Margarida Serra

Process engineering of human pluripotent stem cells for clinical application

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells, constitute an extremely attractive tool for cell therapy. However, flexible platforms for the large-scale production and storage of hPSCs in tightly controlled conditions are necessary to deliver high-quality cells in relevant quantities to satisfy clinical demands. Here we discuss the main principles for the bioprocessing of hPSCs, highlighting the impact of environmental factors, novel 3D culturing approaches and integrated bioreactor strategies for controlling hPSC culture outcome. Knowledge on hPSC bioprocessing accumulated during recent years provides important insights for the establishment of more robust production platforms and should potentiate the implementation of novel hPSC-based therapies.

Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells.

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.

Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control

The successful transfer of human embryonic stem cell (hESC) technology and cellular products into clinical and industrial applications needs to address issues of automation, standardization and the generation of relevant cell numbers of high quality. In this study, we combined microcarrier technology and controlled stirred tank bioreactors, to develop an efficient and scalable system for expansion of pluripotent hESCs. We demonstrate the importance of controlling pO(2) at 30% air saturation to improve hESCs growth. This concentration allowed for a higher energetic cell metabolism, increased growth rate and maximum cell concentration in contrast to 5% pO(2) where a shift to anaerobic metabolism was observed, decreasing cell expansion 3-fold. Importantly, the incorporation of an automated perfusion system in the bioreactor enhanced culture performance and allowed the continuous addition of small molecules assuring higher cell concentrations for a longer time period. The expanded hESCs retained their undifferentiated phenotype and pluripotency. Our results show, for the first time, that the use of controlled bioreactors is critical to ensure the production of high quality hESCs. When compared to the standard colony culture, our strategy improves the final yield of hESCs by 12-fold, providing a potential bioprocess to be transferred to clinical and industrial applications.

Human liver cell spheroids in extended perfusion bioreactor culture for repeated‐dose drug testing

Primary cultures of human hepatocyte spheroids are a promising in vitro model for long‐term studies of hepatic metabolism and cytotoxicity. The lack of robust methodologies to culture cell spheroids, as well as a poor characterization of human hepatocyte spheroid architecture and liver‐specific functionality, have hampered a widespread adoption of this three‐dimensional culture format. In this work, an automated perfusion bioreactor was used to obtain and maintain human hepatocyte spheroids. These spheroids were cultured for 3‐4 weeks in serum‐free conditions, sustaining their phase I enzyme expression and permitting repeated induction during long culture times; rate of albumin and urea synthesis, as well as phase I and II drug‐metabolizing enzyme gene expression and activity of spheroid hepatocyte cultures, presented reproducible profiles, despite basal interdonor variability (n = 3 donors). Immunofluorescence microscopy of human hepatocyte spheroids after 3‐4 weeks of long‐term culture confirmed the presence of the liver‐specific markers, hepatocyte nuclear factor 4α, albumin, cytokeratin 18, and cytochrome P450 3A. Moreover, immunostaining of the atypical protein kinase C apical marker, as well as the excretion of a fluorescent dye, evidenced that these spheroids spontaneously assemble a functional bile canaliculi network, extending from the surface to the interior of the spheroids, after 3‐4 weeks of culture. Conclusion: Perfusion bioreactor cultures of primary human hepatocyte spheroids maintain a liver‐specific activity and architecture and are thus suitable for drug testing in a long‐term, repeated‐dose format. (HEPATOLOGY 2012)

A multi-organ chip co-culture of neurospheres and liver equivalents for long-term substance testing

Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, often do not accurately predict drug toxicity, leading to high attrition rates in clinical studies (Paul et al., 2010). The phylogenetic distance between humans and laboratory animals is enormous, this affects the transferability of animal data on the efficacy of neuroprotective drugs. Therefore, many neuroprotective treatments that have shown promise in animals have not been successful when transferred to humans (Dragunow, 2008; Gibbons and Dragunow, 2010). We present a multi-organ chip capable of maintaining 3D tissues derived from various cell sources in a combined media circuit which bridges the gap in systemic and human tests. A steady state co-culture of human artificial liver microtissues and human neurospheres exposed to fluid flow over two weeks in the multi-organ chip has successfully proven its long-term performance. Daily lactate dehydrogenase activity measurements of the medium and immunofluorescence end-point staining proved the viability of the tissues and the maintenance of differentiated cellular phenotypes. Moreover, the lactate production and glucose consumption values of the tissues cultured indicated that a stable steady-state was achieved after 6 days of co-cultivation. The neurospheres remained differentiated neurons over the two-week cultivation in the multi-organ chip, proven by qPCR and immunofluorescence of the neuronal markers βIII-tubulin and microtubule-associated protein-2. Additionally, a two-week toxicity assay with a repeated substance exposure to the neurotoxic 2,5-hexanedione in two different concentrations induced high apoptosis within the neurospheres and liver microtissues, as shown by a strong increase of lactate dehydrogenase activity in the medium. The principal finding of the exposure of the co-culture to 2,5-hexanedione was that not only toxicity profiles of two different doses could be discriminated, but also that the co-cultures were more sensitive to the substance compared to respective single-tissue cultures in the multi-organ-chip. Thus, we provide here a new in vitro tool which might be utilized to predict the safety and efficacy of substances in clinical studies more accurately in the future.

Stirred bioreactors for the expansion of adult pancreatic stem cells

Adult pluripotent stem cells are a cellular resource representing unprecedented potential for cell therapy and tissue engineering. Complementary to this promise, there is a need for efficient bioprocesses for their large scale expansion and/or differentiation. With this goal in mind, our work focused on the development of three-dimensional (3-D) culture systems for controlled expansion of adult pancreatic stem cells (PSCs). For this purpose, two different culturing strategies were evaluated, using spinner vessels: cell aggregated cultures versus microcarrier technology. The use of microcarrier supports (Cytodex 1 and Cytodex 3) rendered expanded cell populations which retained their self-renewal ability, cell marker, and the potential to differentiate into adipocytes. This strategy surmounted the drawbacks of aggregates in culture which were demonstrably unfeasible as cells clumped together did not proliferate and lost PSC marker expression. Furthermore, the results obtained showed that although both microcarriers tested here were suitable for sustaining cell expansion, Cytodex 3 provided a better substrate for the promotion of cell adherence and growth. For the latter approach, the potential of bioreactor technology was combined with the efficient Cytodex 3 strategy under controlled environmental conditions (pH-7.2, pO2-30% and temperature-37 degrees C); cell growth was more efficient, as shown by faster doubling time, higher growth rate and higher fold increase in cell concentration, when compared to spinner cultures. This study describes a robust bioprocess for the controlled expansion of adult PSC, representing an efficient starting point for the development of novel technologies for cell therapy.

Novel culture strategy for human stem cell proliferation and neuronal differentiation.

Embryonal carcinoma (EC) stem cells derived from germ cell tumors closely resemble embryonic stem (ES) cells and are valuable tools for the study of embryogenesis. Human pluripotent NT2 cell line, derived from a teratocarcinoma, can be induced to differentiate into neurons (NT2‐N) after retinoic acid treatment. To realize the full potential of stem cells, developing in vitro methods for stem cell proliferation and differentiation is a key challenge. Herein, a novel culture strategy for NT2 neuronal differentiation was developed to expand NT2‐N neurons, reduce the time required for the differentiation process, and increase the final yields of NT2‐N neurons. NT2 cells were cultured as 3D cell aggregates (“neurospheres”) in the presence of retinoic acid, using small‐scale stirred bioreactors; it was possible to obtain a homogeneous neurosphere population, which can be transferred for further neuronal selection onto coated surfaces. This culturing strategy yields higher amounts of NT2‐N neurons with increased purity compared with the amounts routinely obtained with static cultures. Moreover, mechanical and enzymatic methods for neurosphere dissociation were evaluated for their ability to recover neurons, trypsin digestion yielding the best results. Nevertheless, the highest recoveries were obtained when neurospheres were collected directly to treated surfaces without dissociation steps. This novel culture strategy allows drastic improvement in the neuronal differentiation efficiency of NT2 cells, insofar as a fourfold increase was obtained, reducing simultaneously the time needed for the differentiation process. The culture method described herein ensures efficient, reproducible, and scaleable ES cell proliferation and differentiation, contributing to the usefulness of stem cell bioengineering.

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization

Human embryonic stem cells (hESCs) have an enormous potential as a source for cell replacement therapies, tissue engineering, and in vitro toxicology applications. The lack of standardized and robust bioprocesses for hESC expansion has hindered the application of hESCs and their derivatives in clinical settings. We developed a robust and well‐characterized bioprocess for hESC expansion under fully defined conditions and explored the potential of transcriptomic and metabolomic tools for a more comprehensive assessment of culture system impact on cell proliferation, metabolism, and phenotype. Two different hESC lines (feeder‐dependent and feeder‐free lines) were efficiently expanded on xeno‐free microcarriers in stirred culture systems. Both hESC lines maintained the expression of stemness markers such as Oct‐4, Nanog, SSEA‐4, and TRA1‐60 and the ability to spontaneously differentiate into the three germ layers. Whole‐genome transcriptome profiling revealed a phenotypic convergence between both hESC lines along the expansion process in stirred‐tank bioreactor cultures, providing strong evidence of the robustness of the cultivation process to homogenize cellular phenotype. Under low‐oxygen tension, results showed metabolic rearrangement with upregulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg‐effect‐like phenotype, with no evidence of hypoxic stress response, in contrast to two‐dimensional culture. Overall, we report a standardized expansion bioprocess that can guarantee maximal product quality. Furthermore, the “omics” tools used provided relevant findings on the physiological and metabolic changes during hESC expansion in environmentally controlled stirred‐tank bioreactors, which can contribute to improved scale‐up production systems.

Production of oncolytic adenovirus and human mesenchymal stem cells in a single-use, Vertical-Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier-based cell culture processes.

Anchorage‐dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large‐scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage‐dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single‐use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical‐Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage‐dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow‐derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical‐Wheel bioreactors (PBS‐VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS‐VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS‐VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA‐DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS‐VW, and scale‐up was successfully carried out for two different microcarrier‐based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical‐Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier‐based cell cultures of complex biopharmaceuticals.

Modeling human neural functionality in vitro: three-dimensional culture for dopaminergic differentiation.

Advances in mechanistic knowledge of human neurological disorders have been hindered by the lack of adequate human in vitro models. Three-dimensional (3D) cellular models displaying higher biological relevance are gaining momentum; however, their lack of robustness and scarcity of analytical tools adapted to three dimensions hampers their widespread implementation. Herein we show that human midbrain-derived neural progenitor cells, cultured as 3D neurospheres in stirred culture systems, reproducibly differentiate into complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Moreover, an extensive toolbox of analytical methodologies has been adapted to 3D neural cell models, allowing molecular and phenotypic profiling and interrogation. The generated neurons underwent synaptogenesis and elicit spontaneous Ca(2+) transients. Synaptic vesicle trafficking and release of dopamine in response to depolarizing stimuli was also observed. Under whole-cell current-and-voltage clamp, recordings showed polarized neurons (Vm=-70 mV) and voltage-dependent potassium currents, which included A-type-like currents. Glutamate-induced currents sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate antagonists revealed the existence of functional glutamate receptors. Molecular and phenotypic profiling showed recapitulation of midbrain patterning events, and remodeling toward increased similarity to human brain features, such as extracellular matrix composition and metabolic signature. We have developed a robust and reproducible human 3D neural cell model, which may be extended to patient-derived induced pluripotent stem cells, broadening the applicability of this model.