Intranasal Delivery of Therapeutic Neural Stem Cells (NSCs) in Murine Model: A Procedure for Tumor-targeted Delivery of Oncolytic Virus Loaded NSCs into Brain Tumor Bearing Mouse Model

Overview

This video demonstrates the intranasal delivery of oncolytic virus loaded therapeutic neural stem cells into a brain-tumor bearing mouse model. The non-invasive intranasal administration of stem cells bypasses the blood brain barrier, facilitating their delivery to the tumor site in the brain for therapeutic applications.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Hypoxic Preconditioning of Human NSCs (Figure 1A)

1. Plate cultured NSCs in T75 culture flasks until 90% confluency is reached.
2. Place cell culture flasks inside a humidified CO₂ chamber/incubator with maintained 1% O₂ levels for 24 h, via N₂ gas supply controlled by an automated built-in flow meter. These cells are referred to as ^HNSCs. Control ^NNSCs are generated as:
   1. Maintain human NSCs (clone HB1.F3.CD) in DMEM medium with 10% FBS and 1% penicillin-streptomycin in a humidified 37 °C incubator under 95% room air and 5% CO₂.

2. Loading of Human NSCs with Oncolytic Virus (Figure 1B-1H)

1. Infect ^NNSCs, ^HNSCs with 50 conditional replicative adenovirus (CRAd)-S-pk7 viral particles/cell.
2. After 2 h of incubation at room temperature with periodic tapping, wash cells three times with media to remove unbound viral particles and suspend cells in sterile saline for injection.
   CAUTION: Carry out all virus-related in vitro cell culture procedures in facilities with the appropriate biosafety level designation, such as BSL2. Appropriate personal protective equipment must be worn by researchers handling the oncolytic virus per institutional animal facility guidelines.

3. Intranasal Delivery of Therapeutic NSCs (Figures 1D and Figure 2D)

1. Culture and collect NSCs (^NNSCs, ^HNSCs).
2. Anesthetize mice via i.p. injection of a ketamine and xylazine mixture according to the procedure:
   1. Anesthetize the animal based on body weight using the approved reagents, i.e., either via an intraperitoneal (i.p.) injection of a mixture of ketamine (50–100 mg/kg; consult institutional IACUC for approved dose range) and xylazine (5–10 mg/kg; consult institutional IACUC for approved dose range) in 200–250 µL sterile saline, or via 4–5% isoflurane anesthesia using an inhalational device.
3. Place mice on a clean drape facing up with a heating pad underneath to maintain body temperature. Adjust a padded pillow made of rolled up paper towels with tapes to ensure the upright angle of the nostrils when placed under the head of the mouse.
4. Using a micropipette, dispense 2 µL of NSC suspension to each nostril of the mouse. Visually confirm the aspiration of the droplet before moving to the next mouse. The total number of cells delivered can be determined by the user; use 62,500–125,000 cells/µL, such that 0.5–1 million cells can be delivered in 4 x 2 µL doses.
5. Use a timer to ensure 5 min intervals between each cell suspension administration, up to 4 times.
6. Allow animals to regain mobility in a recovery chamber, with the supine position maintained throughout.

Representative Results

Figure 1: A flow chart for general stem cell preparation procedures involved prior to intranasal delivery. (A–C) Hypoxic pretreatment or genetic modifications of stem cells to overexpress CXCR4. (B–D) Washing, enzymatic dissociation, cell collection, and counting steps are performed for subsequent loading of stem cells with therapeutic or diagnostic cargo. (E) Therapeutic reagents, such as oncolytic viruses, or diagnostic tracers, such as MPIO particles, can be loaded in collected stem cells in appropriate concentrations with periodic shaking and gentle agitation (F). (G–I) The loaded stem cells can then be washed and counted for intranasal delivery into mice.

Figure 2: Schematic flow chart of the general animal procedures involved. (A) Tumor models are created via xenograft of patient derived glioma cells. (B) Tumor growth is monitored via BLI. (C) Head-only irradiation therapy is delivered (D) Supine and head-tilt posture is maintained during the therapeutic intranasal stem cell delivery. (E) Post therapy survival monitoring, small animal imaging and histological analysis of tissue if necessary are followed up.

Materials

Name	Company	Catalog Number	Comments
Cell culture supplies (Plastics)	ThermoFisher Scientific	Varies	Replaceable with any source
Legend Micro 21R Refrigerated Microcentrifuge	ThermoFisher Scientific	75002490	Replaceable with any source
Bench centrifuge Sorvall ST16R	ThermoFisher Scientific	75004240	Replaceable with any source