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

Summary

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.

Abstract

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.

Introduction

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 influence....

Protocol

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 plasticware that require special handling. First, whenever inserts are manipulated, avoid touching the fragile membrane, which tears easily and could theref....

Results

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

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI-1640 medium	Sigma	R8755	Warm in 37 °C water bath before use
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham	Sigma	D6421	Warm in 37 °C water bath before use, must be supplemented with 0.365 gm/L L-glutamine
Horse serum	ATCC	30-2040	Warm in 37 °C water bath before use
Fetal bovine serum	MultiCell	80350	Warm in 37 °C water bath before use
Nerve Growth Factor-7S from murine submaxillary gland	Sigma	N0513	Reconstitute the lyophilized powder in a solution of buffered saline or tissue culture medium containing 0.1–1.0% bovine serum albumin or 1-10% serum
Trypsin-EDTA solution	Sigma	T3924	Warm in 37 °C water bath before use
Lipopolysaccharides from Escherichia coli 055:B5	Sigma	L2880	Toxic
Cell culture inserts for use with 24-well plates	BD Falcon	353095	0.4 μm pores
24-well plates	TrueLine	TR5002	Coat with collagen before use
Routine PC12 cell culture medium			Routine N9 cell culture medium
-       85% RPMI medium			-       90% DMEM-F12 medium
-       10% heat-inactivated horse serum			-       10% heat-inactivated horse serum
-       5% heat-inactivated fetal bovine serum
PC12 differentiation medium			N9 treatment medium
-        99% RPMI medium			-       99% DMEM-F12 medium
-        1% heat-inactivated fetal bovine serum			-       1% heat-inactivated fetal bovine serum
-        50 ng/mL nerve growth factor
PC12 treatment medium
-        99% RPMI medium
-        1% heat-inactivated fetal bovine serum