Cold Storage of Rat Hepatocyte Suspensions for One Week in a Customized Cold Storage Solution – Preservation of Cell Attachment and Metabolism

Abstract

Background & AimsPrimary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells.MethodsRat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production) of adherent cells were assessed.ResultsCold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage.ConclusionIn the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.

Supercooling as a Viable Non-Freezing Cell Preservation Method of Rat Hepatocytes

“…To the best of our knowledge apart from the differences in ionic concentrations and use of different buffers and impermeants, both solutions focus on preserving the intracellular balance; both solutions contain lactobionate which has been implied as the key component to avoid injury to liver cells in cold storage [26,27]. While the UW solution is rich in potassium (K+), the HTS-FRS solution is rich in sodium (Na+); nevertheless recent findings [26,27] suggest that this is not a distinguishing feature for long term hypothermic storage. Similarly although the osmotic pressure of the two solutions differ slightly (~350 mOsm for HTS-FRS and ~320 mOsm for UW) and this can cause a small variation in cell shrinkage during preservation, this effect has also been found to have minimal impact on cold storage of rat hepatocytes [27].…”

“…While the UW solution is rich in potassium (K+), the HTS-FRS solution is rich in sodium (Na+); nevertheless recent findings [26,27] suggest that this is not a distinguishing feature for long term hypothermic storage. Similarly although the osmotic pressure of the two solutions differ slightly (~350 mOsm for HTS-FRS and ~320 mOsm for UW) and this can cause a small variation in cell shrinkage during preservation, this effect has also been found to have minimal impact on cold storage of rat hepatocytes [27]. …”

Characterization of injury in isolated rat proximal tubules during cold incubation and rewarming

“…In particular, a chloride-dependent component of injury has been described in rat hepatocytes [16, 22], while chloride-poor solutions accentuated damage in endothelial cells [23, 24]. The role of chloride in cold-induced injury of renal tubules has not yet been addressed.…”

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

“…In contrast to porcine aortic endothelial cells and rat renal proximal tubules, rat hepatocytes display a chloride-dependent cell injury after cold (4°C) storage in chloride-rich solutions and are therefore significantly better protected in chloride-poor solutions. 19,20 This chloride-dependent cold-induced injury appears to involve lysosomal permeabilization. 52 In this setting, substitution of chloride by lactobionate might favour the maintenance of a slightly alkaline cytosolic pH (via inhibition of bicarbonate extrusion by the HCO 3 − /Cl − antiporter), thus limiting the adverse effects of released lysosomal enzymes.…”

“…18 TiProtec solution (and modifications thereof) have the additional advantage that they are serum-free and contain no albumin. In contrast to porcine aortic endothelial cells, 10 rat hepatocytes display a chloride-dependent cold-induced cell injury, 19,20 i.e. the chloride-poor TiProtec modification is superior to TiProtec for both, cold storage 19 and cryopreservation 18 of rat hepatocytes.…”