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Imaged single\u2010cell deposition followed by whole\u2010well imaging using specialized instruments has, in many cell line development labs, replaced the \u201cgold standard\u201d of two rounds of limiting dilution due to its increased speed and the assurance of clonality provided by orthogonal images. However, there is still a lack of information on how the procedures used to define these clonal cell lines perform. Here we use a mixture of two distinguishable Chinese hamster ovary (CHO) cells to document that a greater than 99% probability of clonality can be obtained from our single\u2010cell cloning method that uses our preparation procedures, the VIPS\u00ae single\u2010cell deposition instrument, the Cell Metric\u00ae whole\u2010well imager, and a comprehensive visual review. Together with the assurance of cell\/well images, the determination of the probability of clonality of our VIPS+Cell Metric method provides a strong package of evidence of single\u2010cell derivation of a recombinant CHO cell line.<\/jats:p>","DOI":"10.1002\/btpr.70012","type":"journal-article","created":{"date-parts":[[2025,3,11]],"date-time":"2025-03-11T21:28:13Z","timestamp":1741728493000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Assessing the probability of clonality achieved by single\u2010cell cloning of\n                    <scp>CHO<\/scp>\n                    cells through cell deposition combined with imaging using distinguishable cells"],"prefix":"10.1002","volume":"41","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3695-153X","authenticated-orcid":false,"given":"Genevieve H.","family":"Nonet","sequence":"first","affiliation":[{"name":"Resilience, Inc.  Alameda California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena","family":"Scut","sequence":"additional","affiliation":[{"name":"Resilience, Inc.  Alameda California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raymond","family":"Ogawa","sequence":"additional","affiliation":[{"name":"Resilience, Inc.  Alameda California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Milan T.","family":"Tomic","sequence":"additional","affiliation":[{"name":"Resilience, Inc.  Alameda California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,3,4]]},"reference":[{"key":"e_1_2_8_2_1","unstructured":"European Medicines Agency.ICH Harmonized Tripartite Guideline: Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological\/Biological Products Q5D. International Conference on Harmonization. Accessed November 30 2023.https:\/\/www.ema.europa.eu\/en\/documents\/scientific\u2010guideline\/ich\u2010q\u20105\u2010d\u2010derivation\u2010and\u2010characterisation\u2010cell\u2010substrates\u2010used\u2010production\u2010biotechnologicalbiological\u2010products\u2010step\u20105_en.pdf"},{"key":"e_1_2_8_3_1","unstructured":"US Food and Drug Administration.Points to consider in the manufacture and testing of monoclonal antibody products for human use.1997. Accessed September 2 2024.https:\/\/www.fda.gov\/regulatory\u2010information\/search\u2010fda\u2010guidance\u2010documents\/points\u2010consider\u2010manufacture\u2010and\u2010testing\u2010monoclonal\u2010antibody\u2010products\u2010human\u2010use"},{"key":"e_1_2_8_4_1","unstructured":"World Health Organization.Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks Annex 3 TRS No 978.2013. Accessed March 7 2024.https:\/\/www.who.int\/publications\/m\/item\/animal-cell-culture-trs-no-978-annex3"},{"key":"e_1_2_8_5_1","unstructured":"KennettS.Establishing clonal cell lines\u2010 a regulatory perspective. Presented at well\u2010characterized biotechnology products symposium.2014. Accessed August 20 2024.docplayer.net\/47822551-Establishing-clonal-cell-lines-a-regulatory-perspective.html"},{"key":"e_1_2_8_6_1","unstructured":"WelchJ.Tilting at clones: a regulatory perspective on the importance of \u201cClonality\u201d of mammalian cell banks.https:\/\/www.trio-biotech.com\/wp-content\/uploads\/2017\/06\/Joel-Welch.pdf-Amsterdam-April-2017.pdf"},{"key":"e_1_2_8_7_1","unstructured":"World Health Organization.Guidelines on the quality safety and efficacy of biotherapeutic protein products prepared by recombinant DNA technology Annex 4 TRS No 987.2014. 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