Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

Nienow, Alvin W.; Hewitt, Christopher J.; Heathman, Thomas R.J.; Glyn, Veronica A.M.; Fonte, Gonҫalo N.; Hanga, Mariana P.; Coopman, Karen; Rafiq, Qasim A.

doi:10.1016/j.bej.2015.08.003

Cited by 77 publications

(82 citation statements)

References 17 publications

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“…Briefly, the harvesting procedure involved a two‐step process: (1) detachment of the cells from the microcarriers using an increased agitation rate of 150 rpm for 7 min during enzymatic reagent exposure and (2) separation of the cells and microcarriers prior to cell concentration by filtration. After detachment and separation, >95% cells with >95% viability were recovered (Nienow et al, ).…”

Section: Methodsmentioning

confidence: 99%

Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

Rafiq

Hanga

Heathman

et al. 2017

Biotech & Bioengineering

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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 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Section: Methodsmentioning

confidence: 99%

Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

Rafiq

Hanga

Heathman

et al. 2017

Biotech & Bioengineering

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“…To overcome these challenges, clinical research and commercial groups have used the use of rocking motion bioreactor systems (e.g., GE Healthcare's WAVE bioreactor or BIOSTAT RM from Sartorius Stedim Biotech), which improve the homogeneity of the culture, sampling and implementation of process control strategies (Marsh et al, 2017;Vormittag et al, 2018). However, there have been extensive studies in the literature which have demonstrated the potential to culture numerous mammalian and human cells under both transitional and turbulent fluid dynamic conditions for both free suspension cells (Nienow, 2006) and adherent cells on microcarriers (Chen, Chew, Tan, Reuveny, & Weng Oh, 2015;Nienow et al, 2016;Rafiq et al, 2017). Some early work was successful in demonstrating the growth of T-cells in both spinner flasks and small bioreactors (Bohnenkamp, Hilbert, & Noll, 2002;Carswell & Papoutsakis, 2000) but neither used Dynabeads for cell activation.…”

Section: Introductionmentioning

confidence: 99%

Establishing the scalable manufacture of primary human T‐cells in an automated stirred‐tank bioreactor

Costariol

Rotondi

Amini

et al. 2019

Biotech & Bioengineering

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Advanced cell and gene therapies such as chimeric antigen receptor T-cell immunotherapies (CAR-T), present a novel therapeutic modality for the treatment of acute and chronic conditions including acute lymphoblastic leukemia and non-Hodgkin lymphoma. However, the development of such immunotherapies requires the manufacture of large numbers of T-cells, which remains a major translational and commercial bottleneck due to the manual, small-scale, and often static culturing systems used for their production. Such systems are used because there is an unsubstantiated concern that primary T-cells are shear sensitive, or prefer static conditions, and therefore do not grow as effectively in more scalable, agitated systems, such as stirred-tank bioreactors, as compared with T-flasks and culture bags.In this study, we demonstrate that not only T-cells can be cultivated in an automated stirred-tank bioreactor system (ambr ® 250), but that their growth is consistently and significantly better than that in T-flask static culture, with equivalent cell quality.Moreover, we demonstrate that at progressively higher agitation rates over the range studied here, and thereby, higher specific power inputs (P/M W kg −1 ), the higher the final viable T-cell density; that is, a cell density of 4.65 ± 0.24 × 10 6 viable cells ml −1 obtained at the highest P/M of 74 × 10 −4 W kg −1 in comparison with 0.91 ± 0.07 × 10 6 viable cells ml −1 at the lowest P/M of 3.1 × 10 −4 W kg −1 . We posit that this improvement is due to the inability at the lower agitation rates to effectively suspend the Dynabeads ® , which are required to activate the T-cells; and that contact between them is improved at the higher agitation rates. Importantly, from the data obtained, there is no indication that T-cells prefer being grown under static conditions or are sensitive to fluid dynamic stresses within a stirred-tank bioreactor system at the agitation speeds investigated. Indeed, the opposite has proven to be the case, whereby, the cells grow better under higher agitation speeds while maintaining their quality. This study is the first demonstration of primary T-cell ex vivo manufacture activated by Dynabeads ® in an automated stirred-tank bioreactor system such as the ambr ® 250 and the findings have the potential to be applied to multiple other cell candidates for advanced therapy applications. K E Y W O R D Sbioprocessing, immunotherapy, manufacture, scale-up, stirred-tank bioreactor, T-cell

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“…enzymes, chelators), magnetic or even stimuli‐responsive (e.g. thermo‐responsive, pH‐responsive, electro‐responsive, photo‐responsive).…”

Section: Introductionmentioning

confidence: 99%

Expansion of bone marrow-derived human mesenchymal stem/stromal cells (hMSCs) using a two-phase liquid/liquid system

Hanga

Murasiewicz

Pacek

et al. 2017

J. Chem. Technol. Biotechnol

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BACKGROUNDHuman 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.RESULTSAn 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.CONCLUSIONSThese 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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Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

Cited by 77 publications

References 17 publications

Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

Establishing the scalable manufacture of primary human T‐cells in an automated stirred‐tank bioreactor

Expansion of bone marrow-derived human mesenchymal stem/stromal cells (hMSCs) using a two-phase liquid/liquid system

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