Effect Of Various Culture Conditions Research Articles

Favorable culture conditions for spermatogonial propagation in human and non-human primate primary testicular cell cultures: a systematic review and meta-analysis.

Introduction: Autologous transplantation of spermatogonial stem cells (SSCs) isolated from cryopreserved testicular biopsies obtained before oncological treatment could restore fertility in male childhood cancer survivors. There is a clear necessity for in vitro propagation of the limited SSCs from the testicular biopsy prior to transplantation due to limited numbers of spermatogonia in a cryopreserved testicular biopsy. Still, there is no consensus regarding their optimal culture method. Methods: We performed a systematic review and meta-analysis of studies reporting primary testicular cell cultures of human and non-human primate origin through use of Pubmed, EMBASE, and Web of Science core collection databases. Of 760 records, we included 42 articles for qualitative and quantitative analysis. To quantify in vitro spermatogonial propagation, spermatogonial colony doubling time (CDT) was calculated, which measures the increase in the number of spermatogonial colonies over time. A generalized linear mixed model analysis was used to assess the statistical effect of various culture conditions on CDT. Results: Our analysis indicates decreased CDTs, indicating faster spermatogonial propagation in cultures with a low culture temperature (32°C); with use of non-cellular matrices; use of StemPro-34 medium instead of DMEM; use of Knockout Serum Replacement; and when omitting additional growth factors in the culture medium. Discussion: The use of various methods and markers to detect the presence of spermatogonia within the reported cultures could result in detection bias, thereby potentially influencing comparability between studies. However, through use of CDT in the quantitative analysis this bias was reduced. Our results provide insight into critical culture conditions to further optimize human spermatogonial propagation in vitro, and effectively propagate and utilize these cells in a future fertility restoration therapy and restore hope of biological fatherhood for childhood cancer survivors.

Effect of various culture conditions on shoot multiplication and GC–MS analysis of Plumbago zeylanica accessions for plumbagin production

Plumbago zeylanica, a pharmaceutically important medicinal plant, contains a wide range of phytocompounds. Culture parameters like carbon source, nitrogen source, and culture media are essential for the development and growth of explants. In this investigation, the influence of various carbon sources (sucrose, glucose, and fructose at 3% concentration), nitrogen source (ammonium nitrate, sodium nitrate, and potassium nitrate) and plant tissue culture media (MS medium, Gamborg’s B5 medium, White medium and Nitsch medium) on shoot multiplication of five different accessions was studied. Optimum growth of all five accessions was observed in MS media containing 3% sucrose and ammonium nitrate as a source of carbon and nitrogen. Out of five accessions, IC-524441 showed the highest shoot multiplication. Further, methanolic extracts of all accessions (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source) were prepared and comparison of extracts in DPPH assay indicated that accession number IC-524441 was the most effective free radical scavenging agent. Total phenolic, flavonoid and tannin content ranges were from 20 to 70 µg/ml, 40 to 100 µg/ml and 55 to 120 µg/ml, respectively, and the highest amount was found in accession number IC-524441. Sucrose and ammonium nitrate content may be responsible for increased antioxidant activity, flavonoids content, phenolic content, and tannin content in accession number IC-524441. GC–MS of ethyl acetate extract of all five accessions of P. zeylanica was conducted (grown in MS media containing 3% sucrose and ammonium nitrate as nitrogen source). GC–MS analysis of the aerial part showed the presence of various phytocompounds, which include 1,4-naphthalenedione, 3-eicosene, 5-eicosene, phthalic acid, o-anisic acid, thioctic acid, 1-octadecene, 5-t-butyl-cycloheptene, 2-benzoyl-1,2-dihydro-1-isoquinolinecarbonitrile, octadecanal, silane, 3-methoxy-2-methyl-2-(1-phenyl-ethylamino)-propionic acid, and 1-nonadecene. Accession number IC-524441 contains the highest amount of plumbagin, i.e. 14.19 ± 0.5 µg/ml as compared to the others.

Strain and Vibration in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into any mesenchymal tissue, including bone, cartilage, muscle, and fat. MSC differentiation can be influenced by a variety of stimuli, including environmental and mechanical stimulation, scaffold physical properties, or applied loads. Numerous studies have evaluated the effects of vibration or cyclic tensile strain on MSCs towards developing a mechanically based method of differentiation, but there is no consensus between studies and each investigation uses different culture conditions, which also influence MSC fate. Here we present an overview of the response of MSCs to vibration and cyclic tension, focusing on the effect of various culture conditions and strain or vibration parameters. Our review reveals that scaffold type (e.g., natural versus synthetic; 2D versus 3D) can influence cell response to vibration and strain to the same degree as loading parameters. Hence, in the efforts to use mechanical loading as a reliable method to differentiate cells, scaffold selection is as important as method of loading.

Effect of Various Culture Conditions on the Production of Mycotoxin by Aspergillus sp.

Effect of Various Culture Conditions on the Production of Mycotoxin by Aspergillus sp.

Statistical Analysis and Modeling of Pelletized Cultivation of Mucor circinelloides for Microbial Lipid Accumulation

Microbial oil accumulation via oleaginous fungi has some potential benefits because filamentous fungi can form pellets during cell growth and these pellets are easier to harvest from the culture broth than individual cells. This research studied the effect of various culture conditions on the pelletized cell growth of Mucor circinelloides and its lipid accumulation. The results showed that cell pelletization was positively correlated to biomass accumulation; however, pellet size was negatively correlated to the oil content of the fungal biomass, possibly due to the mass transfer barriers generated by the pellet structure. How to control the size of the pellet is the key to the success of the pelletized microbial oil accumulation process.

Studies on Optimization of Growth Parameters for L-Asparaginase Production byStreptomyces ginsengisoli

A species of Streptomyces, Streptomyces ginsengisoli, a river isolate, was evaluated for production of an enzyme, L-asparaginase, with multiple functions mainly anticancer activity. The actinomycete was subjected to submerged fermentation by “shake flask” method. The quantity of L-asparaginase produced was estimated as 3.23 μmol/mL/min. The effect of various culture conditions on L-asparaginase production was studied by adopting a method of variation in one factor at a time. Of the various conditions tested, glucose (followed by starch) and peptone served as good carbon and nitrogen sources, respectively, for maximal production of enzyme at pH 8. The temperature of 30°C and an incubation period of 5 days with 0.05 g% asparagine concentration were found to be optimum for L-asparaginase production.

Factor influencing the production and properties of extracellur protease from Streptomyces bikiniensis

The actinomycete Streptomyces bikiniensis collected from laboratory stock culture, Department of Microbiology, University of Chittagong was found to be a protease producer. Effect of various culture conditions for the production of protease from S. bikiniensis and its properties were determined in the present study. The organism showed maximum protease yield at temperature 37°C with an initial culture, pH 7.0 after 4 days of incubation period under stationary condition. The crude enzyme preparation exhibited highest activity at substrate (casein) concentration of 1.0% with a reaction time of 75 min, at 40°C and pH 6.0. EDTA was found to a potent inhibitor of protease of the isolate while, 1,2- epoxy-3-(P-nitrophenoxy) propane exhibited low inhibitory effect on the enzyme activity. The enzyme was partially purified by precipitation method using ammonium sulfate fractionation and the maximum amount of the enzyme was precipitated at 60% saturation. DOI: http://dx.doi.org/10.3329/cujbs.v4i1.13399 The Chittagong Univ. J. B. Sci.,Vol. 4(1&2):153-163, 2009

Production of the α-glycosidase inhibitor 1-deoxynojirimycin from Bacillus species

1-Deoxynojirimycin (DNJ), a potent α-glycosidase inhibitor, has therapeutic applications in treatments of HIV, Gaucher’s disease, and diabetes. DNJ has been extracted from natural sources (mulberry leaves) for therapeutic purposes; however, DNJ ingredients are in limited supply and are costly to obtain on a large scale. Since certain strains of Bacillus and Streptomyces species reportedly produce DNJ, they may serve as potential sources for high-yield DNJ production. In this study, we obtained evidence for a DNJ production in Bacillus subtilis DSM704 by hydrophilic interaction chromatography-tandem mass spectrometry. In addition, from a screen of 750 microorganisms, we identified additional Bacillus strains (Bacillus amyloliquefaciens AS385 and Bacillus subtilis B4) that produce DNJ in large quantities. Investigation of the effect of various culture conditions, using Bacillus subtilis DSM704 and the DNJ high-production Bacillus strains, provided evidence for the importance of sorbitol supplementation on the yield of the DNJ precursor, 2-amino-2-deoxy-D-mannitol, thereby increasing DNJ production. The role of sorbitol in increased DNJ production was supported by an observed increase in mRNA expression of the biosynthetic gene, gabT1. When Bacillus amyloliquefaciens AS385 was cultured in medium supplemented with sorbitol, extracellular DNJ concentration reached a maximum of 460mg/l of medium (equivalent to 9.20mg/g of freeze-dried medium), indicating that this strain can serve as a source for food- and drug-grade products. These findings not only lead to a further understanding of the DNJ biosynthetic pathway, but also suggest a method for microbial mass production of DNJ for therapeutic applications.

Development of Monascus fermentation technology for high hypolipidemic effect

Monascus species has been used as the traditional food fungus in Eastern Asia for several centuries. Monascus-fermented products are gradually developed as the popular functional food for the prevention of cardiovascular disease, but we know that culture condition affects the hypolipidemic effect of Monascus-fermented product. In the past, the cholesterol-lowering agent--monacolin K--is regarded as the most important hypolipidemic agent. Two natural yellow pigments--monascin and ankaflavin--are also proven as novel hypolipidemic agents in recent years. However, the hypolipidemic effect of Monascus-fermented product should contribute from monacolin K, monascin, ankaflavin, and other unknown functional ingredients. In addition to hypolipidemic effect, the safety concern of Monascus-fermented product is involved in the levels of mycotoxin--citrinin. The hypolipidemic effect and the production of these functional metabolites or mycotoxin are influenced by many factors such as the choice of culture substrates, carbon and nitrogen source, pH value, extra nutrients, and so on. Therefore, this review focused on the effect of various culture conditions and nutrients on the functional metabolites production, hypolipidemic effect as well as citrinin concentration, and further organized the fermentation technologies used by previous studies for the promotion of hypolipidemic effect and safety.

In vitro Shoot Regeneration and Control of Shoot Tip Necrosis in Tissue Cultures of Soymida febrifuga (Roxb.) A. Juss.

Soymida febrifuga (Roxb.) A. Juss., (Meliaceae) an indigenous lofty deciduous tree, endemic to India, has extensive pharmacological activity. Continuous destruction of plants due to environmental and geopolitical instability has posed a major threat to endemic tree species. In vitro propagation techniques are also problematic due to high incidence of shoot tip necrosis (STN). The effect of various culture conditions on regeneration and STN of Soymida febrifuga were investigated. Nodal segments from field grown plants were used as explants and cultured on MS supplemented with different concentrations of BA, KN, NAA and IAA. The synergistic effect of BA (2 mg/l) and NAA (0.2 mg/l) induced a mean of 6.3 ± 0.09 shoots from the nodal explants with a frequency of 80.4%. Frequent subculturing of nodal explants from in vitro derived shoots increases the number of multiple shoots, but the regenerated shoots exhibited the symptoms of STN. Various factors such as strength of the media, different carbon sources activated charcoal and different calcium sources have been investigated for reducing the incidence of shoot necrosis. STN can easily be recovered by increasing the levels of calcium. Transfer of shoots showing early signs of necrosis to half strength MS supplemented with CN (556 mg/l), CP (1.0 mg/l), AC (20 mg/l) and fructose (100 mg/l) facilitated recovery of more than 98% of the shoots. Rooted plantlets produced, using the optimized protocol, were acclimatized successfully. Key words: Soymida febrifuga, Shoot regeneration, Shoot tip necrosis D.O.I. 10.3329/ptcb.v21i1.9559 Plant Tissue Cult. & Biotech. 21(1): 11-25, 2011 (June)

Responses of PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in MCF-7 cells are culture condition dependent

To compare the effects of the food toxin 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and estradiol in hormone-responsive MCF-7 cells, the cells were exposed to different concentrations of either PhIP or estradiol. The effect of various culture conditions (e.g. phenol red, FBS, vehicle (DMSO/EtOH) and seeding density) on responses was studied. Cells were continuously grown with steroid-containing or -deprived medium, or switched from steroid-containing to -deprived medium for the experiments to minimize the effect of background estrogenicity. Effects of PhIP and estradiol on cell viability and proliferation were determined by ATP analysis and Ki-67 immunocytochemistry. Expression of estrogen receptor alpha, cell stress markers (p53 and ERK) and estrogen responsive proteins (c-myc and ERK) were immunoblotted. All concentrations of estradiol induced cell proliferation, viability and changes in protein expression, typical for estrogenic responses. PhIP, however, increased viability only at low concentrations and depending on culture conditions. No changes in protein expressions by PhIP were noted, not even when switching cells from steroid-containing to -deprived medium which down-regulated the expression of proteins at basal level. Vehicle affected significantly viability, especially after exposure to PhIP, but not protein expression while medium changes affected both. In conclusion, the effects of PhIP and estradiol in MCF-7 cells are dependent on culture conditions. The detected PhIP-induced changes are weaker compared to those induced by estradiol.

Preparation of Novel Culinary-Medicinal Mushroom Products Using Solid-State Fermentation and Their Taste Quality

Four species of culinary-medicinal mushrooms and fungi (Cordyceps sinensis, Termitomyces albuminosus, Antrodia camphorata, and A. salmonea) were individually inoculated into cooked grains, and new products were formed after fungal fermentation and their nonvolatile taste components studied. The effect of various culture conditions on the mycelial growth in fungus-fermented products was also studied. The grains and their corresponding products showed similar proximate profiles. Contents of total soluble sugars and polyols in Cordyceps sinensis-fermented rice (CFR) and Termitomyces albuminosus-fermented wheat (TFW) were as low as those in the grain. After fungal fermentation, those contents in Antrodia camphorata-fermented embryo rice (ACFR) and A. salmonea-fermented oat (ASFO) increased significantly. Contents of total free amino acids in mycelia ranged from 2.49 to 27.46 mg/g. Contents of total free amino acids in CFR and ACFR were higher than those in rice and embryo rice. Contents of flavor 5'-nucleotides showed three different levels. Equivalent umami concentrations of T. albuminosus and C. sinensis mycelia were significantly higher than those of other mycelia, four products and four grains. On the basis of the results obtained, the four products were relatively weak in the umami taste.

The effect of medium, carbon source and explant on regeneration and control of shoot-tip necrosis in Harpagophytum procumbens

Harpagophytum procumbens, an indigenous plant to southern Africa, has extensive pharmacological activity. It is traded internationally but is difficult to cultivate. In vitro propagation techniques are also problematic due to a high incidence of shoot-tip necrosis (STN). The effect of various culture conditions on regeneration and STN of H. procumbens were investigated. In this paper the effects of medium type and strength, sucrose concentration, sugar types, callus explant and sub-culturing were studied. Of the different media types tested, half-strength Murashige and Skoog (MS) medium reduced STN significantly without affecting shoot multiplication and growth. Sucrose at 0.086 M (3%) was the preferred carbon source both in terms of growth and preventing STN compared to glucose, maltose and fructose at equimolar concentrations. Indirect organogenesis of this species was possible via callus obtained from nodal explants with BA at 4.4 μM and IAA at 5.4 μM being the best hormone combination but the plants were hyperhydric. Transferring cultures to fresh medium at two-weekly intervals helped to reduce STN and enhanced growth.

Impact of submerged culture conditions on growth and bioactive metabolite produced by endophyte Hypocrea spp. NSF-08 isolated from Dillenia indica Linn. in North-East India

Summary An endophytic fungus, associated with tropical tree species Dillenia indica Linn., has a broad-spectrum antimicrobial property against human and plant pathogenic microorganisms. The fungus was identified as Hypocrea spp. NSF-08, a teleomorphic genus of Trichoderma, based on morphological, sporulation and molecular characteristics. The effect of various culture conditions, supplementary carbon, nitrogen sources and amino acid amendment on growth and antimicrobial agent production by the fungus was determined. The basal medium, enriched with glycerol (2%) as carbon source and ammonium nitrate as nitrogen source, promoted the biosynthesis of secondary metabolite. Optimum temperature required for maximum production of antimicrobial agent was 25 °C and pH 5.5, respectively, in an incubation period of eight days, while specific rate of product formation was at maximum on the 4th day. Amendment of amino acid alanine with glucose and glutamic acid in combination with glycerol, sucrose and fructose enhanced the production of bioactive metabolite by the fungus. The antimicrobial agent produced by Hypocrea spp. was extracted with ethyl acetate as solvent and purified by TLC. The purified active compound with UV λ-max 242 nm got the lowest minimum inhibitory concentration (MIC) against Staphylococcus aureus and Fusarium oxysporum, whereas no activity recorded against Pseudomonas aeruginosa.

Differentiation of a Fibrin Gel Encapsulated Chondrogenic Cell Line

Hyaline cartilage has very limited regenerative capacity following damage. Therefore engineered tissue substitutes have been the focus of much research. Our objective was to develop a fibrin-based scaffold as a cell delivery vehicle and template for hyaline cartilage regeneration, and compare its cellular properties against monolayer and pellet culture for chondrogenic cells. The chondrogenic precursor cell line, RCJ 3.1C5.18 (C5.18), was chosen as a test system for evaluating the effect of various culture conditions, including cell encapsulation, on articular chondrogenic cell differentiation. The C5.18 cells in monolayer showed elevated expression of collagen II, an articular chondrogenic marker, but also markers for fibrocartilage differentiation (collagen I and versican) when cultured with chondrogenic medium as compared to basic maintenance medium. Pellets of C5.18 cells cultured in chondrogenic medium were histologically more organized in structure than pellets cultured in control maintenance medium. The chondrogenic medium cultured pellets also secreted an extracellular matrix that was comprised of type II with very little type I collagen, indicating a trend towards a more hyaline-like cartilage. Moreover, when cultured in chondrogenic medium, fibrin-encapsulated C5.18 cells elaborated an extracellular matrix containing type II collagen, as well as aggrecan, which are both components of hyaline cartilage. This indicated a more articular-like chondrogenic differentiation for fibrin encapsulated C5.18 cells. The results of these experiments provide evidence that the C5.18 cell line can be used as a tool to evaluate potential scaffolds for articular cartilage tissue engineering.

Effect of culture conditions on lipid and gamma-linolenic acid production by mucoraceous fungi

The effect of various culture conditions on growth, lipid production and fatty acid composition in Mucor rouxii and Mucor sp.1b were studied. Total lipid production was higher in media containing potassium nitrate for both the cultures (30%) and cultures grown on plant seed oil produced more than 44% lipid. Among the carbon sources tested, γ-linolenic acid (GLA) production was maximal in cultures grown on glucose. The major fatty acids produced by these two cultures were palmitic, stearic and oleic acids. Levels of GLA in M. rouxii and M. sp.1b was in the range of 3–17% under different culture conditions. Lactose was a poor promoter for biomass and lipid production in both cultures. No GLA was found in fungal cultures grown on sesame oil. The optimal conditions for the production of GLA was standardised in these cultures.

Long-Term Human T-Cell Culture System Supporting Hepatitis C Virus Replication

We recently found that human T-cell leukemia virus type I-infected cloned MT-2C cells could support the replication of hepatitis C virus (HCV) up to 30 days postinoculation (p.i.). To obtain a more persistent HCV replication system using MT-2C cells, we examined the effect of various culture conditions after virus inoculation. We found that persistent HCV infection (at least 80 days) could be reproducibly achieved by reducing the temperature from 37°C to 32°C. Under this culture condition, HCV RNA could be detected in the cells up to 198 days p.i. Sequence analysis of HCV hypervariable region 1 revealed that certain HCV species became predominant during the culture period. We also demonstrated that virus transmission by the cell-free mode could be successfully repeated at least 4 times at 32°C as opposed to only twice at 37°C. This HCV-infected culture system will be useful for the various biological studies, including investigations into the mechanisms of HCV replication and multiplication.

Time lapse video recordings of highly purified human hematopoietic progenitor cells in culture.

Major hurdles in studies of stem cell biology include the low frequency and heterogeneity of human hematopoietic precursor cells in bone marrow and the difficulty of directly studying the effect of various culture conditions and growth factors on such cells. We have adapted the cell analyzer imaging system for monitoring and recording the morphology of limited numbers of cells under various culture conditions. Hematopoietic progenitor cells with a CD34+ CD45RAlo CD71lo phenotype were purified from previously frozen organ donor bone marrow by fluorescence activated cell sorting. Cultures of such cells were analyzed with the imaging system composed of an inverted microscope contained in an incubator, a video camera, an optical memory disk recorder and a computer-controlled motorized microscope XYZ precision stage. Fully computer-controlled video images at defined XYZ positions were captured at selected time intervals and recorded at a predetermined sequence on an optical memory disk. In this study, the cell analyzer system was used to obtain descriptions and measurements of hematopoietic cell behavior, like cell motility, cell interactions, cell shape, cell division, cell cycle time and cell size changes under different culture conditions.

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae.

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent Km values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent Km values were 0.1 mM for alpha-ketoglutarate and 0.22 mM for glutamine.

フグ毒産生細菌の細胞内フグ毒産生に及ぼす培養条件の影響

The effect of various culture conditions on tetrodotoxins formation was studied using a strain F-9 isolated from the puffer fish Takifugu rubripes. The strain was identified as that similar to Vibrio nereis. The TTXs formation by the strain was proved by GC/MS analysis and the major toxin was identified as 4-epi TTX by HPLC. ORI medium gave a high toxin formation. Cells in the stationary phase showed a high toxicity. As compared with static culture, shaking culture was suitable for high toxin formation. Optimum temperatures for both growth and toxin formation were 27.5°C, but optimum NaCl concentrations for growth and toxin formation were 3 and 5%, respectively. These results suggested that various culture conditions influenced the productive ability of tetrodotoxins in the strain.