Device Bonding and Antibody Binding: Developments for On-Chip Differentiation of Induced Pluripotent Stem Cells into Hepatocytes
Sammanfattning: Abstract This thesis describes the bonding optimization of a liver-on-a-chip microfluidic device for evaluation of cultured induced pluripotent stem cell (iPSC)-derived definitive endoderm (DE) cells during their targeted differentiation into mature hepatocytes. Furthermore, it describes the optimization of a number of antibody assays for use during said differentiation, for the purpose of studying the cellular phenotype. The device was used to carry out such an on-chip differentiation in order to study whether cells differentiated from definitive endoderm into hepatocytes on-chip would exhibit a different secretion of albumin than cells differentiated in conventional multiwell plates. In order to enable intracellular assays and facilitate immunofluorescent staining of the cells being cultured in the liver-on-a-chip device, the plasma treatment used for assembling the device was adjusted. A 3-second exposure to oxygen plasma induced by 5W radiofrequency excitation was found to produce a partial covalent bond between the device and its glass slide that was strong enough to resist leakage under 400µl/s flow while still being detachable from the glass slide without injury to the cells cultured inside. A panel of antibodies for immunofluorescent (IF) staining were tested and optimized on iPSC-derived DE cells grown in 96-well plates, along with periodic acid and Schiff's reagent (PAS) staining and enzyme-linked immunosorbent assays (ELISAs) in order to deremine how best to assay the maturity of the cells being cultured in the devices. It was found that while IF and PAS staining offer valuable information about the cells' maturity and functionality, the fact that they are terminal, less quantitative than ELISAs and more difficult to perform make them a less appealing option than the latter. iPSC-derived DE cells were differentiated on-chip and in 96-well plates, and their supernatant collected and assayed for albumin by means of ELISA. The device-cultured cells were found to have secreted noticeably less albumin than their well plate counterparts, relative to their initial cell number. This stems, at least in part, from the devices having far fewer cells than the well plates as a consequence of practical limitations of the device seeding procedure. Still, the presence of albumin in the supernatant of both cultures shows that they successfully reached maturity in the on-chip cultures as well as in the well plates. Once the practical limitations have been overcome through changes to the device design and/or cell culture protocol, the device offers a promising platform for differentiation of iPSC-derived cells in a physiologically relevant 3D environment.
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