In situ monitoring of 3D in vitro cell aggregation using an optical imaging system

Abstract

Bioreactor systems that maintain cells and tissues in suspension are increasingly popular for culturing 3D constructs to avoid the loss of in vivo cell function associated with traditional 2D culture methods. There is a need for the online monitoring of such systems to provide better understanding and control of the processes involved and to prevent the disruption of these processes caused by offline sampling and endpoint analysis. We describe a system for the imaging and analysis of cell aggregation, over long periods, within a high aspect rotating vessel (HARV). The system exploits side illumination, using an adjustable beam pattern, to restrict the detected light to that scattered by the cell aggregates, thus eliminating the need for the fluorescent labeling of the cells. The in situ aggregation of mammalian cells (MCF-7 breast carcinoma cells) was monitored over an 8 h period and image sequences showing the growth and motion of the aggregates within the bioreactor were obtained. Detailed size and population data have been derived characterizing the development of the aggregates during this time. We show how the number of resolvable aggregates increases to reach a peak and then declines as these aggregates merge. Once formed, remaining aggregates are found to consolidate to form more tightly packed bodies, typically reducing in cross-sectional area by one third. These results provide the basis for the development of an automated feedback system to control the growth of 3D cell cultures for repeatable, reliable, and quality controlled experimentation.

Matrix compositions and the development of breast acini and ducts in 3D cultures

“…Nevertheless, the establishment of in vitro heterotypic 3D tissue-like culture conditions defining matrix composition and density, cell ratios, and concentrations would provide invaluable tools to test drugs and validate a patient's personalized therapy course (Weigelt and Bissell 2008). However, such achievement will require the development and characterization of reliable 3D culture conditions in which the stromal-epithelial interactions are integrated and appropriate outcomes are measured (Mueller et al 1999;Wozniak and Keely 2005;Kherlopian et al 2008;Krause et al 2008;Sawyer et al 2008;Weigelt and Bissell 2008;Legant et al 2009). Those conditions may also allow the development of 3D cultures to model the early steps of breast cancer progression (Li et al 2008).…”

“…The challenges with heterotypic 3D cultures are considerable and include the definition of the cell type and their respective ratios, and the physical and chemical microenvironment conditions comprising extracellular matrix (ECM) nature and density, and signaling molecule concentrations within the culture medium (Kim et al 2004). Furthermore, analysis methods suitable for 3D cultures are limited, particularly in providing minimally invasive and/or real-time monitoring throughout the duration of a culture (Sawyer et al 2008).…”

“…Numerous 3D cell culture conditions have been and are currently used, but the development of an automated feedback system to control the growth of 3D cell cultures for repeatable, reliable, and quality controlled experimentation has yet to be achieved (Sawyer et al 2008). However, little is known about the consequences of variations in these conditions on breast tissue-like structures and breast cancer progression parameters in 3D cell cultures.…”

Tracking large solid constructs suspended in a rotating bioreactor: A combined experimental and theoretical study

“…The whole assembly is mounted and attached to a motor that can rotate at variable angular speeds V Ã (see Table II). Experiments were carried out for several different rotation rates, and the resulting disc trajectories recorded at 30 frames per second (see Sawyer et al, 2008 for details of the imaging system).…”

Monitoring of 3D breast carcinoma cell culture using proton magnetic resonance imaging

“…Two-dimensional (2D) breast cancer cell cultures have been used for many years for in vitro applications. However, 2D culture does not reveal function of cells observed in a 3D culture (Abbot, 2003) or a concentration which is high enough to be able to detect them by MRI (Sawyer et al, 2008).…”