Advantages of Automated Cell Culture using CELLSTAR® AutoFlask™

Cell based assays are nowadays one of the most important techniques in drug discovery and toxicology screens, facilitating in vivo analysis of promising drug compounds or toxic agents. Researchers in both the academic and pharmaceutical markets are increasingly adopting a cell-based approach to their drug discovery research, shifting their focus away from biochemical analysis of discrete cellular components to evaluate cellular function, including tissue and organ activity, through live cell imaging.These types of assays -categorized as high content screening (HCS) - allow researchers to study how drugs, proteins or other biological materials affect cell viability and gene expression, or the respective signaling pathways. Therefore the utilization of living cells leads to more comprehensive data, which complement results from biochemical and other assays used in the drug discovery processes.

During the last decade drug discovery has become a fully integrated industrial process leading to an increasing need for cultivated cells and automated solutions for their production. This purpose has initiated the development of both products and systems designed to satisfy the stringent requirements for consistency, sterility and quality demanded by cell culture processes. 

By replacing laborious and time-consuming manual processes, automation reduces costs, increases throughput and enables round the clock production. Consistency and the high level of process flexibility provided by the computer-based systems are still the prime reason for the use of robotics. Thus applying automation to tissue culture techniques can improve assay development as well as cell maintenance and expansion, increasing the efficiency of drug discovery efforts.

Greiner Bio-One, in collaboration with the Genomics Institute of the Novartis Research Foundation (GNF); San Diego (USA) has developed the CELLSTAR® AutoFlask™, a unique cell culture flask for automated tissue culture. 

Incorporating a number of novel design features the CELLSTAR® AutoFlask™ will increase the range of possible applications for devices of this type as well as the respective robotic system.

The external dimensions correspond to ANSI Standards, making the flask suitable for use on a wide range of both existing cell culture and liquid handling systems.  One of the unique design features of the AutoFlask™ is the in built centrifugation pocket which enables separation of cells from supernatant inside the flask.  Upon horizontal centrifugation cells are collected inside the pocket whereas the medium remains in the bottom vessel of the flask.  The robot accessible pre-cut multiple entry septum enables sterile media and solution exchange and the removal of cell based products.  Pre-cutting of the septum provides a number of benefits such as the prevention of coring by the injection needle, permits re-closing of the septum slit and ensures sterility of the flask content throughout the complete cultivation process.  The septum is located at the A1 position to ease programming and piercing with any liquid handling system. Therefore the CELLSTAR® AutoFlask™ has not to be moved to an upright position for access. Consequently it can remain in its horizontal position during the entire cultivation period to minimize any cell stress due to flask movement.

The integrated filter membrane facilitates gas exchange while providing a sterile barrier against contamination. The hydrophobic membrane is PTFE-coated to prevent wetting of the filter from the internal flask contents. The venting area design together with the high airflow rate of the filter material provides an optimized oxygen supply which will allow the cultivation of even very sensitive cells.
To show the impact of these CELLSTAR® AutoFlask™ features on specific cell line growth, a comparison was made with competitive automated cell culture flasks.  As growth areas of these flasks differ, the initial seeding concentrations were adjusted accordingly to ensure the evaluation was conducted on a like for like basis (see table below).

1. Vitality of SKNMC and CHO

Cultivation of SKNMC and CHO cells using CELLSTAR® AutoFlask™ as well as competitive automated cell culture flaks under the above described settings led to comparable cell vitality values, indicating appropriate cell culture conditions for each experimental setup.

Figure 1: Vitality of SKNMC and CHO cells after 72 hrs of cultivation.
Cells have been seeded in the appropriate media and cultivated at 37°C and 5% CO2. After 72hrs of cultivation cells have been harvested by Trypsin digestion and vitality has been determined using the electric sensing zone method [CASY® Cell Counter; Schärfe System; Germany].

To analyse cell proliferation processes in more detail a defined starting concentration of SKNMC and CHO cells was seeded in the respective media and maximum cell numbers were analysed on each day of cultivation.

2. CHO growth curve

Detailed analysis of CHO cell propagation revealed accelerated cell proliferation after the second day in vitro in the CELLSTAR® AutoFlask™ when compared to competitor 1 and 2. Maximum cell numbers dependent on the provided cell growth area were achieved between 72 and 96 hours of cultivation, whereas these were not achieved in the same timeframe using the flasks from competitor 1 or 2. This indicates cell splitting and expansion can already be initiated after 72hours when using CELLSTAR® AutoFlask™, enabling a significant time saving throughout the entire cell preparation process.
Figure 2: Growth Curve CHO.
CHO cells have been cultivated at 37°C and 5% CO2 for 96hrs.  Each 24hrs cells have been harvested by Trypsin digestion and maximum cell number has been determined using the electric sensing zone method [CASY® Cell Counter; Schärfe System; Germany].

3. SKNMC growth curve
SKNMC cells cultivated in CELLSTAR® AutoFlask™ demonstrated a similar effect as CHO cells, although less prominent due to a slower proliferation rate compared to CHO cells. Despite this inhibition in cell division, CELLSTAR® AutoFlask™ provided exceptional cultivation conditions leading to increased cell numbers after only four days in vitro.

Figure 3: Growth Curve SKNMC.
SKNMC cells have been cultivated at 37°C and 5% CO2 for 96hrs.  Each 24hrs cells have been harvested by Trypsin digestion and maximum cell number has been determined using the electric sensing zone method [CASY® Cell Counter; Schärfe System; Germany].

Conclusion: 

Different experimental approaches using Greiner Bio-One’s CELLSTAR® AutoFlask™ revealed optimal cell growth conditions for both common and very sensitive cell lines leading to accelerated cell proliferation under these circumstances. 
The CELLSTAR® AutoFlask™ provides unique features such as the centrifugation pocket and the easy to access septum as well as exceptional cultivation conditions assuring reliable supply of cells with consistent quality in automated systems. 
Greiner Bio-One’s CELLSTAR® AutoFlask™ therefore not only offers an extensive range of applications and compatibility to various cell culture and liquid handling systems but also facilitates earlier cell harvest and time savings in the entire cultivation process.