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David Zamora, Jennifer Wehmeyer, Heuy-Ching Wang, Robert Christy, Anthony Johnson; Development of an Amniotic Membrane-Based Cell Scaffold Using Super Critical CO2 Technology. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4703.
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Current ocular wound dressings on the market address such basic needs as wound protection, hydration, and healing. In addition to these criteria, there is a tremendous need for a dressing that is also biocompatible, can be used to deliver antibiotics for infection control or growth factors for healing, as well as deliver cells for therapeutic healing. Commercially available human amniotic membrane (AM) meets some of these criteria, but current techniques for processing confer undesirable tissue architecture and contain chemical preservatives that can decrease healing activity. We hypothesize that supercritical carbon dioxide (SCCO2) technology can be used as a single step technique to process and sterilize AM tissues suitable to support the growth and potential delivery of therapeutic cells to the eye.
Fresh AM was obtained from University Hospital, San Antonio, TX (CRADA for Material Transfer #W81XWH-11-0441). The AM was processed for 10 minutes with 0.004% peracetic acid for sterilization using a Nova 2200 supercritical carbon dioxide pressure chamber, and analyzed by routine histology. Human cornea epithelial cells (HCE), obtained originally from Dr. Ashok Kumar, were cultured (P8) using keratinocyte-serum free medium (GIBCO) supplemented with bovine pituitary extract and epidermal growth factor. HCE were fluorescently labeled using 10mM CFSE and then seeded onto the prepared AM for 0, 24, and 48 hrs and imaged using fluorescent microscopy. MTT viability assays were also performed and proliferation rate determined at these time points.
SCCO2 treatment preserved the structural integrity of AM, while successfully sterilizing the tissue. HCE were successfully seeded onto the surface of the AM and the tissue was able to support migration and proliferation of these cells, as determined by fluorescent microscopy and MTT viability assays.
We have successfully combined existing SCCO2 technology and the healing properties of AM to create an “off-the-shelf,” sterile product for use in the clinic, as well as deployed with military medics. This newly developed scaffold serves the dual purpose of being a biocompatible, biodegradable bandage that can also be used as a platform to deliver cells to the eye.
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