Biointerfaces

Our current research on biointerfaces mainly focuses on two areas, namely, (1) design and applications of psuedo three-dimensional cell-culture substrates, and (2) effects of substrate topography on cell behavior.
Resources
Book chapter: Yu, K.N., Nikezic, D., "Alpha-Particle Radiobiological Experiments Involving Solid State Nuclear Track Detectors as Substrates", 2009, in Nuclear Track Detectors: Design, Methods and Applications, Eds. Maksim Sidorov and Oleg Ivanov, (Nova Science Publishers: New York) p. 133-154. (download pdf version) (purchase book)

 

Design and applications of psuedo three-dimensional cell-culture substrates

Representative SEM image of cells grown in a pore.

In vitro experiments rely heavily on tissue culture, e.g., to study the cell differentiation, proliferation and function, etc. These in vitro experiments usually involve flat culture substrates, e.g., through the use of Petri-dishes and flasks, and are thus convenient for routine growth of cells. However, such conventional cell culture will normally generate only two-dimensional (2D) cell monolayers. Such 2D monolayers will lead to highly abnormal geometric and mechanical pressures on many types of cells, which are far from the realistic conditions and complexities of three-dimensional (3D) tissues.

We explored the fabrication of three-dimensional (3D) substrates by creating micrometer-size pores on polyallyldiglycol carbonate (or PADC) polymer films through irradiation of the film by alpha particles and subsequent chemical etching. HeLa cells cultured on these 3D substrates were observed using scanning electron microscope. Multiple directions and multiple layers of HeLa cells were observed to have grown in the pores, with normal nuclei and cell membranes as well as good cell spreading. For the cells cultured in 3D substrates with or without additional small pores, no significant differences were observed between their vinculin expression profiles, which was in contrast to the observation made for cells cultured on 2D substrates showing that small pores could enhance vinculin expression. The presence of the large pores and/or the enhanced biocompatibility of the substrate in the present experiments might be the reasons. The protrusions of cells were confined by the small pores, which was similar to the observation made for cells cultured on 2D substrates.
 

Spatial expression of vinculin (Green), F-actin (Red: labeled by rhodamine–phalloidin) and nuclei (Blue: stained with Hoechest 33342) on HeLa cells obtained using the confocal microscope in different focal planes (4 µm between each plane) in the superposition of vinculin, F-actin, nuclei, and transmission mode optical images. Bar = 25 µm. Animations: the focal plane gets progressively deeper.
 

The above figure shows the images on the focal planes containing the HeLa cells grown on the PADC film substrate which has both large and small pores. Multiple directions and layers of cells are seen in the pores, with good conditions of cells, including good spreading and normal nuclei. 

Publication:

Ng, C.K.M., Cheng, J.P., Cheng, S.H., Yu, K.N., 2010. Convenient fabrication of three-dimensional cell-culture substrates through introduction of micrometer-size pores on polyallyldiglycol carbonate polymer films. Nuclear Instruments and Methods in Physics Research A, 619, 401-407.

 

Effects of substrate topography on cell behavior

Superposition of HeLa cells and track-etch pits generated from irradiation of 3 MeV alpha particles for 3 h, etched for 3 h in 6.25 N aqueous NaOH at 70 oC and then for 5 min in 1 N NaOH/ethanol at 40 oC.

It is well established that pores introduce topographies onto the substrates, while substrate topographies will control the nature and degree of cell-cell and cell-matrix interactions and determine the morphology and functional induction of cultured cells in vivo. However, it would be sometimes difficult to separate the relative contribution of topography and porosity from pores. 

We explored the feasibility of using pits created on the surface of a polymer (polyallyldiglycol carbonate or PADC) by alpha-particle irradiation and subsequent chemical etching to study the substrate topographical effects on behaviors of cells (HeLa cervix cancer cells). The pits were purposely not etched-through, so that topographical effects alone (excluding porosity effects) can be studied. The average opening diameter and depth of the track-etch pits after etching in NaOH/H2O with the additional etching by NaOH/ethanol for 5 min determined from their lateral images after polishing the edge of the film were 4.8±0.1 and 6.6±0.3 mm, respectively.

 

 

Spatial expression of vinculin on HeLa cells on (a) raw and unetched PADC film; (b) etched blank PADC film; (c) PADC film irradiated by 3 MeV alpha particles for 3 h and etched; (d) PADC film irradiated by 3 MeV alpha particles for 6 h and etched. For (b), (c) and (d), etching was performed for 3 h in 6.25 N aqueous NaOH at 70 oC and then for 5 min in 1 N NaOH/ethanol at 40 oC. Bar = 20 mm; (a) to (d) are at identical magnification. For (a) to (d), upper left: images from confocal microscope; upper right: transmission mode optical images; lower left: superposition of upper left and upper right images.
 

The presence of focal adhesions and cytoskeletonal reorganization were assessed through expression of vinculin. The figure above shows the expression of vinculin on HeLa cells cultured on the PADC films subjected to different treatments. We observe overall increases in vinculin expression for HeLa cells cultured on PADC films with track-etch pits. On the contrary, vinculin-rich regions were not revealed in cells cultured on the raw (unetched) as well as the blank (etched) PADC films.

Moreover, a closer look at the cells cultured on PADC films with track-etch pits revealed that the cells were largely contained by the track-etch pits. In other words, the cell membrane edges tended to be in contact with the pits. Inhibition of membrane protrusion at the pores could explain this phenomenon.

Publications:

  • Ng, C.K.M., Poon, W.L., Li, W.Y., Cheung, T., Cheng, S.H., Yu, K.N., "Study of substrate topographical effects on epithelial cell behavior using etched alpha-particle tracks on PADC films", 2008, Nuclear Instruments and Methods in Physics Research B, 266, 3247-3256.
  • Ng, C.K.M., Chan, K.F., Li, W.Y., Tse, A.K.W., Fong, W.F., Cheung, T., Yu, K.N., "Biocompatibility enhancement of chemically etched CR-39 SSNTDs through superficial pore formation by alpha-particle irradiation", 2008, Radiation Measurements, 43 (Suppl. 1), S537-S540.
  • Ng, C.K.M., Wong, M.Y.P., Lam, R.K.K., Ho, J.P.Y., Yu, K.N., 2011. Fabrication of pseudo three-dimensional PADC cell culture substrates for dosimetric studies. Radiation Measurements 46, 1790-1794.
  • Ng, C.K.M., Yu, K.N., 2012. Proliferation of Epithelial Cells on PDMS Substrates with Micropillars Fabricated with Different Curvature Characteristics. Biointerphases, Volume 7, Numbers 1-4, 21, DOI: 10.1007/s13758-012-0021-2
  • Ng, C.K.M., Tjhin, V.T., Lin, A.C.C., Cheng, J.P., Cheng, S.H., Yu, K.N., 2012. Fabrication of substrates with curvature for cell cultivation by alpha-particle irradiation and chemical etching of PADC films. Nuclear Instruments and Methods in Physics Research B 278, 15-19.
  • Ng, C.K.M., Chong, E.Y.W., Roy, V.A.L., Cheung K.M.C., Yueng, K.W.K., Yu, K.N., 2012. Fabrication of micropillar substrates using replicas of alpha-particle irradiated and chemically etched PADC films. Applied Radiation and Isotopes, 70, 1432–1435.

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Nuclear Radiation Unit
Department of Physics and Materials Science
City University of Hong Kong
Tat Chee Ave, Kowloon Tong, Hong Kong
Email: apnru@cityu.edu.hk

 

Page last modified on 13-Jun-2012

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