Hostname: page-component-cd9895bd7-lnqnp Total loading time: 0 Render date: 2024-12-27T08:53:16.486Z Has data issue: false hasContentIssue false
  • English
  • Français

A Method for Quick, Low-Cost Automated Confluency Measurements

Published online by Cambridge University Press:  28 October 2011

Gil Topman ,
Orna Sharabani-Yosef  and
Amit Gefen
Gil Topman
Affiliation:
Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel
Orna Sharabani-Yosef
Affiliation:
Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel
Amit Gefen*
Affiliation:
Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel
*
Corresponding author. E-mail: gefen@eng.tau.ac.il
Get access

Abstract

A culture's confluency is a fundamental measure in the field of biology, and routine quantification of confluence in cell culture protocols, biological assays and tissue engineering work is important. However, current techniques for obtaining confluency are either subjective, destructive, not simple enough, or time-consuming. We developed an image processing method for automated confluency measurement from a single microscope image without any chemical staining. To demonstrate utility we monitored the confluency of three cell types: NIH3T3 fibroblasts, C2C12 myoblasts, and 3T3L1 pre-adipocytes for 5 days, twice a day. The captured micrographs had different and uneven illumination, the cell types varied in cell-to-background contrast, and the confluency ranged between 10% and 100%. Despite these variable conditions, our method was shown to be practical, economic, and easy to implement, providing quantitative confluency measurements over time in each culture case. The method is hence suitable for routine automatic determination of confluency to standardize handling of cells, achieve reproducibility across trials, and improve accuracy in experimental outcome measures.

Keywords

image analysissegmentationtexturecell culturemicroscopytissue engineering
Type
Software and Techniques Development
Information
Microscopy and Microanalysis , Volume 17 , Issue 6 , December 2011 , pp. 915 - 922
Copyright
Copyright © Microscopy Society of America 2011

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Ahmed, A.S., Gogal, R.M. Jr. & Walsh, J.E. (1994). A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: An alternative to [3H]thymidine incorporation assay. J Immunol Methods 170, 211–224.CrossRefGoogle ScholarPubMed
Curl, C.L., Harris, T., Harris, P.J., Allman, B.E., Bellair, C.J., Stewart, A.G. & Delbridge, L.M.D. (2004). Quantitative phase microscopy: A new tool for measurement of cell culture growth and confluency in situ. Pflugers Arch 448, 462–468.CrossRefGoogle ScholarPubMed
Ginty, P.J., Howard, D., Rose, F.R., Whitaker, M.J., Barry, J.J., Tighe, P., Mutch, S.R., Serhatkulu, G., Oreffo, R.O., Howdle, S.M. & Shakesheff, K.M. (2006). Mammalian cell survival and processing in supercritical CO(2). Proc Natl Acad Sci USA 103, 7426–7431.CrossRefGoogle ScholarPubMed
Liang, C.C., Park, A.Y. & Guan, J.L. (2007). In vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2, 329–333.CrossRefGoogle ScholarPubMed
Malpica, N., Santos, A., Tejedor, A., Torres, A., Castilla, M., Garcia-Barreno, P. & Desco, M. (2003). Automatic quantification of viability in epithelial cell cultures by texture analysis. J Microsc 209, 34–40.CrossRefGoogle ScholarPubMed
Mölder, A., Sebesta, M., Gustafsson, M., Gisselson, L., Wingren, A. & Alm, K. (2008). Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography. J Microsc 232, 240–247.CrossRefGoogle ScholarPubMed
Or-Tzadikario, S., Sopher, R. & Gefen, A. (2010). Quantitative monitoring of lipid accumulation over time in cultured adipocytes as function of culture conditions: Toward controlled adipose tissue engineering. Tissue Eng Part C Methods 16, 1167–1181.CrossRefGoogle ScholarPubMed
Roehm, N.W., Rodgers, G.H., Hatfield, S.M. & Glasebrook, A.L. (1991). An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods 142, 257–265.CrossRefGoogle ScholarPubMed
Rubin, H. (1981). Growth regulation, reverse transformation, and adaptability of 3T3 cells in decreased Mg2+ concentration. Proc Natl Acad Sci USA 78, 328–332.CrossRefGoogle ScholarPubMed
Selinummi, J., Ruusuvuori, P., Podolsky, I., Ozinsky, A., Gold, E., Yli-Harja, O., Aderem, A. & Shmulevich, I. (2009). Bright field microscopy as an alternative to whole cell fluorescence in automated analysis of macrophage images. PLoS ONE 4, e7497.CrossRefGoogle ScholarPubMed