Upscaling manufacture of osteogenically differentiated mesenchymal stem cells using nanoscale vibration and microcarrier suspension culture

Purpose/Objectives: Bone is the second most transplanted tissue following blood, and over 2 million procedures are performed each year involving bone grafts, which poses issues of supply and efficacy. Nano-amplitude sinusoidal vibration (1 kHz, 30 nm) delivered continuously in a pistonic manner - by our bespoke nano-amplitude vibrational bioreactor - drives the differentiation of human mesenchymal stem cells (hMSCs) towards an osteoblast lineage, in both 2D and 3D environments, further maturation has been confirmed by the presence of mineralisation. Upscaling the nanovibrational cell stimulation process is key for supplying the large quantities of phenotypically controlled nanovibrated hMSCs needed for cellular therapies and orthopaedic use, e.g. for repair of critical-sized or non-union bone defects. The vibrational bioreactor can currently deliver precise vibration to T150 flasks and multi-layer CellSTACKs. However, microcarrier suspension culture systems offer almost unbound upscaling potential to provide sufficient numbers of cells for clinical and commercial viability of a new therapy.
*Methodology: In this present work an experimental setup designed to deliver nanoscale vibration to hMSCs adhered to polystyrene microcarriers, suspended in a 250 mL spinner flask, was developed, optimised, characterised, and tested (Figure 1A). The spinner flask was adapted such that the impeller was driven with an overhead motor, to enable application of nanovibration from its base. This osteogenic vibration is within a precise range, laser interferometry was used to quantify, shift and reduce as far as possible motor-induced vibration. Further, scanning laser vibrometry was used to confirm that suitable nanovibration was delivered by measuring the motion of the fluid’s top surface. With this validated setup, the potential to upscale the nanovibrational cell production process was assessed. Specifically, hMSCs were seeded onto microcarriers, 24 hours later the spinner flask was continuously nanovibrated and stimulation was applied for 3 weeks. Seeding efficiency, proliferation, stemness and osteogenic markers were assessed and compared to non-vibrated control spinner flasks, using a variety of techniques including RT-qPCR, immunofluorescence, alizarin red, micro-computed tomography (microCT) and flow cytometry.
*Results: Vibration analysis enabled fine-tuning of the setup to ensure that only lower frequency (<400 Hz) and lower amplitude (5 nm) vibration was introduced to setup due to motor-driven impeller rotation. Further, the fluid vibrated pistonically at 1 kHz (replicating prior results of 2D multiwell plate nanovibration). Proliferation, assessed via MTT assay showed slower growth in the vibrated flask. Osteogenesis and mineralisation on microcarriers were assessed with mRNA expression of relevant osteogenic markers (osteocalcin, osteopontin, RUNX2), osteocalcin immunofluorescence, alizarin red and quantifiable amounts of mineralisation detected with microCT (Figure 1B).
*Conclusion/Significance: The integration of nanoscale vibration and microcarrier suspension culture represents one of the first attempt to exploit mechanotransduction for cell therapy manufacture. Microcarrier culture is readily exploitable within good manufacturing practice (GMP) for advanced therapy medicinal products (ATMPs) and offers the potential to provide large quantities (billions) of nanovibrated “osteoprimed” hMSCs per batch. This development will be crucial to make a commercially viable and clinically relevant cell therapy for the repair of critical-sized (non-healing) bone defects.