title: 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.
authors.affiliation: InvivoSciences, Inc.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Conway
authors.affiliation: InvivoSciences, Inc.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gerger
authors.affiliation: InvivoSciences, Inc.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Balay
authors.affiliation: InvivoSciences, Inc.
authors.forename: Rachel
authors.initials: R
authors.lastname: O'Connell
authors.affiliation: Gilson, Inc.
authors.forename: Seth
authors.initials: S
authors.lastname: Hanson
authors.affiliation: InvivoSciences, Inc.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Daily
authors.affiliation: InvivoSciences, Inc.; tetsuro@invivosciences.com.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Wakatsuki
mesh_terms: D000273:Adipose Tissue / Q000166:cytology; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D039904:Pluripotent Stem Cells / Q000166:cytology; D012371:Robotics / Q000295:instrumentation / Q000379:methods*