Chromatophores immobilization

Here we will present the results obtained by investigating three different types of microcarriers namely, glass, polystyrene, and gelatin beads for their efficiency in binding fish chromatophores. Various amounts of ferromagnetic powder of iron (II, III oxide) were incorporated in gelatin beads used for chromatophore immobilization. 

Figure 32.9 presents experimental video-image output at time intervals t = 0, 50, 180, 360 s after addition of toxin to fish chromatophores immobilized on gelatin microcarrier with 10% of ferromagnetic material. The aggregation of pigment granules induced by toxin in cells is obvious. 

Mojovic, L. and Jovanovic, G.N., Development of a microbiosensor based on fish chromatophores immobilized on ferromagnetic gelatin beads. Food Technol Biotechnol. 43 (1), 1-7, 2005. 

Hara M, Ajiki S, Miyake J. Topological characterization and immobilization of a chromatophore membrane from Rhodopseudomonas viridis or application as a photoelectrical device. Supramo-lecular Science 1998 5 717-721. 

Immobilization of chromatophores on the surface of the microbeads represents a key step in biosensor development. Fish chromatophores are anchorage-dependent cells that require compatible surface for attachment, subsequent spreading, and growth [5]. Generally, immobilization on the surface of small beads or microcarriers is a suitable method for the cultivation of anchorage-dependent cells. Van Wezel [13] first reported immobilization of cells on mammalian cells on diethyla-minoethyl (DEAE)-Sephadex A50. Subsequently this type of carrier was improved by optimizing... 

For these experiments, in order to protect the cells from environmental shear stress, an 5 p.m thin membrane made of alginate and poly-L-lysine was created around the immobilized micro-carrier [31]. The movement of microcapsules with immobilized chromatophores was observed microscopically in a glass microtube (d = 700 pm 1 = 5 cm) with the magnetic field conduit embedded in the wall. The experimental setup is presented in Figure 32.4. Fluid (L-15 medium) velocities applied were in the range from 1.6 to 6.4mm/s and corresponded to the predicted operational fluid velocities of the biosensor [11]. Fluid flow was provided by microsyringe pump (LabTronix, USA). 

The effect of the cell/bead ratio (A = number of cells/bead) on the cell attachment rate constant and on the viability of immobilized cells was determined for gelatin beads with 10% of ferromagnetic material (Table 32.1). It is important to insure a large initial cell-to-bead ratio, which would not affect cell viability, and also to minimize the proportion of unoccupied beads during the immobilization process. Fish chromatophores are terminally differentiated cells and do not replicate in tissue culture, thus the initial ceU/bead ratio will not increase with time, as reported for some other proliferating animal cells like Vero cells [10]. By microscopic examination, we observed that immobilized fish chromatophores stay functional, for example, responsive to cloni-dine for 2-4 weeks, although a small decrease in cell/bead ratio occurred due to apoptosis or cell death. 

For each initial cell/bead ratio observed, viable immobilized cell/bead ratio was calculated from the total number of cells immobilized and measured after 24 h. Typical results are presented in Table 32.1. These results indicate that A = 70 is an optimum value to use for immobilization of fish chromatophores. Higher initial cell/bead ratio causes lower cell viability, and therefore lower viable immobilized cell/bead ratio is achieved. Lower viability is most probably due to the shortage of living space and due to higher competition of cells for nutrients. At A = 70, microscopic examination did not show the presence of unoccupied beads. 

Effect of Cell/Bead Ratio on Fish Chromatophore Attachment Rate Constant k and on Cell Viability of Immobilized Chromatophores ... 

Field intensities needed to capture microcapsules of immobilized chromatophores with 10% of ferromagnetic material that are considered to be the most appropriate for use in biosensor, were found to be in the range from 954 to 1092 A/m for the applied fluid velocities from 1.6 to 6.4 mm/s. These magnetic field intensities may be easily provided by solenoid or by some other type of magnetic field conduit. 

FIGURE 32.10 Decrease of the cell area of immobilized chromatophores with time, as a response to clonidine (c = 50 nM). Experimental data ( ), and model data (-), cr = 0.009%. 

The response of immobilized chromatophores to neurotoxin clonidine was monitored by measuring cell area covered by pigment. Percentage of area change is dose-dependent for this... 

Mojovic, L., Upson, R., Willard, C., Chaplen, F.W.R., and Jovanovic, G.N., Immobilization of fish chromatophores onto gelatin-based microcarriers, in Proceedings of the sixth World Congress of Chemical Engineering, ShoUcross, D., Ed., Melbourne, Australia, September 23-27, 2001 (CD edition) ISBN 0 7340 2201 8 AICHE, Melbourne, 2001 1-7. 

Subsaturating photobleaching spectra were taken on chromatophores as described previously (Farchaus et al, 1990a). Room temperature absorption spectra were taken on a Shimadzu UV-160 spectrophotometer or an Aminco DW2a spectrophotometer. Low temperature absorption and linear dichroism (LD) spectra (lOK) were taken on a previously described apparatus (Breton, 1985) of RCs immobilized in polyacrylamide gels and squeezed uniaxially (Breton, 1988). 

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