Dynamic cultivation of human mesenchymal stem/stromal cells for the production of extracellular vesicles in a 3D bioreactor system.

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology Letters Pub Date : 2024-04-01 Epub Date: 2024-02-13 DOI:10.1007/s10529-024-03465-4

Ciarra Almeria, René Weiss, Maike Keck, Viktoria Weber, Cornelia Kasper, Dominik Egger

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Abstract

Purpose: 3D cell culture and hypoxia have been demonstrated to increase the therapeutic effects of mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs). In this study, a process for the production of MSC-EVs in a novel 3D bioreactor system under normoxic and hypoxic conditions was established and the resulting EVs were characterized.

Methods: Human adipose-derived MSCs were seeded and cultured on a 3D membrane in the VITVO® bioreactor system for 7 days. Afterwards, MSC-EVs were isolated and characterized via fluorescence nanoparticle tracking analysis, flow cytometry with staining against annexin V (Anx5) as a marker for EVs exposing phosphatidylserine, as well as CD73 and CD90 as MSC surface markers.

Results: Cultivation of MSC in the VITVO® bioreactor system demonstrated a higher concentration of MSC-EVs from the 3D bioreactor (9.1 × 10⁹ ± 1.5 × 10⁹ and 9.7 × 10⁹ ± 3.1 × 10⁹ particles/mL) compared to static 2D culture (4.2 × 10⁹ ± 7.5 × 10⁸ and 3.9 × 10⁹ ± 3.0 × 10⁸ particles/mL) under normoxic and hypoxic conditions, respectively. Also, the particle-to-protein ratio as a measure for the purity of EVs increased from 3.3 × 10⁷ ± 1.1 × 10⁷ particles/µg protein in 2D to 1.6 × 10⁸ ± 8.3 × 10⁶ particles/µg protein in 3D. Total MSC-EVs as well as CD73^-CD90⁺ MSC-EVs were elevated in 2D normoxic conditions. The EV concentration and size did not differ significantly between normoxic and hypoxic conditions.

Conclusion: The production of MSC-EVs in a 3D bioreactor system under hypoxic conditions resulted in increased EV concentration and purity. This system could be especially useful in screening culture conditions for the production of 3D-derived MSC-EVs.

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在三维生物反应器系统中动态培养人类间充质干细胞/基质细胞以生产细胞外囊泡。

目的：三维细胞培养和缺氧已被证明能提高间充质干/基质细胞（MSCs）衍生的细胞外囊泡（EVs）的治疗效果。本研究建立了一种在常氧和缺氧条件下在新型三维生物反应器系统中生产间充质干细胞-细胞外囊泡的工艺，并对所产生的细胞外囊泡进行了表征。随后，通过荧光纳米粒子追踪分析、流式细胞术（以附件素 V（Anx5）作为暴露于磷脂酰丝氨酸的 EVs 的标记）以及作为间充质干细胞表面标记的 CD73 和 CD90，对间充质干细胞-EVs 进行分离和表征：结果：在 VITVO® 生物反应器系统中培养间充质干细胞显示，在常氧和缺氧条件下，三维生物反应器中的间充质干细胞-EV（9.1 × 109 ± 1.5 × 109 和 9.7 × 109 ± 3.1 × 109 颗粒/毫升）浓度高于静态二维培养（4.2 × 109 ± 7.5 × 108 和 3.9 × 109 ± 3.0 × 108 颗粒/毫升）。此外，作为EVs纯度的衡量标准，颗粒与蛋白的比率从二维的3.3 × 107 ± 1.1 × 107颗粒/微克蛋白增加到三维的1.6 × 108 ± 8.3 × 106颗粒/微克蛋白。在二维常氧条件下，间充质干细胞EVs总量以及CD73-CD90+间充质干细胞EVs都有所增加。EV的浓度和大小在常氧和缺氧条件下没有显著差异：结论：在缺氧条件下的三维生物反应器系统中生产间充质干细胞EV可提高EV的浓度和纯度。结论：在缺氧条件下的三维生物反应器系统中生产间充质干细胞EV可提高EV的浓度和纯度，该系统对筛选生产三维间充质干细胞EV的培养条件特别有用。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.