PMID- 29584778 OWN - NLM STAT- MEDLINE DCOM- 20180709 LR - 20181114 IS - 1932-6203 (Electronic) IS - 1932-6203 (Linking) VI - 13 IP - 3 DP - 2018 TI - The enzymatic de-epithelialization technique determines denuded amniotic membrane integrity and viability of harvested epithelial cells. PG - e0194820 LID - 10.1371/journal.pone.0194820 [doi] LID - e0194820 AB - The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin alpha5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture. FAU - Trosan, Peter AU - Trosan P AUID- ORCID: 0000-0002-4836-0887 AD - Laboratory of the Biology and Pathology of the Eye, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic. AD - Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic. FAU - Smeringaiova, Ingrida AU - Smeringaiova I AD - Laboratory of the Biology and Pathology of the Eye, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic. AD - Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic. FAU - Brejchova, Kristyna AU - Brejchova K AD - Laboratory of the Biology and Pathology of the Eye, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic. FAU - Bednar, Jan AU - Bednar J AD - Laboratory of the Biology and Pathology of the Eye, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic. AD - Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic. FAU - Benada, Oldrich AU - Benada O AD - Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic. FAU - Kofronova, Olga AU - Kofronova O AD - Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic. FAU - Jirsova, Katerina AU - Jirsova K AD - Laboratory of the Biology and Pathology of the Eye, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic. AD - Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20180327 PL - United States TA - PLoS One JT - PloS one JID - 101285081 RN - 0 (Collagen Type IV) RN - 0 (Laminin) RN - 0 (Nanog Homeobox Protein) RN - 0 (SOXB1 Transcription Factors) RN - 0 (laminin alpha5) RN - 9007-49-2 (DNA) RN - 9G34HU7RV0 (Edetic Acid) RN - EC 3.4.21.4 (Trypsin) SB - IM MH - Amnion/cytology/*metabolism/pathology MH - Cell Proliferation MH - Cell Survival MH - Cells, Cultured MH - Collagen Type IV/metabolism MH - DNA/analysis/isolation & purification MH - Edetic Acid/chemistry MH - Epithelial Cells/cytology/*metabolism/pathology MH - Humans MH - Laminin/metabolism MH - Microscopy, Electron, Scanning MH - Nanog Homeobox Protein/metabolism MH - Re-Epithelialization MH - SOXB1 Transcription Factors/metabolism MH - Trypsin/metabolism PMC - PMC5870984 COIS- Competing Interests: The authors have declared that no competing interests exist. EDAT- 2018/03/28 06:00 MHDA- 2018/07/10 06:00 PMCR- 2018/03/27 CRDT- 2018/03/28 06:00 PHST- 2017/10/06 00:00 [received] PHST- 2018/03/09 00:00 [accepted] PHST- 2018/03/28 06:00 [entrez] PHST- 2018/03/28 06:00 [pubmed] PHST- 2018/07/10 06:00 [medline] PHST- 2018/03/27 00:00 [pmc-release] AID - PONE-D-17-36030 [pii] AID - 10.1371/journal.pone.0194820 [doi] PST - epublish SO - PLoS One. 2018 Mar 27;13(3):e0194820. doi: 10.1371/journal.pone.0194820. eCollection 2018.