Surgically produced, controllable immunocompromised pigs CURRENT STATUS: POSTED

Majority of research on human regenerative medicine has focused on the use of human mature cells/tissues and stem cell derived products. To ensure their safety and efficacy for clinical application, preclinical testing in large animals such as pigs, using pre-clinical and clinical matching protocols, is required. However, presently, there is no universal, stable, adjustable immunosuppressed pig model in which human regenerative cell and tissue products may be evaluated. Consequently, we established a controllable immunocompromised pig model by surgical excision of immune organs and varying the administration of immunosuppressive agents based on the physical condition and pharmacokinetic profile. Here, we describe the precise experimental procedure and provide information on immunosuppression therapies in different breeds of laboratory pigs. We provide practical information for the production of operational SCID pigs. We believe that our procedure may be immediately implemented in human-to-pig experiments, resulting in cost-benefit perspectives and furthering the development of new regenerative medicine.


Introduction
In recent years, a variety of human cells and tissues have been created from human-derived embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), and efforts are underway to utilize them in regenerative medicine. Trilineage differentiation tests of these stem cells and safety tests of human cells induced to differentiate from these cells are conducted in immunocompromised mice. However, the efficacy of the human-derived regenerative medical products thus obtained should ideally be investigated by preclinical tests in large animals such as pigs.
From this perspective, attempts have been made worldwide to create pigs with severe combined immunodeficiency (SCID pigs). However, these animals can only be reared in an extremely strictly controlled environment 1,2 . For the same reason, numerous experiments have been carried out in which human-derived cells have been transplanted into pigs and maintained by means of immunosuppressant administration, but there are concerns that pigs of different ages and breeds may respond differently 3 , and a stable, adjustable immunosuppression protocol that can be widely used has yet to be developed. We have surgically removed the main immunoregulatory organs from large numbers of laboratory pigs to induce a state of severe immunodeficiency, enabling their longterm acceptance of human cells, and reported the results in a previous paper 4 .
In this paper, we give a step-by-step description of the experimental procedure that is the core element of this technique, including key tips for its success. We provide precise information on immunosuppression therapies for different breeds of laboratory pig. This information will be of use to many researchers whose aim is to demonstrate the safety and efficacy of human regenerative medical products, and we hope that it will be immediately implemented in animal experiments from the animal protection and cost-benefit perspectives.

A: Thymectomy
The adult porcine thymus consists of the left and right cervical lobes and the thoracic lobe, which occupies the entire mediastinum. Total thymectomy can be performed comparatively simply soon after birth in newborn piglets by making an incision to a point close to the sternum and pulling out the thymus 7 . However, in adult pigs the following procedure is used to perform total thymectomy without disarticulating the sternum. Standing on the left of the animal's head, the operator frees the left and right cervical lobes of the thymus from the cranial side and passes the right cervical lobe under the anterior neck muscles to enable traction to be applied on both the left and right cervical lobes simultaneously (Supplementary Information, Video 1). Traction is then applied to pull the left and right cervical lobes to the cranial side, and the thoracic lobe in the anterior mediastinum is carefully dissected and removed (Fig. 1a).

B: Splenectomy
The spleen contains large quantities of B cells involved in antibody production, and in pigs, in particular its volume is extremely large. Splenectomy in pigs is the subject of particular attention for 4 liver regeneration 8 that changes perfusion in the portal region, and for the production of diabetic model animals 9 . In splenectomy in pigs, it is important that the splenic body omental vessels, short gastric vessels, and the splenic vessels, all of which are large, are carefully ligated (Fig. 1b).

C: Gastrostomy formation
When producing long-term immunosuppression, tube insertion in the stomach can be a source of infection, and great care and attention are required. Because pigs are quadrupeds, it is important that the gastrostomy be inserted at a different site from the greater curvature of the central gastric corpus or the anterior surface, the locations used in humans. It must be inserted high up, near the cardiac part of the stomach, to take account of the animal's position standing on four legs. To avoid the creation of dead space, a Witzel tube is worked through the serosa (Fig. 1c) and then attached directly to the abdominal wall, the tube is brought out in the back via a subcutaneous tunnel, and a drug injection port is attached.

Immunosuppressant administration to laboratory pigs
Porcine models have long been used for the preclinical validation of organ transplants, and the effective concentrations of immunosuppressants are known to differ greatly from those in humans.
The IC 50 values for cyclosporine, tacrolimus, and other calcineurin inhibitors in in vitro tests of peripheral blood lymphocyte stimulation with phytohemagglutinin (PHA) are 13-19-fold higher, and even for mycophenolate mofetil (MMF) the concentration is 1.5-fold higher. For methylprednisone, however, the value is 0.4-fold, indicating that unlike other laboratory animals, pigs respond well to steroids 10 . An immunosuppression protocol based on the oral administration of tacrolimus and MMF 11 and a steroid tapering method 12 has been reported for use in porcine allogeneic organ transplants, that is, transplanting an organ from one pig into another.
However, not enough is known about the xenogeneic transplantation of human-derived cells or tissues into pigs. Numerous xenogeneic transplantations of porcine organs and tissues into monkeys 5 have been conducted to enable their potential use in humans, but our present model is the opposite combination, i.e., transplantation of human cells to pigs. Kawamura et al. administered three different immunosuppressants when evaluating human-derived myocardial sheets in laboratory mini-pigs (CLAWN mini-pigs), but long-term graft survival was not achieved 13 . Our method uses the major porcine immune organs. We removed the thymus and spleen, and carefully administered the abovementioned three immunosuppressants via a gastrostomy tube.
We established an experimental animal model, comprising three-way crossbred pigs, Göttingen minipigs 4 , CLAWN mini-pigs, and micro mini-pigs 6 , and produced a protocol capable of inducing the longterm survival of human-derived cells and tissues ( Table 1).
The most crucial and helpful parameter in keeping pigs under immunosuppressive treatment is the change in body weight. Many medications cause asitia and often result in vomiting and diarrhea subsequently. For instance, MMF is well known to have such side effects. Vomiting interferes with dosing and, importantly, diarrhea increases the blood level of tacrolimus, the principal immunosuppressant used as the primary medication. Careful observation of body weight, as well as the behavior, enables detection of early signs of gastrointestinal disorders. It will be effective to feed piglets with 100 mL of milk substitute (Sanikko one, Marubeni Nisshin Feed Co., Ltd.), twice a day, to prevent dehydration and ameliorate vomiting and/or diarrhea. To alleviate the side effects, divided administration with uneven doses also deserves consideration as a chronopharmacological approach 14 .
We are confident that the information in this paper will be useful to many researchers for non-clinical and clinical application validation studies in human-sized animals of human-derived regenerative medical products produced by the latest techniques, from both the efficacy and safety perspectives.

MATERIALS ▲CRITICAL
The reagents and surgical instruments listed below are those used in our laboratory.
Similar equipment and reagents can be used as alternatives due to investigator availability.
Experimental animals: The adult pigs used were either three-way-crossbred pigs obtained from 6 livestock farmers or Göttingen pigs or micro mini-pigs or CLAWN miniature pigs, which are breeds developed for laboratory use. Table 1 shows their sexes, ages, and weights.     ▲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. 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  Table 2.

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 × 10 4 cells -2 × 10 5  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.  Table 2.

Anticipated Results
We estimated the dose of immunosuppressant with reference to the 100% inhibitory concentration of peripheral blood mononuclear cells (PBMC). PBMC proliferation studies demonstrated that 0.1 ug/mL of mycophenolic acid (MPA) completely inhibited PHA-induced PBMC proliferation (Fig. 3). After estimation of the target plasma concentration of MPA, which is equivalent to the 0.1 µg/mL on free dug theory 15 in micro mini-pigs, we administered MMF via a gastrostomy tube and measured the plasma MPA concentration. Plasma concentrations of MPA were higher than the target concentration throughout the day (Fig. 4).
The dose estimated from the in vitro efficacy and pharmacokinetics was very similar to empirical doses used for post-operative immunosuppressant administration in our studies (Table1). assistance and advice on pharmacokinetics and in vitro efficacy studies.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. Table2 Troubleshooting Table.pdf Vedeo1_Thymectomy of an adult mini-pig.mov Table 1 Profile of pigs and post-operative immunosuppressant regimens in the various laboratories..pdf Development of an immunodeficient pig model allowing longterm accommodation of artificial human vascular tubes by Manabu Itoh, Yosuke Mukae, Takahiro Kitsuka, +13