2.4 Transduction
of MSC with BV
2.4.1 Thawing
and Cultivating MSCs
l
Cryovial with 1 Â 10
6 hMSCs (e.g., from bone marrow, ATCCPCS-500-012).
l
Water bath (37
C) (WNB 22, Memmert, Schwabach, GER).
l
T25-flasks (Sarstedt, Nu ¨mbrecht, GER).
l
Growth medium (Dulbecco’s MEM supplemented with 10%
fetal calf serum and 2 mM L-alanyl-L-glutamine) (Biochrom,
Berlin, GER).
l
Trypsin–EDTA solution (Biochrom, Berlin, GER).
l
Phosphate-buffered saline (PBS), w/o Ca
2+ and Mg
2+ (#L1825
[Biochrom, Berlin, GER]).
l
Serological pipettes and holder (Sarstedt, Nu ¨mbrecht, GER).
l
Sterile pipette tips and pipette (Eppendorf, Hamburg, GER).
l
1.5 mL tubes (Eppendorf, Hamburg, GER).
l
15 mL and/or 50 mL centrifuge tube (Sarstedt,
Nu ¨mbrecht, GER).
l
Aspiration system (Vacusafe™, [Integra, Biebertal, GER]).
l
Incubator (HERAcell™ 240i [Thermo Fisher Scientific]).
l
Centrifuge (Heraeus™ Megafuge™ X1R [Thermo Fisher Scientific, Darmstadt, GER]).
2.4.2 Determination
of MSC Concentration
and Viability
l
Hemocytometer/counting chamber, Neubauer improved (Marienfeld, Ko ¨nigshofen, GER).
l
PBS (#L1825, Biochrom, Berlin, GER).
l
Incident light microscope (DM1i, Leica, Wetzlar, GER).
l
Sterile pipette and pipette tips (Sarstedt, Nu ¨mbrecht, GER).
l
0.4% trypan blue solution (Sigma-Aldrich, Hamburg, GER).
2.4.3 Transduction
l
hMSCs cultured in growth medium (from Subheading 2.4.1).
l
Psuedotyped BV (P2 viral stock).
l
T-flasks (Sarstedt, Nu ¨mbrecht, GER).
l
Growth medium (Dulbecco’s MEM supplemented with 10%
fetal calf serum and 2 mM L-alanyl-L-glutamine).
l
Incubator (HERAcell™ 240i, Thermo Fisher Scientific).
l
PBS with Ca
2+ and Mg
2+ (Merck Millipore, Burlington,
MA, USA).
l
Trypsin (Biochrom, Berlin, GER).
l
Cytometer (Guava
® easyCyte™ 6HT 2 L [Merck Millipore,
Darmstadt, GER]).
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