Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures.

Area of Science:

• Biotechnology
• Cell Biology
• Proteomics

Background:

• 3D cell cultures offer more physiologically relevant models than 2D.
• Clinostat incubators provide a microgravity environment for enhanced 3D cell growth.
• Standard protocols for 3D cell culture analysis are limited.

Purpose of the Study:

• To present a protocol for 3D cell culture using a clinostat incubator.
• To demonstrate the advantages of clinostat incubation for spheroid formation and monitoring.
• To establish a pipeline for phenotypic and proteomics analysis of 3D cell cultures.

Main Methods:

• Utilized a standalone clinostat incubator for 3D cell culture (HepG2/C3A and THLE-3 cell lines).
• Employed integrated cameras for monitoring spheroid growth, counting, and measurement.
• Performed proteomics analysis on collected spheroids without trypsinization.
• Developed a semi-automated system for spheroid quantification.

Main Results:

• Achieved highly reproducible spheroids with low shear forces and active nutrient diffusion.
• Demonstrated that hepatocytes require 3 weeks to develop liver-specific functionalities in 3D culture.
• Showcased successful proteomics analysis for monitoring liver phenotypes like respiratory chain metabolism and metal detoxification.
• Validated the protocol for both cancerous and non-cancerous hepatocyte cell lines.

Conclusions:

• The clinostat incubator protocol facilitates robust 3D cell culture, monitoring, and analysis.
• This method enables comprehensive phenotypic characterization and proteomics studies on 3D models.
• The protocol offers a valuable toolbox for advancing research in liver biology and disease modeling.