Propagation and Harvesting of Oncolytic Herpes Simplex Virus in Mammalian Cells

الملخص

Source: Nguyen, H., et al. Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus. J. Vis. Exp. (2021).

This video demonstrates the propagation and harvesting of oncolytic herpes simplex virus in mammalian cell cultures, highlighting viral entry, replication, and the recovery of virus-loaded cells. It further shows methods for preserving infected cells for downstream processing.

البروتوكول

1. Oncolytic herpes simplex virus (oHSV) growth

NOTE: Ensure institutional biosafety committee approval before working with oHSV. This study was conducted under approved IBC Protocol no. 18007. Maintain BSL2 precautions: bleach all pipets, tips, tubes, and other materials that come into contact with the virus. Spray gloves with 70% isopropyl alcohol before hands leave the BSL2 cell culture hood. Always thoroughly wash hands with soap water after working with a virus.

1. On day -1, seed low-passage Vero cells in 20 T-150 cm² flasks at a density of 7-8 × 10⁶ cells/flask in regular Vero cell medium.
   NOTE: Vero medium is prepared by supplementing Dulbecco's modified eagle medium (DMEM) with 10% heat-inactivated fetal calf serum (IFCS).
2. On Day 0 (cells are 80-90% confluent), add oHSV inoculum on the Vero cells.
   1. Preparation of virus inoculum
      1. Use a multiplicity of infection (MOI) of 0.01 (MOI can vary from 0.01 to 0.1 depending on the replication capacity of the virus) for virus amplification. Use the formula below to calculate the amount of virus (mL) for 20 flasks.Amount of virus (mL) for 20 flasks = amount of virus needed (pfu)/titer of virus stock (pfu/mL).
      2. Use high-glucose Dulbecco's phosphate-buffered saline (DPBS) supplemented with 1% IFCS to prepare the virus inoculum (7 mL per T-150 cm² flask, i.e., ~140 mL for 20 flasks). Add the required amount of oHSV to 140 mL of high-glucose DPBS/1% IFCS solution, vortex for 1 min, and keep the mixture ready for addition to Vero cells.
   2. Wash the T-150 cm² flasks 2x with high-glucose DPBS supplemented with 1% IFCS (10 mL/wash). Aspirate the DPBS/1% IFCS and add 7 mL of virus inoculum/T-150 cm² flask.
   3. Gently rock the flasks for 5 min using a flask rocker for proper distribution of the inoculum over the Vero monolayer, and then incubate the flasks at 37 °C for 1.5-2 h. Make sure the incubator shelf is level.
   4. Remove the inoculum and add DMEM supplemented with 1% IFCS (25 mL/flask). Incubate the flasks for 2-4 days.
3. Check the flasks daily for 90-100% cytopathic effect (CPE) (see Figure 1).
4. Harvest the oHSV-infected Vero cells.
   1. Collect the culture supernatant (~20 mL) from each flask (leave ~5 mL in each flask) in 50 mL conical centrifuge tubes or media container.
   2. Use a cell scraper to scrape the cells from the bottom of the flasks gently.
      NOTE: The cells should quickly come off the flasks.
   3. Add ~15 mL of the culture supernatant (collected in step 1.4.1) to each flask (which brings the volume of ~20 mL in each flask, i.e., 400 mL for 20 flasks) and gently wash the bottom of the flasks a few times using a 10 mL sterile serological pipet.
      NOTE: Do not pipet vigorously. Aim to keep all cells intact.
   4. Collect the cells (+ medium) into 50 mL conical centrifuge tubes on ice (use 8 tubes to hold 400 mL of harvested cells from 20 flasks).
   5. Spin the cells at 300 g for 10 min at 4 °C and aspirate the supernatant.
   6. Add 1.25 mL (50%) of Virus Buffer (VB) and 1.25 mL (50%) of culture supernatant (collected in step 1.4.1) to each centrifuge tube and re-suspend each pellet thoroughly. Transfer the re-suspended cells from eight 50 mL centrifuge tubes to one 50 mL conical centrifuge tube.
      NOTE: Refer to the preparation of VB solution in Table 1. Filter-sterilize the VB solution using a media sterilization filter. Use 0.5 mL of VB + 0.5 mL of supernatant per T-150 cm² flask for re-suspension, i.e., for 20 flasks (20 mL), use 10 mL of VB and 10 mL of culture supernatant.
   7. Snap-freeze the re-suspended cells using dry ice/100% ethanol and store at -80 °C.

Table 1: Solution composition

Solution Composition
Preparation of virus buffer	Quantity
1 M Sodium chloride	15 mL
1 M Tris-hydrochloride	3 mL
Purified water	132 mL
	adjust pH to 6.8
Preparation of X-gal solution (~6.5 mL for one 6-well plate)
250 mM potassium ferricyanide	130 µL
250 mM potassium ferrocyanide	130 µL
1 M Magnesium chloride	13 µL
X-gal pre-dissolved (20 mg/mL) in dimethyl sulfoxide (DMSO)	162.5 µL
PBS	6064.5 µL
Preparation of Neutral Red solution for one 6-well plate
Neutral Red solution	100 µL
Methanol	1 mL
Purified water	7 mL

النتائج

Figure 1: Cytopathic effect in Vero cells after oHSV infection. Vero cells were inoculated with oHSV coding for mCherry (shown in red fluorescence) at an MOI of 0.01 and imaged (10x magnification) at 36, 48, and 72 h post-virus infection. ...

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
1.7 mL Centrifuge tubes	Sigma	CLS3620
15 mL Polypropylene centrifuge tubes	Falcon	352097
5 mL Polypropylene tubes	Falcon	352063
50 mL Polypropylene centrifuge tubes	Falcon	352098
Cell scraper	Fisher Scientific	179693
Dimethyl sulfoxide	Sigma	D2650-100ML
Dulbecco’s Modified Eagle Medium	Corning	MT-10-013-CV
Dulbecco’s Phosphate Buffered Saline	Corning	MT-21-031-CV
Fetal Bovine Serum	Hyclone	SH3007003
High-Glucose Dulbecco’s Phosphate-buffered Saline	Sigma	D4031
Plate rocker	Fisher	88861043
Sorvall ST 16R Centrifuge	ThermoFisher Scientific	75004381
Sorvall ST 21R Centrifuge	ThermoFisher Scientific	75002446
Sterile Microcentrifuge Tubes with Screw Caps	Fisher Scientific	02-681-371
Sucrose	Fisher Scientific	BP220-1
T150 culture flask	Falcon	355001