Nutrient Regulation by Continuous Feeding Removes Limitations on Cell Yield in the Large-Scale Expansion of Mammalian Cell Spheroids

Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods. Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed. Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and SSB culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products. Continuously fed bioreactor systems combined with nutrient regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies and will facilitate the translation of cell culture from the research lab to clinical applications.     

Role of lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K(m) values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K(m) about 10% of wild-type TPL), is activated by K(+), and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-Cys or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain 1 mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.     

Oxidative aromatization of Hantzsch 1,4-dihydropyridines in the presence of mixed-addenda vanadomolybdophosphate heteropolyacid, H6PMo9V3O40

A variety of Hantzsch 1,4-dihydropyridines were oxidized to the corresponding pyridines in high yields in the presence of H(6)PMo(9)V(3)O(40), a Keggin type heteropolyacid, in refluxing acetic acid. The heteropolyacid was found to be reusable.     

CYP17, SRD5A2, CYP1B1, and CYP2D6 gene polymorphisms with prostate cancer risk in North Indian population

To investigate the involvement of the CYP17, SRD5A2, CYP1B1, and CYP2D6 variants with prostate cancer, a case-control study of 100 patients and an equal number of age-matched control men was conducted. There appears to be a nonsignificant increase with risk of prostate cancer for individuals carrying one copy of the CYP17 A2 allele (OR, 1.80; 95% CI, 0.99-3.29, P=0.05). The risk was increased in individuals having two A2 alleles (OR; 2.81, 95% CI, 1.06-7.40, P=0.03). Compared with men having the VV genotype of SRD5A2 gene, there was no significant association between the VL genotype and the risk of prostate cancer (OR; 0.54, 95% CI; 0.29-1.03, P=0.06). There was no difference in the occurrence of the genotype LL between controls and prostate cancer patients (OR; 0.90, 95% CI; 0.43-1.89, P=0.79). There was a nonsignificant increased risk of prostate cancer for individuals carrying the CYP1B1Leu/Val genotype (OR, 1.70, 95% CI, 0.91-3.17, P =0.09), which was increased in those having the Val/Val allele (OR, 3.38; 95% CI, 1.13-10.07, P=0.02). Relative to men homozygous for the wild-type allele in CYP2D6 gene, those heterozygous for the B allele had an odds ratio of 1.78 (95% CI, 0.76-4.17, P=0.18) for patients, and for homozygous individuals, it was 1.95 (0.55-6.93, P=0.30). These observations have suggested that the CYP17 A2/A2, CYP1B1 Val/Val, and CYP2D6 genotypes may be associated with an altered risk of prostate cancer, while the CYP2D6 and SRD5A2 V89L polymorphism have no association with its risk in the North Indian population.     

Lead Accumulation Potential in Acacia victoria

To assess the potential of Pb⁺² accumulation in different parts of Acacia victoria, one year old A. victoria seedlings were exposed to Pb²⁺(NO₃)₂ in 5 different concentrations: 0, 50, 250, 500 and 1000 (mg Pb²⁺ L^?1) for 45 days. Subsequently, Pb²⁺ uptake was quantified in roots, shoots and leaves of the seedlings by Atomic Absorption Spectroscopy (AAS). In addition, some physiological parameters such as biomass production, shoots and roots length, plant appearance, tissue concentrations and chlorophyll content were examined. Tissue concentrations increased as Pb²⁺ concentration increased for A. victoria. The visible toxicity symptoms (chlorosis and necrosis) appeared only to the highest concentration (1000 mg Pb²⁺ L^?1), resulting in photosynthesis decrease, plant height, root length and dry biomass reduction. Almost 70% (up to 3580 mg Kg^?1 of dry tissue) from the Pb²⁺ was accumulated in the entire plant tissues was retained in the roots in the seedlings exposed to 1000 mg Pb²⁺ L^?1. The seedlings accumulated between 403 to 913 mg Kg^?1 of Pb²⁺ in shoots and 286 to 650 mg Kg^?1 of Pb²⁺ in leaves at different treatments. Bioconcentration and translocation factors were determined 5.14 and 0.255, respectively. The results show that A. victoria is suitable for lead-phytostabilization in Pb²⁺-contaminated soil.     

Responses of wheat plants to interactions of 24-epibrassinolide and <Emphasis Type="Italic">Glomus mosseae</Emphasis> in saline condition

This study was designed to investigate the possible effects of 24-Epibrassinolide (BR), arbuscular mycorrhizal (AM) fungus, Glomus mosseae, singularly and collectively under salt stress in wheat (Triticum aestivum L.) plants. After foliar spraying of mycorrhizal and non-mycorrhizal plants by 5 µM epibrassinolide (24-Epi), they were treated with 0 and 150 mM NaCl for 2 weeks and then harvested. The results showed interactions of G. mosseae and 24-Epi could alleviate the adverse effects of salinity by improving relative water content (RWC) of leaves (62%), relative growth rate (40.74%), shoot fresh weights (39.83%) and shoot phosphorous content (63.93%), stimulating leaf enzymatic antioxidant activities including catalase (2.24 fold) and ascorbate peroxidase (2.18 fold) as well as malondialdehyde (36.17%) and H₂O₂ concentrations (49.74%) as compared to those of NaCl treatments. Moreover, mycorrhizal dependency of root dry weight (2%) and phosphorus concentration (0.4%) increased with AM infection and 24-Epi application under saline condition. Leaf RWC, also, negatively correlated with membrane electrolyte leakage. Furthermore, the greatest mitigating effects were observed in mycorrhizal plants subjected to NaCl and 24-Epi. This study indicated that 24-Epi application and AM fungi may synergistically mitigate harmful impacts of salinity in wheat plants.     

Novel method to obtain highly enriched cultures of adult rat Schwann cells

Schwann cells (SCs) can be used to repair both the peripheral and central nervous systems. Therefore, establishment of a procedure to obtain activated, highly proliferative SCs, in an appropriate time for clinical applications, is a prerequisite. Purification is complicated by contamination with fibroblasts which often become the predominant cell type in an in vitro SC culture. This study describes a novel and efficient method to enrich SCs by utilizing the differential detachment properties of the two cell types. In culture, cells were treated with two different media and the chelator, EGTA, which detached SCs faster than fibroblasts and allowed for easy isolation of SCs. Within seven days, high yields of SCs with a purity of greater than 99% were achieved. This was confirmed by immunostaining characterization and flow-cytometric analyses using an antibody against the p75 low affinity nerve growth factor receptor (p75LNGFR). The entire procedure was completed in approximately 21 days. This method has the advantage of being technically easier, faster, and more efficient than other previously described methods. An SC culture that was about 99% homogenous was achieved.