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

Abstract

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.

The effect of glycerol as a cryoprotective agent in the cryopreservation of adipose tissue

“…Several studies have reported that using DMSO or DMSO + FBS as CPAs was effective in protecting cell survival and retention volume of the adipose tissue compared with the absence of CPA [ 9 , 22 – 25 ]. However, the use of DMSO and FBS has the risk of toxicity and zoonotic infection [ 18 ]. Furthermore, due to the integrity and complexity of tissue compared with isolated cells, thorough elution of DMSO may be difficult [ 26 , 27 ].…”

“…The multi-lineage differentiation capacity of the ASCs was detected as previously described [ 18 ]. Adipogenic differentiation was cultured at a concentration of 10 6 cells/mL in the adipogenic induction medium followed by culture for 14 days.…”

Optimized Protocol for Proportionate CNS Cell Retrieval as a Versatile Platform for Cellular and Molecular Phenomapping in Aging and Neurodegeneration

“…Physiologically, in unicellular organisms, trehalose is secreted as part of a stress response elicited by environmental discomfort, e.g., through exposure to thermal stress or low humidity, in order to preserve cellular integrity [ 53 ]. Moreover, it has achieved broad applications due to its favorable properties regarding osmoregulation, cryoprotection, and desiccation tolerance [ 54 , 55 ]. According to current biochemical models, trehalose is assumed to either build a protective, cocoon-like matrix around nearby protein structures, to entrap water molecules that are remote from protein structures and reduce protein–water interferences, or stabilize them by the establishment of hydrogen-bonds substituted from water molecules [ 56 ].…”

Time‐Dependent Hydrogen Bond Network Formation in Glycerol‐Based Deep Eutectic Solvents

“…[3,4] Because glycerol molecules can penetrate cell membranes, [5] their presence inside of cells, together with an extensive mixed hydrogen bond (HB) network formation, can result in the dehydration of those cells, slowing down the dynamics of water molecules and preventing the ice-formation during freezing and/or thawing. [6][7][8][9][10] Recently, a contemporary class of green solvents, Deep Eutectic Solvents (DESs), introduced by Abbott et al, [11] are regarded as a potential new type of cryoprotectants. [12,13] DESs are frequently classified as Ionic Liquids' (ILs) analogues.…”

“…Because glycerol molecules can penetrate cell membranes, [5] their presence inside of cells, together with an extensive mixed hydrogen bond (HB) network formation, can result in the dehydration of those cells, slowing down the dynamics of water molecules and preventing the ice‐formation during freezing and/or thawing [6–10] …”