Fluorescent Labeling of Glioma Cells: A Lentiviral Vector-based Transfection Method to Obtain Glioma Cells Expressing Fluorescent Protein for Deep Tissue Imaging

Overview

In this video, we describe a protocol for genetically engineering glioma cells to express infrared fluorescent protein or iRFP. These fluorescent-tagged glioma cells hold utility in deep tissue imaging to understand the development of brain tumors.

Protocol

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

1. Labeling of Glioblastoma Cells with FP635 or FP720 Construct

1. Produce and purify lentivirus according to the protocol described below:
   1. Observe the dishes seeded on Day 1. When cells are 80–90% confluent they are ready for transfection.
      ​NOTE: The cells should still have room to undergo 1–2 cell divisions
   2. Prepare the plasmid mix by aliquoting the four plasmids into a 50 mL tube. For a 12 × 15 cm dish preparation, use 270 µg of transfer vector, 176 µg of pMDL (Gag/Pol), 95 µg of pVSVG (vesicular stomatitis virus glycoprotein) and 68 µg of pREV.
   3. Prepare 13.5 mL of 0.25M CaCl₂ (dilute with filtered ddH₂O) and add to the plasmid mix. Add 13.5 mL 2× BBS solution. Mix gently by inverting several times and incubate 15 min at room temperature (22–26°).
   4. Add the transfection mixture (spreading in drops) to each plate (2.25 mL per plate). Swirl the plates gently and incubate at 3% CO₂, 37 °C overnight (16–20 h).
   5. Observe the cells. They should be reaching confluency. If a visible marker (such as GFP) is present in the lentivector plasmid, transfection efficiency may be assessed visually. Ideally, transfection efficiency should be >90%.
   6. Remove media, add 15 mL of fresh DMEM + 2% FBS to each dish and transfer to 10% CO₂, 37 °C. Incubate overnight.
   7. Collect first harvest of supernatant: Collect and pool supernatant (12 dishes × 15 ml/dish = 180 mL) from dishes.
   8. Add 15 mL of fresh DMEM + 2% FBS to each dish. Incubate dishes at 10% CO₂, 37 °C overnight. The vessel containing the lentiviral supernatant should be put in a secondary containment (a sealed plastic bag will do) and stored at 4 °C.
   9. Concentrate the viral preparation: Concentrate the viral particles by ultracentrifugation. Centrifuge the supernatant at 70,000 x g for 2 h at 20 °C. Conical tubes (as opposed to round bottom tubes) and swinging bucket rotors (as opposed to fixed angle rotors) are recommended (simply because this combination makes the almost invisible pellet easier to locate). We use conical tubes with a Beckman SW28 rotor (capacity for six tubes) at 19,400 r.p.m. for 2 h at 20 °C. Fill the tubes with 30 mL of supernatant. The total volume of supernatant from a 12 × 15 cm dish preparation is 360 mL; therefore, processing the full volume will require two consecutive spins. Load the tubes, do the first spin, pour out the supernatant without disturbing the pellet, reload with fresh viral supernatant and carry out the second spin.
   10. Pour off the supernatant and allow the remaining liquid to drain by resting the inverted tubes on paper towels. Siphon off remaining droplets using an aspirator in order to remove all liquid from the pellet. The pellet should be barely visible as a small translucent spot.
   11. Resuspend the viral pellets in 100 µL of 1× HBSS. Avoid frothing. Rinse the tubes with 100 µL of 1× HBSS. Pool both volumes to obtain 200 µL final volume.
   12. Transfer to a screw-cap microfuge tube, wrap in parafilm and vortex at low speed for 15–30 min. The resuspended viral preparation will look anywhere from clear to slightly milky.
   13. Clear the suspension by spinning for 10 s on a tabletop microcentrifuge. Transfer the supernatant to a fresh screw-cap microfuge tube. Make 20 µL aliquots of the supernatant. This viral supernatant is of in vitro grade quality.
2. Culture approximately 2.0 x 10⁶ cells from a human glioma cell line with DMEM plus 10% fetal bovine serum (FBS) in a 15 cm dish; or culture 2.0 x 10⁶ human glioblastoma patient-derived (GBM-PDX) spheres with DMEM/F12 1:1 medium with B27 supplement plus human recombinant epidermal growth factor (EGF; 20 ng/mL) and FGF (10 ng/mL) in a T75 flask.
3. Maintain all cells at 37 °C, 5% CO₂, and 100% relative humidity.
4. Dissociate the cells.
   1. For the glioma cells: remove the supernatant from the plates and adding 3-5 mL of trypsin 1X to the glioma cells.
   2. For the GBM spheres: collect the cells by pipetting the cells into a 15 mL tube.
      1. Spin down the GBM spheres at 200 x g for 5 min using a tabletop centrifuge at room temperature.
      2. Remove the supernatant and add 3–5 mL of cell detachment solution.
   3. Incubate all cells at 37 °C for 10–15 min.
5. Carefully dissociate the cells by pipetting up and down several times (ensure that spheres and glioma cells are completed dissociated) and spin down at 200 x g for 5 min.
6. Remove the supernatant and resuspend the cells with 5 mL of medium by pipetting up and down several times. Determine the cell viability by trypan blue exclusion.
7. Plate 1.0 x 10⁶ of target cells in a 10 cm dish with 5 mL of medium by pipetting.
8. Transduce cells with lentivirus expressing fluorescent proteins at multiplicity of infection (MOI) 5 and incubate at 37 °C.
9. After 24 h, remove the medium from the transduced cells, add 5 mL of fresh medium, and incubate for additional 48 h at 37 °C.
10. Dissociate the infected cells into a single-cell suspension using trypsin as described above. Spin down the cells at 200 x g for 5 min and resuspend in 500 µL of sorting solution (phosphate-buffered saline (PBS) with 1% FBS).
11. Pipette the cell suspension into FACS tubes. Co-stain the cells with 1 µL/mL of 4',6-diamidino-2-phenylindole (DAPI) dihydrochloride to exclude dead cells. Negative controls are required to set up the flow cytometer gates (non-transduced cells and non-transduced cells/ DAPI stained).
12. Sort the fluorescent-positive/DAPI-negative cells into sorting solution and collect the sorted cells into a 15 mL conical tube.
13. Spin down the sorted cells at 200 x g for 5 min, remove the supernatant, and resuspend in 5 mL of culture medium. Seed the sorted cells in a 10-cm dish by pipetting. Incubate at 37 °C for at least 48–72 h.
14. Expand the sorted cells by dissociating the cells as described above and plating into multiple dishes for in vivo experiments.

Materials

Name	Company	Catalog Number	Comments
DMEM/High Glucose	HyClone/GE	SH30022.1
DMEM/F12 1:1	Gibco	11320-082
FBS	HyClone/GE	SH30071.03
Accutase	Innovative cell technologies	AT-104
Trypsin	HyClone/GE	SH30236.01
B27 supplement	Gibco	17504044
Human recombinant EGF	Stemcell Technologies	2633
Human recombinant FGF	Stemcell Technologies	2634
DPBS	Corning	21-031-00
FACS tubes	Falcon	352235
DAPI	ThermoFisher Scientific	62248
p24 ELISA	Clontech	632200
Cell Sorter	Sony	SH8007
Ultra-centrifuge Optima L-80 XP	Beckman Coulter	392049
Tissue Culture 100mm Dishes	Olympus Plastics	25-202
Tissue Culture 150mm Dishes	Olympus Plastics	25-203
Tissue Culture Flasks T75	Corning	430720U
50 mL conical tubes	Corning	430290
Plasmids: lentiviral transfer vector	Verma Lab Salk Institute for Biological Studies
Lentiviral packaging vectors	pMDL pRev and pVSVG	K4975-00
Endotoxin free maxipreps	Endo-free Maxiprep Kit Qiagen	12632
1X— HBSS	Invitrogen	14025-092
15 cm tissue culture dishes
0.45 µm filter units 500 mL capacity
Conical bottom ultracentrifugation tubes	Beckman	358126
Round bottom ultracentrifugation tubes	Beckman	326819
24-well tissue culture plates
Fluorescent microscope
Multichannel pipettor
ELISA microplate reader