Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for
transplantation to patients with
limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with
EDTA and/or
proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for
DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral
buffer. Cell removal was more thorough and uniform than with
EDTA, or
EDTA plus mechanical scraping with an electric toothbrush, or
n-heptanol plus
EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or
thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including
laminin α2, γ1-γ3 chains, α1/α2 and α6
type IV collagen chains,
fibronectin, nidogen-2, and
perlecan.
Sodium hydroxide treatment was efficient with fresh or cryopreserved (10%
dimethyl sulfoxide or 50%
glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before
transplantation to patients.