Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs. Introduction Mesenchymal stem/stromal cells (MSCs)-based therapies have potential utility in the treatment of inflammatory diseases, the direct regeneration of mesenchymal tissues, or the up-regulation of innate tissue repair processes [1]. The most widely studied and best characterized MSCs are derived from bone marrow [2]. However, MSCs can be isolated from other tissues that may be more accessible, including placenta, adipose tissue and umbilical cord [3C5]. Placental-derived MSCs (pMSCs) are an attractive source of MSCs, as they not only behave similarly to bone marrow derived MSCs [5], but a single placenta (500C700 g tissue) is sufficient for manufacturing several hundred units of allogeneic MSCs [6]. Regardless of the tissue source, MSC populations will require expansion to generate clinically relevant cell numbers. Many promising therapies require single or multiple doses of approximately 2 x 106 cells/kg [7]. For MSC-based therapies to become a routine and economically viable treatment approach, the most efficient and cost effective method for their large-scale manufacture will require an automated closed-system bioreactor. Bioreactor designs used for MSC expansion include micro-carrier suspensions in spinner flasks, stirred tank reactors, and perfusion reactors, such as fixed beds or hollow fibre bioreactors [6,8C10]. Simple micro-carrier suspension cultures achieve a large surface area for adherent cell culture. However, there is no connectivity between individual micro-carriers, and empty micro-carriers do not contribute to the total surface area available to the culture. As a result, some micro-carriers rapidly reach confluence, whilst others remain empty; this requires frequent passaging to overcome localized space limitations [6,11]. Micro-carrier cultures also Neostigmine bromide IC50 require mixing to enable nutrient exchange and prevent concentration gradients. The shear forces arising from Neostigmine bromide IC50 mixing must be carefully modulated, as this can compromise MSC stemness characteristics during expansion [12,13]. Packed bed bioreactors potentially solve both problems by providing a continuous and connected surface area with no need for mixing. However, the maximum perfusion flow velocity cannot exceed 3 x 10?4 m/s without compromising the growth rate [14]. This greatly limits the scalability, as both soluble nutrients and oxygen must be supplied by medium perfusion alone. The bioreactor design described here overcomes these problems by incorporating a gas permeable polydimethylsiloxane (PDMS) shell, which decouples the bulk medium perfusion from the supply of oxygen. This allows a reduced perfusion flow rate or even a single pass medium supply. Fused polystyrene pellets are used to create a scaffold, that is definitely consequently air flow plasma treated to generate charged practical organizations on the surface, which promotes cell attachment related to commercial cells tradition polystyrene (TCP) [15]. Bubble formation within the bioreactor caused by pressure drops and temp changes across the bioreactor was prevented by pressurizing the waste tank to 2 PSI. The system was in the beginning optimised using an immortalized murine MSC human Neostigmine bromide IC50 population, and then the system was shown to become appropriate for the direct remoteness of pMSCs from placental cells break down and Neostigmine bromide IC50 subsequent development. Materials and Methods Solitary Pass Small-Scale Bioreactor Design This system contained a 1.5 cm diameter by 7.5 cm long scaffold providing a total surface area of 160 cm2, connected to a single complete circuit (Fig 1A). A 5 mm solid polydimethylsiloxane (PDMS, Dow Corning, MI, USA) tube was moulded to just match the polystyrene scaffold with an additional 1 cm head space to function as a bubble capture. Perfused medium was driven by a syringe pump (New Era Pump Systems Inc., NE-1800, Farmingdale, NY, Neostigmine bromide IC50 Col4a6 USA) that was managed outside the incubator. Medium was firstly perfused through a 30 cm size of 16.