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To reduce phenolic pollutants in the environment, many countries have imposed firm restrictions on industrial wastewater discharge. In addition, the current industrial process of phenolic resin production uses phenol and formaldehyde as the reactants to perform a polycondensation reaction. Due to the toxicity of formaldehyde and phenolic pollutants, the main purpose of this research was to design a green process using horseradish peroxidase (HRP) enzymatic polymerization to remove phenols and to produce formaldehyde-free phenolic polymers. In this study, the optimal reaction conditions, such as reaction temperature, pH, initial phenol concentration and initial ratio of phenol, and H2O2, were examined. Then, the parameters of the enzyme kinetics were determined. To solve the restriction of enzyme inactivation, several nonionic surfactants were selected to improve the phenol removal efficiency, and the optimal operation conditions in a surfactant-containing system were also confirmed. Importantly, the molecular weight of the synthetic phenolic polymers could be controlled by adjusting the ratio of phenol and H2O2. The content of biphenols in the products was almost undetectable. Collectively, a green chemistry process was proposed in this study and would benefit the treatment of phenol-containing wastewater and the production of formaldehyde-free phenolic resin in the future.

In this study, Saccharomyces cerevisiae was engineered to degrade and utilize xylan, one of the major polysaccharide chains present in hemicellulose. Different hemicellulases from Trichoderma reesei, namely: endoxylanase, β-xylosidase, acetylxylan esterase, α- d -glucuronidase and α- l -arabinofuranosidase, were heterologously secreted by S. cerevisiae. A mixture experimental design was adapted to statistically describe the synergistic interactions between the hemicellulases and to determine the optimum formulations for the hydrolysis of xylan substrates. The hydrolytic activities of the hemicellulase mixtures were then improved by displaying the hemicellulases on the yeast surface as whole-cell biocatalysts. The engineered yeast strains displaying hemicellulases were further engineered to express xylose-utilization genes xylose isomerase (XI) and xylulokinase (XK) which enable its utilization of xylose as a sole carbon source. The resulting consortium was then able to grow and produce ethanol from different xylan substrates.

The aim of this study was to utilize a new and highly effective bioreactor system, i.e., simultaneous saccharification and fermentation (SSCF), for bioethanol production by the cocultivation of Trichoderma reesei, Aspergillus niger, and Zymomonas mobilis by using a direct conversion process of pretreated-wood dust medium. Wood dust has been effectively used to obtain reducing sugars (glucose, xylose, and other byproducts) through the use of either a supercritical fluid extraction (SFE) or steam explosion (SE) pretreatment step. In addition, experimental results showed that polyurethane as a porous carrier could enhance total saccharification enzyme activity at an inoculum proportion of 1/1 of T. reesei, and A. niger and at a total inoculum concentration of 6.5 × 106 spores/ml. Furthermore, the concentration of alginate beads (3%) and immobilized proportion of Z. mobilis to alginate beads (1:4) were also examined. In accordance with previous reports, bioethanol production was carried out in a SSCF bioreactor by the cocultivation of T. reesei and A. niger in the polyurethane carrier and Z. mobilis immobilized in alginate beads using pretreated-wood dust medium. Experimental results revealed that, after 24 h of cultivation, the yield of bioethanol produced using pretreated-wood dust medium (1%) were 0.069 g/g and 0.049 g/g, for SFE and SE, respectively. Meanwhile, the sugar conversion rate reached 20.72% and 24.39% for SFE and SE, respectively. Thus, the results of this study show that pretreated-wood dust medium has significant potential for use in bioethanol production.

Recently, we demonstrated the feasibility of subcutaneous transplantation of MIN6 cells embedded in a scaffold with poly(ethylene glycol) methyl ether (mPEG)-poly(Ala) hydrogels. In this study, we further tracked these grafts using magnetic resonance (MR) and bioluminescence imaging. After being incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles and then mixed with mPEG-poly(Ala) hydrogels, MIN6 cells appeared as dark spots on MR scans. For in vivo experiments, we transfected MIN6 cells with luciferase and/or incubated them overnight with CSPIO overnight, 5 × 106 MIN6 cells embedded in mPEG-poly(Ala) hydrogels were transplanted into the subcutaneous space of each nude mouse. The graft of CSPIO-labeled MIN6 cells was visualized as a distinct hypointense area on MR images located at the implantation site before day 21. However, this area became hyperintense on MR scans for up to 64 days. In addition, positive bioluminescence images were also observed for up to 64 days after transplantation. The histology of removed grafts showed positive insulin and iron staining. These results indicate mPEG-poly(Ala) is a suitable scaffold for β-cell encapsulation and transplantation. Moreover, MR and bioluminescence imaging are useful noninvasive tools for detecting and monitoring mPEG-poly(Ala) hydrogel-embedded MIN6 cells at a subcutaneous site.

Herein, we report the synthesis and characterization of a novel positively charged thermosensitive hydrogel prepared from poloxamer (PLX)-poly(L-alanine-lysine) (Lys-Ala-PLX-Ala-Lys) that demonstrates potential in biomedical applications including tissue engineering and drug delivery. At a dilute concentration, the copolymer self-assembled into micelles that were sensitive to temperature elevation. In the concentration range of 3–7 wt%, the aqueous copolymer solution underwent sol–gel transition near the physiological temperature and exhibited changes in its secondary structure content, as evidenced by FT-IR. Excellent viability of cells cultured within the scaffold was observed after 72 h of incubation. On the other hand, negatively charged bovine serum albumin was incorporated into the hydrogel without diminishing material integrity. Over the course of seven days, the protein underwent extended zero-order release. These findings highlight the potential of this system as a cell scaffold as well as a depot for the extended delivery of drugs and proteins, specifically those which bear a charge opposite to that of the hydrogel.

The liver is the current site of choice for clinical islet transplantation, but many intraportal grafts were lost in the early stage of transplantation. Other disadvantages include the difficulty of monitoring the islets and procedure-associated complications. In contrast, the subcutaneous space offers the advantages of a large space, a less invasive procedure and easy graft monitoring and removal. However, islet transplantation into an unmodified subcutaneous site has poor efficacy; possibly due to poor oxygenation and inadequate vascularization of the transplanted tissue. In this study, we tested the feasibility of subcutaneous transplantation of MIN6 beta cells embedded in temperature-sensitive poly(ethylene glycol) methyl ether (mPEG)-Ala hydrogels as scaffolds. After culturing for 14 days, the viability of MIN6 beta cells in mPEG-Ala hydrogel was comparable with those in medium, either assayed by MTT or LIVE/DEAD. Static incubation showed that both had comparable insulin secretion. For in vivo experiments, we infected MIN6 beta cells with luciferase by AAV and then transplanted 5*106 cells embedded in mPEG-Ala hydrogel into the subcutaneous space of each nude mouse’s upper back. Positive In Vivo Imaging System (IVIS) images and positive insulin staining of tissue histology were observed up to 41 days after transplantation. Meanwhile, we incubated MIN6 beta cells with chitosan-coated superparamagnetic iron oxide (CSPIO) overnight and then transplanted 5*106 MIN6 beta cells into the subcutaneous tissue of each nude mouse’s left flank. The graft of CSPIO-labeled MIN6 beta cells was visualized on Magnetic Resonance (MR) scans as distinct hypointense spots on T2-weighted images located at the transplantation site at day 6 and 20. These results indicate that subcutaneous transplantation of MIN6 beta cells embedded in temperature-sensitive mPEG-Ala hydrogels is feasible. Moreover, the IVIS and MR imaging are useful tools for detecting and monitoring beta cells at the subcutaneous site. Disclosure J. Juang: None.H. Lin: None.C. Chen: None.C. Kao: None.S. Wu: None.S. Lin: None.C. Shen: None.J. Wang: None.I. Chu: None.

Cupriavidus taiwanensis 187 is reportedly efficient in achieving the degradation of phenol and accumulation of polyhydroxybutyrate (PHB). This study attempted to optimize the cultivation conditions and fermentation strategies for phenol degradation and PHA accumulation by C. taiwanensis 187. After the cultivation conditions were optimized, the conditions required for achieving phenol degradation (100%) and PHB accumulation (51 mg/L) by C. taiwanensis 187 were identified as 30 °C and 200 rpm, when the cultivation time was around 7 h. The accumulation of PHB was further increased from 72 to 213 mg/L by feeding phenol in three rounds into the fermenter along with the exhaustion of dissolved oxygen, which could totally degrade the phenol at around 1500 mg/L. Production of PHB by C. taiwanensis 187 was confirmed by GC, 1H-NMR, and 13C-NMR analyses. Each analytical result proved that C. taiwanensis 187 was able to use phenol as the sole carbon source for producing PHB. Finally, these results revealed that the phenol degraded by C. taiwanensis 187 mainly contributed to cell growth rather than PHB accumulation. These results indicated that the strain C. taiwanensis 187 could be used to degrade phenol to obtain usable biological polyesters.

The aim of this work was to study the feasibility of surface displaying synthetic phytochelatin (EC) on Saccharomyces cerevisiae to overcome the inhibitory effect of heavy metals on ethanol production. Via the fusion of a gene encoding EC to an α-agglutinin gene, the engineered S. cerevisiae was able to successfully display EC on its surface. This surface engineered yeast strain exhibited an efficient cadmium adsorption capability and a remarkably enhanced cadmium tolerance. Moreover, its ethanol production efficiency was significantly improved as compared to a control strain in the presence of cadmium. Similar results could also be observed in the presence of other metals, such as nickel, lead and copper. Overall, this method allows simultaneous biorefinery and heavy metal removal when using heavy metal-contaminated biomass as raw materials.

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable β-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. Moreover, we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigel-coated surface. Thus, we provided a totally defined condition from hESC culture to insulin-producing cell differentiation, and the derived cells could be a therapeutic resource for diabetic patients in the future.

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, are an important source of cells for regenerative medicine. The immediate obstacles that need to be addressed are the poor cell survival rate of hESCs and their cell quality after cryopreservation. In this study, we used the Cell Alive System (CAS) which combines a programmed freezer with an oscillating magnetic field to reduce cryo-injury during the freezing process. The hESC clumps suspended in freezing medium were divided into three groups: (i) cells frozen by a conventional freezing container, Mr. Frosty and kept in a −80 °C freezer (MF); (ii) cells frozen to −32 °C by CAS, and then transferred to a −80 °C freezer (CAS); (iii) cells frozen to −32 °C by CAS, and then transferred to a pre-cooled Mr. Frosty and kept in a −80 °C freezer (CAS-MF) for overnight. All cryovials were placed in liquid nitrogen for one week, and hESCs were then thawed and cultured on feeder for 7 days. The results of alkaline phosphatase (AP) staining showed that the attachment efficiency of the cells cryopreserved by CAS and CAS-MF was significantly higher (29.0% and 44.0%) than in the MF method (7.0%). Furthermore, we confirmed the cells cryopreserved using CAS-MF could be subcultured while expressing pluripotent markers, differentiate into three germ layers, and maintain a normal karyotype. These results demonstrate that the use of CAS-MF offers an efficient method of hESC banking.

The aim of this study was to use a modified bioreactor system for simultaneous saccharification of cellulose and bioethanol production. We tested Aspergillus niger and Trichoderma reesei for cellulose saccharification and Zymomonas mobilis for bioethanol production simultaneously in this modified bioreactor. The results showed that various carboxymethylcellulose (CMC) concentrations (10, 15, or 20 g/L) as a substrate for A. niger and T. reesei yielded bioethanol production of 0.51, 0.78, and 0.89 g/L and a CMC conversion rate of 10.2%, 10.7%, and 8.89%, respectively. These data suggested that at 10 g/L CMC as a substrate, the CMC conversion rate was higher than that at the other concentrations. When CMC concentration exceeded 15 g/L, bioethanol production was prolonged to 40 h. These results were attributed to the viscosity of CMC. This study also tested Napier grass (an agricultural byproduct) for bioethanol production. The results revealed bioethanol production and the theoretical Napier grass conversion rate were 0.38 g/L and 12.6%, respectively, for 13-h cultivation when the feeding concentration of Napier grass was 10 g/L. When Napier grass concentration was increased to 15 g/L, bioethanol production and the Napier grass conversion rate reached 0.51 g/L and 23%, respectively, after 14-h cultivation. Eventually, the experimental results indicated using agricultural waste for bioethanol production has been become a potential strategy.

During the fermentation process, dissolved oxygen values and carbon-to-nitrogen ratios are critical factors influencing DHA productivity. This study employed an intermittent oxygen feeding method to maintain a 50% dissolved oxygen level and produced a dissolved oxygen fluctuation environment to facilitate both Aurantiochytrium limacinum SR21 growth and lipid accumulation. Study results indicated that at a 1.25 C:N ratio and medium composition of 100 g L −1 glycerol, 40 g L −1 yeast extract, and 40 g L −1 peptone, A. limacinum SR21 achieved biomass at 61.76 g L −1 , lipid content at 65.2%, DHA concentration at 20.3 g L −1 , and DHA productivity at 122.62 mg L −1 h −1 , this result were better than most similar researches. Dissolved oxygen fluctuation environment also altered the fatty acid composition of A. limacinum SR21. In the late period of the fermentation process, C16:0 fatty acid ratios decreased significantly to below 5%, and C22:6 fatty acid ratios increased to 70%.

Chondroitin sulfate C (CSC) is an important extracellular matrix (ECM) component of native cartilage tissue. Since ECM is considered to play an important role in guiding proper cellular functions, such as proliferation, differentiation, migration, synthesis or degradation of ECM, in specific tissues, we would like to elucidate the effects of CSC on chondrocytes are cultured on type-II collagen (COL II) scaffolds in this study. In particular, we want to investigate if the oligosaccharides of CSC (O-CSC) have much stronger effects on the chondrocytes. In this in vitro study, human articular chondrocytes were cultured on porous scaffolds made of COL II, cross-linked by genipin. Media containing different molecular weights of CSC were used to cultivate the cells. The results were examined mainly from the gene expression profiles of the cultured cells. The expression levels of several genes were examined by real-time PCR. These included genes of COL II, aggrecan, SRY-related high mobility group-box gene 9 (SOX9) and cartilage oligo matrix protein (COMP), alkaine phosphtase (ALP), a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS-4), a disintegrin and metalloproteinases with thrombospondin motifs 5 (ADAMTS-5), matrix metalloproteinases 3 (MMP-3) and tissue inhibitors of metalloproteinases 3 (TIMP-3). The results suggested that O-CSC is more potent in upregulating genes that promote chondrogenesis and downregulating genes that degrade cartilage ECM. The results suggest low-molecular-weight glycosaminoglycans may have therapeutic values in osteoarthritis treatment and may lead to further understanding of the basic mechanism of the interactions between the chondrocytes and their ECM.

Ex vivo generation of megakaryocytes from hematopoietic stem cells (HSCs) is crucial to HSC research and has important clinical potential for thrombocytopenia patients to rapid platelet reconstruction. In this study, factorial design and steepest ascent method were used to screen and optimize the effective cytokines (10.2 ng/ml TPO, 4.3 ng/ml IL-3, 15.0 ng/ml SCF, 5.6 ng/ml IL-6, 2.8 ng/ml FL, 2.8 ng/ml IL-9, and 2.8 ng/ml GM-CSF) in megakaryocyte induction medium that facilitate ex vivo megakaryopoiesis from CD34(+) cells. After induction, the maximum fold expansion for accumulated megakaryocytes was almost 5000-fold, and the induced megakaryocytes were characterized by analysis of gene expression, polyploidy and platelet activation ability. Furthermore, the combination of megakaryocyte induction medium and HSC expansion medium can induce and expand a large amount of functional megakaryocytes efficiently, and might be a promising source of megakaryocytes and platelets for cell therapy in the future.

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for alpha-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of alpha-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l(-1) maltose, 66 g l(-1) yeast extract, and 5 g l(-1) K(2)HPO(4) at 30 degrees C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l(-1) ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l(-1) h(-1) and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l(-1), and the productivity was 1.5 g l(-1) h(-1). The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l(-1) of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l(-1) h(-1) was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.

Shih-Heng Tseng,1,2 Min-Yuan Chou,2 I-Ming Chu1 1Department of Chemical Engineering, National Tsing Hua University, 2Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan Abstract: We have developed a theranostic nanoparticle, ie, cet-PEG-dexSPIONs, by conjugation of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody, cetuximab, to dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) via periodate oxidation. Approximately 31 antibody molecules were conjugated to each nanoparticle. Cet-PEG-dexSPIONs specifically bind to EGFR-expressing tumor cells and enhance image contrast on magnetic resonance imaging. Cet-PEG-dexSPION-treated A431 cells showed significant inhibition of epidermal growth factor-induced EGFR phosphorylation and enhancement of EGFR internalization and degradation. In addition, a significant increase in apoptosis was detected in EGFR-overexpressing cell lines, A431 and 32D/EGFR, after 24hours of incubation at 37°C with cet-PEG-dexSPIONs compared with cetuximab alone. The antibody-dependent cell-mediated cytotoxicity of cetuximab was observed in cet-PEG-dexSPIONs. The results demonstrated that cet-PEG-dexSPIONs retained the therapeutic effect of cetuximab in addition to having the ability to target and image EGFR-expressing tumors. Cet-PEG-dexSPIONs represent a promising targeted magnetic probe for early detection and treatment of EGFR-expressing tumor cells. Keywords: epidermal growth factor receptor, cetuximab, superparamagnetic iron oxide nanoparticle, magnetic resonance imaging, sodium periodate, polyethylene glycol

S-acetylthio-2-methylpropionic acid (S-AMPA) is an important chiral intermediary for numerous hypertension drugs such as captopril. S-AMPA can be produced by hydrolyzing the corresponding racemic methyl MAMP (S,R-methyl-β-acetylthioisobutyrate) by lipases or esterases that have the appropriate stereo specificity. Psudomonas fluorescens IFO 12055 possessing a highly specific lipase was used to process this reaction in the form of immobilized cells. Reaction kinetic and immobilization methods were also studied. Strong product inhibition was observed, that is, at 3% S-AMPA (namely 183 mM), activity was reduced by 50%. Spontaneous hydrolysis of the ester and thioester bonds was also observed, and was independent of the cells. Thus, reaction selectivity and yield must be optimized through adjusting the substrate concentration and total biocatalyst activity. Conventional calcium alginate (3% w/w) encapsulation was modified by adding 3% w/w polyethyleneimine (PEI) and cross-linked by a biologically derived agent, genipin (5.6 mM). This method was found to be satisfactory to produce stable and functioning biocatalyst and can maintain high reactivity for repeated 25 batches with e.e. values above 90%.

The development of ex vivo culture systems that facilitate the expansion of hematopoietic stem and progenitor cells is crucial to stem cell research and clinical application. In this study, a serum-free, stroma-free and cytokine-containing culture system for CD34+ and colony-forming cell (CFC) expansion was systematically developed and optimized using the two-level factorial design and steepest ascent methods. The experimental results show that the optimal compositions of the serum substitutes and the cytokine cocktail were BIT2 (1.5 g/l BSA, 4.39 μg/ml insulin, 60 μg/ml transferrin, and 25.94 μM 2-ME), and CC-S6 (8.46 ng/ml TPO, 4.09 ng/ml IL-3, 15 ng/ml SCF, 6.73 ng/ml FL, 0.78 ng/ml IL-6, 3.17 ng/ml G-CSF, and 1.30 ng/ml GM-CSF) in the Iscove’s modified Dulbecco’s medium, respectively. After one-week culture, the increases in the total number of white blood cells (WBC), CD34+cells and CFC were 64-, 27- and 22-fold, respectively. Its expansion ability of CD34+ cells and CFC was comparable to that of X-vivo 20™, Stemline™, and Stemspan™ commercial media. These systematic methodologies are helpful in improving the ex vivo expansion system for hematopoietic stem cell and progenitor cells.

A serum free medium for recombinant CHO NTHU 108 cell growth and fusion protein (CD20 linked to a human IgG-Fc γ4 fragment) synthesis were systematically developed using factorial designs combined with the steepest ascent method. Experimental results indicate that the optimal composition of serum replacement for specific fusion protein production was 1% SITE (selenium, insulin, transferrin, ethanolamine), 0.3 g/L yeast extract, and 0.09% linoleic acid-BSA. Cell growth and fusion protein production of the adapted CHO NTHU 108 cultured in Iscove’s modified Dulbecco’s medium supplemented with these serum substitutes were comparable to those in the Ex-Cell 301 commercial serum-free medium. These serum substitutes can also promote CHO cell growth and fusion protein production in nine kinds of commercial media. The low protein content of the developed medium facilitates downstream processing and product purification.

Dimethyl sulfoxide (DMSO) (1% v/v) stimulated stable transformed Chinese hamster ovary (CHO) cells to synthesize different recombinant proteins and repress their proliferation rate. The expressions of a fusion protein and β-galactosidase were increased 1.6- and 1.4-fold after adding DMSO. The expression of fusion protein was increased by up to 2.8-fold that of uninduced control by the simultaneous addition of DMSO and pentanoic acid. However, DMSO did not increase the production of the monoclonal antibody (immunoglobulin G) of three hybridoma cell lines (OKM1, OKT4 and HyGPD-YK-1-1), although it could inhibit the growth rates of the hybridoma.

When aurintricarboxylic acid (ATA) was added at a concentration of 30 mg/l in DME/F12 medium to Chinese hamster ovary (CHO) NTHU-108 cells in static six-well plates, some of the cells exhibited adherent growth while others grew in suspension. Beyond the critical concentration of ATA, CHO cells grew in single-cell suspension. In the same serum-free medium, insulin at a level of 0.5 mg/l was found to be the most important protein ingredient promoting cell growth. We used the respective kinase inhibitors to investigate their influence on the cell proliferation induces by ATA and insulin. It is interesting that the inhibition by seven kinase inhibitors of ATA-induced proliferation is similar to that of insulin-induced proliferation. It is possible that ATA mimics insulin and influences the mitogen activated signal transduction to induce the proliferation of CHO cells. Although the actual mechanism of the proliferation of CHO cells by ATA is unclear, ATA supported the long-term proliferation of CHO cells under serum-free conditions and thus could be used as a good substitute for insulin in the formulation of protein-free media.

Nine carboxylic acids were tested to evaluate their effects on recombinant fusion protein production and cell growth of Chinese Hamster Ovary (CHO) cells. Pentanoic acid was demonstrated to have the highest enhancement effect on the protein biosynthesis of CHO cells among the acids tested. Pentanoic acid also had less growth suppression effects compared with butyrate. The optimal induction time and concentration of pentanoic acid for a 120-h batch culture were 72 h and 1 mM, respectively. Apoptosis (programmed cell death) was observed in the serum-free batch culture of CHO cells using a cell death detection ELISA kit. The addition of butyrate accelerated the rate of apoptosis of CHO cells whereas the addition of pentanoate did not. These results confirmed that pentanoic acid was a better stimulant for protein biosynthesis in animal cell culture than butyrate.

Paclitaxel is an anticancer agent with low aqueous solubility. More extensive clinical use of this drug is somewhat delayed due to lack of appropriate delivery vehicles. An attempt was made to adopt an o/w emulsion as the drug carrier which incorporated paclitaxel in the triacylglycerol stabilized by a mixed-emulsifier system. A suitable formulation was found in this study: 0.75 mg/ml paclitaxel, 10% (w/v) oil blend, 4% (w/v) EPC, 3% (w/v) Tween 80 in 2.25% (w/v) glycerol solution. The formulated emulsion has very good stability when stored at 4 degrees C, and the paclitaxel containment efficiency can be maintained above 95% and the mean emulsion diameter around 150 nm for at least 3 months. Paclitaxel-emulsion displayed cytotoxicity against HeLa cells with IC50 at 30 nM. The average life span of ascitic-tumor-bearing mice was prolonged significantly by the treatment of paclitaxel-emulsion (P0.05). The formulated emulsion is a promising carrier for paclitaxel and other lipophilic drugs.

Production of a lipopeptide surfactant, surfactin, by Bacillus subtilis ATCC 21332 was highly enhanced when iron concentration in the medium was raised from 4 μ m to the few m m level. This iron enrichment of the medium also resulted in increased biomass concentration. With 1.7 m m iron sulfate added to the media of different initial iron concentrations or at different stages of growth, surfactin concentration can be raised to levels of several hundred mg l −1 in general. Acidification of the broth was usually observed following the iron addition, and surfactin in the broth disappeared rapidly due to precipitate as soon as the pH level dropped below 5.0; however, if this acidification phenomenon was delayed, avoided (for reasons still unknown), or reversed (by alkaline addition), surfactin production was highly enhanced to as high as 3,500 mg l −1 which was almost ten times over the previously reported level for this strain and higher than most reported values for genetically improved strains.

Two positively charged derivatives of poly(ethylene glycol) (PEG) were coated onto alginate-poly( l -lysine)-alginate (A-P-A) microcapsules by allowing them to interact them with the negatively charged alginate on the capsule surface. The polymers are methoxypolyoxyethylene amine (PEGA1) and polyoxyethylene bis(amine) (PEGA2), which contain charged amine groups at one or both ends, respectively, with PEG as the backbone. The coating of the microcapsules with PEG-amine resulted in a much smoother capsule surface than A-P-A microcapsule surfaces as examined under a scanning electron microscope. The diffusivity of bovine serum albumin into the microcapsules remained the same after PEGA1 coating. But the diffusivity decreased to less than one-fifth that in A-P-A microcapsules coated with PEGA2. The biocompatibility of the microcapsules also improved as investigated by an in vivo study. Microcapsules were implanted in the peritoneal cavity of BALB c mice and retrieved 120 d after implantation. The fibrotic action against A-P-A microcapsules was severe and the capsules retrieved by peritoneal lavage aggregated into clusters. In contrast, the surface-modified capsules were free-flowing and free of cell overgrowth. Secretion of insulin from rat islets within A-P-A-PEGA microcapsules responded well to changes in glucose concentration in a static glucose test. Intraperitoneal transplantation of the microencapsulated islets into streptozotocin-induced diabetic mice could maintain normal blood glucose levels in test animals for up to 200 d without immunosuppression.

The long term in vivo biocompatibility is an essential feature for the design and development of sustained drug release carriers. In the recent intraocular drug delivery studies, hydrogels were suggested as sustained release carriers. The biocompatibility test for these hydrogels, however, was commonly performed only through in vitro cell culture examination, which is insufficient before the clinical applications. We compared three thermosensitive hydrogels that have been suggested as the carriers for drugs by their gel-solution phase-change properties. A new block terpolymer (PEOz-PCL-PEOz, ECE) and two commercial products (Matrigel® and Pluronic F127) were studied. The results demonstrated that the ocular media remained translucent for ECE and Pluronic F127 in the first 2 weeks, but cataract formation for Matrigel occurred in 2 weeks and for Pluronic F127 in 1 month, while turbid media was observed for both Matrigel and Pluronic F127 in 2 months. The electrophysiology examinations showed significant neuroretinal toxicity of Matrigel and Pluronic F127 but good biocompatibility of ECE. The neuroretinal toxicity of Matrigel and Pluronic F127 and superior biocompatibility of ECE hydrogel suggests ECE as more appropriate biomaterial for use in research and potentially in intraocular application.

Magnetic beads coated with monoclonal antibodies (MAb) were used to specifically remove cells carrying the common acute lymphoblastic leukemia antigen (cALLA). The target-cell elimination effectiveness (TEE) was found to be kinetically controled, and can be quantitatively described by a model where antigen-carrying cells and MAb-coated magnetic beads are described as colloidal particles. This model can correctly explain the temperature effect on the immunomagnetic cell adhesion where models based on antigen-antibody interaction failed.

The production of alkaline protease from Bacillus subtilis ATCC 14416 was studied. Inorganic nitrogen compounds in the medium were found to show inhibitory effects on the enzyme formation. The pH variations of the culture broth gave indications of the start and the end of the enzyme production period and thus provided a useful indicator for process control. The rapid deactivation of protease after the end of the production period was studied, and ways to stabilize the enzyme were discussed.

Owing to of the limited repair capacity of articular cartilage, it is essential to develop tissue-engineered cartilage for patients suffering from joint disease. Chondroitin sulfate (CS) and hyaluronan (HA) are the components of the cartilage extracellular matrix (ECM) and are known to influence the proliferation and differentiation of chondrocytes. Scaffolds composed of type-II collagen, CS, and HA may create an environment that can preserve the normal phenotype of cells to promote regeneration of cartilage-like constructs. In this investigation, we prepared and characterized 3-dimensional type-II collagen scaffolds both with and without HA and CS. Porous composite scaffolds fabricated by freeze-drying showed interconnected pores with mean diameters of 140+/-30 microm and porosities of 92-95% after cross-linking with genipin. After a 14-day in vitro culture, morphologically round chondrocytes were found to be uniformly distributed throughout the sponges. Expression of genes of aggrecan, type-II collagen and cartilage oligomeric matrix protein (COMP) was statistically and significantly increased on scaffolds with CS and HA than those without CS and HA. Furthermore, there was a markedly greater accumulation of proteoglycans (PGs) on the scaffolds with CS and HA.

A method of rapid determination of chloro- phyll a and lipid contents of microalgae based on colori- metric analysis of the digital images of the microalgae is proposed. The color variation of microalgae during cultiva- tion is evaluated by the brightness of the three primary colors (red, green, and blue). The brightness values of the three primary colors are modeled as two linear correlation functions (RGB model) for microalgal chlorophyll a and lipid contents, respectively. The chlorophyll a and lipid contents predicted by the proposed model are compared with that determined by the standard methods. The good agreement of the model predictions with experimental results is demonstrated with a squared correlation coeffi- cient (R 2 ) of 0.99 for chlorophyll a and lipid. The reliability of the RGB model was verified in real cultivations of the microalgae in a photobioreactor. Growth dynamics, con- tents of chlorophyll a and lipid corresponded very well with previously reported studies. Biotechnol. Bioeng. 2008;99: 1034-1039. 2007 Wiley Periodicals, Inc.

The development of ex vivo expansion of hematopoietic stem cells (HSCs) is a promising approach to restore the required bone marrow function of patients with hematological disorders. Previously, we have reported the development of an optimized serum-free and cytokines-limited defined medium using statistic methodology for umbilical cord blood-derived HSC expansion. The aim of this study was to analyze further the characteristics and functions of cells in vitro and in vivo when cultured in this defined medium. After a 7-day batch culture, the average absolute fold expansions for CD133(+) cells, CD34(+)CD133(+) cells, CD34(+)CD38() cells, CD133(+)CD38(-) cells, CD34(+)CXCR4(+) cells, CD133(+)CXCR4(+) cells, and long-term culture-initiating cells were 21-, 20-, 723-, 618-, 160-, 384-, and 8-fold, respectively. The high enrichment of CD38(-) cells and CXCR4(+) cells of the CD34(+) subpopulation provided a very early uncommitted HSC proliferation and homing ability. Furthermore, the expanded cells showed a high level of telomerase activity to maintain their telomere length and repopulated the lethally irradiated NOD/SCID mice in vivo. These results indicated that the cytokines limited expanded cells from CD133(+) cells could substantially support simultaneous expansion of various stem/progenitor cells and engraft with the expanded cells from a low number of HSCs initially.

Objective In this study, a serum-free, stroma-free, and chemically defined medium for hematopoietic stem cell (HSC) expansion was systematically developed and optimized using factorial design and the steepest ascent method. Materials and methods Mononuclear cells (MNCs) were isolated from umbilical cord blood (UCB). HSCs were stimulated to proliferate ex vivo in the MNC culture system with variable serum substitutes, cytokines, and basal media according to experimental design. The expanded cells were assessed for cellular characteristics by surface antigen analysis, colony-forming cell assay (CFC assay), and long-term culture-initiating cell assay (LTC-IC assay). Results The optimal compositions of serum substitutes and the cytokine cocktail for HSC expansion in the MNC culture system were BIT (4 g/L BSA, 0.71 μg/mL insulin, and 27.81 μg/mL transferrin), and CC-9 (5.53 ng/mL TPO, 2.03 ng/mL IL-3, 16 ng/mL SCF, 4.43 ng/mL FL, 2.36 ng/mL IL-6, 1.91 ng/mL G-CSF, 1.56 ng/mL GM-CSF, 2.64 ng/mL SCGF, and 0.69 ng/mL IL-11) in the Iscove's modified Dulbecco's medium. After 6-day culture, the absolute fold expansions for white blood cells, CD34 + cells, CD34 + CD38 − cells, CFC, and LTC-IC were 1.4-, 30.4-, 63.9-, 10.7-, 2.8-fold, respectively. Conclusion Using the statistic methodology to develop HSC medium, our formula had lower cytokine concentrations comparing to other literatures and commercial media, but had superior or comparable expansion ability on HSC growth.

Yu-Shiang Peng,1,* Po-Liang Lai,2,* Sydney Peng,1 His-Chin Wu,3 Siang Yu,1 Tsan-Yun Tseng,4 Li-Fang Wang,5 I-Ming Chu1 1Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 2Department of Orthopedic Surgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, 3Department of Materials Engineering, Tatung University, Taipei, 4Graduate School of Biotechnology and Bioengineering, College of Engineering, Yuan Ze University, Chung-Li, 5Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan *Yu-Shiang Peng and Po-Liang Lai contributed equally to this work Abstract: Parkinson’s disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF) have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood–brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI) is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI) and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol) of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively) by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810). This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA) polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the polymer:DNA weight ratio of 10:1, which was 1.7-fold higher than the maximal GDNF expression of PEI1800-g-CHI/DNA. The low toxicity and high transfection efficiency of PEI600-g-CHI make it ideal for application to GDNF gene therapy, which has potential for the treatment of Parkinson’s disease. Keywords: chitosan, gene transfection, glial cell, Parkinson’s disease, polyethylene imine

Objective A complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34 + cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34 + cells under a complete and optimal serum-free condition for complying with clinical regulations. Materials and Methods Serum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in nonobese diabetic/severe combined immunodeficient mice. Results The optimal serum-free megakaryocyte induction medium was Iscove's modified Dulbecco's medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated nonobese diabetic/severe combined immunodeficient mice. Conclusion In conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy.

Magnetic beads coupled with secondary monoclonal antibodies were used to remove cells expressing the common acute lymphoblastic leukemia antigen by adsorption. The influences of the bead-to-cell ratio, temperature and the amout of antibodies used on the effectiveness of the immunomagnetic cell isolation were studied.

A new methylotrophic strain (T15), which employs the ribulose monophosphate (RuMP) cycle of formaldehyde assimilation, was isolated on the basis of high in vitro activities of formaldehyde and formate dehydrogenases (19 and 678 mU per mg protein, respectively). Serial subculturing of the strain in batch cultures, on 4 g/l CH3OH for 6 months, led to loss of substantial percentages of the NAD-linked formaldehyde (25%) and formate (98%) dehydrogenases. The activities of these two enzymes were partially recovered when cells were grown continuously at very low dilution rate (0.03 h−1). We found large variations (40 to 1000%) in the activities of other key enzymes of carbon-substrate oxidation (both linear and cyclic) and assimilation, in batch cultures with pure and mixed substrates, and in continuous cultures of different dilution rates. Key intracellular reaction rates, including those of the cyclic and linear substrate oxidation, were measured in vivo using a 14C-tracer technique in both continuous and batch cultures. The results indicate significant variations in these reaction rates, particularly those of linear and cyclic carbon oxidation. Overall, the cyclic oxidation appears to be employed to a larger (although not predominant) extent in strain T15 compared with another RuMP strain (L3) we have previously examined. T15 exhibits high biomass yields (up to 0.63 g cells per g CH3OH) and growth rates (up to 0.46 h−1) on CH3OH in batch cultures. CH3NH2 can also be utilized as a substrate. In continuous culture, T15 could be grown at dilution rates up to 0.36 h−1 with a corresponding biomass yield of 0.4. Examination of a large number of data on the biomass yields of strains T15 and L3 reveals that the large variations in yields derive from the variable branching of carbon flow between linear and cyclic oxidation and assimilation, rather than changes in the biosynthetic efficiency of carbon incorporation into biomass.

A technique using C-14 isotope tracers to probe the branching of carbon flow in methylotrophic bacteria has been devised and applied to continuous steady-state cultures. Methylomonas L3, a strain which utilizes the KDPG/TA variant of the ribulose monophosphate cycle for carbon fixation, was employed in the experimental studies. The actual in vivo rates of substrate-carbon incorporation into biomass, both direct and via CO(2), and of the two carbon oxidation schemes were determined in three different steady-state cultures. The results show that the carbon substrate is oxidized predominantly via formate (the linear oxidation scheme), and that the cyclic scheme of oxidation is minimally, if at all, utilized. The carbon incorporation and oxidation patterns appear to vary considerably with the dilution rate and the inoculum history. The experimental accuracy of the new technique is discussed in detail.

The two components, delta pH and delta psi, of the membrane protonmotive force (delta p) effect and are affected by the transport of many substrates and metabolites. Because the integrity (or restoration) of the delta p requires the expenditure of metabolic energy, such transport processes affect the overall cell bioenergetics. However, the transport or high concentrations of certain substrates and metabolites can have more serious effects on cell metabolism because they partially or completely abolish either or both the delta pH and delta psi. If the cells cannot eventually restore the collapsed component(s) of the delta p, complete growth inhibition and cell death become inevitable. In the butanol/acetone fermentation of Clostridium acetobutylicum, the transport and the presence of key metabolites (acetic and butyric acids, and butanol) have serious and some necessary effects on the delta p. Acetic and butyric acids act as uncouplers of the delta pH, thereby reducing the internal pH. Using other acid uncouplers (such as acetoacetate, which is metabolized by the cells, or FCCP, which is not metabolized by the cells), we found that a lower pHo combined with the metabolic-energy drain of the uncoupling effect and high internal acid concentrations are implicated in the mechanism(s) of solventogenesis. Thus, the production or presence (or both) of the two acids (acetic and butyric) is beneficial to the initiation of solvent production. The transport mechanisms of CH3OH, CH2O, and HCOOH in obligate CH3OH utilizers (methylotrophs) were also discussed in detail. We showed that CH3OH is actively transported by the cells at the expense of metabolic energy and that its transport significantly affects the dynamics of continuous bioreactors. The accumulation of CH2O was found to be driven by the membrane delta p. Finally, formate was accumulated by the delta pH according to the general transport mechanism of short-chain fatty acids. The inhibition of growth by formate was explained by its uncoupling effect on the cells. Growth inhibition by CH3OH appeared to be related to the severe reduction of the membrane delta pH and cell pHi by relatively low CH3OH concentrations.

A recently developed methodology of directly measuring the oxidation and incorporation patterns of carbon substrate in continuous cultures of RuMP-type methylotrophs is extended to batch cultures of the obligate methylotrophMethylomonas L3. The ratio of cyclic to total substrate oxidation varies with the initial methanol concentration from 0 to 68%. Formaldehyde, as a methanol cosubstrate, enhances the net substrate oxidation. The substrate oxidation and incorporation pattern is also affected by the state of the culture inoculum.

High methanol concentrations have a negative effect on the growth rate and the biomass yield of growth transients induced by methanol pulses in continuous cultures of Methylomonas L3. The physiological basis of this effect is investigated by measuring the effect of the methanol pulse on the cell energy charge (EC) and ATP, ADP, and AMP concentrations, and by comparing the results of the pulse transients against an unstructured model. The methanol pulse is shown to lead to increased values of the cell EC and ATP concentration, and thus, inhibition and reduced availability of biosynthetic energy are excluded as causes of inhibition. When the biomass and methanol profiles of the transient experiments are compared in phase-plane diagrams against computer simulations based on the model, satisfactory agreement between experimental data and model predictions is found in single-substrate, high-dilution-rate experiments. Conversely, poor agreement between experimental data and simulation results indicates a more severe growth inhibition than the model predicts at low dilution rates and a less severe one in mixed-substrate experiments. Based on these findings and other relevant physiological information, we propose that the large variations in the negative effect of methanol on growth result from the fact that cells accumulate methanol to widely different concentrations depending on their physiological state. In their effort to detoxify from the high intracellular methanol and formaldehyde concentrations, cells oxidize considerably more methanol than they can incorporate into biomass. This leads to a useless ATP surplus, which the cells must hydrolyze without doing any useful biosynthetic work, and this results in lower biomass yields.

The dynamic behavior of the Ribulose Monophosphate-type Methylomonas L3 in continuous cultures was studied, using methanol pulses to induce fast transients in steady-state cultures of single (methanol) and mixed (methanol plus formaldehyde) substrates. In several experiments, the methanol-uptake rate (MUR) profiles displayed negative MUR values for a time period following the methanol pulse, and significant amounts of methanol disappeared immediately following the pulse. These phenomena suggested the accumulation of methanol in the cells upon pulsing, apparently due to an active transport system. Accordingly, and in order to estimate the potential of the transport system for methanol accumulation, accumulation profiles were calculated for several pulse experiments. The calculations are based on a methanol balance and experimentally determined values of the cell volume and the true transient biomass yields. It is calculated that methanol accumulates up to 200-fold to very high intracellular concentrations. The accumulation is calculated to be much higher in single- (methanol) substrate cultures of low dilution rate than in cultures of high dilution rate or of mixed substrates. The specific growth rate immediately following the methanol pulse decreased in single-substrate cultures and increased in mixed-substrate ones. The biomass yield decreased after the methanol addition in all experiments; however, the drop was less severe in the mixed-substrate experiments. It is also suggested that formaldehyde as a methanol cosubstrate may be an effective means of providing more stable biomass yields and growth rates in reactors with imperfect mixing, and of protecting the reactor against accidentally induced methanol accumulation.