# Human Amniotic Membrane Procurement Protocol and Evaluation of a Simplified Alkaline Decellularization Method

## TL;DR

This paper compares two methods for removing cells from human amniotic membrane, finding a simplified alkaline method to be efficient and practical, though with some trade-offs in tissue structure preservation.

## Contribution

The study introduces a simplified alkaline decellularization protocol that reduces cost and time while maintaining tissue integrity.

## Key findings

- The alkaline method achieved high nuclear clearance but partially preserved cellular structures.
- Detergent-based methods preserved collagen intensity closer to native tissue.
- The simplified alkaline method is more practical but causes slight ECM alterations.

## Abstract

Amniotic membrane (AM) has gained wide application in regenerative medicine due to its biocompatibility and extracellular matrix (ECM) composition. Effective decellularization is essential to minimize immunogenicity while preserving tissue architecture. This study standardized AM procurement and compared a simplified alkaline-based decellularization protocol with a conventional detergent–alkaline method, emphasizing practicality, histological integrity, and collagen preservation. Methods: Human AM was aseptically obtained from placental tissue and processed using either method. Histological analysis with hematoxylin eosin and Masson’s trichrome staining quantified nuclear content and collagen integrity. Results: The alkaline method achieved the greatest nuclear clearance but retained epithelial outlines, indicating partial persistence of cellular structures. In contrast, the detergent method achieved complete morphological decellularization but showed slightly higher residual nuclear signal. Masson’s trichrome staining revealed that the detergent-based method preserved collagen intensity most closely to native tissue (mean gray values: 128.3 ± 28.2 vs. 140.2 ± 23.4), while the alkaline group exhibited significantly reduced staining (177.8 ± 17.2; p < 0.001). Conclusions: the simplified alkaline method provided efficient decellularization with reduced cost, time, and cytotoxic risk, making it a practical approach for AM processing. However, partial ECM alteration suggests that detergent-based methods remain preferable when optimal structural preservation is required.