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

Mehrnoosh Saghizadeh,Homayon Ghiasi,Ezra Maguen,Alexander V Ljubimov,Andrei A Kramerov,Michael A Winkler,William J Brunken,Katerina Jirsova,Dhruv Sareen,David M Hemmati,Slobodan D Dimitrijevich,Yaron S Rabinowitz,Clive N Svendsen,Loren Ornelas,Chantelle A Ghiam,Deepak Shukla

doi:10.1371/journal.pone.0079632

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.

Highlights

Limbal epithelial stem cells (LESC) in the human cornea reside at its periphery known as the corneoscleral limbus and continuously renew the corneal epithelium [1,2,3]
To better visualize the debridement, human amniotic membrane (HAM) was stained with Trypan blue (TB) that is excluded by live cells
HAM presented a monolayer of tightly packed epithelial cells, all stained by TB (Fig. 2A,B)

Summary

Introduction

Limbal epithelial stem cells (LESC) in the human cornea reside at its periphery known as the corneoscleral limbus and continuously renew the corneal epithelium [1,2,3] In some conditions these cells degenerate and/or die, leading to limbal epithelial stem cell deficiency (LSCD). LSCD results in corneal erosions and vascularization, conjunctival ingrowth (conjunctivalization), and scarring, causing compromised corneal transparency and gradual vision loss [4,5]. This condition may be hard to treat especially in cases of total stem cell deficiency [4]

Methods

Results

Conclusion