Biomedical Engineering Reference
In-Depth Information
the main drawback of these in vitro techniques. It has been suggested that active and
paracellular solute transport is not compromised during these in vitro experiments
[212] . Along with their drawbacks, these techniques have several advantages over
in vivo and other in vitro methods. Advantages over in vivo methods include bypass-
ing drug dissolution and stomach-emptying steps, avoiding degradation of peptides
and proteins in the stomach, and allowing control over drug input and choice of the
intestinal region to be perfused. In these techniques, lymphatic and blood vessels
remain intact for solute uptake and have relatively extended tissue viability as com-
pared to other in vitro techniques.
10.7.1.3 Brush Border Membrane Vesicles
Brush border membrane vesicles are isolated from human intestinal epithelial cells.
These vesicles are further purified and used to evaluate drugs and their dosage
form for intestinal transport studies [213,214] . The isolation and purification pro-
cedure of brush border membrane vesicles was described by Schmitz et al. [215] .
However, this process was complex in nature and was further simplified by Kessler
et al. [216] . During preparation, brush border fragments should be separated from
microsomal fragments (endoplasmic reticulum) using calcium ions (Ca 2 ). Brush
border fragments are not readily separable from microsomal fragments (endoplasmic
reticulum) unless Ca 2 ions are used [217] . Endoplasmic reticulum and the mito-
chondria are converted in the larger particulate form after aggregation with the help
of Ca 2 . Particles thus formed are easily separated by slow speed centrifugation at
about 2000  g . Supernatant containing brush border fragment is centrifuged at
20,000  g to get a pellet of brush border fragment [215] .
10.7.1.4 Cell Line Studies
Cell line studies have gained plenty of attention from researchers because of their
extended viability. Intestinal cells are grown in the form of monolayers and used to
study intestinal drug transport [218-222] . These monolayers have extended viability
when kept in nutrient- and oxygen-rich culture media and hence are preferred over
other in vitro methods. Generally, intestinal cells are difficult to culture because of
the unavailability of a well-differentiated human intestinal cell line, but the CACO-
2 cell line, obtained from the human colon, is isolated, differentiated, and grown in
culture media. When CACO-2 cells are cultured in a medium, they undergo entero-
cytic differentiation and develop occluding junctional complexes between adjacent
cells within 3 days, and develop morphological characteristics of the small intestine,
such as microvilli, desmosomes, occluding junctions, and cell polarity after about
15 days. Due to extended viability, cells grown on microporus membranes can be
directly used for drug diffusion studies for at least 20 days. Generally, polycarbon-
ate and nitrocellulose membranes are used because of their translucent nature, which
allows microscopic examination, and also because they are permeable to both hydro-
philic and hydrophobic solutes, whatever their MW [223] .
The transport of proteins and peptides across intestinal membrane is studied using
CACO-2 monolayers. More realistic results are obtained from these studies because
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