RESUMO
BACKGROUND: Traditional large-scale culture systems for human mesenchymal stem/stromal cells (hMSCs) use solid microcarriers as attachment substrates. Although the use of such substrates is advantageous because of the high surface-to-volume ratio, cell harvest from the same substrates is a challenge as it requires enzymatic treatment, often combined with agitation. Here, we investigated a two-phase system for expansion and non-enzymatic recovery of hMSCs. Perfluorocarbon droplets were dispersed in a protein-rich growth medium and were used as temporary liquid microcarriers for hMSC culture. RESULTS: hMSCs successfully attached to these liquid microcarriers, exhibiting similar morphologies to those cultured on solid ones. Fold increases of 3.03 ± 0.98 (hMSC1) and 3.81 ± 0.29 (hMSC2) were achieved on day 9. However, the maximum expansion folds were recorded on day 4 (4.79 ± 0.47 (hMSC1) and 4.856 ± 0.7 (hMSC2)). This decrease was caused by cell aggregation upon reaching confluency due to the contraction of the interface between the two phases. Cell quality, as assessed by differentiation, cell surface marker expression and clonogenic ability, was retained post expansion on the liquid microcarriers. Cell harvesting was achieved non-enzymatically in two steps: first by inducing droplet coalescence and then aspirating the interface. Quality characteristics of hMSCs continued to be retained even after inducing droplet coalescence. CONCLUSION: The prospect of a temporary microcarrier that can be used to expand cells and then 'disappear' for cell release without using proteolytic enzymes is a very exciting one. Here, we have demonstrated that hMSCs can attach and proliferate on these perfluorocarbon liquid microcarriers while, very importantly, retaining their quality.
RESUMO
Traditional farm-based products based on livestock are one of the main contributors to greenhouse gas emissions. Cultivated meat is an alternative that mimics animal meat, being produced in a bioreactor under controlled conditions rather than through the slaughtering of animals. The first step in the production of cultivated meat is the generation of sufficient reserves of starting cells. In this study, bovine adipose-derived stem cells (bASCs) were used as starting cells due to their ability to differentiate towards both fat and muscle, two cell types found in meat. A bioprocess for the expansion of these cells on microcarriers in spinner flasks was developed. Different cell seeding densities (1,500, 3,000, and 6,000 cells/cm2 ) and feeding strategies (80%, 65%, 50%, and combined 80%/50% medium exchanges) were investigated. Cell characterization was assessed pre- and postbioprocessing to ensure that bioprocessing did not negatively affect bASC quality. The best growth was obtained with the lowest cell seeding density (1,500 cells/cm2 ) with an 80% medium exchange performed (p < .0001) which yielded a 28-fold expansion. The ability to differentiate towards adipogenic, osteogenic, and chondrogenic lineages was retained postbioprocessing and no significant difference (p > .5) was found in clonogenicity pre- or postbioprocessing in any of the feeding regimes tested.
Assuntos
Reatores Biológicos/normas , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Abastecimento de Alimentos/métodos , Carne/provisão & distribuição , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Animais , Bovinos , Contagem de Células , Células-Tronco Mesenquimais/metabolismoRESUMO
Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS . The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06-0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253-2266. © 2017 Wiley Periodicals, Inc.
Assuntos
Técnicas de Cultura Celular por Lotes/instrumentação , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Microfluídica/instrumentação , Técnicas de Cultura Celular por Lotes/métodos , Técnicas de Cultura de Células/métodos , Proliferação de Células/fisiologia , Células Cultivadas , Meios de Cultura Livres de Soro/metabolismo , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Microfluídica/métodos , Miniaturização , Projetos Piloto , Robótica/instrumentaçãoRESUMO
BACKGROUND: Human mesenchymal stem/stromal cells (hMSCs) are at the forefront of regenerative medicine applications due to their relatively easy isolation and availability in adults, potential to differentiate and to secrete a range of trophic factors that could determine specialised tissue regeneration. To date, hMSCs have been successfully cultured in vitro on substrates such as polystyrene dishes (TCPS) or microcarriers. However, hMSC sub-cultivation and harvest typically employs proteolytic enzymes that act by cleaving important cell membrane proteins resulting in long-term cell damage. In a process where the cells themselves are the product, a non-enzymatic and non-damaging harvesting approach is desirable. RESULTS: An alternative system for hMSC expansion and subsequent non-enzymatic harvest was investigated here. A liquid/liquid two-phase system was proposed, comprising a selected perfluorocarbon (FC40) and growth medium (DMEM). The cells exhibited similar cell morphologies compared with TCPS. Moreover, they retained their identity and differentiation potential post-expansion and post-harvest. Further, no significant difference was found when culturing hMSCs in the culture systems prepared with either fresh or recycled FC40 perfluorocarbon. CONCLUSIONS: These findings make the FC40/DMEM system an attractive alternative for traditional cell culture substrates due to their ease of cell recovery and recyclability, the latter impacting on overall process costs. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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Advanced therapeutic medicinal products (ATMPs) have emerged as novel therapies for untreatable diseases, generating the need for large volumes of high-quality, clinically-compliant GMP cells to replace costly, high-risk and limited scale manual expansion processes. We present the design of a fully automated, robot-assisted platform incorporating the use of multiliter stirred tank bioreactors for scalable production of adherent human stem cells. The design addresses a needle-to-needle closed process incorporating automated bone marrow collection, cell isolation, expansion, and collection into cryovials for patient delivery. AUTOSTEM, a modular, adaptable, fully closed system ensures no direct operator interaction with biological material; all commands are performed through a graphic interface. Seeding of source material, process monitoring, feeding, sampling, harvesting and cryopreservation are automated within the closed platform, comprising two clean room levels enabling both open and closed processes. A bioprocess based on human MSCs expanded on microcarriers was used for proof of concept. Utilizing equivalent culture parameters, the AUTOSTEM robot-assisted platform successfully performed cell expansion at the liter scale, generating results comparable to manual production, while maintaining cell quality postprocessing.