Culture of Hoffa fat pad mesenchymal stem/stromal cells on microcarrier suspension in vertical wheel bioreactor for extracellular vesicle production

Alexander Otahal*, Karina Kramer, Markus Neubauer, Slavomira Gulová, Zsombor Lacza, Stefan Nehrer, Andrea De Luna

*Corresponding author for this work

Research output: Journal article (peer-reviewed)Journal article


BACKGROUND: Mesenchymal stromal cells (MSCs) are increasingly employed in regenerative medicine approaches for their immunomodulatory and anti-inflammatory properties, which are encoded in their secretome including extracellular vesicles (EVs). The Hoffa fat pad (HFP) located infrapatellarly harbours MSCs that could assist in tissue homeostasis in osteoarthritic joints. Intraarticular injection therapies based on blood products could modulate the populations of released HFP-MSC-EVs in a quantitative manner.

METHODS: To obtain amounts of HFP-MSC-derived EVs that allow pre-clinical evaluation, suitable EV production systems need to be developed. This work investigates the release of EVs from primary HFP-MSCs cultivated in a 3D environment using microcarrier suspension culture in a vertical wheel bioreactor in comparison to conventional 2D culture. To simulate an intraarticular blood product therapy, cultures were treated with citrate-anticoagulated platelet-rich plasma (CPRP) or hyperacute serum (hypACT) before EV collection. HFP-MSC-EVs are enriched via ultrafiltration and characterised via Western Blot, nanoparticle tracking analysis in scatter as well as fluorescence mode. EV potency was determined via RT-qPCR analysing the expression of type II and X collagen (COL2 and COL10), as well as inducible nitric oxide synthase (iNOS) in primary OA chondrocytes.

RESULTS: Blood product supplementation elevated HFP-MSC metabolic activity as determined via XTT assay over the course of 14 days. 3D culture resulted in a roughly 100-fold EV yield compared to 2D culture and elevated number of EVs released per cell. Total protein content correlated with the EV concentration. While typical EV marker proteins such as CD9, CD63 or Alix were detected in total protein extracts, CD9 and CD73 colocalised on individual EVs highlighting their cell origin. The type of blood product treatment did not affect the size or concentration of EVs obtained from HFP-MSCs. Assessing potency of 3D culture EVs in comparison to 2D EVs revealed superior biological activity with regard to inhibition of inflammation, inhibition of chondrocyte hypertrophy and induction of cartilage-specific ECM production.

CONCLUSIONS: HFP-MSCs proliferate in presence of human blood products indicating that animal serum in culture media can be avoided in the future. The culture of HFP-MSCs in the employed bioreactor was successfully used to generate quantities of EVs that could allow evaluation of HFP-MSC-EV-mediated effects in pre-clinical settings. In addition, EV potency of 3D EVs is superior to EVs obtained in conventional 2D culture flasks.

Original languageEnglish
Article number61
Pages (from-to)61
JournalStem Cell Research and Therapy
Issue number1
Publication statusPublished - 05 Mar 2024


  • Animals
  • Humans
  • Suspensions
  • Mesenchymal Stem Cells
  • Adipose Tissue
  • Biological Assay
  • Bioreactors


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