Evaluation of Functional and Structural Alterations in Muscle Tissue after Short-Term Cold Storage in a New Tissue Preservation Solution

Abstract

Storage of muscle preparations in vitro is required for the diagnosis of neuromuscular disorders and for electrophysiological tests. The current standard protocols for muscle storage or transport, i.e. placement on 0.9% NaCl-moistened gauze, lead to impaired function and structural alterations. For other tissues, however, improved preservation methods and solutions have recently been described. In this study, functional and structural alterations in the murine diaphragm were compared after storage on 0.9% NaCl-moistened gauze and after storage in different modifications of the new vascular preservation solution TiProtec®. Muscle force generation after nerve stimulation, histological parameters and ATP levels were investigated after 2.5 h of cold storage at 4°C in the different media and 0.5 h of rewarming at 25°C in Tyrode buffer. Murine diaphragms were injured during cold storage and rewarming, with the degree of the alteration being dependent on the type of solution used. There were no histological alterations and no caspase 3 activation in all groups. In contrast, diaphragms stored in the modified TiProtec solution showed markedly better performance concerning force generation after nerve stimulation (7.1 ± 1.1 cN · s) as well as higher ATP content (2.4 ± 0.7 µmol/g) and were superior to storage on 0.9% NaCl-moistened gauze (1.4 ± 0.4 cN · s; 0.3 ± 0.1 µmol/g). In conclusion, the modified TiProtec preservation solution showed promising results for short-term cold storage of murine diaphragms. For further evaluation, the transferability of these positive findings to storage conditions for muscles of other species, especially human muscle tissue, needs to be investigated.

Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions

“…The basic cold storage solution used in the experiments is a chloride-poor variant (8.1 mM Cl − , 2 mM α-ketoglutarate, 5 mM aspartate, 95 mM lactobionate, 1 mM H 2 PO 4 − , 16 mM Na + , 93 mM K + , 8 mM Mg 2+ , 0.05 mM Ca 2+ , 30 mM N -acetylhistidine, 10 mM glycine, 5 mM alanine, 2 mM tryptophan, 20 mM sucrose, 10 mM glucose, and pH 7.0; solution 6 of Pless-Petig et al 14 without iron chelators and adenosine) of the vascular storage solution TiProtec ® , 12,13 which was developed in our laboratory. Only the chloride-poor modification was used in the experiments to exclude the injurious influence of chloride we described earlier for cold storage of rat hepatocytes.…”

“…Cold storage of hepatocyte suspensions is usually performed in buffered salt solutions, cell culture medium, or in organ preservation solutions. 8 – 11 In previous experiments, we showed largely improved rat hepatocyte storage in a chloride-poor modification of the tissue preservation solution TiProtec, 12 , 13 which contains, besides other components, iron chelators for protection against iron-dependent cold-induced cell injury and the small amino acids glycine and alanine. In contrast to cold storage in cell culture medium and organ preservation solutions, hepatocyte viability was mainly unchanged after 1 wk of cold storage in this modified TiProtec solution, both with and without iron chelators, and did not notably decrease during early rewarming.…”

Optimization of long-term cold storage of rat precision-cut lung slices with a tissue preservation solution

“…At all examined time points, no significant differences between solutions with and without addition of the chelators LK 614 and deferoxamine were observed(Figure 2). Iron chelators showed beneficial effects for cold storage of vascular endothelial cells (26) and muscle tissue (25), which are also present in PCLS. Additionally, iron chelators showed a tendency for higher viability in the current study (see above) and did not exert negative effects.…”

“…A more "gentle" way to preserve tissue slices is long term hypothermic (cold) storage at 4 °C, which does not necessitate special devices and was already optimized for use in precision cut liver and kidney slices (21)(22)(23), but to the best of our knowledge has not been investigated yet for PCLS storage. Recently developed cold storage solutions have been successfully used for the preservation of human lung epithelial cells (A549), hepatocytes and tissues such as blood vessels and striated muscles (24,25,27). Thus, we here set out to analyze effects of long-term cold storage in standard culture medium and two optimized preservation solutions (either with high potassium chloride concentrations or a chloride-poor, lactobionate-rich analog) on cellular viability, mitochondrial membrane potential, cell composition, inflammatory activation and bronchoconstriction in rat PCLS.…”

Organ Preservation into the 2020s: The Era of Dynamic Intervention

“…Chelation of the iron by deferoxamine and lipophilic membrane-permeable LK-614 protects the myocardium and endothelium against iron-dependent hypothermic injury [87]. The authors have developed TiProtec, a new preservation solution, in which the role of antioxidants refers to iron chelators [88, 89]. TiProtec is the modified Custodiol® (HTK) solution, which besides iron chelators (deferoxamine and LK-614) contains higher potassium concentration, amino acids (L-glycine and L-alanine) to inhibit hypoxic injury, arginine to increase NO supply, and N-acetyl-histidine instead of histidine as a buffer.…”