Gene expression and functional analyses of primary rat hepatocytes on nanofiber matrices.
Long term culture of primary hepatocytes is valuable for diagnostic and therapeutic applications. However, standard monolayer culture of primary hepatocytes on tissue culture plastic (TCP) - either uncoated or coated with a biological material such as collagen or laminin - is problematic. Thus, novel support matrices are under development to better maintain gene expression and differentiated function of primary hepatocytes in vitro. In this study, a fabricated nanofiber matrix was compared to control conditions of uncoated and laminin-coated TCP. Gene expression and biochemical analyses were performed to compare functional abilities of the hepatocytes in the different conditions. Hepatocytes cultured on nanofibers maintained higher cytochrome P450 1A activity (0.49 +/- 0.08 ng resorufin/ml/min) compared to hepatocytes on laminin (0.11 +/- 0.05 ng resorufin/ml/min). In addition, albumin production of hepatocytes on nanofibers was greater than twice the production of hepatocytes on laminin (day 14, 34.4 +/- 1.8 vs. 15.9 +/- 4.5 microg albumin/ml/day). Hepatocytes demonstrated the ability to generate urea from ammonia in all conditions; however, hepatocytes performed ureagenesis more effectively on nanofibers than on laminin (0.55 +/- 0.25 microM vs. 0.36 +/- 0.24 microM urea, day 14). Gene expression of hepatocytes cultured on nanofiber and laminin conditions were similar on a per cell basis determined by analysis using a custom microarray of 250 genes expressed in hepatocytes. Similar cell attachment data between conditions and similar numbers of cells expressing the hepatocyte marker hepatocyte nuclear factor 4alphaindicates that hepatocytes grown on nanofibers only marginally display improved hepatic functions compared to laminin control conditions.
Brophy
Colleen M
CM
Department of Biomedical Engineering, Physiology, Mayo Clinic, Rochester, MN 55905, USA.
Luebke-Wheeler
Jennifer L
JL
Amiot
Bruce P
BP
Remmel
Rory P
RP
Rinaldo
Piero
P
Nyberg
Scott L
SL
D000418:Albumins / Q000096:biosynthesis; D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D015870:Gene Expression; D020869:Gene Expression Profiling; D051557:Hepatocyte Nuclear Factor 4 / Q000096:biosynthesis; D022781:Hepatocytes / Q000378:metabolism*; D007797:Laminin / Q000494:pharmacology; D008297:Male; D057139:Nanofibers; D051381:Rats; D017207:Rats, Sprague-Dawley; D014508:Urea / Q000378:metabolism
D000418:Albumins; D051557:Hepatocyte Nuclear Factor 4; D007797:Laminin; D014508:Urea; D019363:Cytochrome P-450 CYP1A1