Marcos F.Q. Sousa

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.

Perfusion of 3D encapsulated hepatocytes--a synergistic effect enhancing long-term functionality in bioreactors.

Long‐term primary cultures of hepatocytes are essential for bioartificial liver (BAL) devices and to reduce and replace animal tests in lead candidate optimization in drug discovery and toxicology tests. The aim of this work was to improve bioreactor cultures of hepatocyte spheroids by adding a more physiological perfusion feeding regime to these bioreactor systems. A continuous perfusion feeding was compared with 50% medium replacement (routinely used for in vitro tests) at the same dilution rate, 0.125 day−1, for three operative weeks. Perfusion feeding led to a 10‐fold improvement in albumin synthesis in bioreactors containing non‐encapsulated hepatocyte spheroids; no significant improvement was observed in phase I drug metabolizing activity. When ultra high viscous alginate encapsulated spheroids were cultured in perfusion, urea synthesis, phase I drug metabolizing activity and oxygen consumption had a threefold improvement over the 50% medium replacement regime; albumin production was the same for both feeding regimes. The effective diffusion of albumin in the alginate capsules was 7.75.10−9 cm2 s−1 and no diffusion limitation for this protein was observed using these alginate capsules under our operational conditions. In conclusion, perfusion feeding coupled with alginate encapsulation of hepatocyte spheroids showed a synergistic effect with a threefold improvement in three independent liver‐specific functions of long‐term hepatocyte spheroid cultures. Biotechnol. Bioeng. 2011; 108:41–49.

Carbon Monoxide Modulates Apoptosis by Reinforcing Oxidative Metabolism in Astrocytes: ROLE OF Bcl-2*

Background: Low doses of carbon monoxide (CO) prevent apoptosis in several cell models, including astrocytes. Results: CO improves cytochrome c oxidase (COX) activity and induces mitochondrial biogenesis. Bcl-2 expression and interaction with COX is involved in CO signaling. Conclusion: CO stimulates oxidative phosphorylation, improves metabolism, and prevents astrocytic apoptosis. Significance: Metabolism modulation can be a potential strategy against cerebral ischemia. Modulation of cerebral cell metabolism for improving the outcome of hypoxia-ischemia and reperfusion is a strategy yet to be explored. Because carbon monoxide (CO) is known to prevent cerebral cell death; herein the role of CO in the modulation of astrocytic metabolism, in particular, at the level of mitochondria was investigated. Low concentrations of CO partially inhibited oxidative stress-induced apoptosis in astrocytes, by preventing caspase-3 activation, mitochondrial potential depolarization, and plasmatic membrane permeability. CO exposure enhanced intracellular ATP generation, which was accompanied by an increase on specific oxygen consumption, a decrease on lactate production, and a reduction of glucose use, indicating an improvement of oxidative phosphorylation. Accordingly, CO increased cytochrome c oxidase (COX) enzymatic specific activity and stimulated mitochondrial biogenesis. In astrocytes, COX interacts with Bcl-2, which was verified by immunoprecipitation; this interaction is superior after 24 h of CO treatment. Furthermore, CO enhanced Bcl-2 expression in astrocytes. By silencing Bcl-2 expression with siRNA transfection, CO effects in astrocytes were prevented, namely: (i) inhibition of apoptosis, (ii) increase on ATP generation, (iii) stimulation of COX activity, and (iv) mitochondrial biogenesis. Thus, Bcl-2 expression is crucial for CO modulation of oxidative metabolism and for conferring cytoprotection. In conclusion, CO protects astrocytes against oxidative stress-induced apoptosis by improving metabolism functioning, particularly mitochondrial oxidative phosphorylation.

Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: Improved in vitro models for toxicological applications

During the last years an increasing number of in vitro models have been developed for drug screening and toxicity testing. Primary cultures of hepatocytes are, by far, the model of choice for those high-throughput studies but their spontaneous dedifferentiation after some time in culture hinders long-term studies. Thus, novel cell culture systems allowing extended hepatocyte maintenance and more predictive long term in vitro studies are required. It has been shown that hepatocytes functionality can be improved and extended in time when cultured as 3D-cell aggregates in environmental controlled stirred bioreactors. In this work, aiming at further improving hepatocytes functionality in such 3D cellular structures, co-cultures with fibroblasts were performed. An inoculum concentration of 1.2×10(5) cell/mL and a 1:2 hepatocyte:mouse embryonic fibroblast ratio allowed to improve significantly the albumin secretion rate and both ECOD (phase I) and UGT (phase II) enzymatic activities in 3D co-cultures, as compared to the routinely used 2D hepatocyte monocultures. Significant improvements were also observed in relation to 3D monocultures of hepatocytes. Furthermore, hepatocytes were able to respond to the addition of beta-Naphtoflavone by increasing ECOD activity showing CYP1A inducibility. The dependence of CYP activity on oxygen concentration was also observed. In summary, the improved hepatocyte specific functions during long term incubation of 3D co-cultures of hepatocytes with fibroblasts indicate that this system is a promising in vitro model for long term toxicological studies.

Towards purification of adenoviral vectors based on membrane technology.

Given increasing applications of recombinant adenoviruses for gene therapy and vaccination, there is a need for highly robust and fast purification platforms for their large scale manufacture. Traditional chromatographic methods using resins as matrices have several limitations such as high‐pressure drops and slow processing rates due to pore diffusion and channelling of the feed through the bed. In contrast, membrane adsorbers offer the advantage of fast, gentle, and effective isolation. Furthermore, membranes are easy to use, no column packing is needed and, when used as disposables, no cleaning validation is necessary, representing a substantial advantage to meet cGMP requirements. In this work, a strategy for purification of adenovirus vectors from cell‐culture bulks fully based on membrane devices is presented. Ultrafiltration membranes with molecular weight cutoffs of 300, 500, and 750 kDa were tested for the concentration of cell‐culture supernatant after an initial clarification step. The results show that the use of ultrafiltration/diafiltration membranes not only concentrates the virus but also leads to the removal of 90% of host cell DNA and proteins in the retentate. Two membrane adsorbers (Sartobind Q and Sartobind anion direct) were evaluated for adenovirus vectors capture and purification. To define the best operating conditions, the effect of pH, conductivity, and recirculation of load bulk on the recovery yield of infectious adenoviruses were evaluated. Sartobind anion direct allows for higher recovery yields (up to 62%) of infectious adenoviruses than Sartobind Q; identical ratios between total and infectious adenoviruses (TP/IP) were achieved for both membrane adsorbers. The overall recovery yield of the process is ∼52%; this work credits membrane technology as an alternative for the concentration and purification of adenoviruses and as a promising solution for downstream processing of other viral vectors.

Kinetic Analysis of L1 Homophilic Interaction ROLE OF THE FIRST FOUR IMMUNOGLOBULIN DOMAINS AND IMPLICATIONS ON BINDING MECHANISM

L1 is a cell adhesion molecule of the immunoglobulin (Ig) superfamily, critical for central nervous system development, and involved in several neuronal biological events. It is a type I membrane glycoprotein. The L1 ectodomain, composed of six Ig-like and five fibronectin (Fn) type-III domains, is involved in homophilic binding. Here, co-immunoprecipitation studies between recombinant truncated forms of human L1 expressed and purified from insect Spodoptera frugiperda Sf9 cells, and endogenous full-length L1 from human NT2N neurons, showed that the L1 ectodomain (L1/ECD) and L1/Ig1–4 interacted homophilically in trans, contrary to mutants L1/Ig1–3 and L1/Ig2-Fn5. All mutants were correctly folded as evaluated by combination of far-UV CD and fluorescence spectroscopy. Surface plasmon resonance analysis showed comparable dissociation constants of 116 ± 2 and 130 ± 6 nm for L1/ECD-L1/ECD and L1/ECD-L1/Ig1–4, respectively, whereas deletion mutants for Ig1 or Ig4 did not interact. Accordingly, in vivo, Sf9 cells stably expressing L1 were found to adhere only to L1/ECD- and L1/Ig1–4-coated surfaces. Furthermore, only these mutants bound to HEK293 cells overexpressing L1 at the cell surface. Enhancement of neurite outgrowth, which is the consequence of signaling events caused by L1 homophilic binding, was comparable between L1/ECD and L1/Ig1–4. Altogether, these results showed that domains Ig1 to Ig4 are necessary and sufficient for L1 homophilic binding in trans, and that the rest of the molecule does not contribute to the affinity under the conditions of the current study. Furthermore, they are compatible with a cooperative interaction between modules Ig1–Ig4 in a horseshoe conformation.

Metabolic alterations induced by ischemia in primary cultures of astrocytes: merging 13C NMR spectroscopy and metabolic flux analysis.

J. Neurochem. (2010) 113, 735–748.

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.

Scalable culture systems using different cell lines for the production of Peste des Petits ruminants vaccine

Peste des Petits ruminants (PPR) is considered as one of the major constraints to the productivity of small ruminants in Africa and Asian countries. Currently PPR control is done by vaccination with an attenuated PPR strain (Nigeria 75/1) produced in monolayers of Vero cells grown in roller bottles or static flasks. This work focuses on the production of a PPR vaccine strain using stirred conditions as an advanced option for process scale-up. Non-porous microcarriers (Cytodex-1) were used to support Vero cell growth in suspension cultures. The use of Ex-Cell medium could improve cell specific productivities obtained with standard serum containing medium, independently of the type of system used, i.e. static as well as suspension stirred cultures. As an alternative, several cell lines adapted to grow as single cells in suspension (CHO-K1, BHK-21A and 293) and another anchorage-dependent (MRC-5) were evaluated in their capacity to produce a PPR vaccine. BHK-21A and 293 cells grown as single-cell suspension in serum free medium were both suited to produce PPR vaccine with productivities similar to Vero cells, namely 10(6)TCID(50)/mL. However, for the 293 cells, these results were only obtained 2-3 days later. CHO-K1 and MRC-5 cells have shown not to be suitable to adequately produce this virus. These results provide further insights into the feasibility of applying microcarrier cell culture technology to produce PPR vaccine in Vero cells as well as in the alternative use of single-cell suspension cultures of BHK-21A, significantly simplifying the existing production process.

Carboxylesterase 2 production and characterization in human cells: new insights into enzyme oligomerization and activity

Carboxylesterase 2 (CES2), the main carboxylesterase expressed in human intestine, is an increasingly important enzyme in anti-cancer combined therapies for the treatment of different pathologies like colon adenocarcinoma and malignant glioma. The production of human recombinant CES2, in human embryonic kidney cells (HEK-293T cells) using serum-free media, is herein described. CES2 secretion to the media was achieved by the simple addition of an in-frame C-terminal 10× histidine tag (CES2-10xHis) without the need of addition of extra N-terminal signalling sequences or the mutation or deletion of the C-terminal HTEL motif responsible for retaining the protein in the lumen of endoplasmic reticulum. This secretion allowed a fourfold increase in CES2 production. The characterization of human recombinant CES2 showed that this protein exists in other active and inactive forms than the described 60 kDa monomer.