Cultivation of infective forms ofTrypanosoma congolensefrom trypanosomes in the proboscis ofGlossina morsitans

Parasitology ◽  
1981 ◽  
Vol 82 (1) ◽  
pp. 81-95 ◽  
Author(s):  
M. A. Gray ◽  
I. Cunningham ◽  
P. R. Gardiner ◽  
A. M. Taylor ◽  
A. G. Luckins
Keyword(s):  
Culture Medium ◽  
Primary Cultures ◽  
Calf Serum ◽  
Tsetse Flies ◽  
Foetal Calf Serum ◽  
Culture Vessel ◽  
Minimum Essential Medium ◽  
Glossina Morsitans ◽  
Plastic Surface ◽  
Dermal Collagen

SUMMARYTwo stocks ofTrypanosoma congolensewere established in culture at 28 °C using trypanosomes from the proboscides of infectiveGlossina morsitans. Successful primary cultures were initiated by placing an infected tsetse proboscis beside a bovine dermal collagen explant in Eagle's minimum essential medium supplemented with foetal calf serum. The trypanosomes multiplied rapidly in the medium and also gradually formed an adherent layer on the plastic surface of the culture vessel. Three primary cultures produced organisms infective for mice from 14, 20 and 35 days after initiation and thereafter continuously until days 76, 76 and 52 when they were discarded. Four attempts to initiate infective cultures using infected tsetse proboscides in medium without dermal explants were unsuccessful. When trypanosomes from primary cultures were placed in culture medium with proboscides from uninfected tsetse flies, the parasites multiplied, formed an adherent layer in the culture flasks and were seen in the proboscides within 24 h. A line of 1 stock was serially sub-passaged in this way 4 times during a period of 215 days. Infectivity titrations in mice indicated that primary and sub-passaged cultures each contained similar numbers of infective organisms. Another line of the same stock was also sub-passaged 4 times in medium alone over a period of 186 days. These sub-cultures again retained infectivity for mice, but titrations showed a decrease in infective organism production in the 4th sub-culture. Primary and sub-passaged cultures all included a variety of morphologically different developmental forms ofT. congolense, closely resembling those described in the labrum and hypopharynx ofGlossinaby previous workers. Short metacyclic-like trypanosomes and organisms with proteinaceous surface coats were present in infective cultures. Cultures were successfully re-established after cryopreservation at −196 °C and retained the ability to produce infective organisms.

scholarly journals Immunoreactive insulin from mouse brain cells in culture and whole rat brain

1984 ◽  
Vol 218 (1) ◽  
pp. 19-27 ◽  
Author(s):  
N P Birch ◽  
D L Christie ◽  
A G C Renwick
Keyword(s):  
Rat Brain ◽  
Mouse Brain ◽  
Culture Medium ◽  
Calf Serum ◽  
Brain Cells ◽  
Immunoreactive Insulin ◽  
Foetal Calf Serum ◽  
Cells In Culture ◽  
Pancreatic Insulin ◽  
Contrast Microscopy

Foetal mouse brain cells were cultured as described previously [Sotelo, Gibbs, Gajdusek, Toh & Wurth (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 653-657] without added insulin and without foetal calf serum after 12 days in culture. Examination by phase-contrast microscopy showed that these modifications did not appear to affect growth and development of the cells adversely. Silver impregnation of the cultures and indirect immunofluorescence following reaction with tetanus toxin showed that a high proportion of the cells resembled neurones. Analysis of concentrated culture medium by radioimmunoassay and high-pressure liquid chromatography (h.p.l.c.) revealed that the cells produced two main forms of immunoreactive insulin which differed from authentic pancreatic insulin in retention time. Immunoreactive somatostatin was also produced in culture and this was resolved into at least three forms by h.p.l.c. Immunoreactive insulin was also extracted from whole rat brain by using two published procedures. The method of Havrankova, Schmechel, Roth & Brownstein [Proc. Natl. Acad. Sci. U.S.A. (1978) 75, 5737-5741] consistently gave greater yields of insulin than did that of Eng & Yalow [Diabetes (1980) 29, 105-109] and the concentration was about three times that of plasma. The extracted insulin was further characterized by h.p.l.c. in each case and was found to behave like authentic pancreatic insulin. The production of insulin and somatostatin by foetal mouse brain cells in culture suggests that they may be a useful model system for studies of neuropeptide biosynthesis.

scholarly journals Infectivity of Theileria parva sporozoites following cryopreservation in four suspension media and multiple refreezing : evaluation by in vitro titration

2006 ◽  
Vol 73 (3) ◽  
Author(s):  
V. Mbao ◽  
D. Berkvens ◽  
T. Dolan ◽  
N. Speybroeck ◽  
J. Brandt ◽  
...  
Keyword(s):  
East Coast ◽  
Calf Serum ◽  
Roswell Park Memorial Institute ◽  
Foetal Calf Serum ◽  
Theileria Parva ◽  
Minimum Essential Medium ◽  
Freezing Medium ◽  
In Vitro Technique ◽  
Phosphate Buffered Saline

Theileria parva sporozoite stabilates are used for immunizing cattle against East Coast fever and in in vitro sporozoite neutralization assays. In this study, we attempted to identify a cheaper freezing medium and quantified the infectivity loss of sporozoites due to refreezing of stabilates, using an in vitro technique. Pools of stabilates prepared using Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI 1640), foetal calf serum (FCS) and phosphate-buffered saline (PBS) were compared. All were supplemented with bovine serum albumin except the FCS. RPMI 1640 was as effective as MEM in maintaining sporozoite infectivity while the infectivity in PBS and FCS reached only 59 % and 67 %, respectively. In a second experiment, a stabilate based on MEM was subjected to several freeze-thaw cycles including various holding times on ice between thawing and refreezing. Refrozen stabilate gave an average sporozoite infectivity loss of 35 % per cycle. The results indicate that RPMI can be used as a cheaper freezing medium for T. parva stabilates and that refrozen stabilate doses need to be adjusted for the 35 % loss of infectivity.

Cultivation of Blastocrithidia triatomae (Trypanosomatidae) on a cell line of its host Triatoma infestans (Reduviidae)

Parasitology ◽  
1989 ◽  
Vol 98 (3) ◽  
pp. 387-393 ◽  
Author(s):  
Dagmar Reduth ◽  
G. A. Schaub ◽  
Mary Pudney
Keyword(s):  
Cell Line ◽  
Primary Cultures ◽  
Calf Serum ◽  
Host Cells ◽  
Triatoma Infestans ◽  
Foetal Calf Serum ◽  
Tryptose Phosphate Broth ◽  
A Cell ◽  
Vigorous Growth

SUMMARYBlastocrithidia triatomae parasitizes vectors of Chagas' disease and is very difficult to cultivate in conventional media. However, co-cultivation with a cell line of its host Triatoma infestans (TI-32; in Schneider's Drosophila medium supplemented with 20% foetal calf serum and 10% tryptose phosphate broth) led to vigorous growth at 24 or 28 °C without an adaptation phase. More than 60 primary cultures were initiated successfully without any failures. Subcultures could be started immediately at weekly intervals. The doubling time was similar in both the primary cultures (41 h) and the 33rd subculture (39 h). The importance of the reduviid cells for B. triatomae became clear after removal of the insect cells, when multiplication of epimastigotes stopped and mainly cysts were formed. Cysts produced in vitro were infective for reduviids. Scanning electron microscopy showed that B. triatomae attached to the host cells, inserted its flagellum into them and destroyed them.

Megakaryocytic Progenitors in Long Term Cultures from MDS Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4924-4924
Author(s):  
Maria Juliana Majado ◽  
María I. Macizo ◽  
Consuelo González-García ◽  
Eduardo Salido ◽  
José A. Molina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Culture Medium ◽  
Horse Serum ◽  
Calf Serum ◽  
Methyl Cellulose ◽  
Refractory Anemia ◽  
Foetal Calf Serum ◽  
Cell Counts ◽  
Control Bone

Abstract Introduction: bone marrow long-term cultures (LTC) have been reported to produce very poor adherent stromal layer in MDS, but there are not many studies about the behaviour of the progenitor cells in supernatant culture medium. In this study we report the results of megakaryocytic progenitors (CFU-MK) in the supernatant of LTC of patients with refractory anemia (RA). Material and method: LTCs were performed to 11 RA patients and to 13 normal multiorgan donors as control. Bone marrow red cells were sedimented with 1% methyl-cellulose and seeded for LTC in 10ml flasks (Nalge Nunc International), with Iscover medium supplemented with horse serum, foetal calf serum, hydrocortisone and carbonic air, at a final concentration of 1x106 per ml. Flasks were placed in an incubator for 8 weeks. Half culture medium volume was renovated weekly, cell counts and assays of CFU-MK (Megacult C, Stem Cell Technologies), were performed. CFU-MK colonies were separated in three groups, containing 3–20 cells, 21–50 cells and more than 50 cells. Student t-test was used for statistical comparisons. Results: Are in the following table, expressed as mean + standard desviation. After the third week no colony growth was observed in normal such as in MDS. Growth of CFU-MK colonies containing more than 50 cells was higher in basal control bone marrow, no statistical differences were found in the rest of the cultures. Results CFU-MK(3–30 Cells) CFU-MK(20–50 Cells) CFU-MK (>50 Cells) CFU-MK total w: week Control basal 15.75±15.79 3.33±5.42 4.88±7.44 22.42±26.43 RA basal 7.85±8.66 0.80±1.01 0.70±0.98 9.45±9.46 t-St ns ns 0.047 ns Control w 1 3.54±4.42 0.7±1.44 0.74±1.48 4.98±6.62 RA w 1 3.29±4.52 0.29±0.93 0.25±0.67 3.82±5.65 t-St ns ns ns ns Control w 2 0.55±1.14 0.07±0.24 0.05±0.25 0.67±1.43 RA w 2 0.18±0.32 0.00±0.00 0.04±0.13 0.21±0.38 t-St ns ns ns ns Control w 3 0.18±0.53 0.00±0.00 0.00±0.00 0.18±0.53 RA w 3 0.00±0.21 0.00±0.00 0.00±0.00 0.00±0.21 t-St ns ns Discussion: No important difference was found in LTC supernatant CFU-MK in our RA patients, and this supports the idea that the stromal damage is more important, in the pathophysyology of MDS, than that of the stem cells.

Neutrophil chemotactic factors secreted fromToxoplasma gondii

Parasitology ◽  
1991 ◽  
Vol 103 (1) ◽  
pp. 29-34 ◽  
Author(s):  
M. Nakao ◽  
E. Konishi
Keyword(s):  
Toxoplasma Gondii ◽  
Calf Serum ◽  
Culture Fluid ◽  
Chemotactic Activity ◽  
Boyden Chamber ◽  
Foetal Calf Serum ◽  
Neutrophil Chemotaxis ◽  
Minimum Essential Medium ◽  
Chemotactic Factors ◽  
Neutrophil Chemotactic Activity

Neutrophil chemotactic activity was detected in the fluid ofToxoplasma gondiicultures by the agarose plate and the Boyden chamber methods.Toxoplasmaculture fluid was obtained by incubating the tachyzoites at 37 °C in a 5% CO2atmosphere for 6 h in Dulbecco's modified Eagle's minimum essential medium containing 10% heat-inactivated foetal calf serum. Soluble extracts from tachyzoites had negligible activity, indicating that the chemotactic factors were metabolites secreted from tachyzoites. The chemotactic activity inToxoplasmaculture fluid depended on the number of tachyzoites and the period of incubation. The substances responsible for neutrophil chemotaxis were two heat-labile peptides with estimatedMr4·5 and 14 kDa with N-terminal groups free.

The primary culture of epithelia from the secretory coil and collecting duct of normal human and cystic fibrotic eccrine sweat glands

1986 ◽  
Vol 83 (1) ◽  
pp. 103-118
Author(s):  
C.M. Lee ◽  
F. Carpenter ◽  
T. Coaker ◽  
T. Kealey
Keyword(s):  
Ion Transport ◽  
Collecting Duct ◽  
Serum Insulin ◽  
Primary Cultures ◽  
Calf Serum ◽  
Sweat Glands ◽  
Foetal Calf Serum ◽  
Secretory Coil ◽  
Eccrine Sweat Glands ◽  
Eccrine Sweat

Isolated human eccrine sweat glands have been microdissected into their secretory and reabsorptive components. Complete separation of these epithelia was confirmed by differential uptake of Neutral Red stain by an intermediate section of gland containing the junction between the secretory coil and the collecting duct. Primary cultures were obtained from explants of both tissues in medium RPMI-1640 or Williams E supplemented with foetal calf serum, insulin, transferrin, epidermal growth factor and hydrocortisone. The cells in the initial coil cultures had an elongated morphology while those of ductal origin were polyhedral. After 10 days both cultures were composed of polyhedral cells of varying diameter. All these morphological types were of epithelial lineage, as demonstrated by the binding of a monoclonal antibody to cytokeratin, the intermediate filament specific for epithelial cells. Outgrowth from both secretory and reabsorptive epithelia were multilayered, with plentiful desmosomal connections and an underlying basal lamina. Ultrastructural features typical of the epithelial cell types present in intact eccrine sweat glands were absent in a high proportion of the proliferating cells but domes, indicative of transepithelial active ion transport, were present in dense cultures from the reabsorptive duct. Outgrowth was also obtained from the secretory and reabsorptive epithelia of sweat glands from two cystic fibrotic patients. Since the most characteristic malfunction of cystic fibrosis is the impaired ion transport in the eccrine sweat gland, the availability of cultured epithelia should provide a useful model for study of the disease.

scholarly journals Comparison of two vitrification methods for cryopreservation of porcine embryos

2011 ◽  
Vol 49 (No. 5) ◽  
pp. 183-189
Author(s):  
J. Říha ◽  
J. Vejnar
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Artificial Insemination ◽  
Culture Medium ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Post Mortem ◽  
Donor Insemination ◽  
Viable Embryo ◽  
Fresh Embryos

The aim of this study was to compare two vitrification methods of porcine perihatching blastocysts with regard to the success of transfer of these embryos to the recipients. Expanded, hatching, or hatched blastocysts were recovered post mortem from superovulated donors in 5.5 to 6.0 days after artificial insemination of donor gilts with homospermic doses. In protocol VS I, the embryos in perihatching developmental stage were equilibrated in a culture medium H-MEMD with 10% v/v of glycerol (1.37M solution of glycerol in medium) for 10 min and placed in a vitrification medium for 1.5 min max. (vitrification medium contained 50% v/v 2M sucrose in tridistilled water, 30% v/v of glycerol, and 20% v/v of foetal calf serum – FCS). Then they were dropped with micropipette and stored in liquid nitrogen vapour. For protocol VS II, we used H-MEMD culture medium supplemented with 20% v/v of FCS, 25% v/v ethylene glycol, and 25% v/v dimethyl sulphoxide (DMSO). Embryos were equilibrated for 10 min in a mixture of the vitrification medium and culture medium (1 : 1), and were kept in the vitrification medium for 1.5 minutes. Then they were dropped with micropipette and stored in liquid nitrogen vapour. Embryos were thawed by immersing the drop with the embryo in H-MEMD culture medium with 0.8M sucrose for 10 minutes. After thawing and washing in the medium with sucrose, all embryos were washed three times in a fresh medium and prepared for transfer. Recipients were synchronized either using Regumate-feeding followed by treatment with PMSG and HCG (gilts) or using piglet weaning (sows – 1st and 2nd parity). Recipients showing standing heat at the time of donor insemination were used for laparoscopic and non-surgical ET on day 5.5–6.0 of the cycle. The fraction of viable embryo vitrified under VS I or VS II protocol was 85% and 80%, compared to 95% in control fresh embryos (P > 0.05). Pregnancy of recipients was 57.3% (5/7), 67.0% (4/6) for VS I or VS II group and 42.7% (10/23) for control (P < 0.001). We can conclude on the basis of our data that both protocols for vitrification yielded similar results and can be used for cryopreservation of porcine embryos.  

The differentiation of mouse gonads in vitro: a light and electron microscopical study

Development ◽  
1986 ◽  
Vol 97 (1) ◽  
pp. 189-199
Author(s):  
Sarah Mackay ◽  
Robert A. Smith
Keyword(s):  
Electron Microscopy ◽  
Culture Medium ◽  
Light And Electron Microscopy ◽  
Calf Serum ◽  
Microscopical Study ◽  
Electron Microscopical Study ◽  
The Other ◽  
Foetal Calf Serum ◽  
Electron Microscopical

Indifferent urogenital complexes were excised from mouse foetuses assessed by developmental criteria as day 10·5 or 11. After 4 or 6–7 days in culture, complexes were fixed and examined by light and electron microscopy. The effect of culturing sexed complexes in mixed sex groups was investigated. The effect of the presence or absence of foetal calf serum in the culture medium was considered. No evidence of inhibition of one sex by the other was found. Ovaries developed further in cultures than testes.

The culture of mouse blastocysts in the presence of uterine flushings collected during normal pregnancy, delayed implantation and pro-oestrus

Development ◽  
1977 ◽  
Vol 41 (1) ◽  
pp. 295-300
Author(s):  
R. J. Aitken
Keyword(s):  
Culture Medium ◽  
Cell Transformation ◽  
Calf Serum ◽  
Normal Pregnancy ◽  
Foetal Calf Serum ◽  
Delayed Implantation ◽  
Simple Medium ◽  
Uterine Proteins ◽  
Stimulation Of ◽  
Blastocyst Hatching

When day-3 mouse embryos were cultured in a simple medium supplemented with uterine fluids of mice autopsied on day 4 of pregnancy, 48 h after administration of oestradiol, or during pro-oestrus, the percentage of blastocysts hatching from the zona pellucida was significantly greater than in unsupplemented medium. In the presence of uterine fluids recovered during delayed implantation this stimulation of blastocyst hatching was not observed. When the culture medium was supplemented with dialysed uterine flushings containing 20 or 30 μg protein/ml, both ‘day 4’ and ‘delay’ uterine proteins were equally effective in enhancing hatching frequency (P < 00·5). The results suggested that ‘delay’ uterine fluids may contain a dialysable inhibitor of blastocyst activity. The putative inhibitor was not effective in the presence of serum, since uterine fluids recovered both on day 4 of pregnancy and during delayed implantation significantly increased the size attained by lastocyst outgrowths in the presence of foetal calf serum (P < 0·001). The percentage of blastocysts exhibiting giant cell transformation and outgrowth was also increased (P < 0·02) by these uterine fluids when the concentration of FCS in the medium was minimal (0·25 %).

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