Cryopreservation Preserves the Structural Integrity, Biochemical Components and Biologic Function of Amniotic Membrane Tissue

Abstract

Purpose: The therapeutic potential of amniotic membrane (AM) has been examined in a variety of clinical indications, particularly in ophthalmology where AM has been used extensively for indications such as pterygium, conjuntivochalasis, and corneal defects. To date, there has been no comprehensive study comparing cryopreserved amniotic membrane processed with the CryoTek™ method and fresh AM.

Methods: Fresh AM, either thin or thick, and fresh amniochorion were compared to CryoTek™ processed thin and thick AM and amniochorion. Histological properties were assessed by hematoxylin and eosin, Masson’s Trichrome, and Safranin O staining. Biochemical properties were measured by contents of protein, albumin, and hyaluronan (HA) with their molecular weight spectrum. Functional assays compared macrophage viability and proliferation, and human corneal fibroblast TGF-β1 induction.

Results: Histochemical staining confirmed that the cryopreservation process did not alter the tissue architecture or collagen and glycosaminoglycan content. Biochemically, cryopreservation reduced total protein and human serum albumin contents, but retained high molecular weight hyaluronan species including HC-HA complex, known to exert anti-inflammatory and anti-scarring effects. Both fresh and cryopreserved AM water-soluble extracts similarly suppressed viability and proliferation of RAW264.7 macrophages, and dose-dependently inhibited the TGF-β1 promoter activity in human corneal fibroblasts.

Conclusions: These results collectively indicate that cryopreservation by the CryoTek™ method effectively preserves histological, biochemical and functional components of the AM tissue.

Keywords: 765 wound healing • 687 regeneration • 480 cornea: basic science