Comparative studies of cellular viability levels on 2D and 3D in vitro culture matrices

Abstract

In this study, the cellular viability and function of immortalized human cervical and dermal cells are monitored and compared in conventional 2D and two commercial 3D membranes, Collagen and Geltrex, of varying working concentration and volume. Viability was monitored with the aid of the Alamar Blue assay, cellular morphology was monitored with confocal microscopy, and cell cycle studies and cell death mechanism studies were performed with flow cytometry. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to 3D environment causing alterations to effective resazurin concentration, uptake and conversion rates, which was dependent on exposure time, but also due to the effect of the membrane itself on cellular function. These effects were verified by flow cytometry, in which no significant differences in viable cell numbers between 2D and 3D systems were observed after 24 h culture. The results showed the observed effect was different after shorter exposure periods, was also dependent on working concentration of the 3D system and could be mediated by altering the culture vessel size. Cell cycle analysis revealed cellular function could be altered by growth on the 3D substrates and the alterations were noted to be dependent on 3D membrane concentration. The use of 3D culture matrices has been widely interpreted to result in "improved viability levels" or "reduced" toxicity or cellular "resistance" compared to cells cultured on traditional 2D systems. The results of this study show that cellular health and viability levels are not altered by culture in 3D environments, but their normal cycle can be altered as indicated in the cell cycle studies performed and such variations must be accounted for in studies employing 3D membranes for in vitro cellular screening.

Keywords: 3D matrices; Collagen I; Confocal microscopy; Geltrex®; In vitro screening.

Fig. 1

a HeLa cells were seeded on 2D culture for 24 h, nuclei were stained with the nuclear stain NucRed. b HaCaT cells were seeded on 2D culture for 24 h, nuclei were stained with the nuclear stain NucRed. c HeLa cells were seeded on 3D culture (Collagen Rat tile) for 24 h and nuclei stained with NucRed. d HaCaT cells were seeded on 3D culture (Collagen Rat tile) for 24 h and nuclei stained with NucRed. e HeLa cells were seeded on 3D culture (Geltrex) for 24 h and nuclei stained with NucRed. f HaCaT cells were seeded on 3D culture (Geltrex) for 24 h and nuclei stained with NucRed (scale bar 20 µm)

Fig. 2

Alamar Blue response following 24, 48 and 72 h growth on both 2D and 3D culture (Collagen) of HeLa and HaCaT cells on both a 6 well plate and b a 24 well plate. Data are expressed as a percentage of three independent experiments ± SD of three individual experiments and relative to a 2D culture control. Statistically significant differences between the 3D culture membrane viability responses and that of the 2D cultures are denoted by *P < 0.05 and **P < 0.01

Fig. 3

Alamar Blue response following 24, 48 and 72 h growth on both 2D and 3D culture (Geltrex) of HeLa and HaCat cells on both a 6 well plate and b a 24 well plate. Data are expressed as a percentage of three independent experiments ± SD of three individual experiments and relative to a 2D culture control. Statistically significant differences between the 3D culture membrane viability responses and that of the 2D cultures are denoted by *P < 0.05 and **P < 0.01

Fig. 4

YOPRO and PI stained flow cytometry live, apoptotic and necrotic assay for HeLa (a) and HaCaT (b) cells grown on Collagen (3D) in different concentration and cells grown on plastic (2D) culture. Data are expressed as a percentage of three independent experiments ± SD of three individual experiments. Statistically significant differences between the 3D culture membrane live/dead cell analyses and that of the 2D cultures are denoted by *P < 0.05 and **P < 0.01

Fig. 5

YOPRO and PI stained flow cytommetery live, apoptotic and necrotic assay for Hela (a) and HaCaT (b) cells grown on Geltrex^® (3D) in different concentration and cells grown on plastic (2D) culture. Data are expressed as a percentage of three independent experiments ± SD of three individual experiments. Statistically significant differences between the 3D culture membrane live/dead cell analyses and those of the 2D cultures are denoted by *P < 0.05 and **P < 0.01

Fig. 6

Cell cycle analysis of HeLa (left) and HaCaT (right) cells grown on three different concentrations of Collagen gel (3D) and cells grown on plastic (2D) culture, and percentage of cells at G0/G1, S, and G2/M phases of cell cycle. Data are expressed as a percentage of three independent experiments ± SD of three individual experiments. Statistically significant differences between the 3D culture membrane cell cycle analyses and that of the 2D cultures are denoted by *P < 0.05 and **P < 0.01

Fig. 7

Cell cycle analysis of HeLa and HaCaT cell grown on two different concentration of Geltrex (3D) and cells grown on plastic (2D) culture, and percentage of cells at G0/G1, S and G2/M phases of cell cycle. Data are expressed as a percentage of three independent experiments ± SD of three individual experiments. Statistically significant differences between the 3D culture membrane cell cycle analyses and that of the 2D cultures are denoted by *P < 0.05 and **P < 0.01