Serum-Free Cryopreservation of Primary Rat Hepatocytes in a Modified Cold Storage Solution: Improvement of Cell Attachment and Function

Abstract

In summary, significant improvement of cell attachment and function compared to cell culture medium was achieved after cryopreservation in serum-free hepatocyte cold storage solution.

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

“…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. In contrast to the findings at 4°C, after cryopreservation, cell survival of both, rat hepatocytes 18 and porcine aortic endothelial cell monolayers (Figures 1A and 3A) was higher in the chloride-poor solutions compared to their chloride-rich counterparts. Cryopreservation in chloride-poor solutions also seemed to improve protection of the endothelial layer of aortic segments (Figure 7), while high potassium concentrations had no beneficial effect (Figure 1, 2, 3, similar results in solutions 2 and 3).…”

“…Viability after freezing in solution 1 was only slightly higher than after freezing in cell culture medium directly after thawing (Figure 1A), but delayed cell death was markedly lower (Figure 1B) and an intact monolayer with only few detached cells was observed after 3 h of re-culture (Figure 2C). In the chloride-poor solution 2 (with balanced sodium and potassium concentrations), a solution that yielded good results in the cryopreservation of rat hepatocytes 18 and in solution 3, the chloride-poor analogue of TiProtec (potassium-rich), initial viability after thawing was higher than in the other solutions (Figure 1A). Decrease of viability during re-culture was similar to that of cells frozen in solution 1 yielding a final viability of around 50% (Figure 1B).…”

“…Solution 2 is a chloride-poor variant with balanced Na + /K + concentrations, as used earlier for cryopreservation of rat hepatocytes. 18 For direct comparison to TiProtec in terms of chloride concentration, solution 3 was used, which resembles solution 1 with respect to cation composition (high potassium concentration) but is chloride-poor (Table 1). N -acetylhistidine and the iron chelator LK 614 were kindly provided by Dr. F. Köhler Chemie; all solutions (composition see Table 1) were prepared in our laboratory.…”

“…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. Since porcine aortic endothelial cells are better protected in chloride-rich TiProtec at 4°C cold storage, 10 the question arises whether, for these cells, better cryopreservation results can be achieved in the original TiProtec or in chloride-poor modifications.…”

“…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.…”