Dissolvable Microcarriers Allow Scalable Expansion And Harvesting Of Human Induced Pluripotent Stem Cells Under Xeno-Free Conditions.

The development of bioprocesses capable of producing large numbers of human induced pluripotent stem cells (hiPSC) in a robust and safe manner is critical for the application of these cells in biotechnological and medical applications. Scalable expansion of hiPSC is often performed using polystyrene microcarriers, which have to be removed from the cell suspension using a separation step that causes loss of viable cells. In this study, application of novel xeno-free dissolvable microcarriers (DM) for an efficient and integrated expansion and harvesting of hiPSC is demonstrated. After an initial screening under static conditions, hiPSC culture using DM is performed in dynamic culture, using spinner-flasks. A maximum 4.0 ± 0.8-fold expansion is achieved after 5 days of culture. These results are validated with a second cell line and the culture is successfully adapted to fully xeno-free conditions. Afterwards, cell recovery is made within the spinner flask, being obtained a 92 ± 4% harvesting yield, which is significantly higher than the one obtained for the conventional filtration-based method (45 ± 3%). Importantly, the expanded and harvested hiPSC maintain their pluripotency and multilineage differentiation potential. The results here described represent a significant improvement of the downstream processing after microcarrier-based hiPSC expansion, leading to a more cost-effective and efficient bioprocess.

Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems.

Human pluripotent stem cells (hPSC) have attracted a great attention as an unlimited source of cells for cell therapies and other in vitro biomedical applications such as drug screening, toxicology assays and disease modeling. The implementation of scalable culture platforms for the large-scale production of hPSC and their derivatives is mandatory to fulfill the requirement of obtaining large numbers of cells for these applications. Microcarrier technology has been emerging as an effective approach for the large scale ex vivo hPSC expansion and differentiation. This review presents recent achievements in hPSC microcarrier-based culture systems and discusses the crucial aspects that influence the performance of these culture platforms. Recent progress includes addressing chemically-defined culture conditions for manufacturing of hPSC and their derivatives, with the development of xeno-free media and microcarrier coatings to meet good manufacturing practice (GMP) quality requirements. Finally, examples of integrated platforms including hPSC expansion and directed differentiation to specific lineages are also presented in this review.

Correction: Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems.

[This corrects the article DOI: 10.1371/journal.pone.0151264.].

Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems.

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.

Scalable expansion of human-induced pluripotent stem cells in xeno-free microcarriers.

The expansion of human-induced pluripotent stem cells (hiPSCs) is commonly performed using feeder layers of mouse embryonic fibroblasts or in feeder-free conditions in two-dimensional culture platforms, which are associated with low production yields and lack of process control. Robust large-scale production of these cells under defined conditions has been one of the major challenges to fulfil the large cell number requirement for drug screening applications, toxicology assays, disease modeling and potential cellular therapies. Microcarrier-based systems, in particular, are a promising culture format since they provide a high surface-to-volume ratio and allow the scale-up of the process to stirred suspension bioreactors. In this context, this chapter describes a detailed methodology for the scalable expansion of hiPSCs in spinner flasks and using xeno-free microcarriers to allow further translation to Good Manufacturing Practice (GMP) conditions.

Kinetics and mechanism of the cutinase-catalyzed transesterification of oils in AOT reversed micellar system.

The kinetics of the enzymatic transesterification between a mixture of triglycerides (oils) and methanol for biodiesel production in a bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane reversed micellar system, using recombinant cutinase from Fusarium solani pisi as a catalyst, was investigated. In order to describe the results that were obtained, a mechanistic scheme was proposed, based on the literature and on the experimental data. This scheme includes the following reaction steps: the formation of the active enzyme-substrate complex, the addition of an alcohol molecule to the complex followed by the separation of a molecule of the fatty acid alkyl ester and a glycerol moiety, and release of the active enzyme. Enzyme inhibition and deactivation effects due to methanol and glycerol were incorporated in the model. This kinetic model was fitted to the concentration profiles of the fatty acid methyl esters (the components of biodiesel), tri-, di- and monoglycerides, obtained for a 24 h transesterification reaction performed in a stirred batch reactor under different reaction conditions of enzyme and initial substrates concentration.

Performance of a cutinase membrane reactor for the production of biodiesel in organic media.

The enzymatic transesterification of oils with an alcohol, using recombinant cutinase of Fusarium solani pisi microencapsulated in sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane reversed micelles, was performed in a membrane bioreactor (MBR). A tubular ceramic membrane with a nominal molecular weight cut off of 15,000 Da was used to retain the enzyme, and characterized in terms of rejection coefficients of the reaction components by transmission experiments. The performance of the MBR in a total recirculation-batch mode was compared with results obtained in a stirred batch tank reactor. The continuous operation of the MBR was also evaluated and the influence of the alcohol type and permeate flow rate on conversion degree and productivity (up to 500 g(product) /day/g(enzyme) was attained) were analyzed. Cutinase wild type and mutant T179C were tested for this process and the high long-term operational stability of the cutinase mutant demonstrated its potential as biocatalyst for the enzymatic continuous production of biodiesel.

Transesterification of oil mixtures catalyzed by microencapsulated cutinase in reversed micelles.

Recombinant cutinase from Fusarium solani pisi was used to catalyze the transesterification reaction between a mixture of triglycerides (oils) and methanol in reversed micelles of bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane for the purposes of producing biodiesel. The use of a bi-phase lipase-catalyzed system brings advantages in terms of catalyst re-use and the control of water activity in the medium and around the enzyme micro-environment. Small-scale batch studies were performed to study the influence of the initial enzyme and alcohol concentrations, and the substrates molar ratio. Conversions in excess of 75 were obtained with reaction times under 24 h, which makes this enzymatic process highly competitive when compared to similar lipase catalyzed reactions for biodiesel production using methanol.