Imaging Transgenic Beta-Cells in an Immunodeficient Mouse Challenged with Diabetic Splenocytes

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

1. Engineering and maintenance of NIT-1 cell lines

1. Maintain NIT-1 cell lines, which share a genetic background with non-obese diabetic (NOD) mice, and 293FT cells in tissue culture-treated dishes in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/L glucose supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a 37 °C incubator with 5% CO₂.
2. Grow the 293FT cells to 70%-80% confluence for transfection.
3. Per 10 cm dish of 293FT cells, combine 10 µg of pLenti-luciferase-blast (see Supplemental File 1), which expresses the firefly luciferase transgene (luc2) under the control of the constitutively active EF1α promotor, 2 µg each of the packaging plasmids pMD2.G, pMDLg/pRRE, and pRSV-Rev, 4 µg of the envelope protein plasmid pCMV-VSV-G, and 60 µg of linear polyethylenimine (PEI) in 1 mL of serum-free DMEM. Adjust the amounts based on the number of cells to be transfected.
4. Leave the DNA, PEI, and DMEM at room temperature (RT) for 20 min, and then add them to the 293FT cells.
5. Collect the medium containing the lentiviral particles 48 h post-transfection.
6. Transduce the NIT-1 cells at ~80% confluency with 1 mL of medium containing lentiviral particles per 10⁶ cells for 48-72 h.
7. Select the luciferase-expressing cells with 5 µg/mL blasticidin for 48 h.
8. Further, genetically modify the luciferase-expressing cells to suit the experimental question. Include a luciferase-expressing wild-type or non-targeting control cell line for comparison.       
   NOTE: Examples of target gene modifications include clustered regularly interspaced short palindromic repeats (CRISPR) knockout, CRISPRa/i, shRNA knockdown, and overexpression.

2. Preparation of NIT-1 cells for transplant

1. Grow the luciferase-expressing cells until they are 80%-90% confluent.
2. Remove the growth medium and wash the cells with phosphate-buffered saline (PBS, 5 mL for a 10 cm dish).
3. Add 1 mL of 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) to each dish for 2 min.
4. Neutralize the trypsin with 5 mL of growth medium.
5. Use a pipette to wash the cells off the dish and transfer them to a conical tube.
   NOTE: Cells from multiple dishes of the same cell line can be combined.
6. Count the cells using a stain such as trypan blue manually with a hemocytometer or automatically with an automatic cell counter.
7. Centrifuge at 250 × g for 5 min at RT and remove the supernatant with an aspirating pipette.
8. Resuspend the cell pellet in PBS at a concentration of 5 × 10⁷ cells/mL (10⁷ cells per cell line per mouse will be transplanted in a volume of 200 µL).
9. Keep the cells on ice (for up to 3 h) until transplantation.

3. Transplantation of NIT-1 cells into nonobese diabetic-severe combined immunodeficiency (NOD-scid) mice

1. Anesthetize the recipient mice (8-10-week-old NOD-scid mice of the same sex as the mice used to isolate the splenocytes) by isoflurane inhalation in a knockdown chamber. Deliver 2.5% isoflurane into the chamber at a flow rate of 1.5 L/min and an evacuation rate of 9 L/min. 
2. Lubricate the animal's eyes with ophthalmic ointment to prevent drying.
3. Using an electric shaver with a guard, remove the hair from the back (dorsal side) of the mice to expose the skin. The shaved transplant area will be approximately 1 in x 2 in. Return the shaved mice to the knockdown chamber until the transplant.
4. Transfer the mice one at a time to a clean surface with an isoflurane nose cone. Gently guide the mouse's head into the nose cone. Use an isoflurane flow rate of 0.25 L/min to maintain the anesthesia during the transplant.
5. Wipe the transplant area with an isopropyl alcohol prep pad to remove loose hair and disinfect the skin.
6. Ensure the cells are fully resuspended before loading the syringe. Draw an excess volume (>300 µL) of cells into a 1 mL sterile syringe with a 26 G needle. Remove any bubbles; then return excess cells to the tube so that 300 µL of cell suspension remains in the syringe.
7. Using a pair of curved forceps held in the non-dominant hand, gently lift the skin on one side (left or right) of the mouse's back to allow for easier access to the subcutaneous space.
8. Using the dominant hand, position the syringe parallel to the coronal and sagittal planes of the mouse's body. With the needle pointed toward the head of the mouse, insert the needle into the skin near the hindquarters and gently guide it into the subcutaneous space. Ensure that the entire needle remains under the skin and does not poke through.
9. Adjust the forceps to gently hold the skin around the base of the needle. Slowly dispense a small amount of cell suspension (<50 µL) and confirm that a small bulge forms under the skin at the site of the injection. Continue injecting the cell suspension until 200 µL has been delivered to the subcutaneous space.
10. Holding the forceps in place and keeping the needle parallel to the mouse's body, slowly withdraw the needle. After the needle has been removed, hold the skin closed with forceps for a few seconds to prevent the cells from leaking out of the puncture wound.
11. If a genetic modification is being evaluated, repeat the transplant on the opposite side of the mouse using a different syringe so that both mutant and control cells are injected into each mouse, with one cell line on the left and the other on the right. If only one cell line is being transplanted, inject the cells on one side only.
12. Following the transplant, transfer each mouse to a fresh cage. Allow each mouse to fully recover from the anesthesia before adding additional mice to the cage.
    NOTE: Mice are recovered when they resume normal activities and do not show signs of lethargy or impaired movement.
13. Evaluate the health status of the mice daily for the duration of the experiment following the transplant. If the grafts form a large bulge or the mice become weak and lethargic, measure their blood glucose and provide 10% (w/w) sucrose water ad libitum to all the hypoglycemic mice. Follow all veterinary recommendations and euthanize any mice with continued poor body condition as per institutional guidelines. In this study, mice were euthanized by CO₂ inhalation followed by cervical dislocation.

4. Isolation and purification of autoreactive splenocytes

1. Identify 10-16-week-old male or female NOD mice with recent-onset (less than 10 days) diabetes by using urine (or blood) glucose test strips. NOD mice are considered diabetic when they have urine/blood glucose ≥250 mg/dL on at least 2 consecutive days.       
   NOTE: For each NOD-scid mouse, 10 million splenocytes are needed; one spleen typically yields 50 million to 150 million splenocytes. The splenocytes were isolated from pathogen-free mice housed in a sentinal-monitored facility.
2. Euthanize the appropriate number of diabetic NOD mice.
   NOTE: In this study, the mice were euthanized by CO₂ inhalation followed by cervical dislocation to ensure death.
3. With the mouse on its back, make a vertical incision approximately 2 in in length in the skin with surgical scissors. Open the right side of the mouse from the researcher's point of view and locate the bright red spleen. Gently cut the spleen away from the pink pancreas and transfer it to a sterile 10 cm Petri dish containing 5 mL of sterile PBS. Repeat the dissection with as many mice as needed.
   NOTE: Spleens from multiple mice can be combined in one dish. Conduct the following steps using sterile reagents and equipment in a sterile hood.
4. Mash the spleen(s) with the flat top of a sterile syringe plunger.
5. Place a 40 µm or 70 µm strainer in a 50 mL conical tube and wash the strainer with 5 mL of PBS to prime it. Transfer the spleen(s) suspension into the strainer and continue gentle mashing through the strainer. Wash the dish with 10 mL of PBS and transfer the wash to the strainer. Continue mashing until the red color is gone from the strainer.
6. Discard the strainer and spin the tube at 500 × g for 5 min at RT. Remove the supernatant with an aspirating pipette.
7. Resuspend the cell pellet in 5 mL (for up to three spleens) of ACK lysis buffer prewarmed to RT and lyse the red blood cells for 4 min. Increase the volume by 1-2 mL per spleen if additional spleens are needed.
8. Stop the reaction with 5 mL of NIT-1 cell media (described above) per 5 mL of lysis buffer. Pass the cell suspension through a fresh strainer to remove clumps. Discard the strainer, and spin at 500 × g for 5 min at RT.
9. Resuspend the cell pellet in 20 mL of PBS, and count the cells. Spin at 500 × g for 5 min at RT. Resuspend the cells in sterile PBS at a concentration of 1 × 10⁸ cells/mL (the injection volume is 0.1 mL/mouse) in a 1.5 mL safe-lock reaction tube.
10. Store the cells on ice (for not more than 1 h) or keep the cells at RT, and proceed to the tail vein injection immediately.

5. Intravenous injection of diabetic splenocytes via the lateral tail vein

1. Warm up the body of the adult recipient NOD-scid mice (e.g., by using a heat lamp for ~5-10 min) to vasodilate the veins (this helps for visualization and injection into the vein).
   NOTE: To avoid the overheating of the mice, do not exceed this time. Always monitor the health status. If the mice appear exhausted or immobile, shut off the heat lamp immediately.
2. Warm up the cell suspension. Prepare a sterile syringe (0.3-1.0 mL) with a sterile needle (27-30 G, 0.3 mm/0.5 in or smaller), or use a sterile 0.5 mL insulin syringe with a 0.3 mm (0.5 in) needle. Always keep the needle sterile and use a sterile tray if the syringe must be put down between injections.
3. Resuspend the splenocyte solution prior to each injection. For each mouse, draw 100 µL of the prewarmed and mixed splenocyte suspension into the syringe. Make sure that no air bubbles are present in the syringe or in the suspension.
   NOTE: Make sure to have all the equipment and supplies prepared (alcohol wipes for disinfection, a syringe loaded with the splenocytes to be injected, and a fresh cage for separating injected mice) before placing the mouse in the restraint device.
4. Place the mouse in the restraint device. Capture the tail with the non-dominant hand and locate one of the two lateral tail veins. Gently rotate the tail if needed. Wipe the tail with a disinfection wipe (70% isopropyl alcohol) to clean the skin and increase the visibility of the vein.
5. Insert the needle at an acute angle into the central region of the tail with the dominant hand. With the bevel of the needle facing up, slide the needle a few millimeters through the skin.       
   NOTE: Make sure the needle is parallel to the vein and placed just slightly under the skin. Be prepared for sudden movements of the mouse/tail directly after penetrating the skin/vessel wall. A successful insertion of the needle should feel like a "smooth slide" into the vein. If another attempt is needed, move further up the tail toward the body.
6. Apply gentle pressure to the syringe to inject the splenocyte suspension. Do not allow the needle to move further in or out when injecting. Successful injections are smooth without feeling any back pressure during injection and are indicated by a transparent/white blood flow immediately following injection.
7. Gently release the needle out of the vein and apply light pressure to the tail with a disinfection wipe until the bleeding stops. Release the mouse from the restraint device, and gently transfer it to a freshly prepared cage.  
   NOTE: Inject two to three control mice that do not receive a NIT-1 cell transplant with splenocytes to confirm the potential of the autoreactive splenocytes to induce diabetes, which takes approximately 2-4 weeks post injection.

6. In vivo bioluminescent imaging of NIT-1 grafts

NOTE: Image the grafts one to two times per week. On the day of the transplant, wait at least 2 h after the transplant to allow the grafts to settle and ensure stable luciferase expression. If time is a limiting factor, the initial measurement may be taken on Day 1 instead. A recommended initial imaging schedule is Day 0 or Day 1 post injection, Day 5, Day 10, Day 14, Day 18, and Day 25. Adjust the schedule, however, based on the progress of autoimmunity as judged by the loss of bioluminescent signal.

1. Prepare a 15 mg/mL D-luciferin solution in Dulbecco's PBS. Agitate at RT to dissolve, and sterile filter (0.22 µm). Store 1 mL aliquots at −20 °C, and thaw as needed.
   NOTE: The aliquots can be refrozen.
2. At least 5 min prior to imaging, inject the mice intraperitoneally using a 1 mL syringe and a 26 G needle with D-luciferin solution at a dose of 150 mg/kg. 
   NOTE: Use a fresh syringe and needle for each mouse. 
3. Anesthetize the mice by isoflurane inhalation according to institutional guidelines. The recommended conditions are 2.5% isoflurane with a flow rate of 1.5 L/min into the knockdown chamber and an evacuation rate of 9 L/min.
4. Lubricate the animal's eyes with ophthalmic ointment to prevent drying.
5. Open the software associated with the imaging instrument. Create a new user and/or login. On the control panel on the bottom right, select Initialize (Figure 1A). To auto-save the imaging data, create the appropriate folders on the computer, and then select Acquisition | Auto-save to… (Figure 1A). Once the instrument has finished initializing, set the exposure time to 1 min.    
   NOTE: The complete imaging parameters are shown in Figure 1B.
6. Transfer the mice one at a time from the knockdown chamber to the imaging instrument. Place the mouse on its stomach with its limbs splayed, and gently guide its head into the nose cone. An isoflurane flow rate of 0.25 L/min delivered via a nose cone is appropriate to maintain anesthesia during imaging. Gently flatten the mouse by pressing the center of its back with both hands and then spreading the hands outward and apart.
7. Select Acquire (Figure 1B). Record any relevant details of the experiment in the pop-up window (Figure 1C).      
   NOTE: See Figure 2A for representative images of three 8-week-old female NOD-scid mice transplanted with two grafts at various time points.