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Oncolytic measles virus (MeV) is a promising anti-cancer treatment. However, the production of high titers of infectious MeV (typically 107–109 TCID50 per dose) is challenging because the virus is unstable under typical production conditions. The objective of this study was to investigate how the multiplicity of infection (MOI) and different media—a serum-containing medium (SCM), a serum-free medium (SFM) and two chemically defined media (CDM)—affect MeV production. We infected Vero cells at MOIs of 0.02, 0.2 or 2 TCID50 cell−1 and the lowest MOI resulted in the largest number of infected cells towards the end of the production period. However, this did not equate to higher maximum MeV titers, which were similar for all the MOIs. The medium had a moderate effect, generating maximum titers of 0.89–2.17 × 106, 1.08–1.25 × 106 and 4.58–9.90 × 105 TCID50 mL−1 for the SCM, SFM and CDM, respectively. Infection at a low MOI often required longer process times to reach maximum yields. On the other hand, a high MOI requires a large amount of MeV stock. We would therefore recommend a mid-range MOI of 0.2 TCID50 cell−1 for MeV production. Our findings show that SCM, SFM and CDM are equally suitable for MeV production in terms of yield and process time. This will allow MeV production in serum-free conditions, addressing the safety risks and ethical concerns associated with the use of serum. [ABSTRACT FROM AUTHOR]

The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up, we transferred HLSC expansion to multi-plate and stirred-tank bioreactor systems to determine their limitations. A seeding density of 4000 cells cm−2 was needed for efficient expansion. Although growth was not significantly affected by antibiotics, the concentrations of lactate and ammonia were important. A maximum expansion capacity of at least 20 cumulative population doublings (cPDs) was observed, confirming HLSC growth, identity and functionality. For the expansion of HLSCs in the multi-plate bioreactor system Xpansion (XPN), the oxygen supply strategy was optimized due to a low kLa of 0.076 h−1. The XPN bioreactor yielded a final mean cell density of 94 ± 8 × 103 cells cm−2, more than double that of the standard process in T-flasks. However, in the larger XPN50 device, HLSC density reached only 28 ± 0.9 × 103 cells cm−2, while the glucose consumption rate increased 8-fold. In a fully-controlled 2 L stirred-tank bioreactor (STR), HLSCs expanded at a comparable rate to the T-flask and XPN50 processes in a homogeneous microenvironment using advanced process analytical technology. Ultimately, the scale-up of HLSCs was successful using two different bioreactor systems, resulting in sufficient numbers of viable, functional and undifferentiated HLSCs for therapeutic applications. [ABSTRACT FROM AUTHOR]

Mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) are considered a promising therapeutic tool in cell therapy due to their immunomodulatory, regenerative and angiogenic capabilities. However, there is a lack of process knowledge, particularly for a large-scale production of MSC-EV using fully controlled stirred tank bioreactor (STR) systems. For the establishment of a STR-based process, we investigated dynamic process set-ups in spinner flasks, using three different microcarriers, as well as in shaking flasks, using microcarrier-free spheroids. An immortalized cell line (hMSC-TERT) and a particle-free chemically defined medium was used for all approaches. Cell characteristics (e.g., growth, metabolism, cell-specific particle production rates), MSC-EV epitope markers and MSC-EV potency in migration assays were analyzed. We showed that the transfer to a dynamic system (non-porous microcarrier, spinner flask) significantly increased the cell-specific particle production rate (6-fold) and the expression of EV-specific markers. Moreover, MSC proliferation and, most importantly, the therapeutic potency of MSC-derived particles including EVs was maintained. We demonstrated that high cell-specific particle production rates were associated with an increased glucose consumption rate rather than cell growth, which can be utilized for future process development. Furthermore, we showed that dynamic conditions of a controlled 1 L STR significantly increased the cell-specific particle production rate (24-fold) as well as the final concentration (3-fold) of potent MSC-derived particles including EVs. This indicates that fully controlled STRs are an efficient production system for MSC-derived particles including EVs that may open and facilitate the path for clinical applications. [ABSTRACT FROM AUTHOR]

Cancer therapy with oncolytic measles virus (MV) requires high titers of pure, infectious virus particles (~108 times more particles per dose than the measles vaccine). A gentle but efficient downstream purification process is therefore essential. Here we investigated the suitability of ion-exchange chromatography for the purification of MV using resin-based stationary phases. The success of purification was evaluated based on the infectious virus yield, the ratio of infectious to total MV particles, and the quantity of major contaminants (host cell DNA and total protein). After testing different anion-exchange, cation-exchange and hybrid materials, we found that the cation-exchanger Eshmuno ® CPX (Merck) was the most promising resin. By optimizing the pH of the feed and buffers, and reducing the flow rate, we achieved a yield of 80.7% infectious MV particles while reducing the ratio of infectious to total MV particles by only 16.8%. We also removed 98.3% of the total protein and 80.5% of the host cell DNA. Although resin-based chromatography is not yet typically used for virus purification, we have developed a promising new approach to improve the efficiency of MV purification during downstream processing. [ABSTRACT FROM AUTHOR]