A Novel Method for Faster Formation of Rat Liver Cell Spheroids

Abstract

Hepatocyte spheroids are expected to be the main component of the artificial liver bioreactor for their higher function. The preparation of hepatocyte spheroids, however, can require as many as 24 to 96 h. To reduce this time, we investigated a method employing a new technique of rat hepatocyte preparation and a dynamic culture. The modified Seglen's method for standard hepatocyte isolation was altered by elimination of ethyleneglycol bis(aminoethylether) tetraacetate from the first perfusate and calcium from the second perfusate. Isolated hepatocytes were cultured in a spinner flask by spinning at 120 rpm. The modified Seglen's method was used as a control. Cells obtained by the new method were more cohesive and formed a higher proportion of cell aggregates than control cells. In the spinning culture, hepatocytes had a tendency to aggregate and 80% of cells formed spheroids within 6 h of culturing. The mean size of spheroids was 68.5 +/- 18.5 microm. Confocal laser scanning microscopy revealed that individual spheroids contained approximately 30% of nonparenchymal cells over their surface. Using the new hepatocyte preparation method followed by a spinning culture, we were able to produce hepatocyte spheroids in as few as 6 h.

Multicellular Tumor Spheroids for Evaluation of Cytotoxicity and Tumor Growth Inhibitory Effects of Nanomedicines In Vitro: A Comparison of Docetaxel-Loaded Block Copolymer Micelles and Taxotere®

“…In contrast, 3-D MCTS better approximate the state of cancer cells in their native environment and thus can be used to more accurately estimate a drug’s therapeutic potential. A variety of methods have been used to grow MCTS for use in cancer research including spinning culture flasks [41], hanging drops [42], liquid overlay on agarose [43], micropatterned plates [44], and recently, using inter-cellular linkers [45]. However, many of these techniques are impractical, time-consuming, and involve delicate handling procedures, limiting the use of the MCTS model in drug screening and development.…”

Mesenchymal stem cells and Interleukin-6 attenuate liver fibrosis in mice

“…Hepatocytes were isolated from C57BL/6 mice (n=20) according to the two step perfusion method as described previously [18]. Isolated hepatocytes were plated at a concentration of 1 x 10 4 cells/cm 2 in collagen coated plates (Becton Dickinson, USA) in RPMI 1640 medium (Sigma Aldrich, USA) supplemented with 100 ug/ml streptomycin (MP Biomedicals, USA), 100 units/ml penicillin (MP Biomedicals, USA) and 10% fetal bovine serum (Sigma Aldrich, USA) in a humidified incubator at 5% CO 2 and 37°C temperature.…”

Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size

“…Several techniques for spheroid formation have been reported, including the spinner flask, hanging drop, and liquid overlay methods. [18][19][20] However, a common problem with these techniques is the difficulty in controlling spheroid size. In contrast, microfabrication is suitable for the development of sizecontrolled microwells with narrow size distribution.…”