Perfluorocarbon facilitated O2 transport in a hepatic hollow fiber bioreactor

A mathematical model describing O₂ transport in a hepatic hollow fiber (HF) bioreactor supplemented with perfluorocarbons (PFCs) in the circulating cell culture media was developed to explore the potential of PFCs in properly oxygenating a bioartificial liver assist device (BLAD). The 2‐dimensional model is based on the geometry of a commercial HF bioreactor operated under steady‐state conditions. The O₂ transport model considers fluid motion of a homogeneous mixture of cell culture media and PFCs, and mass transport of dissolved O₂ in a single HF. Each HF consists of three distinct regions: (1) the lumen (conducts the homogeneous mixture of cell culture media and PFCs), (2) the membrane (physically separates the lumen from the extracapillary space (ECS), and (3) the ECS (hepatic cells reside in this compartment). In a single HF, dissolved O₂ is predominantly transported in the lumen via convection in the axial direction and via diffusion in the radial direction through the membrane and ECS. The resulting transport equations are solved using the finite element method. The calculated O₂ transfer flux showed that supplementation of the cell culture media with PFCs can significantly enhance O₂ transport to the ECS of the HF when compared with a control with no PFC supplementation. Moreover, the O₂ distribution and subsequent analysis of ECS zonation demonstrate that limited in vivo‐like O₂ gradients can be recapitulated with proper selection of the operational settings of the HF bioreactor. Taken together, this model can also be used to optimize the operating conditions for future BLAD development that aim to fully recapitulate the liver's varied functions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

红细胞伪装纳米颗粒：一个具有潜力的药物及疫苗递送系统

红细胞伪装纳米颗粒是一种以红细胞或红细胞膜纳米囊泡为载体在体内递送药物、酶、多肽和抗原等物质的系统,具有生物相容性好、循环周期长、靶向性强等优势。本文从红细胞载体的种类、发展历程、递送策略应用以及其局限性和未来的挑战等方面进行了详细阐述,并展望了其未来的发展方向。     

Comparison of cell growth in T-flasks, in micro hollow fiber bioreactors, and in an industrial scale hollow fiber bioreactor system

In this article, cell growth in a novel micro hollow fiberbioreactor was compared to that in a T-flask and theAcuSyst-Maximizer®, a large scale industrial hollowfiber bioreactor system. In T-flasks, there was relativelylittle difference in the growth rates of one murine hybridomacultured in three different media and for three other murinehybridomas cultured in one medium. However, substantialdifferences were seen in the growth rates of cells in themicro bioreactor under these same conditions. These differencecorrelated well with the corresponding rates of initial cellexpansion in the Maximizer. Quantitative prediction of thesteady-state antibody production rate in the Maximizer was moreproblematic. However, conditions which lead to faster initialcell growth and higher viable cell densities in the microbioreactor correlated with better performance of a cell line inthe Maximizer. These results demonstrate that the microbioreactor is more useful than a T-flask for determining optimalconditions for cell growth in a large scale hollow fiberbioreactor system.     

Protein-free human-human hybridoma cultures in an intercalated-spiral alternate-dead-ended hollow fiber bioreactor

Batch cell cultures of a human-human hybridoma line in a convective flow dominant intercalated-spiral altetnate-dead-ended hollow fiber are compared with those using conventional axial-flow hollow fiber bioreactors and a stirred-tank bioreactor. Relatively short-term fed-batch and perfusion cell cultures were also employed for the intercalated-spiral bioreactor. When operating conditions of a batch intercalated-spiral bioreactor were properly chosen, the cell growth and substrate consumption paralleled that of a batch stirred-tank culture. The results verified the premise of the intercalated-spiral hollow fiber bioreactor that nutrient transport limitations can be eliminated when the convective flux through the extracapillary space is sufficiently high.(c) John Wiley & Sons, Inc.     

Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor

Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described. Hep G2 cells (a human hepatoma cell line) were cultivated in an Acysyst-P^® (Endotronic) with a total fiber surface area of 7.2 m² (6×1.2 m²) to produce Hep G2 crude conditioned medium (CCM). Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells. We have succeeded in growing the Hep G2 cells in an antibiotics-and serum-free IMDM medium, supplemented with 50g/ml of Hep G2 CCM protein at inoculation. The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS). The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated. Hep G2 CCM (20–40 g protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro. The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents.     

Polymerization of human hemoglobin using the crosslinker 1,11‐bis(maleimido)triethylene glycol for use as an oxygen carrier

Vasoconstriction and systemic hypertension are the main side effects associated with transfusion of current commercial polymerized hemoglobins (PolyHbs). It is hypothesized that the presence of free tetrameric hemoglobin (Hb) in the PolyHb solution is the root cause of these side effects. Therefore, increasing the size of PolyHbs and reducing the amount of free Hb in solution should dually reduce the extent of vasoconstriction and systemic hypertension. However, all current commercial PolyHb preparations have a small fraction of free Hb in solution. Hence, there is an urgent need to develop novel chemical strategies to synthesize large PolyHb molecules with a higher degree of polymerization without free Hb in solution. In this study, a Hb‐based oxygen carrier was synthesized by polymerizing human Hb using a dimaleimide poly(ethylene glycol) derivative (1,11‐bis(maleimido)triethylene glycol). The resultant PolyHb has a weight‐averaged molecular weight of 1.49 ± 0.62 MDa, O₂ affinity (P₅₀) of 2.75 ± 0.55 mm Hg, and Hill coefficient (n) of 0.97 ± 0.07. Light scattering analysis of the PolyHb dispersion confirmed the absence of free Hb monomers, dimers, and tetramers in solution. This work is significant, as it should enable future engineering of nonvasoactive PolyHbs with potential applications in transfusion medicine. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

CB.Hep-1 hybridoma growth and antibody production using protein-free medium in a hollow fiber bioreactor

The protein-free medium TurboDoma HP.1 (THP.1) was used to produce the CB.Hep-1 monoclonal antibody (mAb) in a CP-1000 hollow fiber bioreactor (HFB). This mAb is used for the immunopurification of recombinant hepatitis B surface antigen (rHBsAg), which is included in a vaccine preparation against the Hepatitis B Virus. By using the experimental conditions tested in this work we were able to generate more than 433 mg of IgG in 43 days. The maximum antibody concentration obtained was about 2.4 mg ml^-1and the IgG production per day was approximately 11 mg of monoclonal antibody, which constitutes a good concentration value in comparison to the results obtained in ascitic fluid, where concentration for this hybridoma was around 3 mg ml^-1. We used different analytical methods to control the quality of mAbs, obtained from the in vitro system. They included affinity constant determination, analysis of N-glycan structures, immunoaffinity chromatography and antigen binding properties. The results obtained suggest that no significant changes occurred in the mean characteristics of the mAb harvested from the bioreactor during the 43 days of cultivation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor™. Part I. Effect of the cell density on the process

High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor? using external hollow fiber filter as cell separation device. Both “classical” tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF‐ and ATF‐based cultures was shown at 20–35 × 10⁶ cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by‐product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9–1.3 × 10⁸ cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 × 10⁸ cells/mL, achieved for the first time in a wave‐induced bioreactor, and 1.32 × 10⁸ cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO₂ level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 × 10⁸ cell/mL based on the analysis of the theoretical distance between the cells for the present cell line. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:754–767, 2013     

Enhancing Cell Viability with Pulsating Flow in a Hollow Fiber Bioartificial Liver

A pulsating flow of medium was used to alleviate diffusion and transport limitations in a hollow fiber bioreactor containing a human hepatoblastoma cell line. The strategy is easy to implement but effective. The pulsating flow is introduced by a solenoid pinch valve at the outlet of the bioreactor and regulated by a timing circuit. In a permeability test, the system with pulsating flow had much less membrane fouling as compared to the control, a conventional hollow fiber unit. In hepatocyte culture test runs, the pulsating-flow bioreactor demonstrated the ability to maintain a higher cell viability. Histological sections indicated significantly smaller necrotic regions in the pulsating-flow bioreactor as compared to the conventional unit.