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

Abstract

Patient-specific induced pluripotent stem cells (iPSCs) have tremendous potential for development of regenerative medicine, disease modeling, and drug discovery. However, the processes of reprogramming, maintenance, and differentiation are labor intensive and subject to intertechnician variability. To address these issues, we established and optimized protocols to allow for the automated maintenance of reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) using a customized TECAN Freedom EVO. Generation of iPSCs was performed offline by nucleofection followed by selection of TRA-1-60-positive cells using a Miltenyi MultiMACS24 Separator. Pluripotency markers were assessed to confirm pluripotency of the generated iPSCs. Passaging was performed using an enzyme-free dissociation method. Proof of concept of differentiation was obtained by differentiating human PSCs into cells of the retinal lineage. Key advantages of this automated approach are the ability to increase sample size, reduce variability during reprogramming or differentiation, and enable medium- to high-throughput analysis of human PSCs and derivatives. These techniques will become increasingly important with the emergence of clinical trials using stem cells.

Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media

“…Following TRA-1-60 selection, 10 lines were maintained in StemFlex medium and 10 lines in TeSR-E8 medium ( Figure 1 A). We had already adapted automation for the culture of pluripotent stem cells using TeSR-E8 ( Crombie et al., 2017 ), and StemFlex also allowed maintenance on the automated platform, with colonies showing typical pluripotent stem cell morphology ( Figures 1 B and S1 ) and similar levels of expression of TRA-1-60 ( Figures 1 C and 1D). After 8 passages, all iPSC lines for both media expressed the pluripotency markers OCT-4 and TRA-1-60 ( Figures 2 A–2L).…”

“…Indeed, human variability is a main source of differences observed between cell lines generated and maintained in various laboratories ( Allegrucci and Young, 2006 , Allegrucci et al., 2007 ), with genetic background and methodologies also having a significant contribution ( Kilpinen et al., 2016 ). The automation of pluripotent stem cell culture provides an efficient way to improve these aspects, increasing throughput and standardizing many aspects of cell culture, including in iPSC generation, maintenance, passaging, and differentiation into progeny cells of interest ( Crombie et al., 2017 , Konagaya et al., 2015 , Paull et al., 2015 ). These automated steps allow minimal variation in each procedure, reduce inter-sample variability, and hence increase robustness of cell culture procedures ( Daniszewski et al., 2017 ).…”

Automation in the Life Science Research Laboratory

“…The advent of new thermostable polymerases obsoleted a whole generation of Polymerase Chain Reaction machinery designed upon a more repetitive protocol (Hawker et al, 2018). Despite these difficulties, with considerate design allowing for reconfiguration and modification premature obsolescence can be delayed (Harrison et al, 2007;Crombie et al, 2017), referred to in some industries as future-proofing. Understanding and planning for obsolescence is therefore an important part of any automation strategy.…”

“…Matching commercially available automation equipment to these requirements is often not a feasible option with fixed componentry and locked-in software frequently being the limiting factors. Automated cell culture is an example where the available systems can be insufficiently flexible to accommodate the specific cell culture requirements of an individual laboratory (Crombie et al, 2017), with some requiring a broad range of cell culture types and others having more focussed needs. A high level of experimental process variation is therefore more likely to require a bespoke automation system, the development of which will have an associated time and financial cost.…”

“…A range of ingenious methods have been developed to build low-cost automation solutions, including the integration of Lego into microscopy automation (Almada et al, 2019), microfluidics for DNA assembly (Shih et al, 2015) and rapid synthesis and testing of small molecule libraries (Baranczak et al, 2017). Laboratories with novel protocols that are nearly but not quite suited to existing automation equipment have been able to successfully upgrade commercially available systems for their specific needs (McGraw et al, 2014;Richter et al, 2015;Zhang et al, 2016;Crombie et al, 2017;Konczal and Gray, 2017). Repurposing existing equipment in this fashion either through software or hardware modification is a cost-and time-efficient method of obtaining higher levels of protocol automation without the arduous task of designing and building an entirely novel system.…”