Traditionally, cell cultures are monitored by time consuming microscopy, manual imaging and image processing. Conventional time-lapse recordings have provided new data of living cell behavior and growth. Despite the advantages, the technique has drawbacks in particular the length of follow-up time. In addition, monitored areas of interest are limited, and analysis of data is not automated. We tested a new technology platform for continuous monitoring and analysis of living cell cultures. The system applies machine vision technology to analyze and quantify morphologic traits. Events such as apoptosis, cell division, cellular movement, attachment, and the number of single cells can be continuously observed and recorded for up to several weeks, if needed. The optical design utilizes a dynamic z-stack to produce all-in-focus 123 information rich images enabling detailed analysis by generating in-depth images not previously seen with other techniques. Machine vision technology has traditionally been employed in the fields of medical imaging, precision robotics, and on factory assembly lines for consistently differentiating shape, size, position, patterns and movements 45.

Human embryonic stem cells (hESCs) are pluripotent cells capable of self-renewal and differentiation into all cell types in the body 67. They hold great potential for regenerative medicine. Significant quantities of hESCs are needed for differentiation to a final phenotype for cell transplantation. Current culture methods are not capable of producing adequate quantities of hESCs at an acceptable quality and price for cell transplantation. Improved automated culture analyses for undifferentiated hESCs are desired. In this report we describe, for the first time, the growth dynamics of hESCs using a new automated culture and monitoring system. Using this system, we compare conventional culture medium (containing animal protein serum replacement) to a xeno-free culture medium.

We applied and further developed the new automated monitoring and analysis platform with the goal of comparing hESC cultures in two different culture media. The platform enabled us to monitor growing hESC colonies. A grid-format picture capture system allowed the monitoring of living hESC cultures ranging in size from a few hundred micrometers to cell colonies of several millimetres at a defined picture capture cycle rate. The captured images were visualized with analysis software as movies, which were further used for the analysis of the colony growth.

The hESCs were cultured in a defined atmosphere (5% CO₂ and 36.5°C) from days 1 to 7 after passaging (Figure 2a). The hESC colonies attached to the feeders on days 1 to 2 after passaging (Figure 2b), and outgrowth of the hESC colonies started at day 3 (Figure 2c). At day 7, the hESC colonies had reached a size where they needed to be passaged (Figure 2e). Using the automated system, continuous monitoring of hESC colony growth was feasible [see Additional file 4]. We analyzed the total area and the areas of differentiated and undifferentiated cells in the hESC colonies at day 6 after passaging [see Additional file 3]. Totally differentiated and non-growing hESC colonies were excluded from the analysis. The average size of the HS237 and HS293 colonies at day 6 was 0.99 mm² (± 0.61, n = 24), with 91% undifferentiated cells and 9% differentiated cells, according to the automated analysis protocol.

To confirm the results of the automated analysis, some of the hESC colonies were also analyzed by FACS, and the rest were stained immunohistochemically. Analysis by FACS of the pooled HS237 colonies (n = 143), with a total of 3.0 million cells at day 7, revealed that an average size colony of 1.04 mm² contained ~21000 cells (n = 29). The analysis (in triplicate) revealed that 81% of the HS237 cells were positive for SSEA4 (Figure 2d). This result was consistent with the automated area protocol result, which indicated that 86.9% of the HS237 colony area (n = 29) was undifferentiated. Immunohistochemical staining of the automatically monitored colonies showed that these colonies were positive for Nanog and that SSEA1 was expressed in only a small proportion (3%) of the cells (Figure 2f). Both Nanog and SSEA-4 are widely considered as markers for undifferentiated hESCs although they may not always monitor the same cell population. SSEA-1 was used as common marker for differentiated cells since the aim was not to address towards which lineages the cells were differentiating.

Parallel cell culture plates were cultured in conventional incubators in otherwise similar conditions. The colonies grown in parallel plates were observed by an experienced observer similarly with routine every day evaluation practise. There were no differences in the growth rates or in the areas of undifferentiated and differentiated cells between the hESC colonies cultured in a standard incubator or in the automated system based on the colony size. Typically, spontaneous differentiation of undifferentiated colonies started six to eight days after passaging in the center of the colonies (data not shown). This phenomenon occurred similarly in the colonies grown in the Cell-IQ system and in a common incubator. Also, there were similar portion of totally differentiated and non-growing hESC colonies in the plates that were grown in Cell-IQ system and in a common incubator. As FACS and immunohistochemical analyses confirmed that the automated analysis protocol recognized undifferentiated and differentiated areas reliably, we further examined the growth parameters of HS237 and HS293 lines. There was no difference in the average colony area between lines HS237 (0.79 ± 0.32 mm²) and HS293 (1.20 ± 0.80 mm²) at day 6 after passaging. The growth rates of the undifferentiated areas (from days 5 to 6) of HS237 (215 ± 94 μm²/min) and HS293 (328 ± 187 μm²/min) were also similar. There were more undifferentiated areas in HS293 colonies than in HS237 colonies (P < 0.01, Figure 3).

The development of animal component-free culture conditions for hESCs is a major challenge 12. In this study, we tested whether the automated culturing and monitoring system could be used for the testing of culture media. Two different media were used: conventional hESC culture medium and an X-vivo 10-based, xeno-free culture medium. Both HS237 and HS293 colonies grew better in conventional medium than in X-vivo 10 medium [see Additional file 5]. The average colony size (both HS237 and HS293) in the X-vivo 10 medium was smaller than in conventional hESC medium (0.44 ± 0.31 vs. 0.99 mm² ± 0.61, P < 0.01, Figure 3), and the undifferentiated cell area was smaller in colonies cultured in the X-vivo 10 medium (64%) than in conventional hESC medium (91%, P < 0.01).

The continuous monitoring of living cells revealed more information than conventional microscopic observation of cultures. With the aid of the automated system we were able to observe cell behavior that would be impossible to discover by conventional microscopic observation. Traditionally, the growth rates of hESC colonies have been manually counted from single cell suspensions as doubling time during the exponential growth phase 13 or as an expansion of the area of a single colony from microscopic images 14. The automated monitoring system allowed a reliable area measurement of live, unlabeled, intact cell colonies. Hence, we obtained continuous quantitative data from the cultures with constant real-time feedback. In culture medium testing, the automated machine vision culture platform, revealed that both hESC lines grew better in conventional hESC medium than in X-vivo 10 medium.

The automated cell culturing and analysis system provides an optimal tool for the evaluation of hESC cultures, allowing continuous well-to-well comparison of the effects of different culture media or different growth factor concentrations on cell growth and behaviour – such as differentiation. In addition, it can also be used as a tool in the optimization of differentiation protocols for hESCs. Whether this system can be applied to monitoring and analysis of 3D cell differentiation remains to be studied. Our data clearly demonstrate that continuous monitoring of living cell cultures without disturbing the cells can reveal more information than conventional microscopic observation of cultures. The automated system enables rapid and reliable analysis of undifferentiated growth dynamics of hESCs.

The author(s) declare that they have no competing interests.

S.N. performed the Cell-IQ experiments and data analyses and contributed to writing the manuscript. K.R. performed the cell culture and characterization experiments and contributed to writing the manuscript. H.P. and R.S. contributed to the conception and design of the experiments. O.H. contributed to the conception, and design of the experiments, interpretation of data, and critically revising the manuscript for important intellectual content. H.S. contributed to the conception, and design of the experiments, interpretation of data, and to writing the manuscript.