Analysis of clonogenic growth in vitro.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
11 2021
11 2021
Historique:
received:
23
12
2020
accepted:
10
08
2021
pubmed:
27
10
2021
medline:
18
11
2021
entrez:
26
10
2021
Statut:
ppublish
Résumé
The clonogenic assay measures the capacity of single cells to form colonies in vitro. It is widely used to identify and quantify self-renewing mammalian cells derived from in vitro cultures as well as from ex vivo tissue preparations of different origins. Varying research questions and the heterogeneous growth requirements of individual cell model systems led to the development of several assay principles and formats that differ with regard to their conceptual setup, 2D or 3D culture conditions, optional cytotoxic treatments and subsequent mathematical analysis. The protocol presented here is based on the initial clonogenic assay protocol as developed by Puck and Marcus more than 60 years ago. It updates and extends the 2006 Nature Protocols article by Franken et al. It discusses different strategies and principles to analyze clonogenic growth in vitro and presents the clonogenic assay in a modular protocol framework enabling a diversity of formats and measures to optimize determination of clonogenic growth parameters. We put particular focus on the phenomenon of cellular cooperation and consideration of how this can affect the mathematical analysis of survival data. This protocol is applicable to any mammalian cell model system from which single-cell suspensions can be prepared and which contains at least a small fraction of cells with self-renewing capacity in vitro. Depending on the cell system used, the entire procedure takes ~2-10 weeks, with a total hands-on time of <20 h per biological replicate.
Identifiants
pubmed: 34697469
doi: 10.1038/s41596-021-00615-0
pii: 10.1038/s41596-021-00615-0
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
4963-4991Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.
Références
Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J. & van Bree, C. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319 (2006).
pubmed: 17406473
doi: 10.1038/nprot.2006.339
Puck, T. T. & Marcus, P. I. Action of x-rays on mammalian cells. J. Exp. Med. 103, 653–666 (1956).
pubmed: 13319584
pmcid: 2136626
doi: 10.1084/jem.103.5.653
Puck, T. T., Marcus, P. I. & Cieciura, S. J. Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer. J. Exp. Med. 103, 273–283 (1956).
pubmed: 13286432
doi: 10.1084/jem.103.2.273
pmcid: 2136583
Puck, T. T. & Marcus, P. I. A rapid method for viable cell titration and clone production with hela cells in tissue culture: the use of X-irradiated cells to supply conditioning factors. Proc. Natl Acad. Sci. USA 41, 432–437 (1955).
pubmed: 16589695
pmcid: 528114
doi: 10.1073/pnas.41.7.432
Rheinwald, J. G. & Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–343 (1975).
pubmed: 1052771
doi: 10.1016/S0092-8674(75)80001-8
Reynolds, B. A. & Weiss, S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710 (1992).
pubmed: 1553558
doi: 10.1126/science.1553558
Phillips, T. M., McBride, W. H. & Pajonk, F. The response of CD24
pubmed: 17179479
doi: 10.1093/jnci/djj495
Wink, D. A. et al. Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc. Natl Acad. Sci. U. S. A. 90, 9813–9817 (1993).
pubmed: 8234317
pmcid: 47662
doi: 10.1073/pnas.90.21.9813
Liebmann, J. E. et al. Cytotoxic studies of paclitaxel (Taxol) in human tumour cell lines. Br. J. Cancer 68, 1104–1109 (1993).
pubmed: 7903152
pmcid: 1968657
doi: 10.1038/bjc.1993.488
Wang, Q. et al. UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J. Natl Cancer Inst. 88, 956–965 (1996).
pubmed: 8667426
doi: 10.1093/jnci/88.14.956
Elkind, M. M. & Sutton, H. Radiation response of mammalian cells grown in culture. 1. Repair of X-ray damage in surviving Chinese hamster cells. Radiat. Res. 13, 556–593 (1960).
pubmed: 13726391
doi: 10.2307/3570945
Huang, P. et al. Folic acid-conjugated silica-modified gold nanorods for X-ray/CT imaging-guided dual-mode radiation and photo-thermal therapy. Biomaterials 32, 9796–9809 (2011).
pubmed: 21917309
doi: 10.1016/j.biomaterials.2011.08.086
Elgendy, M., Sheridan, C., Brumatti, G. & Martin, S. J. Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival. Mol. Cell 42, 23–35 (2011).
pubmed: 21353614
doi: 10.1016/j.molcel.2011.02.009
Alimova, I. N. et al. Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle 8, 909–915 (2009).
pubmed: 19221498
doi: 10.4161/cc.8.6.7933
Rochat, A., Kobayashi, K. & Barrandon, Y. Location of stem cells of human hair follicles by clonal analysis. Cell 76, 1063–1073 (1994).
pubmed: 8137423
doi: 10.1016/0092-8674(94)90383-2
Sanford, K. K., Earle, W. R. & Likely, G. D. The growth in vitro of single isolated tissue cells. J. Natl Cancer Inst. 9, 229–246 (1948).
pubmed: 18105872
Brix, N. et al. The clonogenic assay: robustness of plating efficiency-based analysis is strongly compromised by cellular cooperation. Radiat. Oncol. 15, 248 (2020).
pubmed: 33121517
pmcid: 7597001
doi: 10.1186/s13014-020-01697-y
Orth, M., Unger, K., Schoetz, U., Belka, C. & Lauber, K. Taxane-mediated radiosensitization derives from chromosomal missegregation on tripolar mitotic spindles orchestrated by AURKA and TPX2. Oncogene 37, 52–62 (2018).
pubmed: 28869599
doi: 10.1038/onc.2017.304
Hess, J. et al. Genomic amplification of Fanconi anemia complementation group A (FancA) in head and neck squamous cell carcinoma (HNSCC): cellular mechanisms of radioresistance and clinical relevance. Cancer Lett. 386, 87–99 (2017).
pubmed: 27867017
doi: 10.1016/j.canlet.2016.11.014
Puck, T. T., Cieciura, S. J. & Fisher, H. W. Clonal growth in vitro of human cells with fibroblastic morphology; comparison of growth and genetic characteristics of single epithelioid and fibroblast-like cells from a variety of human organs. J. Exp. Med. 106, 145–158 (1957).
pubmed: 13439121
pmcid: 2136737
doi: 10.1084/jem.106.1.145
Fisher, H. W. & Puck, T. T. On the functions of x-irradiated “feeder” cells in supporting growth of single mammalian cells. Proc. Natl Acad. Sci. USA 42, 900–906 (1956).
pubmed: 16589971
pmcid: 528367
doi: 10.1073/pnas.42.12.900
Terasima, T. & Tolmach, L. J. Changes in x-ray sensitivity of HeLa cells during the division cycle. Nature 190, 1210–1211 (1961).
pubmed: 13775960
doi: 10.1038/1901210a0
Son, J. et al. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway. Nature 496, 101–105 (2013).
pubmed: 23535601
pmcid: 3656466
doi: 10.1038/nature12040
Varfolomeev, E. et al. IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 131, 669–681 (2007).
pubmed: 18022362
doi: 10.1016/j.cell.2007.10.030
Till, J. E. & McCulloch, C. E. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213–222 (1961).
pubmed: 13776896
doi: 10.2307/3570892
Becker, A. J., McCulloch, C. E. & Till, J. E. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197, 452–454 (1963).
pubmed: 13970094
doi: 10.1038/197452a0
Park, C. H., Bergsagel, D. E. & McCulloch, E. A. Mouse myeloma tumor stem cells: a primary cell culture assay. J. Natl Cancer Inst. 46, 411–422 (1971).
pubmed: 5115909
Courtenay, V. D. & Mills, J. An in vitro colony assay for human tumours grown in immune-suppressed mice and treated in vivo with cytotoxic agents. Br. J. Cancer 37, 261–268 (1978).
pubmed: 343804
pmcid: 2009601
doi: 10.1038/bjc.1978.35
Hamburger, A. W. & Salmon, S. E. Primary bioassay of human tumor stem cells. Science 197, 461–463 (1977).
pubmed: 560061
doi: 10.1126/science.560061
Whitlock, C. A. & Witte, O. N. Long-term culture of B lymphocytes and their precursors from murine bone marrow. Proc. Natl Acad. Sci. USA 79, 3608–3612 (1982).
pubmed: 6980414
pmcid: 346472
doi: 10.1073/pnas.79.11.3608
Kondo, M., Weissman, I. L. & Akashi, K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672 (1997).
pubmed: 9393859
doi: 10.1016/S0092-8674(00)80453-5
Matsui, W. et al. Characterization of clonogenic multiple myeloma cells. Blood 103, 2332–2336 (2004).
pubmed: 14630803
doi: 10.1182/blood-2003-09-3064
Eliason, J. F., Aapro, M. S., Decrey, D. & Brink-Petersen, M. Non-linearity of colony formation by human tumour cells from biopsy samples. Br. J. Cancer 52, 311–318 (1985).
pubmed: 4041360
pmcid: 1977209
doi: 10.1038/bjc.1985.195
Pomp, J. et al. Cell density dependent plating efficiency affects outcome and interpretation of colony forming assays. Radiother. Oncol. 40, 121–125 (1996).
pubmed: 8884965
doi: 10.1016/0167-8140(96)01767-7
Veldwijk, M. R., Zhang, B., Wenz, F. & Herskind, C. The biological effect of large single doses: a possible role for non-targeted effects in cell inactivation. PLoS One 9, e84991 (2014).
pubmed: 24465461
pmcid: 3898915
doi: 10.1371/journal.pone.0084991
Adrian, G., Ceberg, C., Carneiro, A. & Ekblad, L. Rescue effect inherited in colony formation assays affects radiation response. Radiat. Res. 189, 44–52 (2018).
pubmed: 29136392
doi: 10.1667/RR14842.1
Dakhore, S., Nayer, B. & Hasegawa, K. Human pluripotent stem cell culture: current status, challenges, and advancement. Stem Cells Int. 2018, 7396905 (2018).
pubmed: 30595701
pmcid: 6282144
doi: 10.1155/2018/7396905
Repetto, G., del Peso, A. & Zurita, J. L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat. Protoc. 3, 1125–1131 (2008).
pubmed: 18600217
doi: 10.1038/nprot.2008.75
Lindhagen, E., Nygren, P. & Larsson, R. The fluorometric microculture cytotoxicity assay. Nat. Protoc. 3, 1364–1369 (2008).
pubmed: 18714304
doi: 10.1038/nprot.2008.114
Riss, T. L. et al. Cell viability assays. in Assay Guidance Manual (eds. Markossian S. et al.) (Eli Lilly and the National Center for Advancing Translational Sciences, 2004).
Rampersad, S. N. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors (Basel) 12, 12347–12360 (2012).
doi: 10.3390/s120912347
Lundin, A., Hasenson, M., Persson, J. & Pousette, A. Estimation of biomass in growing cell lines by adenosine triphosphate assay. Methods Enzymol. 133, 27–42 (1986).
pubmed: 3821540
doi: 10.1016/0076-6879(86)33053-2
Temple, S. Division and differentiation of isolated CNS blast cells in microculture. Nature 340, 471–473 (1989).
pubmed: 2755510
doi: 10.1038/340471a0
Davis, A. A. & Temple, S. A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 372, 263–266 (1994).
pubmed: 7969470
doi: 10.1038/372263a0
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
pubmed: 17529971
doi: 10.1038/nbt1310
Ploemacher, R. E., van der Sluijs, J. P., Voerman, J. S. & Brons, N. H. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74, 2755–2763 (1989).
pubmed: 2819245
doi: 10.1182/blood.V74.8.2755.2755
Sutherland, H. J., Lansdorp, P. M., Henkelman, D. H., Eaves, A. C. & Eaves, C. J. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc. Natl Acad. Sci. USA 87, 3584–3588 (1990).
pubmed: 2333304
pmcid: 53946
doi: 10.1073/pnas.87.9.3584
Fogg, D. K. et al. A clonogenic bone marrow progenitor specific for macrophages and dendritic cells. Science 311, 83–87 (2006).
pubmed: 16322423
doi: 10.1126/science.1117729
Riether, C. et al. Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents. Nat. Med. 26, 1459–1467 (2020).
pubmed: 32601337
doi: 10.1038/s41591-020-0910-8
Hu, Y. & Smyth, G. K. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009).
pubmed: 19567251
doi: 10.1016/j.jim.2009.06.008
Fiebig, H. H., Maier, A. & Burger, A. M. Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery. Eur. J. Cancer 40, 802–820 (2004).
pubmed: 15120036
doi: 10.1016/j.ejca.2004.01.009
Borowicz, S. et al. The soft agar colony formation assay. J. Vis. Exp. 92, e51998 (2014).
Leivas, A. et al. Natural killer cells efficiently target multiple myeloma clonogenic tumor cells. Cancer Immunol. Immunother. (in the press).
Kukreja, A. et al. Enhancement of clonogenicity of human multiple myeloma by dendritic cells. J. Exp. Med. 203, 1859–1865 (2006).
pubmed: 16880256
pmcid: 2034506
doi: 10.1084/jem.20052136
Eke, I., Hehlgans, S., Sandfort, V. & Cordes, N. 3D matrix-based cell cultures: automated analysis of tumor cell survival and proliferation. Int. J. Oncol 48, 313–321 (2016).
pubmed: 26549537
doi: 10.3892/ijo.2015.3230
Nielson, L., Smyth, G. & Greenfield, P. Hemacytometer cell count distributions: implications of non-Poisson behavior. Biotechnol. Prog. 7, 560–563 (1991).
doi: 10.1021/bp00012a600
Harnicek, D. et al. Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair. Int. J. Cancer 139, 467–479 (2016).
pubmed: 26933761
doi: 10.1002/ijc.30070
Weiss, E. M. et al. High hydrostatic pressure treatment generates inactivated mammalian tumor cells with immunogeneic features. J. Immunotoxicol. 7, 194–204 (2010).
pubmed: 20205624
doi: 10.3109/15476911003657414
Preciado, S. et al. The incorporation of extracellular vesicles from mesenchymal stromal cells into CD34
pubmed: 31184411
pmcid: 6852558
doi: 10.1002/stem.3032
Yao, T. & Asayama, Y. Animal-cell culture media: history, characteristics, and current issues. Reprod. Med. Biol. 16, 99–117 (2017).
pubmed: 29259457
pmcid: 5661806
doi: 10.1002/rmb2.12024
Nuryadi, E., Mayang Permata, T. B., Komatsu, S., Oike, T. & Nakano, T. Inter-assay precision of clonogenic assays for radiosensitivity in cancer cell line A549. Oncotarget 9, 13706–13712 (2018).
pubmed: 29568388
pmcid: 5862609
doi: 10.18632/oncotarget.24448
Lacerda, L. et al. Simvastatin radiosensitizes differentiated and stem-like breast cancer cell lines and is associated with improved local control in inflammatory breast cancer patients treated with postmastectomy radiation. Stem Cells Transl. Med. 3, 849–856 (2014).
pubmed: 24833589
pmcid: 4073823
doi: 10.5966/sctm.2013-0204
Dahle, J., Kakar, M., Steen, H. B. & Kaalhus, O. Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing. Cytometry A 60, 182–188 (2004).
pubmed: 15290719
doi: 10.1002/cyto.a.20038
Choudhry, P. High-throughput method for automated colony and cell counting by digital image analysis based on edge detection. PLoS One 11, e0148469 (2016).
pubmed: 26848849
pmcid: 4746068
doi: 10.1371/journal.pone.0148469
Bewes, J. M., Suchowerska, N. & McKenzie, D. R. Automated cell colony counting and analysis using the circular Hough image transform algorithm (CHiTA). Phys. Med. Biol. 53, 5991–6008 (2008).
pubmed: 18836215
doi: 10.1088/0031-9155/53/21/007
Meijering, E., Carpenter, A. E., Peng, H., Hamprecht, F. A. & Olivo-Marin, J. C. Imagining the future of bioimage analysis. Nat. Biotechnol. 34, 1250–1255 (2016).
pubmed: 27926723
doi: 10.1038/nbt.3722
Moen, E. et al. Deep learning for cellular image analysis. Nat. Methods 16, 1233–1246 (2019).
pubmed: 31133758
doi: 10.1038/s41592-019-0403-1
pmcid: 8759575
Segebarth, D. et al. On the objectivity, reliability, and validity of deep learning enabled bioimage analyses. eLife 9, e59780 (2020).
pubmed: 33074102
pmcid: 7710359
doi: 10.7554/eLife.59780
Parker, W. S. & Risbey, J. S. False precision, surprise and improved uncertainty assessment. Philos. Trans. A Math. Phys. Eng. Sci. 373, 20140453 (2015).
pubmed: 26460113
Kellerer, A. M. & Rossi, H. H. A generalized formulation of dual radiation action. Radiat. Res. 75, 471–488 (1978).
doi: 10.2307/3574835
Unkel, S., Belka, C. & Lauber, K. On the analysis of clonogenic survival data: statistical alternatives to the linear-quadratic model. Radiat. Oncol. 11, 11 (2016).
pubmed: 26822015
pmcid: 4730743
doi: 10.1186/s13014-016-0584-z
Vembadi, A., Menachery, A. & Qasaimeh, M. A. Cell cytometry: review and perspective on biotechnological advances. Front. Bioeng. Biotechnol. 7, 147 (2019).
pubmed: 31275933
pmcid: 6591278
doi: 10.3389/fbioe.2019.00147
Gerweck, L. E., Dullea, R., Zaidi, S. T., Budach, W. & Hartford, A. Influence of experimental factors on intrinsic radiosensitivity assays at low doses of radiation: cell multiplicity. Radiat. Res. 138, 361–366 (1994).
pubmed: 8184010
doi: 10.2307/3578684
Rockwell, S. Effects of clumps and clusters on survival measurements with clonogenic assays. Cancer Res. 45, 1601–1607 (1985).
pubmed: 3978627