INTRODUCTION B IOMEDICAL RESEARCH is evolving toward the simula- tion of in vivo environments with in vitro cell cul- ture systems. In doing so, researchers have realized that there are vast differences between conventional two-di- mensional (2D) petri dish cultures and three-dimensional (3D) cultures. 1,2 This sparked interest in cell behavior in 3D cultures spanning various fields of research includ- ing cancer, 3 developmental biology, 4 and tissue engi- neering, where 3D cell–scaffold constructs 5 and cell sheet engineering 6,7 are studied. Cell proliferation ranks high among the many parameters of cellular behavior studied, reflected by an 20% increase in publication numbers related to the use of cell proliferation assays—455 in 1994 to 552 in 2003 (PubMed). However, these assays are sometimes erroneously assumed to be applicable for measuring cell proliferation in high cell density or 3D cultures, an oversight our group has also committed. 8 Through our experiences thereafter, we found inconsis- tent correlations between cell proliferation assays under those culture conditions, 9 and hence we decided to study this phenomenon in more detail. As a simple model, low to high cell densities in 2D culture plates were used to evaluate the validity of com- mon cell proliferation assays. Extending the observations to complex 3D environments allows greater appreciation of the complexities in assaying cell proliferation. The proliferation assays often utilized include metabolic as- says (alamarBlue, CellTiter 96 AQ ueous MTS assay), DNA quantification (PicoGreen), radioactive labeling ([ 3 H]thymidine), and manual cell counting (hemocy- TISSUE ENGINEERING Volume 11, Number 1/2, 2005 © Mary Ann Liebert, Inc. The Challenge to Measure Cell Proliferation in Two and Three Dimensions KEE W. NG, M.Eng., 1,* DAVID T.W. LEONG, B.Eng, 2,* and DIETMAR W. HUTMACHER, M.B.A., Ph.D. 3,4 ABSTRACT Various assays, using different strategies, are available for assessing cultured cell proliferation. These include measurement of metabolic activity (tetrazolium salts and alamarBlue), DNA quantification using fluorophores (Hoechst 33258 and PicoGreen), uptake of radioactively-labeled DNA precur- sors such as [ 3 H]thymidine, and physical counting (hemocytometer). These assays are well estab- lished in characterizing cell proliferation in two-dimensional (2D), monolayer cultures of low cell densities. However, increasing interest in 3D cultures has prompted the need to evaluate the effec- tiveness of using these assays in high cell density or 3D cultures. We show here that typical cell pro- liferation assays do not necessarily correlate linearly with increasing cell densities or between 2D and 3D cultures, and are either not suitable or only rough approximations in quantifying actual cell numbers in a culture. Prudent choice of techniques and careful interpretation of data are therefore recommended when measuring cell proliferation in high cell density and 3D cultures. 1 Department of Surgery, 2 Department of Biological Sciences, 3 Division of Bioengineering, and 4 Department of Orthopedics Surgery, National University of Singapore, Singapore. * K.W.N. and D.T.W.L. contributed equally to this work. 182