Development of a cellular analysis platform featuring arrays of patterned microwells fabricated atop permeable supports

• Main Author: Ornoff, Douglas M
• Format: Dissertation
• Language: English
• Published: Ann Arbor ProQuest Dissertations & Theses 2014
• Subjects: Biomedical engineering; Materials science; Physiology
• ISBN: 9781321142570; 1321142579

Many human pulmonary diseases lead to accumulation of fluid in the alveoli, the air sacs located in the distal lung and at which gas exchange occurs. The most serious example of alveolar fluid buildup is in Acute Respiratory Distress Syndrome (ARDS), in which an insult to the lung results in injury to the cells lining the alveolus, leading to compromise of the alveolar capillary barrier and impaired gas exchange. Current ARDS mortality rates lie at 30-40%. Worsening this problem is the lack of disease-specific therapies for treating ARDS: the cornerstone of treatment is merely supportive respiratory care via mechanical ventilation.
Further investigations into treating ARDS have been hampered by unresolved questions about the normal physiology of alveolar fluid transport. Therefore, new insights are needed in order to develop more effective ARDS therapies. The alveolus is lined by two types of cells: squamous alveolar type 1 cells that cover 98% of the alveolar surface area and small cuboidal alveolar type 2 cells. While studies have examined AT2 cells, the ion transport properties of AT1 cells remain unknown. Recent attempts to culture AT1 cells in bulk monolayers for ion and fluid transport studies have been unsuccessful. It was therefore hypothesized that a microscale device to grow single AT1 cells in conditions that mimic the native lung would enable study of AT1 ion transport.
This dissertation describes the development of microfabricated devices that feature an array of microwells patterned atop a porous or otherwise permeable support. First, a method for fabricating 1002F photoresist into a freestanding microwell array is explored. Next, a strategy to co-fabricate freestanding 1002F films with the hydrogel chitosan to form microwells with a permeable support is described. Review of the literature suggests that this is the first reported co-fabrication of hydrogel and photoresist into a freestanding film. Lastly, an approach to micropattern commercially available permeable supports, etched with submicron-scale cylindrical pores, is presented. Together, these platforms offer potential for growth and analysis of not only primary alveolar cells, but a range of other cell types in a variety of research endeavors, pulmonary and otherwise.