Growth of cells in a new defined protein-free medium

The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum.Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR.     

Serial Subcultivation of Giardia lamblia in Keister's Modified TYI-S-33 Medium Containing Ultroser G

ABSTRACT. The ability of Giardia strains of the duodenalis type to grow in Keister's modified TYI-S-33 medium varies with serum lot. Recently, strains of Giardia including MR4, WB, and Human-1-Portland, have been cultivated in Keister's modified TYI-S-33 medium containing the serum substitute Ultroser G and have been cultured serially at least 40 times. An optimal concentration of 8% Ultroser G promotes maximal growth in Keister's modified TYI-S-33 medium for all three strains. This concentration of Ultroser G will produce a two-log increase in the number of trophozoites in approximately three days post-inoculation. Generation times for the trophozoites ranging from 6 to 11 h have been achieved in Keister's modified TYI-S-33 containing 10% adult bovine serum and from 8 to 13 h in Keister's modified TYI-S-33 with 8% Ultroser G. Despite the excellent growth of Giardia strains in medium containing Ultroser G, the maximum trophozoite density is only about half of that achieved in Keister's modified TYI-S-33 medium supplemented with 10% adult bovine serum. Comparisons of trophozoites grown with serum or the serum substitute reveal no discernable differences in morphology and motility. Additionally, these strains have been successfully cryopreserved and revived in Keister's modified TYI-S-33 medium supplemented with Ultroser G. Because Ultroser G is a characterized mixture of six main groups of ingredients (growth factors, adhesion factors, mineral trace elements, hormones, binding proteins, and vitamins), the variability in cell proliferation that may occur when changing serum lots should be minimized when using this product.     

Overall assessment of antimicrobial peptides in piglets: a set of meta-analyses

Developing alternatives to antibiotics is an urgent need in livestock production. Antimicrobial peptides (AMPs) are regarded as powerful antibiotic substitutes (ASs) because AMPs have broad-spectrum antimicrobial activities and growth-promoting ability. Here, we aimed to comprehensively assess the effects of AMPs on the growth performance, diarrhea rate, intestinal morphology and immunity of healthy or challenged piglets, compared with an antibiotics group or negative control group. We performed a set of meta-analyses of feeding trials from database inception to 27 May 2019. Among the 1379 identified studies, 20 were included in our meta-analyses (56 arms and 4067 piglets). The meta-analyses revealed that (1) compared with the negative control group, AMPs significantly improved the healthy piglets’ average daily gain (ADG), average daily feed intake (ADFI), gain : feed ratio (G/F), levels of immune globulin (Ig) IgM and IgG, and intestinal villus height : crypt depth ratio (V/C) (P < 0.05). Meanwhile, AMPs significantly increased the challenged piglets’ ADG, ADFI, G/F and V/C of the jejunum and ileum, and notably deceased the diarrhea rate (P < 0.05); (2) compared with antibiotics group, the effects of AMPs were slightly weaker than those of antibiotics in the healthy piglets, but AMPs have similar effects to those of antibiotics in challenged piglets. In a higher purity, the optimal dose of AMPs may be approximately 0.01%. Our findings indicate that AMPs can improve piglet growth performance, enhance immunity, benefit intestinal morphology and decrease the diarrheal rate. AMPs could be great ASs especially under infection conditions.     

Optimization of the processing of porcine platelet-rich plasma and its application on human mesenchymal stem cell cultivation

Because of ethical and scientific controversy, the utilization of fetal bovine serum (FBS) for cell culture medium must be minimized. This study develops porcine platelet-rich plasma (P-PRP) as a FBS substitute for human mesenchymal stem cell (hMSC) cultivation. Concentrating porcine blood by serial centrifugation to obtain P-PRP leads to activation by different agonist combinations to stimulate the secretion of growth factors. The concentration of growth factor in P-PRP is significantly increased by activation (p < 0.05). The concentration of PDGF, KGF and TGF-β in activated P-PRP is significantly higher than that in FBS. Design-expert was used to decide whether Co−T+Ca−, Co+T−Ca−, and Co+T+Ca− are optimal agonist formulations. MSC cultivation shows that the attachment rate, proliferation rate and viability of P-PRP supplemented media are significantly higher than those for FBS-supplemented and commercial media (p < 0.05). The results demonstrate that P-PRP is an optimal FBS substitute that supports in vitro h-MSCs expansion for subsequent biomedical applications.     

Bone grafts,bone substitutes and orthobiologics

The biology of fracture healing is better understood than ever before, with advancements such as the locking screw leading to more predictable and less eventful osseous healing. However, at times one’s intrinsic biological response, and even concurrent surgical stabilization, is inadequate. In hopes of facilitating osseous union, bone grafts, bone substitutes and orthobiologics are being relied on more than ever before. The osteoinductive, osteoconductive and osteogenic properties of these substrates have been elucidated in the basic science literature and validated in clinical orthopaedic practice. Furthermore, an industry built around these items is more successful and in demand than ever before. This review provides a comprehensive overview of the basic science, clinical utility and economics of bone grafts, bone substitutes and orthobiologics.     

Molecular modelling of a template substitute and monomers used in molecular imprinting for aflatoxin B1 micro-HPLC analysis

The preparation of molecularly imprinted polymers (MIPs) involves the polymerisation of functional monomers in the presence of template molecules. 5,7-Dimethoxycoumarin (DMC) was found to be a structural analogue for aflatoxin B1 (AB1) and serves as its substitute in a grafting solution for the MIP synthesis. It was found that both methacrylic acid and allylamine are functional monomers which could provide a similar binding towards AB1 and DMC.     

Culture of an epithelial cell line (MDCK) with a serum substitute

This paper reports the suitability of culturing a line of dog kidney epithelial cells, MDCK, in the presence of a serum substitute, Ultroser G. Serial subcultivation with this product was possible for at least 10 passages without any change in cell shape and size, saturation density, dome-forming ability, transepithelial resistance, and growth curve. Adhesion of newly plated cells to plastic was somewhat lower than in fetal calf serum but the trypsin-harvesting kinetics were essentially the same. However, the membrane ion transport systems was alterd: cell sodium influx was greatly diminished, suggesting a deep change in the amiloride-sensitive Na⁺ channels: sodium efflux was highly enhanced (both active and passive).     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration,flower and plant formation from petiolar and nodal explants of culantro (<Emphasis Type="Italic">Eryngium foetidum</Emphasis> L.)

Summary A procedure for plant regeneration, flower and plant formation from petiolar and inflorescence nodal explants of culantro is discribed. Leaf petioles were excised from young leaves of non-flowering plants while nodal explants were excised from the inflorescence. Explants were cultured in Murashige and Skoog (MS) medium alone or supplemented with 0.5μM naphthaleneacetic acid (NAA) and 0.9, 1.8, 4.5, or 9 μM thidiazuron (TDZ). All explants produced multiple shoots. In addition, nodal explants formed flowers. Shoot number, flower number and shoot length were influenced by TDZ and NAA. Rooted shoots from both types of explants were transferred to soil where plants were successfully established.     

A solid phase adsorption method for preparation of bovine serum albumin-bovine hemoglobin conjugate

A solid phase adsorption method was proposed to prepare well-defined bovine serum albumin–bovine hemoglobin (Hb) conjugate. After adsorption by the solid phase, Q Sepharose Fast Flow media, bovine serum albumin (BSA) molecules were allowed to react with glutaraldehyde. The spacing out of BSA molecules on the solid phase was assumed to limit polymerization of BSA molecules, except some molecules bound closely on the solid phase resulting in minor dimer formation. Following the elution procedure, the activated monomeric BSA was separated from the dimers by gel filtration chromatography on Superdex 200 and then reacted with bovine Hb at 4 °C and pH 9.5. The 1:1 (BSA:Hb) conjugate was obtained with the yield of 64%. The P₅₀ values of the conjugates, prepared under anaerobic and aerobic conditions, were 19.1 and 14.2 mmHg, respectively. The dependence of the P₅₀ on chloride ions for the conjugate was slightly diminished, presumably due to covalent attachment of BSA to bovine Hb.     

Influence of the scaffolding protein Zonula Occludens (ZOs) on membrane channels

Zonula Occludens (ZO) proteins are ubiquitous scaffolding proteins providing the structural basis for the assembly of multiprotein complexes at the cytoplasmic surface of the plasma membrane and linking transmembrane proteins to the filamentous cytoskeleton. They belong to the large family of membrane-associated guanylate kinase (MAGUK)-like proteins comprising a number of subfamilies based on domain content and sequence similarity. ZO proteins were originally described to localize specifically to tight junctions, or Zonulae Occludentes, but this notion was rapidly reconsidered since ZO proteins were found to associate with adherens junctions as well as with gap junctions, particularly with connexin-made intercellular channels, and also with a few other membrane channels. Accumulating evidence reveals that in addition to having passive scaffolding functions in organizing gap junction complexes, including connexins and cytoskeletals, ZO proteins (particularly ZO-1) also actively take part in the dynamic function as well as in the remodeling of junctional complexes in a number of cellular systems. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.