Supplementary MaterialsSupplementary Information 41598_2018_31019_MOESM1_ESM

Supplementary MaterialsSupplementary Information 41598_2018_31019_MOESM1_ESM. due to Rabbit Polyclonal to TEAD1 distinctive features and fragility of these cell products. This work demonstrates a novel alternative approach which utilizes inertial focusing to separate microcarriers (MCs) from the final cell suspension. First, we systematically investigated MC focusing dynamics inside scaled-up curved channels with trapezoidal and rectangular cross-sections. A trapezoidal spiral channel with ultra-low-slope (Tan()?=?0.0375) was found to contribute to strong MC focusing (~300? ?Re? ?~400) while managing high MC volume fractions up to ~1.68%. Accordingly, the high-throughput trapezoidal spiral channel successfully separated MCs from hMSC suspension with total cell yield~94% (after two passes) at a high volumetric flow rate of ~30?mL/min (Re~326.5). Introduction Off-the-shelf (allogeneic) therapies transplanting human mesenchymal stem cells (hMSCs), derived mainly from bone-marrow, adipose tissue, and umbilical cord blood tissue1, are widely adopted due to hMSCs regenerative, immunosuppressive, and multipotent features2,3. The clinical demand for hMSCs is rising significantly, with more than 400 registered clinical trials4,5, and the required doses per patient can reach up to 109 cells1,6,7. For instance, the number of cells is estimated to be ~1012 cells per lot for diseases that need high doses of ~108-109 cells to be delivered. Using multilayer tissue culture flasks cannot meet the demand efficiently for cell therapy products beyond the scale of 100 billion cells1,8,9. Thus, embracing alternative methods for cell expansion is necessary. Bioreactors, for scaling up the cultures in 3D rather than scaling out the cell culture flask in 2D, are used as an efficient and cost-effective approach to commercialization10C12. Among different adherent cell bioreactors, employing suspension scaffolds so-called microcarriers (MCs), ~100C300?m in diameter, within a stirred tank has been widely recognized7,13; recently it was demonstrated within a 50-L bioreactor that a 43-fold expansion of hMSCs could be reached in 11 days14. Using microcarriers, however, necessitates clarification of cell suspension bulk and downstream removal of MCs. Following cell expansion and detachment from microcarriers, existing systems for separation of MCs and cells are tangential flow filtrations (TFF), counter-flow centrifugation elutriations (CCE), and dead-end sieving8. However, clogging (cake formation) and high shear stress for sieve-based systems15,16, as well as high operative costs due to bulkiness and rotating parts for CEE systems such as KSep platform (Sartorious), pose disadvantages. Herein, we report on the advancement of an alternative method using inertial focusing C shown recently to be scalable for filtration of large-scale lot size in the order of liter per min17C20. The inertial focusing phenomenon is only reliant on hydrodynamic forces, therefore, it gives rise to the relatively ease of parallelization to scale out the throughput. A high-throughput cell retention device was recently introduced; it utilized spiral channels for perfusion bioreactors while the projected device footprint for overall ~1000?L perfusion rate during one day was approximated to be 100?mm??80?mm??300?mm17,18, noticeably smaller when Daidzein compared to other CEE systems. Furthermore, the inertial-based filtration is a continuous clog-free (or membrane-less) system thereby sustaining reliable Daidzein steady performance without declining during long-term operation, and obviating the need for filter replacement. In this work, we first systematically investigated inertial focusing of microcarriers in scaled-up spiral channels (channel size ?0.5?mm). Afterward, removal of microcarriers from hMSCs suspension was accomplished by inertial focusing with ~99% purity while cell harvest yield reached ~94%. Design Principle Inertial focusing for neutrally-buoyant particles flowing inside a channel occurs when the particle radius is comparable to the channel hydraulic diameter, where Re is channel Reynolds number, DH and R are channel hydraulic diameter and radius of curvature respectively) by 60% across the spiral channels. In Daidzein other words, the difference in positive secondary flow between two spirals increases particularly at the downstream loops (3rd to 4th loop), as shown in Fig.?2c. This illustrates the enhanced secondary flow drag (FD~UD where UD is secondary velocity) sweeping particles (microcarriers) toward the inner wall to establish focusing only in an ultra-low-slope trapezoidal spiral (Results?Section). Because inertial focusing of MCs near the inner wall cannot be interpreted solely as a result of positive secondary flow without considering the shear force; we investigated MC focusing dynamics experimentally due to the lack of a shear-gradient force model exclusively for spiral channels. Material and Methods Channel fabrication Aluminum master molds were fabricated via micro-milling technique (Whits Technologies, Singapore). After casting the mixed polydimethylsiloxane polymer (PDMS, Sylgard 184 Silicone Elastomer Kit, Dow Corning) and curing agent (10:1 ratio) into the mold, it was cured for 30?min in an oven with 80?C. To boost bonding, we used semi-cured PDMS.