An automated continuous monitoring system: a useful tool for monitoring neuronal differentiation of human embryonic stem cells

Abstract

The currently used cell culturing and differentiation procedures are both time- and laborintensive. Automation of some of these procedures will increase the efficiency of commonly used cell differentiation protocols. We used a particular cell culture platform to rapidly and efficiently screen the neuronal differentiation of human embryonic stem cells (hESC). Continuous live monitoring and analysis of non-labeled cells using this system allowed us to characterize neuronal populations over the entire neuronal differentiation process. The differentiation of individual cells from early progenitor cells to neurons and glial cells could be monitored continuously using this system with sub-confluent cell cultures. The imaged data was collected and analyzed with a specially designed cell recognition protocol, which resulted in a quantitative neuronal cell count. The analysis results were confirmed using conventional laboratory methods such as manual counting and flow cytometry. Our findings suggest that an automated culture platform combined with automated monitoring and analysis systems is a reliable method for developing enhanced cell differentiation procedures or as part of an automated quality control system for existing protocols

Comparative Analysis of Targeted Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) and Human Embryonic Stem Cells Reveals Variability Associated With Incomplete Transgene Silencing in Retrovirally Derived hiPSC Lines

“…The cells were monitored with time‐lapse imaging at 4 and 8 weeks during the neural differentiation. Quantitative analysis of time‐lapse imaging data was performed by Cell‐IQ analysis software (Chip‐Man Technologies Ltd., Tampere, Finland) [26], but the accurate neuronal cell number could not be reliably determined because of confluence of the cultures (supplemental online Fig. 7).…”

Automated image analysis with the potential for process quality control applications in stem cell maintenance and differentiation

“…It further requires sample sacrifice or careful assurance that clinical safety or efficacy is not altered by analytical techniques which could trigger cellular abnormalities, a particular issue for small scale high value processes . Noninvasive PAT, ideally automated, real‐time and continual, would reduce these issues . It may also reduce or remove post product analysis and reduce production failure through allowing reaction at the point of process deviation.…”

“…12 Noninvasive PAT, ideally automated, real-time and continual, would reduce these issues. [13][14][15] It may also reduce or remove post product analysis and reduce production failure through allowing reaction at the point of process deviation. This would have additional benefits for short shelf life products coupled with onerous post production testing time requirements.…”

Effects of inflammatory cytokines IFN-γ, TNF-α and IL-6 on the viability and functionality of human pluripotent stem cell-derived neural cells

“…In previous studies, neurons and neurites outgrowing from the plated aggregates were counted at a single timepoint. Here, we took advantage of automated time-lapse imaging and analysis (Huttunen et al, 2011) and performed continuous follow-up for 72 h after different cytokine treatments. IFN-γ slightly reduced the neuronal outgrowth rate from aggregates at a concentration of 10 ng/ml, while cell migration was not affected with the highest concentrations.…”

“…1.) (Narkilahti et al, 2007;Nat et al, 2007;Huttunen et al, 2011). One day after the cells were plated, the 24-well plates were transferred into a time-lapse imaging system and the cells were imaged for 24 h to obtain a baseline recording.…”