Cold-induced cell injury and its aggravation by RPMI 1640
Vero-B4 cells stored 168 h at 4°C in Krebs-Henseleit buffer (KH) showed little release of lactate dehydrogenase (LDH) at the end of cold incubation (Figure ). However, LDH release increased rapidly during rewarming, especially in the first hour. Addition of glucose to KH (KHG) decreased the cell injury during rewarming. Therefore, glucose was added in most of the following experiments to rule out any influence of this rewarming component most likely related to substrate/energy depletion.
Figure 1 Cold-induced injury to Vero-B4 cells. Vero-B4 cells were incubated in RPMI 1640 (RPMI), Krebs-Henseleit buffer (KH) and KH + 11.1 mM D-glucose (KHG) at 4°C for 168 hours and then rewarmed in RPMI 1640 at 37°C for 3 hours. Part of the cells (more ...)
In contrast to the results in KH and KHG, very high LDH release was observed at the end of cold incubation of the cells in (complete) RPMI 1640, a cell culture medium containing glucose (Figure ). Addition of deferoxamine (1 mM) to the media before cold incubation prevented cold-induced cell damage in RPMI 1640 and in KHG but not the rewarming injury in KH without glucose.
Although RPMI 1640 aggravated cell injury in the cold, there was no evidence of a toxicity of RPMI 1640: During warm incubation in RPMI 1640, which is the cell culture medium recommended by the German Collection of Microorganisms and Cell Cultures (DMSZ) for this cell line [29
], Vero-B4 cells proliferated normally and showed normal morphology. Using RPMI 1640 from a different company (Sigma instead of Gibco) did not change the amount of cold-induced cell injury seen after cold storage in RPMI 1640 (Table ).
Comparison of RPMI 1640 from two different companies
Role of medium supplements
Comparison of “complete” RPMI 1640 supplemented with foetal bovine serum and penicillin/streptomycin as described in the Methods section, versus “pure” RPMI 1640 showed that the supplements were not responsible for the strong cold-induced injury in RPMI 1640 (Table ).
Other cell culture media
The cell culture media DMEM, L-15 and M199 were compared to RPMI 1640 to evaluate whether enhancement of cold-induced injury is particular for RPMI 1640 or is an effect caused by cell culture media in general. Only cells stored in RPMI 1640 showed a strong cell injury directly after cold incubation (Figure ). Storage in the other media resulted in far less injury during cold storage; best cell survival was seen in M199. Deferoxamine offered nearly complete protection in all tested media.
Figure 2 Cold-induced injury to Vero-B4 cells after cold incubation in different cell culture media. Vero-B4 cells were incubated in RPMI 1640, DMEM, L-15 and M199 at 4°C for 168 hours and then rewarmed in RPMI 1640 at 37°C for 3 hours. Cell injury (more ...)
Medium components potentially responsible for the RPMI 1640 effect
As aggravation of cold-induced cell injury was no general effect of cell culture media, we compared the composition of RPMI 1640 with that of the other media and KHG to identify substances or differences in concentrations of substances which could cause the RPMI 1640 effect (Table ): there were 14 compounds with concentrations in RPMI 1640 outside the range of concentrations in the other media/solutions; of these, the concentrations of Ca2+, inorganic phosphate (Pi) and the components vitamin B12, i-inositol, biotin and p-aminobenzoic acid appeared as most likely culprits to cause enhanced cell injury in RPMI 1640. We added vitamin B12, i-inositol, biotin and p-aminobenzoic acid to KHG, but none of them proved to be responsible for the enhancing RPMI 1640 effect (Table ).
Composition of different cell culture media and Krebs-Henseleit buffer
Effects of components potentially responsible for the injurious effect of RPMI 1640
Effects of calcium and inorganic phosphate concentrations
RPMI 1640 contains a lower concentration of Ca2+ and a higher concentration of inorganic phosphate than the other media and KHG buffer. Therefore, KHG (with 11.1 mM glucose) was modified to resemble RPMI 1640 medium in these respects (KHG(Ca-,P+); with 0.4 mM Ca2+ and 5.6 mM HPO42- as in RPMI 1640). Cold storage in this modified KHG resulted in a similar aggravation of cold-induced cell injury as seen in RPMI 1640 (Figure ). Further modifications of KH with or without glucose combined with low calcium and/or high phosphate concentrations showed that a combination of all three was necessary to achieve the effect seen in RPMI 1640: The addition of glucose alone (KHG) provoked only a very slight increase in cold-induced cell injury (in comparison to KH), which was similar in the presence of high phosphate (KHG(P+)) and moderately aggravated at low calcium concentrations (KHG(Ca-)). In the absence of glucose, cold storage in all of the modified solutions (KH(Ca-), KH(P+), KH(Ca-,P+)) resulted in little cold-induced injury, similar to that in KH (Figure ). Only the combination of a reduced Ca2+ concentration with an increased concentration of inorganic phosphate in the presence of glucose, KHG(Ca-,P+), evoked a cell injury corresponding to that observed in RPMI 1640. Addition of deferoxamine showed protection in all solutions.
Figure 3 Influence of modified Krebs-Henseleit buffer on cold-induced injury to Vero-B4 cells. Vero-B4 cells were stored at 4°C for 168 hours in RPMI 1640, Krebs-Henseleit buffer (KH), modified KH buffer with either 11.1 mM glucose (KHG), low Ca2+ concentration (more ...)
Assessment of the metabolic activity (resazurin reduction) of Vero-B4 cells incubated for 168 h at 4°C and rewarmed for 3 h confirmed these results: Cells cold stored in RPMI 1640 medium and in the triply modified buffer KHG(Ca-,P+) showed hardly any resazurin reduction (Figure ). Loss of metabolic activity could be inhibited completely by the addition of deferoxamine during cold incubation. Metabolic activity of cells cold stored in buffer with single or double modifications was only slightly decreased compared to control cells (Figure ).
Figure 4 Reductive metabolism of Vero-B4 cells after cold storage and rewarming. Vero-B4 cells were incubated at 4°C for 168 hours in RPMI 1640, Krebs-Henseleit (KH) buffer containing glucose (KHG), or modified buffers containing a low Ca2+ concentration (more ...)
Assessment of cell morphology also confirmed that the triple combination of glucose, low calcium and high inorganic phosphate is the culprit for the enhancement of cold-induced injury in RPMI 1640: Vero-B4 cells cold-incubated in KHG appeared confluent, and displayed a regular shape after cold storage and rewarming (Figure ). After cold incubation in RPMI 1640 medium and subsequent rewarming, cells were rounded and partially detached, had small, dark nuclei and pronounced bleb formation could be observed. Similar changes were observed after cold incubation in the triply modified buffer KHG(Ca-,P+) and subsequent rewarming. Addition of deferoxamine to RPMI 1640 and to KHG(Ca-,P+) prevented detachment, nuclear alterations and bleb formation, and a normal monolayer was observed after rewarming (data not shown).
Figure 5 Morphology of Vero-B4 cells after cold storage/rewarming. Vero-B4 cells were incubated at 4°C for 168 hours in Krebs-Henseleit buffer with 11.1 mM glucose (KHG), RPMI 1640 (RPMI) and modified KHG buffer containing Ca2+ and inorganic phosphate (more ...)
Evidence for an involvement of the mitochondria
Iron-dependent cold-induced cell injury is mediated by a mitochondrial permeability transition (MPT) [13
]. The MPT inhibitor combination trifluoperazine plus fructose [30
] prevented enhancement of cold-induced cell injury in both RPMI 1640 and the triply modified KHG(Ca-,P+) (Figure ).
Figure 6 Blockade of mitochondrial permeability transition (MPT). Vero-B4 cells were incubated in RPMI 1640 or a modified KH buffer containing glucose, a low Ca2+ concentration and a high inorganic phosphate concentration (KHG(Ca-,P+)) at 4°C for 168 hours (more ...)
Other cell types
Further experiments were performed with LLC-PK1 cells, porcine aortic endothelial cells and rat hepatocytes in an analogous fashion in order to assess whether the enhancement of cold-induced injury by glucose/low calcium/high phosphate is specific to Vero-B4 or kidney cells.
LLC-PK1 kidney cells showed slightly higher injury after cold storage in RPMI 1640 and L-15 medium than after cold storage in MEM and M199 and slightly increased injury in the triply modified KH compared to KH, but these effects were neither marked nor significant (Table ). As described for other cells, the iron chelators deferoxamine (1 mM) and the MPT inhibitor combination trifluoperazine (tfp; 20 μM) plus fructose (10 mM) inhibited cold-induced injury in all solutions.
Cold-induced cell injury to LLC-PK1 cells
In rat hepatocytes, cold incubation in KH buffer with triple modification (KHG(Ca-,P+)) only slightly increased cell injury after 14 h of cold storage compared to KHG (Figure ). However, when Pi concentration was increased to 25 mM and calcium was nominally absent (KH(Ca--,P++), as is present in the University of Wisconsin organ preservation solution, cell injury increased severely, regardless of the presence of glucose. However, the enforced triple combination KHG(Ca--,P++) did not only aggravate cold-induced injury but also increased cell injury at 37°C during 14 h incubation (KHG: 10 ± 3%, KHG(Ca--,P++): 41 ± 20%).
Figure 7 Influence of modified Krebs-Henseleit buffers on cold-induced injury of hepatocytes and aortic endothelial cells. Rat hepatocytes (A; n = 5) and porcine aortic endothelial cells (B; n = 4) were incubated in Krebs-Henseleit buffer with (KHG) or without (more ...)
In porcine aortic endothelial cells, there was also no increase in cold-induced cell injury in the triply modified KHG(Ca-,P+). However, in the enhanced triple combination KHG(Ca--,P++), cell injury strongly increased during 24 h cold incubation (Figure ), but not during 24 h warm incubation (KHG: 4 ± 3%, KHG(Ca--,P++): 16 ± 14%).