We are indebted to the patients for their participation in the tissue collection for these experiments. A DoD OCRP Ovarian Cancer Academy Award (W81XWH-15-0253) and a pilot award from Colleen&#39;s Dream Foundation to Anirban K. Mitra supported this research.

The protocol follows the guidelines of the Institutional Regulatory Board of Indiana University.
1. Cell Preparation
<ol>
	<li>Isolation and culture of human primary mesothelial cells
	<ol>
		<li>Isolate human primary mesothelial cells (HPMCs) from human omentum as described previously17,18 and grow them in complete growth medium [Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1% non-essential amino acids, and 1% vitamins] at 37 &#176;C and 5% CO2. 
		NOTE: The HPMCs are typically grown to 100% confluence (Figure 1A).</li>
	</ol>
	</li>
	<li>Growth conditions of ovarian cancer cells
	<ol>
		<li>Grow HeyA8 ovarian cancer cells in complete growth medium at 37 &#176;C and 5% CO2. 
		NOTE: Use HeyA8 cells grown to about 80% confluence (Figure 1B).</li>
	</ol>
	</li>
</ol>
2. Cell Culture
<ol>
	<li>Proximal culture set-up
	<ol>
		<li>Use Transwell inserts for 6-well plates with a 10 &#956;m-thick tissue-culture-treated polycarbonate membrane with 0.4 &#956;m pores (108 pores/cm2) and a 4.67 cm2 growth area. If needed, optimize for different insert sizes by scaling the number of cells seeded according to the growth surface area. Warm complete growth medium, trypsin, and phosphate-buffered saline (PBS) to 37 &#176;C before use.</li>
	</ol>
	</li>
	<li>Preparing the inserts
	<ol>
		<li>To seed the HeyA8 cells on the lower surface of the membrane of the insert, remove the insert from its packaging using sterile forceps and place it inverted in a sterile 15 cm culture dish.</li>
		<li>Use a 15 cm dish, as it has the depth to accommodate the inverted insert, or substitute it with any appropriate sterile container. Depending on the experiment, place the required number of inserts in the 15 cm dish.</li>
		<li>Label the three controls and three experimental conditions accordingly with a marker on the edge of the inserts.</li>
	</ol>
	</li>
	<li>Preparing the cancer cells
	<ol>
		<li>To trypsinize the HeyA8 cells grown at 80% confluence in a 25 cm2 flask (~2 x 106 cells), add 1 mL of trypsin (0.25%) for 40&#8211;60 s and neutralize the trypsin with 6 mL of complete growth medium.</li>
		<li>Centrifuge the cells at 500 x g at room temperature (RT) for 3 min, discard the supernatant, and resuspend the cell pellet in 5 mL of complete growth medium. Count the live cells using the trypan blue dye exclusion method.</li>
	</ol>
	</li>
	<li>Seeding the cancer cells
	<ol>
		<li>Suspend 100,000 cells/mL of live HeyA8 cells in complete growth medium. Carefully seed 80,000 HeyA8 cells suspended in 800 &#956;L of complete growth medium on the bottom of the insert (which is now facing up, since it is inverted).</li>
		<li>Seed the cells to form a dome-like shape so the cells remain on the insert (Figure 1C). Start from the center of the membrane and move outward in concentric circles while slowly pipetting. Avoid going more than 3 mm from the edge to prevent any spilling of the medium. 
		NOTE: The number of cells seeded will depend on the growth rate of the cells and the duration of the experiment. The number of HeyA8 cells seeded for this proximal culture were optimized and the cells will be 80&#8211;90% confluent at the end of the third day.</li>
	</ol>
	</li>
	<li>Cancer cell attachment
	<ol>
		<li>Cover the 15 cm dish and carefully move the dish containing the inserts to the CO2 incubator. It is critical not to disrupt the drop of medium and cells on the insert at this step. Leave the cells at 37 &#176;C for 4 h to allow the cancer cells to attach to the insert.</li>
	</ol>
	</li>
	<li>Preparing the mesothelial cells
	<ol>
		<li>After around 4 h, trypsinize the HPMCs grown at 100% confluence in a 75 cm2 flask (~4 x 106 cells) with 2 mL of trypsin (0.25%) for 1&#8211;2 min and neutralize the trypsin with 12 mL of complete growth medium.</li>
		<li>Centrifuge the cells at 500 x g at RT for 3 min, resuspend the cell pellet in 10 mL of complete growth medium, and count the live cells using the trypan blue dye exclusion method.</li>
	</ol>
	</li>
	<li>Seeding the mesothelial cells
	<ol>
		<li>Suspend 300,000 cells/mL of live HPMCs in complete growth medium. Add 2.5 mL of fresh complete growth media to each well of a 6-well plate. Carefully bring the 15 cm dish containing the inserts out to the biosafety hood. Using sterile forceps, flip the insert and place it in the well of the 6-well plate so that it is upright, with the HeyA8 cells attached to the lower surface immersed in the complete growth medium (Figure 1C).</li>
		<li>Seed 450,000 HPMCs suspended in 1.5 mL of growth medium in the inside of the inserts with the HeyA8 cells attached to the surface facing the bottom of the well (Figure 1C) or to inserts without any HeyA8 cells for the HPMC controls. Add 1.5 mL of growth medium without cells to the HeyA8 controls.</li>
	</ol>
	</li>
	<li>Growing the proximal culture
	<ol>
		<li>Let the proximal culture grow in the CO2 incubator at 37 &#176;C and 5% CO2 for 72 h. If required, the medium can be replenished as follows.
		<ol>
			<li>For the inside of the insert, remove 750 &#181;L and add 750 &#181;L of fresh complete growth medium.</li>
			<li>For the outside of the insert (the 6-well plate), remove 1 mL and add 1 mL of fresh complete growth medium. The 1 mL was chosen for the convenience of changing the medium in one step. If desired, 1.25 mL can be removed and replaced instead.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Collecting the cells
	<ol>
		<li>Collect the cells after 72 h of proximal culture. Take special care at this step to prevent cross-contamination of the HeyA8 cells and HPMCs. Trypsinize the cells, centrifuge them, and lyse the pellet instead of lysing the cells on the membrane to prevent a cross-contamination.</li>
	</ol>
	</li>
	<li>Trypsinizing the cells
	<ol>
		<li>Rinse both sides of the insert with 1x PBS.</li>
		<li>Transfer the inserts to a new well and add trypsin. Add 0.5 mL of trypsin to the inside of the insert and add 2 mL of trypsin to the 6-well plate to trypsinize the cancer cells attached to the lower surface of the insert. Incubate the plate for 2 min at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Neutralizing the trypsin
	<ol>
		<li>Add 1 mL of complete growth medium to the inside of the insert to neutralize the trypsin, pipette up and down, and then take the cells and transfer them to a labeled collection tube. Add an additional 2 mL of complete growth medium for a complete neutralization. Care should be taken not to pierce the membrane while pipetting to prevent a cross-contamination of the cells.</li>
		<li>To collect the cells on the bottom of the insert, pipette the 2 mL trypsin that was added in step 12.2 onto the bottom surface 2&#8211;3x so that the cells fall into the bottom of the 6-well plate. Neutralize the trypsin on the bottom of the plate with 6 mL of growth medium and transfer the contents of the plate to a labeled collection tube.</li>
	</ol>
	</li>
	<li>Collecting and lysing the cells 
	<ol>
		<li>Centrifuge the cells at 500 x g for 3 min and aspirate the media. Resuspend and lyse the cell pellets with 0.7 mL of phenol- and guanidine-thiocyanate-based lysis reagent for the RNA extraction. Isolate the RNA using any suitable kit.</li>
	</ol>
	</li>
</ol>
3. Quantitative Real-time Polymerase Chain Reaction
NOTE: The following steps describe a quantitative real-time polymerase chain reaction (qPCR) to study the gene expressions changes as a result of the proximal culture.
<ol>
	<li>Prepare cDNA from the RNA samples for qPCR. Use any suitable reverse transcriptase with a corresponding buffer and random primers to ensure the reverse transcription of all mRNA (see Table of Materials).</li>
	<li>Assess the changes in the mRNA expression levels of fibronectin (FN1) and E-cadherin (CDH1) and transform growth factor beta 1 (TGF&#946;1) by qPCR with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. It is possible to use standardized commercially available gene expression assays (see Table of Materials) or to design primers.</li>
	<li>Compare the HeyA8 cells grown in proximal culture with HPMCs with the HeyA8 controls and the HPMCs grown in proximal culture with the HPMC controls.</li>
</ol>

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The authors have nothing to disclose.

Understanding the mechanism of paracrine and juxtacrine signaling between cells is essential for developing a better knowledge of normal tissue homeostasis and disease conditions7,8. Most paracrine signaling studies are conducted by collecting conditioned medium from one cell type and using it to treat the other cell type. This method has an advantage in its inherent simplicity. However, it does not accurately recapitulate the localized concentrations of the secr...

The role of productive reciprocal interactions between cancer cells and the tumor microenvironment in tumor progression has been well established and has become a major focus of research in cancer biology1. Similar instances of bidirectional signaling are crucial during wound healing, immune responses, angiogenesis, stem cell niches, and during development2,3,4,5,6,7,8. A common theme in all these biological processes is that cells respond in various ways to extracellular cues from their microenvironment which determine cell fate, tissue physiology, and disease progression. Therefore, the focus has increasingly turned toward developing a better understanding of the mechanisms involved in such cell-cell communications. A majority of such interactions involve paracrine or juxtacrine signaling between cells. Paracrine signaling involves the secretion of specific signaling factors by one cell which are perceived by corresponding receptors on another cell in the vicinity, triggering a response in it9,10, whereas juxtacrine signaling requires direct contact between cellular components of the two cells involved11,12.
Such signaling is a crucial component in tissue homeostasis, as well as in the tumor microenvironment. The cancer cells benefit from paracrine and juxtacrine factors from cells in the tumor stroma, including cancer-associated fibroblasts (CAFs), immune cells, and adipocytes13,14,15,16. The paracrine signaling can be mediated by growth factors, cytokines, chemokines, etc., while the juxtacrine signaling involves juxtaposed ligands and receptors as in Notch signaling, or interactions between integrins and their respective extracellular matrix proteins. We have demonstrated the importance of reciprocal interactions between ovarian cancer cells and CAFs in tumor progression and metastasis14. Similarly, the interactions of metastasizing ovarian cancer cells with the mesothelial cells covering the site of metastasis regulate key microRNAs and transcription factors in the cancer cells which promote metastatic colonization17,18.
Most studies on paracrine signaling involve the use of a conditioned medium collected from one cell type to treat the second cell type with. While this approach has been widely used, it does not effectively replicate the localized high-concentration levels of the secreted factor in the microenvironment of the receiving cell. It also fails to reproduce the kinetics of the continuous flow of the secreted factor being produced by one cell and received by the neighboring cell. Paracrine signaling is effective over short distances as the secreted factors are at the required concentrations only in the vicinity of the source cell and tend to diffuse and dilute out as the distance increases. This localized high concentration of the secreted factor is essential to trigger a response in the receptor cell. Moreover, the response in the recipient cells is also dependent on the balance of newly secreted factors and their continuous depletion through degradation, binding, and internalization in the recipient cells and diffusion away from the source cell. Conditioned medium can be concentrated to account for the higher localized concentrations present in the microenvironment, but that cannot accurately replicate the exact concentrations. Moreover, it cannot mimic the natural kinetics of production and depletion of the factor involved. To more accurately replicate paracrine signaling and separate it from juxtacrine signaling mechanisms, we have devised a novel proximal culture method, which involves growing the two cell types on either surface of a porous membrane. The pores are small enough to prevent juxtacrine interactions and yet allow the exchange of secreted factors at localized high concentrations. In that way, this system retains the kinetics of production and depletion of the paracrine factors.

<table><tbody><tr><td>24 mm Transwell permeable support with 0.4 &amp;micro;m Pore Polycarbonate Membrane Insert</td><td>Corning (Costar)</td><td>3412</td><td>&amp;bull; 10 &amp;micro;m thick translucent polycarbonate membrane&lt;br/&gt; &amp;bull; Treated for optimal cell attachment&lt;br/&gt; &amp;bull; Packaged 6 inserts in a 6 well plate, 4 plates per case&lt;br/&gt; &amp;bull; Membrane must be stained for cell visibility&lt;br/&gt; &amp;bull; Sterilized by gamma radiation</td></tr><tr><td>6 well plate</td><td>Corning (Falcon)</td><td>353046</td><td>Flat Bottom, TC-treated, sterile, with Lid</td></tr><tr><td>15 cm culture dish</td><td>Corning (Falcon)</td><td>353025</td><td>Sterile, TC-treated Cell Culture Dish</td></tr><tr><td>DMEM</td><td>Corning (Cellgro)</td><td>10-013-CV</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>MEM Nonessential amino acids</td><td>Corning (Cellgro)</td><td>25-025-CI</td><td></td></tr><tr><td>MEM Vitamins</td><td>Corning (Cellgro)</td><td>25-020-CI</td><td></td></tr><tr><td>0.25% Trypsin, 2.21 mM EDTA</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>Pipets</td><td></td><td></td><td>Any make is fine</td></tr><tr><td>CO2 Incubator</td><td></td><td></td><td>Any make is fine</td></tr><tr><td>Biosafety level II cabinet</td><td></td><td></td><td>Any make is fine</td></tr><tr><td>FN1 TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs01549976_m1</td><td></td></tr><tr><td>TGFB1 TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs00998133_m1</td><td></td></tr><tr><td>CDH1 TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs01023895_m1</td><td></td></tr><tr><td>GAPDH TaqMan Gene Expression Assay</td><td>ThermoFisher Scientific</td><td>Hs99999905_m1</td><td></td></tr><tr><td>miRNeasy mini RNA isolation Kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher Scientific</td><td>43-688-13</td><td></td></tr><tr><td>HeyA8 ovarian cancer cells</td><td>Obtained from Ernst Lengyel Lab, University of Chicago</td><td></td><td></td></tr><tr><td>TGF&amp;beta; Neutralizing Antibody</td><td>R&amp;amp;D Systems</td><td>MAB1835-100</td><td></td></tr></tbody></table>

a proximal culture method to study paracrine signaling between cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Metastasizing ovarian cancer cells encounter mesothelial cells at the site of metastasis within the peritoneal cavity19. Productive paracrine and juxtacrine interactions with the mesothelial cells help in inducing adaptive responses in the ovarian cancer cells, which enable successful metastasis17,18,20,21. To test the effectiveness of th...

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A Proximal Culture Method to Study Paracrine Signaling Between Cells

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

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9.4K Views.  Indiana University School of Medicine. Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

Video: A Proximal Culture Method to Study Paracrine Signaling Between Cells

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Cancer Research

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Developmental Biology

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches.
This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

The present study outlines a highly reproducible and tractable method to study paracrine noncanonical Wnt signaling events in vitro. This protocol was applied to evaluate the impact of paracrine Wnt5a signaling in murine neural crest cells and myoblasts.

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This ...

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

Immunology and Infection

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Medicine

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...

3D Co-culture with Direct Interaction: Co-culturing Cancer Cells with Monocytes to Study Their Interaction

Source: Espinoza-S&#225;nchez, et al. <a href="https://www.jove.com/v/56589/analyzing-communication-between-monocytes-primary-breast-cancer-cells">Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System.</a> J. Vis. Exp. (2018).
This video describes the 3D co-culture of breast cancer cells with monocytes to study their interaction in an extracellular matrix extract (ECME) based environment. This method helps in studying specific features such as collagen degradation, immune cell recruitment, and cell invasion.

Source: Espinoza-S&#225;nchez, et al. Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ...

JoVE Encyclopedia of Experiments

Breast Cancer

In vitro studies

Proximal Culture System: An In Vitro Culture Technique to  Study Paracrine Signaling Between Cells

Source: Dasari, S. et al. <a href="https://www.jove.com/t/58144">A Proximal Culture Method to Study Paracrine Signaling Between Cells.</a>&#160;J. Vis. Exp. (2018)
In this video, we develop a proximal co-culture model of ovarian cancer cells and primary human mesothelial cells. This physiologically relevant model allows the study of paracrine signaling, where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

Source: Dasari, S. et al. A Proximal Culture Method to Study Paracrine Signaling Between Cells.&#160;J. Vis. Exp. (2018)
In this video, we develop a ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Summary

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Chapters in this video

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

A Cell Culture Model of Resistance Arteries

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

3D Co-culture with Direct Interaction: Co-culturing Cancer Cells with Monocytes to Study Their Interaction

Proximal Culture System: An In Vitro Culture Technique to Study Paracrine Signaling Between Cells