The microfluidic chip used in these experiments was previously reported by us (Vollertsen et al. 2020). In short, the PDMS chip features 64 independently addressable chambers for cell culturing with a volume of 30 nL each. Figure 1a shows a photograph of the chip where chambers in each quarter are filled with a different color of food dye. The chambers can be addressed using a valve-based combinatorial multiplexer (Hua et al. 2006) as shown in Fig. 1b. The multiplexer is mirrored on the other side of the chambers to prevent a net flow through non-addressed chambers where the multiplexer valves may peristaltically push or pull liquid as a side effect of other chambers being addressed. A purge channel (Fig. 1b) located between the multiplexer and the chambers connects the channels so that these can be purged of their content without contaminating the chambers. This aspect is important for both the surface modification of the chip and the removal of unwanted cells in the channels after cell seeding in the chambers. Figure 1c shows a brightfield image of four of the chambers containing hESC after 24 h of automated cell culturing.
The low volumes of the chip are an essential aspect for reducing the cost of screening for optimal conditions for stem cell differentiation, as the main cost factor for such experiments is the cell culture medium and its components. Clearly, our experimental microfluidic platform currently costs a lot more to fabricate than commercial well plates, but the cost of manufacturing could be reduced dramatically by automating fabrication.
Another aspect to consider in future chip fabrication is the use of PDMS. This material has been widely discussed and reviewed due to both its absorption of small hydrophobic molecules and potential release of uncrosslinked polymers (Halldorsson et al. 2015; van Meer et al. 2017). For full certainty that these material properties don’t influence the stem cell differentiation by changing the ratio of externally-added to cell-secreted factors it would be best to use devices made from polymers such as polystyrene or PMMA. The adverse effects of PDMS should be noted in particular for testing differentiation pathways towards certain cell types. For example, human neuronal stem cell culture has been shown to result in widespread cell death in PDMS devices (Kajtez et al. 2020). However, for stem cell differentiation towards cardiac mesodermal cells (or even full cardiomyocytes), we do not expect the PDMS absorption of molecules to greatly hinder the differentiation. It has been previously shown that cardiomyocytes can be derived from stem cells in PDMS devices (Giobbe et al. 2015). Furthermore, we have also successfully derived cardiomyocytes from stem cells on PDMS (though admittedly a different brand) in well plates (data not shown).
HESC population change depending on different medium exchange intervals
The effect of culturing hESCs in the 30 nL chambers with different medium exchange rates was investigated by monitoring the cells’ proliferation rates. For easier representation, the exchange frequencies are henceforth expressed as intervals between medium exchanges (e.g. every 5 h instead of 4.8 d−1). Four different medium exchange intervals (2 h, 3 h, 5 h, and 8 h) with 14–16 replicates per interval were tested on a single chip. On average, there was less medium per cell per hour in the chambers for all conditions than in the control 6-well plate (Table 1). These time intervals were chosen to allow more time for endogenous factors produced by the cells to accumulate in the chambers between medium refreshments, since these factors are removed along with every medium refreshment.
Figure 2a shows the mean number of cells per chamber for each medium exchange interval at 1 h, 16 h, and 24 h after seeding. After seeding, most chambers contained between 70 and 120 cells. The cell counts at 16 h and 24 h after seeding show cell proliferation for all four intervals. However, it also becomes apparent that the average cell number per chamber at the beginning of the experiment (1 h) differs by up to 27% between the four conditions. For example, the 8 h chambers have an average of 101 cells per chamber, while the 3 h chambers only have an average of 74 cells per chamber. As a result, there is more medium available per cell (in between medium exchanges) in some of the conditions, which may also have an effect on the proliferation. Therefore, a subset of the chambers is shown in Fig. 2b, where all chambers contained on average approximately 90 cells at 1 h after seeding. The subset contains between n = 7 (2 h) and n = 12 (5 h) chambers per condition. A repeated measures ANOVA (analysis of variance) showed no statistically significant difference between the conditions. In addition, a second experiment with 1h, 2h, 3h, and 5h medium exchange intervals was performed, which likewise showed no statistically significant difference between conditions (Suppl. Information 1).
The results show that the hESCs remain highly proliferative for at least the first 24 h even with only tens of nanoliters of medium and long exchange intervals (5-8 h). Furthermore, they show that frequent medium exchanges at low shear stress τ (τ < 0.13 Pa) directly after cell seeding do not result in decreased cell proliferation. This suggests that the common practice of leaving hESCs to attach overnight in microfluidic devices without medium refreshment may not be necessary.
Pluripotency staining of hESCs in chambers
We confirmed that the hESCs remained pluripotent while they were proliferating in the microfluidic chip. HESC pluripotency on day 0 (24 h after seeding) in the microchambers was confirmed by immunostaining for the pluripotency markers SOX2 and OCT3/4. On day -1, the cells were seeded at approximately 100–150 cells per chamber and Essential 8 (E8) medium was exchanged every 2 h and every 5 h for half of the chambers, respectively. On day 0, the cells were fixed and stained. As a positive control, hESCs cultured in well plates were also immunostained. The brightfield and fluorescence images are shown in Fig. 3 (the fluorescent images are pseudo-colored whereby the nucleus staining is shown in blue, SOX2 immunostaining in green and OCT3/4 immunostaining in orange). Both the 2 h and 5 h conditions show the presence of SOX2 and OCT3/4 markers, indicating pluripotency. For comparison, cells which had been differentiated towards the cardiac mesoderm (for 3 days) in well plates were also fixed and stained for SOX2 and OCT3/4. As expected, the cardiac mesodermal cells no longer show SOX2 expression and show significantly lower OCT3/4 expression compared to hESCs on day 0. Although OCT3/4 is a marker for pluripotency, OCT4 also regulates the switch from SOX2 to SOX17 expression which directs the cell towards a cardiac fate (Zeineddine et al. 2006; Stefanovic et al. 2009). Therefore, OCT4 is present in the cells for longer than SOX2 in this differentiation process which could explain the residual expression observed in the cardiac mesodermal cells.
Cell re-organization during differentiation to cardiac mesodermal cells
We performed an automated differentiation experiment in which we differentiated hESCs into early cardiac mesoderm. Differentiation medium containing Activin A, BMP-4, and CHIR (ABC medium) replaced the E8 medium in the chambers on day 0. Medium exchange intervals were set to 1 h, 2 h, 3 h and 5 h. Prior to the start of differentiation, the same medium exchange interval (2 h) had been used in all chambers after seeding. Each chamber contained between about 100 and 150 cells on day 0. Table 1 shows the average volume of medium per cell per hour assuming approximately 100 cells per chamber. On average, the cells in the 1 h and 2 h chambers come into contact with roughly the same amount of medium as the cells in the well plate in the three days of differentiation.
Figure 4 shows brightfield images of two of the chambers for each condition on each day. On day 1, a clear difference is seen between the 1 h interval condition and the other three conditions. In the 1 h chambers, the cells are no longer spread evenly throughout the chambers, but instead have condensed in groups to form aggregates. This aggregate formation is also observed in well plates (Supplementary Fig. 2a). In comparison, the cells treated with 2 h and 3 h exchange intervals formed less compact groups with less defined edges. The Supplementary Video shows a time lapse of the cells compacting in the 2 h chambers. In contrast to the 1 h chambers, the cells in these chambers are still individually distinguishable. For the 5 h chambers a continuation of this trend can be observed, as there is only some grouping of cells and several cells remain as single cells. On day 2, the cell aggregates in the 1 h chambers have dispersed and spread throughout the chamber. These cells have a different morphology than the hESCs on day 0. Whereas the hESCs on day 0 are elongated, these day 2 cells are round and circular. Interestingly, the 2 h chambers on day 2 did not form aggregates even though by this time the same amount of ABC medium has flowed through the cells as was flowed through the 1 h chambers by day 1. A possible explanation for this could be the cell proliferation from day 1 to day 2 resulting in less available differentiation factors per cell. In general, the cells in the 2 h, 3 h and 5 h chambers on day 2 show a monolayer with cobblestone-like morphology and only occasional small aggregates at chamber sides (as seen in Fig. 4 in the 3 h chambers). All conditions show high cell proliferation. On day 3 the chambers in all conditions are filled with cells which have dispersed to cover most of the chamber floor. In the 1 h chambers wider gaps between cells are visible, indicating some cell death and a decrease in proliferation. The cell morphology and distribution in the 2 h chambers are comparable to those in the 1 h chambers on day 2. The cells in both the 3 h and 5 h chambers also show high proliferation, resulting in (nearly) confluent chambers. Here the cell morphology has also changed to resemble that of the cells in the 2 h chambers despite the lower total amounts of ABC medium which have passed through the chambers.
In an earlier experiment, differentiation was started when there were about 300–400 cells per chamber. In that case the cells in the 1 h chambers also formed condensed aggregates on day 1. However, these were still present on day 2 and the cells only started to disperse between days 2 and 3. For all medium exchange intervals, the cells greatly overpopulated the chambers by day 3. These images are shown in Supplementary Fig. 3. These results show that a lower seeding density is essential for obtaining a cell monolayer on day 3 when using these growth factor concentrations (20 ng/mL Activin A, 20 ng/mL BMP-4, and 1.5 µM CHIR).
For the experiments in this section, live-cell imaging was the method of choice to be able to show the same cells in the chambers over the course of differentiation. Future studies, for example for more detailed cell morphological changes, would also benefit from immunostaining at different time points.
MESP1 expression on day 3
Differentiation into cardiac mesodermal cells was confirmed by fluorescence imaging of the MESP1mCherry-hESC reporter line (Den Hartogh et al. 2015). Previously, we have shown that MESP1 is transiently activated, peaking at day 3, during cardiac differentiation of hESCs (Den Hartogh et al. 2015). Here we confirm that the cells cultured in the chip also express MESP1mCherry on day 3 by performing flow cytometry and comparing the results to negative controls (hESCs) and positive controls (day 3 cardiac mesodermal cells cultured in a well plate). In order to obtain a sufficient amount of cells, the cells from all chambers were extracted and pooled. The flow cytometry results (Supplementary Fig. 4) show that the percentage of MESP1-expressing cells in the chambers was about twice as high as in the well plates (Table 2). However, since the chip was designed to test several different conditions in parallel, we decided to use fluorescence microscopy for further experiments. Although the method is not suited to determining absolute percentages of MESP1-expressing cells within a population, it is well-suited to making relative comparisons between several different conditions. Moreover, it has the advantage that each chamber can be compared.
MCherry fluorescence was observed using microscopy on day 3 in all of the chambers containing cells, regardless of whether these had 1 h, 2 h, 3 h, or 5 h medium exchange intervals. Figure 5a shows an example image of a chamber from each condition. The fluorescence and brightfield overlay images show that several, but not all cells in each chamber express MESP1. Similar results are also observed in the original well plate protocol (see Supplementary Fig. 2b for comparison).
MESP1mCherry fluorescence is often particularly bright in the 1 h and 2 h chambers. Figure 5b shows an array of images, whereby each image was taken in a different chamber and each row corresponds to a different medium exchange interval. The cells with the highest fluorescence intensity are shown colored, whereby blue and magenta correspond to a pixel value ≥ 100 and ≥ 140, respectively. The thresholds were chosen at about 1.25 and 1.75 times the background noise (values around 80) which overlaps with the fluorescence signal from mCherry in several images. The number of cells per image with intensities above these thresholds were counted and are shown in boxplots in Fig. 5c. The blue boxplots (≥ 100 threshold) show a wide spread for all conditions. The interquartile range for the 1 h and 2 h boxes encompass approximately the same values. For the 3 h and 5 h intervals a clear trend toward decreasing cell numbers is observed. A Kruskal–Wallis Test was performed on the 100 threshold and showed a significant difference between the mean ranks (p = 0.001) of at least one pair of the datasets. Dunn’s pairwise tests revealed a significant difference between the 1 h and 5 h intervals (p = 0.003) as well as between the 2 h and 5 h intervals (p = 0.002), after the Bonferroni correction for multiple comparisons. Due to the low number of cells above the 140 threshold, the test was not performed on this threshold.
The decrease in fluorescence intensity for longer medium exchange intervals (5 h), may be due to a delayed peak MESP1 expression compared to the 1 h and 2 h chambers. In another experiment MESP1mCherry fluorescence was imaged on day 2 of differentiation for 1 h and 2 h medium exchange intervals. Both conditions showed MESP1 expression, demonstrating that at least for these two conditions the cells already enter the cardiac mesoderm stage on day 2. To confirm whether the fluorescence intensity difference is due to differentiation with a higher efficiency or to a time-shifted differentiation, further experiments with live-cell imaging of MESP1mCherry have to be performed.