LABORATORY-scale Automation for CELL Biology

BY MARC FEIGLIN AND ROLAND DURNER, PHD

The Value of Automation
With the increasing amount of cell culture work performed in today’s life sciences laboratory1, the need for automation is evident. Whether it is to provide cells for cell-based assays or cell-based products, the process of culturing large amounts of cells is a time-consuming, laborious process. Over the past decade, a number of large-scale solutions for automating cell maintenance and growth have been introduced to the market. These systems have demonstrated the ability to automate the many aspects of cell culture. Today, this need for automation is expanding into cell biology labs with smaller budgets and throughput needs. However, the price and size of earlier automated solutions make them inaccessible to the large majority of cell biology laboratories.
By utilizing standard laboratory automation components, Tecan Group Ltd. (Maenne-dorf, Switzerland) has been able to design a number of systems designed for various cell biology tasks that meet these criteria including: automated solutions for simple media exchange for feeding of slow-growing cell lines in microplates; automated solutions for cell-line development and transfection; complex fully automated systems for all aspects of flask-based cell and protein production. This article will present examples of some of the available solutions that meet cell biology automation needs and will discuss how these solutions were able to overcome many of the issues that arise when automating cell biology with standard laboratory automation.

Researcher Needs
Whether a researcher is trying to develop a new transfected cell line or grow cells for a high throughput screen, the basic needs for those looking to automated cell biology are similar. First and foremost, researchers need a reliable supply of cells with a consistent quality of cell cultures. In addition, researchers often need to work simultaneously with multiple cell lines that might require different growth conditions. Trying to maintain many different cell lines can be a tedious affair, which is why automation can be such an attractive option. However, today’s automation systems must be affordable, easy to operate, fit into standard laboratories, and be flexible enough to handle researchers’ changing needs.
The goal of developing automated cell culture solutions is to produce tools that fit researchers’ needs while utilizing as many familiar components as possible. These include incubators and cell counters that are adapted for use with automated liquid-handling instrumentation. Systems are designed to fit into biological hoods intended to maintain sterile working conditions. Finally, many of these automation systems are modular so that they can be adapted to changing needs.
Sterility
A sterile working environment is critical for cell culture work. It is even more critical in an automated environment where there may not be any human interaction that could identify sterility issues. In fact, since humans are a common source of cell culture contamination, automated systems can present an advantage for maintaining sterility. Tecan cell culture systems are typically placed in a sterile environment such as a simple positively pressured HEPA sterile bench or a Class II biological hood (Fig. 1). Systems automatically run regular decontamination protocols utilizing ethanol and PBS washes. Media and other liquids are typically kept in sealed environments, exposed only within the hoods for very short periods of time.
In the event a user accidentally causes contamination, automated methods for rapidly detecting mycoplasma contamination have been integrated into the system.

Designing an Automation-friendly Flask
The T-flask is the recognized vessel used by researchers for typical cell growth in the laboratory. The flasks are available with numerous surface chemistries and coatings to promote attachment and growth. On the other hand, T-flasks were designed for humans, not automation. There are a few large-scale, expensive automation systems that can operate with these flasks, but the typical automation used in laboratories cannot work reliably with those available on the market. Standard T-flasks require specialized equipment for uncapping, storage and incubation.
Several major cell culture labware manufacturers helped design an automation-friendly cell culture flask while retaining the advantages of current T-flasks. The new flask had to meet the footprint standard of a microplate2 while also maintaining the height standard3, in order to work with existing automation equipment. A standard microplate footprint would provide about 100 sq. cm of growth area, an adequate amount for such a flask. In addition to a cap for manual access, the flask needed a septum for automated access. Septum piercing is more reliable than automated capping and de-capping of flasks; it also does not require any special automation, as numerous liquid handlers today are capable of piercing septa. The RoboFlask (Fig. 2) from Corning Inc. Life Sciences (Acton, MA) offers all of these features and has been successfully implemented on Tecan automation4. Over two dozen different cell lines have been grown in this flask (Table 1).
The advantages of this automation-friendly flask are obvious. In the horizontal position, the flask can be manipulated like any ordinary microplate, with numerous existing automation options. To gain access into the flask, it must be manoeuvred into a vertical position, where Tecan’s standard liquid-handling arm can pierce the septa and perform the required pipetting action. The Freedom EVO® (Fig. 3), the latest automated liquid-handling instrument, sits on the deck and rotates the flask between the horizontal and vertical positions. This “flipper” device is also used for dissociating adherent cells from the flask surface through a gentle rocking motion or, if required, vigorous knocking.
Among other standard automation options, these flasks can be easily used with the standard CO2 incubators integrated by Tecan. Capacities from 40 to 1,000 flasks are available, depending on the customer’s capacity requirements. Tecan’s CellGEMTM software can automatically determine the ratio of flasks and plates required at any time in the system.

Other Issues with
Automating Cell Growth
Typically, automated liquid-handling systems such as the Freedom EVO are optimized for delivering volumes of a few millilitres or less. In automated cell culture setups, the systems may need to deliver media volumes of as much as 100 ml and down to a few microlitres when transfecting cells with DNA. Tecan’s cell culture systems include optional upgraded system plumbing, which enables the system to accurately deliver volumes throughout this range. Integrated peristaltic and membrane pumps are used for larger volumes, while traditional syringe methods are used for smaller amounts.
Since these systems are often running for extended periods of time, bulk media on the system is kept refrigerated. Integrated heaters automatically warm the media before it is added to the cells. Integrated spinner flasks enable large-scale dilution of cells for systems being used to grow-up larger cell quantities.
A variety of cell-counting options have also been used on Tecan cell culture systems. Recent counters that have been integrated include the Cedex counter from Innovatis AG (Bielefeld, Germany) and the Beckman Coulter Inc. (Fullerton, CA) Vi-Cell™ XR. Both these systems utilize the Trypan Blue method for measuring cell count and viability. Tecan systems have also integrated the Guava Technologies Inc. (Hayward, CA) PCA-96 counter, which offers a number of methods for cell measurements.

Conclusion: Automation to Fill a
Variety of Cell Biology Needs
All of these automated cell culture tools are utilized to build a variety of solutions for researchers. Some of the simplest systems include solutions for automating the exchange of media in plates of Caco-2 cells being prepared for ADMET assays5. Numerous solutions have also been developed for the automated generation and transfection of cell lines6,7. One product in this area is the Cellerity, a fully automated system for maintaining and scale-up growth of multiple cell lines in parallel. This system can be used for producing cells for assaying in systems, such as cell-based assay systems, or for producing protein for structural biology studies using systems protein crystallography workstations. The era of automated cell biology is only beginning.

References
(1)     Garippa, R. 2004. A multi-faceted approach to the advancement of cell-based drug discovery. Drug Discovery World No 5. pp. 43-55
(2)     Society for Biomolecular Screening. Standard ANSI/SBS 1-2004: Microplates - Footprint Dimensions. Available at www.sbsonline.org/msdc
/pdf/ANSI_SBS_1-2004.pdf
(3)     Society for Biomolecular Screening. Standard ANSI/SBS 2-2004: Microplates - Height Dimensions. Available at www.sbsonline.org
/msdc/pdf/ANSI_SBS_2-2004.pdf
(4)     Durner, R. et al. Automated Cell Culturing using Corning RoboFlask on Tecan Cellerity. Program and Abstracts of the LabAutomation 2005 Conference. Abstract TP035
(5)     Gerber, L. et al. 2002. Robotic Feeding Station for Maintenance of CACO-2 Cells. AAPS Pharm Sci Vol. 4 No. 4.
(6)     Jones, R. 2004. Automated Cell Line Generation and DNA Extraction Support Large-Scale Genetic Study. Tecan Journal No. 3. pp. 4-6.
(7)     Pap, E. and S. Grueneberg. 2004. Automated Generation of Cell Lines. Tecan Journal No. 2. pp. 9-11.

Marc Feiglin is chief technology officer, Drug Discovery, and Roland Durner

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