Systematic optimization of human pluripotent stem cells media using Design of Experiments

Abstract

Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin−1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

A human neurodevelopmental model for Williams syndrome

“…After 4 days, transduced DPCs were trypsinized, plated on mouse embryonic fibroblasts and cultured using human embryonic stem cell (hESC) medium. After manually picked and clonally expanded, feeder-free iPSCs were grown on matrigel-coated dishes (BD Bioscience, San Jose, CA, USA) with mTeSR1 (StemCell Technologies) or iDEAL 29 .…”

High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells

“…We hypothesized that our image cytometry method in combination with DOE analysis could be used to rapidly and efficiently screen for the most significant effectors of gene-editing efficiency and to quantify their leverage on the output. DOE is regularly applied in engineering to optimize complex processes and has been applied previously in biological systems to optimize cell culture conditions [37,38]. Use of DOE in this application is particularly prescient as identification of factors that primarily influence gene-editing efficiency can inform future experiments that are designed to optimize only the most important inputs over a much greater range of levels.…”

“…Our platform exploits image cytometry and high content image analysis (HCA) in combination with a customized microcontact printed cell substrate [34,35] to simultaneously monitor non-viral delivery and editing in human cells. We use established image analysis methods [36] and design-of-experiments (DOE) based statistical techniques [37,38] to screen existing liposomal delivery materials and to optimize delivery inputs to maximize gene-editing outcomes. Dynamic tracking of Cas9 protein expression, subcellular localization and gene disruption within subpopulations of cells suggests that the cell number and the level of Cas9 expression within 24 hours of delivery are important predictors of editing.…”

A comparative study of endoderm differentiation in humans and chimpanzees

“…Feeder free iPSC cultures were initially maintained on Growth Factor Reduced Matrigel using Essential 8 Medium (E8) as previously described. After 10 passages in E8, all cell lines were transitioned to iDEAL feeder free medium that was prepared in house as specified previously (39). Cell culture was conducted at 37°C, 5% CO2, and atmospheric O2.…”