Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny

Abstract

Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types.

Chemically controlled aggregation of pluripotent stem cells

“…PSC, pluripotent stem cell [Color figure can be viewed at wileyonlinelibrary.com] mitigate the risk that variability in input parameters will cause unexpected outcomes. Poor repeatability in PSC aggregation is a widespread concern in the field (Hookway, Butts, Lee, Tang, & McDevitt, 2016). To date, the factors that affect human PSC aggregate formation properties are not well understood; however, cell health, confluence, dissociation timing, and other complex factors likely all play a role in this critical quality attribute.…”

Single cell determination of cardiac microtissue structure and function using light sheet microscopy

“…Although CMs and CFs were mixed at a 3:1 ratio and seeded at a total of 2000 cells per tissue, an average of only ~500 cells were identified in the heterotypic microtissues, but the ratio of CMs to CFs was retained at an average of 400 CMs to 127 CFs (~3:1 CM:CF) ( Supplementary Figure 2). The lower-thanexpected cell numbers were likely due to lack of total incorporation of all 2000 cells during the initial tissue formation step-which is to be expected 9, 26,37 . However, the maintained ratio of the heterogeneous cardiac cell populations allowed for the interrogation of intra-tissue spatial interactions.…”

“…Lactate-purified hiPSC-CMs and primary human CFs were dissociated with 0.25% Trypsin for 10min to obtain a single-cell suspension, mixed at a 3:1 CM:CF ratio, and seeded into an array of inverted 400µm pyramidal microwells at a density of ~2000 cells per microwell 9,26 . Cells self-assembled into 3D tissues over the course of 24h and were then transferred from the microwells to rotary orbital suspension culture at a density of ~4000 microtissues per 10cm Petri dish (~8x10 5 cells/mL) and maintained in RPMI/B27+ medium 26 Cell classification and spatial quantification. Imaris image analysis software (version 9.3.1) was used to identify labeled cell nuclei within the microtissues.…”

Bioengineering Considerations for a Nurturing Way to Enhance Scalable Expansion of Human Pluripotent Stem Cells

“…Using the aforementioned knowledge, bioengineers could identify the most important properties that should be incorporated into the ideal design of bioreactor systems to maintain the aggregate formation and growth stability of hPSCs ( Figure 3). These culture methods involve traditional 2D monolayer culture (adherent or monolayers on solid or semi‐solid substrates) and typical 3D suspension culture (cell aggregates in static suspension culture or cell spheroids in dynamic suspension cultures with liquid fluids) . Application of dynamic suspension culture platforms to support hPSC growth in matrix‐free cell aggregates is more compatible with processes with a reduced number of required processing steps.…”