Carlos A. V. Rodrigues

Stem cell cultivation in bioreactors

Cell-based therapies have generated great interest in the scientific and medical communities, and stem cells in particular are very appealing for regenerative medicine, drug screening and other biomedical applications. These unspecialized cells have unlimited self-renewal capacity and the remarkable ability to produce mature cells with specialized functions, such as blood cells, nerve cells or cardiac muscle. However, the actual number of cells that can be obtained from available donors is very low. One possible solution for the generation of relevant numbers of cells for several applications is to scale-up the culture of these cells in vitro. This review describes recent developments in the cultivation of stem cells in bioreactors, particularly considerations regarding critical culture parameters, possible bioreactor configurations, and integration of novel technologies in the bioprocess development stage. We expect that this review will provide updated and detailed information focusing on the systematic production of stem cell products in compliance with regulatory guidelines, while using robust and cost-effective approaches.

Neural stem cell differentiation by electrical stimulation using a cross-linked PEDOT substrate: Expanding the use of biocompatible conjugated conductive polymers for neural tissue engineering.

BACKGROUND The use of conjugated polymers allows versatile interactions between cells and flexible processable materials, while providing a platform for electrical stimulation, which is particularly relevant when targeting differentiation of neural stem cells and further application for therapy or drug screening. METHODS Materials were tested for cytotoxicity following the ISO10993-5. PEDOT PSS was cross-linked. ReNcellVM neural stem cells (NSC) were seeded in laminin coated surfaces, cultured for 4 days in the presence of EGF (20 ng/mL), FGF-2 (20 ng/mL) and B27 (20 μg/mL) and differentiated over eight additional days in the absence of those factors under 100Hz pulsed DC electrical stimulation, 1V with 10 ms pulses. NSC and neuron elongation aspect ratio as well as neurite length were assessed using ImageJ. Cells were immune-stained for Tuj1 and GFAP. RESULTS F8T2, MEH-PPV, P3HT and cross-linked PEDOT PSS (x PEDOT PSS) were assessed as non-cytotoxic. L929 fibroblast population was 1.3 higher for x PEDOT PSS than for glass control, while F8T2 presents moderate proliferation. The population of neurons (Tuj1) was 1.6 times higher with longer neurites (73 vs 108 μm) for cells cultured under electrical stimulus, with cultured NSC. Such stimulus led also to longer neurons. CONCLUSIONS x PEDOT PSS was, for the first time, used to elongate human NSC through the application of pulsed current, impacting on their differentiation towards neurons and contributing to longer neurites. GENERAL SIGNIFICANCE The range of conductive conjugated polymers known as non-cytotoxic was expanded. x PEDOT PSS was introduced as a stable material, easily processed from solution, to interface with biological systems, in particular NSC, without the need of in-situ polymerization.

Hypoxia enhances proliferation of mouse embryonic stem cell‐derived neural stem cells

Neural stem (NS) cells can provide a source of material with potential applications for neural drug testing, developmental studies, or novel treatments for neurodegenerative diseases. Herein, the ex vivo expansion of a model system of mouse embryonic stem (mES) cell‐derived NS cells was characterized and optimized, cells being cultivated under adherent conditions. Culture was first optimized in terms of initial cell plating density and oxygen concentration, known to strongly influence brain‐derived NS cells. To this end, the growth of cells cultured under hypoxic (2%, 5%, and 10% O2) and normoxic (20% O2) conditions was compared. The results showed that 2–5% oxygen, without affecting multipotency, led to fold increase values in total cell number about twice higher than observed under 20% oxygen (20‐fold vs. 10‐fold, respectively) this effect being more pronounced when cells were plated at low density. With an optimal cell density of 104 cells/cm2, the maximum growth rates were 1.9 day−1 under hypoxia versus 1.7 day−1 under normoxia. Cell division kinetics analysis by flow cytometry based on PKH67 tracking showed that when cultured in hypoxia, cells are at least one divisional generation ahead compared to normoxia. In terms of cell cycle, a larger population in a quiescent G0 phase was observed in normoxic conditions. The optimization of NS cell culture performed here represents an important step toward the generation of a large number of neural cells from a reduced initial population, envisaging the potential application of these cells in multiple settings. Biotechnol. Bioeng. 2010;106: 260–270.

Microcarrier expansion of mouse embryonic stem cell‐derived neural stem cells in stirred bioreactors

Neural stem cells (NSCs) are self‐renewing multipotent cells, able to differentiate into the phenotypes present in the central nervous system. Applications of NSCs may include toxicology, fundamental research, or cell therapies. The culture of floating cell clusters, called “neurospheres,” is widely used for the propagation of NSC populations in vitro but shows several limitations, which may be circumvented by expansion under adherent conditions. In particular, the derivation of distinct populations of NSCs from embryonic stem cells capable of long‐term culture under adherent conditions without losing differentiation potential was recently described. However, the expansion of these cells in agitated bioreactors has not been addressed until now and was the aim of this study. Selected microcarriers were tested under dynamic conditions in spinner flasks. Superior performance was observed with polystyrene beads coated with a recombinant peptide containing the Arg–Gly–Asp (RGD) motif (Pronectin F). After optimization of the culture, a 35‐fold increase in cell number was achieved after 6 days. High cellular viability and multipotency were maintained throughout the culture. The study presented here may be the basis for the development of larger scale bioprocesses for expansion of these and other populations of adherent NSCs, either from mouse or human origin.

Nonviral gene delivery to neural stem cells with minicircles by microporation.

The main purpose of this work was to evaluate the transfection of novel DNA vectors, minicircles (mC), on embryonic stem cell-derived neural stem cells (NSC). We demonstrated that by combining microporation with mC, 75% of NSC expressing a transgene is achieved without compromising cell survival, morphology, and differentiation potential. When comparing mC with their plasmid DNA (pDNA) counterparts, both gave rise to similar transfection levels but cells harboring mC showed 10% higher cell viability, maintaining 90% of survival at least for 10 days. Long-term analysis showed that NSC harbor a higher number of mC copies and consequently exhibit higher transgene expression when compared to their pDNA counterpart. Taken together, our results offer the first insights on the use of mC as a novel and safe strategy to genetically engineer NSC envisaging their use as biopharmaceuticals in clinical settings for the treatment of neurodegenerative or neurological diseases.

Clinical‐scale purification of pluripotent stem cell derivatives for cell‐based therapies

Human pluripotent stem cells (hPSCs) have the potential to revolutionize cell‐replacement therapies because of their ability to self renew and differentiate into nearly every cell type in the body. However, safety concerns have delayed the clinical translation of this technology. One cause for this is the capacity that hPSCs have to generate tumors after transplantation. Because of the challenges associated with achieving complete differentiation into clinically relevant cell types, the development of safe and efficient strategies for purifying committed cells is essential for advancing hPSC‐based therapies. Several purification strategies have now succeeded in generating non‐tumorigenic and homogeneous cell‐populations. These techniques typically enrich for cells by either depleting early committed populations from teratoma‐initiating hPSCs or by positively selecting cells after differentiation. Here we review the working principles behind separation methods that have facilitated the safe and controlled application of hPSC‐derived cells in laboratory settings and pre‐clinical research. We underscore the need for improving and integrating purification strategies within differentiation protocols in order to unlock the therapeutic potential of hPSCs.

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.

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.

Microcarrier Culture Systems for Stem Cell Manufacturing

Abstract Stem cells hold a great promise for the generation of new cell therapies for many currently untreatable diseases, for human disease modeling or drug discovery. Many of these applications, however, will require the production of very large numbers of cells. A versatile system for the large-scale production of stem cells under adherent conditions is provided by microcarrier culture technology, which has been originally developed for animal cell culture in suspension bioreactors. This chapter describes the main characteristics of stem cell culture on microcarriers, providing an overview of the different bioprocess development steps. General guidelines and methodologies for the selection of the most appropriate microcarrier and its adaptation for the different stem cell types are provided as well as strategies for cell inoculation, bioreactor operation, and efficient cell harvesting and downstream processing.

Design and operation of bioreactor systems for the expansion of pluripotent stem cell-derived neural stem cells

Neurodegenerative disorders, such as Parkinsons disease, Huntingtons disease, and multiple sclerosis, affect millions of people worldwide, with devastating impact on the patients and families. A reliable method to obtain cells for replacement therapy, using ex-vivo culture techniques, would be of great value for the treatment of these conditions. This project focuses on the development of bioreactor culture systems for the large-scale expansion of neural stem cells, starting with a model line of mouse embryonic stem cell-derived neural stem cells under adherent culture conditions. Adherent conditions are an alternative to conventional culture of NS cells as aggregates and may circumvent problems associated with this system. Small scale stirred bioreactors were successfully used for mNS cell expansion, with microcarriers to support cell adhesion and proliferation, with retention of neural stem/progenitor cell markers. The system was optimized by determining the best values for parameters like stirring speed, microcarrier concentration or feeding regimen. The system here described may open a new door for applications requiring high numbers of mouse neural cells, providing an efficient way for their generation. Furthermore, the know-how obtained with this work may be applied for the development of an equivalent system for human cells, which may find clinical applications.