Automated selection and harvesting of pluripotent stem cell colonies

Abstract

The ability of pluripotent stem cells to differentiate into specialized cells of all three germ layers, their capability to self-renew, and their amenability to genetic modification provide fascinating prospects for the generation of cell lines for biomedical applications. Therefore, stem cells must increasingly suffice in terms of industrial standards, and automation of critical or time-consuming steps becomes a fundamental prerequisite for their routine application. Cumbersome manual picking of individual stem cell colonies still represents the most frequently used method for passaging or derivation of clonal stem cell lines. Here, we explore an automated harvesting system (CellCelector™) for detection, isolation, and propagation of human embryonic stem cells (hESCs) and murine induced pluripotent stem cells (iPSCs). Automatically transferred hESC colonies maintained their specific biological characteristics even after repeated passaging. We also selected and harvested primary iPSCs derived from mouse embryonic fibroblasts expressing the green fluorescent protein (GFP) under the control of the Oct4 promotor using either morphological criteria or GFP fluorescence. About 80% of the selected and harvested primary iPSC colonies gave rise to homogenously GFP-expressing iPSC lines. To validate the iPSC lines, we analyzed the expression of pluripotency-associated markers and multi-germ layer differentiation potential in vitro. Our data indicate that the CellCelector™ technology enables efficient identification and isolation of pluripotent stem cell colonies at the phase contrast or fluorescence level.

Site-specific gene expression analysis using an automated tissue micro-dissection punching system

“…However, whereas tomo-seq requires multiple replicates of the same samples, the information provided still is associated with a low spatial resolution. Recently, a system employing a capillary tube for manually picking up a single cell from a tissue has been reported 19, 20 , but this requires considerable time to capture up to one hundred cells or more. More recently, Ståhl et al .…”

5. Themenbereich Stammzellen: Aktuelle Entwicklungen der Stammzellforschung in Deutschland

“…In diesem Umfeld wurde mittlerweile ein automatisiertes System zur Isolierung und Vermehrung von ES-Zellen sowie anderer pluripotenter Zelltypen entwickelt (Haupt et al, 2012). Zur Überführung bestehender Labormethoden in standardisierte industrielle Verfahren ist die Entwicklung solcher Verfahren wichtig und notwendig.…”

Small Molecule Screening in Human Induced Pluripotent Stem Cell-derived Terminal Cell Types

“…This limited the number of cells that could be produced in both the undifferentiated and differentiated states and created significant culture-to-culture variability (82). The development of single cell dissociation and improved feederfree culture conditions has facilitated the bulk production of undifferentiated cells through reduced manual handling, automation, or bioreactors and enabled the subsequent generation of larger quantities of differentiated cells (78,(83)(84)(85). Efforts have also been made to identify proliferative committed progenitor cells for each in vitro differentiated cell type that can self-renew, be generated in bulk, and be frozen in large qualitycontrolled batches (79,86,87).…”