Entitled “Engineering 3D micro-compartments for highly efficient and scale-independent expansion of human pluripotent stem cells in bioreactors” this paper highlights: Biomimetic features of C-Stem™-generated capsules, allowing for in vivo-like hiPSC self-organization into lumenized rosettes / Unprecedented exponential hiPSC growth in 10L bioreactors: >275-fold within 6.5 days, with high reproducibility / Maintenance of cell quality (pluripotency & genonomic integrity) throughout massive expansion

The lumenized rosette conformation is critical to the behavior of hPSCs in vivo. Replicating this architecture seems a logical approach to achieve in vivo-like exponential growth while preserving cell quality. However, current protocols for hPSC culture in lumenized rosette conformation were not designed for scale-up. The C-Stem™ platform was invented to fill this gap.

Over the past decade, developmental biology entered a golden era, fueled by novel biomimetic approaches to culture human pluripotent stem cells (hPSCs). In vitro recapitulation of the lumenized rosette conformation was described by the group of Magdalena Zernicka-Goetz in 2014 using mice PSCs cultured in micro-wells coated with Matrigel®. This approach was then successfully applied to human ES and iPS cells in 2016 by the same team.

The lumenized rosette architecture emerges during the transition from naïve to primed pluripotency. In response to extracellular matrix cues, naïve PSCs start to polarize. As transcriptional factors trigger primed pluripotency, hPSC form de novo intracellular actin-rich compartments called apicosomes, which accumulate Ca2+ and express apical proteins. As apicosomes regroup, concomitant mechanisms promoting water influx towards the center of the epiblast participate in the establishment of a fluid-filled lumen. The resulting rosette architecture displays apical-basal polarity: all apical domains are facing the lumen, while basal domains are facing the external environment of the epiblast.

While it has been reported that hPSCs cultured in 2D spontaneously form lumens, such rosette-like structures were found to rapidly collapse (in ~5 days or less). hPSCs end up establishing an inverted topology in 2D. As opposed to the rosette architecture, in Petri dishes the apical domain is facing the cell culture media, while the basal pole is established at the bottom of the dish, with limited access to external cues and nutrients. As a result, in 2D culture, signalling molecules secreted at the apical domain are diluted in a large volume of media, which is rinsed at every media change. Developmental biologist Marta Shahbazi estimated a 2,500-fold difference in the concentration of autocrine factors in the lumen of hPSC colonies in rosette conformation versus cell culture medium in standard 2D culture.

Pluripotency is a transient cell property in vivo. At the end of the first week of development, naïve hPSCs transition to primed pluripotency. Within the following 5 to 7 days, the hPSC colony, benefiting from a very fast 15h to 16h cell cycle, grows exponentially to reach over 1,000 cells.