Page 93 - IMO-2-1

Innovative Medicines & Omics Economical method for titering viral vectors

Table 1. Transfection reagents for lentiviral vector (iii) The tank was filled with 7 L of medium collected from
production. (A) tube 1, and (B) tube 2 the transfected 293T cells. Once the medium was
filtered, additional medium was added until 100 mL
(A) Tube 1 remained in the tank, after which the remaining
Transfection reagents Amount/volume in CF2 Size (kb) medium was collected
pRRL.sin.cPPT.LSP. 600 µg 8.512 (iv) 100 mL of OptiMEM 1 was added, and filtration was
IRES.mVenus.WPRE continued for another 15 min. This step was repeated
pMDLg/pRRE 600 µg 8.89 twice
pRSV/REV 320 µg 4.174 (v) The filtered medium was centrifuged at 50,000× g for
pMD2/VSV.G 400 µg 5.924 2 h at 4°C
2M CaCl 2 1.2 mL - (vi) The virus pellet was resuspended in 4 – 6 mL of
dH O To – 8 mL - OptiMEM1 medium, and the filtered viral supernatant
2
was stored at −80°C
Total 8 mL - (vii) The TFF apparatus was cleaned with 1 L of 0.5 M
(B) Tube 2 sodium hydroxide for 30 min, and then the process
Reagents Volume in CF2 was repeated once. Next, the apparatus was washed
1M HEPES 0.4 mL with 2 L of 0.1 M sodium hydroxide for 1 h, followed
2M NaCl 1 mL by two washes (1 h each) with 2 L of PBS each time.
150mM Na HPO 4 120 µL Finally, the TFF was run.
2
dH O 6.508 mL
2 2.3. Methods for titering the lentiviral vector
Total 8 mL
Abbreviations: CaCl : Calcium chloride; CF2: CF2 2.3.1. qPCR lentivirus titer kit method
2
CellSTACK 2 chambers; dH O: Distilled water; CF2: CF2 This methodology took approximately 2.5 h to complete,
2
CellSTACK 2 chambers; dH O: Distilled water; at a cost of Australian dollars (AUD) $1,958.00 per 96-well
2
HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; plate. The cost included the kit, quantitative standards, and
NaCl: Sodium chloride; Na HPO : Disodium phosphate.
2 4 associated freight charges (both international and local).
allow complete precipitation. The mixture was then Following the manufacturer’s instructions for titering
added to OptiMEM 1 medium without FCS; then it lentiviral vectors (ABM kit; LV900; Canada), the supplied
9
8
6
7
was transferred onto the 293T cells. The cells were standards were diluted at 10 , 10 , 10 , and 10 copies/mL
incubated in a humidified chamber at 37°C with for qPCR analysis and the production of a quantitative
5% carbon dioxide for 4 h. After incubation, the standard curve. Lentiviral vectors (1 µL) were diluted
OptiMEM 1 medium (without FCS) was replaced 1:1,000. Viral vector samples that produced large pellets
with fresh OptiMEM 1 medium, and the cells were after centrifugation were diluted 10,000 times. Quantitative
then incubated overnight at 37°C with 5% carbon analysis was performed when the R value of the standard
2
dioxide curve was >0.99, with an R of 0.998 being typical for the
2
(iii) Day 3 – 5 – Virus harvesting: The virus-containing standard curve.
medium was harvested on days 3 – 5, replenished
with fresh OptiMEM 1 medium (without FCS) after 2.3.2. TRIzol method
each harvest. The harvested medium may be stored The following methodology required 1 h for RNA isolation,
at −80°C until ready for filtration through 0.45 µm 2 h 30 min for qPCR, at a cost of AUD$115.20 per 96-well
Nalgene filters to remove cell debris. plate. The TRIzol reagent was priced at $AUD296.00, and
the primers cost approximately AUD$30.00. The total
2.2.1. Vector concentration and purification cost was approximately AUD$300.00 for 500 samples
The TFF system was used for filtration and concentration or $AUD58.00 for 96 samples. This was approximately
of the vector. 34 times less costly than using the ABM kit, which also
(i) Before starting the vector concentration, the flow rate measures 96 samples. The method is illustrated graphically
of the TFF apparatus was ensured to be between 700 in Figure 1.
and 900 mL/min i. The vector (1 µL) was diluted 1:1000 with PBS and
(ii) The TFF was washed with 1,000 mL phosphate- mixed. Depending on the size of the pellet, the vector
buffered saline (PBS), followed by 500 mL OptiMEM may have needed to be diluted between 1:100 and
1 medium 1:10,000

Volume 2 Issue 1 (2025) 87 doi: 10.36922/imo.6552