2 Materials
All materials should be understood as examples. Equipment of
similar function, and reagents with an appropriate purity of a different manufacturer might be used as well.
2.1 Upstream
Processing Based
on an Example
of Vero Cells
2.1.1 Thawing Cells
1. Cells (Vero cells in defined low passage number, # CCL-81,
ATCC, Manassas, VA, USA) in cryovial.
2. Water bath (37
C) (WNB 22, Memmert GmbH + Co. KG,
Schwabach, Germany).
3. Growth medium DMEM-HG (Biochrom, Berlin, Germany)
(see Note 1).
4. 10 mM HEPES buffer (Biochrom, Berlin, Germany).
5. 4 mM L-glutamine (Biochrom, Berlin, Germany).
6. 70% (v/v) ethanol (AppliChem GmbH, Darmstadt, Germany).
7. Sterile serological 2 mL pipette (Sarstedt, Nu ¨mbrecht,
Germany).
8. 15-mL centrifuge tube (Sarstedt, Nu ¨mbrecht, Germany).
9. Centrifuge (Heraeus Megafuge X1R, Thermo Fisher Scientific,
Darmstadt, Germany).
10. T-25 flask (Sarstedt, Nu ¨mbrecht, Germany).
11. 37
C incubator (HeraCell 240, Thermo Fisher Scientific,
Waltham, MA, USA).
12. Sterile workbench (NuAire CellGard ES NU-480, MN, USA).
2.1.2 Cell Passaging
1. Deionized water (Milli-Q Academic, Q-Gard
® 1, Merck
KGaA, Darmstadt, Germany).
2. DMEM-HG (Biochrom, Berlin, Germany).
3. FBS (Biochrom, Berlin, Germany).
4. 10 mM HEPES buffer (Biochrom, Berlin, Germany).
5. 4 mM L-glutamine (Biochrom, Berlin, Germany).
6. 75–175 cm
2 T-flasks (Sarstedt, Nu ¨mbrecht, Germany).
7. Phosphate-buffered saline (PBS), w/o Ca
2+ and Mg
2+
(#L1825, Biochrom, Berlin, Germany) (PBS composition:
NaCl—8000, KCl—200, Na 2 HPO 4 —1150, KH 2 PO 4 —200,
MgCl 2 -6H 2 O—100, CaCl 2 —100, all values in mg/L).
8. Trypsin (Biochrom, Berlin, Germany).
9. 70% (v/v) ethanol (AppliChem GmbH, Darmstadt, Germany)
10. Sterile serological and aspiration pipettes (Sarstedt, Nu ¨mbrecht, Germany).
11. 1.5 mL tubes (Eppendorf, Hamburg, Germany).
224
Keven Lothert et al.