Preparation Instructions
1. Sterilization:
- Immerse the scaffold in 70% ethanol for 15 minutes.
- Rinse thoroughly with sterile PBS (phosphate-buffered saline).
- Allow the scaffold to air dry in a sterile environment.
2. Handling Tips:
- Use sterile forceps to handle the scaffold.
- Avoid excessive bending to maintain structural integrity.
3. Cell Seeding:
- Prepare a single-cell suspension with a concentration optimized for your study.
- Slowly pipette the cell solution directly onto the scaffold surface.
- Incubate for 30 minutes to allow cell adhesion before adding the complete culture medium.
Sample Protocol: 3D Cell Culture for Skin Tissue Engineering
Objective: Establish a 3D epidermal model using primary human keratinocytes.
Materials Required:
- MEW scaffold
- Primary human keratinocytes
- DMEM/F12 culture medium supplemented with growth factors
- Sterile culture dishes
Procedure:
- Pre-condition the scaffold by immersing it in the culture medium overnight.
- Seed keratinocytes at a density of 5 x 10⁵ cells per scaffold.
- Incubate at 37°C with 5% CO₂ for 24 hours to promote initial attachment.
- Change the medium every 48 hours.
- Assess cell proliferation and morphology using microscopy on days 3, 7, and 14.