Characterization of freeze-thaw induced ultrastructural damage to endothelial cells in vitro

Abstract

The pathophysiology of endothelial cells is important to a variety of vascular conditions including coagulation and hemostasis resulting from clinical frostbite. Use of an in vitro model system demonstrated that when bovine endothelial cells were frozen at 1 degrees C or 20 degrees C/min and thawed immediately (20 degrees C/min), a variety of ultrastructural alterations occurred. Membranous structures were most extensively damaged, with mitochondria the most sensitive organelle. Low amplitude mitochondrial swelling, first evident at 0 degrees C, progressed to high amplitude swelling by -10 degrees C (frozen). In addition, the rough endoplasmic reticulum was dilated and formed large vesicles with a homogeneous matrix. Nuclear changes first occurred at -15 degrees C. These included separation and distortion of the nuclear membrane, changes in chromatin distribution, and disruption of the nucleolus. Scanning electron microscopy revealed perforated plasma membranes in some cells at -10 degrees C (frozen) and in most cells by -20 degrees C. Cultures frozen at 20 degrees C/min revealed mostly the same ultrastructural damage noted at 1 degrees C/min except a higher percentage of cells exhibited alterations. Data from the recovery index and lactic dehydrogenase (LDH) release correlated well with observed ultrastructural changes. Early swelling of mitochondria and dilation of rough endoplasmic reticulum was not lethal in the absence of freezing. Increased swelling in cytoplasmic organelles coupled with nuclear alterations at -15 degrees C resulted in a decreased survival rate and release of significant quantities of LDH by -20 degrees C. No unique morphological changes were temperature specific, but the total number of cells that displayed alterations increased as temperature decreased.

Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells

“…It is essential to preserve the native chromatin architecture and the original nucleosome distribution patterns for ATAC-Seq. Freezing samples prior to the purification of nuclei can be detrimental to nuclear integrity and can affect chromatin structures 8 , thus restricting the application of ATAC-Seq to freshly-isolated nuclei. This limits the use of ATAC-Seq on clinical samples, which are typically stored frozen, and represents a major logistical hurdle for long-distance collaborative projects, for which sample freezing is often inevitable.…”

Donmuş Çözünmüş Kanın Farklı Değerlerde Santrifüjünün DNA İzolasyonu Üzerine Etkileri

“…Decreasing hemoglobin and EDTA might increase DNA binding to the membrane of the column. This might increase capturing of fragmented DNA by ice-induced DNA breaks (Heard, 1955;Trusal et al, 1984;Lahiri & Schnabel, 1993). DNA integrity can be important for some studies including comparative genomic hybridization (CGH) and whole-genome amplification approach or targeting long amplification size (Wang et al, 2007;Craig et al, 2012;Lucena-Aguilar et al, 2016).…”

Recent developments in cell shipping methods

“…Potential cell infection or contamination should be avoided, aside from endogenous cell modifications (Jenkins et al, 2012; Yong et al, 2017). Cell membrane damage and apoptosis have long been connected with cryopreservation (Yong et al, 2017; Trusal et al, 1984). Additionally, cryopreservation is time‐consuming and operator‐dependent, and it frequently requires equipment that is not ideal for cell therapy, such as liquid nitrogen, which is not GMP compliant (B. Chen et al, 2013).…”