A Rapid and Accurate Method for the Stem Cell Viability Evaluation: The Case of the Thawed Umbilical Cord Blood

Katsares, V.; Petsa, Anastasia; Felesakis, Antigonos; Paparidis, Zissis; Nikolaidou, Eleni; Gargani, Sofia; Karvounidou, Iliana; Ardelean, Karina-Alina; Grigoriadis, Nikolaos; Grigoriadis, John

doi:10.1309/lmle8bvhywct82cl

Cited by 13 publications

(9 citation statements)

References 16 publications

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“…Cell density was measured after every 24 h interval for nine subsequent days from initial culture. The total number of cells/ml was calculated according to the method explained by Dodds and Roberts (1986) and cell viability was determined by using trypan blue exclusion test (Katsares et al 2009) using the following formulas …”

Section: Methodsmentioning

confidence: 99%

Plant regeneration from cell suspension culture in Saccharum officinarum L. and ascertaining of genetic fidelity through RAPD and ISSR markers

et al. 2017

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The aim of this study was to produce sugarcane plantlets from cell suspension culture and study its genetic fidelity using molecular markers. The study was carried out using sugarcane varieties Co 86032 and Q117. Callus cultures of both the varieties were optimized using six different callus induction media. After screening the growth response of callus on six different callus induction media, it was observed that medium no. VI supplemented with 500 mg l−1 of each PVP, Casein hydrolysate and MES buffer showed high amounts of callus in Co 86032 (79.66 ± 0.44%) and Q117 (82.83 ± 1.69%). Addition of PEG 8000 at 2.5% to this medium had a profound impact on inducing somatic embryogenesis in Co 86032 (54.66 ± 1.76%) and Q117 (66.66 ± 2.60%) as compare to control (24.33 ± 1.76%) and (27.33 ± 2.73%), respectively. Cell suspension cultures were established by culturing embryogenic calli in liquid medium showed well established suspension cultures with fever cell aggregates. There was negligible cell division during initial 2 days of incubation and cell count increased rapidly between 2 and 8 days. Further incubation beyond 8 days resulted in a decrease in cell viability. Enhanced callus proliferation in Q117 while enhanced shoot regeneration in Co 86032 was observed from cell suspension culture. The clonal fidelity of in vitro regenerated plants was assessed by using RAPD and ISSR markers. Analysis of the ten RAPD markers indicated that 90.48 and 86.95% true-to-type regenerated plantlets in Co 86032 and Q117, respectively. However, in the ISSR markers, Co 86032 did not show any polymorphism and in the Q117, 92.18% true-to-type plantlets were found. These results confirmed that somaclonal variation occurs during the process of indirect organogenesis and RAPD and ISSR marker based molecular analysis is a suitable method for an early detection of variation in sugarcane.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0579-3) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Plant regeneration from cell suspension culture in Saccharum officinarum L. and ascertaining of genetic fidelity through RAPD and ISSR markers

et al. 2017

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“…At Day 7, cells were mechanically extracted using a Pasteur pipette with fresh and warm (37°C) medium 23, 26. The obtained cell suspension (100 μL) was diluted at the 1 : 1 ratio using the Trypan Blue (CAS 72‐57‐1, Sigma, Germany) staining solution and live cells were counted by a hematocytometer 23, 26, 40…”

Section: Methodsmentioning

confidence: 99%

A library of tunable agarose carbomer‐based hydrogels for tissue engineering applications: The role of cross‐linkers

Rossi

Perale

Storti

et al. 2011

J of Applied Polymer Sci

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The role of the crosslinking agents was studied for a series of agarose-carbomer-based hydrogels, specifically developed for tissue engineering applications, and was quantified using the most typical polycondensation parameter; the ratio between the reacting moieties, i.e., hydroxyl (A) and carboxyl (B) groups. Because of the bonds among hydrophilic groups, as A/B ratio was increased, the gel network showed higher compactness and less ability to swell. The role of crosslinkers was also further analyzed using environmental scanning electron microscopy (E/SEM) and rheological measurements. SEM analysis underlined the presence of different structures as well as the erosion due to the presence of cosolvents in hydrogel synthesis. Rheological measurements showed the dependence of crossover strain value and yield stress upon the ratio of hydroxyl/carboxyl groups and, generally, a clear pseudoplastic behavior. Such detailed characterizations were essential to investigate the design of an optimized formulation capable of being a proper hosting environment for glial cells, which were here used as they are a promising cell type in several central nervous system repair strategies.

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“…Other factors possibly involved in this discrepancy of cell cryosurvival include the limitations of both methods used to quantify cryosurvival. Although the trypan blue staining method is widely used to measure cell viability, it has been described as highly subjective and possessing significant accuracy errors in numerous studies [31,47]. In this work, PP-50 has been shown to mediate the transport of trehalose, highlighting its use in existing cell cryopreservation protocols and therapeutic applications.…”

Section: Accepted Manuscriptmentioning

confidence: 92%

“…Individual vials were thawed in a 37 °C water bath with continuous and mild agitation for 5 min. Viability was firstly determined by the trypan blue exclusion method using a cell counter [31]. Cells negative for trypan blue were considered live and those stained blue were considered dead.…”

Section: Pp-50/af647 Synthesismentioning

confidence: 99%

Increased cryosurvival of osteosarcoma cells using an amphipathic pH-responsive polymer for trehalose uptake

Mercado

Slater

2016

Cryobiology

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Amphipathic pH-responsive polymers have shown to increase the permeability of cell membranes to trehalose hence improving the cryopreservation of mammalian cells. However, the trafficking of both the polymer and trehalose across the cell membrane has not yet been thoroughly analysed. The objective of this study was to investigate the effect on cryopreservation of the trafficking of the disaccharide trehalose along PP-50, an amphipathic polymer, through an osteosarcoma cell line (SAOS-2). Confocal microscopy analysis confirmed the presence of intracellular labelled trehalose only when incubated in the presence of PP-50. Further analysis confirmed that both trehalose and PP-50 localised in the cytoplasm, accumulated mainly in the perinuclear area. Quantitative analysis of the colocalisation between trehalose and PP-50 showed Pearson and Manders coefficients of 0.862 ± 0.008 and 0.766 ± 0.033, respectively, suggesting a high degree of intracellular colocalisation between these molecules. Cryopreserved cells pre-incubated with trehalose and PP-50 showed increased cryosurvival when compared with cells pre-incubated in the absence of the polymer. PP-50 showed to be directly involved in the uptake of trehalose, a critical characteristic towards use in cryopreservation and biomedical applications.

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A Rapid and Accurate Method for the Stem Cell Viability Evaluation: The Case of the Thawed Umbilical Cord Blood

Cited by 13 publications

References 16 publications

Plant regeneration from cell suspension culture in Saccharum officinarum L. and ascertaining of genetic fidelity through RAPD and ISSR markers

Plant regeneration from cell suspension culture in Saccharum officinarum L. and ascertaining of genetic fidelity through RAPD and ISSR markers

A library of tunable agarose carbomer‐based hydrogels for tissue engineering applications: The role of cross‐linkers

Increased cryosurvival of osteosarcoma cells using an amphipathic pH-responsive polymer for trehalose uptake

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