Ana Fernandes-Platzgummer

A xenogeneic-free bioreactor system for the clinical-scale expansion of human mesenchymal stem/stromal cells

The large cell doses (>1 × 106 cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex‐vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost‐effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)‐free microcarrier‐based culture system was successfully established for bone marrow (BM) MSC and adipose tissue‐derived stem/stromal cell (ASC) cultivation using a 1L‐scale controlled stirred‐tank bioreactor, allowing the production of (1.1 ± 0.1) × 108 and (4.5 ± 0.2) × 107 cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat. Also, the three different feeding regimes studied—(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed‐batch addition of concentrated nutrients and growth factors every 2 days—yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA‐DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier‐based stirred culture system under xeno‐free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP‐compliant clinical‐scale MSC production system. Biotechnol. Bioeng. 2014;111: 1116–1127.

Different stages of pluripotency determine distinct patterns of proliferation, metabolism, and lineage commitment of embryonic stem cells under hypoxia.

Oxygen tension is an important component of the stem cell microenvironment. Herein, we have studied the effect of low oxygen levels (2% O(2)), or hypoxia, in the expansion of mouse embryonic stem (ES) cells. In the presence of leukemia inhibitory factor (LIF), cell proliferation was reduced under hypoxia and a simultaneous reduction in cell viability was also observed. Morphological changes and different cell cycle patterns were observed, suggesting some early differentiation under hypoxic conditions. However, when cells were maintained in a ground state of pluripotency, by inhibition of autocrine FGF4/ERK and GSK3 signaling, hypoxia did not affect cell proliferation, and did not induce early differentiation. As expected, there was an increase in lactate-specific production rate and a significant increase in the glucose consumption under hypoxic conditions. Nevertheless, during neural commitment, low oxygen tension exerted a positive effect on early differentiation of ground-state ES cells, resulting in a faster commitment toward neural progenitors. Overall our results demonstrate the need to specifically regulate the oxygen content, especially hypoxia, along with other culture conditions, when developing new strategies for ES cell expansion and/or controlled differentiation.

Scale-up of mouse embryonic stem cell expansion in stirred bioreactors.

The aim of this study was to develop a robust, quality controlled and reproducible large‐scale culture system using serum‐free (SF) medium to obtain vast numbers of embryonic stem (ES) cells as a starting source for potential applications in tissue regeneration, as well as for drug screening studies. Mouse ES (mES) cells were firstly cultured on microcarriers in spinner flasks to investigate the effect of different parameters such as the agitation rate and the feeding regimen. Cells were successfully expanded at agitation rates up to 60 rpm using the SF medium and no significant differences in terms of growth kinetics or metabolic profiles were found between the two feeding regimens evaluated: 50% medium renewal every 24 h or 25% every 12 h. Overall, cells reached maximum concentrations of (4.2 ± 0.4) and (5.6 ± 0.8) ×106 cells/mL at Day 8 for cells fed once or twice per day; which corresponds to an increase in total cell number of 85 ± 7 and 108 ± 16, respectively. To have a more precise control over culture conditions and to yield a higher number of cells, the scale‐up of the spinner flask culture system was successfully accomplished by using a fully controlled stirred tank bioreactor. In this case, the concentration of mES cells cultured on microcarriers increased 85 ± 15‐fold over 11 days. Importantly, mES cells expanded under stirred conditions, in both spinner flask and fully controlled stirred tank bioreactor, using SF medium, retained the expression of pluripotency markers such as Oct‐4, Nanog, and SSEA‐1 and their differentiation potential into cells of the three embryonic germ layers.

Kinetic and metabolic analysis of mouse embryonic stem cell expansion under serum-free conditions.

There is a need for a deeper understanding of the biochemical events affecting embryonic stem (ES) cell culture by analyzing the expansion of mouse ES cells in terms of both cell growth and metabolic kinetics. The influence of the initial cell density on cell expansion was assessed. Concomitantly, the biochemical profile of the culture was evaluated, which allowed measuring the consumption of important substrates, such as glucose and glutamine, and the production of metabolic byproducts, like lactate. The results suggest a more efficient cell metabolism in serum-free conditions and a preferential use of glutaminolysis as an energy source during cell expansion at low seeding densities. This work contributes to the development of fully-controlled bioprocesses to produce relevant numbers of ES cells for cell therapies and high-throughput drug screening.

A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue.

Human mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and the development of microcarrier-based cultures in scalable bioreactors with well-defined xenogeneic-free components represent important milestones towards the clinical-scale production of these cells. In this work, we optimized our previously developed xeno-free microcarrier-based system for the scalable expansion of human MSC isolated from bone marrow (BM MSC) and adipose-derived stem/stromal cells (ASC). By adapting the agitation/feeding protocol at the initial cell seeding/cultivation stage in spinner flasks, we were able to maximize cell expansion rate and final cell yield. Maximal cell densities of 3.6 × 10(5) and 1.9 × 10(5) cells/mL were obtained for BM MSC (0.60 ± 0.04 day(-1) ) and ASC (0.9 ± 0.1 day(-1) ) cultures, upon seven and eight days of cultivation, respectively. Ready-to-use microcarriers Synthemax® II and Enhanced Attachment® supported identical expansion performance of BM MSC, turning those effective alternatives to the pre-coated plastic microcarriers used in our xeno-free scalable culture system. Importantly, expanded MSC maintained their immunophenotype and multilineage differentiation potential. Moreover, secretome analysis suggested a priming effect of stirred culture conditions on cytokine production by MSC. This culture system yielded considerable final cell densities that can be scaled-up to controlled large-scale bioreactors allowing a more efficient, safe and cost-effective MSC production for clinical settings.

Delta One T cells for immunotherapy of chronic lymphocytic leukemia: clinical-grade expansion/ differentiation and preclinical proof-of-concept

Purpose: The Vδ1+ subset of γδ T lymphocytes is a promising candidate for cancer immunotherapy, but the lack of suitable expansion/differentiation methods has precluded therapeutic application. We set out to develop and test (preclinically) a Vδ1+ T-cell–based protocol that is good manufacturing practice compatible and devoid of feeder cells for prompt clinical translation. Experimental design: We tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (more than 2–3 weeks) Vδ1+ T cells from the peripheral blood of healthy donors and patients with chronic lymphocytic leukemia (CLL). We characterized the phenotype and functional potential of the final cellular product, termed Delta One T (DOT) cells, in vitro and in vivo (xenograft models of CLL). Results: We describe a very robust two-step protocol for the selective expansion (up to 2,000-fold in large clinical-grade cell culture bags) and differentiation of cytotoxic Vδ1+ (DOT) cells. These expressed the natural cytotoxicity receptors, NKp30 and NKp44, which synergized with the T-cell receptor to mediate leukemia cell targeting in vitro. When transferred in vivo, DOT cells infiltrated tumors and peripheral organs, and persisted until the end of the analysis without showing signs of loss of function; indeed, DOT cells proliferated and produced abundant IFNγ and TNFα, but importantly no IL17, in vivo. Critically, DOT cells were capable of inhibiting tumor growth and preventing dissemination in xenograft models of CLL. Conclusions: We provide a clinical-grade method and the preclinical proof of principle for application of a new cellular product, DOT cells, in adoptive immunotherapy of CLL. Clin Cancer Res; 22(23); 5795–804. ©2016 AACR.

Stirred tank bioreactor culture combined with serum-/xenogeneic-free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal stem/stromal cells

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell-based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non-invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)-free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin-based Cultispher(®) S microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. This system enabled the production of 2.4 (±1.1) x10(5) cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier-based stirred culture system, using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates.

Scalable microcarrier-based manufacturing of mesenchymal stem/stromal cells.

Due to their unique features, mesenchymal stem/stromal cells (MSC) have been exploited in clinical settings as therapeutic candidates for the treatment of a variety of diseases. However, the success in obtaining clinically-relevant MSC numbers for cell-based therapies is dependent on efficient isolation and ex vivo expansion protocols, able to comply with good manufacturing practices (GMP). In this context, the 2-dimensional static culture systems typically used for the expansion of these cells present several limitations that may lead to reduced cell numbers and compromise cell functions. Furthermore, many studies in the literature report the expansion of MSC using fetal bovine serum (FBS)-supplemented medium, which has been critically rated by regulatory agencies. Alternative platforms for the scalable manufacturing of MSC have been developed, namely using microcarriers in bioreactors, with also a considerable number of studies now reporting the production of MSC using xenogeneic/serum-free medium formulations. In this review we provide a comprehensive overview on the scalable manufacturing of human mesenchymal stem/stromal cells, depicting the various steps involved in the process from cell isolation to ex vivo expansion, using different cell tissue sources and culture medium formulations and exploiting bioprocess engineering tools namely microcarrier technology and bioreactors.

Scalable ex vivo expansion of human mesenchymal stem/stromal cells in microcarrier-based stirred culture systems.

The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different microcarriers and xeno-free medium formulations.

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells

Umbilical cord matrix (UCM)‐derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non‐invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large‐scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate‐based culture supplement (UltraGROTM, AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC‐like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGROTM‐supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGROTM‐supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13‐fold after 5–6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic‐free microcarrier‐based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright