For virus concentration/purification, the desired product should
be found in the retentate. Virus recovery rates of 70% have been
published [71, 92, 93].
2 Materials
In the following, we describe a protocol to generate pseudotyped
baculoviruses, to produce the BV on a bioreactor scale, and to
purify it via depth filtration and a subsequent TFF. Furthermore,
we describe the transduction of human mesenchymal stem cells
with purified BV. The whole process must be performed sterile;
that is, a clean-bench system (Thermo Fisher Scientific, Waltham,
MA, USA) and an autoclave (Systec, Linden, GER) are prerequisite. The origin of all materials mentioned is exemplary.
2.1 Generation
of the Pseudotyped
Baculovirus
The generation of the pseudotyped BV is mainly based on the
BacMam-System using MAX Efficiency
® DH10-Bac™ (Thermo
Fisher Scientific, Waltham, MA, USA). We only describe the materials needed to generate the recombinant vector-encoding for the
gene of interest. The materials for the generation of recombinant
bacmid DNA and of the baculovirus stock can be found in Chap. 8.
l
Transfer cloning vector (e.g., BacMam pCMV-Dest Vector,
Thermo Fisher Scientific, Waltham, MA, USA).
l
Sequence of the gene of interest (e.g., growth factor).
l
Other sequences of genetic elements (e.g., a reporter gene like
GFP, a secretion signal, etc.).
2.2 Production
of the Baculovirus
on a Bioreactor-Scale
The BV is produced in Sf-9 (immortalized ovary cells from Spodoptera frugiperda) insect cells in a stirred tank bioreactor. All materials
needed for the bioreactor assembly, the process control, the cultivation of Sf-9 cells, the initiation of cultivation, the infection of Sf-9
cells with the baculovirus/BV, and the quantification of the BV, are
described in Chap. 8.
2.3 Downstream
Processing
of the Baculovirus
When “water” is required, this always refers to ultrapure water
(Milli-Q Academic, Merck Millipore, Burlington, MA, USA).
2.3.1 Quantification
of Baculoviruses
Quantification methods for BV are described in detail in Chap. 8.
This chapter covers the quantification of baculoviral DNA (vDNA),
more specifically, DNA extraction and subsequent quantification by
PCR, as well as the quantification of an infectious titer by an
end-point dilution assay, that is, the 50% tissue culture infection
dose (TCID 50 ), and a plaque assay. Although, in this chapter, these
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be found in the retentate. Virus recovery rates of 70% have been
published [71, 92, 93].
2 Materials
In the following, we describe a protocol to generate pseudotyped
baculoviruses, to produce the BV on a bioreactor scale, and to
purify it via depth filtration and a subsequent TFF. Furthermore,
we describe the transduction of human mesenchymal stem cells
with purified BV. The whole process must be performed sterile;
that is, a clean-bench system (Thermo Fisher Scientific, Waltham,
MA, USA) and an autoclave (Systec, Linden, GER) are prerequisite. The origin of all materials mentioned is exemplary.
2.1 Generation
of the Pseudotyped
Baculovirus
The generation of the pseudotyped BV is mainly based on the
BacMam-System using MAX Efficiency
® DH10-Bac™ (Thermo
Fisher Scientific, Waltham, MA, USA). We only describe the materials needed to generate the recombinant vector-encoding for the
gene of interest. The materials for the generation of recombinant
bacmid DNA and of the baculovirus stock can be found in Chap. 8.
l
Transfer cloning vector (e.g., BacMam pCMV-Dest Vector,
Thermo Fisher Scientific, Waltham, MA, USA).
l
Sequence of the gene of interest (e.g., growth factor).
l
Other sequences of genetic elements (e.g., a reporter gene like
GFP, a secretion signal, etc.).
2.2 Production
of the Baculovirus
on a Bioreactor-Scale
The BV is produced in Sf-9 (immortalized ovary cells from Spodoptera frugiperda) insect cells in a stirred tank bioreactor. All materials
needed for the bioreactor assembly, the process control, the cultivation of Sf-9 cells, the initiation of cultivation, the infection of Sf-9
cells with the baculovirus/BV, and the quantification of the BV, are
described in Chap. 8.
2.3 Downstream
Processing
of the Baculovirus
When “water” is required, this always refers to ultrapure water
(Milli-Q Academic, Merck Millipore, Burlington, MA, USA).
2.3.1 Quantification
of Baculoviruses
Quantification methods for BV are described in detail in Chap. 8.
This chapter covers the quantification of baculoviral DNA (vDNA),
more specifically, DNA extraction and subsequent quantification by
PCR, as well as the quantification of an infectious titer by an
end-point dilution assay, that is, the 50% tissue culture infection
dose (TCID 50 ), and a plaque assay. Although, in this chapter, these
372
Friederike Eilts et al.