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 ...

microfluidic-co-culture-models-for-dissecting-immune-response-in

Research

JoVE Journal

Immunology and Infection

4.5K Views.  CNR Institute for Photonics and Nanotechnology. 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.

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...