Continuous in vitro culture of Babesia divergens in a serum-free medium

Parasitology ◽  
1997 ◽  
Vol 115 (1) ◽  
pp. 81-89 ◽  
Author(s):  
N. GRANDE ◽  
E. PRECIGOUT ◽  
M. L. ANCELIN ◽  
K. MOUBRI ◽  
B. CARCY ◽  
...  
Keyword(s):  
Human Serum ◽  
Reduced Glutathione ◽  
Lipid Fraction ◽  
Basal Medium ◽  
Defined Medium ◽  
Specific Lipids ◽  
Serum Free ◽  
Babesia Divergens ◽  
Lipid Fractions

Babesia divergens was cultivated in RPMI 1640 (25 mM HEPES) supplemented with 10% human serum (RPMI-10% HS) with a high percentage of parasitized erythrocytes (PPE) ([ges ]40%). Standardization of in vitro tests, purification of exoantigens, biochemical studies and the safety of the culture handler motivated the development of a serum-free defined medium. Removal of serum greatly reduced the PPE but, after a period of adaptation, the culture was continuous and the parasite was able to develop a 3% routine PPE. Addition of vitamins or reduced glutathione in basal medium (RPMI) did not improve the PPE. The supplementation of basal medium with lipidic carrier (Albumax I or bovine serum albumin–Cohn's fraction V) promoted the growth of B. divergens with high PPE (>30%) close to those obtained in RPMI–10% HS. Neither protein nor lipid fractions alone were able to restore the growth of B. divergens. Nevertheless, the whole lipid fraction from serum or Albumax I added to delipidated albumin partially restored the growth (7% PPE), indicating that the presentation of specific lipids by a carrier is crucial for the parasite. All the data indicate that Albumax I can replace human serum offering the advantages of safety, standardization for chemosensitivity tests, and exoantigen purification.

scholarly journals Bioactive Lipids of Marine Microalga Chlorococcum sp. SABC 012504 with Anti-Inflammatory and Anti-thrombotic Activities

Marine Drugs ◽  
2021 ◽  
Vol 19 (1) ◽  
pp. 28
Author(s):  
Katie Shiels ◽  
Alexandros Tsoupras ◽  
Ronan Lordan ◽  
Constantina Nasopoulou ◽  
Ioannis Zabetakis ◽  
...  
Keyword(s):  
Essential Fatty Acids ◽  
Lipid Fraction ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Bioactive Molecules ◽  
Bioactive Lipids ◽  
Alternative Source ◽  
Lipid Fractions ◽  
Anti Inflammatory

Microalgae are at the start of the food chain, and many are known producers of a significant amount of lipids with essential fatty acids. However, the bioactivity of microalgal lipids for anti-inflammatory and antithrombotic activities have rarely been investigated. Therefore, for a sustainable source of the above bioactive lipids, the present study was undertaken. The total lipids of microalga Chlorococcum sp., isolated from the Irish coast, were fractionated into neutral-, glyco-, and phospho-lipids, and were tested in vitro for their anti-inflammatory and antithrombotic activities. All tested lipid fractions showed strong anti-platelet-activating factor (PAF) and antithrombin activities in human platelets (half maximal inhibitory concentration (IC50) values ranging ~25–200 μg of lipid) with the highest activities in glyco- and phospho-lipid fractions. The structural analysis of the bioactive lipid fraction-2 revealed the presence of specific sulfoquinovosyl diacylglycerols (SQDG) bioactive molecules and the HexCer-t36:2 (t18:1/18:1 and 18:2/18:0) cerebrosides with a phytosphingosine (4-hydrosphinganine) base, while fraction-3 contained bioactive phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules. These novel bioactive lipids of Chlorococcum sp. with putative health benefits may indicate that marine microalgae can be a sustainable alternative source for bioactive lipids production for food supplements and nutraceutical applications. However, further studies are required towards the commercial technology pathways development and biosafety analysis for the use of the microalga.

scholarly journals In-vitro Assessment of Biological Properties of Lipids Isolated from Gonads of Stomopneustes variolaris

2020 ◽  
Vol 2 (1) ◽  
pp. 66
Keyword(s):  
Biological Effects ◽  
Lipid Fraction ◽  
Radical Scavenging ◽  
Biological Properties ◽  
Total Phenolic ◽  
Klebsiella Pneumonia ◽  
Spectroscopic Techniques ◽  
Lipid Fractions ◽  
Anti Fungal

Lipid fractions of gonads present in sea urchins serves as a source of bioactive agents with potent pharmaceutical properties. The present study reports the in-vitro biological effects of lipids isolated from gonads of sea urchin: Stomopneustes variolaris collected from the East coast of India. The extracted lipids were characterized by spectroscopic techniques such as GCMS and FTIR and tested for in-vitro biological effects. GCMS analysis of the lipid extract detected high levels of hexa triacontane (17.023 %), tetratetracontane (15.913%), and octacosane (15.628%) and low concentrations of oleic acid (2.206%) and sulfurous acid, pentadecy 2-propyl ester (1.744%). FTIR analysis identified rich composition of functional groups present in the lipids such as 3418.93 cm-1 (hydroxyl), 2921.08cm-1 and 2854.81 cm-1 (alkane), 2660.69 cm-1 (carboxylic acid), 1596.11 cm-1 (amine), 1291.76 cm-1 (aromatic amine). The lipid fraction evaluated by agar diffusion assay measured in terms of zone of inhibition showed bactericidal effects against gram-positive bacteria: Streptococcus aureus (30 mm); Pseudomonas aeruginosa (28.5 mm) and gram-negative bacteria: Escherichia coli (29.5 mm); Klebsiella pneumonia (27.5 mm) and Vibrio cholera (28 mm) respectively. The lipid fraction also showed an effective anti-fungal effect against C.albicans (25 mm). Further, the lipid fractions showed good radical scavenging effect against total phenolic, flavonoid content (15.12 mg GAE/g and 32.72 mg QE/g), and hydrogen peroxide radicals (IC50- 48.28mg/ml) confirming its anti-oxidant potential. Based on the observed results, it was identified that the lipid fraction of gonads of Stomopneustes variolaris demonstrated various biological effects such as bactericidal, anti-fungal and radical scavenging activities which could have a great scope in the formulation of biopharmaceutical agents.

scholarly journals 319IN VITRO MATURATION OF EQUINE OOCYTES IN A COMPLETELY DEFINED MEDIUM SUPPLEMENTED WITH PROGESTERONE

2004 ◽  
Vol 16 (2) ◽  
pp. 279
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
F. Prati ◽  
G. Mari
Keyword(s):  
Polar Body ◽  
Basal Medium ◽  
Defined Medium ◽  
Chi Square ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Chi Square Test ◽  
The Media ◽  
Cytoplasmic Maturation

A completely defined medium for in vitro maturation (IVM) of equine oocytes has not yet been developed, since most of the media used for IVM are supplemented with serum or BSA. Furthermore, in this species there is no report about the influence of progesterone on maturation, although it has already been used as supplement (500ngmL−1) in EMMI (Maclellan LJ et al., 2001, Theriogenolgy 55, 310 abst). The aims of this study were to develop a completely defined medium for equine oocyte maturation and to investigate the effect of progesterone on nuclear maturation. Equine oocytes were collected by follicular scraping of abattoir-derived ovaries between April and June. The basal medium for maturation was SOFaa supplemented with pFSH-LH 0.1IUmL−1 (Pluset, Laboratorios Calier, Barcelona, Spain), EGF* 50ngmL−1, ITS (Insulin, Transferrin, Sodium selenite), L-cysteine 1.2mM, Maturation SOF (MSOF). Compact cumulus-oocyte complexes were selected, washed three times in H-SOF and matured in one of the following media (15–20 oocytesmL−1): (1) MSOF+FCS 10% (MSOF-FCS), (2) MSOF+progesterone 100ngmL−1 (MSOF-P4), (3) MSOF. After 24h of culture in 5% CO2 in air at 38.5°C, the oocytes were denuded by gently pipetting in a 0.25% trypsin solution, washed and stained with Hoechst 33258 (10μgmL−1 in PBS) for 30min at room temperature. Oocytes were examined under a fluorescent microscope to assess nuclear maturation. Only oocytes with an evident polar body and metaphase II plate (MII) were considered mature. The experiment was done in 6 replicates. Chi Square test was used for statistical analysis (Statistica for Windows – Stat Soft Inc., Tusla, OK, USA). Significance was assessed for P<0.05. The results of this study show that MSOF can be considered a suitable completely defined medium for IVM of equine oocytes. Adding progesterone significantly (P<0.05) increases the nuclear maturation rate at 24h of culture. It can be speculated that although cumuls cells produce this hormone, supplementation is useful to reach progesterone concentrations similar to those present in follicular fluid (early dominant 63.4±19.3ngmL−1, healthy preovulatory follicle 1094.3±170.9ngmL−1; Gerard N et al., 2002, Reproduction 124, 241–248). Further studies are needed to investigate the influence of progesterone on cytoplasmic maturation and to test the effect of different progesterone concentrations and time of maturation in a completely defined system.*All chemicals were purchased from Sigma, St. Louis, MO, USA, unless otherwise stated. Table 1 Maturation of equine oocytes in different media

Role of Organic Phosphate in Mineralization of Bone in vitro

1981 ◽  
Vol 60 (C) ◽  
pp. 1586-1586 ◽  
Author(s):  
H. C. Tenenbaum
Keyword(s):  
Defined Medium ◽  
Organic Phosphate ◽  
Limiting Factor ◽  
Serum Free ◽  
Osteogenic Cells ◽  
Inorganic Phosphates

Calvarial periostea were dissected from 17-day-old embryonic chicks and folded with the osteogenic cells in apposition. The folded explains were cultured for up to six d on serum and plasma clots or in serum-free hormone-supplemented completely-defined medium. Osteoid consistently formed in such cultures in both types of media, and this osteoid mineralized when appropriate levels of β-glycerophosphate were added to each type of medium. The data presented suggest that the levels of organic phosphate might be more important than inorganic phosphates as a limiting factor in the initiation of mineralization of bone in vitro.

scholarly journals PREPARATION FROM HUMAN SERUM OF AN ALPHA-ONE PROTEIN WHICH INDUCES THE IMMEDIATE GROWTH OF UNADAPTED CELLS IN VITRO

1967 ◽  
Vol 32 (2) ◽  
pp. 297-308 ◽  
Author(s):  
Richard Holmes
Keyword(s):  
Human Serum ◽  
Small Molecules ◽  
Gel Electrophoresis ◽  
Human Heart ◽  
Biological Response ◽  
Defined Medium ◽  
Heart Cells ◽  
Chemically Defined Medium ◽  
Carbamyl Phosphate

An alpha-one protein is separated from human serum on a microbead column. This nondialyzable protein induces the immediate growth of unadapted cells placed in chemically defined Medium A2 + APG. HeLa, conjunctiva and human heart cells, which stop growing if the protein is removed, continue to grow only if the protein is returned or the cells are permitted to adapt to the defined medium. A 90–120 day period is required for adaptation. The spreading and growth of fully adapted cells is also stimulated by the addition of the protein. As little as 0.4 µg per ml of medium is effective. The protein analyzed by paper, starch, and discontinuous acrylamide gel electrophoresis appears to be a single component. The protein is periodate-Schiff positive and readily binds small molecules which are removed, without loss of biological activity, by precipitating the protein in 50% saturated ammonium sulfate. The protein is adsorbed on the microbead column as a complex with the beta lipoprotein fraction of human serum; it cannot be separated from bovine or equinesera by this method; and it is not identical with fetuin. Its biological response is not duplicated by insulin, carbamyl phosphate, putrescine, or linoleic acid.

Insulin influences developmental competence of bovine oocytes cultured in α-MEM plus follicle-simulating hormone

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 563-572 ◽  
Author(s):  
Gustavo Bruno Mota ◽  
Ingrid Oliveira e Silva ◽  
Danielle Kaiser de Souza ◽  
Flavia Tuany ◽  
Michele Munk Pereira ◽  
...  
Keyword(s):  
Basal Medium ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Serum Free ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Blastocyst Rate ◽  
Defined Culture ◽  
Bovine Oocytes

SummaryThe aim of this study was to evaluate the dose–response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus–oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus–oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P < 0.05) than the DCM control. Insulin concentrations of 1, 10, or 100 ng/ml decreased the relative levels of GDF9 and HSP70-1 transcripts in oocytes at the end of IVM (P < 0.05). The transcripts levels of PRDX1 decreased (P < 0.05) only when 10 or 100 ng/ml insulin was added to the DCM medium. No difference in levels of GLUT1 transcripts (P > 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.

The in vitro growth and metabolism of Acer saccharum tissue

1971 ◽  
Vol 49 (4) ◽  
pp. 495-500 ◽  
Author(s):  
Martin C. Mathes ◽  
Mariafranca Morselli ◽  
J. W. Marvin
Keyword(s):  
Acer Saccharum ◽  
Sugar Maple ◽  
Culture Medium ◽  
Basal Medium ◽  
Soluble Starch ◽  
Defined Medium ◽  
Tissue Growth ◽  
Starch Digestion ◽  
Callus Tissues

Isolated sugar maple callus was grown on defined medium containing 0.2 kinetin and 2.0 ppm indoleacetic acid. Selected components of the basal medium were varied but did not result in increased tissue growth. The presence of 1, 5, or 25 ppm gibberellic acid in the culture medium did not increase the secretion of starch digesting materials by the tissue. The diameter of the starch digestion zone was not influenced by changing the acidity of the medium in the range of pH which supported tissue growth. The ability of isolated callus tissues from various species to digest extracellular starch was examined. It was found that equal amounts of various tissues do not possess an equal capacity for the in vitro secretion of extracellular materials, most probably enzymes, into the culture medium. It was found that isolated sugar maple tissue metabolized sucrose, galactose, and cellobiose while mannose and rhamnose were not used.Isolated sugar maple tissue which had been subcultured on the same medium for a long period was found to undergo nutritional changes which enabled the tissue to adapt to the medium. The starch-digestion enzyme activity of the tissue was found to increase when sugar maple tissue was placed on soluble starch medium.

342 DEVELOPMENT OF PIG OOCYTES MATURED AND FERTILIZED IN CHEMICALLY DEFINED MEDIUM CONTAINING PVA, PVP, AND PFF

2006 ◽  
Vol 18 (2) ◽  
pp. 278
Author(s):  
H.-B. Seok ◽  
I.-D. Kim
Keyword(s):  
Basal Medium ◽  
Defined Medium ◽  
Sodium Lactate ◽  
Chemically Defined Medium ◽  
Sodium Pyruvate ◽  
Hoechst 33342 ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Vitro Development

This study was conducted to develop a serum-free, defined medium for IVM of pig oocytes. TCM-199 with supplemented with polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and porcine follicular fluid (pFF) were used as basal medium. The effects of the these additives on the rates of maturity and development during in vitro fertilization and in vitro culture were examined and subsequently considered as possibile substitutes for bovine serum albumin (BSA). In an experiment that contained nine replicates, pig oocytes from abattoir-derived ovaries were matured in TCM-199 containing pyruvic acid, gentamycin, L-cysteine, �-estradiol, and FSH, and supplemented with 0.1% PVA, 0.1% PVP, 10% pFF, or 0.1% BSA (control) for 44 h at 39�C, 5% CO2. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate, and the same indicated supplements for 2 days. Maturation, fertilization, and morula and blastocyst formation were examined at 44 h after IVM and at 12, 48, and 120 h after IVF, respectively. Morphology of oocytes and cleaved cells was desplayed by staining with Hoechst 33342. Maturation rates of pig oocytes in IVM media containing PVA (82.4%), pFF (89.4%), or BSA (90.0%) were significantly higher (P < 0.05) than that with PVP (78.6%). Cleavage rates after IVF with PVP (64%) was significantly lower (P < 0.05) than those in PVA (73%), pFF (77%), or BSA (73%) supplements. In vitro development rates to morulae and blastocysts with PVP (54%) were also significantly lower (P < 0.05) than those with the supplements of PVA (63%), pFF (69%), or BSA (65%). In comparison of maturation and fertilization rates of pig oocytes in each of the supplements, the maturity rates with PVA (82.4%), pFF (89.4%), or BSA (90.0%) were significantly lower (P < 0.05) than that with PVP (72.4%) and the fertilization rates with pFF (87.1%) or BSA (89.1%) were significantly higher (P < 0.05) than those with PVA (78.0%) or PVP (70.6%). It may be concluded that PVA and pFF can be substituted for BSA in medium for culturing pig oocytes, and that PVP would not be an acceptable substitute for BSA.

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