Hemoglobin regulates the metabolic and synthetic function of rat insulinoma cells cultured in a hollow fiber bioreactor

Pancreatic islet transplantation continues to benefit patients with type 1 diabetes by normalizing glucose metabolism and improving other complications of diabetes. However, islet transplantation therapy is limited by the inadequate availability of pancreatic islets. In order to address this concern, this work investigated the expansion of rat insulinoma cells (INS‐1) and their ability to generate insulin in a hollow fiber bioreactor (HFB). The long‐term goal of this project is to develop a bioartificial pancreas. HFBs were incubated at two different oxygenation conditions (10% and 19% O₂) to determine the best scenario for O₂ transport to cultured cells. Also, bovine hemoglobin (BvHb) was supplemented in the cell culture media of the HFBs in order to increase O₂ transport under both oxygenation conditions. Our results show that INS‐1 cells expanded under all oxygenation conditions after 2 weeks of culture, with a slightly higher cell expansion under normoxic oxygenation (19% O₂) for both control HFBs and BvHb HFBs. In addition, cellular insulin production remained steady throughout the study for normoxic control HFBs and BvHb HFBs, while it increased under hypoxic oxygenation (10% O₂) for both types of HFBs but to different extents. Under the two different oxygenation conditions, cellular insulin production was more uniform with time in BvHb HFBs versus control HFBs. These results, along with qRT‐PCR analysis, suggest a possible dysregulation of the insulin‐signaling pathway under hypoxic culture conditions. In conclusion, the HFB culture system is an environment capable of expanding insulinomas while maintaining their viability and insulin production capabilities. Biotechnol. Bioeng. 2010;107: 582–592. © 2010 Wiley Periodicals, Inc.     

Optimising Cell Aggregate Expansion in a Perfused Hollow Fibre Bioreactor via Mathematical Modelling

The need for efficient and controlled expansion of cell populations is paramount in tissue engineering. Hollow fibre bioreactors (HFBs) have the potential to meet this need, but only with improved understanding of how operating conditions and cell seeding strategy affect cell proliferation in the bioreactor. This study is designed to assess the effects of two key operating parameters (the flow rate of culture medium into the fibre lumen and the fluid pressure imposed at the lumen outlet), together with the cell seeding distribution, on cell population growth in a single-fibre HFB. This is achieved using mathematical modelling and numerical methods to simulate the growth of cell aggregates along the outer surface of the fibre in response to the local oxygen concentration and fluid shear stress. The oxygen delivery to the cell aggregates and the fluid shear stress increase as the flow rate and pressure imposed at the lumen outlet are increased. Although the increased oxygen delivery promotes growth, the higher fluid shear stress can lead to cell death. For a given cell type and initial aggregate distribution, the operating parameters that give the most rapid overall growth can be identified from simulations. For example, when aggregates of rat cardiomyocytes that can tolerate shear stresses of up to are evenly distributed along the fibre, the inlet flow rate and outlet pressure that maximise the overall growth rate are predicted to be in the ranges to (equivalent to to ) and to (or 15.6 psi to 15.7 psi) respectively. The combined effects of the seeding distribution and flow on the growth are also investigated and the optimal conditions for growth found to depend on the shear tolerance and oxygen demands of the cells.     

The pleiotropic functions of intracellular hydrophobins in aerial hyphae and fungal spores

Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins–hydrophobins (HFBs)–that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.     

Two Novel Class II Hydrophobins from Trichoderma spp. Stimulate Enzymatic Hydrolysis of Poly(Ethylene Terephthalate) when Expressed as Fusion Proteins

Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET.     

Rapid Selection and Proliferation of CD133(+) Cells from Cancer Cell Lines: Chemotherapeutic Implications

Cancer stem cells (CSCs) are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133(+)] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB) (Celdyne, Houston, TX). For comparison, another bioreactor, the rotary cell culture system (RCCS) manufactured by Synthecon (Houston, TX) was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133(+) cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a (+)15-fold proliferation of the CD133(+) cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (−)4.8-fold decrease in the CD133(+)cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133(+) cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.     

Economic comparison of diagnostic antibody production in perfusion stirred tank and in hollow fiber bioreactor processes

The total operating costs of small-scale monoclonal antibody production were calculated for two different upstream options and general downstream procedure based on protein A chromatography. The upstream options were a spin-filter equipped stirred-tank bioreactor (STR) and a hollow fiber bioreactor (HFB). Both the bioreactors were operated in perfusion mode. The total operating costs of the processes were 6,900 €/g for STR option and 6,400 €/g for the HFB option. In the both systems, the costs were dominated by expenses derived from the downstream section (almost 80%) that was almost identical in the both systems. In the upstream section, the investment depreciation was the largest cost item. The lower total costs of the HFB option were a result of lower investment costs and more concentrated product that led into savings also in downstream section. This study brings out the HFB as on viable alternative for stirred-tank bioreactor, especially in small-scale diagnostic monoclonal antibody production.     

Detoxification of organophosphate nerve agents by immobilized dual functional biocatalysts in a cellulose hollow fiber bioreactor

A whole-cell technology for detoxification of organophosphates based on genetically engineered Escherichia coli cell expressing both cellulose-binding domain (CBD) and organophosphorus hydrolase (OPH) onto cell surface was reported recently (Wang et al., 2002). This study reports the application of these biocatalysts when immobilized in a cellulose hollow fiber bioreactor (HFB) for the biodetoxification of a model organophosphate, paraoxon, in a continuous flow mode. In 24 h, 0.79 mg wet cell/cm2 fiber surface were immobilized onto cellulose fibers specifically and strongly through the cellulose binding domain, forming a monolayer demonstrated by Scanning Electronic Micrograph, and essentially no cell was washed away by washing buffer. The immobilized biocatalyst had a high performance of detoxifying paraoxon solution of 5,220 mumol/h x L reactor or 990 mumol/h x m2 reactor. The immobilized biocatalysts maintained a stable degradation capacity for 15 uses over a period of 48 days with only 10% decline in degradation efficiency under operating and storage conditions. In addition, the bioreactor was easily regenerated by washing with 1% sodium dodecyl sulfate (SDS), with 86.7% immobilization capacity and 93.9% degradation efficiency recovery. This is the first report using the HFB in a non-traditional way, immobilizing whole-cell biocatalysts by specific adhesion thus rendering the catalysis operation the advantages of low pressure drop, low shear force, and low energy requirement. The successful application of this genetically engineered dual functional E. coli strain in a model bioreactor shows its promise in large-scale detoxification of organophosphate nerve agents in bulk liquid phase.     

Production of recombinant human interleukin-2 with BHK cells in a hollow fibre and a stirred tank reactor with protein-free medium.

For the production of recombinant human interleukin-2 (IL-2) two different culture processes, a 1-2 liter homogeneous stirred bubble-free aerated system and a dense cell hollow fibre bioreactor were compared. Cultivations were carried out with serum- or protein-free medium formulations. In the stirred culture 0.75 mg IL-2 were produced with 1 l of perfused medium at a maximum cell number of 3 X 10(10). The product yield in the hollow fibre module was only 0.23 mg l-1 at a maximum cell number of 6 X 10(10). In contrast to results with hybridoma or EBV-transformed cell lines, in which hollow fibre bioreactors showed comparable efficiency to perfused stirred tank reactors, the tissue-like cell density is disadvantageous as adherent cells tend to stick together leaving insufficient intercellular space for removal of product.     

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.     

Generation of human embryonic stem cell-derived mesoderm and cardiac cells using size-specified aggregates in an oxygen-controlled bioreactor

The ability to generate human pluripotent stem cell-derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and biopharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called "embryoid bodies" (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro-printing strategy to generate large numbers of size-specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size-control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT-PCR data for markers such as Brachyury, LIM domain homeobox gene Isl-1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells.     

利用生物反应器培养植物细胞的研究进展(Ⅱ)

介绍了当前用于植物细胞培养的生物反应器类型(搅拌式、气升式、转鼓式和鼓泡式生物反应器)及其特点,对各种类型的反应器进行了比较与选择;并进一步介绍了植物细胞固定化培养,提出今后利用反应器大规模培养植物细胞的发展研究方向。     

利用生物反应器培养植物细胞的研究进展（Ⅱ）

介绍了当前用于植物细胞培养的生物反应器类型（搅拌式、气升式、转鼓式和鼓泡式生物反应器）及其特点,对各种类型的反应器进行了比较与选择;并进一步介绍了植物细胞固定化培养,提出今后利用反应器大规模培养植物细胞的发展研究方向。     

Hollow fiber bioreactor: new development for the study of contrast agent transport into hepatocytes by magnetic resonance imaging

The aim of our study was to develop a magnetic resonance (MR)-compatible in vitro model containing freshly isolated rat hepatocytes to study the transport of hepatobiliary contrast agents (CA) by MR imaging (MRI). We set up a perfusion system including a perfusion circuit, a heating device, an oxygenator, and a hollow fiber bioreactor (HFB). The role of the porosity and surface of the hollow fiber (HF) as well as the perfusate flow rate applied on the diffusion of CAs and O2 was determined. Hepatocytes were isolated and injected in the extracapillary space of the HFB (4 x 10(7) cells/mL). The hepatocyte HFB was perfused with an extracellular CA, gadopentetate dimeglumine (Gd-DTPA), and gadobenate dimeglumine (Gd-BOPTA), which also enters into hepatocytes. The HFB was imaged in the MR room using a dynamic T1-weighed sequence. No adsorption of CAs was detected in the perfusion system without hepatocytes. The use of a membrane with a high porosity (0.5 microm) and surface (420 cm2), and a high flow rate perfusion (100 mL/min) resulted in a rapid filling of the HFB with CAs. The cellular viability of hepatocytes in the HFB was greater than 85% and the O2 consumption was maintained over the experimental period. The kinetics of MR signal intensity (SI) clearly showed the different behavior of Gd-BOPTA that enters into hepatocytes and Gd-DTPA that remains extracellular. Thus, these results show that our newly developed in vitro model is an interesting tool to investigate the transport kinetics of hepatobiliary CAs by measuring the MR SI over time.     

Bioreactors for 3-dimensional high-density culture of human cells

A bioreactor was developed as an instrument to culture human or animal cells that require attachment in a large quantity or at a high density. The purpose for developing such a bioreactor is two-fold: to produce a large quantity of animal or human cells that have been modified by gene recombination technology to accommodate manufacture of physiologically-active substances or human proteins on an industrial scale; and for research to culture animal cells to form a high-density 3-dimensional structure as a morphological or functional tissue or organ entity. In the current report, the circulatory flow bioreactor and radial flow bioreactor (RFB) are introduced, in which the former can be scaled up. As a small bioreactor produced for the latter purpose, a rotary cell culture system and novel multicoaxial hollow-fiber bioreactor are introduced. Finally, a small RFB culture system that was scaled down by the present author and his collaborators for the study of a 3-dimensional high density culture system is described. The RFB can be readily scaled up for manufacturing or scaled down for research purposes. This is a cell culturing system that can induce the functions of human tissues by preparing a high density 3-dimensional organization of cells of human origin.     

Poly(lactic-co-glycolic acid) hollow fibre membranes for use as a tissue engineering scaffold

Mass transfer limitations of scaffolds are currently hindering the development of 3-dimensional, clinically viable, tissue engineered constructs. We have developed a poly(lactide-co-glycolide) (PLGA) hollow fibre membrane scaffold that will provide support for cell culture, allow psuedovascularisation in vitro and provide channels for angiogenesis in vivo. We produced P(DL)LGA flat sheet membranes using 1, 4-dioxane and 1-methyl-2-pyrrolidinone (NMP) as solvents and water as the nonsolvent, and hollow fibre membranes, using NMP and water, by dry/wet- and wet-spinning. The resulting fibres had an outer diameter of 700 micro m and an inner diameter of 250 micro m with 0.2-1.0 micro m pores on the culture surface. It was shown that varying the air gap and temperature when spinning changed the morphology of the fibres. The introduction of a 50 mm air gap caused a dense skin of 5 micro m thick to form, compared to a skin of 0.5 micro m thick without an air gap. Spinning at 40 degrees C produced fibres with a more open central section in the wall that contained more, larger macrovoids compared to fibres spun at 20 degrees C. Culture of the immortalised osteogenic cell line 560pZIPv.neo (pZIP) was carried out on the P(DL)LGA flat sheets in static culture and in a P(DL)LGA hollow fibre bioreactor under counter-current flow conditions. Attachment and proliferation was statistically similar to tissue culture polystyrene on the flat sheets and was also successful in the hollow fibre bioreactor. The P(DL)LGA hollow fibres are a promising scaffold to address the size limitations currently seen in tissue engineered constructs.     

Glycosylation of an immunoglobulin produced from a murine hybridoma cell line: the effect of culture mode and the anti-apoptotic gene, bcl-2

The impact of bcl-2 over-expression on the glycosylation pattern of an antibody produced by a bcl-2 transfected hybridoma cell line (TB/C3.bcl-2) was investigated in suspension batch, continuous and high cell density culture (Flat hollow fibre, Tecnomouse system). In all culture modes bcl-2 over-expression resulted in higher cell viability. Analysis of the glycans from the IgG of batch cultures showed that >95% of the structures were neutral core fucosylated asialo biantennary oligosaccharides with variable terminal galactosylation (G0f, G1f and G2f) consistent with previous analysis of glycans from the conserved site at Asn-297 of the IgG protein. The galactosylation index (GI) was determined as an indicator of the glycan profile (=(G2 + 0.5* G1)/(G0 + G1 + G2)). GI values in control cultures were comparable to bcl-2 cultures during exponential growth (0.53) but declined toward the end of the culture when there was a loss in cell viability. Low dilution rates in chemostat culture were associated with reduced galactosylation of the IgG glycans in both cell lines. However, at the higher dilution rates the GI for IgG was consistently higher in the TB/C3.bcl-2 cultures. In the hollow fibre bioreactor the galactosylation of the IgG glycans was considerably lower than in suspension batch or continuous cultures with GI values averaging 0.38. Similar low galactosylation values have been found previously for high density cell cultures and these are consistent with the low values obtained when the dissolved oxygen level is maintained at a low value (10%) in controlled suspension cultures of hybridomas.     

动物细胞培养用生物反应器及相关技术

动物细胞大量培养是生产生物制品的重要途径,它用到的关键设备是生物反应器。根据培养细胞、培养载体、培养液混合方式的不同,生物反应器主要有搅拌式、气升式、中空纤维式、回转式等,其中搅拌式规模最大。回转式是NASA于20世纪90年代中期开发的一种新型生物反应器,被誉为空间生物反应器,可用于组织工程研究。与生物反应器配套的技术主要有灌注、微载体、多孔微球、转入抗凋亡基因等,可以有效地提高细胞密度,增加生物制品产量,提高质量。今后生物反应器研制主要朝两个方向发展:一是,以高密度培养动物细胞生产蛋白质药物为目的,二是以三维培养动物细胞(主要是人类细胞)再生组织或器官为目的。     

The efficacy of new colchicine derivatives and viability of the T-Lymphoblastoid cells in three-dimensional culture using F MRI and HPLC-UV ex vivo

The paper describes ex vivo applications of colchicine derivatives for the treatment of human T-Lymphoblastoid (CEM) cells. Moreover, the role of the substitutions of ring A at C-1 and C-7 side chain of colchicine analogues was probed by the synthesis and examination of their effects on the three-dimensional (3-D) CEM cells’ growth. The CEM cells were cultured in the hollow fiber bioreactor (HFB) device. We used ¹H and ¹⁹F magnetic resonance imaging (MRI) to monitor changes in 3-D CEM cell culture. ¹⁹F MRI was used for visualization of the cellular uptake of new fluorine derivatives. Before and after treatment CEM cells profile was investigated with high performance liquid chromatography (HPLC-UV).     

Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture

Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.     

Characterization of a perfusion reactor utilizing mammalian cells on microcarrier beads

Our overall objective is to develop a cell culture analogue bioreactor (CCA) that can be used together with a corresponding physiologically based pharmacokinetic model (PBPK) to evaluate molecular mechanisms of toxicity. The PBPK is a mathematical model that divides the body into compartments representing organs, integrating the kinetic, thermodynamic, and anatomical parameters of the animal. The CCA bioreactor is a physical replica of the PBPK; where the PBPK specifies organs, the CCA bioreactor contains compartments with a corresponding cell type that mimics some of the characteristic metabolism of that organ. The device is a continuous, dynamic system composed of multiple cell types that interact through a common circulating cell culture medium. The CCA bioreactor and the model can be coupled to evaluate the plausibility of the molecular mechanism that is input into the model. This paper focuses on the design, development, and characterization of a CCA bioreactor to be used in naphthalene dose response studies. A CCA bioreactor prototype developed previously is improved upon by culturing the cells on microcarrier beads. Microcarrier beads with cells attached can form packed beds with cell culture medium perfusing the beds. In this study, two packed beds of cells, one with L2 cells (rat lung) and one with H4IIE cells (rat hepatoma), are linked in a physiologically relevant arrangement by a common recirculating cell culture medium. Studies of this CCA bioreactor presented here include mixing profiles, effect of reactor environment on cell viability and intracellular glutathione, naphthalene distribution profile, and initial naphthalene dosing studies. Unlike the prototype system there is no detectable response to naphthalene addition; in a companion paper we show that this discrepancy can be explained by differences in liquid residence times in the organ compartments. The perfusion reactor design is shown to have significant operating improvements over prototype designs.