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Automated selective dissociation of cells from different regions of multicellular spheroids

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Summary

In this report we describe a new apparatus which has been developed for the automated selective dissociation of multicellular spheroids into fractions of viable cells from different locations in the spheroid. This device is based on the exposure of spheroids to a 0.25% solution of trypsin under carefully controlled conditions, such that the cells are released from the outer spheroid surface in successive layers. Study of the spheroid size, number of cells per spheroid, and sections through the spheroid with increasing exposure to trypsin demonstrate the effectiveness of this technique. The technique has been successfully used on spheroids from five different cell lines over a wide range of spheroid diameters. We also present data detailing the effect of varying the dissociation temperature, the mixing speed, the trypsin concentration, and the number of spheroids being dissociated. The new apparatus has several advantages over previous selective dissociation methods and other techniques for isolating cells from different regions in spheroids, including: a) precise control over dissociation conditions, improving reproducibility; b) short time to recover cell fractions; c) ability to isolate large numbers of cells from many different spheroid locations; d) use of common, inexpensive laboratory equipment; and e) easy adaptability to new cell lines or various spheroid sizes. Applications of this method are demonstrated, including the measurement of nutrient consumption rates, regrowth kinetics, and radiation survivals of cells from different spheroid regions.

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References

  1. Acker, H. Microenvironmental conditions in multicellular spheroids grown under liquid-overlay conditions. In: Acker, H.; Carlsson, J.; Durand, R., et al. Recent results in cancer research: spheroids in cancer research, vol. 95. Berlin: Springer-Verlag; 1984:116–133.

    Google Scholar 

  2. Bauer, K. D.; Keng, P. C.; Sutherland, R. M. Isolation of quiescent cells from multicellular spheroids using centrifugal elutriation. Cancer Res. 42:72–76; 1982.

    PubMed  CAS  Google Scholar 

  3. Chen, T. R. In situ determination of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104:255–262; 1977.

    Article  PubMed  CAS  Google Scholar 

  4. Durand, R. E. Isolation of cell subpopulations from in vitro tumor models according to sedimentation velocity. Cancer Res. 35:1295–1300; 1975.

    PubMed  CAS  Google Scholar 

  5. Durand, R. E.; Olive, P. L. Cytotoxicity, mutagenicity and DNA damage by Hoechst 33342. J. Histochem. Cytochem. 30:111–116; 1982.

    PubMed  CAS  Google Scholar 

  6. Durand, R. E. Use of Hoechst 33342 for cell selection from multicell systems. J. Histochem. Cytochem. 30:117–122; 1982.

    PubMed  CAS  Google Scholar 

  7. Durand, R. E. Oxygen enhancement ratio in V79 spheroids. Radiat. Res. 96:322–334; 1983.

    Article  PubMed  CAS  Google Scholar 

  8. Durand, R. E. Chemosensitivity testing in V79 spheroids: drug delivery and cellular microenvironment. JNCI 77:247–252; 1986.

    PubMed  CAS  Google Scholar 

  9. Durand, R. E. Use of a cell sorter for assays of cell clonogenicity. Cancer Res. 46:2775–2778; 1986.

    PubMed  CAS  Google Scholar 

  10. Erba, E.; Ubezio, P.; Broggini, M., et al DNA damage, cytotoxic effect and cell cycle perturbation of Hoechst 33342 on L1210 cells in vitro. Cytometry 9:1–6; 1988.

    Article  PubMed  CAS  Google Scholar 

  11. Erlichman, C.; Tannock, I. F. Growth and characterization of multicellular tumor spheroids of human bladder carcinoma origin. In Vitro 22:449–456; 1986.

    CAS  Google Scholar 

  12. Freyer, J. P.; Sutherland, R. M. Selective dissociation and characterization of cells from different regions of multicell tumor spheroids. Cancer Res. 40:3956–3965; 1980.

    PubMed  CAS  Google Scholar 

  13. Freyer, J. P.; Wilder, M. E., Raju, M. R. Coulter volume cell sorting to improve the precision of radiation survival assays. Radiat. Res. 97:608–614; 1984.

    Article  PubMed  CAS  Google Scholar 

  14. Freyer, J. P.; Sutherland, R. M. A reduction in the in situ rates of oxygen and glucose consumption of cells in EMT6/Ro spheroids during growth. J. Cell. Physiol. 124:516–524; 1985.

    Article  PubMed  CAS  Google Scholar 

  15. Freyer, J. P.; Sutherland, R. M. Regulation of growth saturation and the development of necrosis in EMT6/Ro multicellular spheroids by the glucose and oxygen supply. Cancer Res. 46:3504–3512; 1986.

    PubMed  CAS  Google Scholar 

  16. Freyer, J. P.; Sutherland, R. M. Proliferative and clonogenic heterogeneity of cells from EMT6/Ro multicellular spheroids induced by the glucose and oxygen supply. Cancer Res. 46:3513–3520; 1986.

    PubMed  CAS  Google Scholar 

  17. Freyer, J. P.; Sillerud, L. O.; Mattingly, M. High-resolution NMR imaging of conditions inside an intact multicellular tumor spheroid. Presented at the Conference on Prediction of Tumor Treatment Response, Banff, Canada, April 21–24; 1987.

  18. Freyer, J. P.; Wilder, M. E.; Raju, M. R. Rapid assay for cell age response to radiation by electronic volume flow cell sorting. Int. J. Radiat. Biol. 52:91–106; 1987.

    CAS  Google Scholar 

  19. Freyer, J. P. Role of necrosis in regulating the growth saturation of multicellular spheroids. Cancer Res. 48:2432–2439; 1988.

    PubMed  CAS  Google Scholar 

  20. Fried, J. D.; Doblin, J.; Takamoto, S., et al. Effects of Hoechst 33342 on survival and growth of two tumor cell lines and on hematopoietically normal bone marrow cells. Cytometry 3:42–47; 1982.

    Article  PubMed  CAS  Google Scholar 

  21. Giesbrecht, J. L.; Wilson, W. R.; Hill, R. P. Radiobiological studies of cells in multicellular spheroids using a sequential trypsinizing technique. Radiat. Res. 86:368–386; 1981.

    Article  PubMed  CAS  Google Scholar 

  22. Holm, D. M.; Cram, L. S. An improved flow microfluorometer for rapid measurement of cell fluorescence. Exp. Cell Res. 80:105–110; 1973.

    Article  PubMed  CAS  Google Scholar 

  23. House, W.; Waddell, A. Detection of mycoplasmas in cell cultures, J. Pathol. Bacteriol. 93:125–132 1967.

    Article  PubMed  CAS  Google Scholar 

  24. Jett, J. H.; Gurley, L. An improved sum-of-normals technique for cell cycle distribution analysis of flow cytometric DNA content histograms. Cell Tissue Kinet. 14:413–423; 1981.

    PubMed  CAS  Google Scholar 

  25. Luk, C. K.; Sutherland, R. M. Influence of growth phase, nutrition and hypoxia on heterogeneity of cellular buoyant densities in in vitro model systems. Int. J. Cancer 37:883–890; 1986.

    Article  PubMed  CAS  Google Scholar 

  26. Luk, C. K.; Keng, P. C.; Sutherland, R. M. Radiation response of proliferating and quiescent subpopulations isolated from multicellular spheroids. Br. J. Cancer 54:25–32; 1986.

    PubMed  CAS  Google Scholar 

  27. Mueller-Klieser, W. Microelectrode measurements of oxygen tension distributions in multicellular spheroids cultured in spinner flasks. In: Acker, H.; Carlsson, J.; Durand, R., et al., eds. Recent results in cancer research: spheroids in cancer research, vol. 95. Berlin: Springer-Verlag; 1984:134–149.

    Google Scholar 

  28. Mueller-Klieser, W. Multicellular spheroids: a review on cellular aggregates in cancer research. J. Cancer Res. Clin. Oncol. 113:101–122; 1987.

    Article  PubMed  CAS  Google Scholar 

  29. Mueller-Klieser, W. Tumor physiology and cellular microen-vironments. Presented at the Conference on Prediction of Tumor Treatment Response, Banff, Canada, April 21–24; 1987.

  30. Siemann, D. W.; Keng, P. C. Cell cycle specific toxicity of the Hoechst 33342 stain in untreated and irradiated murine tumor cells. Cancer Res. 46:3556–3559; 1986.

    PubMed  CAS  Google Scholar 

  31. Sutherland, R. M. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240:177–184; 1988.

    Article  PubMed  CAS  Google Scholar 

  32. Sutherland, R. M.; Buchegger, F.; Schreyer, M., et al. Penetration and binding of radiolabeled anti-CEA monoclonal antibodies and their F(ab′) and Fab fragments in human colon multicellular spheroids. Cancer Res. 47:1627–1633; 1987.

    PubMed  CAS  Google Scholar 

  33. Van Zant, G.; Fry, C. G. Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells. Cytometry 4:40–46; 1983.

    Article  PubMed  Google Scholar 

  34. Wibe, E.; Lindmo, T.; Kaalhus, O. Cell kinetic characteristics in different parts of multicellular spheroids of human origin. Cell Tissue Kinet. 4:639–651; 1981.

    Google Scholar 

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Authors and Affiliations

  1. Life Sciences Division, Los Alamos National Laboratory, Cell Biology Group LS-4, Mail Stop M888, 87545, Los Alamos, New Mexico

    James P. Freyer & Patricia L. Schor

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  1. James P. Freyer
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  2. Patricia L. Schor
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Additional information

This work was supported by grants CA-36535, CA-22585, and RR-02845 from the National Institutes of Health, Bethesda, MD, the National Flow Cytometry Resource (NIH grant RR-01315), and by the Department of Energy, Washington, DC.

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Freyer, J.P., Schor, P.L. Automated selective dissociation of cells from different regions of multicellular spheroids. In Vitro Cell Dev Biol 25, 9–19 (1989). https://doi.org/10.1007/BF02624405

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  • DOI: https://doi.org/10.1007/BF02624405

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