TY - JOUR
T1 - Ultrastructural and functional investigations of adult hepatocyte spheroids during in vitro cultivation
AU - Dvir-Ginzberg, Mona
AU - Elkayam, Tsiona
AU - Aflalo, Eliahu D.
AU - Agbaria, Riad
AU - Cohen, Smadar
PY - 2004/1/1
Y1 - 2004/1/1
N2 - Cultivation of primary adult hepatocytes creates a challenge because of their loss of hepatocellular functions if prevented from attaining polarized cell-cell and cell-matrix interactions. Thus, when hepatocytes are seeded in nonadhesive porous alginate scaffolds, they form 100-μm-diameter spheroids with enhanced cell-cell interactions. Using transmission electron microcopy (TEM), histology, and functional studies, we investigated the state of hepatocyte spheroids during in vitro cultivation. TEM of day 3 spheroids revealed multiple cell layers, with tight junctions between adjacent cells and microvillus-lined channels that resembled bile canaliculi, both structurally and functionally. When copper ions were added to the external medium, the spheroidal hepatocytes performed endocytosis and eventually secreted the heavy metal ions into the bile lumens. From day 8 on, however, there was a rapid decline in cell viability. Histology and TEM analysis of day 13 spheroids revealed a necrotic center, with one viable cell layer on the outskirts. The absence of DNA laddering and negative results in TUNEL assay indicated that apoptosis is not the main process leading to cell death. Cell necrosis may be a result of accumulated bile secretions in the compacted spheroids. Collectively, our results suggest that spheroids derived from adult hepatocytes may have limited utility in long-term applications.
AB - Cultivation of primary adult hepatocytes creates a challenge because of their loss of hepatocellular functions if prevented from attaining polarized cell-cell and cell-matrix interactions. Thus, when hepatocytes are seeded in nonadhesive porous alginate scaffolds, they form 100-μm-diameter spheroids with enhanced cell-cell interactions. Using transmission electron microcopy (TEM), histology, and functional studies, we investigated the state of hepatocyte spheroids during in vitro cultivation. TEM of day 3 spheroids revealed multiple cell layers, with tight junctions between adjacent cells and microvillus-lined channels that resembled bile canaliculi, both structurally and functionally. When copper ions were added to the external medium, the spheroidal hepatocytes performed endocytosis and eventually secreted the heavy metal ions into the bile lumens. From day 8 on, however, there was a rapid decline in cell viability. Histology and TEM analysis of day 13 spheroids revealed a necrotic center, with one viable cell layer on the outskirts. The absence of DNA laddering and negative results in TUNEL assay indicated that apoptosis is not the main process leading to cell death. Cell necrosis may be a result of accumulated bile secretions in the compacted spheroids. Collectively, our results suggest that spheroids derived from adult hepatocytes may have limited utility in long-term applications.
UR - http://www.scopus.com/inward/record.url?scp=11144228413&partnerID=8YFLogxK
U2 - 10.1089/ten.2004.10.1806
DO - 10.1089/ten.2004.10.1806
M3 - Article
C2 - 15684689
AN - SCOPUS:11144228413
SN - 1076-3279
VL - 10
SP - 1806
EP - 1817
JO - Tissue Engineering
JF - Tissue Engineering
IS - 11-12
ER -