The procedure consists of a pre-operative step (Step 1-7), thymectomy (Step 8-15), splenectomy (Step 16-21), gastrostomy (Step 22-36), and treatment with immunosuppressants (Step 37-38). Additionally, in vitro PBMC proliferation (Step 39-61) and pharmacokinetic assessments (Step 62-79) are performed.
(A) Surgical operations
Pre-operative STEP ●Timing 45 min
▲CRITICAL STEP It is important to perform splenectomy and gastrostomy after fasting for more than 24 hours.
1. Sedate the pig with midazolam (0.3 mg/kg, i.m.), and atropine (0.04 mg/kg, i.m.) and wait until the pig is completely calm. This sedation can be given before transfer to the operating room.
2. Administer buprenorphine (0.01~0.02 mg/kg, i.m.) for 30 min before the start of surgery
3. In the surgical preparation room, shave the puncture site under inhaled isoflurane with 2 liter/min oxygen via a face mask. (3~5%).
4. Insert a 22-gauge angiocatheter into posterior auricular vein to access i.v. and place a secure airway.
▲CRITICAL STEP Endotracheal intubation with a 6.5- to 7.5-mm tube is preferred for airway protection.
5. After shaving, move to the operating room, place in the supine position.
6. Deliver ongoing maintenance anesthesia with 1–2% isoflurane through a nose cone and mechanically ventilated closed-loop anesthesia machine.
▲CRITICAL STEP Pulse oximetry, body temperature and heart rate must be monitored through anesthesia induction, maintenance and recovery.
7. Disinfect the operation area.
Thymectomy ●Timing 20-30 min
8. Use sterile surgical instruments
9. Perform incision of the skin on the upper manubrium of the sternum from near the midline neck hyoid bone (approximately 7 cm)
10. Perform dissection of subcutaneous tissue while avoiding the carotid artery and vagus nerve on both sides of the sternohyoid muscle
11. Pinch the ends of the thymus on both sides of the sternohyoid muscle with forceps, and when dissecting the thymus and subcutaneous tissue, perform dissection to the back of the manubrium of the sternum
12. Pass the right cervical lobe under the anterior neck muscles to enable traction to be applied on both the left and right cervical lobes simultaneously
13. Traction is then applied to pull the left and right cervical lobes to the cranial side
14. The thoracic lobe in the anterior mediastinum is carefully dissected and removed
15. After thymectomy, suture the subcutaneous tissue and skin
Splenectomy ●Timing 20-30 min
16. Perform a midline incision from the region proximal to the xiphoid process to the lower abdomen
17. Perform incision to the muscle layer using a scalpel, and perform incision of the peritoneum using an electric scalpel
18. After performing peritoneal incision, secure field of view of the perisplenic region by using a retractor
19. Lift the spleen to a position where the three blood vessels (splenic body omental vessels, short gastric vessels, and the splenic vessels) of the spleen are visible
▲CRITICAL STEP When lifting the spleen, dissect the renal fascia in advance, and after dissection, by putting gauze in the space created, the three blood vessels in the spleen are easily visible
20. Perform ligation at one place of the splenic artery and short gastric artery on the splenic side and two places on the abdominal side. Subsequently, resect the section between the ligation points
▲CRITICAL STEP Dissect the part away from the ligation position to prevent hemorrhage
21. After performing vessel dissection, remove the spleen
Gastrostomy ●Timing 20-30 min
22. Perform gastric fistula tube attachment after splenectomy
23. Prepare an intravenous tube with about 1/3 of the drip chamber remaining (Fig 2a)
24. Secure a field of view of the region surrounding the short stomach artery ligature of the stomach
25. Put a thread around the planned incision site on the upper part on the side of the greater curvature of the stomach (for performing purse‐string suturing in step 27)
26. Using an electric scalpel, make a 1 to 1.5 cm incision required for gastrostomy tube insertion
▲CRITICAL STEP The surgical assistant lifts the both sides of the incision with tweezers, etc. so that the gastric contents do not leak when the stomach is incised.
? TROUBLESHOOTING In comparison with humans and dogs, pigs may have delayed gastric emptying, so the gastric contents may remain in the stomach. On the day before surgery, give easily digestible feed such as liquid feed
27. After inserting the drip chamber side of the processed gastrostomy tube into the stomach, perform purse‐string suture to prevent the tube from coming out of the stomach (Fig. 2b)
28. Fasten tube to stomach by Witzel sutures (Fig 2c)
29. The tip of the gastrostomy tube is pulled out from the region surrounding the costal arch
30. Join the fixed parts of the stomach tube with the abdominal wall at 4 to 5 places by 3-0 nylon thread
31. In order to perform fixation of the gastric fistula tube, fix the opening of the subcutaneous tissue of the back with 4-0 nylon thread (Fig. 2d)
32. Fill the tube with PG water jelly to prevent backflow of the gastric contents and close the tip with an injection cap or a screw-clamp compressor (Fig.2e)
33. Place a jacket on the pig and store the gastrostomy tube in the pocket
34. Postoperatively, further administer additional doses of Buprenorphine and meloxicam (0.2 mg/kg, s.c.) and continue observation until emergence from anesthesia. Return the pig to the breeding cage after emergence from anesthesia
35. Give analgesic at the same dose for 3 days, including the day of surgery. Twice daily in the case of buprenorphine, and once daily in the case of meloxicam.
36. Postoperatively, disinfect the gastrostomy subcutaneous opening every two to three days
Administration of immunosuppressants
37. Dissolve tacrolimus tablets in hot water at 50 to 60°C to create a 25 mg/mL solution. Prepare Prednisolone as a 5 mg/mL solution in the same manner. CELLCEPT powder for oral suspension in 31.8% (Chugai pharmaceutical Co.,Ltd.) was used as mycophenolate mofetil (MMF). Adjust the administration volume according to the administration dose. Table 1 shows the immunosuppressant recipe implemented at each facility.
38. Perform immunosuppressant administration as follows
① Push the jelly filled in the tube into the stomach using 30 mL of heated water
② Immunosuppressant administration
③ Push the immunosuppressant remaining in the tube into the stomach using 30 mL of heated water
④ Fill the gastrostomy tube with 30 mL of PG water
▲CRITICAL STEP Perform daily feeding and administration at a uniform time, as changes in immunosuppressant serum concentration levels differ between fasting and non-fasting states
? TROUBLESHOOTING The problems and solutions during immunosuppressant administration are summarized in Table 2.
(B) In vitro and pharmacokinetics studies
PBMC proliferation studies
FBS The serum is thawed and heat-inactivated for 30 min at 56 ℃. The heat-inactivated serum is stored in 50-mL aliquots at -20 ℃.
RPMI-1640 (Culture medium) For culturing PBMC derived from MMP peripheral blood, RPMI-1640 is supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin-streptomycin. The medium is stored at 4 ℃ and warmed to at least room temperature before use.
PHA stock solution PHA powder is dissolved in PBS to a final concentration of 1 mg/mL. To make 1 mg/mL of stock solution, 10 mg of the powder is suspended in 10 mL of PBS. This solution can be stored in suitably sized aliquots at -20℃ for several month.
Cell Titer Glo Reagent The 100 mL CellTiter-Glo buffer is transferred into the amber bottle containing CellTiter-Glo substrate to reconstitute the lyophilized enzyme/substrate mixture. Mix by gently vortexing. This reagent can be stored in suitably sized aliquots at -20℃ for several month.
Before conducting the PBMC proliferation study, we optimized the assay system as below. Cell proliferation depends on cell density, stimulant concentration and incubation time. In order to determine the optimal assay condition for evaluating the immunosuppressant, we investigated these factors. We examined the following three conditions; (1) cell density: 1 × 104 cells - 2 × 105 cells / well, (2) stimulant (PHA): 0.1 – 10 μg/mL, (3) incubation time: 48 - 96 h. As a result, the sub-maximum proliferative activity was observed under the conditions of cell density of 5 × 104 cells / well in 96 well round bottom plate with 3 μg/mL PHA after 72 h incubation.
(A) Isolation of PBMC ● TIMING 1.5 h
39. Collect approximately 7 mL of blood with a sodium heparinized 10-ml syringe from male micro mini-pig.
40. Transfer 5 mL of fresh blood into 15-mL conical tubes containing 5 mL of PBS and gently mix by drawing the blood and buffer in and out of a pipette.
41. Aliquot 5 mL of Ficoll-Paque PLUS into two 15-mL conical tubes (one Ficoll tube for every 5 mL of diluted blood).
42. Place the tip of the pipette against inside of the tube, carefully layer 5 mL of diluted blood onto the 5 mL of Ficoll-Paque PLUS already filled.
43. Centrifuge the tubes at 400 g for 30 min at room temperature.
44. Using a clean pipette, carefully collect the white layer of PBMC between plasma and Ficoll-Paque PLUS and transfer the cells to a clean 50 mL centrifuge tube containing 20 mL of RPMI-1640.
45. After gentle pipetting, centrifuge the tubes at 400g for 10 min at room temperature and discard the supernatant.
46. Resuspend the cells thoroughly in 20 mL RPMI-1640 by pipetting.
47. Count cells using a 1: 2 dilution with trypan blue. We typically see 20 - 50 × 106 PBMCs isolated from 5 mL of blood.
■ PAUSE POINT If needed, PBMCs can be stocked frozen by using CELLBANKER 1 plus. Although the survival rate decreases to 50-70% by cryopreservation of cells, the reactivity to PHA does not change. When using, dissolve promptly in a 37℃ hot bath, wash with culture medium and count the number of live cells before use.
(B) Plating and proliferation assay
Preparation and plating for in vitro simulation takes 1.5 h. Cells are usually harvested 3 d after the stimulation.
48. Centrifuge the tubes at 1,400 rpm for 5 min at room temperature.
49. Resuspend cells in culture medium at a final concentration of 5 × 105 cells per mL.
50. Transfer 100 μL (5 × 104 cells) per well of cell suspension to 96 well round-bottom plate.
51. Add 60 μL of culture media and 20 μL of 10-fold concentration of MPA to the cells.
▲CRITICAL STEP To make 10-fold concentration of MPA serial dilution for dose-response curve, dilute MPA solubilized in MeOH serially with MeOH and further dilute 100 times with culture media. As a result, the carry-in of MEOH into the culture system is 0.1%.
52. Pre-incubate the cells for 30 min at 37℃.
53. Add 20 μL of 30 μg/mL PHA to the cells at a final concentration of 3 μg/mL.
54. Incubate the cells for 3 days at 37℃.
▲CRITICAL STEP To reduce the edge effect in culture plate, avoid using the outer wells of the plate for PBMC cultures, as these are most subject to evaporation and temperature distribution over period of incubation. The outer wells can be filled with PBS.
(C) Cell viability assay ● TIMING 1 h
55. Equilibrate the plate and its contents at RT for 30 min.
56. Remove 100 μL of culture media from each well.
57. Add 100 μL of CellTiter-Glo Reagent.
58. Mix contents on an orbital shaker to induce cell lysis.
59. Allow the plate to incubate for 10 min at RT to stabilize luminescent signal.
60. Transfer 100 μL of cell lysate to opaque-walled multiwell plates.
61. Measure luminescence by micro plate reader.
(A) Central venous catheter (CVC) placement ●TIMING 1h
62. Perform catheterization surgery one week before pharmacokinetics study in male micro mini-pig
63. The preoperative procedure is performed in the same manner as steps 1 to 4
64. Incise the skin and subcutaneous muscular layer 2-3 cm from the cervical ventral midline approximately 3 -4 cm to the head side along the body axis from approximately 2 cm from the anterior border of scapula to the sternum
65. Dissect the internal jugular vein using artery Forceps
66. After dissection, ligate the blood vessels on the head side and thread catheter fixation thread in chest side blood vessel
67. Insert CVC 5-8 cm into the vessel and perform fixation with a thread so that the catheter does not move. In addition, perform fixation of the muscular layer of the neck
▲CRITICAL STEP Adjust the insertion depth of the catheter based on the size of the animal.
68. After confirming that the blood can be collected with a catheter, inject 2 mL of heparinized saline from the end of the catheter and attach an injection cap
69. Suture the muscular layer, subcutaneous tissue and the incision in the skin
70. Attach the clip that came with kit at approximately 1 cm from the incision to the catheter that came out of the body through the incision on the skin, and perform fixation of the clip by suturing on the skin
71. Store in the pocket of the jacket from the clip fixation part to the end part
(B) Treatment with mycophenolate mofetil
● TIMING It takes 30 min to prepare the MMF solution and administer the drug.
72. CELLCEPT powder for oral suspension in 31.8% (Chugai pharmaceutical Co.,Ltd.) was used as mycophenolate mofetil (MMF). Alternatively, it is possible to use CELLCEPT Capsule 250 (Chugai pharmaceutical co.,Ltd)
73. The suspension is administered via gastrostomy.
74. The dose of MMF was 20 mg/kg in the morning, 30 mg/kg afternoon.
75. Administration in the morning is carried out after 15minutes after feeding. !CAUTION It is necessary to adjust of feeding time because PK profile would be change due to the fed/fast condition.
(C) Blood sampling and measurement of plasma mycophenolic acid concentration
● TIMING It takes 15min to collect the blood and prepare the plasma at each point.
76. The pharmacokinetic studies of the MMF were carried out 7 days after the start of administration.
77. Approximately 0.5 mL of blood was collected with a sodium heparinized syringe from CV at each sampling point (before dosing, 0.5, 1, 2, 4, 8, 9, 10, 12 and 24h after dosing.) !CAUTION Blood sampling at 8h. was performed just before afternoon treatment
78. The blood samples were centrifuged at 1,200 x G for 5 min at 25℃. Isolated plasma was stored at -20 ~ -30℃ until analysis.
■PAUSE POINT Plasma sample can be stored at -30℃ for several months
79. Plasma concentration of mycophenolic acid was measured by LC/MS/MS method