Dynamic Cultivation of Human Mesenchymal Stem/stromal Cells for the Production of Extracellular Vesicles in a 3D Bioreactor System

Overview

Journal Biotechnol Lett

Specialties Biochemistry
Biotechnology

Date 2024 Feb 13

PMID 38349512

Authors

Ciarra Almeria

Rene Weiss

Maike Keck

Viktoria Weber

Cornelia Kasper

Dominik Egger

Affiliations

Soon will be listed here.

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 × 10 ± 1.5 × 10 and 9.7 × 10 ± 3.1 × 10 particles/mL) compared to static 2D culture (4.2 × 10 ± 7.5 × 10 and 3.9 × 10 ± 3.0 × 10 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 × 10 ± 1.1 × 10 particles/µg protein in 2D to 1.6 × 10 ± 8.3 × 10 particles/µg protein in 3D. Total MSC-EVs as well as CD73CD90 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.

References

Guo S, Debbi L, Zohar B, Samuel R, Arzi R, Fried A . Stimulating Extracellular Vesicles Production from Engineered Tissues by Mechanical Forces. Nano Lett. 2021; 21(6):2497-2504. DOI: 10.1021/acs.nanolett.0c04834. View

Huang T, Jia Z, Fang L, Cheng Z, Qian J, Xiong F . Extracellular vesicle-derived miR-511-3p from hypoxia preconditioned adipose mesenchymal stem cells ameliorates spinal cord injury through the TRAF6/S1P axis. Brain Res Bull. 2022; 180:73-85. DOI: 10.1016/j.brainresbull.2021.12.015. View

Tkach M, Thery C . Communication by Extracellular Vesicles: Where We Are and Where We Need to Go. Cell. 2016; 164(6):1226-1232. DOI: 10.1016/j.cell.2016.01.043. View

Kim H, Lee M, Bae E, Ryu J, Kaur G, Kim H . Comprehensive Molecular Profiles of Functionally Effective MSC-Derived Extracellular Vesicles in Immunomodulation. Mol Ther. 2020; 28(7):1628-1644. PMC: 7335740. DOI: 10.1016/j.ymthe.2020.04.020. View

Kang T, Jones T, Naddell C, Bacanamwo M, Calvert J, Thompson W . Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31. Stem Cells Transl Med. 2016; 5(4):440-50. PMC: 4798737. DOI: 10.5966/sctm.2015-0177. View

Lai R, Chen T, Lim S . Mesenchymal stem cell exosome: a novel stem cell-based therapy for cardiovascular disease. Regen Med. 2011; 6(4):481-92. DOI: 10.2217/rme.11.35. View

Almeria C, Kress S, Weber V, Egger D, Kasper C . Heterogeneity of mesenchymal stem cell-derived extracellular vesicles is highly impacted by the tissue/cell source and culture conditions. Cell Biosci. 2022; 12(1):51. PMC: 9063275. DOI: 10.1186/s13578-022-00786-7. View

Almeria C, Weiss R, Roy M, Tripisciano C, Kasper C, Weber V . Hypoxia Conditioned Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Increased Vascular Tube Formation . Front Bioeng Biotechnol. 2019; 7:292. PMC: 6819375. DOI: 10.3389/fbioe.2019.00292. View

Wang Y, Zhang Z, Chi Y, Zhang Q, Xu F, Yang Z . Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation. Cell Death Dis. 2013; 4:e950. PMC: 3877551. DOI: 10.1038/cddis.2013.480. View

10.

Camussi G, Deregibus M, Cantaluppi V . Role of stem-cell-derived microvesicles in the paracrine action of stem cells. Biochem Soc Trans. 2013; 41(1):283-7. DOI: 10.1042/BST20120192. View

11.

Ullah I, Subbarao R, Rho G . Human mesenchymal stem cells - current trends and future prospective. Biosci Rep. 2015; 35(2). PMC: 4413017. DOI: 10.1042/BSR20150025. View

12.

Yin K, Wang S, Zhao R . Exosomes from mesenchymal stem/stromal cells: a new therapeutic paradigm. Biomark Res. 2019; 7:8. PMC: 6450000. DOI: 10.1186/s40364-019-0159-x. View

13.

Yuan X, Sun L, Jeske R, Nkosi D, York S, Liu Y . Engineering extracellular vesicles by three-dimensional dynamic culture of human mesenchymal stem cells. J Extracell Vesicles. 2022; 11(6):e12235. PMC: 9206229. DOI: 10.1002/jev2.12235. View

14.

Cheng N, Chen S, Li J, Young T . Short-term spheroid formation enhances the regenerative capacity of adipose-derived stem cells by promoting stemness, angiogenesis, and chemotaxis. Stem Cells Transl Med. 2013; 2(8):584-94. PMC: 3726138. DOI: 10.5966/sctm.2013-0007. View

15.

Kouroupis D, Correa D . Increased Mesenchymal Stem Cell Functionalization in Three-Dimensional Manufacturing Settings for Enhanced Therapeutic Applications. Front Bioeng Biotechnol. 2021; 9:621748. PMC: 7907607. DOI: 10.3389/fbioe.2021.621748. View

16.

Mao C, Zhang T, Li D, Zhou E, Fan Y, He Q . Extracellular vesicles from hypoxia-preconditioned mesenchymal stem cells alleviates myocardial injury by targeting thioredoxin-interacting protein-mediated hypoxia-inducible factor-1α pathway. World J Stem Cells. 2022; 14(2):183-199. PMC: 8963381. DOI: 10.4252/wjsc.v14.i2.183. View

17.

Zhang X, Wang N, Huang Y, Li Y, Li G, Lin Y . Extracellular vesicles from three dimensional culture of human placental mesenchymal stem cells ameliorated renal ischemia/reperfusion injury. Int J Artif Organs. 2021; 45(2):181-192. DOI: 10.1177/0391398820986809. View

18.

Witwer K, van Balkom B, Bruno S, Choo A, Dominici M, Gimona M . Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications. J Extracell Vesicles. 2019; 8(1):1609206. PMC: 6493293. DOI: 10.1080/20013078.2019.1609206. View

19.

Fuzeta M, Bernardes N, Oliveira F, Costa A, Fernandes-Platzgummer A, Farinha J . Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System. Front Cell Dev Biol. 2020; 8:553444. PMC: 7669752. DOI: 10.3389/fcell.2020.553444. View

20.

Gardiner C, Ferreira Y, Dragovic R, Redman C, Sargent I . Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J Extracell Vesicles. 2013; 2. PMC: 3760643. DOI: 10.3402/jev.v2i0.19671. View