Live cell catapulting and recultivation does not change the karyotype of HCT116 tumor cells

Langer, Sabine; Geigl, Jochen B.; Ehnle, Susanne; Gangnus, Rainer; Speicher, Michael R.

doi:10.1016/j.cancergencyto.2005.01.013

Cited by 23 publications

(16 citation statements)

References 11 publications

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“…LMPC is a robust technology that offers an innovative method to isolate homogeneous cell types from heterogeneous populations [59]. This technology has been improved with the new PALM Microbeam HT platform (Carl Zeiss MicroImaging); this platform allows for the isolation of cells in a viable state, which in turn permits subsequent reculturing [15, 16, 57]. Thus, not only will we be able to effectively isolate individual or groups of GFP+, hepatocyte‐like cells from a mixed population of lentivirus‐transduced cells, but in the future, our aim will be to reculture this enriched cell population following the LMPC procedure.…”

Section: Discussionmentioning

confidence: 99%

Differentiation and Enrichment of Hepatocyte-Like Cells from Human Embryonic Stem Cells In Vitro and In Vivo

et al. 2007

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Human embryonic stem cells (hESC) may provide a cell source for functional hepatocytes. The aim of this study is to establish a viable human hepatocyte-like cell line from hESC that can be used for cell-based therapies. The differentiated hESC were enriched by transducing with a lentivirus vector containing the green fluorescent protein (GFP) gene driven by the ␣1-antitrypsin promoter; the GFP gene is expressed in committed hepatocyte progenitors and hepatocytes. GFP؉ hESC were purified by laser microdissection and pressure catapulting. In addition, differentiated hESC that were transduced with a lentivirus triple-fusion vector were transplanted into NOD-SCID mice, and the luciferase-induced bioluminescence in the livers was evaluated by a charge-coupled device camera. GFP؉ hESC expressed a large series of liver-specific genes, and expression levels of these genes were significantly improved by purifying GFP؉ hESC; our results demonstrated that purified differentiated hESC express nearly physiological levels of liver-specific genes and have liver-specific functions that are comparable to those of primary human hepatocytes. The differentiated hESC survived and engrafted in mouse livers, and human liverspecific mRNA and protein species were detected in the transplanted mouse liver and serum at 3 weeks after transplantation. This is the first time that human albumin generated by hESC-derived hepatocytes was detected in the serum of an animal model. This also represents the first successful transplantation of differentiated hESC in an animal liver and the first bioluminescence imaging of hESC in the liver. This study is an initial step in establishing a viable hepatocyte-like cell line from hESC. STEM CELLS 2007;25:3058 -3068 Disclosure of potential conflicts of interest is found at the end of this article.

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Section: Discussionmentioning

confidence: 99%

Differentiation and Enrichment of Hepatocyte-Like Cells from Human Embryonic Stem Cells In Vitro and In Vivo

et al. 2007

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“…Single cells were isolated using the PALM MicroBeam System (PALM Microlaser Technologies, Bernried, Germany) as described previously (11,16,17) and collected in a 200 μl Eppendorf tube cap containing 10 μl lysis and fragmentation buffer as detailed below.…”

Section: Methodsmentioning

confidence: 99%

High resolution array-CGH analysis of single cells

Fiegler

Geigl

Langer³

et al. 2006

Nucleic Acids Research

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137

105

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Heterogeneity in the genome copy number of tissues is of particular importance in solid tumor biology. Furthermore, many clinical applications such as pre-implantation and non-invasive prenatal diagnosis would benefit from the ability to characterize individual single cells. As the amount of DNA from single cells is so small, several PCR protocols have been developed in an attempt to achieve unbiased amplification. Many of these approaches are suitable for subsequent cytogenetic analyses using conventional methodologies such as comparative genomic hybridization (CGH) to metaphase spreads. However, attempts to harness array-CGH for single-cell analysis to provide improved resolution have been disappointing. Here we describe a strategy that combines single-cell amplification using GenomePlex library technology (GenomePlex® Single Cell Whole Genome Amplification Kit, Sigma-Aldrich, UK) and detailed analysis of genomic copy number changes by high-resolution array-CGH. We show that single copy changes as small as 8.3 Mb in single cells are detected reliably with single cells derived from various tumor cell lines as well as patients presenting with trisomy 21 and Prader–Willi syndrome. Our results demonstrate the potential of this technology for studies of tumor biology and for clinical diagnostics.

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“…[4][5][6] However, recently a protocol for life cell catapulting and recultivation has been developed 7,8 that extends its possibilities into the fields of stem cell research and tissue engineering without changing the cells. 9,10 The protocol is designed to handle adherent cells growing on a polymer membrane in a culture dish and makes it possible to separate specific cell types or clusters out of a heterogeneous cell population. Cell separation is thus inherently a two-step procedure, with identification and dissection of the cells of interest preceding the transport step and recultivation.…”

Section: Introductionmentioning

confidence: 99%

Principles of laser-induced separation and transport of living cells

2007

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Separation and transport of defined populations of living cells grown on a thin membrane can be achieved by laser microdissection (LMD) of the sample of interest, followed by a laser-induced forward transport process [laser pressure "catapulting" (LPC)] of the dissected cell cluster. We investigate the dynamics of LMD and LPC with focused and defocused UV-A laser pulses by means of time-resolved photography. Catapulting is driven by plasma formation when tightly focused pulses are used, and by confined thermal ablation at the bottom of the sample for defocused catapulting. With both modalities, the initial specimen velocity amounts to about 50 to 60 ms. Time-resolved photography of live cell catapulting reveals that in defocused catapulting, strong shear forces arise when the sample is accelerated out of the culture medium covering the cells. By contrast, pulses focused at the periphery of the specimen cause a fast rotational movement that minimizes the flow of culture medium parallel to the sample surface, and thus the resulting shear stresses. Therefore, the recultivation rate of catapulted cells is much higher when focused pulses are used. Compared to collateral damage by mechanical forces, side effects by heat and UV exposure of the cells play only a minor role.

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Live cell catapulting and recultivation does not change the karyotype of HCT116 tumor cells

Cited by 23 publications

References 11 publications

Differentiation and Enrichment of Hepatocyte-Like Cells from Human Embryonic Stem Cells In Vitro and In Vivo

Differentiation and Enrichment of Hepatocyte-Like Cells from Human Embryonic Stem Cells In Vitro and In Vivo

High resolution array-CGH analysis of single cells

Principles of laser-induced separation and transport of living cells

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