Three dimensional cell reconstructions for morphological analysis and modelling

• Main Author: Ratcliffe, Jonathan Albert
• Published: University of Nottingham 2011
• Subjects: 571.6; TP Chemical technology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546460

It is highly desirable to devise a systematic approach to predict cell – material interactions, especially for novel biomaterial surfaces, and to further understanding in the complex area of attachment and spreading. The aim of this research was to produce a new method of studying morphology in real time, whereby data from live spreading cells can be collected for mathematical modelling. There is an abundance of models for sub-cellular elements, however, there are few calibrated models of whole cells; in particular, three-dimensional models predicting attachment, spreading and cell morphology have yet to be produced. Live HOS cells were imaged using LavaCell membrane stain and CLSM every 5 min for a period of 75 min in this study, capturing sufficient detail to produce three dimensional representations of cells during initial attachment and spreading. In order for the contact line to be measured, the interface between the cell membrane and the substrate had to be imaged in sufficient resolution for accurate measurements of the angles to be made. An image processing algorithm developed using Matlab was able to detect the edge of cells in the CLSM z-stack optical sections. These were then used to create contour plots onto which a surface representing the cell membrane could be added. These reconstructions of cells can be easily manipulated to enable the dynamic contact line of attaching cells to be measured for a model based on two-phase poroviscous flow equations. The three dimensional representations not only showed the changing morphology of spreading cells, but gave data on contact radius and area, contact angle and cell height. The main modelling prediction is a near contact line law, which is given by; Θ3 - Φ3 = 3 µ(n)ln(R/λ) (3nV - J(V,n,... )) γ where Θ is the dynamic contact angle (which remains to be determined by experimental means as the cell is spreading), Φ is the static contact angle, n the network density at the contact-line, J is the mass transfer rate from G- to F-actin at contact line and V equals the outward normal velocity of contact line. Once the method had been developed for glass surfaces, the influence on attachment and spreading of various material substrate and protein conditioning layers was investigated. This was achieved by using transparent thin film coated surfaces of titanium nitride and titanium oxide and pre-coating glass with fibronectin and albumin respectively. Three dimensional representations showed the ability to reproduce the different cell response to each surface and gave comparable morphologies to cells fixed for SEM and immunocytochemical staining.