Article

Sorting and expansion of murine embryonic stem cell colonies using micropallet arrays

Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Cytometry Part A (Impact Factor: 3.07). 02/2009; 75(2):121-9. DOI: 10.1002/cyto.a.20672
Source: PubMed

ABSTRACT Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption. Murine embryonic stem cells were cultured on arrays composed of releasable elements termed micropallets created from a biocompatible photoresist. Micropallets containing undifferentiated colonies were released using a laser-based technique followed by cell collection and expansion in culture. The micropallet arrays provided a biocompatible substrate for maintaining undifferentiated murine stem cells in culture. A surface coating of 0.025% gelatin was shown to be optimal for cell culture and collection. Arrays composed of surface-roughened micropallets provided further improvements in culture and isolation. Colonies of viable stem cells were efficiently isolated and collected. Colonies sorted in this manner were shown to remain undifferentiated even after collection and further expansion in culture. Qualitative and quantitative analyses of sorting, collection efficiency, and cell viability after release and expansion of stem cell colonies demonstrated that the micropallet array technology is a promising alternative to conventional sorting methods for stem cell applications.

0 Followers
 · 
79 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Single cell analysis is mainly limited to single time-point measurements, without the possibility to track behavioral changes of a single cell or its descendents. Here, the integration of a spatiotemporal single cell lab-on-a-chip system with an automated cultivation device allows single cell analysis under defined growth conditions and, especially, semiautomated cell retrieval and growth kinetic analysis of the single cell descendants. Performance of the new platform was evaluated using the yeast Saccharomyces cerevisiae. The yeast was singularized in the lab-on-a-chip (Envirostat), which combines the possibility of cell cultivation with cell analysis. Singularized cells were collected in a microtiter plate and cultivated in a semiautomated cultivation device (Bioscreen C). S. cerevisiae showed highly reproducible and glucose concentration independent growth kinetics in population experiments. Yet, growth kinetics in cultures inoculated with only one or few cells exhibited strong variations, because of an unexpected growth phenotype: colony formation in submerse cultures. Interestingly, the colony-like structures grew for more then 60 h and were stable for at least 82 h, despite rigorous shaking. Cell agglomeration due to pseudohyphal growth could be excluded, suggesting changes in cell wall properties of yeast populations starting from a single yeast. Single cell analysis still exhibits unexpected obstacles. Nevertheless, this new single cell analysis platform can be used for studying cellular dynamics of single cells and expanded cell populations thereof.
    Cytometry Part A 02/2009; 75(2):130-9. DOI:10.1002/cyto.a.20684 · 3.07 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Editorial
    Cytometry Part A 02/2009; 75(2):83-5. DOI:10.1002/cyto.a.20702 · 3.07 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Positive selection, sorting, and collection of single cells from within a heterogeneous population are required for many biological studies. We recently demonstrated a miniaturized cell array for this purpose; however, on-chip pre-enrichment and isolation of specific target cells would provide significant value for cell isolation. In the current work, mixed cell samples of fewer than 30,000 cells were used for panning by means of on-array antibody-capture to pre-enrich the target population. The cell surface receptors Fc(epsilon)R(1), c-Kit, and ErbB2 were used for positive selection of RBL, RBL, and SK-BR-3 cells, respectively, from the mixed population. The capture efficiency, selectivity, and enrichment for the target cells were calculated and compared with fibronectin-coated controls. As expected, the capture efficiency depended on the frequency of the target cell in the mixed population over the range of 0.3-33%. For a frequency of 5% target cells, the capture efficiency was 39%-53% for the three conditions, while the selectivity varied between 78% and 98% with 16-20-fold enrichment. Furthermore, single-cell cloning studies demonstrated a high cloning efficiency of target cells selectively isolated from the array. Antibody-based pre-enrichment in combination with micropallet-based cell selection will be a valuable tool for isolation and expansion of rare cells from small heterogeneous populations.
    Cytometry Part A 07/2009; 75(7):609-18. DOI:10.1002/cyto.a.20741 · 3.07 Impact Factor
Show more

Preview

Download
0 Downloads
Available from