Comparison of impact of two decontamination solutions on the viability of the cells in human amnion

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 (BASE·128) 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. 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 BASE·128 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 BASE·128 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.

Distribution of an analgesic palmitoylethanolamide and other N-acylethanolamines in human placental membranes

“…In respect of wound healing, PEA and AEA are the most interesting of the NAEs studied here because their anti-inflammatory, analgesic, anti-fibrotic and neuroprotective properties [17,34,40] strongly overlap similar features described in AM/ACM [2,3,6,56,64]. Thus, they can be at least partially responsible for the positive effects associated with AM/ACM in the healing process.…”

“…The tissue was repeatedly rinsed by physiological solution and blood clots were removed. Then the placental membranes (AM, ACM) were manually separated from the residual placental tissue, dissected, transferred to a mesh support (Sanatyl, Tylex Letovice, Czech Republic) and divided into 4 cm 2 samples [6,56]. AM and ACM were processed as either fresh tissue or decontaminated tissue (AM-d, ACM-d) with BASE 128 for 24 h at 4-8˚C, according to standard decontamination procedures used for placental membranes in clinical practice [6,56].…”

“…Then the placental membranes (AM, ACM) were manually separated from the residual placental tissue, dissected, transferred to a mesh support (Sanatyl, Tylex Letovice, Czech Republic) and divided into 4 cm 2 samples [6,56]. AM and ACM were processed as either fresh tissue or decontaminated tissue (AM-d, ACM-d) with BASE 128 for 24 h at 4-8˚C, according to standard decontamination procedures used for placental membranes in clinical practice [6,56]. As concentrations of some proteins are not homogeneously distributed within placenta and AM [57], we collected samples from two different areas of placental disc (PL): from the area near the umbilical cord described as central amniotic membrane or placenta (AM1, PL1) and the area at the edge of placental disc (periphery of the placenta, AM2, PL2) [58,59].…”

Inter-placental variability is not a major factor affecting the healing efficiency of amniotic membrane when used for treating chronic non-healing wounds

“…The formers are responsible for both donor-dependent (inter-placental) variations (Hopkinson et al 2006a , b ; Krabcova et al 2014 ) and intra-placental sub-region variations of AM composition (Deihim et al 2016 ; Litwiniuk et al 2018 ; Moraes et al 2021 ). They can be influenced by the donor’s overall physiological status, genetic predisposition, the presence of pathology, or even by the week of pregnancy at which the placenta was retrieved (Skinner et al 1981 ; Tossetta et al 2014 ).…”

“…Similarly, the study based on the evaluation of TGF β s (1,2,3) presence discovered significant differences in their concentration among individual donors proposing a potential modification of the healing effect based on the donor individuality (Hopkinson et al 2006a , b ; Han et al 2008 ). Another study evaluated the placenta quality dependent on the pregnancy week retrieval (Skinner et al 1981 ). Contrary, no significant difference in pluripotency markers concentration was found between the placental and reflected amnion (Garcia-Lopez et al 2019 ), suggesting the homogeneous distribution of the pluripotency transcription factors, making all regions of AM equal in the regenerative processes effect.…”