Hypoxia priming improves in vitro angiogenic properties of umbilical cord derived-mesenchymal stromal cells expanded in stirred-tank bioreactor

Noronha, N. C. Nadia de Cassia; Mizukami, Amanda; Orellana, Maristela Delgado; Oliveira, Maria Carolina de; Covas, Dimas Tadeu; Swiech, Kamilla; Malmegrim, Kelen Cristina Ribeiro

doi:10.1016/j.bej.2021.107949

Cited by 9 publications

(16 citation statements)

References 59 publications

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“…Under both settings, our harvest yields showed similar or even higher values with respect to previous studies. [ 34,49,53 ] Moreover, the best harvested cell density obtained in these experiments, when translated into manufacturing scale STRs (2.4‐L batch in a 3L‐STR; 18000 cm [2 54 ] ; 4.8 L medium consumed; 1.47 × 10 9 cells), would be able to surpass even the highest harvested cell number reported to date from a hollow‐fiber GMP‐compliant system (standard‐bioreactor; approx. 21000 cm 2 ; 5 L medium consumed; 6.05 × 10 8 cells) [55] …”

Section: Discussionmentioning

confidence: 99%

“…Seeding therefore requires optimization when transitioning from 2D to microcarrier-based cultures, considering options such as refinements to the medium composition and microcarrier coating. [28,29] The majority of studies conducted to date refer to large-scale expansion of MSCs from bone marrow and adipose tissue sources, [30] and only a few have reported the expansion of WJ-MSCs on microcarriers in spinner flasks [17,18,22,[31][32][33] or STRs [17,18,20,31,34] using different experimental approaches based mainly on the use of hPL and SCM.…”

Section: Introductionmentioning

confidence: 99%

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Identification of critical process parameters for expansion of clinical grade human Wharton's jelly‐derived mesenchymal stromal cells in stirred‐tank bioreactors

López‐Fernández,

Garcia‐Gragera,

Lecina

et al. 2024

Biotechnology Journal

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Cell therapies based on multipotent mesenchymal stromal cells (MSCs) are traditionally produced using 2D culture systems and platelet lysate‐ or serum‐containing media (SCM). Although cost‐effective for single‐dose autologous treatments, this approach is not suitable for larger scale manufacturing (e.g., multiple‐dose autologous or allogeneic therapies with banked MSCs); automated, scalable and Good Manufacturing Practices (GMP)‐compliant platforms are urgently needed. The feasibility of transitioning was evaluated from an established Wharton's jelly MSCs (WJ‐MSCs) 2D production strategy to a new one with stirred‐tank bioreactors (STRs). Experimental conditions included four GMP‐compliant xeno‐ and serum‐free media (XSFM) screened in 2D conditions and two GMP‐grade microcarriers assessed in 0.25 L‐STRs using SCM. From the screening, a XSFM was selected and compared against SCM using the best‐performing microcarrier. It was observed that SCM outperformed the 2D‐selected medium in STRs, reinforcing the importance of 2D‐to‐3D transition studies before translation into clinical production settings. It was also found that attachment efficiency and microcarrier colonization were essential to attain higher fold expansions, and were therefore defined as critical process parameters. Nevertheless, WJ‐MSCs were readily expanded in STRs with both media, preserving critical quality attributes in terms of identity, viability and differentiation potency, and yielding up to 1.47 × 109 cells in a real‐scale 2.4‐L batch.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of critical process parameters for expansion of clinical grade human Wharton's jelly‐derived mesenchymal stromal cells in stirred‐tank bioreactors

López‐Fernández,

Garcia‐Gragera,

Lecina

et al. 2024

Biotechnology Journal

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“…Importantly, we investigated the effects of larger-scale manufacturing on MSC functional response to priming and found that hit priming conditions differentially impacted MSC-EV identity and potency in bioreactors. This work represents one of few studies investigating MSC priming in a bioreactor (107) and likely the first study evaluating EVs in this context. Our result that +CTL, Hit 2, and Hit 4 significantly impacted MSC-EV protein and particle yield agrees with previous evidence of increased MSC-EV protein and particle yield with priming as well as a synergistic effect of combinatorial priming (27, 29, 31, 37, 44–47, 49–52, 108–112).…”

Section: Discussionmentioning

confidence: 99%

High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia

Larey,

Spoerer,

Daga

et al. 2023

Preprint

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Due to their immunomodulatory function, mesenchymal stromal cells (MSCs) are a promising therapeutic with the potential to treat neuroinflammation associated with neurodegenerative diseases. This function can be mediated by secreted extracellular vesicles (MSC-EVs). Despite established safety, MSC clinical translation has been unsuccessful due to inconsistent clinical outcomes resulting from functional heterogeneity. Current approaches to mitigate functional heterogeneity include ‘priming’ MSCs with inflammatory signals to enhance function. However, comprehensive evaluation of priming and its effects on MSC-EV function has not been performed. Clinical translation of MSC-EV therapies requires significant manufacturing scale-up, yet few studies have investigated the effects of priming in bioreactors. As MSC morphology has been shown to predict their immunomodulatory function, we screened MSC morphological response to an array of priming signals and evaluated MSC-EV identity and potency in response to priming in flasks and bioreactors. We identified unique priming conditions corresponding to distinct morphologies. These conditions demonstrated a range of MSC-EV preparation quality and lipidome, allowing us to discover a novel MSC-EV manufacturing condition, as well as gain insight into potential mechanisms of MSC-EV microglia modulation. Our novel screening approach and application of priming to MSC-EV bioreactor manufacturing informs refinement of larger-scale manufacturing and enhancement of MSC-EV function.Graphical AbstractHighlightsMSCs morphologically respond to inflammatory priming conditions.Priming ‘hits’ identified from morphological screen increase MSC-EV production.Priming MSCs in bioreactors enhances MSC-EV modulation of microglia.Changes in MSC-EV production and potency reflected by lipid content.First demonstration of effects of priming on MSC production of EVs in a bioreactor.

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“…In general, there are several ways to count cells in microcarrier culture: (1) Cell counting after cells have detached from microcarriers [26,29,37,50]; (2) Cell counting after microcarriers have dissolved [35,46,48]; (3) Nuclei counting after cells lysis with microcarriers [27,28,36]; (4) DNA quantification [18,29]; (5) Metabolic assays [34,44,47,91]. Counting methods with simple steps and high throughput are ideal and more favorable than assaybased approaches.…”

Section: Sampling and Cell Countingmentioning

confidence: 99%

“…Even though the theory is simply to count all the cells and viability is determined by the integrity of cell membrane, different automated cell counters may yield diverse results [92]. Trypan blue enhances cell counts with viability information [27,41,50] while fluorescence dyes, such as acridine orange, calcein, DAPI, propidium iodine and others can show higher color contrast for counting live and dead cells with or without lysis buffers by automated cell and nuclear counters [17,31,36,48]. These automated counters principally analyze the images of the stained cells/nuclei and count the number of particles.…”

Section: Sampling and Cell Countingmentioning

confidence: 99%

Bioprocessing of Human Mesenchymal Stem Cells: From Planar Culture to Microcarrier-Based Bioreactors

Tsai

Pacak

2021

Bioengineering

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Human mesenchymal stem cells (hMSCs) have demonstrated great potential to be used as therapies for many types of diseases. Due to their immunoprivileged status, allogeneic hMSCs therapies are particularly attractive options and methodologies to improve their scaling and manufacturing are needed. Microcarrier-based bioreactor systems provide higher volumetric hMSC production in automated closed systems than conventional planar cultures. However, more sophisticated bioprocesses are necessary to successfully convert from planar culture to microcarriers. This article summarizes key steps involved in the planar culture to microcarrier hMSC manufacturing scheme, from seed train, inoculation, expansion and harvest. Important bioreactor parameters, such as temperature, pH, dissolved oxygen (DO), mixing, feeding strategies and cell counting techniques, are also discussed.

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Hypoxia priming improves in vitro angiogenic properties of umbilical cord derived-mesenchymal stromal cells expanded in stirred-tank bioreactor

Cited by 9 publications

References 59 publications

Identification of critical process parameters for expansion of clinical grade human Wharton's jelly‐derived mesenchymal stromal cells in stirred‐tank bioreactors

Identification of critical process parameters for expansion of clinical grade human Wharton's jelly‐derived mesenchymal stromal cells in stirred‐tank bioreactors

High throughput screening of mesenchymal stromal cell morphological response to inflammatory signals for bioreactor-based manufacturing of extracellular vesicles that modulate microglia

Bioprocessing of Human Mesenchymal Stem Cells: From Planar Culture to Microcarrier-Based Bioreactors

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