Cell attachment after one week of cold storage in different solutions.

<p>(A) Control culture of non-stored primary rat hepatocytes. (B-D) Cell cultures from cell suspensions stored for one week in cell culture medium (B), chloride-rich, potassium-rich solution 1 (C) and chloride-poor solution 2 with balanced Na⁺/K⁺ concentrations (D). (E) Quantification of adherent, viable cells after cold storage and 24 h of cell culture as percentage of cultures of non-stored control cells. (F) Reductive metabolism of cultures obtained from cold-stored cell suspensions, given as percentage of non-stored control cell cultures (n = 8, *p<0.0001).</p

Influence of ion composition and iron chelators on cell volume.

<p>Histograms of cell volume distribution as assessed by flow cytometry after one week of cold storage in different solutions. Storage in cell culture medium induced marked cell swelling (A), storage in chloride-rich solutions containing iron chelators slight swelling (B, C). Cell swelling in chloride-rich basic solutions was as pronounced as in cell culture medium, irrespective of sodium concentration (D), substitution of chloride by lactobionate induced marked cell shrinkage (B, C). Cell swelling was inhibited by the addition of iron chelators (E, F; basic solution 3 vs. solution 3 w/o adenosine, solution 3 vs. basic solution 3+ adenosine), but not influenced by addition of adenosine (E, F).</p

Cell viability after cold storage of primary rat hepatocytes in different solutions.

<p>(A) Progression of cell death during cold storage (4°C) in cell culture medium and Krebs-Henseleit-buffer (KH). (B) Cell viability after one week of cold storage in cell culture medium, cold storage solutions 1 and 2 (n = 7;* p = 0.0003). (C-F) Original FACS plots showing cell viability after one week of cold storage. Cells were stained with propidium iodide (PI, 5 µg/mL) and viability was assessed by flow cytometry. C: Non-stored control cells. Cells after cold storage in cell culture medium (D), chloride-rich solution 1 (E) or chloride-poor solution 2 (F). Red dots indicate dead (i.e. propidium iodide-positive) cells. The percentage of viable cells is given in each panel.</p

Comparison of solutions 2 and 6 to established organ preservation solutions.

<p>Viability (A), cell attachment (B) and resazurin conversion (as measure for general reductive metabolism, C) after one week of cold storage in solution 2, University of Wisconsin solution (UW), histidine-tryptophan-ketoglutarate solution (HTK) and Celsior (n = 8). Urea production (D) and forskolin-triggered glucose release (E) of cultures from cells stored in solutions 2 and 6, UW and Celsior for one week (n = 6). Activities are expressed as percentage of cultures from non-stored control cells; *p≤0.0001.</p

Influence of cold storage solution components on cell viability after cold storage.

<p>‘Basic solution’ refers to solutions without iron chelators (deferoxamine + LK 614) and without adenosine. In supplemented basic solution 7, NaCl addition was reduced to maintain osmolarity. Data is given as median (25/75% percentile).</p>*<p>significantly different from cell culture medium.</p>†<p>significantly different from basic solution 7 (n = 6).</p

Composition of cold storage solutions.

<p>KH: Krebs-Henseleit buffer; L-15: Leibovitz L-15 cell culture medium.</p><p>All concentrations are given in mM. Cold storage solutions 1–7 without iron chelators (LK 614 and deferoxamine) and without adenosine are referred to as <b>‘basic solutions’</b> in the text. L-15 cell culture medium contains amino acids, trace elements etc. not listed in this table (to maintain lucidity) and was additionally supplemented (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040444#s2" target="_blank">Materials and Methods</a>) prior to use. Calculated osmolarity is given in mosm/L.</p>*<p>Before use, calculated osmolarity was raised to 311 mosm/L (basic solution: 305.2 msom/L) by addition of NaCl.</p

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

<div><p>Organ shortage leads to an increased utilization of marginal organs which are particularly sensitive to storage-associated damage. Cold incubation and rewarming-induced injury is iron-dependent in many cell types. In addition, a chloride-dependent component of injury has been described. This work examines the injury induced by cold incubation and rewarming in isolated rat renal proximal tubules. The tissue storage solution TiProtec^® and a chloride-poor modification, each with and without iron chelators, were used for cold incubation. Incubation was performed 4°C for up to 168 h, followed by rewarming in an extracellular buffer (3 h at 37°C). After 48, 120 and 168 h of cold incubation LDH release was lower in solutions containing iron chelators. After rewarming, injury increased especially after cold incubation in chelator-free solutions. Without addition of iron chelators LDH release showed a tendency to be higher in chloride-poor solutions. Following rewarming after 48 h of cold incubation lipid peroxidation was significantly decreased and metabolic activity was tendentially better in tubules incubated with iron chelators. Morphological alterations included mitochondrial swelling and fragmentation being partially reversible during rewarming. ATP content was better preserved in chloride-rich solutions. During rewarming, there was a further decline of ATP content in the so far best conditions and minor alterations under the other conditions, while oxygen consumption was not significantly different compared to non-stored control tubules. Results show an iron-dependent component of preservation injury during cold incubation and rewarming in rat proximal renal tubules and reveal a benefit of chloride for the maintenance of tubular energy state during cold incubation.</p></div

Mitochondrial alterations after cold incuabtion and rewarming.

<p>Freshly isolated rat proximal renal tubules (A, F: control) were incubated at 4°C in chloride-rich (B, D, G, I) or chloride-poor (C, E, H, J) cold storage solution with addition of iron chelators for 48 h. Mitochondria were stained with 300 nM MitoTracker Red at the beginning of cold storage and confocal laser scanning images were taken after fixation at the end of cold storage (B, C; detail: G, H) or after 3 h of rewarming (D, E; detail: I, J). Arrows: filamentous mitochondria; arrow heads: mitochondrial fragmentation; stars: mitochondrial swelling.</p

Oxygen consumption of renal proximal tubules.

<p>Isolated tubules were incubated at 4°C in the chloride-rich solution 1 in the presence of the iron chelators deferoxamine (0.5 mM) and LK 614 (20 μM) for 48 h. Tubules were rewarmed in extra-cellular buffer at 37°C for up to three hours. Oxygen consumption was measured at baseline (control) and after 48 h of cold incubation followed by 2 h or 3 h of rewarming in extra-cellular buffer. Respiration was measured in extra-cellular buffer at 37°C under continuous stirring using an O2K-oxygraph. Results summarize data from 20 measurements. Two measurements each were performed with utilization of tubules from 10 individual preparations. *p <0.05.</p

Lipid peroxidation in isolated proximal renal tubules after cold incubation and rewarming.

<p>Isolated tubules were incubated at 4°C in the chloride-rich solution 1 (intermediate grey bars) or its chloride-poor counterpart solution 2 (dark grey bars) each in the absence or presence of the iron chelators deferoxamine (0.5 mM) and LK 614 (20 μM; striped bars) for 48 h under gentle agitation. Thereafter, tubules were spun down, resuspended in pre-warmed extracellular buffer and incubated under gentle motion at 37°C for two hours. Thiobarbituric acid-reactive substances (TBARS) were determined in the supernatants at the end of cold incubation (darker part of the bars) and at the end of rewarming (light grey part of the bars). *p<0.05 and ***p<0.001 vs. respective solution without iron chelators at the end of cold incubation; ##p<0.01 and ###p<0.001 vs. respective solution without iron chelators at the end of rewarming (n = 7).</p