| Summary: | Replication of biological cells for the purpose of imaging and analysis under
electron and scanning probe microscopy has facilitated the opportunity to
study and examine some molecular processes and structures of living cells in
a manner that were not possible before. The difficulties faced in direct
cellular analysis when using and operating Atomic Force Microscopy (AFM)
in situ for morphological studies of biological cells have led to the
development of a novel method for biological cell studies based on
nanoimprint lithography. The realization of the full potential of high
resolution AFM imaging has revealed some very important biological events
such as exocytosis and endocytosis. In this work, a soft lithography
Bioimprint replication technique, which involved simple fabrication steps,
was used to form a hard replica of the cell employing a newly developed
biocompatible polymer that has fast curing time at room temperature
essential for this process. The structure and topography of the rat muscle cell
and the endometrial (Ishikawa) cancer cell were investigated in this study.
Cells were cultured and incubated in accordance with standard biological
culturing procedures and protocols approved by the Human Ethics
Committee, University of Otago. An impression of the cell profile was
created by applying a layer of the polymer onto the cells attached to a
substrate and rapidly cured under UV-light. Fast UV radiation helps to lock
cellular processes within seconds after exposure and replicas of the cancer
cells exhibit ultra-cellular structures and features down to nanometer scale.
Elimination of the AFM tip damping effects due to probing of the soft
biological tissue allows imaging with unprecedented resolution. Highxx
resolution AFM imagery provides the opportunity to examine the structure
and topography of the cells closely so that any abnormalities can be
identified. Craters that resemble granules and features down to 100 nm were
observed. These represent steps on a transitional series of sequential
structures that indicate either an endocytotic or exocytotic processes, which
were evident on the replicas. These events, together with exocytosis, play a
very significant part in the tumorigenesis of these cancer cells. By forming
cell replica impressions, not only have they the potential to understand
biological cell conditions, but may also benefit in synthesizing three
dimensional (3-D) scaffolds for natural growth of biological cells and
providing an improvement over standard cell growth conditions. Further
examinations by observing the characteristic behaviour of the plasma
membrane when the cells were induced by certain compound such as cobalt
chloride (CoCl2) under control and stimulated conditions have brought in the
opportunity to examine the effect of this stimulant in inducing apoptosis in
many different kinds of cells. Numbers of pores formed on the cells
membrane were found to increase significantly after the cells where induced
with CoCl2 that correlated well with the level of vascular endothelial growth
factor (VEGF) receptors expression, which contributed to tumour growth.
This indicates CoCl2 has exaggerated the expression of the VEGF growth
factor. Investigations were also done to the cells using functionalized nanoparticles
as bio-markers to establish the connection between exocytosis with
nanopores found on the membrane surfaces of the cells. These microbeads
were found attached to sites surrounding the nucleus of the cell and higher
numbers of visible beads would confirm that there was an up-regulation of
the VEGF expression in cells induced by CoCl2. All these can contribute to
expanding the knowledge about exocytosis and fundamental physiology of
cells, and also assist in understanding diseases especially cancer.
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