Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Abstract

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells

“…Furthermore, a lack of standardization in methodology between laboratories likely introduces further variability 9 . While previous work has automated the expansion of individual lines [10][11][12][13] , we reasoned that developing a fully automated, modular platform for parallel iPSC derivation, expansion and differentiation would allow us to identify, and minimize, factors contributing to variability in iPSC behavior as well as provide a platform for large-scale in vitro iPSC studies.…”

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of <em>Arabidopsis thaliana</em>

“…Additional limitations of this technique include the difficulty of using this piece of equipment with aseptic techniques; however, we did not encounter high percentages of contamination due to ½ MS media lacking sucrose. One could circumvent any contamination issue by placing the robot in a sterile room, allowing for sterile conditions and cell culture, or by using a liquid handling robot with a sterile chamber 24,25 . Another limitation is tip size and seed aspiration.…”

Development of a Modular Automated System for Maintenance and Differentiation of Adherent Human Pluripotent Stem Cells

“…This system allows for large sample-sized research, reduced variability, and future high-throughput analysis of the transcriptome and metabolome of progeny cells derived from patient iPSCs. The performance of our system could be deemed to lie between that of simple liquid handlers 11 and a more complex fully integrated platform. 9 Our system enables the maintenance of human fibroblasts undergoing reprogramming; allows for the long-term maintenance, passaging, and differentiation of iPSCs, which are not available with smaller liquid handlers; and offers an economical alternative to more complete equipment.…”