How to Properly Seed 6-Well Transwell Inserts for Epithelial Cells: A Step-by-Step Guide

How to Properly Seed 6-Well Transwell Inserts for Epithelial Cells

Proper seeding in 6-well transwell inserts requires attention to cell density, media volume, and cell viability factors to ensure optimal growth and function of epithelial cells. The procedure involves preparing the insert and cells carefully to avoid overcrowding and dehydration.

1. Preparing the Transwell Inserts

• Use sterile 6-well transwell inserts with appropriate membrane pore size, commonly 0.4 μm for epithelial cells.
• Pre-wet the membrane by adding culture media to both apical and basal chambers and incubate for 30 minutes at 37°C.
• Remove media from the apical side before seeding.

2. Cell Suspension and Density

Cell density is crucial to avoid overcrowding or under-seeding. Overcrowding reduces nutrient access and oxygen exchange, which harms viability. Insufficient cells cause slow monolayer formation.

• Harvest epithelial cells in log-phase growth for best viability.
• Count cells accurately using a hemocytometer or an automated counter.
• Seed at a density of approximately 1-2 × 105 cells per cm2. For a 6-well transwell insert (~4.67 cm2), this corresponds to roughly 5 × 105 to 1 × 106 cells per insert.
• Resuspend cells in a small volume (100–200 μL) of complete culture medium for even distribution on the membrane surface.

3. Media Composition and Supplements

Maintain cell viability by using appropriate culture media supplemented with necessary factors.

• Use epithelial-specific culture media tailored to the cell line.
• Consider adding supplements to enhance proliferation: for example, ROCK inhibitor at a 1:500 dilution may improve proliferation and survival, inspired by protocols in stem cell cultures.

4. Seeding Procedure

1. Carefully dispense the cell suspension onto the apical side of the insert.
2. Avoid disturbing the membrane; dispense slowly to ensure uniform coverage.
3. Add culture medium to the basolateral chamber to support nutrition from beneath.
4. Incubate at 37°C, 5% CO2 without disturbance for at least 24 hours to allow cells to adhere.

5. Monitoring and Maintenance

Check cells regularly for confluency and morphology changes.

• Avoid media changes in the first 24 hours to prevent detachment.
• Refresh media every 2-3 days carefully, replacing media in both apical and basolateral compartments.

Summary of Key Steps

• Pre-wet transwell membranes before seeding.
• Seed at optimal density (~5 × 105 cells per insert) to prevent overcrowding or sparse growth.
• Use epithelial-specific media; consider supplements like ROCK inhibitor to enhance proliferation.
• Seed cells gently and maintain culture conditions to support viability.
• Regularly monitor and change media carefully.