Name: development of an insert co-culture system of two cellular types in the absence of cell-cell contact
Uploaded: 2016-07-17T15:00:00
Description: Watch this Scientific Journal Video about Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact at JoVE.com

This work was funded by a Natural Sciences and Engineering Research Council (NSERC) Canada grant to MGM. JR is a NSERC-Vanier student fellow.

N.B.: Each of the following steps should be performed under sterile conditions in a laminar flow hood as required for mammalian cell culture. In addition, the general guidelines for optimal sterile cell cultivation apply, e.g., discarding tips any time they may lead to cross-contamination, reducing the amount of time cells are exposed to the air when performing entire media changes, properly but gently stirring all cell suspensions to ensure their homogenous pipetting, etc. Moreover, inserts are a kind of plasti...

The most critical step of any insert co-culture system experiment actually dwells in choosing the proper insert to use. Pore size and membrane material must be taken into thorough account, without forgetting to consider the type of cells that will be seeded and the purpose of the experiment. For example, chemotaxic assays may use the same type of membrane than cell co-cultures to analyze cell behavior modulations induced by secreted soluble factors in the absence of cell-cell contact. However, both types of experiments r...

The study of tissues, organs or systems in vitro is an attempt to simplify the very complex relationships existing between the several cellular subtypes that comprise multicellular organisms. Indeed, in vitro studies make it possible to acquire a detailed understanding of single cell populations. There are two major advantages of conducting in vitro experiments: 1) reduced cellular interactions, and 2) the ability to readily manipulate the cellular environment. Hence, these two advantages have allowed scientists to predict the behavior of specific cell types in vivo, leading to the ability to regulate outcomes of extrinsic influences in whole organisms. In that sense, in vitro cell culture often works as a bridge connecting basic and applied life sciences. Nonetheless, there are also several disadvantages of working in vitro, the most important one being that a certain reservation may dwell in the physiological relevance of observed phenotypes. Indeed, when a single cell type is grown in a vessel, the culture loses, to a various extent, its cell-cell connections with other cell types, its contribution to the humoral environment from the tissue and organism of origin, and the anchors within the tissue that enabled it to uphold a particular three-dimensional structure sometimes crucial for cell function.
The question of cell-cell relationships has been addressed by the development of mixed culture techniques. In this method, two or more cell populations are grown together in the same culture vessel. However, these mixed cultures bear important inconveniences. On one hand, some cell subtypes do not physically interact with one another in the tissue of origin and rely solely on paracrine communications sustained by secreted soluble factors and nearby receptors. This is the case for several inflammatory processes that depend on proximal cytokine signaling. In mixed cultures, physical interactions are unavoidable and make it impossible to study paracrine communications in the absence of cell-cell contacts that can yield altered results. On the other hand, achieving cell-specific interpretations from within a mixed population becomes unfeasible without the use of harsh separation techniques that could significantly affect results.
To solve these important issues, the use of conditioned media has been developed as a technique allowing for compartmentalized cultures and the study of paracrine signaling. This method requires the transfer of the supernatant of one cell type, thus named conditioned medium, to wells containing another population of cells. Yet, an important drawback is that short-lived molecules do not survive long enough in the conditioned medium to be transferred to the wells of the second population of cells. Even long-lived molecules will be greatly diluted over time due to diffusion. Furthermore, both cell populations only participate in unidirectional paracrine communication rather than in active bidirectional communication. This leads to the absence of feedback signaling that is vital in recreating accurate multicellular relationships as they exist in vivo.
As a consequence and driven by the need to better simulate the original in vivo conditions in the in vitro cellular environment, several advances in cell culture techniques have been achieved over the years. One of the most significant advancements has been the use of permeable supports with microporous membranes for compartmentalizing cell cultures, used for the first time by Grobstein in 19531. Such permeable supports have been tailored over the years to accommodate numerous cell types and to be used in several different applications. Nowadays, these supports exist as hollow inserts that are designed to rest in wells from a multiwell tissue culture plate or in circular dishes. In a co-culture system, the insert contains one cell type whereas the well or dish contains the other cellular population, allowing to study the contribution of two different populations of cells on their humoral environment (Figure 1). As a result, cellular polarity (basolateral vs apical secretion or signal reception) is preserved, thus conferring insert co-culture systems an important advantage over mixed cultures and conditioned medium techniques. Several types of membrane materials are available, the most common ones being polyester (PET), polycarbonate (PC) or collagen-coated polytetrafluoroethylene (PTFE), and they exist in different pore sizes ranging from 0.4 &#181;m to 12.0 &#181;m. These varieties of materials and pore sizes offer a spectrum of inserts exerting variable features relevant to optical properties, membrane thickness and cell adherence that make them practical at different levels for the following uses not limited to: 
-studying cell differentiation, embryonic development, tumor metastasis and wound repair by chemotaxic assays through permeable membranes; 
-evaluating drug penetration by assessing their transport through epithelial or endothelial monolayers cultured on permeable supports, and; 
-performing cell co-cultures to analyze cell behavior modulations induced by secreted soluble factors in the absence of cell-cell contact.
The purpose of this article is to describe general methodological guidelines to fulfill the third function stated above, that is to evaluate cellular changes mediated by secreted soluble factors in the absence of cell-cell contact using an insert co-culture system. Several different fields of research make use of insert co-culture systems in order to answer questions relevant to the effect of secreted soluble factors on populations of cells. Indeed, paracrine signaling that modulates cellular behavior at various levels is pertinent in all tissues and systems, which makes insert co-culture systems indispensible to ensure advances in these fields. Conversely, the use of inserts can confirm that signal transduction is by direct cell-cell contact and not by secreted factors. One of the most important uses of inserts is in inflammation studies2-14 where the effect of secreted cytokines is evaluated in various cellular players of immunity. In particular, the study of inflammation in the central nervous system (CNS) has greatly profited from insert co-culture studies, which have allowed to better defining the distinct paracrine roles of neurons and microglia in driving neuroinflammation15-21. These systems were also devised to study the anti-inflammatory potential of molecules that relies on their ability to reduce or inhibit the secretion of pro-inflammatory factors22-26. Research pertaining to cancer27-31, in particular the mechanisms underlying angiogenesis32-34 and inflammation35-42 in tumorigenesis, also benefits from insert co-culture systems. Moreover, soluble factors are of prime importance in the processes that drive differentiation and several studies have used inserts to answer questions in that particular field43-50. In the CNS, seeing as neural tissue has a very limited renewal potential, the study of neurotrophism and neuroprotection is fundamental and has been widely ensured by the use of stem cells in co-culture systems51-56. In addition, inserts are also utilized in as diverse fields as nephrology57,58, endothelial interactions and angiogenesis59-62, apoptosis signaling63-65, inflammation in obesity and metabolic syndrome22,23,66-67, inner ear hair cell protection68,69, and even in fungus virulence70,71 and parasitology72,73.
This article offers general methodological guidelines in order to set up an experiment in view of evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. In particular, we will focus our attention on nerve cell co-cultures and their uses in studying neuroinflammatory process. Given the very vast spectrum of experiments that inserts make possible to pilot, it is unbearable to cover every aspect of this cell culture technique. As an example, a specific protocol to measure the effects of cytokines secreted by lipopolysaccharide (LPS)-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RPMI-1640 medium</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">R8755</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Nutrient Mixture F-12 Ham</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D6421</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use, must be supplemented with 0.365 gm/L L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Horse serum</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">30-2040</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Fetal bovine serum</td>
			<td style="width:71px;">MultiCell</td>
			<td style="width:119px;">80350</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:232px;">Nerve Growth Factor-7S from murine submaxillary gland</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">N0513</td>
			<td style="width:328px;">Reconstitute the lyophilized powder in a solution of buffered saline or tissue culture medium containing 0.1&#8211;1.0% bovine serum albumin or 1-10% serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T3924</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Lipopolysaccharides from&#160;Escherichia coli&#160;055:B5</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">L2880</td>
			<td style="width:328px;">Toxic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Cell culture inserts for use with 24-well plates</td>
			<td style="width:71px;">BD Falcon</td>
			<td style="width:119px;">353095</td>
			<td style="width:328px;">0.4 &#956;m pores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">24-well plates</td>
			<td style="width:71px;">TrueLine</td>
			<td style="width:119px;">TR5002</td>
			<td style="width:328px;">Coat with collagen before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Routine PC12 cell culture medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Routine N9 cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160; 85% RPMI medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 90% DMEM-F12 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160; 10% heat-inactivated horse serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 10% heat-inactivated horse serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160; 5% heat-inactivated fetal bovine serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC12 differentiation medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>N9 treatment medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 99% RPMI medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 99% DMEM-F12 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1% heat-inactivated fetal bovine serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 1% heat-inactivated horse serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 50 ng/mL nerve growth factor</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC12 treatment medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 99% RPMI medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1% heat-inactivated fetal bovine serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an insert co-culture system of two cellular types in the absence of cell-cell contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

The use of insert co-culture systems is particularly pertinent in the study of neuroinflammatory processes that showcase paracrine relationships between different cellular players of the CNS. Immunity in the CNS is accomplished mainly by resident cells called microglia that monitor their environment in their resting ramified state (Figure 2A) and are capable of sensing disturbances that could trouble the very precious homeostasis necessary for proper neuronal function<sup...

Watch this Scientific Journal Video about Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact at JoVE.com

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

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