Comparison of impact of two decontamination solutions on the viability of the cells in human amnion
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Comparison of impact of two decontamination solutions on the viability of the cells in human amnion Ingrida Smeringaiova . Peter Trosan . Miluse Berka Mrstinova . Jan Matecha . Jan Burkert . Jan Bednar . Katerina Jirsova
Received: 4 January 2017 / Accepted: 6 June 2017 Ó Springer Science+Business Media B.V. 2017
Abstract Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining.
I. Smeringaiova P. Trosan J. Bednar K. Jirsova (&) Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 2, 128 08 Prague, Czech Republic e-mail: [email protected] M. B. Mrstinova J. Matecha Department of Obstetrics and Gynaecology, Second Faculty of Medicine, Charles University, Prague, Czech Republic J. Burkert K. Jirsova Department of Transplantation and Tissue Bank, Motol University Hospital, Prague, Czech Republic
The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability. Keywords Amniotic membrane Decontamination solution Viability Apoptosis Epithelial and mesenchymal cells
Introduction The human placenta at term has two distinguishable fetal membranes that develop separately: the amniotic membrane (HAM) on the fetal surface of placenta and the chorionic membrane underneath the HAM. The two membranes remain separable due to the existence of a spongy layer in between. HAM
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Cell Tissue Bank
consists of a monolayer of epithelial cells (EC), which resides on a resistant basement membrane, and of a mesenchymal layer
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