A Collaborative Approach to Creating an Automated Cell Culture System

RTS Life Science International is a multifaceted company with a long history of providing automation solutions. With an increasing role in the life science arena, JALA interviewed members of the RTS staff and one of its key pharmaceutical corporate collaborators to provide our readers with an insight into the company and its acCellerator automated cell culture system. The RTS Group, made up of several linked companies, focuses on specialized areas of expertise and provides software, automation systems and equipment for a range of sophisticated scientific processes, including life sciences, nuclear and industrial automation. RTS specializes in custom designed technologies to meet client-specific requirements, using years of automation experience and proven engineering practices. Extensive industrial and scientific experience exists throughout this group of affiliated divisions. The Company operates offices and manufacturing facilities throughout the USA, the UK and Finland.

OVERVIEW OF RTS LIFE SCIENCE INTERNATIONAL

Thurnall PLC, established in 1982, was created to provide automation solutions to industry. RTS was founded in Finland six years ago. Its specialty was high technology robotics and vision software products for use within industrial automation. Three years ago, the companies merged to form RTS Thurnall. The purpose of this merger was to leverage the knowledge base of the various divisions of the group and combine the professional engineering skills which could then be engaged across various markets. The group now employs over 1,400 people in three countries, the USA, Finland and the UK. Among the many strengths Thurnall brought to the alliance was project management expertise. In 2001, Wright Industries (Nashville, TN, USA) was purchased. The group is now quoted on London's AIM listing stock exchange, with about $200M turnover. As a group, RTS now has seven different businesses, each in select markets and each with the engineering expertise to provide benefits to other members of the group.

Re-structuring occurred in January 2001 for two principle reasons; to capitalize on the elements of the group operating in synergistic markets and to offer visibility for stock investors who could then see the productivity of each business group. As part of this process, the group has re-branded RTS Thurnall as RTS Life Science International, so the public can better understand its products and focus. It has been working on laboratory automation applications for a decade. Among its areas of expertise are compound storage and retrieval, high throughput screening (HTS), automated cell culture and other bio-science applications. RTS Assembly systems in Nashville, focuses on high precision assembly and large scale automation, while the Flexible Systems Group develops advanced robotic software and vision aided automation, for example combining the latest vision systems with the ABB FlexPicker robot.

The Life Science group consists of 120 people in the UK and 60 people in the USA. The USA offices are located in Mount Laurel, NJ, Denver and Nashville, TN. The company has devised and implemented large compound storage systems for a number of notable clients including Bayer, AstraZeneca, Eli Lilly, Dow Agrosciences, Johnson and Johnson, Pharmacia, Pfizer, and Sphinx. Gary Walsh, Sales and Marketing Director for RTS Life Science, explained to JALA that Compound Storage and Retrieval is a significant part of the Life Science business. RTS Life Science has three products, the Flexi-Store, a large carousel based storage system, Compact-Store, a high density storage for regular ceiling heights and Sample-Store, which provides high density plate storage. The systems can provide random access picking of individual tubes from a library at a rate ranging from a few hundred to approximately 100,000 tubes per day. This far exceeds the range generally required for hit picking (typically 1,000 to 10,000 tubes per day) and allows individual tubes to be picked for primary screening. Careful control of accelerations allows for one sample every two seconds, or 40,000 samples a day for one robot. To complement its sample storage and retrieval systems, the company has developed a powerful process control software platform called Distributed SPRINT (d-SPRINT). The process control and dynamic scheduling features of d-SPRINT also benefits its other automation platforms such as automated liquid handling, manual storage, Kardex Shuttles and carousels, weighing stations, or other manual systems.

In order to accommodate the diverse needs of pharmaceutical and analytical laboratories, RTS Life Science developed the Assay Platform, a standard hardware platform featuring a proprietary industrial robot. The system allows low volume simple and complex assays to run, in high volume, over several days. Compact design was a major issue, so the instruments are stacked to minimize floor space. The flexibility inherent in the system enables fast, dynamic scheduling with the company's SPRINT software. Reproducible instrument placement is assured with the i-PLATE placement system. The Molecular Devices FLIPR, Aurora VIPR, Wallac Viewlux and Amersham IN Cell Analyzer plate readers are all being integrated for use with the Assay Platform.

AUTOMATED CELL CULTURE SYSTEM

It has been clear to the pharmaceutical industry that the demand for cultured cells is rising at a dramatic rate. The basic need of the industry is to increase cell production while reducing the labor necessary to produce cells of consistent quality through reliable methods. Equally important is consistent media delivery, reproducible cell passaging conditions and incubation times. Finally, the production of reproducible 96 well or 384 well microplates containing viable cultured cells is the ultimate goal of a cell culture system.

RTS Life Science has created acCellerator in response to specific market desires. The acCellerator provides a product that compliments other automated cell culture systems on the market and meets different market demands (for a review of automated cell culture technology see the forthcoming April/May, 2002, issue of JALA). A unique feature of the acCellerator is the ability to provide HT performance through the use of multiple robots. In addition, the system incorporates reliable cell counting and viability determinations. This complex process is managed by the sophisticated and dynamic scheduler and process manager software platform, SPRINT.

The specific hardware features of the acCellerator include Heraeus Cytomat flask and plate incubators, Essen ‘Pipeline’ dispensers, an Innovartis cell counter and a Multidrop within a Class II biological safety cabinet. The current design has six Cytomat flasks and two plate incubators with a capacity of 144 T225 flasks and 378 plates to support the system. The acCellerator is capable of working with the Cytomat incubators 24 hours per day. Incubator capacity on the system is expandable if required.

At the start of the day, an order will be loaded, the media put on the system and the system started. A flask-picking robot picks flasks out of the incubator and places them into the cabinet. Spent media is aspirated by an aspiration station, after which the cells are washed in saline using a dispenser. This is followed by the addition of cell dissociation solution. The flasks can be warmed, if necessary, on dry block heaters that give uniform heating across the plate to facilitate enzymatic release of the cells. A specialized tapping device helps provide a reproducible dissociation of the cells from the flask, much in the same way that technologists tap a flask against the palm of their hand. Cells are mixed (aspirated and dispensed through a pipette tip) to dissociate cell clumps. A Cedex cell counter is used to quantitate cell number and viability. Media are stacked on racks in bottles that can be removed, decapped, put into place for pipetting, and then removed when empty. A plate handling area is used to dispense cells into plates. Magnetic stirring bars in the ‘cell soup’ collection bottles ensure that cells remain suspended throughout dispensing. Cell plate production is performed by a Multidrop, sample distribution device. The Multidrop can deliver user-defined aliquots of cell suspension (maintained on a magnetic stirrer) to either 96 or 384 well plates. Cell concentrations are maintained by dilution of the original “cell soup”. Dilution calculations are automatically performed by the system based on data obtained from the cell counter.

Sterility is maintained by a Class II biological safety cabinet, custom designed to house robots and peripheral hardware. The cabinet allows both operators and robots to access the peripherals. Cytomat plate incubators are used, as they are the industry standard and have a reputation for high precision temperature control. They have rapid temperature and humidity recovery rates owing to the small robot access door. The robot has access to the flasks and plates one at a time, while operators can remove large numbers of flasks or plates mounted in stacks. This approach allows an entire HTS series of plates to be relocated to another laboratory very quickly.

One of the most challenging steps in cell culture is the delivery of media, phosphate buffered saline (PBS), and enzymatic digestion solution (e.g. trypsin), in a sterile manner, while simultaneously opening and closing flasks. To achieve this, a special dispenser was developed and supplied by Essen Instruments which contains a disposable and sterile pipette. Pipeline combines the peristaltic pump tubing, as well as the pipette. In addition, access to the interior of the cell culture flask is through a separate access point for the pipette system, designed for easier robot access and reduced risk of contamination. This flask was designed in collaboration with Corning Inc. (Acton, MA) specifically for automation and is based around a T225 flask to maximize surface area.

The system is controlled using SPRINT, which is intuitive to use and dynamically schedules multiple automated activities. Individual cell flasks are fully tracked and managed by the system to assure the operator of flask and plate location and process progression. Throughput is maximized by multi-tasking using three Staubli robots, multiple dispensers, and multiple dry block heaters. One significant advantage is the use of dry block heaters in the system, obviating the need to go back to the incubator during trypsinization.

The system has the flexibility to work in a number of modes, for example operating overnight to produce two cell lines with an output of 189 plates (one incubator load) each. Another scenario would be the production of 15 cell lines with 20 plates each. For each cell line, the user is able to select the number of plates and cell flasks that should be produced. Generally, 20 cell lines is more than adequate for most HTS laboratories, since about 10–20 cell lines tend to define the main use for HTS. However, the system is able to maintain and plate much larger numbers of cell lines if required. It is also designed to allow for periods of unattended operation. The limit to unattended operation is governed by the plate and flask incubator capacity.

HOW THE ACCELLERATOR MEETS INDUSTRY REQUIREMENTS (A CLIENT'S PERSPECTIVE)

RTS believes in a highly collaborative approach to product development which means that each client is involved from conception to implementation. RTS produces its own software and 3D designs. It also possesses an engineering team that understands the needs of the pharmaceutical industry.

As part of the preparation of this report, the JALA staff interviewed Drs. Gary Allenby and Elaine Anderson, AstraZeneca (Charnwood, U.K.). They have been involved with RTS throughout the process and were able to provide us with a client's perspective on the development of acCellerator. AstraZeneca decided to collaborate with RTS to obtain specific niche applications that were not available elsewhere. There are three major models for the use of the products of a cell culture facility in the pharmaceutical industry. The lead discovery section is responsible for generation of a robust screening platform for pharmaceutical targets. Optimization of cell culture is a key component of developing an ideal HTS. Thus, one model is to house the cell culture facility within the lead generation group. A second model is to centralize cell culture and ask this group to be responsible for providing both cells and isolated membrane preparations to many parts of the business. The third model is to purchase cells for screening from outside vendors.

On average, 15–30 targets are screened through a typical HTS group per company per year. Up to 50% of these targets can be functional HTS assays. Each company screens upwards of 0.5 million compounds in 96 or 384 well plates with 10,000–20,000 cells per well. As an example, it takes two trained people to screen a FLIPR assay and 1.5 people to run tissue culture for such an assay. An automated FLIPR may run 100 to 200 plates per day, depending on assay read times. Producing this quantity of plates per day is a tedious, time-consuming task that does not allow the technologist any time for further assay development. In addition, adherent cells require at least 24 hour incubation in plates prior to screening. Therefore, at best, a four day per week screening regime can be implemented. Furthermore, new screening hardware is making increasing use of cell-based assays. For example, HT patch clamping (Ion Works, Essen Instruments), laser scanning con-focal microscopy on live cells in plates (IN Cell Analyzer formerly LCAS, Amersham Biosciences), and whole cells binding assays (FMAT, Applied Biosystems).

WHAT DID ASTRAZENECA WANT?

AstraZeneca required the potential to address adherent cell lines in flasks. As previously mentioned, this included eight to 12 cell lines at once in Lead Generation and two to four cell lines at once in HTS. Of these, up to two cell lines should be produced for HTS without operator intervention (i.e. at the weekend work). Ideally, operator intervention should be on a daily basis but minimal. The automated system had to be capable of harvesting cells, then passaging and/or plating cells. High system capacity was also important, together with flexibility in equipment utilization and simple operator intervention for processing cells with rapid turnaround time. The system needed to be designed to minimize or prevent contamination (operator or cells) or cross contamination (between cell lines), with rapid harvesting and plating of cells to minimize variability in batch processing.

APPROACH TO AUTOMATION

AstraZeneca's approach was, where possible, to identify tried and tested laboratory equipment and use an integration company with high quality engineering expertise (RTS Life Science). The company tried to identify the risk factors in the process and minimize risk by designing around sterile liquid handling (Pipeline-Essen Instruments), cell-culture flask design (Corning Inc), using a modified automated incubator for flasks (Heraeus Kendro), and ensuring sterile conditions (Bigneat).

ADVANTAGES OBTAINED

AcCellerator models the process performed manually, therefore there is no re-validation of growing or assay conditions. The growing surface of the large flask (T225cm²) allows for a high cell density. The expandable flask and plate incubator capacity, which are customisable, provides for future adaptation. The interchangeable stackers between automated and manual incubators save space, money and time in transferring flasks and plates. The use of three robots results in parallel processing of flasks for harvesting and plating or passaging, improving speed and therefore throughput and cell viability. The cabinet allows space for integration of other bulk reagent dispensers for 1536 well dispensing (e.g. PreSYS-Cartesian or FlexiSpense-Asys), a barcode printer, or a washer for media replacement in serum starvation assays. Other advantages were: easily interchangeable dispensers (Pipeline, Multidrop), dry block heaters for quick error recovery from breakdowns and a quick and simple dispenser disposable (Peripette for Pipeline) for operators to replace. The system can dissociate the functions of passaging and plating so that the automatic passaging of one cell line can occur while another ‘cell soup’, prepared manually, can be plated simultaneously. There are no cross contamination issues between cell lines. Where necessary, all equipment is either disposable or washable automatically (70% ethanol). Cell viability counts are performed on-line, thereby giving the operator the opportunity to decide to continue or discard the batch. This function may be implemented automatically with user-defined viability thresholds. Data suggest that dry block heaters are better at maintaining temperature of the harvesting reagent than a ‘regular’ incubator.

Another advantage is that the flask tapper mimics the action performed by the operator in physically tapping the flask to remove cells. Also, using Pipeline, large void volumes associated with long lengths of peristaltic tubing are avoided. In addition, disposable ‘cell soup’ bottles (Nalgene 1 litre, sterile) are easily replaceable, with no risk of cross contamination. All operations in which cells are exposed to the environment are performed under Class II conditions, protecting the work and the operator. Plate stackers are compatible with an increasingly utilized incubator on automated HTS systems for cell-based assays (Cytomat 6000). Furthermore, acCellerator has a paging system for breakdowns and order completion notification with a data management package to record all data associated with harvesting, plating and passaging for easy quality control.

Drs. Allenby and Anderson were asked by the JALA staff to recommend a “checklist” of questions other companies interested in obtaining an automated cell culture system should ask themselves. The following are their suggestions:

1. Does the system use off-the-shelf components? If so, this can help avoid down-time through breakdowns, replacing components with spares actually in manual use.

2. As technology improves, is it possible to simply upgrade these components?

3. Are any consumables available from notable and maintainable suppliers at HTS levels of consumption?

4. Class II facility? For health and safety reasons, in most pharmaceutical environments, Class II is absolutely necessary for working with human derived cells.

5. Is it possible to clean the system adequately for maintenance and servicing? Can one formaldehyde ‘bomb’ it?

6. Can the system multi-task? The total automated and operator intervention throughput must be examined for both cell harvesting and cell plating, in addition to the rate of batch processing and its relationship to cell viability.

7. Does the system maximize the use of relatively expensive incubator real estate? Can incubation for cell dissociation occur outside the incubator and therefore save processing time and increase throughput?

8. Setup time - how long will it take to configure and re-configure the system for different cell lines?

9. Does the system generate media ‘on-the-fly’ as this is an important time saver?

10. How does the system handle disposables?

11. Has the cell production been optimized relative to system price?

12. Does the system and its disposables (including the flask) maximize cell production?

13. What is the total cell production per day?

14. Is the software user friendly and intuitive to a cell culture expert?

CONCLUSIONS

It was very clear from the meetings with personnel at RTS Life Science and AstraZeneca that the collaborative nature of their venture was the key to success. Both companies felt that they had benefited and learned a great deal in the process. For RTS, the system they developed, and modified versions of it, are highly marketable. The process also places RTS on an even stronger footing within the life science arena. With enormous growth potential in this industry, RTS has developed a new product at a propitious time. JALA looks forward to continuing to bring you updates on the future of RTS Life Science and to observe its development as a significant player in the industry.