BIOSTAT® CultiBag RM Culturing Convenience
batch, serum free cultivation of CHOXM 111 suspension
Dipl. Ing. Irina Bauer*, Prof. Dr. Regine Eibl*,
Introduction
Generally, the inoculum for the bioreactor
is prepared by pooling T-flasks. The pre-
protocol for the propagation of the model
(obtained from Prof. Dr. Martin Fussenegger,
was used for maintenance of the culture.
Zurich) in selective, chemically defined,
T-flasks, the CHO suspension cells are incu-
bated at 37°C in a humidified atmosphere
bioreactor BIOSTAT CultiBag RM 20 basic.
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 2. Methods a. Schedule
Establishment of preculture I in T-75 flask with rapidly growing, healthy CHO XM 111 suspension cells characterized by logarithmic
Feeding of preculture I with ChoMaster FMX-8 growth medium
Establishment of preculture II (T-175 flask) from preculture I
Passage cells into T-175 (minimal seeding density of 2-3*105 viable
cells/mL), if cell density has reached 1x106 viable cells/mL
Pooling of preculture II, inoculation and starting-up BIOSTAT CultiBag
RM 20 with the disposable bioreactor bag CultiBag RM 2L operating
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)
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
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
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%.
– Reaction tubes and sample vials (1.5 mL)
b. Fermentor|Bioreactor preparation
In order to obtain the desired seeding cell
density of about 5*105 viable cells/mL for
um containing 0.2 % Pluronic are filled in
the CultiBag RM 2L, harvest of 5*107 viable
Alternatively for other cell lines, the cells
cells from T-flasks, pooling of the cell
can be centrifuged at maximum 200 g.
pellets and resuspension in 100 mL freshChoMaster HP-1 growth medium have to
Keep in mind that there is no need to use
was then aspirated and replaced with fresh
HP-1 growth medium (pH 7.3, 37°C) in the
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-
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 Acid:
medium:Used antibiotics to keep cells under selec-
f. Culture conditions
dihydrochloride, 2.5 mg mL –1 tetracyclinehydrochloride.
filter-sterilized, conditioned (37 °C, pH 7.3)
ChoMaster HP-1- and HP-5 growth medium (Cell Culture Technologies)
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- tion time. g. Inoculation 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
prepared cell suspension (see section 2d)
(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
Sample 3: Analytics (section 5), feeding with 200 mL HP-5
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
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 5. Analytics
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
of traditional, time-consuming, manual cell
– The BIOSTAT CultiBag RM basic station
pH (1 mL sample) by use of Nova BioProfile
Analyzer 100 or its successor. Alternatively,other automized analyzers (e.g. YSI 2700
Bio-chemistry Analyzer, YSI Incorporated,
and Eppendorf Ebio plus) or also test kits
(for example, from Roche Diagnostics) are available.
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 Asia | Pacific
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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
Carlos E. Crespo-Hernández, Ph.D. Case Western Reserve University Department of Chemistry 10900 Euclid Avenue, Cleveland, OH 44106 Phone: (216)-368-1911 Faculty Website: ht p:/ www.case.edu/artsci/chem/faculty/crespo/ Email: [email protected] Group Website: ht p:/ www.case.edu/artsci/chem/faculty/crespo/group Professional B.S. in Chemistry Preparation University
Poate fi folosit numai de catre persoane care au implinit 18 ani si care sunt deja fumatori. Nicotina este FOARTE OTRAVITOARE in caz ca este inghitita. NU PASTRATI ACEST PRODUS LA INDEMANA COPIILOR. Cititi instructiunile inainte de folosire. RoHS Tigarile obisnuite - informatii reale Numeroase studii stiintifice au demonstrat ca fumul de tigara contine aprox. 4.800 substante c