Enhancing mesenchymal stem cells cultivated on microcarriers in spinner flasks via impeller design optimization for aggregated suspensions.

During the ex vivo expansion of umbilical cord-derived mesenchymal stem cells (hUCMSCs) in a stirred tank bioreactor, the formation of cell-microcarrier aggregates significantly affects cell proliferation and physiological activity, making it difficult to meet the quantity and quality requirements for in vitro research and clinical applications. In this study, computational fluid dynamic (CFD) simulations were used to investigate the effect of an impeller structure in a commercial spinner flask on flow field structure, aggregate formation, and cellular physiological activity. By designing a modified impeller, the aggregate size was reduced, which promoted cell proliferation and stemness maintenance. This study showed that increasing the stirring speed reduced the size of hUCMSC-microcarrier aggregates with the original impeller. However, it also inhibited cell proliferation, decreased activity, and led to spontaneous differentiation. Compared to low stirring speeds, high stirring speeds did not alter the radial flow characteristics and vortex distribution of the flow field, but did generate higher shear rates. The new impeller's design changed the flow field from radial to axial. The use of the novel impeller with an increased axial pumping rate (Qz) at a similar shear rate compared to the original impeller resulted in a 43.7% reduction in aggregate size, a 37.4% increase in cell density, and a better preservation of the expression of stemness markers (SOX2, OCT4 and NANOG). Increasing the Qz was a key factor in promoting aggregate suspension and size reduction. The results of this study have significant implications for the design of reactors, the optimisation of operating parameters, and the regulation of cellular physiological activity during MSC expansion.

Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor.

The higher ethanol titer inevitably requires higher solids loading during the simultaneous enzymatic saccharification and fermentation (SSF) using lignocellulose as the feedstock. The mixing between the solid lignocellulose and the liquid enzyme is crucially important. In this study, a bioreactor with a novel helical impeller was designed and applied to the SSF operation of the steam explosion pretreated corn stover under different solids loadings and different enzyme dosages. The performances using the helical impeller and the common Rushton impeller were compared and analyzed by measuring rheological properties and the mixing energy consumption. The results showed that the new designed stirring system had better performances in the saccharification yield, ethanol titer, and energy cost than those of the Rushton impeller stirring. The mixing energy consumption under different solids loadings and enzyme dosages during SSF operation were analyzed and compared to the thermal energy in the ethanol produced. A balance for achieving the optimal energy cost between the increased mixing energy cost and the reduced distillation energy cost at the high solids loading should be made. The potentials of the new bioreactor were tested under various SSF conditions for obtaining optimal ethanol yield and titer.

Affinity Chromatography of Urokinase Using Cefazoline as a Ligand.

Sepharose 4B-based affinity adsorbent was prepared using cefazoline as a ligand, and epichlorohydrin as an activator. The density of the ligand in the affinity adsorbents was about 43 &mgr;mol/g wet gel. The optimal adsorption pH for urokinase was 6.0, and the optimal adsorption salt concentration was 1.0 mol/L NaCl. The optimal conditions of elution were 0.1 mol/L glycine buffer, pH 9.0, containing 0.5 mol/L NaCl. A crude urokinase with specific activity 500 u/mg protein was purified on an affinity chromatography column (1 cm x 12 cm), yielding a product of specific activity of 49 300 u/mg with 78% recovery and 98.6-fold purification.

[Process strategy for ethanol production from lignocellulose feedstock under extremely low water usage and high solids loading conditions].

The massive water and steam are consumed in the production of cellulose ethanol, which correspondingly results in the significant increase of energy cost, waster water discharge and production cost as well. In this study, the process strategy under extremely low water usage and high solids loading of corn stover was investigated experimentally and computationally. The novel pretreatment technology with zero waste water discharge was developed; in which a unique biodetoxification method using a kerosene fungus strain Amorphotheca resinae ZN1 to degrade the lignocellulose derived inhibitors was applied. With high solids loading of pretreated corn stover, high ethanol titer was achieved in the simultaneous saccharification and fermentation process, and the scale-up principles were studied. Furthermore, the flowsheet simulation of the whole process was carried out with the Aspen plus based physical database, and the integrated process developed was tested in the biorefinery mini-plant. Finally, the core technologies were applied in the cellulose ethanol demonstration plant, which paved a way for the establishment of an energy saving and environment friendly technology of lignocellulose biotransformation with industry application potential.

[Perfusion culture of hematopoietic cells in a stirred tank bioreactor].

To optimize the culture environment and protocol of hematopoietic cells' expansion, avoiding the fluctuation caused by medium changing in stirred culture and concentration gradient in static culture, the hematopoietic cells from cord blood (CB) were cultured in a stirred bioreactor connected with a cell retention system, which is a gravity sedimentation settler designed for hematopoietic cell. Total cells expanded 11.5 and 18.6 fold respectively in the twice perfusion stirred cultures, in which CFU-Mix was expanded 23.2 and 20.4 fold, CFU-GM 13.9 fold and 21.5 fold, BFU-E 8.0 fold and 6.9 fold, CD34+ cells 17.1 fold and 15.4 fold. After 12-day culture, it was obtained that 1082 x 10(6) total cells, 6.31 x 10(6) CFU-GM, 6.2 x 10(6) CFU-Mix and 23 x 10(6) CD34+ cells from 267 x 10(6) CB mononuclear cells (MNC) in the first culture, and 1080 x 10(6) total cells, 4.65 x 10(6) CFU-GM, 11.0 x 10(6) CFU-Mix, and 25.0 x 10(6) CD34+ cells from 180 x 10(6) CB MNC. These two cultures met to the clinical scale. Due to the optimized dissolved oxygen (DO) and stable culture environment, the rate of stem/progenitor cells to total cells in the perfusion culture was higher than that in T-flask cell-retention feeding culture. But the cell growth was inhibited in the later phase of perfusion culture, when the cell density is high. The inhibition should be attribute to the high cell density itself. The perfusion culture environment in bioreactor with optimal DO and pH controlling is more favorable for stem/progenitor cells' maintenance and expansion, and the expanded cells' number has reached a clinical scale. But the high cell density in the later phase of perfusion culture caused inhibition to mature hematopoietic cell's growth.

[In vitro suspension and bioreactor culture of hematopoietic cells].

Stirred culture offers a number of advantages over static systems as it maintains a stable, homogeneous culture environment and is easy to scale-up. This paper focused on the development and application of stirred tank bioreactor to culture hematopoietic cells. Preliminary study of stirred culture of hematopoietic cells was carried out in cord blood mononuclear cells culture in spinner flask. The results showed that the amplification rates of total cell, CFU-GM and BFU-E, with the exception of CFU-Mk, were greater in spinner flask than T-flask. The number of total cells increased 20 fold after 14 days incubation in spinner flask. The amplification rates of CFU-GM, CFU-Mk and BFU-E reached maximum at 10th day, 10th day and 7th day respectively, and the maximal amplification rates were 9.2-fold, 5.5-fold and 2.4-fold respectively, whereas the rate of CD34+ cells in spinner flask was (6.7 +/- 4.0)-fold at day 10. These results indicated that the stirred culture system is better than the static culture systems for hematopoietic cell proliferation. The biocompatibility of cord blood MNC to different types of materials used in bioreactors was also tested. The results showed that glass, stainless steel 316L and polytetraflouroethylene (PTFE) supported the growth of hematopoietic cells well. A higher cell density was reached in stirred bioreactors with controlled pH and DO than static culture. These findings suggested that the controlled large-scale culture could be used to overcome the clinical shortage of hematopoietic cells.