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

Abstract

Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/106 cells after cold storage, 5 ± 3 nmol/106 cells after rewarming vs. control 29 ± 6 nmol/106 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec−1 per 106 cells after rewarming vs. control 232 ± 83 pmol sec−1 per 106 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/106 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production—as elicited by an inhibitor of the respiratory chain, antimycin A—can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment with subsequent energy deficiency is a key factor for the lack of attachment of cold-stored hepatocyte suspensions.

Assessment of albumin and aspartate levels as a simple indicator of the efficacy of cryopreservation

“…Most common damage in the sub cellular level is mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP), and loss of iron, free radical injury. 11 In our study, the mean age was 42 years, patients above 60 years were excluded from the study owing to the excessive friability of the cells. 12 Patients with excessive steatosis and proven cirrhosis have shown to have decreased yield and viability.…”

Plunge-Freezing Cryopreservation of Tendons

“…7−10 Cold storage, typically at 4 °C, is unsuitable for long-term preservation as it does not halt cellular metabolism completely. 11 Freeze-drying may compromise the integrity of tissue's collagen and fiber structure, thereby significantly reducing its translatability. 12 In contrast, cryopreservation at −196 °C is a promising technology for long-term tissue preservation that can effectively cease metabolic activities and may ensure the availability of viable tissues off-the-shelf.…”

Use of the new preservation solution Custodiol‐MP for ex vivo reconditioning of kidney grafts

“…[12][13][14] Rewarming creates an injurious challenge to the cells both by speeding up injurious processes that were already initiated during cold storage [12][13][14] and by abruptly increasing metabolism and energy demand in cells with yet dysfunctional mitochondria. 11,15,16 However, recent innovations in perfusion technologies are not paralleled by concomitant developments regarding the perfusates used for machine perfusion. While the last decades have seen a variety of solutions proposed for static cold storage (eg, UW, HTK, IGL1, and Custodiol-N), 17 machine perfusion is still relying on Belzer's MPS solution developed in the 1980s.…”

“…The technique of slow rewarming of the graft seems to be operative in preventing the temperature paradox upon abrupt reperfusion, 11 on the cellular level described as “rewarming injury” 12‐14 . Rewarming creates an injurious challenge to the cells both by speeding up injurious processes that were already initiated during cold storage 12‐14 and by abruptly increasing metabolism and energy demand in cells with yet dysfunctional mitochondria 11,15,16 …”