Over the last decades, mesenchymal stromal cells (MSC) have been the focus of intense research by academia and industry due to their unique features. MSC can be easily isolated and expanded through ...in vitro culture by taking full advantage of their self-renewing capacity. In addition, MSC exert immunomodulatory effects and can be differentiated into various lineages, which makes them highly attractive for clinical applications in cell-based therapies. In this review, we attempt to provide a brief historical overview of MSC discovery, characterization, and the first clinical studies conducted. The current MSC manufacturing platforms are reviewed with special attention regarding the use of bioreactors for the production of GMP-compliant clinically relevant cell numbers. The first commercial MSC-based products are also addressed, as well as the remaining challenges to the widespread use of MSC-derived products.
Multipotent mesenchymal stromal cells (MSC) have been widely explored for cell-based therapy of immune-mediated, inflammatory, and degenerative diseases, due to their immunosuppressive, ...immunomodulatory, and regenerative potentials. Preclinical studies and clinical trials have demonstrated promising therapeutic results although these have been somewhat limited. Aspects such as low in vivo MSC survival in inhospitable disease microenvironments, requirements for ex vivo cell overexpansion prior to infusions, intrinsic differences between MSC and different sources and donors, variability of culturing protocols, and potency assays to evaluate MSC products have been described as limitations in the field. In recent years, priming approaches to empower MSC have been investigated, thereby generating cellular products with improved potential for different clinical applications. Herein, we review the current priming approaches that aim to increase MSC therapeutic efficacy. Priming with cytokines and growth factors, hypoxia, pharmacological drugs, biomaterials, and different culture conditions, as well as other diverse molecules, are revised from current and future perspectives.
Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic ...purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC‐EV in suspension cultures using spinner flasks and human collagen‐coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T‐flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold‐increase was 5.7‐fold for the 3D culture system and 4.6‐fold for the 2D culture system, signifying a fold‐change of 1.2 (calculated as the ratio of fold‐increase 3D to fold‐increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T‐flask cultures. The total cell production in the spinner flask cultures was 5.2‐fold higher than that in T‐flask cultures. While the EV specific production (particles/cell) in T‐flask cultures (4.40 ± 1.21 × 108 particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 108 particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC‐EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC‐EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno‐free conditions.
Abstract Human Embryonic Kidney 293T cells (HEK-293T) are the most common host for viral vector production and are also widely employed for recombinant protein production. These cells are typically ...cultured in monolayer (adherent culture) using culture medium containing fetal bovine serum (FBS), which impairs batch-to-batch reproducibility and scale-up. The adaptation of adherent cell culture to suspension culture in chemically defined serum-free culture medium is an attractive approach for large-scale bioprocess implementation while aiming for a Good Manufacturing Practice (GMP) compliant production process. Therefore, in the present study, our goal was to adapt HEK-293T cells to serum-free suspension culture conditions and evaluate the feasibility of adapted cells to be transfected using different plasmid vectors for recombinant protein production. Firstly, the cells were efficiently adapted to serum-free conditions by sequential adaptation (FBS-containing medium weaning). During the whole process, parameters such as cell growth, viability and doubling time were evaluated and compared to the control (adherent serum-supplemented HEK-293T cell culture). Afterwards, these cells were adapted to suspension culture by using Erlenmeyer flasks in an orbital shaker platform, being able to achieve meaningful cell density with high viability. Adapted cells presented a transfection efficiency of approximately 50% for all vector constructs used (1054-GFP, Factor-VIII and Factor-IX). Overall, it was possible to successfully adapt HEK-293T cells to suspension and serum-free conditions, which represents an important step towards the development of a scalable and GMP-compliant production process. In addition, adapted cells efficiently expressed the different transgene tested, opening up possibilities for its use in recombinant protein production.
The therapeutic potential of mesenchymal stem/stromal cells (MSC) is widely recognized for the treatment of several diseases, including acute graft-vs.-host disease (GVHD), hematological ...malignancies, cardiovascular, bone, and cartilage diseases. More recently, this therapeutic efficacy has been attributed to the bioactive molecules that these cells secrete (secretome), now being referred as medicinal signaling cells. This fact raises the opportunity of therapeutically using MSC-derived soluble factors rather than cells themselves, enabling their translation into the clinic. Indeed, many clinical trials are now studying the effects of MSC-secretome in the context of cell-free therapy. MSC secretome profile varies between donors, source, and culture conditions, making their therapeutic use very challenging. Therefore, identifying these soluble proteins and evaluating their production in a reproducible and scalable manner is even more relevant. In this work, we analyzed the global profile of proteins secreted by umbilical cord matrix (UCM) derived-MSC in static conditions by using mass spectrometry, enabling the identification of thousands of proteins. Afterwards, relevant proteins were chosen and monitored in the supernatant of a fully-controllable, closed and scalable system (bioreactor) by using multiple reaction monitoring (MRM) mass spectrometric technique in a time-dependent manner. The results showed that the majority of interesting proteins were enriched through time in culture, with the last day of culture being the ideal time for supernatant collection. The use of this regenerative "soup," which is frequently discarded, could represent a step toward a safe, robust and reproducible cell-free product to be used in the medical therapeutic field. The future use of chemically defined culture-media will certainly facilitate secretome production according to Good Manufacturing Practice (GMP) standards.
Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the immunotherapy field with high rate complete responses especially for hematological diseases. Despite the diversity of tumor ...specific-antigens, the manufacturing process is consistent and involves multiple steps, including selection of T cells, activation, genetic modification, and in vitro expansion. Among these complex manufacturing phases, the choice of culture system to generate a high number of functional cells needs to be evaluated and optimized. Flasks, bags, and rocking motion bioreactor are the most used platforms for CAR-T cell expansion in the current clinical trials but are far from being standardized. New processing options are available and a systematic effort seeking automation, standardization and the increase of production scale, would certainly help to bring the costs down and ultimately democratize this personalized therapy. In this review, we describe different cell expansion platforms available as well as the quality control requirements for clinical-grade production.
Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical ...trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production.
In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells.
Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3-ζ endodomain.
In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progression in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice.
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