Transwell® Permeable Supports | Guidelines for Use | Corning

Transwell permeable supports are convenient and easy-to-use devices for studies of both anchorage-dependent and anchorage-independent cells. These inserts provide independent access to both sides of a monolayer, thus giving researchers a versatile tool to study transport and other metabolic activities in vitro.

Below you will find guidelines for use for Transwell permeable supports, as well as other helpful tips and techniques.

1. Transwell inserts are used by first adding medium to the multiwell plate, followed by adding the Transwell inserts, and lastly adding the medium and cells to the inside compartment. Recommended medium volumes are shown in the table below.
   
   
2. An initial equilibrium period may be used to improve cell attachment by adding medium to the multiple well plate well and then to the Transwell insert. The plate should then be incubated for at least one hour or overnight at the same temperature that will be used to grow the cells. The cells are then added in fresh medium to the Transwell insert and returned to the incubator.
   
   
3. The medium level should be checked periodically and fresh medium added as required.
   
   
4. Transwell inserts have three openings for standard pipet tips to allow samples to be added or removed from the lower compartment.
   
   
5. Cell monolayers may be fixed and stained while in the Transwell insert using standard cytological techniques. Avoid using solvents that dissolve polystyrene, polycarbonate, or polyester membrane materials. Processing steps may be carried out by sequentially moving the Transwell insert through a series of multiple well multiwell plate wells containing the appropriate reagents. Protocols for fixing and staining Transwell® inserts are available in our online technical library.
   
   
6. If it is necessary to remove cells from Transwell membranes, we recommend rinsing both the Transwell insert and the plate well. Then the dissociating solution should be added to both the well and the Transwell insert and incubated until the cells begin to come off. A protocol, Trypsinization Procedure for Corning® Transwell® Inserts, for removing cells from Transwell inserts is available in the technical section of the Corning Life Sciences website.
   
   
7. The polycarbonate or polyester membrane with the fixed and stained cells attached may be removed from the Transwell insert by carefully cutting around the membrane edges with a scalpel.
   
   
8. The collagen-coated PTFE membrane is fragile and requires careful handling during removal. A wetted cellulosic membrane filter should be placed in direct contact with the underside of the Transwell insert membrane before it is cut out with a scalpel. The wetted, more rigid, cellulosic filter will serve as a support for the collagen-coated membrane.

• Cell morphology and cell densities on permeable supports are influenced by filter pore size.
• Larger pore sizes may permit some cell types to migrate through the pores on the permeable support.
• Cells grown on permeable supports are often sensitive to initial seeding density for good cell attachment. On first use, try bracketing a range of seeding densities for optimum growth.
• Cell attachment and spreading may be improved by pre-incubating permeable supports in medium prior to seeding.
• Cells requiring extracellular matrix coatings on plastic substrates will also require them on permeable supports.
• The Transwell-Clear insert contains a transparent tissue culture-treated polyester membrane that allows easy viewing of cells using phase contrast microscopy.
• The Transwell-COL insert contains a PTFE membrane that has been treated with an equimolar mixture of types I and III bovine placental collagens. This results in a biologically stabilized collagen matrix covering the fibrils of the filter membrane. These Transwell inserts are excellent for the growth of cells requiring a biological coating.

Transwell permeable supports are available in three membrane materials: polycarbonate, polyester (PET), and collagen-coated polytetrafluoroethylene (PTFE). Polycarbonate Transwell inserts feature thin, translucent membranes available in four pore sizes ranging from 0.4 µm to 8.0 µm. Most are treated for optimal cell attachment. They are supplied sterile and come preloaded in multiple well plates or dishes. The polycarbonate membrane is compatible with most organic fixatives and stains. Polyester (PET) Transwell-Clear inserts have microscopically transparent polyester membranes that are tissue culture-treated for optimal cell attachment and growth. Transwell-Clear inserts provide better cell visibility under phase contrast microscopy and allow assessment of cell viability and monolayer formation. Transwell-COL inserts have transparent (when wet), collagen-treated PTFE membranes that promote cell attachment and spreading and allow cells to be visualized during culture. The Transwell-COL membranes are coated with an equimolar mixture of types I and III collagen derived from bovine placentas. Unlike traditional coating techniques that result in occluding film layers, Corning’s coating process results in a biologically stable collagen that covers every fibril of the filter matrix, thereby retaining the porosity of the membrane.

Chemotaxis and invasion assays are instrumental in studying wound repair, cell differentiation, cellular communication, embryonic development, and tumor metastasis. This technical report highlights important factors to consider for optimization and to help you obtain the most accurate results with chemotaxis and invasion assays using permeable supports.

See how using Transwell permeable supports at the air liquid interface can help you achieve more in vivo-like morphology, metabolic activities, and cellular differentiation.