l
Rapidly transfer the stained cells to the prepared Neubauer
counting chamber and count the cells using the inverse light
microscope.
l
Calculate the cell concentration and viability according to the
manufacturer’s instructions.
3.4.3 Transduction
of hMSC with BV
l
Plate 30,000 hMSCs/cm
2 in a T25 flask in the growth medium.
l
Incubate cells overnight at 37
C and 5% CO 2 in an incubator
before proceeding to the transduction (see Note 27).
l
Mix the pseudotyped BV (P2 viral stock) solution several times
by inversion to ensure a homogeneous solution (see Note 28).
l
Add the calculated volume of the pseudotyped BV (P2 viral
stock) solution directly to the cells in the complete growth
medium (see Note 29).
l
Return the cells into the incubator and the incubate cells overnight at 37
C and 5% CO 2.
l
Detect your protein of interest or your reporter protein 16–48 h
after transduction (e.g., GFP via fluorescence microscopy or
flow cytometry).
4 Notes
1. www.clinicaltrial.gov; last checked 29. August 2019; Search:
“gene therapy”; Recruitment status: “recruiting; enrolling by
invitation; active, not recruiting; completed”.
2. Quantification of residual DNA is performed using dyes that
bind to double-stranded nucleic acids. As the baculovirus is a
DNA virus, no discrimination between free viral DNA (vDNA)
and host cell DNA (hcDNA) can be accomplished.
3. Instead of the BCA Assay, a Bradford Assay can be used. The
BCA Assay is more sensitive, as it relies on a protein-copper
chelation, measured by the detection of reduced copper. The
Bradford Assay, on the contrary, is measured by a color shift
due to protein-dye binding.
4. The baculovirus/BV is very shear-sensitive [98].
5. Different membranes may be used. PES membranes with a
MWCO of 300 kDa have proven to be suitable for baculovirus
purification [92]. Depending on alterations in the process
(e.g., genetic modification or cell line), different materials
may be more appropriate.
6. Store the BV stocks at 4
C excluded from light, as the virus is
very susceptible to photodegradation and aggregation due to
382
Friederike Eilts et al.
Rapidly transfer the stained cells to the prepared Neubauer
counting chamber and count the cells using the inverse light
microscope.
l
Calculate the cell concentration and viability according to the
manufacturer’s instructions.
3.4.3 Transduction
of hMSC with BV
l
Plate 30,000 hMSCs/cm
2 in a T25 flask in the growth medium.
l
Incubate cells overnight at 37
C and 5% CO 2 in an incubator
before proceeding to the transduction (see Note 27).
l
Mix the pseudotyped BV (P2 viral stock) solution several times
by inversion to ensure a homogeneous solution (see Note 28).
l
Add the calculated volume of the pseudotyped BV (P2 viral
stock) solution directly to the cells in the complete growth
medium (see Note 29).
l
Return the cells into the incubator and the incubate cells overnight at 37
C and 5% CO 2.
l
Detect your protein of interest or your reporter protein 16–48 h
after transduction (e.g., GFP via fluorescence microscopy or
flow cytometry).
4 Notes
1. www.clinicaltrial.gov; last checked 29. August 2019; Search:
“gene therapy”; Recruitment status: “recruiting; enrolling by
invitation; active, not recruiting; completed”.
2. Quantification of residual DNA is performed using dyes that
bind to double-stranded nucleic acids. As the baculovirus is a
DNA virus, no discrimination between free viral DNA (vDNA)
and host cell DNA (hcDNA) can be accomplished.
3. Instead of the BCA Assay, a Bradford Assay can be used. The
BCA Assay is more sensitive, as it relies on a protein-copper
chelation, measured by the detection of reduced copper. The
Bradford Assay, on the contrary, is measured by a color shift
due to protein-dye binding.
4. The baculovirus/BV is very shear-sensitive [98].
5. Different membranes may be used. PES membranes with a
MWCO of 300 kDa have proven to be suitable for baculovirus
purification [92]. Depending on alterations in the process
(e.g., genetic modification or cell line), different materials
may be more appropriate.
6. Store the BV stocks at 4
C excluded from light, as the virus is
very susceptible to photodegradation and aggregation due to
382
Friederike Eilts et al.