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Novel low shear 3D bioreactor for high purity mesenchymal stem cell production.

ABSTRACT: Bone marrow derived human Mesenchymal Stem Cells (hMSCs) are an attractive candidate for regenerative medicine. However, their harvest can be invasive, painful, and expensive, making it difficult to supply the enormous amount of pure hMSCs needed for future allogeneic therapies. Because of this, a robust method of scaled bioreactor culture must be designed to supply the need for high purity, high density hMSC yields. Here we test a scaled down model of a novel bioreactor consisting of an unsubmerged 3D printed Polylactic Acid (PLA) lattice matrix wetted by culture media. The growth matrix is uniform, replicable, and biocompatible, enabling homogenous cell culture in three dimensions. The goal of this study was to prove that hMSCs would culture well in this novel bioreactor design. The system tested resulted in comparable stem cell yields to other cell culture systems using bone marrow derived hMSCs, while maintaining viability (96.54% ±2.82), high purity (>98% expression of combined positive markers), and differentiation potential.

SUBMITTER: Burns AB

PROVIDER: S-EPMC8208585 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Novel low shear 3D bioreactor for high purity mesenchymal stem cell production.

Burns Andrew B AB Doris Corinna C Vehar Kevin K Saxena Vinit V Bardliving Cameron C Shamlou Parviz A PA Phillips M Ian MI

PloS one 20210616 6

Bone marrow derived human Mesenchymal Stem Cells (hMSCs) are an attractive candidate for regenerative medicine. However, their harvest can be invasive, painful, and expensive, making it difficult to supply the enormous amount of pure hMSCs needed for future allogeneic therapies. Because of this, a robust method of scaled bioreactor culture must be designed to supply the need for high purity, high density hMSC yields. Here we test a scaled down model of a novel bioreactor consisting of an unsubme ...[more]

PMID: 34133442

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Project description:BackgroundMesenchymal 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.MethodsTo 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.ResultsBlood 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.ConclusionsHFP-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.

Dataset Information

Novel low shear 3D bioreactor for high purity mesenchymal stem cell production.

Publications

Novel low shear 3D bioreactor for high purity mesenchymal stem cell production.

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