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

Abstract

BackgroundIn 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.ResultsCold 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.ConclusionThese 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.

Second Generation of Mannich Base-Type Derivatives with in Vivo Activity against Trypanosoma cruzi

“…Vero cells (EACC number 84113001) from monkey kidney were grown in humidified 95% air, 5% CO 2 atmosphere at 37 °C in RPMI (Gibco) with 10% (v/v) heat-inactivated FBS …”

Synthesis and Biological in vitro and in vivo Evaluation of 2‐(5‐Nitroindazol‐1‐yl)ethylamines and Related Compounds as Potential Therapeutic Alternatives for Chagas Disease

“…In vitro activity assays; amastigote forms : Vero cells (EACC number 84113001) from monkey kidney were grown in humidified 95 % air, 5 % CO 2 atmosphere at 37 °C in RPMI (Gibco®) with 10 % ( v / v ) fetal bovine serum (heat‐inactivated) and were collected first by trypsinization and then by centrifugation at 400 g for 10 min. Assays were performed in microtiter plates (24‐well plates) at 1×10 4 Vero cells per well with rounded coverslips on the bottom; 24 h later the cells were infected with culture‐derived trypomastigotes of T. cruzi (strains SN3, Arequipa and Tulahuen) at a multiplicity of infection (MOI) ratio of 1:10 during 24 h. Non‐phagocyted parasites were removed by washing, and after addition of the compounds at dosages of 50 to 0.1 μ m , cultured in 500 μL per well volumes in RPMI with 1% ( v / v ) fetal bovine serum (heat‐inactivated) in a humidified 95 % air, 5 % CO 2 atmosphere at 37 °C.…”

“…Vero cells culture and cytotoxicity tests:V ero cells (EACC number 84113001) from monkey kidney were grown in humidified 95 %a ir, 5% CO 2 atmosphere at 37 8Ci nR PMI (Gibco)w ith 10 %( v/v)f etal bovine serum (heat-inactivated). [58] Vero cells were collected first by trypsinization and then by centrifugation at 400 g for 10 min. The compounds to be tested were dissolved as mentioned above.…”

Effects of immediate versus delayed ex-vivo lung perfusion in a porcine cardiac arrest donation model

“…Extracellular lactate dehydrogenase (LDH) was measured photometrically by a standard assay in the EVLP perfusate (VITALAB Selectra E; Vital Scientific NV, Dieren, The Netherlands). 14 To correct for LDH derived from damaged erythrocytes, perfusate was centrifuged and free hemoglobin was measured in the supernatant by ultraviolet/visible spectroscopy (UV/VIS) analysis (CLARIOstar; BMG Labtech, Ortenberg, Germany). Samples of lysed pig erythrocytes were used to generate a standard curve of LDH activity released per µmol hemoglobin released and measured perfusate LDH activity was corrected for LDH released from damaged erythrocytes (using this curve) in order to properly reflect injury to lung tissue.…”