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Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the qua...

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Autores principales: Kusakawa, Shinji, Yasuda, Satoshi, Kuroda, Takuya, Kawamata, Shin, Sato, Yoji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672267/
https://www.ncbi.nlm.nih.gov/pubmed/26644244
http://dx.doi.org/10.1038/srep17892
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author Kusakawa, Shinji
Yasuda, Satoshi
Kuroda, Takuya
Kawamata, Shin
Sato, Yoji
author_facet Kusakawa, Shinji
Yasuda, Satoshi
Kuroda, Takuya
Kawamata, Shin
Sato, Yoji
author_sort Kusakawa, Shinji
collection PubMed
description Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process.
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spelling pubmed-46722672015-12-11 Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation Kusakawa, Shinji Yasuda, Satoshi Kuroda, Takuya Kawamata, Shin Sato, Yoji Sci Rep Article Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process. Nature Publishing Group 2015-12-08 /pmc/articles/PMC4672267/ /pubmed/26644244 http://dx.doi.org/10.1038/srep17892 Text en Copyright © 2015, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Kusakawa, Shinji
Yasuda, Satoshi
Kuroda, Takuya
Kawamata, Shin
Sato, Yoji
Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation
title Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation
title_full Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation
title_fullStr Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation
title_full_unstemmed Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation
title_short Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation
title_sort ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672267/
https://www.ncbi.nlm.nih.gov/pubmed/26644244
http://dx.doi.org/10.1038/srep17892
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