Trehalose to cryopreserve human pluripotent stem cells

Ntai, Aikaterini; Spada, Alberto; Blasio, Pasquale De; Biunno, Ida

doi:10.1016/j.scr.2018.07.021

Cited by 34 publications

(16 citation statements)

References 59 publications

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“…Moreover, Ntai et al also reported that the time of preservation was no obvious influence on preservation. Using different CPAs to preserve pluripotent stem cells, the authors found that the viability did not differ between cells preserved for 7 days and those preserved for 30 days [ 39 ]. Therefore, we evaluated the outcome at 30 days of preservation in the present study.…”

Section: Discussionmentioning

confidence: 99%

The combination of trehalose and glycerol: an effective and non-toxic recipe for cryopreservation of human adipose-derived stem cells

et al. 2020

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Background Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPAs) protect cells from cryodamage during cryopreservation. Safe and efficient cryopreservation of ADSCs is critical for cell-based therapy in clinical applications. However, most CPAs are used at toxic concentrations, limiting their clinical application. Objective The aim of this study is to develop a non-toxic xeno-free novel CPA aiming at achieving high-efficiency and low-risk ADSC cryopreservation. Methods We explored different concentrations of trehalose (0.3 M, 0.6 M, 1.0 M, and 1.25 M) and glycerol (10%, 20%, and 30% v/v) for optimization and evaluated and compared the outcomes of ADSCs cryopreservation between a combination of trehalose and glycerol and the commonly used CPA DMSO (10%) + FBS (90%). All samples were slowly frozen and stored in liquid nitrogen for 30 days. The effectiveness was evaluated by the viability, proliferation, migration, and multi-potential differentiation of the ADSCs after thawing. Results Compared with the groups treated with individual reagents, the 1.0 M trehalose (Tre) + 20% glycerol (Gly) group showed significantly higher efficiency in preserving ADSC activities after thawing, with better outcomes in both cell viability and proliferation capacity. Compared with the 10% DMSO + 90% FBS treatment, the ADSCs preserved in 1.0 M Tre + 20% Gly showed similar cell viability, surface markers, and multi-potential differentiation but a significantly higher migration capability. The results indicated that cell function preservation can be improved by 1.0 M Tre + 20% Gly. Conclusions The 1.0 M Tre + 20% Gly treatment preserved ADSCs with a higher migration capability than 10% DMSO + 90% FBS and with viability higher than that with trehalose or glycerol alone but similar to that with 10% DMSO + 90% FBS and fresh cells. Moreover, the new CPA achieves stemness and multi-potential differentiation similar to those in fresh cells. Our results demonstrate that 1.0 M Tre + 20% Gly can more efficiently cryopreserve ADSCs and is a non-toxic CPA that may be suitable for clinical applications.

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Section: Discussionmentioning

confidence: 99%

The combination of trehalose and glycerol: an effective and non-toxic recipe for cryopreservation of human adipose-derived stem cells

et al. 2020

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“…In contrast, we observed an improvement in the quality of the recovered cells over time. Many researchers have reported the substitution or combination of DMSO with other cryoprotectants, such as ethylene glycol or polyethylene glycol [ 55 , 63 , 68 , 69 , 70 , 71 ]. However, these cryoprotectants were shown to be more genotoxic than DMSO when cryopreserving human oocytes [ 72 ].…”

Section: Discussionmentioning

confidence: 99%

Insights into Species Preservation: Cryobanking of Rabbit Somatic and Pluripotent Stem Cells

Gavin-Plagne

Perold

Osteil

et al. 2020

IJMS

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Induced pluripotent stem cells (iPSCs) are obtained by genetically reprogramming adult somatic cells via the overexpression of specific pluripotent genes. The resulting cells possess the same differentiation properties as blastocyst-stage embryonic stem cells (ESCs) and can be used to produce new individuals by embryonic complementation, nuclear transfer cloning, or in vitro fertilization after differentiation into male or female gametes. Therefore, iPSCs are highly valuable for preserving biodiversity and, together with somatic cells, can enlarge the pool of reproductive samples for cryobanking. In this study, we subjected rabbit iPSCs (rbiPSCs) and rabbit ear tissues to several cryopreservation conditions with the aim of defining safe and non-toxic slow-freezing protocols. We compared a commercial synthetic medium (STEM ALPHA.CRYO3) with a biological medium based on fetal bovine serum (FBS) together with low (0–5%) and high (10%) concentrations of dimethyl sulfoxide (DMSO). Our data demonstrated the efficacy of a CRYO3-based medium containing 4% DMSO for the cryopreservation of skin tissues and rbiPSCs. Specifically, this medium provided similar or even better biological results than the commonly used freezing medium composed of FBS and 10% DMSO. The results of this study therefore represent an encouraging first step towards the use of iPSCs for species preservation.

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“…Owing to multiple cryoprotective properties, selected disaccharides, such as sucrose and trehalose, are widely used in cryopreservation practice as additives to various freezing [2,51] and vitrification solutions [15,33,83]. Conceptually, combined introduction of sugars into cryopreservation media and their pre-freeze loading into cells serves as an alternative to conventional cryopreservation workflow.…”

Section: Discussionmentioning

confidence: 99%

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

et al. 2019

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Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me 2 SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me 2 SO-and serum-free biopreservation strategies due to safety concerns over Me 2 SO-induced side effects and immunogenicity of animal serum.In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay).Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me 2 SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at À 80 � C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me 2 SO-and serumfree cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells.

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Trehalose to cryopreserve human pluripotent stem cells

Cited by 34 publications

References 59 publications

The combination of trehalose and glycerol: an effective and non-toxic recipe for cryopreservation of human adipose-derived stem cells

The combination of trehalose and glycerol: an effective and non-toxic recipe for cryopreservation of human adipose-derived stem cells

Insights into Species Preservation: Cryobanking of Rabbit Somatic and Pluripotent Stem Cells

Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars

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