OWLS APPLICATION NOTES NO-004

QUANTIFICATION OF CELL ADHESIVITY
using OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY (OWLS) detection

Abstract
The molecular interactions and cell-biological mechanisms behind the adhesive behavior of a cell are not properly understood. In the last decades, the development of bio-protheses and the hope for elaboration of successful cell replacement therapies accelerated the search for appropriate scaffold materials and conditions which may help to keep cells alive and functioning on artificial surfaces. For understanding some crucial steps of cell-attachment, the initial phases of attachment of two different types of cloned cells – NE-4C neural stem and MDCK epithelial cells - were analyzed by optical waveguide light mode spectroscopic (OWLS) methods,

Application of OWLS sensors
as selective detectors of substrate-near cell components
Distance from the surface
10 0

Detection of substrate-dependent alterations in initial cell attachment
SiO2/Ti 39 Absorbed mass [arb.units / 34 0 29 0 24 19 0 14 0 9 4 0 PL

Attachment points

Evanescent light

100

Lamini

Waveguide

PLL+F

OWLS methods allow quantifying the deposition of material in a thin (<150 nm) layer above the solid sensor surface. In terms of cells, it can provide data on focal contacts and adhesion sites, while the rest of the cellular mass remains out of the field of detection.

23 2

23 7

24 2

24 7

25 Time 2 [min]

25 7

26 2

26 7

27 2

Surface chemistry, sensor surface

sensitization

of

the

Bare or amino-functionalized sensor surfaces were coated with different attachment-molecules including poly-L-lysine, laminin, fibronectin, and RGD peptide-mimetics.

The length of the latency period depends on the adhesivity of the provided substrate

Cell preparations
Absorbed mass [arb.units / area]
55 65

Cell suspensions in protein-free, artificial cerebro-spinal fluid were introduced into the measuring cuvette, and the cell - substrate interactions were monitored up to 80 – 120 min. For control, the “attachment” of cells fixed with paraformaldehyde, poisoned by Cytochalasin B, or sedimented at +4 oC were also assayed.
300

NE-4C

300 250 200 150 100 50 0

MDCK

D e p o s ite d m a te r ia l [a r b .u n its ]

200 150 100 50 0 5 15 25

D e p o s itie d m a te ri a l [a r b .u n its ]

250

Deposition of NE 4C cells on laminin 34 0 Active processes 29 37oC 0 24 0 latency 19 0 14 0 9 6oC 0 4 0 0 10 20 30 40 50 60 70 10 Time [min]

Conclusion
5 15 25 35 45

Time [min]

35

45

55

Time [min]

The ∆N versus time function clearly distinguished the active attachment from passive sedimentation. The results indicate that OWLS techniques allow rapid evaluation of cell – matrix interactions and provide tools to characterize the composition of sets of adhesion molecules on cell surfaces.

OWLS signals in the first 60 min of attachment

References
1. Vörös J. et al., Optical grating coupler biosensors. Biomaterials 23 (2002) 3699-3710. 2. Madarasz E. et al., (2006): Attachment of NE-4C neuroectodermal stem cells to different surfaces: evaluation of cell – substrate interactions by optical waveguide light mode spectroscopy (OWLS) FEBS Letts. In press 3. Madarasz E. et al., (2006):Label-free OWLS assays on cell adhesivity. SBS Conference Seattle 4. www.owls-sensors.com

OWLS recordings revealed characteristic differences in cellbehavior between the two types of cells

MicroVacuum Ltd. H-1147, Kerékgyártó u. 10., Budapest, Hungary
Web: http://www.owls-sensors.com/; E-mail: info@owls-sensors.com

Phone: + 36 1 252 1991; +36 1 467 0108

Fax: +36 1 221 7996

Copyright @ Microvacuum, 2006

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