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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
2. Methods
– 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
Acid:
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-
tion time.
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.
fill/harvest port.
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.
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