Cold storage of rat hepatocyte suspensions for one week in a customized cold storage solution--preservation of cell attachment and metabolism.

BACKGROUND & AIMS: Primary 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. METHODS: Rat 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. RESULTS: Cold 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. CONCLUSION: In 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

Aggravation of cold-induced injury in Vero-B4 cells by RPMI 1640 medium – Identification of the responsible medium components

Abstract Background In modern biotechnology, there is a need for pausing cell lines by cold storage to adapt large-scale cell cultures to the variable demand for their products. We compared various cell culture media/solutions for cold storage of Vero-B4 kidney cells, a cell line widely used in biotechnology. Results Cold storage in RPMI 1640 medium, a recommended cell culture medium for Vero-B4 cells, surprisingly, strongly enhanced cold-induced cell injury in these cells in comparison to cold storage in Krebs-Henseleit buffer or other cell culture media (DMEM, L-15 and M199). Manufacturer, batch, medium supplements and the most likely components with concentrations outside the range of the other media/solutions (vitamin B12, inositol, biotin, p-aminobenzoic acid) did not cause this aggravation of cold-induced injury in RPMI 1640. However, a modified Krebs-Henseleit buffer with a low calcium concentration (0.42 mM), a high concentration of inorganic phosphate (5.6 mM), and glucose (11.1 mM; i.e. concentrations as in RPMI 1640) evoked a cell injury and loss of metabolic function corresponding to that observed in RPMI 1640. Deferoxamine improved cell survival and preserved metabolic function in modified Krebs-Henseleit buffer as well as in RPMI 1640. Similar Ca2+ and phosphate concentrations did not increase cold-induced cell injury in the kidney cell line LLC-PK1, porcine aortic endothelial cells or rat hepatocytes. However, more extreme conditions (Ca2+ was nominally absent and phosphate concentration raised to 25 mM as in the organ preservation solution University of Wisconsin solution) also increased cold-induced injury in rat hepatocytes and porcine aortic endothelial cells. Conclusion These data suggest that the combination of low calcium and high phosphate concentrations in the presence of glucose enhances cold-induced, iron-dependent injury drastically in Vero-B4 cells, and that a tendency for this pathomechanism also exists in other cell types.</p

Aggravation of cold-induced injury in Vero-B4 cells by RPMI 1640 medium – Identification of the responsible medium components

Background In modern biotechnology, there is a need for pausing cell lines by cold storage to adapt large-scale cell cultures to the variable demand for their products. We compared various cell culture media/solutions for cold storage of Vero-B4 kidney cells, a cell line widely used in biotechnology. Results Cold storage in RPMI 1640 medium, a recommended cell culture medium for Vero-B4 cells, surprisingly, strongly enhanced cold-induced cell injury in these cells in comparison to cold storage in Krebs-Henseleit buffer or other cell culture media (DMEM, L-15 and M199). Manufacturer, batch, medium supplements and the most likely components with concentrations outside the range of the other media/solutions (vitamin B12, inositol, biotin, p-aminobenzoic acid) did not cause this aggravation of cold-induced injury in RPMI 1640. However, a modified Krebs-Henseleit buffer with a low calcium concentration (0.42 mM), a high concentration of inorganic phosphate (5.6 mM), and glucose (11.1 mM; i.e. concentrations as in RPMI 1640) evoked a cell injury and loss of metabolic function corresponding to that observed in RPMI 1640. Deferoxamine improved cell survival and preserved metabolic function in modified Krebs-Henseleit buffer as well as in RPMI 1640. Similar Ca2+ and phosphate concentrations did not increase cold-induced cell injury in the kidney cell line LLC-PK1, porcine aortic endothelial cells or rat hepatocytes. However, more extreme conditions (Ca2+ was nominally absent and phosphate concentration raised to 25 mM as in the organ preservation solution University of Wisconsin solution) also increased cold-induced injury in rat hepatocytes and porcine aortic endothelial cells. Conclusion These data suggest that the combination of low calcium and high phosphate concentrations in the presence of glucose enhances cold-induced, iron-dependent injury drastically in Vero-B4 cells, and that a tendency for this pathomechanism also exists in other cell types

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 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

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

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

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