Journal of Laboratory Automation

The CompacT SelecT is the latest generation automated cell culture system in the SelecT product line allowing incubation of up to 90 T-175 flasks and preparation of 210 assay-ready plates. We have successfully implemented the CompacT SelecT in support of a number of cell-based assays used in our Alzheimer's disease (AD) lead optimization programs. One of the distinguishing features of AD pathology is deposition of two neurotoxic forms of the beta-amyloid peptide (Aβ₄₀ and Aβ₄₂) in the brains of patients. It is thought that specifically lowering Aβ₄₀ and Aβ₄₂ in the brains of patients will halt the progression of the disease. The generation of Aβ requires sequential cleavage of the type-I integral membrane amyloid precursor protein (APP) by two proteases, β-secretase (BACE) then γ-secretase. In the specific examples presented here, we have transitioned two cell lines supporting drug discovery efforts for identifying β- and γ-secretase inhibitors (GSIs) from manual cell culture protocols to fully automation using CompacT SelecT. In Chinese hamster ovary (CHO) cells which over express wild-type APP (CHO^APP cells), robust secretion of Aβ₄₀ was observed from cells cultured manually and with CompacT SelecT with signal:background ratios of 54–99 and 23–47, respectively. Despite the reduced signal:background observed with the CompacT SelecT cultured cells, the rank order of potency for a series of 18 BACE inhibitors in reducing Aβ₄₀ secretion was identical when manually cultured cells were compared with CompacT SelecT cultured cells. The correlation coefficient when comparing the two sets of EC₅₀ values was r ² = 0.99. Similarly, CHO cells that over express a reporter construct for the Swedish mutant APP that generates high levels of Aβ peptide (CHO^APP-NL cells) cultured with CompacT SelecT exhibited a reduced secretion of Aβ₄₀ and Aβ₄₂ when compared with manually cultured cells; signal:background ratios for secretion of Aβ₄₀ from CHO^APP-NL cells were 57–71 and 16–31, and for Aβ₄₂ secretion 8–18 and 4–11 comparing manual and automated cell culture, respectively. Similar to the observations in CHO^APP cells, an identical rank order of potency for a series of GSIs was observed when CHO^APP-NL cells cultured manually and in CompacT SelecT were compared; r ² = 0.99 and 0.98, respectively, for EC₅₀ values for inhibition of Aβ₄₀ and Aβ₄₂ secretion. Taken together, the robust signal:background and identical pharmacological profile of inhibitors provide validation for the implementation of automated cell culture for CHO^APP and CHO^APP-NL cells in support of β- and γ-secretase-targeted drug discovery.

As the drug discovery process evolves and demands more challenging and relevant assays, we have experienced a recent and significant escalation in the number of cell-based high-throughput assays for both small molecule and target identification screens. This has resulted in an increased need for the reproducible production of high quality cells in large quantities. Historically, manual cell culture was the only option available for providing cells in sufficient numbers for small molecule ultra-high-throughput screens (uHTSs), representing a technology gap in our automated cell culture process.

Recently, we have applied an automated solution in the form of a novel 10-layer tissue culture flask, the HYPERFlask (Corning, Lowell, MA). This technology, when introduced as an upgrade to the SelecT (an automated cell culture system manufactured by The Automation Partnership, Ltd., Royston, England), provides a new approach to automating production of the high number of cells required for uHTS and consequently a highly desirable alternative to manual cell culture.

The HYPERFlask has a surface area of 1720 cm² and can yield up to 3 × 10⁸ cells after 3 days in culture. This can be compared to a typical yield of 2 × 10⁷ cells from a T175 flask (with a surface area of 175 cm²), the current standard flask type for automated cell culture on the SelecT. Cells grown in both flask types are of comparable quality, as demonstrated by equivalent cell viability, yield per cm², functional response, and pharmacology.

Selection of the optimum cell line from large populations can be time consuming and laborious. Compromises are often necessary to meet challenging timelines, but can limit the parameters or cell line construction strategies that can be evaluated. This article describes a new automated cell culture system for cell line selection and characterization. The technologies enable data to be obtained from large numbers of cells lines allowing users to evaluate, in parallel, a wider range of molecular approaches and cell culture processes. This facilitates more rapid and efficient cell line development. Also, presented are initial biological testing data for the new system and descriptions of how highly parallel processing can contribute to significantly reduced cycle times and better decision making through the rapid identification of optimum cell lines and culture conditions.

The translation of experimental cell-based therapies to volume produced commercially successful clinical products that satisfy the regulator requires the development of automated manufacturing processes to achieve capable and scaleable processes that are both economic and able to meet the unpredictable demands of the market place. The Healthcare Engineering group at Loughborough has conducted novel demonstrators of the transfer of manual human cell culture processes to the CompacT SelecT (The Automation Partnership) automated cell culture platform, including an osteoblast cell line, embryonic carcinoma cell line, primary bone marrow-derived mesenchymal stem cells, primary umbilical cord-derived progenitor cells, and human embryonic stem cells. The work aims to develop and optimize automated cell culture processes for manufacturing cell-based therapies in a quality system and current good manufacturing practice (cGMP) compliant manner and is underpinned by the application of a six-sigma inspired quality engineering approach.

In this technical brief, we outline the need for automated cell culture systems and automated process engineering for the manufacture of cell populations for therapeutic applications. We review the transfer of a manual cell culture process to an automated process and the subsequent methodology for process improvement using examples from our laboratory of the application of these principles to an important regenerative medicine cell type, the human mesenchymal stem cell. We believe that systematic process improvement methodologies combined with the process stability provided by automation are essential to engineer optimized cGMP compliant manufacturing processes that will be required to realize the promise of cell-based therapies.

An automated cell-culture platform becomes the nucleus of an organization performing cell-based research. However, every cell-based project placed on the system brings unique challenges. With each cell line comes millions of years of evolutionary encumbrance and a genetic inclination driving unique phenotypic peculiarities. In vivo, diverse eukaryotic cells rely on their “mammalian host” for survival. An automated system must perform in vitro, the myriad actions needed to sustain multiple cell lines as well, hence becoming an “automated host.” Cells invariably, will endeavor to do as they please. Molding these cells into the operational bounds of a man-made system requires insight into the relationship between cell and machine.

Citing our own experiences, we will describe herein the use of the SelecT automated cell-culture platform (The Automation Partnership, Hertfordshire, England) in our discovery and preclinical profiling programs at Novartis. Achieving the balance between cells and the automated environment, and accommodating variable cell dynamics are discussed.

In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high-throughput screening (HTS) arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the HTS drug discovery platform, with special consideration being given to assay miniaturization, liquid-handling complications, and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell-plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry over, and incubator-induced artifacts.