Info-Broschüre Myanmar 24 Tage 2007 – Djoser Reisen GmbH Informationen für unsere Myanmar - Reisenden (24 Tage) Das ‚Land der 1000 Pagoden' galt einst als das reichste Land Südostasiens. Kein Reisender wird je genügend Zeit finden, alle Pagoden zu besuchen, denn neben diesen religiösen Schätzen der Vergangenheit entdeckt man in Myanmar außergewöhnliche Naturlandschaften und unberührte Strände. Das Wertvollste jedoch sind die Menschen, die durch ihre Wärme und Gelassenheit diese Reise zu einer ganz besonderen Erfahrung werden lassen.
On the partner website Treatment methods of erectile dysfunction given a complete description how to take these tablets. Be sure to check before use.Je l'ai acheté le médicament cialis prix deux ou trois fois, l'effet est des pilules superbes, je ne ne nous a pas déçus même si je suis au dernier étage sur la pilule. Männer werden empfohlen, für mindestens 30 Minuten für den angeblichen Geschlechtsverkehr durchschnittliche Rendite von cialis 20mg zu verwenden.
BIOSTAT® CultiBag RM Culturing Convenience batch, serum free cultivation of CHOXM 111 suspension cells in the BIOSTATCultiBag RM 20 turning science into solutions
Dipl. Ing. Irina Bauer*, Prof. Dr. Regine Eibl*, Generally, the inoculum for the bioreactor Dr. Thorsten Adams** In this application note, we describe a is prepared by pooling T-flasks. The pre- protocol for the propagation of the model culture is routinely realized in 75 cm2 * Zurich University of Applied Sciences protein secreted alkaline phosphatase and 175 cm2 culture flasks containing (ZHAW), Waedenswil, Switzerland expressing CHO XM 111 suspension cells CHOMaster FMX-8 growth medium, which ** Sartorius Stedim Biotech GmbH, (obtained from Prof. Dr. Martin Fussenegger, was used for maintenance of the culture. August-Spindler-Str.11, Swiss Federal Institute of Technology, In order to ensure optimum growth in 37079, Goettingen, Germany Zurich) in selective, chemically defined, T-flasks, the CHO suspension cells are incu- protein and peptide-free ChoMaster media bated at 37°C in a humidified atmosphere (HP-1 and HP-5) using the disposable of 10% CO in air. Cells are seeded at a bioreactor BIOSTAT CultiBag RM 20 basic. minimal density of 2–3 + 105 viable Fig. 1: BIOSTAT® CultiBag RM20 basic.
cells/mL and subcultured or inoculated inthe larger scale when cell densities havereached values around 1*106 viablecells/mL.
In our experience, this method describedfor CHO XM 111 suspension cells can alsobe successfully applied to other animal celllines such as non-transfected CHO suspen-sion cells, Sf-9/Sf-21 suspension cells(DSMZ), and engineered HEK-293 EBNAsuspension cells (Cytos Biotechnology AG,Switzerland). Modifications mainly concernthe culture medium.
1. Equipment and Material
– BIOSTAT® CultiBag RM 20 basic a. Schedule
(Sartorius Stedim Biotech GmbH) Establishment of preculture I in T-75 flask with rapidly growing, healthy CHO XM 111 suspension cells characterized by logarithmic – CultiBag RM 2L growth and doubling times < 24 hours (with Sartofluor 300 Capsule, 0.2 μm; Feeding of preculture I with ChoMaster FMX-8 growth medium Sartorius Stedim Biotech GmbH) Establishment of preculture II (T-175 flask) from preculture I – CHOMaster media HP-1/HP-5 Passage cells into T-175 (minimal seeding density of 2-3*105 viable (Cell Culture Technologies GmbH) cells/mL), if cell density has reached 1x106 viable cells/mL – Cedex Cell Counter (Innovatis AG) Pooling of preculture II, inoculation and starting-up BIOSTAT CultiBag or Cedex HiRes (Innovatis AG) RM 20 with the disposable bioreactor bag CultiBag RM 2L operating or NucleoCounter (ChemoMetec A/S) with 100 mL cell suspension (1*106 viable cells/mL) and 100 mL ChoMaster HP-1 growth medium (see section 2d, 2e, 2f and 2g) – Bioprofile Analyzer 100 or BioProfile Fermentor/Bioreactor and medium preparation (see section 2b and 2c) Analyzer 100+ (Nova Biomedical) Day 8, 9, 10, 11: Sampling, successive feeding of ChoMaster growth medium (up to cell densities of 1.2*106 viable cells/mL HP-1, subsequent feeding of HP-5 – T-flasks (T-75, T-175) growth medium), increase of rocking rate and IPC (see section 3 and 5).
The feeding procedure should be also done in such a mode that glucose levels below 1.0 g/L are avoided.
Partial or complete harvest of cells (see section 4). Cell densities between 2 and 4*106 viable cells/mL may be achievable.
Aim at viabilities above 95%.
– Magnetic stirrer – Pipetboy (Integra Biosciences AG) – Peristaltic-pump (e.g. Dose-it 803, Vitaris AG) – Sterile syringes (10mL, 50 mL) – Serological pipettes – Reaction tubes and sample vials (1.5 mL) – Sterile bottles – Sterile aluminium foil – Safety cabinet class II – Laminar flow module – Roll-Boy with tripod b. Fermentor Bioreactor preparation
In order to obtain the desired seeding cell incubated (CO incubator) for 3 hours 100 mL of ChoMaster HP-1 growth medi- density of about 5*105 viable cells/mL for in order to allow the cells to settle. um containing 0.2 % Pluronic are filled in the CultiBag RM 2L, harvest of 5*107 viable Alternatively for other cell lines, the cells the CultiBag RM 2L in the safety cabinet cells from T-flasks, pooling of the cell can be centrifuged at maximum 200 g.
(clamped air filters) pellets and resuspension in 100 mL freshChoMaster HP-1 growth medium have to The consumed growth medium (FMX-8) Keep in mind that there is no need to use be carried out.
was then aspirated and replaced with fresh Pluronic in media containing serum.
HP-1 growth medium (pH 7.3, 37°C) in the Consequently, the cells were transferred safety cabinet. After cell density check the from T-175 into a sterile beaker (pipetting) cell suspension in the sterile beaker was Selective medium for T-flasks: filter-steril- covered with a sterile aluminum foil and ready for its use in CultiBag RM 2L.
ized, conditioned (37 °C, pH 7.3) ChoMasterFMX-8 growth medium (Cell Culture Tech-nologies).
e. Corrective agent
Additional supplements for FMX-8 medium:Used antibiotics to keep cells under selec- f. Culture conditions
tion pressure, support cell growth and Starting culture volume: prevent SEAP expression: 100 mg mL –1 Final culture volume: G418 sulphate, 5 mg mL –1 puromycin dihydrochloride, 2.5 mg mL –1 tetracyclinehydrochloride.
Medium for BIOSTAT CultiBag RM 20: filter-sterilized, conditioned (37 °C, pH 7.3) ChoMaster HP-1- and HP-5 growth medium (Cell Culture Technologies) Start cell density: 5 x 105 viable cells/mL Final cell density: 3-4 x 106 viable cells/mL Additional supplements for HP-1 Cultivation time: and HP-5 medium:2.5 mg mL –1 tetracycline hydrochloride,supports cell growth and prevents SEAPexpression and 0.2 % Pluronic F68 solution(Sigma) protects cells against shear (onlynecessary in serum-free media!) d) Preculture and Inoculum
for CultiBag RM 2L
For establishing the preculture II (T-175)
representing the subsequent inoculum
after pooling procedure approximately
4 days are required. In case of use of cryop-
reserved vials we recommend a previous
T-flask cultivation of 14 days. In other
words, the use of cryopreserved vials
instead of T-75 will prolong the precultiva-
3. Start-up and operation of BIOSTAT CultiBag RM 20
– By inserting a syringe into the CultiBag s luer lock inoculation port, 100 mL of the Sample 0: Analytics (section 5) prepared cell suspension (see section 2d) were added in the safety cabinet Sample 1: Analytics (section 5) (exhaust air filter was clamped off).
– The filled CultiBag (100 mL HP-1 growth Sample 2: Analytics (section 5), feeding with 200 mL HP-1 growth medium medium, see section 2b, and 100 mL cell and rocking rate increase (16 rpm) suspension, see section 2d) was put back on the tray (clamped air filters), fixed Sample 3: Analytics (section 5), feeding with 200 mL HP-5 and rocking rate increase (18 rpm) – The filter heater was installed and switched on.
Sample 4: Analytics (section 5), feeding with 200 mL HP-5 and rocking rate increase 25 rpm – Air filter lines were opened and aeration (0.2 vvm), rocking (14 rpm, 6°) and heat- Sample 5: Analytics (section 5), feeding with 200 mL HP-5 ing (37 °C) were switched on. and rocking rate increase 30 rpm 6 or 7 days: Sample 6 and 7: Analytics (section 5), partial or complete cell harvest (section 4). In case of partial cell suspension harvest, the adequate amount of fresh HP-5 growthmedium is fed.
4. Complete cell harvest or Scale-up
– 200 mL of HP-5 growth medium 5. Analytics
For harvesting the cells/product, one Daily one 2 mL sample is taken in order of the attached ports can be used. For the scale-up into a larger volume the following – For about 3 hours the cells were allowed Cell growth and viability (1 mL sample) procedure can be used: to settle on the bottom of the CultiBag by use of Cedex or NucleoCounter instead of traditional, time-consuming, manual cell – The BIOSTAT CultiBag RM basic station counting (hemocytometer, Trypan Blue) was switched off.
– The medium was removed from Glucose, lactate, glutamine, glutamate, the CultiBag using the tube of the pH (1 mL sample) by use of Nova BioProfile – The air filters were closed.
Analyzer 100 or its successor. Alternatively,other automized analyzers (e.g. YSI 2700 – The CultiBag RM 2L was removed from Bio-chemistry Analyzer, YSI Incorporated, the tray and transferred to a laminar and Eppendorf Ebio plus) or also test kits flow module.
(for example, from Roche Diagnostics) are available.
– The CultiBag was hung on a tripod standing on a Roll-Boy in the laminarflow module.
– The exhaust filter of the CultiBag was opened whereas inlet filter was closed.
Sales and Service Contacts
For further contacts, visit www.sartorius-stedim.com
Sartorius Stedim Biotech GmbH Sartorius Stedim Austria GmbH Sartorius Stedim North America Inc.
Sartorius Stedim Beijing Franzosengraben 12 Representative Office Bohemia, NY 11716 No. 33, Yu'an Road,Airport Industrial Zone B, Shunyi District Phone +49.551.308.0 Phone +43.1.7965763.18 Fax +49.551.308.3289 Fax +43.1.796576344 Fax +1.631.254.4253 Phone +86.10.80426516 Fax +86.10.80426580 Sartorius Stedim SUS Inc.
Sartorius Stedim Belgium N.V.
Sartorius Stedim Systems GmbH Leuvensesteenweg, 248/B Concord, CA 94520 Sartorius Stedim Shanghai Schwarzenberger Weg 73–79 Represantative Office Phone +1.925.689.6650 Phone +32.2.756.06.80 Room 618, Tower 1, German Centre, Toll Free +1.800.914.6644 Phone +49.5661.71.3400 Fax +32.2.756.06.81 Shanghai, PRC., 201203 Fax +1.925.689.6988 Fax +49.5661.71.3702 Phone +86.21.28986393 Fax +86.21.28986392.11 Sartorius Stedim Systems Inc. Sartorius Stedim Nordic A/S 201 South Ingram Mill Road Hoerskaetten 6D, 1.
Springfield, MO 65802 Sartorius Stedim Guangzhou Office Room 704, Broadway Plaza, Sartorius Stedim Biotech S.A.
Phone +1.417.873.9636 No. 233–234 Dong Feng West Road Phone +45.7023.4400 Fax +1.417.873.9275 Avenue de Jouques – BP 1051 Fax +45.4630.4030 13781 Aubagne Cedex Phone +86.20.8351.7921 Fax +86.20.8351.7931 Phone +33.442.845600 Sartorius Argentina S.A. Fax +33.442.845619 Sartorius Stedim Italy S.p.A.
Int. A. Avalos 4251 Via dell'Antella, 76/A 50012 Antella-Bagno a Ripoli (FI) Sartorius Stedim India Pvt. Ltd.
Sartorius Stedim France SAS Phone +39.055.63.40.41 10, 6th Main, 3rd Phase Peenya Phone +54.11.4721.0505 Fax +39.055.63.40.526 KIADB Industrial Area Fax +54.11.4762.2333 Avenue de Jouques – CS 71058 Bangalore – 560 058 13781 Aubagne Cedex Phone +91.80.2839.1963 0461 Phone +33.442.845600 . W/sart-000 · G.
Sartorius Stedim Netherlands B.V.
Fax +91.80.2839.8262 Sartorius do Brasil Ltda Fax +33.442.846545 Av. Dom Pedro I, 241 3439 MN Nieuwegein Bairro Vila Pires Phone +31.30.6025080 Sartorius Stedim Japan K.K.
Fax +31.30.6025099 KY Building, 8–11 Kita Shinagawa 1-chomeShinagawa-ku Phone +55.11.4451.6226 Sartorius Stedim Spain SA Fax +55.11.4451.4369 C/Isabel Colbrand 10–12, Phone +81.3.3740.5407 Planta 4, Oficina 121 Fax +81.3.3740.5406 Polígono Industrial de Fuencarral Sartorius de México S.A. de C.V.
Circuito Circunvalación Poniente No. 149 Phone +34.91.3586102 Sartorius Stedim Malaysia Sdn. Bhd.
Fax +34.91.3588804 53100 Naucalpan, Estado de México Lot L3-E-3B, Enterprise 4Technology Park Malaysia Phone +52.5555.62.1102 Fax +52.5555.62.2942 57000 Kuala Lumpur Sartorius Stedim Switzerland GmbHLerzenstrasse 21 Phone +60.3.8996.0622 Fax +60.3.8996.0755 Phone +41.44.741.05.00Fax +41.44.741.05.09 Singapore
Sartorius Stedim Singapore Pte. Ltd.
10, Science Park Road, The Alpha
#02-25, Singapore Science Park 2 Sartorius Stedim UK Limited Longmead Business ParkBlenheim Road, Epsom Phone +65.6872.3966 Fax +65.6778.2494 Phone +44.1372.737159Fax +44.1372.726171 Australia
Sartorius Stedim Australia Pty. Ltd.
Unit 5, 7-11 Rodeo Drive
Dandenong South Vic 3175
Phone +61.3.8762.1800Fax +61.3.8762.1828 Specifications subject to change without notice. Printed in Germany on paper that has been bleached without any use of chlorine Publication No.: SBT1003-e09041 · Order No.: 85034-537-73
NORGESTOMET AND ESTRADIOL VALERATE INDUCED LUTEOLYSIS IS DEPENDENT UPON THE UTERUS C. A. Peterson, J. C. Huhn, and D. J. Kesler SUMMARY Beef heifers were assigned to three groups: 1) untreated controls (n= 4), 2) Syncro-Mate B® (SMB) treated (n= 5), and 3) hysterectomized and SMB treated (n= 4). SMB was administered eight or nine days after estrus, approximately 30 days after hysterectomy. This study was conducted to determine if the uterus was necessary for SMB to induce luteolysis. SMB induced premature luteolysis as only 20% of the intact SMB treated heifers had ≥ .75 ng/mL of progesterone seven days after the time of SMB treatment compared to all (100%) of the untreated heifers (P <.05). By nine days after the time of SMB treatment, 25% of the untreated heifers and none (0%) of the intact SMB treated heifers had ≥ .75 ng/mL of progesterone; however, all (100%) of the hysterectomized SMB treated heifers had ≥ .75 ng/mL of progesterone (P <.05). Therefore, SMB-induced luteolysis required the involvement of the uterus. The luteolysin, prostaglandin F2α, is probably the secretion from the uterus that mediates the SMB-induced luteolysis. SMB treatment, however, required 7-8 days to induce luteolysis. INTRODUCTION Syncro-Mate B® (SMB) is a commercially available procedure to synchronize estrus in beef and dairy cattle. The procedure consists of a norgestomet implant and an intramuscular injection containing norgestomet and estradiol valerate administered at the time of implantation. SMB has three known mechanisms of action. First, an estrus suppression dosage of norgestomet diffuses from the implant during the nine days in situ (Kesler and Favero, 1995). Secondly, the injection causes atresia of antral follicles and recruitment of a new cohort of follicles four to five days after administration (Vasconcelos et al., 1997). Thirdly, the injection causes regression of corpora lutea (Kesler and Favero, 1995). Since the implant is left in place for nine days, the injection is needed to induce regression of corpora lutea in cows during the first half of the estrous cycle. Estradiol-17β, the active metabolite of the estradiol valerate contained within the SMB injection, has been demonstrated to hasten corpus luteum regression (Thatcher et al., 1986). Thatcher et al. (1986) reported spikes of 15-keto- 13, 14-dihydro-prostaglandin F2α (PGFM) in the peripheral blood before luteolysis ensued and concluded that estradiol-17β induced luteolysis by provoking a release of PGF2α from the uterus; however, Thatcher et al. (1986) administered estradiol-17β during the second half of the estrous cycle. Progesterone treatment during metestrus has also been reported to shorten the estrous cycle, but only by four days (Woody et al., 1967; Harms and Malven, 1969; Ginther, 1970; Battista et al., 1984; Garrett et al., 1988). The objective of this study was to determine if the hypothesis that SMB induced luteolysis is dependent upon uterine involvement was correct. MATERIALS AND METHODS Three groups of purebred Angus beef heifers from the University of Illinois beef research unit (Urbana, IL) were included in this study. The control group (n=4) was selected from a larger group of estrus-cycling females administered prostaglandin F2α (25 mg Lutalyse®; Pharmica and Upjohn, Kalamazoo, MI, USA) due to their similar timing of estrus (detectable estrus within 48 hours of each