Preparation and seeding
Note: Cell attachment to the scaffold 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.
Steps:
1. Using aseptic technique, remove the scaffold plate from the packaging in a laminar flow environment.
2. Jellagen scaffolds are packaged in a non-tissue culture plate and as such can be used immediately. However, should the scaffold need to be moved, do so with a sterile instrument and take care not to damage the scaffold as it is being transferred.
Note: Tissue-culture treated plasticware may need to be coated with 2% agarose to prevent cell attachment to the plastic instead of the scaffold.
3. Wash the scaffold with cell media of choice before seeding the cells
4. Suspend cells at desired concentration in media and dispense sufficient volume of cell solution on top of the scaffold placed in the well.
5. Transfer to a 37ºC incubator for about 1 – 2 hours to allow for initial cell attachment.
6. After this time add appropriate media to the well
7. Incubate for a further 24 hours for complete attachment.
8. 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 scaffold. Check the morphology of the cells. Cell adherence and spreading will dictate the time for attachment.
9. Once the cells have adequately attached to the scaffold, increase the media volume to provide adequate medium for the culture system
Changing the media
Change the media once attachment has occurred, approx. 24 hours after seeding. The frequency of changes will be determined by cell type, cell attachment efficiency, pH utilization of medium nutrients available to cultures. More frequent medium changes may be required compared to 2D culture systems.
Harvesting of cells
Note: Digestion with proteases such as trypsin, papain (cysteine protease)1 or collagenase are suitable methods of releasing cells from the Jellagen-3D Scaffolds. The strength of the attachment of the cells to the collagen scaffolds 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 confluency of the cells. Collagenase and/or trypsin may be the preferred method. If using papain, the following method is suggested:
1. Prepare enzyme buffer solution (20mM NaAc pH 6.8, 1mM EDTA, 2mM DTT, 330μg/ml papain)
2. Washing the scaffold with EDTA-PBS may assist the protease digestion. Add sufficient volume to cover the scaffold.
3. Aspirate the EDTA-PBS solution from the well.
4. Add sufficient dissociation solution to the well to fully over the scaffold.
5. Transfer to a 37ºC incubator. Check for cell detachment periodically for cell detachment.
6. Once the cells have fully detached, remove the cells and dispense in a centrifuge tube.
7. Centrifuge the cells as required.
