module of a lot of aspects of biological research. This review summarizes conceptual background and basic techniques of culturing animal cells in a format that is readily easy to get to all researchers in the field. Animal tissue culture functions in therapeutic filed are vast. The evaluation of the cell’s response to chemicals, or as a tool to produce cellular-derived protein products that really helps in medical improvement. An animal tissue culture provides an approach to manufacture monoclonal anti- body that makes it potential to produce antibody that have specificity controlled toward pathogen. In the field of animal cell culture discovery leads to the opportunity of curing various diseases such as AIDS and cancer. Ad- ditional application of animal tissue culture gives a con- ventional, quick and approachable method for manufac- ture of well-tolerated and valuable vaccines. Cell culture seems to come with a lot of important towards medical progression. Other than that, there are still differences between in vitro and in vivo system during drug testing and organ transplantation which make it not totally trustworthy.
The type and design of the sparger affects not only the mass transfer rate, but also the magnitude of cell damage. Typically, drilled hole spargers (DHS) or sintered spargers (SS) are used in mammalian cell bioreactors, producing millimeter to micron-sized bubbles, respectively. The sparger can also be designed in the form of a straight pipe, ring, or concentric rings. Sintered spargers (also known as frit sparger or diffuser) have been associated with higher cell damage and foaming due to the small size of the bubbles, which have larger Laplace pressure. On the other hand, using drilled hole spargers with insufficient number of holes can result in high gas entrance velocity (GEV), which has been empirically shown to have negative impact on mammalian cell culture [46,47]. Therefore, proper sparger design is crucial during the scale-up process. One recent modeling study has shown that increased GEV does not correlate with high EDR. Instead, the authors hypothesized that the cell damage results from normal and shear stresses coupled with liquid phase turbulent velocity gradients, which they quantify with a parameter called the stress- induced turbulent energy production (STEP), a value generated from CFD models .
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Methods: The AgNPs were prepared using C. papaya leaves extract in 1:4 ratio. Synthesized AgNPs were characterized using ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, and scanning electron microscopy. The cytotoxicity of plant NP and 50% tissue culture infective dose of Chikungunya virus (CHIKV) were determined before antiviral assay by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. After that, the maximum non-toxic dose (MNTD) and ½MNTD were calculated. The absorbance values were detected using a microplate reader at 595 nm. Median tissue culture infective dose (TCID 50 ) dose was calculated using the Reed and Muench method. In vitro antiviral activity was
Previously, primary screening isolated 20 xanthones from plants of the Guttiferae family that have inhibitory effects on human herpesvirus 4 (HHV-4) also known as the Epstein-Barr virus (EBV). Xanthones, particularly 1,3,7-tri- hydroxy-2-(3-methyl-2-butenyl) xanthone, dulxanthone-B and latisxanthone-C, seemed to significantly inhibit EBA early antigen (EBV-EA), one of the viral genes required for the initiation complex at the lytic origin of viral replication in Raji cells . Mangiferin with a broad spectrum benefi- cial biological activities, was the first xanthone shown to be pharmacologically effective for the treatment of diseases caused by herpesvirus, . One of the effects of the xan- thones is the inhibition of HIV-1 reverse transcriptase. Among xanthones, prenylated xanthones is restricted to the plant species of the family Guttiferae . Prenylated xanthones namely mangostin and y-mangostin, isolated from G. mangostana are active against HIV-1 protease, pre- venting proteolytic cleavage during retroviral replication . A recent investigation submitted a set of 272 xanthones to molecular docking examination and the re- sults suggested that the xanthones could be suitable key components of agents to possess antiviral properties . In fact, prenyl groups have been predicted to be important in protein-protein binding because of their specialized prenyl-binding domains that facilitate attachment to cell membranes. Therefore, a few lead compounds and their de- rivatizations could be screened using a structural-based docking method, or high-throughput screening methods for serving as a proof of concept for feasible viral target.
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envelope of the IEV, but it remains in the plasma membrane at one pole of the CEV, orchestrating actin tails (73, 94, 102). Second, confocal z-axis analyses and electron microscopy have provided evidence that actin tails form exclusively at the cell surface and not in the cytoplasm (43, 97; E. J. Wolffe and B. Moss, unpublished data). Third, inhibitors of microtubules, but not inhibitors of actin polymerization, reduced movement of VV to the cell surface (43, 97). Therefore, it appears that VV moves via microtubules from virus factories to the TGN, where envelopment occurs, and then microtubules ferry IEV to the cell surface (Fig. 3). Actin polymerization apparently occurs only after fusion of the IEV outer envelope with the plasma membrane and is nucleated around A36R, which remains in the plasma membrane (73, 94) (Fig. 3, inset). This explains the polar nature of actin tails, the absence of A36R in EEV, and the use of plasma membrane tyrosine kinase signaling to ini- tiate actin polymerization. Actin tails project CEV at the tips of microvilli into neighboring cells, and this process is appar- ently required for efficient cell-to-cell spread. This was con- firmed in a recent microscopic analysis showing VV atop actin tails projecting across cell junctions (43). However, it is not yet clear whether there is preferential transport of VV particles to sites of epithelial cell contact or specific production of actin tails there. It certainly makes sense that poxviruses would di- rect progeny specifically to other cells, as do the alphaherpes- viruses, but this remains to be tested.
The need for new antibiotics for TB is particularly acute, given the progressive emergence of drug resistance, recent disappointments in treatment-shortening regi- mens, and the ongoing global toll of infection (14). PZA is one of the most important antibiotics used to treat human disease, but it was discovered serendipitously in the 1950s and indeed would have not been found by current screening approaches based on the sequential study of MICs in broth culture, murine infections, and human disease (17). PZA has a complex mechanism of action, requiring intracellular acidiﬁcation. We demonstrated that M. tuberculosis stress genes were upregulated in microspheres and that PZA killed M. tuberculosis in microspheres at neutral pH but not in standard 2D culture systems at a concentration found in epithelial lining ﬂuid (25). This conﬁrms that, within microspheres, bacilli are in a PZA-sensitive compartment and demonstrates the potential to identify other compounds only active against stressed mycobacteria in the correct microenvironment. Similarly, we found that INH was consistently more effective in the 3D system that in the 2D system, with a more rapid fall in luminescence and no failed treatments, supporting the relevance of the model to human TB. Therefore, the system can identify drug resistance in a more clinically relevant fashion and can be used to study novel regimens with variable concentrations in combination rather than single new agents at static concentrations.
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It has been suggested that the lack of continuous proliferation of aquatic invertebrate cells is due to senescence (Crane, 1999; Mothersill and Austin, 2000), which may result from inactivation of proliferation-promoting genes and/or activation of anti-proliferation genes, a decrease in the level of DNA methylation, telomere shortening, and/or DNA damage and repair mechanisms (Crane, 1999). Inactivation of anti-proliferation genes can occur through genetic alterations or through interaction of gene products with cellular or viral proteins (Lee and Cho, 2002). Inactivation of anti-proliferation genes in primary cells of C. quadricarinatus was attempted by transfecting HPV-16 oncogenes, E6 and E7. These genes interact with key regulators of the cell cycle, p53 and the retinoblastoma (Rb), to stimulate unscheduled DNA synthesis (Putral et al., 2005). Inactivation of p53 is the most prevalent alteration found in human and animal tumours (Ko and Prives, 1996; Levine, 1997). Following transfection of the HPV-16 E6+E7 genes or the HPV-16 E7 gene into the C. quadricarinatus cells, mRNA of the E7 gene was monitored via RT-PCR. Results showed that the cells were successfully transcribing the E7 gene, but still were not
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Today, cell culture media can be divided in different types based on their content of animal-derived products. Serum-free media (SFM) do not require the addition of serum for optimal cell growth but may contain other additives derived from animals such as lactalbumin, casein, insulin, lipids or sterols . Animal-component- free media (ACFM) are media in which none of the com- ponents are animal-derived . Protein-free media (PFM) are free of supplemental polypeptide factors but may contain hydrolyzed peptide fragments from animal or plant sources. Finally, chemically defined media (CDM) comprise well-characterized constituents of low molecular weight and are, in most cases, free of proteins [11, 12]. BHK21 cells have already been adapted to grow in serum- free or animal-component-free cell media for rabies vac- cine production [13, 14]. With adaption to serum-free conditions, BHK21 cells switch from anchorage- dependent to suspension growth [13, 14] and fundamental changes in cell structure take place [15, 16]. On the other hand, selective pressures during the adaption of viral strains to BHK21 cells, whether as adherent or as suspen- sion cells, can lead to capsid alterations that influence the antigenicity and stability of the virus particle.
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of the cell cycle, HPV E6 and E7 genes were transfected into the C. quadricarinatus cells. Successful transfection was demonstrated by the presence of oncogene mRNA by RT-PCR. At day 150, transfected cells remained viable, although cell proliferation was stagnant. It may be that, although transfection of the oncogenes was successful, no proliferation of the C. quadricarinatus cells was evident, due to a lack of telomere maintenance.
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Figure 3.4 Change in TEER across healthy Caco-2 monolayers co-cultured with live or UV-killed F. prausnitzii using 50% BHI as apical medium. ........................................... 128 Figure 3.5 Change in TEER across TNF-α-treated Caco-2 monolayers co-cultured with live or UV-killed F. prausnitzii using 50% BHI as apical medium. ................................ 129 Figure 3.6 Change in TEER across Caco-2 monolayers with or without TNF-α treatment using M199 TEER as apical medium. ............................................................................ 130 Figure 3.7 Change in TEER across healthy Caco-2 monolayers co-cultured with live or UV-killed F. prausnitzii using M199 TEER as apical medium. ...................................... 132 Figure 3.8 Change in TEER across TNF-α-treated Caco-2 monolayers co-cultured with live or UV-killed F. prausnitzii using M199 TEER as apical medium. ........................... 133 Figure 3.9 Change in TEER across TNF-α-treated Caco-2 monolayers co-cultured with live or UV-killed F. prausnitzii using M199 TEER as apical medium. ........................... 134 Figure 3.10 Viability of the three F. prausnitzii strains co-cultured with TNF-α-treated Caco-2 cells using 50% BHI as apical medium. ............................................................. 135 Figure 3.11 Viability of the three F. prausnitzii strains co-cultured with healthy or TNF-α- treated Caco2-cells using M199 TEER as apical medium. ............................................. 137 Figure 4.1 Overview of the stable transfection of HEK-TLR cell lines with a NF-κB inducible luciferase plasmid........................................................................................... 154 Figure 4.2 Schematic diagram of the TLR activation assay. ........................................... 160 Figure 4.3 Adaptation of the TLR activation assay to the apical anaerobic co-culture model. ........................................................................................................................... 164 Figure 4.4 HEK293-TLR-Luc cells in the apical anaerobic co-culture model. ................ 165 Figure 4.5 Methods to measure the DO concentrations in the apical and basal compartments of the co-culture chamber........................................................................ 169 Figure 4.6 Zeocin sensitivity of HEK293-TLR2 cells. ................................................... 174 Figure 4.7 TLR activation assay in conventional conditions. .......................................... 176 Figure 4.8 OD 600nm (A) and normalised change in OD 600nm (%) (B) of the three F.
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Wat er intake i s a f fec ted by : animal fac tors such as age , sex , s i z e , me tabol i s m , phy s iologi cal cond i t i on of the animal , performance , body water conten t , ac t ivi ty , breeds ( be tween and wi thin) ; envi ronmen t al fac tors such as season , a i r t emperature , rainfall ( Cas tle and Wa tson , 197 3 ; Cowan e t al , 1978 ) , relat ive humidi ty , solar rad i a t i on , wind , qual i ty , quan t i ty and temperature o f the wa ter ; nu t r i t i onal fac tors such as diet ( qual i ty , type or form) , feed i n t ake , and animal managemen t fac tors such as type of housing ( Campbel l , 1958 ; Payne , 1966 ; MacFarlane and Howard , 1970 ; Cas t le , 1972 ; Cas t le and Wat s on , 197 3 ; Wrigh t and Jones , 1974 ; Cowan e t al , 1 97 8 ; Odwongo , 1983 ; Ni cholson , 1985 ) . Any fac tor that af fec t s wa ter turnover will influence the drinking requ i rement s of the an imal or viceversa .
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Another use of allogeneic bone marrow transplants is in the treatment of hereditary blood disorders, such as different types of inherited anemia and inborn errors of metabolism. The blood disorders include aplastic anemia, betathalassemia, Blackfan-Diamond syndrome, globoid cell leukodystrophy, sickle-cell anemia, severe combined immunodeficiency, X-linked lymphoproliferative syndrome and Wiskott-Aldrich syndrome. Inborn errors of metabolism that are treated with bone marrow transplants include: Hunter’s syndrome, Hurler’s syndrome, Lesch Nyhan syndrome and osteopetrosis. In place of gene transfer, bone marrow stem cell has been studied whether causing angiogenesis or vasculogenesis. Angiogenesis, which means development from capillary endothelial cells, has been considered as a main cause to increase blood vessels in the adults . On the other hand, angioblast or endothelial progenitor cells (EPC), which differentiates to the endothelial cells, enhances vasculogenesis in the embryogenic stage. These cells surround hematopoietic cells and form a blood island, then develop to blood vessels in the embryo. The advantages using bone marrow monocytes are that infection or immune reaction can not occur different from the technique using gene transfer. The disadvantage is necessity to collect bone marrow blood with a volume of 500 to 600ml under general anesthesia. The EPC and angioblast also exists in the peripheral blood, so they could be collected by using apheresis. However, the numbers of these cells are much lower than in the bone marrow and the efficacy of collection is less. To investigate the effects to human ischemic limb by bone marrow monocyte injection, Japan Trial for Therapeutic Angiogenesis Using Cell Transplantation (J-TACT) started since 2000. Our institute also started therapeutic angiogenesis to use bone marrow monocytes since 2004, independently .
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Confirmation of origin (authentication) A small portion of the sample used for primary culture (or a blood sample or DNA derived from the donor) should be frozen or processed immediately. The tissue or DNA can then be used to demonstrate unequivocally that the cell line is derived from the putative donor. Short tandem repeat (STR) profiling is most useful method for the purpose of authentication and identification, although additional information on genotype seuencing(karyotype, copy number variation (CNV) mapping, or even whole-genome sequence) will sometimes help ensure identity.
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In mammals, extensive PCD begins at the blastocyst stage of development and continues throughout life. PCD serves many functions in development, and one of these is to regulate cell numbers (Jacobson et al., 1997). Cell death can be just as important in controlling cell numbers as cell division. In the developing vertebrate nervous system, for instance, many types of neurons are overproduced and then compete with one another for limiting amounts of survival signals (neurotrophic factors) secreted by the target cells they innervate: only about half get enough to survive, while the others undergo PCD. In this way the numbers of neurons are matched to the numbers of target cells they innervate (Oppenheim, 1991). It seems likely that similar mechanisms operate in many developing organs to help match the numbers of different cell types.
Serum, usually from fetal calves, is an extremely complex mixture of many small and large bio-molecules with different, physiologically balanced growth pro- moting and growing inhibiting activities. A concentration 5% - 20% v/v serum is usually needed for optimum cell growth. Due to the endocrinal differentiation require- ments, the induction of insulin-producing cells from mouse ES cells was carried out in serum-free conditions in the later sections. In the context of developing culture conditions to promote application of stem cells in the clinic, use of FCS is a significant issue as it may contain potentially harmful xenogeneic compounds. Bovine se- rum proteins may be internalized in stem cells stimu- lating immunogenicity [13,14], consequently a host of potential problems can arise including viral trans- mis- sion and immunological reactions due to the bovine pro- tein attachment to cells in culture that act as antigenic substrates once transplanted .
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There are two basic methods for obtaining cells for cell culture to obtain primary cell culture. Explant Culture method where small pieces of tissue are attached to a glass or treated plastic culture vessel and bathed in culture medium. After a few days, individual cells will move from the tissue explant out onto the culture vessel surface or substrate where they will begin to divide and grow. The second, more widely used method, speeds up this process by adding digesting (proteolytic) enzymes, such as trypsin or collagenase, to the tissue fragments to dissolve the cement holding the cells together. This creates a suspension of single cells that are then placed into culture vessels containing culture medium and allowed to grow and divide. This method is called Enzymatic Dissociation 7
Any single animal model is unlikely to completely mimic all rele- vant aspects of human liver regeneration, particularly given that the cellular and molecular pathways mediating regeneration are likely to vary somewhat depending on the nature of the initial injury. Therefore, future studies of hepatic stellate cells in liver regenera- tion will be facilitated by the availability of multiple animal mod- els, which are likely to yield complementary insights. Advantages of rodent models include the ability to isolate, culture, and activate hepatic stellate cells in vitro, facilitating follow-up cell culture stud- ies focused on molecular mechanisms involved in regeneration. On the other hand, the excellent live-imaging technologies available in zebrafish are well suited for studying the cellular interactions at play during the regenerative process. As with rodents, PH or toxic chemicals can be used to induce liver regeneration in zebrafish (reviewed in ref. 74). Genetic tools have enabled the development of additional regeneration models including the nitroreductase/ metronidazole cell ablation system (91) and morpholino-based knockdown of a mitochondrial import gene to induce hepatocyte death (92). One promising approach is to perform high-through- put chemical screens in various zebrafish models of liver injury, seeking drugs that affect stellate cells during liver regeneration (24). Hepatic stellate cells in cancer
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Since their development, more than four decades ago, Cell culture vaccines (CCVs) have proved to be safe and effective in preventing rabies. These vaccines are intended for both pre- and post-exposure prophylaxis and have been administered to millions of people worldwide. But, the affordability to CCVs for intramuscular administration during PEP is a major constraint in developing countries of Asia and Africa. Therefore, World Health Organization (WHO) recommends intradermal route of vaccination with CCVs for these countries to reduce the quantity of vaccine and the cost of vaccination. Considering the large number of animal bite cases in the country and huge demand for CCVs, following the recommendations of WHO and ICMR, the drug controller general of India (DCGI) approved intra dermal administration of rabies vaccines using updated TRC regimen in 2006.
The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to iden- tify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of fil- tered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN- 5 B 1-4 (Tn 5 ; High Five) cells and
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Cell isolation procedures for crustaceans and vertebrates share several similarities. For example, Tong and Miao found that cells isolated from younger “pre-molting” prawns have a higher success rate for survival and growth (Tong and Miao 1996). Cell cultivation studies often attempt isolations from both explanted and enzyme-digested tissues. For the explant method, which is best suited for loose tissues, the target tissue is removed from the animal, rinsed in custom buffer solution, dissected into small sections and allowed to attach to a culture surface. If successful, cells within the tissue will proliferate, migrate, and adhere to the substrate. For the enzyme digestion method, digestion enzymes (e.g., trypsin, collagenase) are incubated with dissected explants to degrade the tissue into a single cell suspension. The slurry is often filtered to remove undigested sections and the enzymes are inhibited after a certain amount of incubation time to prevent cell damage. For cell isolations from most shrimp tissues, the explant method tends to have more success than enzyme-digested method in terms of cell proliferation, although enzyme digestion is preferred for hepatopancreas tissues (Ma, Zeng, and Lu 2017).
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