Preparation and seeding
Note: Temperature, pH, gas exchange and cell concentration can affect the rate and efficiency of cell seeding. Optimum seeding rate depends on the type of cell being cultured.
1. Using aseptic technique, remove the coated plate from the packaging in a laminar flow environment.
2. Prepare the plates by washing the wells with the selected tissue culture medium.
Note: Take care to avoid contact with the collagen coating during washing with utensils.
3. Suspend cells at desired concentration in media and dispense sufficient volume of cell solution into the well.
4. Transfer to a 37ºC incubator for about 1 – 2 hours to allow for initial cell attachment.
5. After 24 hours, remove the plate from the incubator and check for cell attachment. Additional testing may be required to optimise the time it takes for the cells to attach.
Changing the media
Change the media 12 to 24 hours after the initial seeding. The frequency of changes will be determined by cell type, cell attachment efficiency, pH utilisation of medium nutrients available to cultures.
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 Coated Plates.
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 confluence 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 coated plate with EDTA-PBS may assist the protease digestion.
3. Aspirate the EDTA-PBS solution from the well.
4.Add sufficient dissociation solution to the well.
5. Transfer to a 37ºC incubator. Periodically check 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.
1. Eun Song., So Yeon Kim., Taehoon Chun., Hyun-Jung Byun. & Young Moo Lee. 2006. Collagen scaffolds derived from a marine source and their biocompatibility. Biomaterials 2. 2951–2961