Optimizing amniotic membrane tissue banking protocols for ophthalmic use

Abstract

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco's modified eagle's medium (DMEM) and glycerol at -80 °C and at -196 °C for a period of 6 weeks and at 4 °C in 70 % alcohol for 6 weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5 × 5 cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at -196 and -80 °C for 6 weeks. Similar sections were also stored at 4 °C in 70 % alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10 % FBS in humidified atmosphere of 5 % carbon dioxide at 37 °C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8 × 10(6); 1.59, after 6 weeks in DMSO:FBS at -196 °C 3.0 × 10(6); 2.38 and at -80 °C 2.1 × 10(6); 1.60, in DMEM:Glycerol at -196 °C 3.6 × 10(6); 2.33 at -80 °C 23 × 10(6); 1.66 and at 4 °C 3.3 × 10(6); 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at -196 °C showed morphology similar to fresh AM in both preservation media and AM stored at -80 °C showed disruption of the stromal matrix. At 4 °C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at -196 °C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6 weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.

Effects of Preservation Methods in the Composition of the Placental and Reflected Regions of the Human Amniotic Membrane

“…In contrast, Fénelon et al [2018], comparing the impact of preservation methods on AM properties for tissue engineering applications, showed that the epithelial cells presented a slight degree of vacuolar degeneration. Moreover, Hettiarachchi et al [2016] compared the morphology of fresh and cryopreserved AM, demonstrating that AM storage in 1:1 DMEN:glycerol at −80°C disrupted the stromal matrix and the epithelial cell monolayer, presenting patchy areas of epithelial cell loss. The different histological aspects between these 2 works and our study could be due to the cryopreservation media used by Fénelon et al [2018] (e.g., RPIM) and to the longer storage period used by Hettiarachchi et al [2016].…”

A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

“…The release of proteins from dead cells in the protein extracts may have contributed to the diminished efficacy for CSK propagation compared to fresh amnion extracts. Alternative cryopreservation in the presence of dimethylsulfoxide (DMSO), a common cryoprotectant to prevent intracellular ice formation, or under freezedrying with the removal of ice through sublimation, can maintain cell viability [48,49]. However, our earlier report has revealed a significant loss of growth factors and ECM proteins (such as laminin and fibronectin) in freeze-dried amnion, which might produce the protein extract with reduced potential in supporting cell growth [50].…”

Transplantation in ophthalmology—single-center perspectives

“…Corneal transplantation is relatively rare in comparison to amniotic membrane transplantation, which was confirmed by the current single-center study (7). Corneal transplantation restores visual function, especially when the ocular surface is significantly affected, but depends very much on the retrospective consequences and restauration of the normal anatomical relationships between lids, cornea, and conjunctiva (8,9).…”