Cell culture inserts offer a way to grow cells that goes beyond flat, two-dimensional culture dishes. These tools create environments that better mimic the complex, three-dimensional structures and polarized conditions found within living tissues. They enable researchers to cultivate cells in a manner that reflects their natural physiological state. This allows for more accurate studies of cellular behavior and interactions.
Anatomy of an Insert
A cell culture insert consists of a porous membrane within a cylindrical housing that fits into the wells of multi-well plates. This design divides a well into two compartments: an upper, or apical, chamber and a lower, or basolateral, chamber. Cells are seeded onto the upper surface of the membrane, allowing them to receive nutrients and signaling molecules from media on both sides.
The membrane is a thin, permeable barrier, often composed of polycarbonate (PC), polyethylene terephthalate (PET), or polytetrafluoroethylene (PTFE). Polycarbonate membranes offer optical clarity for microscopic observation, while polyethylene terephthalate provides good mechanical strength and a smoother surface. Polytetrafluoroethylene membranes are chosen for their chemical resistance and often have a more opaque appearance.
Why and How They Are Used
Researchers employ cell culture inserts to establish polarized cell layers and create separated environments. The insert’s design allows cells, when grown on the membrane, to develop distinct apical and basolateral surfaces, much like they do in tissues such as the gut lining or kidney tubules. This polarization is achieved by providing access to different media compositions or factors above and below the cell layer.
The separation offered by inserts facilitates indirect co-culture experiments, where different cell types can be grown in the apical and basolateral compartments without direct physical contact. This setup aids in studying cell-to-cell communication mediated by secreted factors, such as hormones or growth factors. Allowing cells to interact through soluble molecules across the porous membrane helps researchers understand processes like tissue development or immune responses. Growing cells on a permeable membrane with access to media from both sides offers a more three-dimensional environment than traditional flat plastic dishes. This configuration better simulates physiological conditions, making it suitable for forming barrier models, where the integrity and permeability of a cell layer can be measured.
Key Applications in Research
Cell culture inserts are used to model biological barriers, offering insights into their function and integrity. They are employed to create epithelial barriers, such as those found in the intestine or kidney, allowing researchers to study nutrient absorption, drug transport, or pathogen entry. Inserts also help form endothelial barriers, like the blood-brain barrier, to investigate drug permeability into the central nervous system or the mechanisms of inflammation. The ability to measure the passage of substances across these cell layers is a direct application of their barrier-forming capacity.
These tools also play a role in co-culture studies, where the interactions between different cell types are explored. For instance, researchers might co-culture immune cells with target cells to understand cytokine signaling or grow stromal cells alongside epithelial cells to study tissue differentiation and morphogenesis. Such setups provide a controlled environment to isolate the effects of soluble factors on cell behavior.
Inserts are utilized in cell migration and invasion assays, particularly in cancer research. Cells are seeded onto the membrane, often coated with an extracellular matrix, and their ability to migrate through the pores or invade a matrix towards a chemoattractant in the lower chamber is assessed. This method helps in understanding metastatic processes or the movement of immune cells during inflammation. In pharmaceutical research, inserts model drug transport and absorption across biological membranes. This application helps predict how drugs will be absorbed in the body or how they might cross specific tissue barriers, aiding in drug discovery and development.
Selecting the Right Insert
Choosing the appropriate cell culture insert for an experiment involves considering several technical specifications. The pore size of the membrane is a primary factor, dictating what can pass through and how cells interact with the surface. Smaller pore sizes (0.4 to 1.0 micrometers) suit tight cellular barriers for permeability studies, restricting cell passage while allowing molecules to diffuse. Larger pore sizes (3.0 to 8.0 micrometers) are preferred for cell migration and invasion assays, as they permit cells to physically move through the membrane.
The membrane material also influences cell attachment and experimental outcomes. Polycarbonate membranes are often chosen for their optical transparency, while polyethylene terephthalate membranes are selected for their mechanical strength and ability to promote cell adhesion. Polytetrafluoroethylene membranes are used for their chemical resistance, though they tend to be opaque.
Compatibility with standard laboratory equipment is another practical consideration, with inserts available to fit multi-well plate formats such as 6-well, 12-well, and 24-well plates. Finally, surface treatment of the membrane is important for cell adhesion; some membranes are tissue culture-treated to enhance cell attachment, while others are untreated for applications where minimal adhesion is desired or specific coatings will be applied by the researcher.