Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution.

IF 1.6 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Organogenesis Pub Date : 2018-01-01 Epub Date: 2018-08-06 DOI:10.1080/15476278.2018.1501136
Gesine Pless-Petig, Sven Knoop, Ursula Rauen
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引用次数: 8

Abstract

Cryopreservation is the only long-term storage option for the storage of vessels and vascular constructs. However, endothelial barrier function is almost completely lost after cryopreservation in most established cryopreservation solutions. We here aimed to improve endothelial function after cryopreservation using the 2D-model of porcine aortic endothelial cell monolayers. The monolayers were cryopreserved in cell culture medium or cold storage solutions based on the 4°C vascular preservation solution TiProtec®, all supplemented with 10% DMSO, using different temperature gradients. After short-term storage at -80°C, monolayers were rapidly thawed and re-cultured in cell culture medium. Thawing after cryopreservation in cell culture medium caused both immediate and delayed cell death, resulting in 11 ± 5% living cells after 24 h of re-culture. After cryopreservation in TiProtec and chloride-poor modifications thereof, the proportion of adherent viable cells was markedly increased compared to cryopreservation in cell culture medium (TiProtec: 38 ± 11%, modified TiProtec solutions ≥ 50%). Using these solutions, cells cryopreserved in a sub-confluent state were able to proliferate during re-culture. Mitochondrial fragmentation was observed in all solutions, but was partially reversible after cryopreservation in TiProtec and almost completely reversible in modified solutions within 3 h of re-culture. The superior protection of TiProtec and its modifications was apparent at all temperature gradients; however, best results were achieved with a cooling rate of -1°C/min. In conclusion, the use of TiProtec or modifications thereof as base solution for cryopreservation greatly improved cryopreservation results for endothelial monolayers in terms of survival and of monolayer and mitochondrial integrity.

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无血清和无白蛋白的内皮单层低温保存新方法。
低温保存是保存血管和血管结构的唯一长期储存方法。然而,在大多数成熟的冷冻保存溶液中,内皮屏障功能在冷冻保存后几乎完全丧失。我们的目的是利用猪主动脉内皮细胞单层的2d模型,在低温保存后改善内皮功能。在细胞培养基或以4°C血管保存液TiProtec®为基础的冷藏液中冷冻保存单层,均添加10% DMSO,使用不同的温度梯度。在-80°C下短期保存后,单层快速解冻并在细胞培养基中重新培养。在细胞培养基中冷冻保存后解冻,细胞立即死亡和延迟死亡,再培养24小时后,活细胞率为11±5%。在TiProtec中低温保存并对其进行无氯修饰后,与在细胞培养基中低温保存相比,贴壁活细胞的比例明显增加(TiProtec: 38±11%,改性TiProtec溶液≥50%)。使用这些溶液,在亚融合状态下冷冻保存的细胞能够在再培养期间增殖。在所有溶液中都观察到线粒体断裂,但在TiProtec中冷冻保存后部分可逆,在再培养3小时后在改性溶液中几乎完全可逆。TiProtec及其改性剂在所有温度梯度下都具有明显的保护作用;然而,当冷却速度为-1°C/min时,效果最好。总之,使用TiProtec或其修饰物作为低温保存的基础溶液,在存活和单层和线粒体完整性方面大大改善了内皮单层的低温保存结果。
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来源期刊
Organogenesis
Organogenesis BIOCHEMISTRY & MOLECULAR BIOLOGY-DEVELOPMENTAL BIOLOGY
CiteScore
4.10
自引率
4.30%
发文量
6
审稿时长
>12 weeks
期刊介绍: Organogenesis is a peer-reviewed journal, available in print and online, that publishes significant advances on all aspects of organ development. The journal covers organogenesis in all multi-cellular organisms and also includes research into tissue engineering, artificial organs and organ substitutes. The overriding criteria for publication in Organogenesis are originality, scientific merit and general interest. The audience of the journal consists primarily of researchers and advanced students of anatomy, developmental biology and tissue engineering. The emphasis of the journal is on experimental papers (full-length and brief communications), but it will also publish reviews, hypotheses and commentaries. The Editors encourage the submission of addenda, which are essentially auto-commentaries on significant research recently published elsewhere with additional insights, new interpretations or speculations on a relevant topic. If you have interesting data or an original hypothesis about organ development or artificial organs, please send a pre-submission inquiry to the Editor-in-Chief. You will normally receive a reply within days. All manuscripts will be subjected to peer review, and accepted manuscripts will be posted to the electronic site of the journal immediately and will appear in print at the earliest opportunity thereafter.
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