The Culture of Dermal Replacements

Collagen-based wound dressings have been used to prevent excessive fluid loss and infection in individuals with severe skin damage, as well as to promote the re-growth and healing of the patient\u27s own skin. In this study, problems encountered with cryopreservation of collagen-based wound dressings were researched. A fibroblast cell-line was expanded in culture and seeded onto collagen sponges. The sponges were then cultured for varying lengths of time before freezing them at -80 degrees C. The sponges were analyzed to assess the total number of cells as a function of time in culture. Toxicity tests were performed using varying concentrations of two cryoprotectants, Simethyl Sulfoxide (DMSO) and Glycerol. Varying concentrations were tested in an effort to find the concentration of each that gave the highest cell survival and minimal toxic effects. Over a four hour period, concentrations of up to 10% DMSO did not have an effect on cell viability. The results obtained at 12% DMSO showed that the viability of the cells began to decrease at three hours, and 15% DMSO had a gradual, increasingly toxic effect over time. At glycerol concentrations of 5, 10, and 15%, it was observed that the total cell number was reduced. Efforts were made to culture sponges to obtain a concentration of 1 x 10 (9) fibroblast cells per sponge in order to provide a detectable signal for analysis by magnetic Resonance Imaging. Using MRI, the mechanism of cell death due to cryopreservants can be observed and a more efficient protocol for storing sponges created. However, the highest number of cells cultured per sponge was less than 1.4 x 10 (8). Additional research is needed to improve either the fibroblast concentration on collagen sponges or the resolution obtainable by MRI analysis

Who Does Eric Doll Think He Is?

A question-and-answer column with senior Eric Doll

The Spray-On Bio-Bandage

Fibrin is a natural polymer already used in extreme clinical settings as an adhesive and for shallow wounds when activated by the enzyme thrombin. However, current formations contain irritants, are slow drying, and are not easily washed off. This study used a spray-on bandage composed of thrombin and fibrinogen on a monolayer of 3T3 mouse fibroblast cells to test the enhancement of the body\u27s natural healing without the risks of current formulations. The viability and growth of scratched cells were analyzed using live/dead stain assays 0, 1, and 3 days post-treatment. The results of this study showed cell growth, fibrinogen formation, and scratching were successful. The bottles\u27 spray exerted too much force and caused additional cell death compared to untreated cells. Additionally, the cells migrated too quickly through the scratch, resulting in rapid gap closure. Further research on cell scaffolding, bandage application, and cell viability is required for a successful spray-on bio-bandage.https://digitalcommons.usu.edu/fsrs2022/1001/thumbnail.jp

Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants

The role of myosin Is in endosomal trafficking and the lysosomal system was investigated in a Dictyostelium discoideum myosin I double mutant myoB-/C-, that has been previously shown to exhibit defects in fluid-phase endocytosis during growth in suspension culture (Novak et al., 1995). Various properties of the endosomal pathway in the myoB-/C- double mutant as well as in the myoB- and myoC- single mutants, including intravesicular pH, and intracellular retention time and exocytosis of a fluid phase marker, were found to be indistinguishable from wild-type parental cells. The intimate connection between the contractile vacuole complex and the endocytic pathway in Dictyostelium, and the localization of a myosin I to the contractile vacuole in Acanthamoeba, led us to also examine the structure and function of this organelle in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in the myoB-, myoC- or myoB-/C- cell lines. The transport, processing, and localization of a lysosomal enzyme, alpha-mannosidase, were also unaltered in all three mutants. However, the myoB- and myoB-/C- cell lines, but not the myoC- cell line, were found to oversecrete the lysosomal enzymes alpha-mannosidase and acid phosphatase, during growth and starvation. None of the mutants oversecreted proteins following the constitutive secretory pathway. Two additional myosin I mutants, myoA- and myoA-/B-, were also found to oversecrete the lysosomally localized enzymes alpha-mannosidase and acid phosphatase. Taken together, these results suggest that these myosins do not play a role in the intracellular movement of vesicles, but that they may participate in controlling events that occur at the actin-rich cortical region of the cell. While no direct evidence has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fidelity of the actin cortex, and changes in cortical organization could influence lysosomal-related membrane events such as internalization or transit of vesicles to the cell surface

Skunk River Review, Fall 2013 and Spring 2014

https://openspace.dmacc.edu/skunkriver/1023/thumbnail.jp

Quantitative Whole Body Biodistribution of Fluorescent-Labeled Agents by Non-Invasive Tomographic Imaging

When small molecules or proteins are injected into live animals, their physical and chemical properties will significantly affect pharmacokinetics, tissue penetration, and the ultimate routes of metabolism and clearance. Fluorescence molecular tomography (FMT) offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the major organ systems of living animals, in a manner analogous to traditional approaches with radiolabeled agents. This approach is best used with biotherapeutics (therapeutic antibodies, or other large proteins) or large-scaffold drug-delivery vectors, that are minimally affected by low-level fluorophore conjugation. Application to small molecule drugs should take into account the significant impact of fluorophore labeling on size and physicochemical properties, however, the presents studies show that this technique is readily applied to small molecule agents developed for far-red (FR) or near infrared (NIR) imaging. Quantification by non-invasive FMT correlated well with both fluorescence from tissue homogenates as well as with planar (2D) fluorescence reflectance imaging of excised intact organs (r2 = 0.996 and 0.969, respectively). Dynamic FMT imaging (multiple times from 0 to 24 h) performed in live mice after the injection of four different FR/NIR-labeled agents, including immunoglobulin, 20–50 nm nanoparticles, a large vascular imaging agent, and a small molecule integrin antagonist, showed clear differences in the percentage of injected dose per gram of tissue (%ID/g) in liver, kidney, and bladder signal. Nanoparticles and IgG1 favored liver over kidney signal, the small molecule integrin-binding agent favored rapid kidney and bladder clearance, and the vascular agent, showed both liver and kidney clearance. Further assessment of the volume of distribution of these agents by fluorescent volume added information regarding their biodistribution and highlighted the relatively poor extravasation into tissue by IgG1. These studies demonstrate the ability of quantitative FMT imaging of FR/NIR agents to non-invasively visualize and quantify the biodistribution of different agents over time