Cell Seeding

• Prepare a cell suspension at 1 × 10⁶ cells/mL.

• Carefully pipette 100 μL of cell suspension (containing 100,000 cells) directly onto the center of each scaffold.

• Allow cells to attach for 1-3 hours at 37°C in the incubator without additional media.
  
  • After incubation, gently add 900 μL of cell culture medium to each well to bring the total volume to 1 mL per well.
  Do not let the samples dry out. As needed, carefully replenish with 10-40 μL of cell culture medium ~each hour without overflowing the scaffold or dissociating the cells from the sample. The incubation time is dependent on the cell type used. Include a 2D control to confirm cell attachment of the specific cell type used. Confirm initial seeding and attachment under an inverted microscope before adding more media.

• After incubation, gently add 900 μL of cell culture medium to each well to bring the total volume to 1 mL per well.

Cell Culturing

1. Incubate the well plate with the cell-seeded samples at 37°C and 5% CO₂.
   Note: The scaffolds are compatible with a wide range of standard CO₂ incubators, tri-gas incubators, and hypoxia chambers designed for cell culture applications. Select the appropriate incubation environment based on the experimental requirements.
2. Replace the culture media according to your standard cell culture protocol (e.g., every 2–3 days).* Carefully aspirate the spent media from the side of each well to avoid disturbing the scaffolds.* Gently add 1 mL of pre-warmed complete DMEM to each well.Note: Ensure all media handling is performed under sterile conditions to maintain culture integrity.
3. Determine culture duration based on the intended downstream application:
   * Short-term culture (24–72 hours): Recommended for studies focused on cell attachment and proliferation.
   * Long-term culture (4–56 days): Suitable for applications involving extracellular matrix production or cellular differentiation.
   Note: Optimal culture duration may vary depending on cell type and experimental design. Always refer to your specific assay requirements.