Transfection of Small Numbers of Human Endothelial Cells by Electroporation and Synthetic Amphiphiles

Leeuwen, E. B. M. van; Veen, Anneke Y. van der; Hoekstra, Dick; Engberts, Jan B. F. N.; Halie, M. R.; Meer, Jan van der; Ruiters, Mhj

doi:10.1053/ejvs.1998.0677

Cited by 9 publications

(5 citation statements)

References 18 publications

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“…Cultured human ASCs (pool of five donors, 1 million at passage 4) were transfected with 1 mg pMC1neo-polyA (Stratagene) which is a plasmid encoding the large T antigen of SV40 with neomycin as selectable marker [27,28]. At 48 h post-transfection, transfected cells were selected by adding 250 mg/ml geneticin (G418) to the medium.…”

Section: Immortalization and Lentiviral Tagging Of Ascsmentioning

confidence: 99%

Adipose tissue-derived ECM hydrogels and their use as 3D culture scaffold

Getova

Dongen

Brouwer

et al. 2019

Artificial Cells, Nanomedicine, and Biotechnology

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Adipose tissue has the therapeutic capacity in the form of a fat graft, for example, for treatment of irradiation-induced scars and difficult to heal dermal wounds. For large-scale clinical application, an offthe-shelf product is warranted. In recent years, ECM-derived hydrogels are postulated to harbour therapeutic capacity and might even replicate the beneficial effects of adipose tissue. In normal homeostasis, the natural ECM acts as a deposit of growth factors, that releases them over time. In the healing of lesions, this might promote cell accumulation and proliferation which in turn stimulates angiogenesis and repair. The decellularization of tissue and the generation of hydrogels may leave cytotoxic traces. Therefore, our research assessed the cytotoxic effect of human adipose tissue-derived ECM hydrogels on connective tissue cells i.e. fibroblasts. The results showed no cytotoxicity, meaning the hydrogels caused no cell death. Cell migration and survival were observed when cultured in ECM hydrogels and followed for 7 days. Cell survival in the hydrogel was confirmed with CFDA staining and also cells showed the ability to penetrate and migrate throughout the gel. We conclude that ECM hydrogels are promising to use as innovative therapy for wound healing. ARTICLE HISTORY

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Section: Immortalization and Lentiviral Tagging Of Ascsmentioning

confidence: 99%

Adipose tissue-derived ECM hydrogels and their use as 3D culture scaffold

Getova

Dongen

Brouwer

et al. 2019

Artificial Cells, Nanomedicine, and Biotechnology

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“…Although various gene delivery methods for primary endothelial cells have been developed based on calcium phosphate, cationic liposome, synthetic amphiphiles, DEAE-dextran or electroporation [1–7], cell toxicity, poor transfection efficiency and cost have limited them to be widely utilized in the cardiovascular community. However, recently developed nucleofection technology has been proven to be a new electroporation method with superior transfection efficiency and cell viability for various primary cultures, including primary endothelial cells [8–12].…”

Section: Introductionmentioning

confidence: 99%

Phosphate-buffered saline-based nucleofection of primary endothelial cells

Kang

Ramu

Lee

et al. 2009

Analytical Biochemistry

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Although various non-viral transfection methods are available, cell-toxicity, low transfection efficiency and high-cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that utilizes a combination of novel electroporation conditions and specific buffer components that stabilize the cells in the electrical field. Despite its superior transfection efficiency and cell viability, high cost of the technology has discouraged the cardiovascular researchers to liberally adopt this new technology. Here, we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular and lymphatic endothelial cells were performed by using PBS. Compared to the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and non-primary cells. Moreover, PBSmediated nucleofection of siRNA showed more than 90% knockdown of the expression of target genes in primary endothelial cells. Together, we demonstrate that PBS can be an unprecedented economical alternative for the high-cost buffers for nucleofection of various primary and non-primary cells.

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“…There are many other proprietary liposomes, mainly cationic lipids, which could be assessed (36)(37)(38)(39). Alternative delivery methods which are commonly used include electroporation (40), dendrimers (41,42), peptides (43,44) and polymers (45,46). All these methods facilitate ODN uptake but are not cell phenotype specific.…”

Section: Discussionmentioning

confidence: 99%

Identification of Potentially Effective Antisense Oligodeoxyribonucleotide (ODN) Sequences for Inhibiting Plasminogen Activator Inhibitor-2 (PAI-2) Production by Monocytes

Humphries¹,

Burnand²,

Cunningham³

et al. 2002

Thromb Haemost

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SummaryMonocyte fibrinolytic activity may influence thrombus resolution. The balance between uPA and PAI-2 could determine the fibrinolytic activity of the monocyte. Inhibiting PAI-2 production using specific antisense sequences might alter this balance. Selecting effective sequences is a problem as prediction of the secondary structure of target mRNA is difficult. This study reports the modification of a cell free system for rapid antisense screening.Five 18-19 mer oligodeoxynucleotides (ODN), sequences A, B, K, T and Q, and their matched scrambled controls were designed and screened using a modified rabbit reticulocyte lysate transcription and translation system (RRL). Intracellular uptake of ODNs was confirmed by fluorescence microscopy, scanning laser confocal microscopy and fluorimetery. Monocytes were transfected with a liposome/ODN complex using sequences A, B, A + B combined, or T and PAI-2 levels measured by ELISA. Inhibition of PAI-2 production was calculated as a percentage of control levels (baseline and scrambled). (i) RRL System – Sequence A was the most effective inhibitor of PAI-2 production in this system (median 63%) compared with sequences, B median 9%, K median 14%, T median 11% and Q median –8% respectively (n = 3). Sequence A was the only sequence, which always inhibited PAI-2. This was confirmed using fluorescently labelled protein (n = 2). (ii) Monocyte transfection Fluorescence microscopy and fluorimetry showed that intracellular delivery of labelled antisense was only achieved when a liposome was used. Transfection of monocytes extracted from 5 subjects showed that sequence A significantly reduced PAI-2 production (mean % 41.4, sem 9.1) compared with sequences B (mean% 3.4, sem 8.9, p = 0.04), A + B (mean % 0.4, sem 7.8, p = 0.04), and T (mean % 5.4, sem 4.9, p = 0.01). Futher studies using sequence A on cells from 10 subjects showed a significant reduction in monocyte PAI-2 production (27.6 ng/ml, sem 3.9) compared with matched scrambled controls (mean 38.3 ng/ml, sem 4.5, p = 0.0112) and baseline (mean 51.4 ng/ml, sem 6.7, p = 0.0009).Use of the RRL screening system allowed the selection of a novel antisense sequence, which significantly reduced PAI-2 production in monocytes.

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Transfection of Small Numbers of Human Endothelial Cells by Electroporation and Synthetic Amphiphiles

Cited by 9 publications

References 18 publications

Adipose tissue-derived ECM hydrogels and their use as 3D culture scaffold

Adipose tissue-derived ECM hydrogels and their use as 3D culture scaffold

Phosphate-buffered saline-based nucleofection of primary endothelial cells

Identification of Potentially Effective Antisense Oligodeoxyribonucleotide (ODN) Sequences for Inhibiting Plasminogen Activator Inhibitor-2 (PAI-2) Production by Monocytes

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