Biomedical Engineering Reference
In-Depth Information
and esophageal epithelia. In the following sections, specific applica-
tionsof cell sheet technology are described.
44.5 Corneal Surface Reconstruction
The complete loss of limbal epithelial stem cells due to severe
trauma (e.g., thermal and chemical burns) or eye disease (e.g.,
Stevens-Johnson syndrome, ocular pemphigoid) prompts adjacent
conjunctival tissues to completely cover the cornea, followed by
corneal vascularization and opacification with severe visual loss.
Although corneal transplantation using donated eyes is the most
common method of treatment, the high risk of graft rejection and
a shortage of donor corneas remain significant inadequacies. From
these aspects, cultured corneal epithelial cells treated with dispase
or fibrin gel have been used for ocular surface reconstruction.
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These methods require a carrier substrate to be surgically placed
on host corneal stroma. In addition, infection, inflammation, and
microtraumaduetoincompletelybiodegradablecompoundsremain
controversial issues with regard to the use of the gel for tissue
engineering.
In contrast, limbal epithelial stem cells can be isolated and
cultured on temperature-responsive culture surfaces. To create
functionalbioengineeredcornealepithelialsheetssuitablefortrans-
plantation, we harvested human corneal limbal epithelial cells, con-
taining corneal epithelial stem cells, from a US eye-bank cornea
(Fig. 44.6).
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These cells were seeded onto temperature-
responsive culture surfaces on which mitomycin C (MMC)-treated
3T3 feeder cells had been plated one day previously. Corneal
epithelial stem cells remained in the presence of feeder cell lay-
ers and other specialized conditions. Under our culture conditions,
epithelial cell colonies grew to reach confluency within one to
two weeks. After a few additional days in culture, a multilayered
corneal epithelial sheet formed spontaneously, with cells on the
sheet surface exhibiting a cobblestone morphology characteristic of
epithelialcells.Thecornealepithelialcellphenotypewasconfirmed
by immunohistochemistry with an antikeratin 3 antibody specific
for corneal epithelial cells. Bromodeoxyuridine (BrdU) uptake was
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