A compact, automated cell culture system for clinical scale cell expansion from primary tissues.
ABSTRACT Despite the growing number of clinically practical automated cell culture systems, demand is also increasing for more compact platforms with greater capabilities to prepare primary cells directly from patient tissue. Here we report the development of an automated cell culture system that is also compact. The machinery consisted of a supply unit, an incubation unit, and a collection unit, which fit within a 70 cm x 60 cm x 86 cm space. The compact size was enabled by our concept of using a single culture vessel from the primary culture steps to final cell harvest instead of scaling up with multiple culture vessels. Human fibroblasts and bone marrow stromal cells (BMSCs) were successfully cultured with this system over 19 days without contamination. From three pieces of gingival tissue (2 mm x 2 mm) or from 10 mL of bone marrow aspirate, the system could produce more than 2.0x10(7) cells and up to 3.0x10(7) cells for fibroblasts and BMSCs, respectively. The BMSCs produced by this system were capable of ectopic bone formation after transplantation into the subcutaneous space of nude mice. Our prototype system will provide a foundation for minimizing automatic culture machinery with clinically relevant cell yields while also expanding the automation capabilities to include primary tissue culture.
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ABSTRACT: Infections with HIV still represent a major human health problem worldwide and a vaccine is the only long-term option to fight efficiently against this virus. Standardized assessments of HIV-specific immune responses in vaccine trials are essential for prioritizing vaccine candidates in preclinical and clinical stages of development. With respect to neutralizing antibodies, assays with HIV-1 Env-pseudotyped viruses are a high priority. To cover the increasing demands of HIV pseudoviruses, a complete cell culture and transfection automation system has been developed. The automation system for HIV pseudovirus production comprises a modified Tecan-based Cellerity system. It covers an area of 5×3 meters and includes a robot platform, a cell counting machine, a CO(2) incubator for cell cultivation and a media refrigerator. The processes for cell handling, transfection and pseudovirus production have been implemented according to manual standard operating procedures and are controlled and scheduled autonomously by the system. The system is housed in a biosafety level II cabinet that guarantees protection of personnel, environment and the product. HIV pseudovirus stocks in a scale from 140 ml to 1000 ml have been produced on the automated system. Parallel manual production of HIV pseudoviruses and comparisons (bridging assays) confirmed that the automated produced pseudoviruses were of equivalent quality as those produced manually. In addition, the automated method was fully validated according to Good Clinical Laboratory Practice (GCLP) guidelines, including the validation parameters accuracy, precision, robustness and specificity. An automated HIV pseudovirus production system has been successfully established. It allows the high quality production of HIV pseudoviruses under GCLP conditions. In its present form, the installed module enables the production of 1000 ml of virus-containing cell culture supernatant per week. Thus, this novel automation facilitates standardized large-scale productions of HIV pseudoviruses for ongoing and upcoming HIV vaccine trials.PLoS ONE 01/2012; 7(12):e51715. · 4.09 Impact Factor
Article: An engineered approach to stem cell culture: automating the decision process for real-time adaptive subculture of stem cells.[show abstract] [hide abstract]
ABSTRACT: Current cell culture practices are dependent upon human operators and remain laborious and highly subjective, resulting in large variations and inconsistent outcomes, especially when using visual assessments of cell confluency to determine the appropriate time to subculture cells. Although efforts to automate cell culture with robotic systems are underway, the majority of such systems still require human intervention to determine when to subculture. Thus, it is necessary to accurately and objectively determine the appropriate time for cell passaging. Optimal stem cell culturing that maintains cell pluripotency while maximizing cell yields will be especially important for efficient, cost-effective stem cell-based therapies. Toward this goal we developed a real-time computer vision-based system that monitors the degree of cell confluency with a precision of 0.791±0.031 and recall of 0.559±0.043. The system consists of an automated phase-contrast time-lapse microscope and a server. Multiple dishes are sequentially imaged and the data is uploaded to the server that performs computer vision processing, predicts when cells will exceed a pre-defined threshold for optimal cell confluency, and provides a Web-based interface for remote cell culture monitoring. Human operators are also notified via text messaging and e-mail 4 hours prior to reaching this threshold and immediately upon reaching this threshold. This system was successfully used to direct the expansion of a paradigm stem cell population, C2C12 cells. Computer-directed and human-directed control subcultures required 3 serial cultures to achieve the theoretical target cell yield of 50 million C2C12 cells and showed no difference for myogenic and osteogenic differentiation. This automated vision-based system has potential as a tool toward adaptive real-time control of subculturing, cell culture optimization and quality assurance/quality control, and it could be integrated with current and developing robotic cell cultures systems to achieve complete automation.PLoS ONE 01/2011; 6(11):e27672. · 4.09 Impact Factor