Suspension culture of hepatocyte‐derived reporter cells in presence of albumin to form stable three‐dimensional spheroids

Abstract

Several studies in the past have formed 3-dimensional (3D) spheroids of primary hepatocytes in suspension culture. Unfortunately, primary hepatocytes in a suspension environment tend to lose their differentiated function over time, generally due to damage from fluid shear stress and eventual spheroid settling. We have therefore created a novel suspension culture system, by seeding H35 rat hepatoma cells, a hepatocyte-derived cell line, in a 24-well tissue culture polystyrene (TCPS) plate placed atop an orbital shaker to create 3D spheroids. To provide stability to the formed spheroids, we used a long-chain polymer, bovine serum albumin (BSA), dissolved in the cell culture medium and/or coated on TCPS surfaces placed in suspension configurations. Our results demonstrate that BSA coating of culture surfaces resulted in uniform and well-defined spheroids with little spheroid settling or "flattening" of cell colonies in either static or suspension configurations. In BSA-coated suspension systems, spheroid size scaled with the amount of BSA dissolved in culture medium. In static uncoated cultures, the normalized rat albumin production levels were enhanced by addition of BSA within culture medium. Thus, both addition of BSA to culture medium and application of BSA as a surface coating appear to be meaningful avenues for tailoring spheroid morphology and function. This 24-well plate suspension culture system may be a valuable tool for high throughput investigations of liver cell behavior in a stable, uniform, 3D spheroid state.

Effect of amine content and chemistry on long‐term, three‐dimensional hepatocyte spheroid culture atop aminated elastin‐like polypeptide coatings

“…They showed that an intact actin network was required for spheroid formation, and particularly, the actin configuration was dependent on the cell–cell interactions with the cells in the center of the spheroids showing cuboidal shape with cortical actin and the cells at the periphery of the spheroids showing spread cells with extended actin filaments . We have observed that the extensive cell–cell interactions in spheroids lead to contact inhibition of cellular proliferation and trigger superior cellular differentiation . Glicklis et al assert that spheroids of 100 µm diameter are ideal for maximizing cell–cell interaction while maintaining adequate oxygen transport to cells located near the spheroid center .…”

“…Nevertheless, irregularly shaped aggregates have been documented in the past, as when Nelson et al cultured primary porcine hepatocytes in low‐shear microgravity within an NASA bioreactor . During our previous studies, H35 hepatoma cells cultured in suspension also produced very large globular aggregates in the presence of bovine serum albumin dissolved within the culture medium …”

“…Relative mRNA expression levels were determined by real‐time reverse transcription quantitative polymerase chain reaction (qRT‐PCR) using an ABI7000 Real‐Time PCR Detection System (Life Technologies, Grand Island, NY). All reactions were performed with Bullseye EvaGreen qPCR MasterMix (Midsci, St Louis, MO) beginning with 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The rat‐specific primers were the same as previously published . Results were analyzed comparing gene expression relative to control Hprt gene expression (ΔΔCT).…”

“…All reactions were performed with Bullseye EvaGreen qPCR MasterMix (Midsci, St Louis, MO) beginning with 958C for 10 min followed by 40 cycles of 958C for 15 s and 608C for 60 s. The rat-specific primers were the same as previously published. 25 Results were analyzed comparing gene expression relative to control Hprt gene expression (DDCT). qPCR data were expressed as mean fold change (2 DDCT ) 6 95% confidence intervals (CI).…”

Dynamic Expression Patterns of Differential Proteins during Early Invasion of Hepatocellular Carcinoma

“…Tumor spheroids exhibit many distinct advantages in resembling the 3D cytoarchitecture and pathophysiological micromilieu of tumors [11]. The established 3D hepatocyte cultures in some studies show better cell structure and liver-specific functions [12]. HCC spheroids also exhibit the most malignant properties of HCC tumors [13].…”