PhD Student University of Massachusetts Lowell, United States
Introduction:: Non-viral transfection is an essential tool for gene transfer but challenges in obtaining a high frequency of gene uptake in hard to transfect cells persist [1]. While many new methods of transfection attempt to increase transfection efficiency, overcoming the physical barrier of the nuclear membrane via cell cycle stage arrest in G2/M holds promise [1] .
Micropatterned polyacrylamide hydrogels provide a tunable platform influencing cell mechanics and altering gene expression through epigenetic mechanisms [2]. Additionally, research connects micropatterned substrates with increased efficiency of non-viral gene transfection through the morphological changes in cell shape, size and elongation, with large and elongated cells resulting in higher nanoparticle uptake and transfection of a GFP plasmid [3]. Cell adhesion studies on micropatterned substrates also demonstrate differences in transfection efficiencies [4]. Drops in tension between cells and between cells and the extracellular matrix are associated with entry into mitosis [5].
The goal of this study is to use micropatterned polyacrylamide hydrogels to optimize cell cycle arrest in G2/M and in turn, increase efficiency of non-viral transfection for traditionally hard to transfect cell.
Materials and Methods:: Gel Preparation: Surfaces were fabricated as described previously [6]. Briefly, a mixture of 5% polyacrylamide and 0.15% bis-acrylamide were created for each desired stiffness. This was conjugated with 0.1% Ammonium Persulfate (APS) and 0.1% Tetramethylenediamine (TEMED). A drop was pipetted onto a hydrophobically treated glass slide and sandwiched between an amino-silanized glass coverslip. After polymerization, gels were immersed in 55% hydrazine hydrate for 2 h and washed in 5% glacial acetic acid for 1 h.
Cell Culture: Fibroblasts were seeded on tissue culture plastic (TCP), or on hydrogels with or without patterns.Click or tap here to enter text.. Cells were cultured as per ATCC cell culture guide .. Confluent cells were harvested, washed and then fixed with 70% Ethanol. Fixed cells were stained with Propidium Iodide and analyzed via flow cytometry for cell cycle stage.
Results, Conclusions, and Discussions:: Fibroblasts were used in pilot study to optimize protocol for understanding the relationship between matrix stiffness and cell cycle. Fibroblasts seeded on TCP were subjected to flow cytometry and the resultant data analysis classified cells in the following cell cycle stages: 30.87% in G1/G0 phases, 60.89% in S phase, and 2.24 in G2/M phases (Figure 1). In comparison, cells grown on unpatterned and patterned hydrogels at stiffnesses of 10kPa, and 100kPa showed a higher percentage of cells accumulating in G2M, with the biggest increase of 16.75% when cells were seeded on 100kPa unpatterned hydrogels (Figure 1). As the cell membrane is dissolving or dissolved during G2/M, the increased accumulation of cells in G2/M on hydrogels is hypothesized to lead to an increase in transfection frequency. The combination of cell cycle stage, hydrogel stiffness and hydrogel patterns and effects on transfection are examined. Future studies will explore additional stiffness and patterns and will focus on cells traditionally hard to transfect such as primary cells and stem cells.
Acknowledgements (Optional): :
References (Optional): : [3]
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