Biology subjects

Laflamme, M. A.

Publications and source records attributed to Laflamme, M. A..

4 recordsLinked to original sources

bioRxiv2026

Scaling-Up Vertical-Wheel Bioreactors Based on Cell Aggregate Exposure to Shear Stress and Energy Dissipation Rate

PurposeLarge quantities of human pluripotent stem cells (hPSCs) are required for clinical applications. 3D suspension cultures are suitable for large scale manufacturing of hPSCs but yield, viability and quality are affected by the hydrodynamic environment. This paper characterizes the hydrodynamic environment inside vertical wheel bioreactors (VWBRs) as a function of size and agitation rates, measures its effect on cell aggregation and proliferation, and proposes the use of Lagrangian-based shear stress and energy dissipation rate (EDR) exposures to support scale-up. MethodsIn silico: Transient, 3D, turbulent flow simulations are conducted for two VWBR sizes (100, 500 mL) at five agitation rates between 20 and 80 rpm. Trajectories of cell aggregates of sizes from 200 to 1,000 microns are calculated, and shear stress and EDR exposures are collected along these trajectories. In vitro: ESI-017 hPSCs were cultured in VWBRs for 6 days. Aggregation efficiency and daily fold ratios were calculated based on cell counts and initial inoculation density. ResultsAggregate size, agitation rate and bioreactor size modulate cell aggregate exposures to EDR and shear stress, which significantly depart from maximum or volume average metrics used for scale-up. Combined in vitro/in silico results show EDR affects aggregation efficiency, cell counts and aggregate size, and has a small effect on daily fold ratios but a significant effect on total fold ratio. ConclusionHistory of trajectory-based cell aggregate exposures to EDRs provide a better scale-up basis for VWBRs than volume-averaged EDR. Shear stress does not significantly affect hPSC aggregation, proliferation and expansion in VWBRs under the tested conditions.

bioengineering↗

bioRxiv2025

Growth Factor-Based Manufacturing of Human Pluripotent Stem Cell-Derived Cardiomyocytes Using the Vertical Wheel Bioreactor System

BackgroundMultiple protocols have been reported for the large-scale generation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in bioreactors using small molecules; however, no comparable bioreactor-based methods have been established using growth factors. This is despite evidence that differentiation with optimized concentrations of BMP4, Activin A, and bFGF offers finer control of cardiomyocyte phenotype. Here, we develop scalable hPSC expansion and growth factor-based cardiac differentiation protocols using the vertical wheel bioreactor (VWBR) system. Methods and ResultsThe expansion of undifferentiated hPSCs was first optimized in 100 mL VWBRs by varying parameters, including starting cell seeding density, agitation rate, and media exchange schedule. Proliferation, viability, aggregate diameter, media metabolites, and pluripotency were assessed during hPSC expansion. Next, we evaluated the effects of undifferentiated hPSC culture conditions on subsequent cardiomyocyte differentiation potential. We found that hPSCs expanded in static culture or in VWBRs at different densities and agitation rates all differentiated into hPSC-CM populations of similar cardiac purity; however, cardiomyocyte yields were initially lower when VWBR-expanded hPSCs were used. We compared the differentiation kinetics of hPSCs expanded in VWBRs to conventional 2D culture and found that the former had accelerated mesodermal commitment and significantly greater cKit+/CXCR4+/PDGRF- cell formation during differentiation. Modifying our aggregation and mesoderm induction steps improved cell yields and enabled reliable production of >1x106 cells/mL cardiac troponin T+ (cTnT) hPSC-CMs. Highlighting the versatility of our growth factor-based system, variation in the BMP4:Activin A ratio enabled a second heart field-like differentiation and generation of atrial-like cardiomyocytes in VWBRs. We further show that our expansion and differentiation protocols are reproducible and economical in 500 mL VWBRs, yielding on average 1.11x106 hPSC-CMs/mL at a mean purity of 93% cTnT+. Characterization of VWBR produced hPSC-CM force generation, action potentials, and intercellular calcium transients confirmed the expected phenotype of ventricular-like cells. Lastly, VWBR produced hPSC-CMs robustly engrafted in the infarcted guinea pig myocardium, supporting use as a cell therapy product. ConclusionsThis novel bioreactor-based protocol will enable cardiac cell therapy and tissue engineering applications by providing scalable and consistent production of hPSC-derived cardiac cell products.

cell biology↗

bioRxiv2025

Human pluripotent stem cell-derived atrioventricular node-like pacemaker cells exhibit biological conduction bridge properties in vitro and in vivo

The atrioventricular node (AVN) ensures synchronized heart contractions by establishing the electrical connection between the atria and ventricles. Dysfunction of the pacemaker cells of the AVN leads to atrioventricular block (AV block), a life-threatening condition managed with electronic pacemakers (EPMs). EPMs have drawbacks that could be overcome by a human pluripotent stem cell (hPSC)-derived biological conduction bridge (BioCB). Recent studies demonstrated the differentiation of AVN-like cells from hPSCs, but their conduction properties upon engraftment in vivo remain unexplored. Here we report the generation of AVN-like pacemaker cells (AVNLPCs) from hPSCs using WNT and BMP signaling modulation. These AVNLPCs transcriptionally resemble fetal AVN pacemaker cells, exhibit pacemaker action potentials, and display unique AVN-like conduction properties. Notably, when transplanted into the guinea pig heart, AVNLPCs replicate the functional properties of the AVN. Our study highlights the potential of an AVNLPC-based BioCB as novel cell therapy to improve treatment for AV block patients.

developmental biology↗

bioRxiv2018

NanoMEA: a versatile platform for high-throughput analysis of structure-function relationships in human stem cell-derived excitable cells and tissues

Somatic cells derived from human pluripotent stem cell (hPSC) sources hold significant potential as a means to improve current in vitro screening assays. However, their inconsistent ability to recapitulate the structural and functional characteristics of native cells has raised questions regarding their ability to accurately predict the functional behavior of human tissues when exposed to chemical or pathological insults. In addition, the lack of cytoskeletal organization within conventional culture platforms prevents analysis of how structural changes in human tissues affect functional performance. Using cation-permeable hydrogels, we describe the production of multiwell nanotopographically-patterned microelectrode arrays (nanoMEAs) for studying the effect of structural organization on hPSC-derived cardiomyocyte and neuronal function in vitro. We demonstrate that nanoscale topographic substrate cues promote the development of more ordered cardiac and neuronal monolayers while simultaneously enhancing cytoskeletal organization, protein expression patterns, and electrophysiological function in these cells. We then show that these phenotypic improvements act to alter the sensitivity of hPSC-derived cardiomyocytes to treatment with arrhythmogenic and conduction-blocking compounds that target structural features of the cardiomyocyte. Similarly, we demonstrate that neuron sensitivity to synaptic blockers is increased when cells are maintained on nanotopographically-patterned Nafion surfaces. The improved structural and functional capacity of hPSC-derived cardiomyocyte and neuronal populations maintained on nanoMEAs may have important implications for improving the predictive capabilities of cell-based electrophysiological assays used in preclinical screening applications.

bioengineering↗