Use of a centrifugal bioreactor for cartilaginous tissue formation from isolated chondrocytes

Although a centrifugal bioreactor (CCBR) supports high-density mammalian suspension cell cultures by balancing drag, buoyancy, and centrifugal forces, to date anchorage-dependent cultures have not been tried. Also, steady or intermittent hydrostatic pressures of 8 to 500 kPa, and shears of 0.02 to 1.4 N/m(2) can be simultaneously applied in the CCBR. This article demonstrates the use of a CCBR to stimulate chondrogenesis in a high-density culture. At 3 weeks, histological results show even distribution of glycosaminoglycan (GAG) and collagen, with 1,890 ± 270 cells/mm(2) cell densities that exceed those of 1,470 ± 270 in pellet cultures. Analysis of collagen content reveals similar levels for all treatment groups; 6.8 ± 3.5 and 5.0 ± 0.4 μg collagen/μg DNA for 0.07 and 0.26 MPa CCBR cultures, respectively, in contrast to 6.6 ± 1.9 values for control pellet cultures. GAG levels of 5.6 ± 1.5 and 4.1 ± 0.9 μg GAG /μg DNA are present for cultures stressed at 0.07 and 0.26 MPa, respectively, in comparison to control pellet cultures at the 8.4 ± 0.9 level. Although results to date have not revealed mechanical stress combinations that stimulate chondrogenesis over unstressed controls, system advantages include continuous culture at cell densities above those in the pellet, precise medium control, the ability to independently vary multiple mechanical stresses over a broad range, and the flexibility for integration of scaffold features for future chondrogenesis stimulation studies.     

Evaluation of oxygen transfer rates in stirred-tank bioreactors for clinical manufacturing

Several methods are available for determining the volumetric oxygen transfer coefficient in bioreactors, though their application in industrial bioprocess has been limited. To be practically useful, mass transfer measurements made in nonfermenting systems must be consistent with observed microbial respiration rates. This report details a procedure for quantifying the relationship between agitation frequency and oxygen transfer rate that was applied in stirred-tank bioreactors used for clinical biologics manufacturing. The intrinsic delay in dissolved oxygen (DO) measurement was evaluated by shifting the bioreactor pressure and fitting a first-order mathematical model to the DO response. The dynamic method was coupled with the DO lag results to determine the oxygen transfer rate in Water for Injection (WFI) and a complete culture medium. A range of agitation frequencies was investigated at a fixed air sparge flow rate, replicating operating conditions used in Pichia pastoris fermentation. Oxygen transfer rates determined by this method were in excellent agreement with off-gas calculations from cultivation of the organism (P = 0.1). Fermentation of Escherichia coli at different operating parameters also produced respiration rates that agreed with the corresponding dynamic method results in WFI (P = 0.02). The consistency of the dynamic method results with the off-gas data suggests that compensation for the delay in DO measurement can be combined with dynamic gassing to provide a practical, viable model of bioreactor oxygen transfer under conditions of microbial fermentation.     

Picoliter DNA sequencing chemistry on an electrowetting-based digital microfluidic platform

The results of investigations into performing DNA sequencing chemistry on a picoliter-scale electrowetting digital microfluidic platform are reported. Pyrosequencing utilizes pyrophosphate produced during nucleotide base addition to initiate a process ending with detection through a chemiluminescence reaction using firefly luciferase. The intensity of light produced during the reaction can be quantified to determine the number of bases added to the DNA strand. The logic-based control and discrete fluid droplets of a digital microfluidic device lend themselves well to the pyrosequencing process. Bead-bound DNA is magnetically held in a single location, and wash or reagent droplets added or split from it to circumvent product dilution. Here we discuss the dispensing, control, and magnetic manipulation of the paramagnetic beads used to hold target DNA. We also demonstrate and characterize the picoliter-scale reaction of luciferase with adenosine triphosphate to represent the detection steps of pyrosequencing and all necessary alterations for working on this scale.     

Efficiency in thin-film liquid system for <Emphasis Type="Italic">Hosta</Emphasis> micropropagation

Three varieties of Hosta (Striptease, Minuteman and Stiletto) at four densities (40, 80, 120 and 200 explants per litre) were micropropagated on semi-solid agar and a thin-film liquid system with intermittent wetting of plant tissue. The mechanics of wetting by a small wave front required a larger rectangular vessel (11 × 27=297 cm ²) compared to the common cylindrical baby food jar (18 cm ²). Plants multiplied more rapidly in the agitated thin-film system than on agar. Lower plant densities increased rates of multiplication in liquid, but had little or no effect on multiplication rate on agar. Increasing plant density lowered the overall multiplication rate, but yielded greater numbers of plants per vessel. Yield, tabulated for utilization of shelf-space in growth room, was greater at all densities in rectangular vessels of liquid than conventional jars of agar media. Increased plant density lowered the sugar residual in media following the culture cycle and liquid media had less residual sugar than agar media. A liquid medium with 50 g l^–1 sucrose was concentrated enough so that sugar depletion did not limit growth, even at the highest densities. The liquid system allows the technician to skip the step of manually spacing and orienting the freshly cut bud tissue at the transfer station. Harvesting 75–100 plants per vessel from the large rectangular vessels resulted in most efficient use of technician time. Plants from liquid and agar acclimatized to greenhouse. Increased multiplication, space utilization, sugar availability and worker efficiency was demonstrated to be greater in thin-film liquid than more conventional agar-based system.     

Mist trickling bioreactor for <Emphasis Type="Italic">Centaurium erythraea</Emphasis> Rafn growth of shoots and production of secoiridoids

Shoots of Centaurium erythraea Rafn were cultivated in 5 l mist trickling bioreactor for 21 and 28 days increasing their dry weight from 0.54 g to 13.7 g and 18.3 g, respectively. About 6880 shoots from 223 initial shoot-tips in 21-day bioreactor producing cycle were produced. The shoots could be successfully rooted and transferred to soil. Secoiridoid accumulation (expressed as a sum of gentiopicroside, sweroside and swertiamarin) in shoots after 21 days of culture reached about 303 mg l⁻¹.     

Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150m and cultured for 40days. Up to 100m MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 m MJ peaked after 10days at 48mgg^–1 dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.Revisions requested; 2 July 2004; Revisions received 30 June 2004; 3 September 2004     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Production of recombinant proteins by vaccinia virus in a microcarrier based mammalian cell perfusion bioreactor

The HeLa cell-vaccinia virus expression system was evaluated for the production of recombinant proteins (enhanced green fluorescent protein (EGFP) and HIV envelope coat protein, gp120) using microcarriers in 1.5 L perfused bioreactor cultures. Perfusion was achieved by use of an alternating tangential flow device (ATF), increasing the length of the exponential phase by 50 h compared to batch culture and increasing the maximum cell density from 1.5x10(6) to 4.4x10(6) cell/mL. A seed train expansion method using cells harvested from microcarrier culture and reseeding onto fresh carriers was developed. EGFP was first used as a model protein to study process parameters affecting protein yield, specifically dissolved oxygen (DO) and temperature during the production phase. The highest level of EGFP, 12+/-1.5 microg/10(6) infected cells, was obtained at 50% DO and 31 degrees C. These setpoints were then used to produce glycoprotein, gp120, which was purified and deglycosylated, revealing a significant amount of N-linked glycosylation. Also, biological activity was assayed, resulting in an ID50 of 3.1 microg/mL, which is comparable to previous reports.     

Quantifying maintenance requirements from the steady-state operation of a two-phase partitioning bioscrubber

An innovative method for directly and explicitly quantifying the maintenance energy requirements of pure cultures growing on volatile organic compound (VOC) substrates in a two-phase partitioning bioscrubber is described. Direct evidence of maintenance energy requirements of Achromobacter xylosoxidans Y234 is provided both through observed reductions in the macroscopic biomass-to-substrate yield with decreasing specific growth rates, but more remarkably through achievement of steady-state operation. The data conclusively show that maintenance activities do occur in the two-phase partitioning bioscrubber and clearly illustrate the importance of this phenomenon to the operation of this process, and similar bioreactor systems. While benzene was selected as the principal, sole substrate of interest in this study, ethylbenzene degradation experiments were also subsequently performed to illustrate and confirm the general applicability of the proposed technique, as well as the potential capabilities of the two-phase partitioning bioscrubber for the continuous treatment of waste gases containing various VOCs. The proposed method has been shown to generate maintenance energy estimates that are consistent with those obtained while employing more widely recognized estimation strategies, further validating its capabilities. The proposed steady-state mode also offers key operational advantages in terms of decreased disposal requirements in two-phase partitioning bioscrubbers. (c) 2005 Wiley Periodicals, Inc.     

Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.