Functionalized Metal-Organic Framework for NADH Regeneration by Hydrogen in a Redox Flow Bioreactor.

The electrochemical regeneration of reduced nicotinamide adenine dinucleotide (NADH) using [Rh(Cp*)(bpy)Cl]+ holds significant promise for the industrial synthesis of chiral chemicals. However, challenges persist due to the high consumption of NADH and the limited efficiency of its cyclic regeneration, which currently hinder widespread application. To address these obstacles, based on in-situ growth of 3D ordered metal-organic framework (NU-1000) on the surface of graphite felt, [Rh(Cp*)(bpy)Cl]+ were immobilized on the Zr6 nodes of NU-1000 by solvent-assisted ligand incorporation (SALI), and applied in a flow bioreactor. Moreover, we employ a gas diffusion electrode (GDE) to oxidize H2, providing a clean proton source for the electrochemical regeneration of NADH. Consequently, highly efficient enzymatic electrocatalytic synthesis of L-lactate was achieved when coupled with L-lactate dehydrogenases (LDH) as a model reaction, and the total turnover number (TTN) reached 19600 and 1750 for [Rh(Cp*)(bpy)Cl]+ and NAD+ after 48 h, corresponding to a high turnover frequency (TOF) of 2350 h-1 and 210 h-1 for [Rh(Cp*)(bpy)Cl]+ and NAD+, respectively. This work provides new insights for the construction of efficient enzymatic electrosynthesis systems in industrial production.

Advances in aldo-keto reductases immobilization for biocatalytic synthesis of chiral alcohols.

Chiral alcohols are essential building blocks of numerous pharmaceuticals and fine chemicals. Aldo-keto reductases (AKRs) constitute a superfamily of oxidoreductases that catalyze the reduction of aldehydes and ketones to their corresponding alcohols using NAD(P)H as a coenzyme. Knowledge about the crucial roles of AKRs immobilization in the biocatalytic synthesis of chiral alcohols is expanding. Herein, we reviewed the characteristics of various AKRs immobilization approaches, the applications of different immobilization materials, and the prospects of continuous flow bioreactor construction by employing these immobilized biocatalysts for synthesizing chiral alcohols. Finally, the opportunities and ongoing challenges for AKR immobilization are discussed and the outlook for this emerging area is analyzed.

Impact of interstitial flow on cartilage matrix synthesis and NF-kB transcription factor mRNA expression in a novel perfusion bioreactor.

This work is focused on designing an easy-to-use novel perfusion system for articular cartilage (AC) tissue engineering and using it to elucidate the mechanism by which interstitial shear upregulates matrix synthesis by articular chondrocytes (AChs). Porous chitosan-agarose (CHAG) scaffolds were synthesized and compared to bulk agarose (AG) scaffolds. Both scaffolds were seeded with osteoarthritic human AChs and cultured in a novel perfusion system with a medium flow velocity of 0.33 mm/s corresponding to 0.4 mPa surfice shear and 40 mPa CHAG interstitial shear. While there were no statistical differences in cell viability for perfusion versus static cultures for either scaffold type, CHAG scaffolds exhibited a 3.3-fold higher (p < 0.005) cell viability compared to AG scaffold cultures. Effects of combined superficial and interstitial perfusion for CHAG showed 150- and 45-fold (p < 0.0001) increases in total collagen (COL) and 13- and 2.2-fold (p < 0.001) increases in glycosaminoglycans (GAGs) over AG non-perfusion and perfusion cultures, respectively, and a 1.5-fold and 3.6-fold (p < 0.005) increase over non-perfusion CHAG cultures. Contrasting CHAG perfusion and static cultures, chondrogenic gene comparisons showed a 3.5-fold increase in collagen type II/type I (COL2A1/COL1A1) mRNA ratio (p < 0.05), and a 1.3-fold increase in aggrecan mRNA. Observed effects are linked to NF-κB signal transduction pathway inhibition as confirmed by a 3.2-fold (p < 0.05) reduction of NF-κB mRNA expression upon exposure to perfusion. Our results demonstrate that pores play a critical role in improving cell viability and that interstitial flow caused by medium perfusion through the porous scaffolds enhances the expression of chondrogenic genes and extracellular matrix through downregulating NF-κB1.

Development and operation of immobilized cell plug flow bioreactor (PFR) for treatment of textile industry effluent and evaluation of its working efficiency.

The release of untreated/partially treated effluent and solid waste from textile dyeing industries, having un-reacted dyes, their hydrolysed products and high total dissolved solids (TDS) over the period of time had led to the deterioration of ecological niches. In an endeavour to develop a sustainable and effective alternative to conventional approaches, a plug flow reactor (PFR) having immobilized cells of consortium of three indigenous bacterial isolates was developed. The reactor was fed with effluent collected from the equalization tank of a textile processing unit located near city of Amritsar, Punjab (India). The PFR over a period of 3 months achieved 97.98 %, 82.22 %, 87.36%, 77.71% and 68.75% lowering of colour, chemical oxygen demand (COD), biological oxygen demand (BOD), total dissolved solids (TDS) and total suspended solids (TSS) respectively. The comparison of the phytotoxicity and genotoxicity of untreated and PFR-treated output samples using plant and animal models indicated significant lowering of respective toxicity potential. This is a first report, as per best of our knowledge, regarding direct treatment of textile industry effluent without any pre-treatment and with minimal nutritional inputs, which can be easily integrated into already existing treatment plant. The successful implementation of this system will lower the cost of coagulants/flocculants and also lowering the sludge generation.

Biogas bioconversion into poly(3-hydroxybutyrate) by a mixed microbial culture in a novel Taylor flow bioreactor.

Biogas-based biopolymer production represents an alternative biogas valorization route with potential to cut down plastic pollution and greenhouse gas emissions. This study investigated for the first time the continuous bioconversion of methane, contained in biogas, into poly(3-hydroxybutyrate) (PHB) by a mixed methanotrophic culture using an innovative high mass-transfer Taylor flow bioreactor. Following a hydrodynamic flow regime mapping, the influence of the gas residence time and the internal gas recirculation on CH4 abatement was assessed under non nutrient limiting conditions. Under optimal operational conditions (gas residence time of 60 min and internal gas recycling ratio of 17), the bioreactor was able to support a CH4 removal efficiency of 63.3%, a robust CH4 elimination capacity (17.2 g-CH4 m-3h-1) and a stable biomass concentration (1.0 g L-1). The simultaneous CH4 abatement and PHB synthesis was investigated under 24-h:24-h nitrogen feast/famine continuous operation. The cyclic nitrogen starvation and the Taylor flow imposed in the bioreactor resulted in a relatively constant biomass concentration of 0.6 g L-1 with PHB contents ranging from 11 to 32% w w-1 (on a dry weight basis), entailing an average PHB productivity of 5.9 g-PHB m-3 d-1 with an associated PHB yield of 19.8 mg-PHB g-CH4-1. Finally, the molecular analysis of the microbial population structure indicated that type II methanotrophs outcompeted non-PHB accumulating type I methanotrophs, with a heterotrophic-methanotrophic consortium enriched in Methylocystis, Hyphomicrobium, Rubinisphaeraceae SH PL14 and Pseudonocardia.

Online Measurement System for Dynamic Flow Bioreactors to Study Barrier Integrity of hiPSC-Based Blood-Brain Barrier In Vitro Models.

Electrochemical impedance spectroscopy (EIS) is a noninvasive, reliable, and efficient method to analyze the barrier integrity of in vitro tissue models. This well-established tool is used most widely to quantify the transendothelial/epithelial resistance (TEER) of Transwell-based models cultured under static conditions. However, dynamic culture in bioreactors can achieve advanced cell culture conditions that mimic a more tissue-specific environment and stimulation. This requires the development of culture systems that also allow for the assessment of barrier integrity under dynamic conditions. Here, we present a bioreactor system that is capable of the automated, continuous, and non-invasive online monitoring of cellular barrier integrity during dynamic culture. Polydimethylsiloxane (PDMS) casting and 3D printing were used for the fabrication of the bioreactors. Additionally, attachable electrodes based on titanium nitride (TiN)-coated steel tubes were developed to perform EIS measurements. In order to test the monitored bioreactor system, blood-brain barrier (BBB) in vitro models derived from human-induced pluripotent stem cells (hiPSC) were cultured for up to 7 days. We applied equivalent electrical circuit fitting to quantify the electrical parameters of the cell layer and observed that TEER gradually decreased over time from 2513 Ω·cm2 to 285 Ω·cm2, as also specified in the static control culture. Our versatile system offers the possibility to be used for various dynamic tissue cultures that require a non-invasive monitoring system for barrier integrity.

Effects of phase separation on dewaterability promotion and heavy metal removal of sewage sludge during bioleaching.

Bioleaching is of increasing interest because of its high efficiency in improving sludge dewaterability and removing heavy metals from sewage sludge. However, in traditional single-phase bioleaching, a high-efficiency level cannot be maintained continuously, wherein the microbial synergistic effect is disrupted at a low pH environment. Therefore, in this study, a series of multi-compartment-baffled flow trials were performed to assess the effects of phase separation on sludge bioleaching by comparing a two-phase trial with two single-phase trials. Energy substrate and part of the bioleached sludge were introduced separately into two compartments to form two phases, namely selection phase and bioleaching phase. The results show that phase separation apparently shortened the start-up duration of sludge bioleaching from 7 days in a single-phase bioleaching to 4 days in two-phase bioleaching. The dewaterability of bioleached sludge was also enhanced by phase separation with relative decreases of 25.0-33.3% for specific resistance to filtration and 14.2% for capillary suction time, which was attributed to lower pH values, zeta potential closer to zero, and less dissolved organic matter in bioleached sludge after two-phase bioleaching. Phase separation generally increased the removal ratios of heavy metals during sludge bioleaching by -0.79 to 2.60%, 11.06 to 15.04%, 4.45 to 11.03%, 17.98 to 23.46%, 7.20 to 9.28%, -9.22 to -2.46%, and -6.72 to -10.68% for As, Cd, Cr, Cu, Ni, Pb, and Zn, respectively. Phase separation also enriched the Acidithiobacillus spp. and reduced the inactivation of acid-tolerant fungi, which can be conducive to better synergistic effect, and therefore maintain long-term stable state in the bioleaching phase of the two-phase bioleaching process.

Impact of Chronic Tetracycline Exposure on Human Intestinal Microbiota in a Continuous Flow Bioreactor Model.

Studying potential dietary exposure to antimicrobial drug residues via meat and dairy products is essential to ensure human health and consumer safety. When studying how antimicrobial residues in food impact the development of antimicrobial drug resistance and disrupt normal bacteria community structure in the intestine, there are diverse methodological challenges to overcome. In this study, traditional cultures and molecular analysis techniques were used to determine the effects of tetracycline at chronic subinhibitory exposure levels on human intestinal microbiota using an in vitro continuous flow bioreactor. Six bioreactor culture vessels containing human fecal suspensions were maintained at 37 °C for 7 days. After a steady state was achieved, the suspensions were dosed with 0, 0.015, 0.15, 1.5, 15, or 150 µg/mL tetracycline, respectively. Exposure to 150 µg/mL tetracycline resulted in a decrease of total anaerobic bacteria from 1.9 × 107 ± 0.3 × 107 down to 2 × 106 ± 0.8 × 106 CFU/mL. Dose-dependent effects of tetracycline were noted for perturbations of tetB and tetD gene expression and changes in acetate and propionate concentrations. Although no-observed-adverse-effect concentrations differed, depending on the traditional cultures and the molecular analysis techniques used, this in vitro continuous flow bioreactor study contributes to the knowledge base regarding the impact of chronic exposure of tetracycline on human intestinal microbiota.

Stem Cell-Derived Insulin-Producing Cells in 3D Engineered Tissue in a Perfusion Flow Bioreactor.

To circumvent the lack of donor pancreas, insulin-producing cells (IPCs) derived from pluripotent stem cells emerged as a viable cell source for the treatment of type 1 diabetes. While it has been shown that IPCs can be derived from pluripotent stem cells using various protocols, the long-term viability and functional stability of IPCs in vitro remains a challenge. Thus, the principles of three-dimensional (3D) tissue engineering and a perfusion flow bioreactor were used in this study to establish 3D microenvironment suitable for long-term in vitro culture of IPCs-derived from mouse embryonic stem cells. It was observed that in static 3D culture of IPCs, the viability decreased gradually with longer time in culture. However, when a low flow (0.02 mL/min) was continuously applied to 3D IPC containing tissues, enhanced survival and function of IPCs were demonstrated. IPCs cultured under low flow exhibited a significantly enhanced glucose responsiveness and upregulation of Ins1 compared to that of static culture. In summary, this study demonstrates the feasibility and benefits of 3D engineered tissue environment combined with perfusion flow in vitro for culturing stem cell-derived IPCs. Impact statement This in vitro three-dimensional tissue system combined with the flow can be used to better understand the role of biophysical cues that facilitates improved function and maturation of stem cell-derived insulin-producing cells, which can ultimately advance the field of pancreatic tissue engineering as well as in diabetes treatment.

An Enzymatic Flow-Based Preparative Route to Vidarabine.

The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A), catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions. Glyoxyl-agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration) the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor for the preparation of other purine nucleosides.

In Vitro Endothelialization of Surface-Integrated Nanofiber Networks for Stretchable Blood Interfaces.

Despite major technological advances within the field of cardiovascular engineering, the risk of thromboembolic events on artificial surfaces in contact with blood remains a major challenge and limits the functionality of ventricular assist devices (VADs) during mid- or long-term therapy. Here, a biomimetic blood-material interface is created via a nanofiber-based approach that promotes the endothelialization capability of elastic silicone surfaces for next-generation VADs under elevated hemodynamic loads. A blend fiber membrane made of elastic polyurethane and low-thrombogenic poly(vinylidene fluoride- co-hexafluoropropylene) was partially embedded into the surface of silicone films. These blend membranes resist fundamental irreversible deformation of the internal structure and are stably attached to the surface, while also exhibiting enhanced antithrombotic properties when compared to bare silicone. The composite material supports the formation of a stable monolayer of endothelial cells within a pulsatile flow bioreactor, resembling the physiological in vivo situation in a VAD. The nanofiber surface modification concept thus presents a promising approach for the future design of advanced elastic composite materials that are particularly interesting for applications in contact with blood.

Tissue-Engineered Tubular Heart Valves Combining a Novel Precontraction Phase with the Self-Assembly Method.

Recently, the tubular shape has been suggested as an effective geometry for tissue-engineered heart valves, allowing easy fabrication, fast implantation, and a minimal crimped footprint from a transcatheter delivery perspective. This simple design is well suited for the self-assembly method, with which the only support for the cells is the extracellular matrix they produce, allowing the tissue to be completely free from exogenous materials during its entire fabrication process. Tubular constructs were produced by rolling self-assembled human fibroblast sheets on plastic mandrels. After maturation, the tubes were transferred onto smaller diameter mandrels and allowed to contract freely. This precontraction phase thickened the tissue and prevented further contraction, while improving fusion between the self-assembled layers and aligning the cells circumferentially. When mounted in a pulsed-flow bioreactor, the valves showed good functionality with large leaflets coaptation and opening area. Although physiological aortic flow conditions were not reached, the leaflets could withstand a 1 Hz pulsed flow with a 300 mL/s peak flow rate and a 70 mmHg peak transvalvular pressure. This study shows that the self-assembly method, which has already proven its potential for the production of small diameter vascular grafts, could also be used to achieve functional tubular heart valves.

Scaffold-free cartilage tissue engineering with a small population of human nasoseptal chondrocytes.

OBJECTIVE: Cartilage tissue engineering is a promising approach to provide suitable materials for nasal reconstruction; however, it typically requires large numbers of cells. We have previously shown that a small number of chondrocytes cultivated within a continuous flow bioreactor can elicit substantial tissue growth, but translation to human chondrocytes is not trivial. Here, we aimed to demonstrate the application of the bioreactor to generate large-sized tissues from a small population of primary human nasoseptal chondrocytes. STUDY DESIGN: Experimental study. METHODS: Chondrocytes were cultured in the bioreactor using different medium compositions, with varying amounts of serum and with or without growth factors. Resulting engineered tissues were analyzed for physical properties, biochemical composition, tissue microstructure, and protein localization. RESULTS: Bioreactor-cultivated constructs grown with serum and growth factors (basic fibroblast growth factor and transforming growth factor beta 2) had greater thickness, as well as DNA and glycosaminoglycan (GAG) contents, compared to low serum and no growth factor controls. These constructs also showed the most intense proteoglycan and collagen II staining. CONCLUSION: The combination of bioreactor conditions, serum, and growth factors allowed the generation of large, thick scaffold-free human cartilaginous tissues that resembled the native nasoseptal cartilage. There also may be implications for patient selection in future clinical applications of these engineered tissues because their GAG content decreased with donor age. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E91-E99, 2017.

Dynamic cultivation with radial flow bioreactor enhances proliferation or differentiation of rat bone marrow cells by fibroblast growth factor or osteogenic differentiation factor.

Dynamic cultivation using a radial flow bioreactor (RFB) has gained increasing interest as a method of achieving bone regeneration. In order to enhance bone generation in large bone defects, it is necessary to use an RFB to expand the primary cells such as bone marrow cells derived from biotissue. The present study aimed to evaluate the cell expansion and osteogenic differentiation of rat bone marrow cells (rBMC) when added to basic fibroblast growth factor containing medium (bFGFM) or osteogenic differentiation factor containing medium (ODM) under dynamic cultivation using an RFB. Cell proliferation was evaluated with a DNA-based cell count method and histological analysis. An alkaline phosphatase (ALP) activity assay and immunohistochemistry staining of osteogenic markers including BMP-2 and osteopontin were used to assess osteogenic differentiation ability. After culture for one week, rBMC cell numbers increased significantly under dynamic cultivation compared with that under static cultivation in all culture media. For different culture media in dynamic cultivation, bFGFM had the highest increase in cell numbers. ALP activity was facilitated by dynamic cultivation with ODM. Furthermore, both BMP-2 and osteopontin were detected in the dynamic cultivation with ODM. These results suggested that bFGFM promotes cell proliferation and ODM promotes osteogenic differentiation of rBMC under dynamic cultivation using an RFB.

Flow perfusion culture of MC3T3-E1 osteogenic cells on gradient calcium polyphosphate scaffolds with different pore sizes.

Calcium polyphosphate is a biodegradable bone substitute. It remains a challenge to prepare porous calcium polyphosphate with desired gradient porous structures. In this study, a modified one-step gravity sintering method was used to prepare calcium polyphosphate scaffolds with desired-gradient-pore-size distribution. The differences of porous structure, mechanical strength, and degradation rate between gradient and homogenous calcium polyphosphate scaffolds were evaluated by micro-computed tomography, scanning electron microscopy, and mechanical testing. Preosteoblastic MC3T3-E1 cells were seeded onto gradient and homogenous calcium polyphosphate scaffolds and cultured in a flow perfusion bioreactor. The distribution, proliferation, and differentiation of the MC3T3-E1 cells were compared to that of homogenous calcium polyphosphate scaffolds. Though no significant difference of cell proliferation was found between the gradient and the homogenous calcium polyphosphate scaffolds, a much higher cell differentiation and mineralization were observed in the gradient calcium polyphosphate scaffolds than that of the homogenous calcium polyphosphate scaffolds, as manifested by increased alkaline phosphatase activity (p < 0.05). The improved distribution and differentiation of cultured cells within gradient scaffolds were further supported by both (18)F-fluorine micro-positron emission tomography scanning and in vitro tetracycline labeling. We conclude that the calcium polyphosphate scaffold with gradient pore sizes enhances osteogenic cell differentiation as well as mineralization. The in vivo performance of gradient calcium polyphosphate scaffolds warrants further investigation in animal bone defect models.

Distribution and Viability of Fetal and Adult Human Bone Marrow Stromal Cells in a Biaxial Rotating Vessel Bioreactor after Seeding on Polymeric 3D Additive Manufactured Scaffolds.

One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation.

Study of bioleaching under different hydraulic retention time for enhancing the dewaterability of digestate.

Dewatering of kitchen waste digestate is a key problem to solve so as to increase the application of kitchen waste after anaerobic digestion. In this study, the effects of bioleaching under different hydraulic retention time (HRT = 2, 2.5, and 3 days) on dewaterability of kitchen waste digestate were evaluated. A 12-stage plug flow bioreactor with 180 L working volume was used for digestate bioleaching. The bioleached digestate under different HRTs were collected and dewatered by plate-and-frame filter press. The results showed that the moisture contents of digestate cakes were 67.87 % at 2 days of HRT, 58.06 % at 2.5 days of HRT, and 54.45 % at 3 days of HRT, respectively, indicating the longer the HRT, the lower the moisture content of filter cake. Balanced between the cost and practical need, 2.5 days can be used as the HRT in engineering application. Under the condition of HRT of 2.5 days, the pH, specific resistance to filtration (SRF), capillary suction time (CST), and sedimentation rate of digestate changed from the initial values of 8.08, 210.6 s, 23.4 × 10(12) m kg(-1) and 10 % to 3.21, 32.7 s, 2.44 × 10(12) m kg(-1) and 76.8 %, respectively. Based on the observations above, the authors conclude that bioleaching technology is an effective method to enhance digestate dewaterability and reduce the cost of subsequent reutilization.

Effect of osteogenic differentiation medium on proliferation and differentiation of human mesenchymal stem cells in three-dimensional culture with radial flow bioreactor.

Human mesenchymal stem cells (hMSCs) are multipotent cells, and have been expanded and differentiated into several kinds of mesodermal tissue in vitro. In order to promote bone repair, enhancement of the proliferation and differentiation of hMSCs into osteoblasts in vitro is recommended prior to therapeutic delivery. However, for clinical applications, it is still unclear which method is more advanced for tissue engineering: to transplant undifferentiated cells or partially differentiated stem cells. Therefore, the present study aimed to investigate how osteogenic differentiation medium (ODM) affects hMSCs cultured in a 3D scaffold using a radial-flow bioreactor (RFB) besides cell growth medium (GM). To produce precultured sheets, the hMSCs were first seeded onto type 1 collagen sheets and incubated for 12 h, after which they were placed in the RFB for scaffold fabrication. The culture medium was circulated at 3 mL/min and the cells dynamically cultured for 1 week at 37 °C. Static cultivation in a culture dish was also carried out. Cell proliferations were evaluated by histological analysis and DNA-based cell count. Alkaline phosphatase (ALP) activity, immunocytochemical analysis with BMP-2, and osteopontin on the hMSCs in the collagen scaffold were performed. After 14 days of ODM culture, a significant increase in cell number and a higher density of cell distribution in the scaffold were observed after both static and dynamic cultivation compared to GM culture. A significant increase in ALP activity after 14 days of ODM was recognized in dynamic cultivation compared with that of static cultivation. Cells that BMP-2 expressed were frequently observed after 14 days in dynamic culture compared with other conditions, and the expression of osteopontin was confirmed in dynamic cultivation after both 7 days and 14 days. The results of this study revealed that both the proliferation and bone differentiation of hMSCs in 3D culture by RFB were accelerated by culture in osteogenic differentiation medium, suggesting an advantageous future clinical applications for RFB cell culture and cell transplantation for tissue engineering.

An efficient system for secretory production of fibrinogen using a hepatocellular carcinoma cell line.

AIM: Despite an increasing demand, blood products are not always safe because most are derived from blood donations. One possible solution is the development and commercialization of recombinant fibrinogen, but this process remains poorly developed. This study aimed to develop an effective production system for producing risk-free fibrinogen using human hepatocellular cell lines and serum-free media. METHODS: Three human liver cancer cell lines (HepG2, FLC-4 and FLC-7) were cultivated in a serum-supplemented medium or two serum-free media (ASF104N and IS-RPMI) to compare their fibrinogen secretion abilities. Fibrinogen subunit gene expression was estimated by quantitative polymerase chain reaction. Massive fibrinogen production was induced using a 5-mL radial flow bioreactor (RFB) while monitoring glucose metabolism. Subsequently, fibrinogen's biochemical characteristics derived from these cells were analyzed. RESULTS: FLC-7 cell culture combined with IS-RPMI resulted in significantly better fibrinogen production (21.6 µg/10(7) cells per day). ASF104N had more positive effects on cell growth compared with IS-RPMI, whereas fibrinogen production was more efficient with IS-RPMI than with ASF104N. Changing the medium from ASF104N to IS-RPMI led to significantly increased fibrinogen gene expression and glucose consumption. In the RFB culture, the fibrinogen secretion rate of FLC-7 cells reached 0.73 µg/mL per day during a 42-day cultivation period. The subunit composition and clot formation activity of FLC-7 cell-derived fibrinogen corresponded to those of plasma fibrinogen. CONCLUSION: The FLC-7 cell culture system is suitable for large-scale fibrinogen preparation production.