Hypoxic Three-Dimensional Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred Tank Reactor

Egger, Dominik; Schwedhelm, Ivo; Hansmann, Jan; Kasper, Cornelia

doi:10.3390/bioengineering4020047

Cited by 31 publications

(40 citation statements)

References 42 publications

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“…Methods for dynamic, collision-based assembly of MSC aggregates include forced aggregation by centrifugation [ 92 ] or mixing mediated by shaker platforms [ 75 , 93 ], spinner flasks [ 56 , 59 ], rotating wall vessels (RWVs) [ 56 ], and stirred tank reactors (STRs) [ 94 ]. Aggregation by centrifugation has mainly been used for chondrogenic differentiation of MSCs [ 95 ] and is also known as pellet or micromass culture.…”

Section: Generation Of Msc Aggregatesmentioning

confidence: 99%

“…Aggregation by centrifugation has mainly been used for chondrogenic differentiation of MSCs [ 95 ] and is also known as pellet or micromass culture. Collision-based assembly by mixing was observed with a seeding density of as low as 2 × 10 4 cells/mL in spinner flasks and RWVs [ 56 ], with 1 × 10 5 cells/mL in a STR [ 94 ] and led to randomly sized spheroids, whereas mixing in ultralow adhesive multiwell plates on a shaker platform [ 75 ] and compression by centrifugation [ 92 ] yielded aggregates with narrower size and homogeneous shape distribution.…”

Section: Generation Of Msc Aggregatesmentioning

confidence: 99%

“…However, only a few studies have harnessed dynamic conditions for the generation and cultivation of MSC aggregates ( Table 1 ), as dynamic cultivation aggregates do not necessarily need to be generated by dynamic collision-based assembly. Studies report formation by centrifugation [ 92 ], in hanging drops [ 75 ], in ultralow adhesive multiwell plates [ 75 ], or in a microwell array [ 82 ] followed by cultivation in a dynamic cultivation system, such as shaker platforms [ 75 , 82 , 92 , 93 ], in spinner flasks [ 56 , 59 ], RWV [ 56 ], or STR [ 94 ]. In microwell arrays, all seeded cells are involved in the formation of aggregates, thus the size and cell number per aggregate can be precisely controlled.…”

Section: Dynamic Cultivation Of Aggregatesmentioning

confidence: 99%

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Dynamic Cultivation of Mesenchymal Stem Cell Aggregates

et al. 2018

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Mesenchymal stem cells (MSCs) are considered as primary candidates for cell-based therapies due to their multiple effects in regenerative medicine. Pre-conditioning of MSCs under physiological conditions—such as hypoxia, three-dimensional environments, and dynamic cultivation—prior to transplantation proved to optimize their therapeutic efficiency. When cultivated as three-dimensional aggregates or spheroids, MSCs display increased angiogenic, anti-inflammatory, and immunomodulatory effects as well as improved stemness and survival rates after transplantation, and cultivation under dynamic conditions can increase their viability, proliferation, and paracrine effects, alike. Only few studies reported to date, however, have utilized dynamic conditions for three-dimensional aggregate cultivation of MSCs. Still, the integration of dynamic bioreactor systems, such as spinner flasks or stirred tank reactors might pave the way for a robust, scalable bulk expansion of MSC aggregates or MSC-derived extracellular vesicles. This review summarizes recent insights into the therapeutic potential of MSC aggregate cultivation and focuses on dynamic generation and cultivation techniques of MSC aggregates.

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Section: Generation Of Msc Aggregatesmentioning

confidence: 99%

Section: Generation Of Msc Aggregatesmentioning

confidence: 99%

Section: Dynamic Cultivation Of Aggregatesmentioning

confidence: 99%

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Dynamic Cultivation of Mesenchymal Stem Cell Aggregates

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“…957/2011, 30 January 2013) and the donor gave written consent. Human adipose tissue-derived MSCs were isolated from a female donor (age 29) within 3 -6 h after surgery as described before [50]. Briefly, adipose tissue was obtained from abdominoplasty, minced with scissors and digested with collagenase type I (Sigma Aldrich, St. Louis, MO, USA).…”

Section: Cell Culturementioning

confidence: 99%

The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

Rampler¹,

Egger²,

Rusz³

et al. 2019

Preprint

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The molecular study of fat cell development in the human body is essential for our understanding of obesity and related diseases. Mesenchymal stem/stromal cells (MSC) are the ideal source to study fat formation as they are the progenitors of adipocytes. In this work, we used human MSCs, received from surgery waste, and differentiate them into fat adipocytes. The combination of several layers of information coming from lipidomics, metabolomics and proteomics enabled network analysis of the biochemical pathways in adipogenesis. Simultaneous analysis of metabolites, lipids and proteins in cell culture is challenging due to the compound's chemical difference so that most studies involve separate analysis with unimolecular strategies. In this study, we employed a multimolecular approach using a two-phase extraction to monitor the crosstalk between lipid metabolism and protein-based signaling in a single sample (~10 5 cells). We developed an innovative analytical workflow including standardization with in-house produced 13 C-isotopically labeled compounds, hyphenated high-end mass spectrometry (high-resolution Orbitrap MS) and chromatography (HILIC, RP) for simultaneous untargeted screening and targeted quantification. Metabolite and lipid concentrations ranged over 3-4 orders of magnitude and were detected down to the low fmol (absolute on column) level. Biological validation and data interpretation of the multiomics workflow was performed based on proteomics network reconstruction, metabolic modelling (MetaboAnalyst 4.0) and pathway analysis (OmicsNet). Comparing MSCs and adipocytes, we observed significant regulation of different metabolites and lipids such as triglycerides, gangliosides and carnitine with 113 fully reprogrammed pathways. The observed changes are in accordance with literature findings dealing with adipogenic differentiation of MSC. These results are a proof of principle for the power of multimolecular extraction combined with orthogonal LC-MS assays and network construction. Considering the analytical and biological validation performed in this study, we conclude that the proposed multiomics workflow is ideally suited for comprehensive follow-up studies on adipogenesis and is fit for purpose for different applications.

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“…However, the isolation procedure and the culture conditions during isolation can select subpopulations of MSCs and affect their function and potency 24 . Furthermore, cultivating MSCs in a 3D environment, either on scaffolds or scaffold-free as aggregates, is known to better reflect the physiological environment of MSCs and to have effects on cellular behavior and functionality [25][26][27] . Still, to the best of our knowledge, only one procedure for the isolation of MSCs into a 3D environment, avoiding selection by plastic adherence on a 2D surface, is available 28 .…”

Section: Introductionmentioning

confidence: 99%

From 3D to 3D: isolation of mesenchymal stem/stromal cells into a three-dimensional human platelet lysate matrix

Egger

Oliveira

Mallinger

et al. 2019

Preprint

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Mesenchymal stem/stromal cells (MSCs) are considered an important candidate in cell therapy and tissue engineering approaches. The culture of stem cells in a 3D environment is known to better resemble the in vivo situation and to promote therapeutically relevant effects in isolated cells. Therefore, the aim of this study was to develop an approach for the isolation of MSCs from adipose tissue into a 3D environment. Furthermore, the use of cryoprotective medium for cryopreservation of whole adipose tissue was evaluated. For the isolation of MSCs, a novel human platelet lysate-based hydrogel was used as matrix and the migration, yield, viability and metabolic activity of cells from the 3D matrix were compared to cells from 2D explant culture. Also, the surface marker profile and differentiation capacity of MSCs from the 3D matrix were evaluated and compared to MSCs from isolation by enzymatic treatment. We found that cryopreservation of whole adipose tissue is feasible, and therefore adipose tissue can be stored and is available for MSC isolation on demand. Also, we demonstrate the isolation of MSCs into the 3D matrix and that cells from this condition display a similar phenotype and differentiation capacity like MSCs derived by traditional isolation procedure. The presented approach allows, for the first time, to isolate MSCs directly into a soft 3D hydrogel environment, avoiding any contact to a 2D plastic culture surface. 3D explant isolation of MSCs 3 Significance Statement:In this paper we present a new method for the isolation of mesenchymal stem cells. Usually, these cells grow on two-dimensional plastic surfaces which is far away from their physiologic environment. Our new method allows for the first time the direct outgrowth of cells from primary tissue into a three-dimensional environment, avoiding any contact to a two-dimensional plastic surface. In future, this will allow an entirely three-dimensional in vitro cultivation of stem cells.Using 3D isolated cells will probably also increase the physiologic relevance of in vitro models. 3D explant isolation of MSCs4

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Hypoxic Three-Dimensional Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred Tank Reactor

Cited by 31 publications

References 42 publications

Dynamic Cultivation of Mesenchymal Stem Cell Aggregates

Dynamic Cultivation of Mesenchymal Stem Cell Aggregates

The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

From 3D to 3D: isolation of mesenchymal stem/stromal cells into a three-dimensional human platelet lysate matrix

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