Discipline: Technology and Engineering
Subcategory: Materials Science
Alexis Scott - Alabama State University
Co-Author(s): Ryan Calcutt, Washington University, Missouri; Richard Vincent, New Jersey Institute of Technology, New Jersey; Treena Arinzeh, New Jersey Institute of Technology, New Jersey; Derrick Dean, Alabama State University, Alabama; Ram Dixit, Washington University, Missouri
Introduction: Artificial scaffolds have greatly facilitated experiments to understand how animal cells exert and respond to mechanical stimuli. However, there are no artificial scaffolds that are compatible for growing morphologically normal plant cells. Here we used tobacco BY-2 cells as a model system to identify artificial scaffolds that plant cells can adhere to and grow on. We cultured BY-2 cells on sterilized artificial scaffolds for four days, then washed away unbound cells, and observed the growth using confocal microscopy. We tested several different kinds of scaffolds that varied in composition, fiber density, orientation, and hydrophobicity.
Materials and Methods: Electrospinning was used to prepare nanofibrous scaffolds. The cultured tobacco By-2 cells are grown for two days before five milliliters of cells are placed in a tube with an artificial scaffold. The scaffold has been sterilized overnight with ethanol. After the ethanol, the scaffold is rinsed with water to ensure the ethanol does not kill the cells. Then the tube is placed on a rotator for four days. After four days, the scaffold is taken out of the tube and washed with water to ensure no cells are just lying on the surface. The scaffold is then stained with FM 4-64 dye and seen under confocal microscopy. This is done to see if cells can be seen. The next time this experiment is done, the cells are stained with DAPI. The DAPI stain illuminates the nuclei. The nuclei is needed in order to count the number of cells present on the scaffold.
Results and Discussion: We found that hydrophobic scaffolds supported abundant cell growth with affecting cell morphology, while hydrophilic samples such as gelatin and polyvinyl alcohol did not show any signs of cell growth.
Conclusions: This data is significant because we are one step closer to understanding how to design artificial scaffolds that are effective in controlling plant morphogenesis. Future experiments will examine the extent of plant cell adhesion to the scaffold by scanning electron microscope experiments and pulling individual fibers of the scaffold to see if the cells move with the fibers.
Scanning electron microscopy experiments should be conducted in order to observe whether the cells are visually attached to scaffold fibers. Pulling apart scaffold fibers will allow us to see if the cells move with the fibers. Atomic force microscopy will measure how the cells will react to forces applied to the scaffolds.
Funder Acknowledgement(s): Center for Engineering Mechanobiology award # CMMI-1548571
Faculty Advisor: Ram Dixit, Derrick Dean, email@example.com
Role: I tested scaffolds and sub cultured BY-2 cells. I stained the scaffolds in order to see the cells under the confocal microscope.