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

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.

Polyampholytes as Emerging Macromolecular Cryoprotectants

“…Compared to cryopreservation of suspended cells, protocols for adherent cell monolayers are significantly lacking. To date, there have been few studies on the slow-freezing (nonvitrified) cryopreservation of monolayered cells. − 3-D cell cultures such as spheroids or organoids present even more complexity, such as the significant impact of spheroid size on recovery (large <40% recovery vs small <70%) along with required equilibration times, and hence result in lower cell yields post-thaw. This is clearly observed with human embryonic kidney cells, where 3-D cryopreservation led to recovery of only 36%, and improved methods are obviously required.…”

Extracellular Antifreeze Protein Significantly Enhances the Cryopreservation of Cell Monolayers

“…The above results suggested that the intra/extracellular localization of AFPIII was not sufficient for enhancing mammalian cell cryopreservation in suspension freezing. While this is the most standard cell cryopreservation format, there is a real need to develop technologies and methods for 2- and 3-D cell models, for tissue storage and organ-on-a-chip applications. − The monolayer freezing protocol is illustrated in Figure A. A549 monolayers were frozen in DMSO alone, or with AFPIII (0.8 mg·mL –1 ), AFPIII (0.8 mg·mL –1 ) plus Pep-1 (0.5 mg·mL –1 )), or Pep-1 alone (0.5 mg·mL –1 ).…”

“…Cryopreservation of cells in monolayer format is currently being investigated as a means to supply cells which can be readily used and do not experience extensive “phenotypic drift” due to time-consuming laboratory processes, such as inoculation and propagation, from frozen vials. Successful monolayer cryopreservation of cells would be revolutionary in minimizing batch-to-batch variation and for the development of 2- and 3-D cell models, tissue storage, viral diagnostics, and organ-on-a-chip applications. − However, the current standard (DMSO) approaches used in suspension freezing are not translatable to monolayer freezing, with evidence suggesting that cells within a 2- and 3-D network (monolayers and spheroids/organoids) experience different modes of cryoinjury. , Thus, development of novel cryoprotectants tailored toward the format of cryopreservation and to replace or reduce DMSO content is pivotal for the future development of cell-based therapies and diagnostics.…”

Cryopreservation of human cerebral microvascular endothelial cells and astrocytes in suspension and monolayers

“…In that work, the authors showed that 5% DMSO and 5% HES in the presence of 90% fetal calf serum resulted in only about 50% viability post-thaw [ 85 ]. Similarly, using an improved media formulation (TiProtec, which proved favourable for cold storage of blood vessels) in the cryopreservation of porcine aortic endothelial cell monolayers enhanced mitochondrial integrity and yielded only 50% viable cells [ 86 ]. Addition of antifreeze proteins to 10% DMSO and slow cooling of A549 epithelial cell line monolayers improved post-thaw recovery from 25% to 60% [ 87 , 88 ].…”