CHARACTERIZATION OF THE ADHESIVE INTERACTIONS BETWEEN CELLS AND BIOMATERIALS - Micro and Nanotechnologies in Engineering Stem Cells and Tissues - page 173

larger cell population, assays that provide a greater distractive force must be
considered.

7.4.2 Centrifugation

For larger cell populations, centrifugation assays offer a simple and reproducible
means to quantify cell adhesion. In general, cells are seeded onto a substrate and
allowed to adhere for a period of time, typically no longer than 1 h. 63 After
adherence, cells are subjected to a perpendicular detachment force generated by
spinning at a specified speed in a standard laboratory centrifuge. 63 A schematic of a
typical centrifugation assay procedure can be seen in Figure 7.2. The ratio of post-
spin cell count to pre-spin cell count results in the adherent fraction of cells at the
designated force set by the centrifugal speed.
Centrifugation assays have also been used to quantify differences between initial
adhesion and “strengthened” adhesion. 64 Strengthened adhesion is defined as
adhesion that occurs while cells are incubated on a substrate. As with the micro-
manipulation assays, adhesion strength increased with longer seeding times (e.g., 30
vs. 60min), 64,70 showing that adhesion is time dependent. Reyes and Garcia 70 further
modified the centrifugation assay and developed the mean adhesion strength value,
which is the force that causes 50% cell detachment, for fibrosarcoma cells seeded on
fibronectin coated 96-well plates. Centrifugation assays offer a simple and repro-
ducible method of characterizing biomaterials based on the ability of the material to
successfully initiate cell adhesion.
Despite the success of centrifugation assays, there are limitations, including the
fact that only one speed can be subjected to cells at a time and that at longer adhesion
times (

1 h), 63 the distraction forces generated by the centrifuge are not large
enough to displace large cell populations.

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7.4.3 Hydrodynamic Shear Stress

Flow systems have been developed to apply a wide range of shear detachment forces
to large adherent cell populations and are generally considered a more reliable
adhesion measurement system. 63 Hydrodynamic shear stress assays are classified
according to the geometry of the flow responsible for generating the shear detachment

Precentrifuge
count bound
cells

Postcentrifuge
count bound
cells

RT 37°C Wash

1 hr. 1 hr. D-PBS

Apply
substrate

Add
cells

Invert plate
and centrifuge

Obj.
lens

Obj.
lens

Adhesive seal

FIGURE 7.2 Schematic of a centrifugation assay procedure.