Comparative Analysis of Gene Expression in Rat Liver Tissue and Monolayer- and Spheroid-Cultured Hepatocytes

Abstract

A culture system with spherical multicellular aggregates (spheroids), which are formed by the rearrangement and compaction of cell aggregates, is reported to be more useful than the traditional monolayer culture system for the culture of primary hepatocytes. By performing real-time polymerase chain reaction, we analyzed the expression of genes encoding key molecules involved in liver-specific functions, namely, cell adhesion molecules (integrin 3, cadherin 1 and connexin 32), transcription factors (hepatic nuclear factor 4α and CCAAT/enhancer-binding protein β), protein and metabolic enzymes (albumin, glucose-6-phosphatase, tryptophan 2,3-dioxygenase, arginase 1 and cytochrome P450 7A1) and transporters (organic anion transporting peptide 1, multidrug resistance-associated protein 2 and bile salt export pump), in spheroids derived from rat hepatocytes. Further, we compared these expression levels with those in a hepatocyte monolayer and in liver tissue. Only the gene encoding glucose-6-phosphatase (required for sugar metabolism) was expressed at a similar level in both the monolayer culture and liver tissue for 10 days of culture; the expression of all the other genes in the monolayer culture either rapidly decreased or completely disappeared as the culture duration increased. Although the expression levels of all the genes in the spheroids tended to decrease gradually with culture time, they were consistently higher than those in the monolayer culture for at least 10 days of culture. These results suggest that hepatocyte spheroids acquire intercellular organization and largely maintain many intercellular metabolic functions. Thus, the hepatocyte spheroid culture system seems to be promising for various in vitro cell-based assays.

Superior oxygen and glucose supply in perfusion cell cultures compared to static cell cultures demonstrated by simulations using the finite element method

“…[3][4][5] We used a commercially available FEM software, COMSOL MULT-IPHYSICS version 3.5a ͑COMSOL; Burlington, MA͒, to make a three-dimensional model of the microwell array chip and to calculate velocity and concentration profiles in the models.…”

“…1͒ considering the symmetry of the circular Petri dish and the square-shaped microwell array. Based on the aforementioned experiments, [3][4][5] the depth of the cell culture medium was assumed to be 2 mm and cells seeded into the microwells were assumed to form layers that were 92.1 m thick at the bottom of the microwells. The thickness was calculated by assuming that the cell density was 1.5ϫ 10 14 cells/ m 3 and that 1 ϫ 10 6 cells were seeded into all of the 1024 microwells.…”

“…1,2 However, one of the drawbacks of conventional cell-based assays in the drug discovery is the lowered function of the cells in comparison to cells in vivo. To overcome this drawback, the use of microprocesses, e.g., microfabrication, [3][4][5] microfluidics, [6][7][8] and micropatterning, [9][10][11] is on the rise, and the microprocesses are making it easier to precisely control the "cell culture environment." 12,13 Recently, we and other research groups have developed the perfusion culture microchamber array chips [6][7][8] and have applied the perfusion culture microchamber array chips to drug toxicity testing.…”

Quality and Availability Evaluation of Human Hepatocytes Isolated from Resected Partial Livers for Toxicology and Drug Metabolism Studies in Korea

“…따라서, 동결 간세포의 특이 유전자 발현 변화는 동결 및 해동 자체보다는 세포 고유 특성에 따른 부착이나 배 양 형태의 변화를 통하여 나타나는 것으로 추측된다. 간세포는 단층 형태로 배양할 때 간세포 고유의 특성을 10 일 이내에 대부분 소실하는 것으로 알려져 있다 [19]. 본 연구 에서도 일반적으로 약물대사 평가 시에 많이 사용되고 있는 단층 배양법을 적용하여 시험하였고, 따라서 간 특이 활성들 도 배양시간에 따라 정도의 차이는 있지만 점차 낮아지게 된 Fig.…”

The Liver Vascular Bed for Hepatocytes Cell Therapy and Tissue Engineering

“…When hepatocytes formed spheroids they kept their function within the matrices for a longer period of time (Lin and Bissell. 1993;Sakai et al 2010). A recent paper described the beneficial effect for cotransplantation in the peritoneum and the importance of spheroids structure is also stressed (Selden et al 2003;Hamazaki et al 2002;Torok et al 2011).…”