Computational fluid dynamic characterization of vertical-wheel bioreactors used for effective scale-up of human induced pluripotent stem cell aggregate culture
24 Aug 2021-Canadian Journal of Chemical Engineering (John Wiley & Sons, Ltd)-Vol. 99, Iss: 11, pp 2536-2553
About: This article is published in Canadian Journal of Chemical Engineering.The article was published on 2021-08-24. It has received 13 citations till now. The article focuses on the topics: Induced pluripotent stem cell.
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TL;DR: This manuscript addresses challenges and presents potential solutions to alleviate the anticipated bottlenecks for commercial-scale manufacturing of high-quality therapeutic cells derived from PSCs.
Abstract: Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to manufacturing PSCs in single-use bioreactors, particularly at larger volumetric scales. This manuscript addresses these challenges and presents potential solutions to alleviate the anticipated bottlenecks for commercial-scale manufacturing of high-quality therapeutic cells derived from PSCs.
5 citations
TL;DR:
Abstract: The demand for large cell numbers for cellular therapies and drug screening applications requires the development of scalable platforms capable of generating high-quality populations of tissue-specific cells derived from human pluripotent stem cells (hPSCs). Here, we studied the ability of Gibco StemScale PSC Suspension Medium to promote the efficient expansion of hPSC cultures as aggregates grown in suspension. We tested human induced pluripotent stem cell (hiPSC) growth in 6-well plates (on orbital shaker platforms) and single-use vertical-wheel bioreactors for a total of three consecutive passages. Up to a 9-fold increase in cell number was observed over 5 days per passage, with a cumulative fold change up to 600 in 15 days. Additionally, we compared neural induction of hiPSCs by using a dual SMAD inhibition protocol with a commercially available neural induction medium, which can potentially yield more than a 30-fold change, including neural progenitor induction and expansion. This system can also be adapted toward the generation of floor plate progenitors, which yields up to an 80-fold change in cell number and generates FOXA2-positive populations. In summary, we developed platforms for hiPSC expansion and neural induction into different brain regions that provide scalability toward producing clinically relevant cell numbers.
1 citations
TL;DR: A novel gravimetric method to measure power was used, and the validity of this method was assessed by measuring power for a standard stirred tank bioreactor with a Rushton impeller and the results found to closely match those derived from traditional methods.
Abstract: Correspondence Sunghoon Jung, PBS Biotech Inc., 4721 Calle Carga, Camarillo, CA, 93012 USA. Email: sjung@pbsbiotech.com Abstract The purpose of this investigation was to develop an initial set of Power number versus Reynolds number results for a family of vertical-wheel bioreactors. These bioreactors are increasingly being used for the manufacture of cells for cell therapy but have not been characterized according to this approach. A novel gravimetric method to measure power was used, and the validity of this method was assessed by measuring power for a standard stirred tank bioreactor with a Rushton impeller. The results of the gravimetric method were found to closely match those derived from traditional methods. The validated method was then used to measure the power draw and develop an initial set of Power number versus Reynolds number results for a family of vertical-wheel bioreactors.
1 citations
1 citations
TL;DR: In this paper , the authors performed pancreatic progenitor cell differentiation with and without AKT/P70 inhibitor AT7867 and characterized the resulting cells at protein and transcript level in vitro and in vivo upon transplantation into diabetic mice.
Abstract: Generation of pure pancreatic progenitor cells (PPs) is critical for clinical translation of stem cell derived islets. Herein, we performed PP differentiation with and without AKT/P70 inhibitor AT7867 and characterized the resulting cells at protein and transcript level in vitro and in vivo upon transplantation into diabetic mice. AT7867 treatment increased the percentage of PDX1+NKX6.1+ (-AT7867: 50.9% [IQR 48.9%-53.8%]; +AT7867: 90.8% [IQR 88.9%-93.7%]; p=0.0021) and PDX1+GP2+ PP cells (-AT7867: 39.22% [IQR 36.7%-44.1%; +AT7867: 90.0% [IQR 88.2%-93.6%]; p=0.0021). Transcriptionally, AT7867 treatment significantly upregulated PDX1 (p=0.0001), NKX6.1 (p=0.0005) and GP2 (p=0.002) expression compared to controls, while off-target markers PODXL (p<0.0001) and TBX2 (p <0.0001) were significantly downregulated. Transplantation of AT7867 treated PPs resulted in faster hyperglycemia reversal in diabetic mice compared to controls (time and group: p<0.0001). Overall, our data shows that AT7867 enhances PP cell differentiation leading to accelerated diabetes reversal.
References
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TL;DR: The progress in applications of iPSC technology that are particularly relevant to drug discovery and regenerative medicine are discussed, and the remaining challenges and the emerging opportunities in the field are considered.
Abstract: Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, human iPSCs have been widely used for disease modelling, drug discovery and cell therapy development. This article discusses progress in applications of iPSC technology that are particularly relevant to drug discovery and regenerative medicine, including the powerful combination of human iPSC technology with recent developments in gene editing.
985 citations
TL;DR: It is demonstrated that heterogeneity in hESC colony and aggregate size produces subsets of appropriate conditions for differentiation into specific cell types, and suggested that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories.
Abstract: To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation, a common approach to mimic some of the spatial and temporal aspects of in vivo development, are affected by three factors: input hESC composition, input hESC colony size, and EB size. Using a microcontact printing approach, size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently, size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover, upon forming EBs from these MP-hESCs, we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MP-EBs generated from endoderm-biased (high Gata6/Pax6) input hESCs, higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely, neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size, typical in most differentiation strategies, produces subsets of appropriate conditions for differentiation into specific cell types. Moreover, our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories.
471 citations
TL;DR: The data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway, according to EB size.
Abstract: Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 μm in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 μm in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.
375 citations
TL;DR: It is shown that ‘shear sensitivity’ due to agitation and bursting bubbles is no longer considered a major problem and some recommendations for bioreactor configuration and operating strategy are given.
Abstract: This article mainly addresses the issues associated with the engineering of large-scale free suspension culture in agitated bioreactors >10,000 L because they have become the system of choice industrially. It is particularly concerned with problems that become increasingly important as the scale increases. However, very few papers have been written that are actually based on such large-scale studies and the few that do rarely address any of the issues quantitatively. Hence, it is necessary very often to extrapolate from small-scale work and this review tries to pull the two types of study together. It is shown that 'shear sensitivity' due to agitation and bursting bubbles is no longer considered a major problem. Homogeneity becomes increasingly important with respect to pH and nutrients at the largest scale and sub-surface feeding is recommended despite 'cleaning in place' concerns. There are still major questions with cell retention/recycle systems at these scales, either because of fouling, of capacity or of potential and different 'shear sensitivity' questions. Fed-batch operation gives rise to cell densities that have led to the use of oxygen and enriched air to meet oxygen demands. This strategy, in turn, gives rise to a CO(2) evolution rate that impacts on pH control, pCO(2) and osmolality. These interactions are difficult to resolve but if higher sparge and agitation intensities could be used to achieve the necessary oxygen transfer, the problem would largely disappear. Thus, the perception of 'shear sensitivity' is still impacting on the development of animal cell culture at the commercial scale. Microcarrier culture is also briefly addressed. Finally, some recommendations for bioreactor configuration and operating strategy are given.
372 citations
TL;DR: The main principles for the bioprocessing of hPSCs are discussed, highlighting the impact of environmental factors, novel 3D culturing approaches and integrated bioreactor strategies for controlling hPSC culture outcome.
275 citations