April 15, 2010 (Vol. 30, No. 8)
Gary J. Lye
Researchers Develop and Refine a System for Higher Consistency and Reproducibility
Traditional methods for manual cultivation of stem cells often lead to high variability and low consistency, making development of robust experimental protocols for stem cell research extremely difficult. This has been a major challenge for commercialization of stem cell technologies, rendering them inaccessible for a majority of applications.
To overcome this obstacle, researchers in the department of biochemical engineering at University College London (UCL) recently developed an automated stem cell culture system in a microplate format, offering higher consistency and reproducibility, better protection of cells from contamination, and improved operator safety. The new system also provides a valuable platform for fundamental studies of the culture microenvironment in stem cell biology.
The regenerative medicine bioprocessing program within the UCL department of biochemical engineering focuses on two areas: regenerative medicine translation and bioprocess engineering of stem cells. Both academic and commercial collaborations are essential in this research, which addresses the complete stem cell process from donor or patient biopsy through to clinical implantation into the patient.
The bioprocess engineering aspect of this work deals with the critical challenge of developing reliable processes for the preparation of homogenous populations of stem cells and their progeny. A project funded by the U.K.’s Technology Strategy Board investigates the production of pluripotent and differentiated stem cells in microplate formats, for applications such as compound library screening for drug discovery.
Limitations of Manual Cultivation
High variability and low consistency are major problems in manual stem cell culture processes, for both growth of stem cells and their differentiation into pre-defined cell types. Variations in environmental conditions during bioprocessing have the potential to significantly impact the outcome of stem cell cultures, making it difficult to reproduce experimental protocols and interpret the results obtained.
Many manual handling steps are needed to obtain the final cell population, requiring multiple passages, media exchanges, and cell transfers, each with the potential for contamination and propagation of errors.
Most significantly, it is practically impossible to control several key parameters during these manipulations—most notably temperature and CO2 concentration—as plates are moved from environmentally controlled incubators at 37ºC and 5% CO2, to a biological safety cabinet at room temperature (20–24ºC) and atmospheric CO2 levels (~0.04%), as illustrated in Figure 1.
As a consequence, rapid cooling and diffusion of CO2 out of the media occur, leading to fluctuations in the pH of the culture (Figure 2). Stem cells are extremely sensitive to such changes in pH, and this leads to poor reproducibility in manual culturing, representing a major obstacle to both furthering our understanding of stem cell biology and the commercialization of stem cell applications.
The Automation Solution
Automation provides a comprehensive solution to inconsistencies in manual handling, ensuring that every step of the process is performed in the same way, for the same duration, every time. In addition to improving the consistency of operations, it is possible to perform the entire culture procedure within a precisely controlled environment, avoiding fluctuation in temperature and pH.
The team at UCL has developed a fully automated stem cell cultivation platform based on a Freedom EVO® liquid-handling workstation (Tecan). This integrated system is enclosed within a specially developed Class 2 biological containment cabinet—allowing complete environmental control—and incorporates all the equipment required for fully automated stem cell culture and manipulation, including pipetting and robotic manipulator arms, a microplate centrifuge, and a CO2 incubator.
The sterile, temperature-controlled environment within the cabinet allows independent regulation of atmospheric gases during culture, particularly O2 and CO2. Conditions inside the cabinet are maintained at 37ºC, 5% CO2, and 21% O2, with no variations in temperature, CO2, or pH as plates are moved from the incubator to the deck of the instrument for processing.
This minimizes fluctuations in environmental conditions that have been shown to impact on both cell growth over multiple passages and on the ultimate differentiation into specific cell types. The entire culture operation is performed using disposable pipette tips and consumables to minimize the risks of carryover and contamination and maximize operator safety when dealing with patient-derived cells.
Consistency and Reproducibility
Using the Tecan platform, the UCL team has shown that automated processing improves the consistency and reproducibility of pluripotent stem cell culture compared to manual processes performed by a highly skilled researcher.
Figure 3 shows the automated culture of pluripotent stem cell populations through several passages. While both techniques gave similar cell numbers, automation improved homogeneity of the stem cell population, with no significant changes in gene-expression profile, cell viability, or cell density between successive passages.
Automation offers reliable and consistent production of pluripotent stem cells and differentiated cell types, allowing the controlled and reproducible study of stem cell biology. It also provides a solution for industrial scale-out production, contributing to the commercial development of stem cell technologies for drug discovery and cell-based therapies.
Farlan Veraitch is lecturer in regenerative medicine bioprocessing and Gary J. Lye is professor of biochemical engineering at University College London biochemeng/industry/regenmed.
The authors would like to acknowledge Waqar Hussain, Ph.D., and Paul Mondragon-Teran who performed the work described here, and also the contributions of Professors Peter Dunnill, Chris Mason, and Mike Hoare, and
Ivan Wall, Ph.D.