CytoForm® 500

1. Preparation and Seeding:

Note: Cell attachment to the disc is generally the most critical step in tissue culture. Temperature, pH, gas exchange and cell concentration can affect the rate and efficiency of attachment. Optimum seeding rate depends on the type of cell being cultured.

1. Aseptically remove the Electrospun Gelatin Discs from the packaging in a laminar flow work station.
2. Carefully place the discs into the wells of a 48 well tissue culture plate or larger using a sterile instrument.  Be careful not to damage the product as it is being transferred.  It is recommended to use non-treated tissue culture plasticware.
   Note: Tissue-coated plasticware may need to be coated with agarose to prevent cell attachment to the plastic and promote attachment to the disc.
3. Suspend cells at desired concentration (approximately 5,000 cells/cm²) and dispense sufficient volume of cell solution on top of the disc placed in the well.

Note: Avoid dispensing the cell solution too rapidly as this may cause damage to the sponge.

4. Transfer to a 37ºC incubator for about 1 – 2 hours to allow for initial cell attachment.
5. After 1 – 2 hour, remove the plate from the incubator and check for cell attachment.   Additional testing may be required to optimize the time it takes for the cells to attach to the discs.  Check the morphology of the cells. Cell adherence and spreading will dictate the time needed for attachment.
6. Once the cells have adequately attached to the disc, increase the final volume in each well to fully cover and provide adequate medium for the culture system.

2. Changing the Media:

1. Change the media 24 to 36 hours after the initial seeding.  The frequency of changes will be determined by cell type, cell attachment efficiency, and pH. More frequent medium changes may be required compared to 2D culture systems.

3. Harvesting of Cells:

Note: Protease digestion is the standard method of releasing cells from the discs. The strength of the attachment of the cells to the discs will vary from cell line to cell line. The enzyme concentration and digestion time will vary depending upon the activity of the enzyme and the confluence of the cells.  Collagenase and/or trypsin may be the preferred method.

1. Washing the discs with EDTA-PBS may assist the protease digestion. Add sufficient volume to cover the disc.
2. Aspirate the EDTA-PBS solution from the well.
3. Add sufficient dissociation solution to the well to fully submerge the discs.
4. Transfer to a 37ºC incubator.  Check for cell detachment periodically.
5. Once the cells have fully detached, remove the cells and dispense in a centrifuge tube.
6. Centrifuge the cells as required.