A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture

Abstract

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.

Differentiation of Human Limbal-Derived Induced Pluripotent Stem Cells Into Limbal-Like Epithelium

“…With written informed consent, human placentas were obtained after cesarean section deliveries (approved Cedars‐Sinai Medical Center institutional review board protocol Pro00019230). HAM isolation and removal of the amniotic epithelium using 0.25 M NaOH have been previously described [34]. For iPSC culture, denuded HAM was placed in CellCrown inserts (Sigma‐Aldrich, St. Louis, MO, http://www.sigmaaldrich.com), with the epithelial side facing up.…”

A Cost-Effective Method to Assemble Biomimetic 3D Cell Culture Platforms

“…These methods required soaking of the membrane in the treating reagents which may affect the integrity of the stroma and cause damage of its matrix proteins, or may render the membrane fragile and difficult to handle. The brief alkaline treatment method we followed proved to be simple, efficient and reproducible [19]. Using our designed cell crown enabled the reproducible preparation of hAM readily in short time.…”

“…We compared the use of trypsin-EDTA with mechanical scraping to the use of sodium hydroxide (NaOH), as reported by Saghizadeh et al[19]. In the trypsin treatment method, trypsin (0.25%, Life technologies; CA, USA) was used to treat the membrane for 90 minutes following a modification of a published protocol [20].…”

Biocompatible surgical meshes based on decellularized human amniotic membrane

“…In order to diminish the immunogenicity originated from cells and improve the in vivo biocompatibility, people always separated its basement layer (containing epithelial cells) and conduct decellularization performance to obtain a denuded human amniotic scaffold with~50 μm thickness [16]. Some attempts have been made to decellularize HAM [17]. For example, surfactants like sodium dodecyl sulfate (SDS), ethylenediaminetetraacetate dehydrate (EDTA), sodium hydrochloride and mechanical scraping were used [18,19].…”

“…Some attempts have been made to decellularize HAM [17]. For example, surfactants like sodium dodecyl sulfate (SDS), ethylenediaminetetraacetate dehydrate (EDTA), sodium hydrochloride and mechanical scraping were used [18,19]. However, these treatments cannot remove amniotic epithelial cells completely and shall be optimized the detail procedures.…”