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.

Figure 1. Glycerol-cryopreserved HAM after thawing, Trypan blue staining and treatment.

A, mesh Sanatyl 20 support for HAM in a Petri dish. B, HAM on Sanatyl 20 after TB staining (the whole membrane is stained as glycerol kills the cells). C, the right half of HAM treated with NaOH (staining is significantly lighter than on untreated left half). D, the same HAM without Sanatyl 20 support; note lack of TB staining (cell removal) on the most part of the treated right half. Low-magnification pictures are presented.

Figure 2. Cell removal by NaOH from cryopreserved HAM.

A, phase contrast of cryopreserved and thawed out HAM showing a monolayer of amniotic epithelial cells. B, the same HAM stained with TB; all cells are stained. C, cotton swab rubbing of HAM leaves many amniotic epithelial cells behind. D, after 30 sec rub with 0.5 M NaOH-soaked cotton swab no cells are left. E, NaOH effectively removes cells from HAM (gray line shows the boundary of the rubbed zone). Bar in A–D = 50 µm; in E, bar = 100 µm.

Figure 3. Laminin γ3 chain and type IV collagen α1/α2 chain expression in intact HAM and after various treatments.

Laminin γ3 (A) and type IV collagen α1/α2 (B) are found in the limbal epithelial BM but not in the central corneal BM. Here and below, single long arrows show denuded parts with no cells (DAPI-counterstained nuclei). Double arrows show places where HAM is disrupted after scraping the membrane with electric toothbrush on low speed. EDTA together with rubbing or n-heptanol leaves a number of epithelial cells still attached to HAM. In contrast, thermolysin and NaOH leave little (single short arrows) to no epithelium on the treated HAM. Except for EDTA+toothbrush scraping, all treatments preserve normal continuous staining patterns of both BM components. Immunohistochemical staining of O.C.T.-embedded and sectioned HAM. Bar = 20 µm.

Figure 4. Laminin γ2 chain and nidogen-2 expression in intact HAM and after various treatments.

Laminin γ2 (A) and nidogen-2 (B) are also expressed in central and limbal epithelial BM. The effects of various treatments are similar to the ones shown on Fig. 3. EDTA treatment results in incomplete cell removal or even HAM damage (double arrows) after extensive scraping. Thermolysin treatment removes cells well but shows some local irregular staining for proteolysis-sensitive laminin γ2 chain (A). NaOH produces HAM virtually devoid of epithelial cells with continuous staining for both BM components. Immunohistochemical staining of O.C.T.-embedded and sectioned HAM. Bar = 20 µm.

Figure 5. Fibronectin and perlecan expression in intact HAM and after various treatments.

The results on fibronectin (A) and perlecan (B) are similar to other BM components. Only thermolysin and NaOH completely remove amniotic epithelium. Immunohistochemical staining of O.C.T.-embedded and sectioned HAM. Bar = 20 µm.

Figure 6. Negative control with omission of primary antibody.

Immunohistochemical staining of OCT-embedded and sectioned HAM treated with NaOH. No staining is seen apart from DAPI nuclear staining of HAM stromal cells. Bar = 20 µm.

Figure 7. Phase contrast of various cells grown on NaOH-denuded HAM.

Note cobblestone monolayer of telomerase-immortalized corneal epithelial cells (tHCEC), LESC, and LESC-derived iPSC. Fibroblast-derived iPSC also show good spreading of cells and growth. Bar = 40 µm.

Figure 8. Marker analysis of LESC-enriched cultures grown on top of NaOH-denuded HAM.

Note expected positive staining for putative LESC markers PAX6, K14 (A, B; double label), K15, and ΔNp63 (C, D; double label). Immunohistochemical staining of O.C.T.-embedded and sectioned HAM. Bar = 20 µm.