Abstract
Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability.
The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.
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References
Altshuler GL, Dziewulski DM, Sowek JA and Belfort G (1986). Continuous hybridoma and monoclonal antibody production in hollow fiber reactors-separators. Biotech. Bioeng. 28: 646–658.
Bienkowsky RS (1983) Intracellular degradation of newly synthesized secretory proteins. Biochem. J. 214: 1–10.
Birch JR, Boraston R and Wood L (1985) Bulk production of monoclonal antibodies in fementers. Trends Biotech. 7: 162–166.
Coco-Martin JM, Oberink JW, van der Velden-de Groot CAM and Beuvery EC (1992) The potential flow cytometric analysis for the characterization of hybridoma cells in suspension cultures. Cytotechnology 8: 65–74 (this issue).
Cook JA and Mitchel JB (1989) Viability measurements in mammalian cell systems. Anal. Biochem. 179: 1–7.
Geaugey V, Pascal F, Engasser JM and Marc A (1990) Influence on the culture oxygenation on the release of LDH by hybridoma cells. Biotech. Technol. 4: 257–262.
Goebel NK, Kuehn R and Flickinger MC (1990) Methods for determination of growth-rate-dependent changes in hybridoma volume, shape and surface structure during continuous culture. Cytotechnology 4: 45–57.
Kangas L, Gronroos M and Nieminen AL (1984) Bioluminescence of cellular ATP: A new method for evaluating cytotoxic agents in vitro. Med. Biol. 62: 338–343.
Kuzmits R, Aiginger P, Muller MM, Steurer G and Linkesch W (1986) Assessment of the sensitivity of leukaemic cells to cytotoxic drugs by bioluminescence measurement of ATP in cultured cells. Clin. Sci. 71: 81–88.
Laiho KU, Shelbume JD and Trump BF (1971) Observations on cell volume, ultrastructure, mitochondrial conformation and vital-dye uptake in Ehrlich ascites tumor cells. Am. J. Path. 65: 203–221.
Loken MR, Sweet RG and Herzenberg LA (1976) Cell discrimination by multiangle light scattering. J. Histochem. Cytochem. 24: 284–291.
Maehara Y, Anai H, Tamada R and Sugimachi K (1987) The ATP assay is more sensitive than the succinate dehydrogenase inhibition test for predicting cell viability. Eur. J. Cancer Clin. Oncol. 23: 273–276.
Martin N, Brennan A, Denome L and Shaevitz J (1987) High productivity in mammalian cell culture. Biotechnology 5: 838–840.
Needham D, Ting-Beall HP and Tran-Son-Tay R (1991) A physical characterization of GAP A3 hybridoma cells: morphology, geometry, and mechanical properties. Biotechnol. Bioeng. 38: 838–852.
Patterson Jr. MK (1979) Measurement of growth and viability of cells in culture. Meth. Enzymol. 58: 141–148.
Pavlik ED, Flanigan RC, van Nagell Jr JR, Hanson MB, Donaldson ES, Keaton K, Doss B, Bartmas J and Kenady D (1985) Esterase activity, exclusion of propidium iodide and proliferation in tumor cells exposed to anticancer agents: Phenomena relevant to chemosensitivity determinations. Cancer Invest. 3: 413–426.
Racher AJ, Looby D and Griffiths JB (1990) Use of lactate dehydrogenase release to assess changes in culture viability. Cytotechnology 3: 301–307.
Rhodes M and Birch J (1988) Large-scale production of proteins from mammalian cells. Biotechnology 6: 518–523.
Roper PR and Drewinko B (1976) Comparison of in vitro methods to determine drug-induced cell lethality. Cancer Res. 36: 2182–2188.
Rotman B and Papermaster BW (1966) Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc. Natl. Acad. Sci. 55: 134–141.
Sasaki DT, Dumas SE and Engleman EG (1987) Discrimination of viable and non-viable cells using propidium iodide in two color immunofluorescence. Cytometry 8: 413–420.
Schols D, Pauwels R, Vanlangendonck F, Balzarini J and De Clercq E (1988) A highly reliable, sensitive, flow cytometric/fluorometric assay for the evaluation of the anti-HIV activity of antiviral compounds in MT-4 cells. J. Immunol. Methods 114: 27–32.
Sen S, Srienc F and Hu W-S (1989) Distinct volume distribution of viable and non-viable hybridoma cells: A flow cytometric study. Cytotechnology 2: 85–94.
Wettermark G, Stymne H, Brolin SE and Petersson B (1975) Substrate analyses in single cells. I. Determination of ATP. Anal. Biochem. 63: 293–307.
Whitehead TP, Kricka LJ, Carter TJN and Thorpe GHG (1979) Analytical Luminescence: Its potential in the clinical laboratory. Clin. Chem. 25: 1531–1546.
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Coco-Martin, J.M., Oberink, J.W., van der Velden-de Groot, T.A.M. et al. Viability measurements of hybridoma cells in suspension cultures. Cytotechnology 8, 57–64 (1992). https://doi.org/10.1007/BF02540030
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DOI: https://doi.org/10.1007/BF02540030