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
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.
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