Expansion of human mesenchymal stem/stromal cells on temporary liquid microcarriers

BACKGROUND: Traditional large-scale culture systems for human mesenchymal stem/stromal cells (hMSCs) use solid microcarriers as attachment substrates. Although the use of such substrates is advantageous because of the high surface-to-volume ratio, cell harvest from the same substrates is a challenge as it requires enzymatic treatment, often combined with agitation. Here, we investigated a two-phase system for expansion and non-enzymatic recovery of hMSCs. Perfluorocarbon droplets were dispersed in a protein-rich growth medium and were used as temporary liquid microcarriers for hMSC culture. RESULTS: hMSCs successfully attached to these liquid microcarriers, exhibiting similar morphologies to those cultured on solid ones. Fold increases of 3.03 ± 0.98 (hMSC1) and 3.81 ± 0.29 (hMSC2) were achieved on day 9. However, the maximum expansion folds were recorded on day 4 (4.79 ± 0.47 (hMSC1) and 4.856 ± 0.7 (hMSC2)). This decrease was caused by cell aggregation upon reaching confluency due to the contraction of the interface between the two phases. Cell quality, as assessed by differentiation, cell surface marker expression and clonogenic ability, was retained post expansion on the liquid microcarriers. Cell harvesting was achieved non-enzymatically in two steps: first by inducing droplet coalescence and then aspirating the interface. Quality characteristics of hMSCs continued to be retained even after inducing droplet coalescence. CONCLUSION: The prospect of a temporary microcarrier that can be used to expand cells and then ‘disappear’ for cell release without using proteolytic enzymes is a very exciting one. Here, we have demonstrated that hMSCs can attach and proliferate on these perfluorocarbon liquid microcarriers while, very importantly, retaining their quality

Process parameters for the high-scale production of alginate-encapsulated stem cells for storage and distribution throughout the cell therapy supply chain

AbstractWith the ever-increasing clinical application of cell-based therapies, it is considered critical to develop systems that facilitate the storage and distribution of cell therapy products (CTPs) between sites of manufacture and the clinic. For such systems to be realized, it is essential that downstream bioprocessing strategies be established that are scalable, reproducible and do not influence the viability or function of the living biologic. To this end, we examined alginate-encapsulation as a method to heighten the preservation of human adipose-derived stem cells (hASCs) during hypothermic storage, and establish a scalable process for high-volume production. A drop-wise method for scalable alginate bead generation, using calcium as the cross-linker, was modified to enable the yield of up to 3500 gelled beads per minute. The effect of alginate concentration on the viscosity of non-gelled sodium alginate and the mechanical properties and internal structure of calcium-crosslinked alginate in response to different alginate and calcium concentrations were investigated. Mechanical strength was chiefly dependent on alginate concentration and 1.2% alginate cross-linked with 100mM calcium chloride could withstand stress in the order of 35kPa. Upon selection of appropriate parameters, we demonstrated the suitability of using this method for immobilizing human stem cells. Encapsulated hASCs demonstrated no loss in cell viability, and had a uniform distribution after high-volume production. Following storage, released cells were able to attach and recover a normal morphology upon return to culture conditions. Thus we present a scalable method for stem cell encapsulation and storage for application within the cell therapy supply chain

The effect of scale and interfacial tension on liquid–liquid dispersion in in-line Silverson rotor–stator mixers

AbstractThe effect of scale, processing conditions, interfacial tension and viscosity of the dispersed phase on power draw and drop size distributions in three in-line Silverson rotor–stator mixers was investigated with the aim to determine the most appropriate scaling up parameter. The largest mixer was a factory scale device, whilst the smallest was a laboratory scale mixer. All the mixers were geometrically similar and were fitted with double rotors and standard double emulsor stators. 1wt.% silicone oils with viscosities of 9.4mPas and 339mPas in aqueous solutions of surfactant or ethanol were emulsified in single and multiple pass modes. The effect of rotor speed, flow rate, dispersed phase viscosity, interfacial tension and scale on drop size distributions was investigated.It was found that for all three scales, power draw is the sum of the rotor and flow contributions, with proportionality constants, PoZ and k1, that are practically scale independent. Sauter mean drop size appeared to correlate better with tip speed than energy dissipation rate. For ethanol/water solutions, mean drop size correlated well with Weber number based on interfacial tension, but for surfactant solutions effective interfacial tension gave better correlations with Weber number

Manufacturing of agarose-based chromatographic adsorbents with controlled pore and particle size

The effect of ionic strength of agarose solution and quenching temperature of the emulsion on the structure and mechanical strength of agarose-based chromatographic adsorbents was investigated. Solutions of agarose containing different amounts of NaCl were emulsified at elevated temperature in mineral oil using a high-shear mixer. The hot emulsion was quenched at different temperatures leading to the gelation of agarose and formation of soft particles. Analysis of Atomic Force Microscopy (AFM) images of particle surfaces shows that pore size of particles increases with ionic strength and/or high quenching temperature. Additionally it has been found that the compressive strength of particles measured by micromanipulation also increases with ionic strength of the emulsion and/or high quenching temperature but these two parameters have no significant effect on the resulting particle size and particle size distribution. Results from both characterization methods were compared with Sepharose 4B, a commercial agarose-based adsorbent. This is the first report examining the effect of ionic strength and cooling conditions on the microstructure of micron-sized agarose beads for bioseparation

High flow, low shear impellers versus high shear impellers; dispersion of oil drops in water and other examples

Flow patterns generated by two ChemShear impellers, CS 2 and CS 4 have been measured and flow numbers calculated; Fl = 0.04 for both impellers. Transient and equilibrium drop sizes, d32 μm of 3 different viscosity silicone oils agitated by a high-shear Rushton turbine, RT, a low-shear, high-flow HE3 impeller and the two ChemShears were determined. The equilibrium d32 are correlated for all impellers, by d32 = 1300(εT )−0.58 max.svv0.14 with an R2 = 0.94. However, the time to reach steady state and the equilibrium size at the same specific power do not match the above descriptors of each impeller’s characteristics. In other literature, these descriptors are also misleading. In the case of mixing time, a high shear RT of the same size as a high flow HE3 requires the same time at the same specific power in vessels of H/T = 1. In bioprocessing, where concern for damage to cells is always present, free suspension animal cell culture with high shear RTs and low-shear impellers is equally effective; and with mycelial fermentations, damage to mycelia is greater with low shear than high. The problems with these descriptors have been known for some time but mixer manufacturers and ill-informed users and researchers continue to employ them