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Biomedical Microdevices 6:4, 325339, 2004C 2004 Kluwer Academic Publishers. Manufactured in The Netherlands.NanoLiterBioReactor: Long-Term Mammalian
Cell Culture at Nanofabricated ScaleAles Prokop,1,2, Zdenka Prokop,1 David Schaffer,3
Eugene Kozlov,2 John Wikswo,4,5,6 David Cliffel,7
and Franz Baudenbacher41NanoDelivery, Inc., Nashville, TN 372112Chemical Engineering, Vanderbilt University, Nashville, TN 372353Mechanical Engineering4Biomedical Engineering5Physics & Astronomy6Molecular Physiology & Biophysics7Chemistry, Nashville, TN 37235, USAE-mail: [email protected]. There is a need for microminiaturized cell-culture environments, i.e. NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian
cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. These devices would
enable the development of a new class of miniature, automated
cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin
exposure.We fabricated NBR prototypes, each of which incorporates a
culture chamber, inlet and outlet ports, and connecting microfluidic conduits. The fluidic components were molded in polydimethylsiloxane (PDMS) using soft-lithography techniques, and sealed via
plasma activation against a glass slide, which served as the primary culture substrate in the NBR. The input and outlet ports were
punched into the PDMS block, and enabled the supply and withdrawal of culture medium into/from the culture chamber (10100
nL volume), as well as cell seeding. Because of the intrinsically high
oxygen permeability of the PDMS material, no additional CO2/air
supply was necessary.The developmental process for the NBR typically employed several iterations of the following steps: Conceptual design, mask generation, photolithography, soft lithography, and proof-of-concept culture assay. We have arrived at several intermediate designs. One
is termed circular NBR with a central post (CP-NBR), another,
perfusion (grid) NBR (PG-NBR), and a third version, multitrap
(cage) NBR (MT-NBR), the last two providing total cell retention.Three cells lines were tested in detail: a fibroblast cell line, CHO
cells, and hepatocytes. Prior to the culturing trials, extensive biocompatibility tests were performed on all materials to be employed
in the NBR design. To delineate the effect of cell seeding density
on cell viability and survival, we conducted separate plating experiments using standard culture protocols in well-plate dishes. In
both experiments, PicoGreen assays were used to evaluate the extent of cell growth achieved in 15 days following the seeding. Low
seeding densities resulted in the absence...