Abstract 17 Comparison of Different Support Materials and Cryoprotectants for Amniotic Membrane Cryopreservation

INTRODUCTION: The amniotic membrane (AM) is a thin membrane derived from the placenta, rich in stem cells and structural components that stimulate tissue regeneration. This tissue has emerged as a biological dressing for several clinical applications. Many preservation techniques have been proposed for AM; however, searching for a method capable of preserving their physical and biological properties remains challenging. OBJECTIVE: This work investigated different ways to support the AM in packing, as well as two different cryoprotectants to preserve its histological, cytological, and biological characteristics. METHODS: Human placentas were processed for AM isolation and stored under various conditions: gauze (G) and petrolatum gauze (PG) were evaluated as support materials for the AM, while DMSO and glycerol were analyzed as cryoprotectants for both materials. In addition, after selecting the best support material, two forms of product presentation were also explored: flat fragment (FF) and roll (R). All protocols were stored in liquid nitrogen for at least 2 weeks. After this period, tissue was thawed, and structural integrity, cellular viability, and secretome analysis were performed. RESULTS: Cellular viability with PG was significantly reduced compared with fresh AM (Fresh: 63.6% ± 11.8%; PG+DMSO: 37.2% ± 17.9%; PG+glycerol: 23.2% ± 18.9%) (Figure 1A). Regarding gauze presentation, no difference in cell viability was found between cryoprotectants in the flat fragment groups (FF+DMSO: 53.4% ± 20.2% and FF+Glycerol: 55.5% ± 15.8%) (Figure 1B). As for the roll groups, cell viability was significantly better for DMSO (R+DMSO: 63.9% ± 11.8% and R+Glycerol: 37.1% ± 19.5V) (Figure 1B). Structural integrity evaluated by tissue immunofluorescence and picrosirius confirmed that the extracellular matrix components, such as laminin (Figure 1D), fibronectin (Figure 1E), and collagen (Figure 1F and 1G), were preserved with both cryoprotectants. Likewise, this protocol conserved progenitor/stem cells, assessed by the CD117 marker (Fresh: 25.4% ± 8.7%, DMSO:18.1% ± 9.5%, and Glycerol: 23.1% ± 11.7%) (Figure 1C). Secretome analysis showed a slight reduction of the proteins secreted by cryopreserved AM compared with fresh AM, although the difference was not significant (Figure 1H). Essential proteins involved in tissue regeneration (fibronectin, laminins, collagens, cadherins, and cytokeratins) and immune and antimicrobial control (PTX3, TMP1, SOD, lysozyme C, and immunoglobulins) could be detected from both AM (Figure 1I). DISCUSSION: PG support was harmful to cell viability. DMSO exhibited better results in cell viability and biological functions regardless of tissue presentation and cryopreservation.