Bioreactor Culture Conditions Research Articles

Production of eurycomanone and polysaccharides through adventitious root culture of Eurycoma longifolia in a bioreactor

Eurycoma longifolia is a precious medicinal plant that currently faces resource shortage. The bioreactor culture of adventitious roots (ARs) can be an efficient route for the rapid and large-scale production of the useful metabolites of this plant species. Eurycomanone and polysaccharides are the important bioactive compounds of E. longifolia; however, the bioreactor AR culture targeting the production of both compounds has not been explored. In this work, E. longifolia ARs were cultured in bubble column bioreactors and the bioreactor culture conditions to produce eurycomanone and polysaccharides were optimized by controlling sucrose concentration, inoculation density, and aeration rate. Kinetic study was subsequently implemented to confirm the culture period. The antioxidant activity of ARs were determined to provide a reference for the further production of related products. Results showed that E. longifolia ARs well grew in the bioreactor, and 40 g/L sucrose, 5 g/L inoculation density, and 0.05 vvm aeration rate were the optimal conditions for the production of ARs containing the highest eurycomanone and polysaccharide yields. Kinetic study revealed that 40 days of culture was the most suitable culture period, that produced 8.8 mg/L eurycomanone and 2.4 g/L polysaccharides. The ARs efficiently scavenged DPPH and ABTS radicals and possessed Fe2+ chelating ability, thereby indicating their potential use in the production of antioxidant related products.

Production of Pleurotus sajor-caju crude enzyme broth and its applicability for the removal of bisphenol A.

Bisphenol A is an endocrine interfering compound, produced and used on a large scale worldwide. Chemical and biologic methods can be used to remove it from the environment. Biological methods are considered less costly, safer and, according to green chemistry definitions, an environmentally correct method. Considering the use of a crude enzyme broth, without any downstream process, the costs could be mostly reduced. Thus, the removal of bisphenol A by Pleurotus sajor-caju crude enzyme broth was investigated. Initially, the agro-industrial wastes were characterized and, the composition of the culture medium and the bioreactor culture conditions were defined. The enzyme produced in the highest concentration was characterized and the crude broth used in the bisphenol A removal assays. The OXI45 culture medium presented the highest laccase activity (1,850.7 U L-1, 350 rpm). Greater laccase stability was observed at 20 - 40 oC and pHs 5 - 7. Vanillin and ferulic acid (considered mediator compounds) were identified in the crude broth, probably helping on the obtention of the high value of removal effectiveness (0.052 mg U-1 h-1). The results indicate the potential use of the Pleurotus sajor-caju crude enzyme broth to obtain an enzymatic formulation for application in the environmental area.

Different Mechanized Minibioreactor Framework for Multifunctional Screening in Biotechnology

The present methods connected in biotechnology permit to acquire many sorts of atoms that must be tried on cell societies (High Throughput Screening HTS). Albeit such tests are generally completed consequently on smaller than normal or microwell plates, the strategies in the preindustrial organize are performed physically on higher volume beneficiaries known as bioreactors. The development conditions in the two phases are totally unique. The screening framework displayed in this work depends on the multiwell test plate’s theory, an expendable different minibioreactor that permits proliferation of modern bioreactor culture conditions: air circulation, blending, temperature, O2, pH and obvious range optical absorbance estimations. It is conceivable to duplicate the development conditions for both suspended and disciple creature cell sorts utilizing 1 to 10 ml vol. bioreactors. On account of microscopic organisms or yeast, it is unrealistic to accomplish a high biomass fixation, because of the diminished head volume air supply.

In vitro cartilage construct generation from silk fibroin- chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition.

Cartilage construct generation includes a scaffold with appropriate composition to mimic matrix of the damaged tissue on which the stem cells grow and differentiate. In this study, umbilical cord blood (UCB) derived human mesenchymal stem cells (hMSCs) were seeded on freeze dried porous silk-fibroin (SF)/chitosan (CS) scaffolds. Influence of static and dynamic (spinner flask bioreactor) culture conditions on the developing cartilage construct were studied by in-vitro characterization for viability, proliferation, distribution, and chondrogenic differentiation of hMSCs over the scaffold. Constructs developed in spinner flask consisted of 62% live cells, and exhibited 543% more cell density at the core than constructs cultured in static system. Quantification of DNA and glycosaminoglycans accumulation after 21 days showed the progression of chondrogenic differentiation of hMSCs was higher in dynamic culture compared to static one. In constructs generated under dynamic condition, histology staining for proteoglycan matrix, and fluorescence staining for collagen-II and aggrecan showed positive correlation between early and late stage chondrogenic markers, which was further confirmed by quantitative PCR analysis, showing low collagen-I expression and highly expressed Sox9, collagen-II and aggrecan. The present study demonstrated that construct generated by combining 3D SF/CS scaffold with UCB-hMSCs under dynamic condition using spinner flask bioreactor can be used for cartilage tissue regeneration for future medical treatments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 397-407, 2018.

Review: bioreactor design towards generation of relevant engineered tissues: focus on clinical translation.

In tissue engineering and regenerative medicine, studies that utilize 3D scaffolds for generating voluminous tissues are mostly confined in the realm of in vitro research and preclinical animal model testing. Bioreactors offer an excellent platform to grow and develop 3D tissues by providing conditions that mimic their native microenvironment. Aligning the bioreactor development process with a focus on patient care will aid in the faster translation of the bioreactor technology to clinics. In this review, we discuss the various factors involved in the design of clinically relevant bioreactors in relation to their respective applications. We explore the functional relevance of tissue grafts generated by bioreactors that have been designed to provide physiologically relevant mechanical cues on the growing tissue. The review discusses the recent trends in non-invasive sensing of the bioreactor culture conditions. It provides an insight to the current technological advancements that enable in situ, non-invasive, qualitative and quantitative evaluation of the tissue grafts grown in a bioreactor system. We summarize the emerging trends in commercial bioreactor design followed by a short discussion on the aspects that hamper the 'push' of bioreactor systems into the commercial market as well as 'pull' factors for stakeholders to embrace and adopt widespread utility of bioreactors in the clinical setting. Copyright © 2017 John Wiley & Sons, Ltd.

Numerical Simulation of Mass Transfer and Three-Dimensional Fabrication of Tissue-Engineered Cartilages Based on Chitosan/Gelatin Hybrid Hydrogel Scaffold in a Rotating Bioreactor.

Cartilage tissue engineering is believed to provide effective cartilage repair post-injuries or diseases. Biomedical materials play a key role in achieving successful culture and fabrication of cartilage. The physical properties of a chitosan/gelatin hybrid hydrogel scaffold make it an ideal cartilage biomimetic material. In this study, a chitosan/gelatin hybrid hydrogel was chosen to fabricate a tissue-engineered cartilage in vitro by inoculating human adipose-derived stem cells (ADSCs) at both dynamic and traditional static culture conditions. A bioreactor that provides a dynamic culture condition has received greater applications in tissue engineering due to its optimal mass transfer efficiency and its ability to simulate an equivalent physical environment compared to human body. In this study, prior to cell-scaffold fabrication experiment, mathematical simulations were confirmed with a mass transfer of glucose and TGF-β2 both in rotating wall vessel bioreactor (RWVB) and static culture conditions in early stage of culture via computational fluid dynamic (CFD) method. To further investigate the feasibility of the mass transfer efficiency of the bioreactor, this RWVB was adopted to fabricate three-dimensional cell-hydrogel cartilage constructs in a dynamic environment. The results showed that the mass transfer efficiency of RWVB was faster in achieving a final equilibrium compared to culture in static culture conditions. ADSCs culturing in RWVB expanded three times more compared to that in static condition over 10days. Induced cell cultivation in a dynamic RWVB showed extensive expression of extracellular matrix, while the cell distribution was found much more uniformly distributing with full infiltration of extracellular matrix inside the porous scaffold. The increased mass transfer efficiency of glucose and TGF-β2 from RWVB promoted cellular proliferation and chondrogenic differentiation of ADSCs inside chitosan/gelatin hybrid hydrogel scaffolds. The improved mass transfer also accelerated a dynamic fabrication of cell-hydrogel constructs, providing an alternative method in tissue engineering cartilage.

Streamlined bioreactor-based production of human cartilage tissues.

Engineered tissue grafts have been manufactured using methods based predominantly on traditional labour-intensive manual benchtop techniques. These methods impart significant regulatory and economic challenges, hindering the successful translation of engineered tissue products to the clinic. Alternatively, bioreactor-based production systems have the potential to overcome such limitations. In this work, we present an innovative manufacturing approach to engineer cartilage tissue within a single bioreactor system, starting from freshly isolated human primary chondrocytes, through the generation of cartilaginous tissue grafts. The limited number of primary chondrocytes that can be isolated from a small clinically-sized cartilage biopsy could be seeded and extensively expanded directly within a 3D scaffold in our perfusion bioreactor (5.4±0.9 doublings in 2 weeks), bypassing conventional 2D expansion in flasks. Chondrocytes expanded in 3D scaffolds better maintained a chondrogenic phenotype than chondrocytes expanded on plastic flasks (collagen type II mRNA, 18-fold; Sox-9, 11-fold). After this "3D expansion" phase, bioreactor culture conditions were changed to subsequently support chondrogenic differentiation for two weeks. Engineered tissues based on 3D-expanded chondrocytes were more cartilaginous than tissues generated from chondrocytes previously expanded in flasks. We then demonstrated that this streamlined bioreactor-based process could be adapted to effectively generate up-scaled cartilage grafts in a size with clinical relevance (50mm diameter). Streamlined and robust tissue engineering processes, as the one described here, may be key for the future manufacturing of grafts for clinical applications, as they facilitate the establishment of compact and closed bioreactor-based production systems, with minimal automation requirements, lower operating costs, and increased compliance to regulatory guidelines.

Compositional changes to synthetic biodegradable scaffolds modulate the influence of hydrostatic pressure on chondrogenesis of mesenchymal stem cells

Mechanical cues such as hydrostatic pressure (HP) are known to regulate mesenchymal stem cell (MSC) differentiation. The fate of such cells is also strongly influenced by their substrate. The objective of this study was to test how different modifications of polycaprolactone (PCL) scaffolds would influence the response of MSCs to HP. Porcine bone marrow derived MSCs were cultured on PCL, PCL-hyaluronic acid (HA) and PCL-Bioglass® (BG) scaffolds for 35 d and stimulated with a HP bioreactor (10 MPa; 1 Hz; 2 h d−1). Scaffold composition was found to modulate the response to HP. MSCs seeded onto both PCL and BG scaffolds responded positively to the application of HP, with increases in cartilage extracellular matrix synthesis and a reduction in type I collagen accumulation. This positive effect was not observed on HA scaffolds. The results of this study demonstrate that changes to scaffold composition can have a notable effect on the response of MSCs to bioreactor culture conditions.

Osteogenic performance of donor-matched human adipose and bone marrow mesenchymal cells under dynamic culture.

Adipose-derived mesenchymal cells (ACs) and bone marrow-derived mesenchymal cells (BMCs) have been widely used for bone regeneration and can be seeded on a variety of rigid scaffolds. However, to date, a direct comparison of mesenchymal cells (MC) harvested from different tissues from the same donor and cultured in identical osteogenic conditions has not been investigated. Indeed, it is unclear whether marrow-derived or fat-derived MC possess intrinsic differences in bone-forming capabilities, since within-patient comparisons have not been previously done. This study aims at comparing ACs and BMCs from three donors ranging in age from neonatal to adult. Matched cells from each donor were studied in three distinct bioreactor settings, to determine the best method to create a viable osseous engineered construct. Human ACs and BMCs were isolated from each donor, cultured, and seeded on decellularized porcine bone (DCB) constructs. The constructs were then subjected to either static or dynamic (stirring or perfusion) bioreactor culture conditions for 7-21 days. Afterward, the constructs were analyzed for cell adhesion and distribution and osteogenic differentiation. ACs demonstrated higher seeding efficiency than BMCs. However, static and dynamic culture significantly increased BMCs proliferation more than ACs. In all conditions, BMCs demonstrated stronger osteogenic activity as compared with ACs, through higher alkaline phosphatase activity and gene expression for various bony markers. Conversely, ACs expressed more collagen I, which is a nonspecific matrix molecule in most connective tissues. Overall, dynamic bioreactor culture conditions enhanced osteogenic gene expression in both ACs and BMCs. Scaffolds seeded with BMCs in dynamic stirring culture conditions exhibit the greatest osteogenic proliferation and function in vitro, proving that marrow-derived MC have superior bone-forming potential as compared with adipose-derived cells.

Bioreactors for plant cells: hardware configuration and internal environment optimization as tools for wider commercialization

Mass production of value-added molecules (including native and heterologous therapeutic proteins and enzymes) by plant cell culture has been demonstrated as an efficient alternative to classical technologies [i.e. natural harvest and chemical (semi)synthesis]. Numerous proof-of-concept studies have demonstrated the feasibility of scaling up plant cell culture-based processes (most notably to produce paclitaxel) and several commercial processes have been established so far. The choice of a suitable bioreactor design (or modification of an existing commercially available reactor) and the optimization of its internal environment have been proven as powerful tools toward successful mass production of desired molecules. This review highlights recent progress (mostly in the last 5years) in hardware configuration and optimization of bioreactor culture conditions for suspended plant cells.

Three-Dimensional Characterization of Tissue-Engineered Constructs by Contrast-Enhanced Nanofocus Computed Tomography

To successfully implement tissue-engineered (TE) constructs as part of a clinical therapy, it is necessary to develop quality control tools that will ensure accurate and consistent TE construct release specifications. Hence, advanced methods to monitor TE construct properties need to be further developed. In this study, we showed proof of concept for contrast-enhanced nanofocus computed tomography (CE-nano-CT) as a whole-construct imaging technique with a noninvasive potential that enables three-dimensional (3D) visualization and quantification of in vitro engineered extracellular matrix (ECM) in TE constructs. In particular, we performed a 3D qualitative and quantitative structural and spatial assessment of the in vitro engineered ECM, formed during static and perfusion bioreactor cell culture in 3D TE scaffolds, using two contrast agents, namely, Hexabrix® and phosphotungstic acid (PTA). To evaluate the potential of CE-nano-CT, a comparison was made to standardly used techniques such as Live/Dead viability/cytotoxicity, Picrosirius Red staining, and to net dry weight measurements of the TE constructs. When using Hexabrix as the contrast agent, the ECM volume fitted linearly with the net dry ECM weight independent from the flow rate used, thus suggesting that it stains most of the ECM. When using PTA as the contrast agent, comparing to net weight measurements showed that PTA only stains a part of the ECM. This was attributed to the binding specificity of this contrast agent. In addition, the PTA-stained CE-nano-CT data showed pronounced distinction between flow conditions when compared to Hexabrix, indicating culture-specific structural ECM differences. This novel type of information can contribute to optimize bioreactor culture conditions and potentially critical quality characteristics of TE constructs such as ECM quantity and homogeneity, facilitating the gradual transformation of TE constructs in well-characterized TE products.

Genomic analysis of a hybridoma batch cell culture metabolic status in a standard laboratory 5 L bioreactor

Currently, there is a gap in the knowledge of the culture responses to controlled bioreactor environment during the course of batch cell culture from early exponential phase to stationary-phase. If available, such information could be used to designate gene transcripts for predicting cell status and as a quality predictor for a controlled bioreactor. In this study, we used oligonucleotide microarrays to obtain baseline gene expression profiles during the time-course of a hybridoma batch cell culture in a 5 L bench-scale bioreactor. Gene expression changes that were up or down modulated from early-to-late in batch culture, as well as invariant gene profiles with significant expression were identified using microarray. Typical cellular functions that seemed to be correlated with transcriptomics were oxidative stress response, DNA damage response, apoptosis, and cellular metabolism. As confirmatory evidence, microarray findings were verified with a more rigorous semiquantitative gene-specific Reverse transcriptase-polymerase chain reaction (RT-PCR). The results of this study suggest that under predefined bioreactor culture conditions, significant gene changes from lag to log to stationary phase could be identified, which could then be used to track the culture state.

Enhanced productivity of biomass and bioactive compounds through bioreactor cultures of Eleutherococcus koreanum Nakai adventitious roots affected by medium salt strength

To develop a protocol of Eleutherococcus koreanum Nakai adventitious root culture for production of biomass and bioactive compounds through bioreactors, different strengths of Murashige and Skoog (MS) medium were tested. After 5 weeks of culture, root growth at low salt strengths (1/4, 1/2, and 3/4 MS) was better than that at high salt strengths (1 and 2 MS), and the highest fresh and dry weight was achieved at 1/2 MS. The roots cultured at strengths exceeding 1 MS showed physiological abnormalities such as shorter, thicker, and less numerous roots compared to other treatments. Strengths over 1 MS caused physical dehydration that stimulated proline accumulation in the roots and decreased water potential in the medium because of high osmotic stress. Total production of 5 target compounds (per 1L medium), eleutheroside B and E, chlorogenic acid, total phenolics, and flavonoids, was decreased with increasing medium salt strength. However, the highest total production of eleutheroside B and E (per 1L medium), the main bioactive compounds in this plant, were observed at 1/2 and 3/4 MS, respectively. Therefore, 1/2 MS is a suitable medium salt strength for both biomass and bioactive compound productions, and optimization of bioreactor culture conditions will benefit the large-scale production of E. koreanum-derived bioactive compounds for commercialization.

A positron emission tomography approach to visualize flow perfusion in hollow-fiber membrane bioreactors

Despite the success of hollow-fiber membrane bioreactors in tissue engineering, few evaluations of steady- and pulsatile-flow perfusion through these bioreactors have been made. Such evaluations are vital to the optimization of bioreactor culture conditions. In this study, positron emission tomography (PET) was proposed and used to visualize steady- and pulsatile-flow perfusion in hollow-fiber membrane bioreactors for tissue-engineering applications. PET is a noninvasive method that allows measuring the spatial distribution of a radioactive tracer by detecting its activity within porous scaffolds. A radioactive tracer, 18-fluoro-deoxy-glucose ((18)FDG), was injected into a fluid circuit having a hollow-fiber membrane bioreactor with gel-devoid or gel-filled extracapillary space. Dynamic PET scans of the inlet section were acquired and followed by volumetric PET scans of the whole bioreactor. Results were used to reconstruct dynamic and volumetric two- and three-dimensional images. Pulsatile inlet flow improved the uniformity of perfusion flow within the bioreactor in comparison to the steady inlet flow. Pulsatile flow also reduced the accumulation of radioactive tracer for both gel-devoid and gel-filled bioreactors compared to the steady flow. The stability of the radioactive tracer for both conditions was evaluated. The potential of the PET approach was demonstrated by the quantification of the imaging results for steady- and pulsatile-flow perfusions that can be used for the development of bioreactors for tissue-engineering applications.

Chondrocytes and bone marrow-derived mesenchymal stem cells undergoing chondrogenesis in agarose hydrogels of solid and channelled architectures respond differentially to dynamic culture conditions

The objective of this study was to investigate how a combination of different scaffold architectures and rotational culture would influence the functional properties of thick cartilaginous tissues engineered using either chondrocytes or bone marrow-derived mesenchymal stem cells (BM-MSCs). Expanded porcine chondrocytes and BM-MSCs were suspended in 2% agarose and cast in custom-designed moulds to produce either regular solid or channelled construct cylinders. The study consisted of three seperate experimental arms. First, chondrocyte and BM-MSC constructs were cultured in free swelling conditions for 9 weeks. Second, constructs were subjected to rotational culture for a period of 3 weeks. Finally, BM-MSC-seeded constructs were subjected to delayed rotational culture, in which constructs were first cultured for 3 weeks in free swelling conditions, followed by an additional 3 weeks in rotating culture conditions. Constructs were supplemented with TGFβ3 during the first 3 weeks of all experiments. The introduction of channels alone had little effect on the spatial patterns of tissue accumulation in either chondrocyte- or BM-MSC-seeded constructs. The two cell types responded differentially to rotational culture, resulting in the formation of a more homogeneous tissue in chondrocyte-seeded constructs, but significantly inhibiting chondrogenesis of BM-MSCs. This inhibition of chondrogenesis in response to dynamic culture conditions was not observed if BM-MSC-seeded constructs were first maintained in free swelling conditions for 3 weeks prior to rotation. The results of this study demonstrate that bioreactor culture conditions that are beneficial for chondrocyte-based cartilage tissue engineering may be suboptimal for BM-MSCs.

Chitosan elicits mono-glucosylated stilbene production and release in fed-batch bioreactor cultures of grape cells

The present paper reports the study of the optimal conditions for grape ( Vitis vinifera cv. Barbera) cell suspensions in batch and fed-batch bioreactor cultures, in order to specifically improve the production of mono-glucosylated stilbenes, which are resveratrol derivatives. These compounds are physiologically as active as free resveratrol in cardio- and chemoprotection, but are more stable and bioavailable after ingestion through diet. In fed-batch conditions the production of mono-glucosides was considerably increased together with that of free resveratrol. For the first time, an elicitor (chitosan) was tested in a bioreactor system, demonstrating its efficacy in inducing the production of stilbenes. The bioreactor culture conditions also allowed the accumulation of other polyphenols, such as catechins. The vast majority of the produced compounds was released into the culture media, which represents a relevant advantage for the recovery of specific molecules or of polyphenol-enriched mixtures. These results represent a further step toward the employment of grape cells in fed-batch cultures, as a promising alternative to whole plant extraction for the industrial production of plant polyphenols, considering the necessity for developing sustainable processes.

Optimization of the culture medium composition using response surface methodology for new recombinant cyprosin B production in bioreactor for cheese production

The optimization of culture medium composition was carried out for improvement the recombinant cyprosin B production, an enzyme with high milk-clotting activity. Response surface methodology (RSM) was applied to evaluate the effect of variables namely glucose, yeast extract (YE) and bactopeptone present in the culture medium, used for recombinant cyprosin B production by transformed Saccharomyces cerevisiae BJ1991 strain in shake-flask and bioreactor culture conditions. The central composite experimental design (CCD) was adopted to derive a statistical model for optimizing the composition of the fermentation medium. The optimal concentration estimated for each variable related to a theoretical maximum of cyprosin B activity (488 U mL−1) was 30 g L−1 glucose, 15 g L−1 YE and 27 g L−1 bactopeptone. The optimized medium composition, based on empirical model, led to a cyprosin B activity of 519 U mL−1, which corresponds to an increase of 46%. The fermentation using optimized culture medium in a 5-L bioreactor allowed a significant increase in biomass (82%) and recombinant cyprosin B production (139%). The improvement in the recombinant cyprosin B production after optimization process can be considered adequate for large-scale applications, and the clotting activity of cyprosin B account for their use in industrial cheese making.

High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris

Human alpha-defensin 5 (HD(5)), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD(5) through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD(5) mature peptide (rmHD(5)) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD(5) by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD(5) into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD(5) vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD(5) nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD(5), a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD(5). The results showed that about 165.0 mg/l of rmHD(5) with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD(5) was recovered after purification. The in vitro experiments revealed that rmHD(5) exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD(5) in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.

Mechanical Stimulation of Osteoblasts Using Steady and Dynamic Fluid Flow

In bone tissue engineering, flow perfusion bioreactors have shown great potential for accelerated production of functional constructs, but bioreactor culture conditions have not been optimized. The goal of this study was to investigate the short-term (1- 49 h) effects of intermittent steady, pulsatile, and oscillatory fluid flow (peak flow rate = 1.0 mL/min) on MC3T3-E1 osteoblast activity within a collagen-glycosaminoglycan scaffold. Bioreactor culture at a continuous low flow rate (0.05 mL/min) was also evaluated. Fluid flow exposure stimulated 8 to 51, 15 to 48, and 1.4 to 2.7 greater cyclooxygenase-2 (COX-2) expression, prostaglandin E2 (PGE2) production, and osteopontin expression, respectively, whereas membrane-associated prostaglandin E synthase-1 was 1.8 greater only under steady flow. Overall, intermittent flow (high flow rate) caused greater stimulation than a continuous low flow rate without a loss in cell number. Pulsatile and oscillatory fluid flow tripled COX-2 expression from 25 to 49 h (p < or =0.04), whereas under steady flow, PGE2 production dropped 52% at 49 h (p = 0.05). These results indicate that intermittent flow is advantageous for mechanically stimulating osteoblasts while maintaining cell viability. In addition, results at 49 h suggest that dynamic (pulsatile and oscillatory) flow may be more stimulatory than steady flow over long-term culture.

Culture-based strategies to enhance cellulase enzyme production from Trichoderma reesei RUT-C30 in bioreactor culture conditions

Filamentous fungi Trichoderma reesei are considered to be one of the most efficient hyper producers of cellulase that is used in industry. Cellulase production by T. reesei was carried out using high concentration of cellulose to substitute glucose with the aim to improve cellulase production while trying to reduce production costs. The experiments were conducted separately as fed batch growth with T. reesei cultured using four media in a 7 L stirred tank bioreactor. A mixture of lactose and lactobionic acid was added into the bioreactor as cellulase inducers. The use of a cellulose–yeast extract culture medium yielded the highest enzyme and cell production with a volumetric enzyme activity of 69.8 U L −1 h −1, a filter paper activity of 5.02 U mL −1, a CMCase activity of 4.2 U mL −1, and a fungal biomass of 14.7 g L −1. The biomass concentration as a function of time was constant with relatively rapid, early growth on easily metabolized growth medium components (yeast extract), followed by a second slower growth phase due to hydrolysis of cellulose, which follow cellulase concentration augmentation. The costs to produce 1 L of production medium in laboratory-scale experiments were calculated to compare the tested media.