Directions for Use

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A. Preparation and Seeding:

Note: Cell attachment to the sponge 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 sponge discs from the packaging in a laminar flow work station.
2. Carefully place the sponges into the wells of a 12 well (21mm sponge) or 96 well (4mm sponge) tissue culture plate using a sterile instrument. Be careful not to damage the sponge 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 instead of the sponge.
3. Suspend cells at desired concentration (1×10⁴– 1×10⁵cells/mL) and dispense sufficient volume of cell solution on top of the sponge placed in the well. Note: An alternative method is to suspend cells in a neutralized collagen solution (such as PureCol® type I collagen Catalog #5005-100ML or PureCol® EZ Gel Catalog #5074-35ML). Dispense collagen/cell solution on top of the sponge placed in the well.
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 sponge. Check the morphology of the cells. Cell adherence and spreading will dictate the time for attachment.
6. Once the cells have adequately attached to the sponge, increase the final volume in each well to fully cover and provide adequate medium for the culture system.

B. Changing the Media:

1. 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 (maintain at pH 7.0 to 7.4) utilization of medium nutrients available to cultures. More frequent medium changes may be required compared to 2D culture systems.

C. Harvesting of Cells:

Note: Protease digestion is the standard method of releasing cells from the sponges. The strength of the attachment of the cells to the collagen sponges 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 sponge with EDTA-PBS may assist the protease digestion. Add sufficient volume to cover the sponge.
2. Aspirate the EDTA-PBS solution from the well.
3. Add sufficient dissociation solution to the well to fully over the sponge.
4. Transfer to a 37ºC incubator. Check for cell detachment periodically for cell detachment.
5. Once the cells have fully detached, remove the cells and dispense in a centrifuge tube.
6. Centrifuge the cells as require.

Product Cell Assay

Endothelial Fibroblasts at 100X magnification. Images taken after 8 days of cell culture within SpongeCol^®. Cells stained with Endothelial RSP.

Product References

References for SpongeCol^®:

1. Mak, W. C., et al. “Thermo-Rheological Responsive Microcapsules for Time-Dependent Controlled Release of Human Mesenchymal Stromal Cells.”Biomater. Sci., vol. 5, no. 11, 2017, pp. 2241–2250., doi:10.1039/c7bm00663b.

2. Udhayakumar, Sivalingam, et al. “l-Arginine Intercedes Bio-Crosslinking of a Collagen–Chitosan 3D-Hybrid Scaffold for Tissue Engineering and Regeneration: in Silico, in Vitro, and in Vivo Studies.”RSC Advances, vol. 7, no. 40, 2017, pp. 25070–25088., doi:10.1039/c7ra02842c.

3. Ferreira, L.p., et al. “Design of Spherically Structured 3D in Vitro Tumor Models -Advances and Prospects.”Acta Biomaterialia, 2018, doi:10.1016/j.actbio.2018.05.034.

4. Hatsell, Sarah J., et al. “ACVR1 R206H Receptor Mutation Causes Fibrodysplasia Ossificans Progressiva by Imparting Responsiveness to Activin A.”Science Translational Medicine, vol. 7, no. 303, Feb. 2015, doi:10.1126/scitranslmed.aac4358.

5. Anton-Sales, I., Beekmann, U., Laromaine, A., Roig, A. & Kralisch, D. Opportunities of Bacterial Cellulose to Treat Epithelial Tissues.Current Drug Targets20,808–822 (2019).

Product Videos

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Safety and Documentation

Safety Data Sheet

Certificate of Origin 4mm

Certificate of Origin 21mm

Declaration of Material Source

Product Disclaimer

This product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.