The extended serial subculture of human diploid fibroblasts on microcarriers using a new medium supplement formulation

Human diploid fibroblasts serially passaged on microcarriers exhibit a decrease in their proliferative capacity with each transfer from microcarrier-to-microcarrier. This phenomenon, which does not occur in the same time scale with cells cultured in T-flasks, has been a serious barrier to the systematic utilization of microcarriers in the scale-up of anchorage-dependent human diploid cell cultures. This decreases in cell growth with each passage is shown to be related to the serum content of the medium, with high serum concentrations resulting in a more rapid decrease in cell growth with each serial transfer. As a result, methods for reducing the serum requirement of the cells were investigated. A new medium supplement mixture, PPRF92, has been developed, which allows the serial passaging of MRC5 cells on Cytodex 1 microcarriers through as many as 13 microcarrier-to-microcarrier tranfers, and at a serum levels as low as 1%, with no decrease in the proliferative capacity of the cells until they approach their reported population doubling limit. This new supplement mixture is a significant improvement to microcarrier technology in that it enables the use of microcarriers in the early stages of inocculum build-up for the production purposes. (c) 1992 John Wiley & Sons, Inc.     

Egg yolk lipoprotein,a new supplement for the growth of mammalian cells in serum-free medium

Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity.     

A preliminary search for alternatives to albumin as a medium supplement for the culture of human sperm

Non-animal macromolecules (Select Phytone? UF, wheat peptone, dextran 40, hydroxyethyl starch and methyl cellulose) as an alternative medium supplement for human spermatozoa were compared to bovine serum albumin. Select Phytone? UF and wheat peptone discolored the medium and smelled like broth, making them unlikely to be acceptable for clinical use, whilst the others were colorless and odorless. All supplements were effective in the yield of spermatozoa isolated by swim-up technique, and maintenance of sperm motility. In summary, there are non-animal macromolecules that will support short-term sperm culture.     

Oxidation-deficient silkworm hemolymph as a medium supplement for insect cell culture

Hemolymph is oxidized and darkens visibly during the collection from silkworms due to the activity of tyrosinase in it. Toxic quinones are produced by the oxidation and consequently inhibit the cell growth. Heat treatment can be used to prevent the oxidation; however, the oxidation may occur during the collection of hemolymph before it is heat-treated. It makes the hemolymph collection difficult especially on a large-scale preparation. Hemolymphs collected from 257 different strains of silkworms were examined to select the slowly oxidized hemolymphs. Hemolymphs collected from mutant strains such as Lemone, TBO, Cre, Y4, and wE^b showed relatively slow color changes. Oxidation rates of the hemolymphs were measured by the absorbance change using a spectrophotometer. The hemolymph of wE^b showed the slowest oxidation. The absorbance of this mutant hemolymph reached the saturation value at 20°C in 450 min, whereas the total oxidation time of the wild-type (Baekokjam) hemolymph at the same temperature was 120 min. We tested if this mutant hemolymph is useful as a medium supplement for insect cell culture. Cell growth rate and final cell concentration in the medium supplemented with the wE^b hemolymph were almost same as those in the medium supplemented with the wild-type hemolymph. Hemolymph is collected on a small scale by clipping the abdominal leg; however, this method is not appropriate for large scale preparation. Centrifugation after chopping the silkworm hemolymph by a blending mixer is a more appropriate procedure for large scale collection. Slowly oxidized wE^b hemolymph resulted in higher cell concentration than the wild-type hemolymph when hemolymph was collected by the large scale preparation method.     

A new culture medium for human skin epithelial cells

Summary A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.     

人巨细胞病毒抗体检测方法的应用研究

目的确定用于筛选人巨细胞病毒(HCMV)IgG阳性血浆的ELISA试剂和用于HCMV免疫球蛋白成品检定的中和试验方法。方法比较目前国内外现有的人巨细胞病毒IgG抗体检测方法(3家ELISA试剂和3种病毒中和试验)的相关性。结果德国赛润和意大利德塞的ELISA试剂与成都蓉生微量中和试验及台湾宝血的蚀斑减少中和试验的相关性很好(r299%)。结论从成本和使用便利性考虑,建议使用意大利德赛的ELISA试剂盒用于原料血浆的筛选,使用成都蓉生的微量中和试验作为成品效价检定的方法。     

Integrin alphavbeta3 is a coreceptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic pathogen that causes birth defects in newborns and severe disease in immunocompromised individuals. The broad tropism of HCMV infection suggests that it uses multiple receptors. We recently showed that the epidermal growth factor receptor (EGFR) serves as a receptor for HCMV. Here we show that HCMV also uses integrin alphavbeta3 as a coreceptor. Upon infection, HCMV glycoproteins gB and gH independently bind to EGFR and alphavbeta3, respectively, to initiate viral entry and signaling. Alphavbeta3 then translocates to lipid rafts where it interacts with EGFR to induce coordinated signaling. The coordination between EGFR and alphavbeta3 is essential for the early events of HCMV infection, including viral entry, RhoA downregulation, stress-fiber disassembly and viral nuclear trafficking. Our findings support a model in which EGFR and alphavbeta3 work together as coreceptors for HCMV entry and signaling. This discovery is fundamental to understanding HCMV pathogenesis and developing treatment strategies targeted to viral receptors.     

Rab27a is required for human cytomegalovirus assembly

Human cytomegalovirus (HCMV) completes its final envelopment on intracellular membranes before it is released from the cell. The mechanisms underlying these processes are not understood. Here we studied the role of Rab27a, a regulator of lysosome-related organelle transport, in HCMV production. HCMV infection increased Rab27a expression, and recruitment of Rab27a to membranous strutures at the assembly site. Immuno-gold labelling demonstrated association of Rab27a with viral envelopes. CMV production was reduced after knock-down of Rab27a, and in Rab27a-deficient ashen melanocytes. This study shows a requirement for Rab27a in the CMV life cycle and suggests that CMV and LRO biogenesis share common molecular mechanisms.     

Design and analysis of rhesus cytomegalovirus IL-10 mutants as a model for novel vaccines against human cytomegalovirus

Background

Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-10). Despite only ∼26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties.

Methods and Findings

Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10.

Conclusion

This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic strategies, to nullify HCMV''s ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary mucosal infection, and (3) establish a lifelong persistent infection.     

Pooled human serum: A new culture supplement for bioreactor-based cell therapies. Preliminary results

Background

Bone Marrow MSCs are an appealing source for several cell-based therapies. Many bioreactors, as the Quantum Cell Expansion System, have been developed to generate a large number of MSCs under Good Manufacturing Practice conditions by using Human Platelet Lysate (HPL). Previously we isolated in the human bone marrow a novel cell population, named Mesodermal Progenitor Cells (MPCs), which we identified as precursors of MSCs. MPCs could represent an important cell source for regenerative medicine applications. As HPL gives rise to a homogeneus MSC population, limiting the harvesting of other cell types, in this study we investigated the efficacy of pooled human AB serum (ABS) to provide clinically relevant numbers of both MSCs and MPCs for regenerative medicine applications by using the Quantum System.

Development and evaluation of a new growth medium for Helicobacter pylori

The present study was aimed at modifying the original formulation of Commercial Eugon agar (CEA) to develop a new H. pylori growth medium. Initial studies were carried out to determine the number of H. pylori colonies recovered on in-house H. pylori agar (IHPA), IHPA without l -cysteine and sodium sulfite (IHPA-NC), IHPA without l -cysteine (IHPA-C), IHPA without sodium sulfite (IHPA-N) and CEA as the control. Significant differences ( P <0.001) in the number of colonies recovered were observed between IHPA-N, IHPA-NC and IHPA-C. Incorporation of sodium sulfite decreased the number of colonies recovered, indicating that sodium sulfite was inhibitory to H. pylori growth. Removal of l -cysteine reduced the number of colonies recovered, suggesting that l -cysteine is necessary for the growth of H. pylori . In the subsequent study, incorporation of K₂HPO₄ further increased the number of colonies recovered compared with IHPA-N ( P <0.001), and 0.25% (w/v) of K₂HPO₄ yielded the highest numbers of colonies ( P ≤0.04). Finally, thirty other H. pylori clinical isolates were evaluated for their growth in the IHPAP-N, a new medium consisting of 1.5% (w/v) pepticase, 0.5% (w/v) peptone, 0.4% (w/v) sodium chloride, 0.03% (w/v) l -cysteine, 0.55% (w/v) dextrose, 0.25% (w/v) K₂HPO₄ and 1.5% (w/v) agar. The number of colonies recovered in IHPAP-N was significantly ( P <0.005) higher than that of CEA. IHPAP-N with 0.25% K₂HPO₄ and without sodium sulfite were adequate solid media for the growth of H. pylori .     

Identification and characterization of new human medium reiteration frequency repeats.

We report nine new families of human medium reiteration frequency interspersed repetitive elements (MER elements). They were identified by computer-assisted analyses. Six of them were independently confirmed as repetitive families by DNA-DNA hybridization, and the number of elements for each of these families was estimated by plaque hybridization assay. The involvement of some of the reported MER elements in genetic rearrangements is demonstrated.     

Comparison of an animal product free medium and normal growth supplement on the growth and barrier integrity of a human corneal epithelial cell line

With the development of defined media for general and specific use with cell cultures, and concern over the use of human cells and over potential prion infections associated with growth factor extracts such as bovine pituitary extract, an animal product-free medium has become available. The basic keratinocyte defined medium can be used with a choice of animal product-containing or animal product-free supplements. Human corneal epithelia cell lines were cultured in the media with these two types of supplement, and compared in terms of their growth rates, their capacity to form tight barriers, and calcium regulation of the location of a junction-associated protein, zonula occludins-1 (ZO-1). The growth rates were not different in the two media, as long as the recommended coating was applied to the culture flask for the animal product-free medium. The barrier function was equally effective for confluent cultures seeded at the same densities. A calcium concentration of 100 microM or above resulted in ZO-1 localisation at the cell membrane in either medium. Hence, cultures in the media are comparable, when the coating is employed. Further experiments are being conducted to establish the comparability of responses to chronic treatment with surfactants.     

Thermoinactivation of human cytomegalovirus

Vonka, Vladimir (Baylor University College of Medicine, Houston, Tex.), and Matilda Benyesh-Melnick. Thermoinactivation of human cytomegalovirus. J. Bacteriol. 91:221-226. 1966.-The inactivation at 4 and 37 C of several strains of human cytomegalovirus was studied. The preliminary findings that freshly harvested cytomegalovirus was inactivated more rapidly at 4 C than at higher temperatures was confirmed. Intracellular virus still within infected cells was found to be more stable at 4 C than virus released by sonic treatment just before incubation at 4 C. The composition of the diluent played an important role. In tris(hydroxymethyl)-aminomethane buffer, virus was unstable at both 4 and 37 C, with the rate of inactivation faster at 4 than at 37 C. Similar results were obtained when bicarbonate-phosphate buffer or Eagle's medium when bicarbonate was used as virus diluent. Calf serum stabilized the virus at 37 C, but not at 4 C. The deletion of bicarbonate from Eagle's medium had a stabilizing effect at both temperatures. An even greater stabilizing effect at both 4 and 37 C was obtained when distilled water was used as virus diluent. Inactivation rates varied from one strain to the next at 4 C but not at 37 C. Differences were found also with virus progeny derived from a single strain, but harvested at different stages during virus multiplication. Virus harvested early was more labile at 4 than at 37 C, whereas the late virus was more labile at the higher temperature. Intracellular and extracellular virus preparations were inactivated at the same rates at either 4 or 37 C.     

Sulfhydrylcellulose: a new medium for chromatography of mercurated polynucleotides

We have synthesized a new medium, sulfhydrylcellulose, for affinity chromatography of mercurated polynucleotides. It is the product of reaction between aminoethylcellulose and N-acetylhomocysteine thiolactone. Sulfhydrylcellulose carries up to 90 mumol of SH groups/g and is inexpensive, easy to prepare, and stable. Because it binds mercurated RNA specifically and reversibly and exhibits no size discrimination, sulfhydrylcellulose should have wide applications.