>
			<td>recombinant human cytokine</td>
		</tr>
		<tr>
			<td>L-Glutamine 100X</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECB3000D</td>
			<td>ancillary for cell culture</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte cell separation media</td>
			<td>Cedarlane Labs, Burlington, Canada</td>
			<td></td>
			<td>Separation of lymphocytes by density gradient centrifugation</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Axis-Shield PoC AS, Oslo, Norway</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning, New York, NY</td>
			<td>354230</td>
			<td>growth factor reduced basement membrane matrix</td>
		</tr>
		<tr>
			<td>MDA-MB-231&#160;</td>
			<td>American Type Culture Collection (ATCC), Manassas, VA</td>
			<td>&#160;HTB-26</td>
			<td>human breast cancer cell line</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin 100X&#160;&#160;</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECB3001D</td>
			<td>ancillary for cell culture</td>
		</tr>
		<tr>
			<td>Pipet aid</td>
			<td>Drummond Scientific Co., Broomall, PA</td>
			<td>4-000-201</td>
			<td>Liquid handling</td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent cell linker</td>
			<td>Millipore-Sigma; St. Louis, MO</td>
			<td>PKH26GL</td>
			<td>red fluorescent cell dye</td>
		</tr>
		<tr>
			<td>PKH67 Green fluorescent cell linker</td>
			<td>Millipore-Sigma; St. Louis, MO</td>
			<td>PKH67GL</td>
			<td>green fluorescent cell dye</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECM2001L</td>
			<td>Culture medium for MDA-MB-231 cells</td>
		</tr>
		<tr>
			<td>serological pipettes (2 mL, 5 mL, 10 mL, 25 mL, 50 mL)</td>
			<td>Corning- Millipore-Sigma; St. Louis, MO</td>
			<td>CLS4486; CLS4487; CLS4488; CLS4489; CLS4490</td>
			<td>Liquid handling</td>
		</tr>
		<tr>
			<td>sterile tips (1-10 &#956;L, 10-20 &#956;L, 20-200 &#956;L, 1000 &#956;L)</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECTD00010; ECTD00020; ECTD00200; ECTD01005</td>
			<td>tips for micropipette</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Thermo Fisher Scientific Inc., Waltham, MA</td>
			<td>15250061</td>
			<td>cell stain to assess cell viability</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>EuroClone Spa, Milan, Italy</td>
			<td>ECM0920D</td>
			<td>dissociation reagent for adherent cells</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS-FL fluorescence microscope</td>
			<td>Thermo Fisher Scientific Inc., Waltham, MA</td>
			<td></td>
			<td>Fluorescent microscope for living cells</td>
		</tr>
		<tr>
			<td>Humified cell culture incubator&#160;</td>
			<td>Thermo Fisher Scientific Inc., Waltham, MA</td>
			<td>311 Forma Direct Heat COIncubator; TC 230</td>
			<td>Incubation of cell cultures at 37 &#176;C, 5% CO2</td>
		</tr>
		<tr>
			<td>Juli Microscope</td>
			<td>Nanoentek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory refrigerator (4 &#176;C)</td>
			<td>FDM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Safety Cabinet (Class II)</td>
			<td>Steril VBH 72 MP</td>
			<td></td>
			<td>Laminar flow hood</td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerable centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microfabrication materials&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-Aminopropyl)triethoxysilane (Aptes)</td>
			<td>Sigma Aldrich</td>
			<td>A3648</td>
			<td>silanizing agent for bonding PDMS to plastic coverslip</td>
		</tr>
		<tr>
			<td>Chromium quartz masks / 4&#34;x4&#34;, HRC / No AZ&#160;</td>
			<td>MB W&#38;A,&#160; Germany</td>
			<td></td>
			<td>optical masks for photolithography</td>
		</tr>
		<tr>
			<td>Glass coverslip, D 263 M Schott glass,&#160; (170 &#177; 5 &#181;m)</td>
			<td>Ibidi, Germany</td>
			<td>10812</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide solution 30%</td>
			<td>Carlo Erba Reagents</td>
			<td>412081</td>
			<td>reagents for piranha solution</td>
		</tr>
		<tr>
			<td>Methyl isobutyl ketone</td>
			<td>Carlo Erba Reagents</td>
			<td>461945</td>
			<td>PMMA e-beam resist developer</td>
		</tr>
		<tr>
			<td>Microscope Glass Slides (Pack of 50 slides) 76.2 mm x 25.4 mm&#160;</td>
			<td>Sail Brand</td>
			<td>7101</td>
			<td>substrates for bonding chips</td>
		</tr>
		<tr>
			<td>Miltex Biopsy Punch with Plunger, ID 1.0mm</td>
			<td>Tedpella</td>
			<td></td>
			<td>dermal biopsy punches for chip reservoirs</td>
		</tr>
		<tr>
			<td>PMMA&#160; 950 kDa</td>
			<td>Allresist,Germany</td>
			<td>AR-P. 679.04</td>
			<td>Positive electronic resists for patterning optical masks</td>
		</tr>
		<tr>
			<td>Polymer untreated coverslips</td>
			<td>Ibidi, Germany</td>
			<td>10813</td>
			<td>substrates for bonding chips</td>
		</tr>
		<tr>
			<td>Prime CZ-Si Wafer,&#160; 4&#8221;, (100), Boron Doped</td>
			<td>Gambetti Xenologia Srl, Italy</td>
			<td>30255</td>
			<td></td>
		</tr>
		<tr>
			<td>Propan-2-ol</td>
			<td>Carlo Erba Reagents</td>
			<td>415238</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene glycol monomethyl ether acetate (PGMEA)</td>
			<td>Sigma Aldrich</td>
			<td>484431-4L</td>
			<td>SU-8 resists developer</td>
		</tr>
		<tr>
			<td>SU-8 3005</td>
			<td>Micro resist technology,Germany</td>
			<td>C1.02.003-0001</td>
			<td>Negative Photoresists</td>
		</tr>
		<tr>
			<td>SU-8 3050</td>
			<td>Micro resist technology,Germany</td>
			<td>C1.02.003-0005</td>
			<td>Negative Photoresists</td>
		</tr>
		<tr>
			<td>Suite of Biopunch, ID 4.0 mm, 6.0 mm, 8.0 mm</td>
			<td>Tedpella</td>
			<td>15111-40, 15111-60, 15111-80</td>
			<td>dermal biopsy punches for chip reservoirs</td>
		</tr>
		<tr>
			<td>Sulfuric acid 96%</td>
			<td>Carlo Erba Reagents</td>
			<td>410381</td>
			<td>reagents for piranha solution</td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Dowsil, Dow Corning</td>
			<td>11-3184-01</td>
			<td>Silicone Elastomer (PDMS)</td>
		</tr>
		<tr>
			<td>Trimethylchlorosilane (TMCS)</td>
			<td>Sigma Aldrich</td>
			<td>92360-100ML</td>
			<td>silanizing agent for SU-8 patterned masters</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microfabrication equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 kV e-beam litography</td>
			<td>Raith-Vistec EBPG 5HR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hotplate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical litography system</td>
			<td>EV-420 double-face contact mask-aligner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive Ion Etching system</td>
			<td>Oxford plasmalab 80 plus system</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum dessicator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic co-culture models for dissecting the immune response in in vitro tumor microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

Tumor immune infiltration is a parameter of the host anti-tumor response. Tumors are heterogeneous in the composition, density, location, and functional state of infiltrating leukocytes which interactions with cancer cells can underlie clinically relevant information to predict disease course and response to therapy. In this sense, microfluidic technologies could be used as complementary and privileged in vitro tools to explore the immune contexture of tumors, as well as to monitor the response to anticancer therapies. T...

Watch this Scientific Journal Video about Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments at JoVE.com

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

This protocol provides experimental settings to perform controllable 2D and 3D co-cultures in micro-devices to visualize and monitor tumor-immune cell crosstalks and analyze the effects of anti-cancer treatments. This technology provides real-time and easy visualization of immune cell recruitment and interactions and can be employed for human and patient-derived samples. It is compatible with most state-of-the-art microscopies.Demonstrating the procedure will be Francesco Noto, PhD student at the Department of Oncology and Molecular Medicine, Istituto Superiore di Sanita, and Nicoleta Manduca and Esther Maccafeo, PhD students at Universita Cattolica del Sacro Cuore, Department of Translational Medicine and Surgery. Chips are previously plasma-activated in a plasma cleaner or reactive iron-etching machine in clean room. Murine-spleen cells and a tumor cell line is used here for mimicking the protocols described in the text.Before loading cell suspensions, withdraw media from all six reservoirs. Then slowly load 1 x 10 to the 5th tumor cells resuspended in 10 to 50 microliters of growth medium in the upper left-hand reservoir and lower well. On the right side, gently pipette 1 x 10 to the 6th floating immune cells resuspended in 50 microliters of growth medium into two wells.When all the cells have been added, fill all six reservoirs of each chip with up to 100 to 150 microliters of growth medium and check that the cells have been correctly distributed within each culture compartment. Incubate the chip for one hour to allow the system to stabilize before the time-lapse recording. For live cell imaging, mount the tumor immune on the chip plate on the microscope stage.Customize the acquisition workflow setting in the microscope software interface such as Incucyte Live-Cell Analysis Software. Select windows of observation, the optimal frame rate and time duration, depending on the appropriate parameters for the experiment and cell types under study. Image the cells for the appropriate experimental time period.Prepare two aliquots of matrigel diluted with medium containing a drug or a combination of drugs for the two experimental conditions using cold tips. Gently pipette live-compatible, fluorescent dye-stained tumor cells suspended in matrix solution on ice to obtain a homogenous distribution of the cells. With the chip plate on a cooling block or on a basket with ice, slowly inject the drug-treated tumor cell matrix solution into the left and right gel ports using cold 10 microliter micropipette tips.Apply gentle pressure to push the matrix solution from one side of the channel to the other. When all tumor cells have been loaded, place the device into the incubator in the upright position for 30 minutes. At the end of the incubation, once the matrix gelation is completed, fill the medium channels of all six reservoirs with 50 microliters of culture medium.Check the polymerized gel integrity and tumor cell distribution under a microscope. Then place the chips in an incubator until immune cell preparation is complete. After incubation, aspirate the medium from each well.Place the tip near the inlet of the middle medium channel and use moderate pressure to gently inject 10 microliters of 10 to the 6th immune cells labeled with a contrasting fluorescent dye. Inject 50 to 100 microliters of the medium into each of the four wells of lateral channels, 40 to 90 microliters of the medium into the upper central well, and 50 to 100 microliters of the medium into the lower central well. When all the wells have been loaded, use a microscope to confirm that the immune cell distribution has remained confined to the central chamber.Then carefully place the device on a level surface in the cell culture incubator until imaging. To calculate the extent of the fluorescently-stained live immune cells infiltrating the tumor compartments, image the device at specific time points of interest by fluorescence microscopy and set the appropriate camera parameters in the microscope imaging software, such as Nikon NIS-Elements. Also set the parameters for labeled immune cells.The movement of leukocytes through suitably built microchannel bridges in the microfluidic platform toward their target cells can be tracked by video microscopy. In this tracking analysis, individual PBMCs were challenged with dying doxorubicin-treated or live PBS-treated cancer cells. Relevant chemotaxis values and migration plots were automatically generated, and a different migratory profile for the immune cells was observed when co-loaded with breast cancer cells exposed to doxorubicin or PBS.When the PBMCs were confronted with apoptotic cancer cells, they crossed microchannels toward the dying and dead cells but did not cross to live, untreated cells. A fraction of leukocytes with increasing density over 24 to 48 hours exhibited long-term contacts with doxo-treated cancer cells. In this analysis, a novel 3D immunocompetent tumor model was used to quantify the recruitment of immune cells in response to anti-cancer combinations of epigenetic drugs.Red-dye-labeled PBMCs were then distributed homogeneously into the central fluidic chamber. The ability of the two tumor masses to attract the PBMCs was then compared, with the PBMCs being robustly recruited in this experiment into the right-side microchannel. For the 3D model, mix the solution with hydrogel and cells on ice, avoiding bubbles and pre-polymerization.Inject it slowly without excessive pressure. Tune polymerization steps according to the matrix used. Live/dead cell assays and cytokine secretion profiling from supernatants can be implemented.Immunofluorescence for confocal imaging can be performed to classify immune subtypes and for expression markers of activation and exhaustion maturation.

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Research

JoVE Journal

Immunology and Infection

4.4K visualizações.  CNR Institute for Photonics and Nanotechnology. Este protocolo fornece configurações experimentais para realizar co-culturas 2D e 3D controláveis em micro-dispositivos para visualizar e monitorar crosstalks tumor-imune celular e analisar os efeitos de tratamentos anti-câncer. Essa tecnologia fornece visualização fácil e em tempo real do recrutamento e interações de células imunes e pode ser empregada para amostras humanas e derivadas de pacientes. É compatível com a maioria das microscopias de última geração.Quem demo...