Name: Procurement of Parathyroid Glands from Living Donor Pigs and Ex Vivo Identification (Video) | JoVE Journal
Uploaded: 2025-08-01T14:50:00.000+00:00
Duration: 8 min 7 s
Description: Learn a non-lethal method for parathyroid gland procurement in pigs using thymus retrieval and transillumination. This technique supports ethical xenotransplantation research for treating hypoparathyroidism.

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5 minutes

We would like to acknowledge the French Society of Diabetes and the Fondation pour la Recherche M&#233;dicale for support and research funding.

All animal studies were approved by the animal ethical committee of the University of Lille (referenced #20491-20190219161313).
1. Porcine anesthesia
<ol>
	<li>Administer buprenorphine (0.015 mg/kg, intramuscularly) 30 min before the first incision to provide preoperative analgesia.</li>
	<li>Perform an intramuscular injection of ketamine (50 mg/kg) and xylazine (3 mg/kg) to induce sedation. Administer 5% isoflurane via inhalation for 3 to 5 min to deepen anesthesia.</li>
	<li>Assess the depth of anesthesia by checking the eyelid closure reflex and the swallowing reflex. Intubate the pig and position it supine on the operating table. Reduce isoflurane concentration to 2% via inhalation for anesthesia maintenance.</li>
	<li>Continuously monitor the heart rate and oxygen saturation using a portable multi-parameter veterinary monitor to ensure optimal anesthetic depth and physiological stability throughout the procedure.</li>
</ol>
2. Exposition of the area of interest
<ol>
	<li>Shave and meticulously clean the cervical region, extending from the submandibular area to the sternum.</li>
	<li>Apply antiseptic soap, followed by chlorhexidine, to ensure a sterile field. Thoroughly dry the entire area.</li>
	<li>Position sterile drapes to isolate the anterior cervical region for surgical intervention.</li>
</ol>
3. Porcine thymus exposure
<ol>
	<li>Perform a 10 cm vertical incision along the white cervical line using a scalpel. Carefully dissect the area with Metzenbaum scissors to separate the prethyroid muscles, creating a clear surgical field.</li>
	<li>Place a self-retaining retractor between the omohyoid muscle and the thyroid cartilage to facilitate dissection of the connective tissue and expose the thymus. 
	NOTE: Avoid excessive pressure on the trachea to prevent injury.</li>
	<li>Gradually expose the cranial part of the thymus and carefully separate it from the carotid sheath, which contains the vagus nerve, internal jugular vein, and common carotid artery. 
	NOTE: To prevent injury, use electrocoagulation to achieve hemostasis.</li>
</ol>
4. Porcine thymus retrieval
<ol>
	<li>Once released, extract the cranial part of the thymus, which typically measures 5 - 8 cm.</li>
	<li>Rinse the extracted thymus in isotonic saline solution to facilitate the identification of the parathyroid gland. 
	NOTE: This step is crucial for enhancing visualization and differentiation of the parathyroid gland&#39;s distinct texture</li>
</ol>
5. Identification of the intrathymic parathyroid gland ex vivo
<ol>
	<li>Gently flatten the thymic tissue to facilitate parathyroid gland identification. 
	NOTE: The parathyroid gland exhibits a distinctive texture, particularly in comparison to the surrounding tissue. The fibrotic capsule of the parathyroid gland is firmer and of a similar size to a pea.</li>
	<li>Use a light source to identify the parathyroid gland via transillumination. 
	NOTE: The vasculature surrounding the parathyroid gland should be clearly visible, aiding in its differentiation.</li>
	<li>Carefully excise the capsule and extract the entire parathyroid gland. Using Debakey forceps for delicate handling and Metzenbaum scissors for precise dissection. Isolate the gland from the surrounding thymic tissue.</li>
	<li>Place the isolated parathyroid gland in cold storage solution and maintain it at 4 &#176;C to preserve its viability for subsequent procedures.</li>
	<li>Embed the tissue in paraffin for further processing.</li>
</ol>
6. Surgical closure and awakening
<ol>
	<li>Following the retrieval of the parathyroid gland, close the surgical site in layers. Suture muscles and subcutaneous tissues using absorbable sutures. Close the skin with non-absorbable sutures.</li>
	<li>Gradually wean the pig off anesthesia. Upon regaining consciousness, monitor the pig for immediate postoperative complications.</li>
	<li>Administer analgesics and antibiotics as per postoperative protocol (buprenorphine 0.03 &#224; 0.05 mg/kg and Clamoxyl 15 mg/kg for 48 h). Return the pigs to their housing facility for recovery under veterinary supervision.</li>
</ol>
7. Immunostaining protocol
<ol>
	<li>Perform deparaffinization and dehydration from paraffin-embedded samples: xylene for 5 min, xylene for 10 min, 100% ethanol for 5 min, 90% ethanol for 7 min 30, 70% ethanol for 7 min 30, 50% ethanol for 5 min, distilled water for 5 min and phosphate-buffer-saline (PBS) for 5 min.</li>
	<li>Perform antigen retrieval by microwaving the samples in citrate buffer pH 6, at 650 W for 9 min. Allow the samples to cool down in the buffer for 20 min. Rinse three times with PBS for 5 min each.</li>
	<li>Incubate the samples, encircled with a Dako-pen, at room temperature for 20 min with protein-block solution to block non-specific binding.</li>
	<li>Incubate the samples overnight at 4&#176;C with the primary antibody (PTH Antibody (H-7), Mouse IgG-&#945;-human, sc-398856, 1:100 ) diluted in 1% Bovine Serum Albumin (BSA). Wash twice with PBS for 5 min each.</li>
	<li>Incubate for 1 h and 30 min at room temperature with the secondary antibody (Alexa Fluor 488, goat-&#945;-Mouse, A11001, 1:500) diluted in 1% bovine serum albumin. Wash twice with PBS and once with double distilled water for 5 min.</li>
	<li>Mount the slides with DAPI solution and store the slides at 4 &#176;C.</li>
</ol>

<ol><li>Gafni, R. I., Collins, M. T. Hypoparathyroidism. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGafni%2C+RI+%22N.+Engl.+J.+Med%22">N. Engl. J. Med</a>. 380, 1738-1747 (2019).</li>
<li>Gosmanova, E. O., Houillier, P., Rejnmark, L., Marelli, C., Bilezikian, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Renal+complications+in+patients+with+chronic+hypoparathyroidism+on+conventional+therapy%3A+a+systematic+literature+review%3A+Renal+disease+in+chronic+hypoparathyroidism%5BTitle%5D)+AND+%22Rev.+Endocr.+Metab.+Disord%22%5BJournal%5D)">Renal complications in patients with chronic hypoparathyroidism on conventional therapy: a systematic literature review: Renal disease in chronic hypoparathyroidism.</a> Rev. Endocr. Metab. Disord. 22, 297-316 (2021).</li>
<li>Frey, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+permanent+post-thyroidectomy+hypoparathyroidism+on+self-evaluation+of+quality+of+life+and+voice%3A+results+from+the+national+qol-hypopara+study%5BTitle%5D)+AND+%22Ann.+Surg%22%5BJournal%5D)">Impact of permanent post-thyroidectomy hypoparathyroidism on self-evaluation of quality of life and voice: results from the national qol-hypopara study.</a> Ann. Surg. 274, 851-858 (2021).</li>
<li>Büttner, M., Krogh, D., Siggelkow, H., Singer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+are+predictors+of+impaired+quality+of+life+in+patients+with+hypoparathyroidism%5BTitle%5D)+AND+%22Clin.+Endocrinol.+%28Oxf%29%22%5BJournal%5D)">What are predictors of impaired quality of life in patients with hypoparathyroidism.</a> Clin. Endocrinol. (Oxf). 97, 268-275 (2022).</li>
<li>Gadelha, D. D., Filho, W. A., Brandão, M. A. J., Montenegro, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+parathyroid+allotransplantation+a+viable+option+in+the+treatment+of+permanent+hypoparathyroidism%3F+A+review+of+the+literature%5BTitle%5D)+AND+%22Endocrine%22%5BJournal%5D)">Is parathyroid allotransplantation a viable option in the treatment of permanent hypoparathyroidism? A review of the literature.</a> Endocrine. 80 (2), 253-265 (2022).</li>
<li>Sakane, E. N., Vieira, M. C. C., Vieira, G. M. M., Maeda, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+options+in+hypoparathyroidism%5BTitle%5D)+AND+%22Arch.+Endocrinol.+Metab%22%5BJournal%5D)">Treatment options in hypoparathyroidism.</a> Arch. Endocrinol. Metab. 66, 651-657 (2022).</li>
<li>Zhang, J. L. H., Appelman-Dijkstra, N. M., Schepers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parathyroid+allotransplantation%3A+A+systematic+review%5BTitle%5D)+AND+%22Med.+Sci%22%5BJournal%5D)">Parathyroid allotransplantation: A systematic review.</a> Med. Sci. 10, 19(2022).</li>
<li>Goncu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Graft+survival+effect+of+HLA-A+allele+matching+parathyroid+allotransplantation%5BTitle%5D)+AND+%22J.+Investig.+Med.+Off.+Publ.+Am.+Fed.+Clin.+Res%22%5BJournal%5D)">Graft survival effect of HLA-A allele matching parathyroid allotransplantation.</a> J. Investig. Med. Off. Publ. Am. Fed. Clin. Res. 69, 785-788 (2021).</li>
<li>Barczyński, M., Gołkowski, F., Nawrot, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parathyroid+transplantation+in+thyroid+surgery%5BTitle%5D)+AND+%22Gland+Surg%22%5BJournal%5D)">Parathyroid transplantation in thyroid surgery.</a> Gland Surg. 6, 530-536 (2017).</li>
<li>Montgomery, R. A., et al. Results of two cases of pig-to-human kidney xenotransplantation. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMontgomery%2C+RA+%22N.+Engl.+J.+Med%22">N. Engl. J. Med</a>. 386, 1889-1898 (2022).</li>
<li>Loupy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+response+after+pig-to-human+kidney+xenotransplantation%3A+a+multimodal+phenotyping+study%5BTitle%5D)+AND+%22The+Lancet%22%5BJournal%5D)">Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study.</a> The Lancet. 402, 1158-1169 (2023).</li>
<li>Goncu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenotransplantation+of+microencapsulated+parathyroid+cells+as+a+potential+treatment+for+autoimmune-related+hypoparathyroidism%5BTitle%5D)+AND+%22Exp.+Clin.+Transplant.+Off.+J.+Middle+East+Soc.+Organ+Transplant%22%5BJournal%5D)">Xenotransplantation of microencapsulated parathyroid cells as a potential treatment for autoimmune-related hypoparathyroidism.</a> Exp. Clin. Transplant. Off. J. Middle East Soc. Organ Transplant. 21, 901-912 (2023).</li>
<li>Hasse, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parathyroid+xenotransplantation+without+immunosuppression+in+experimental+hypoparathyroidism%3A+long-term+in+vivo+function+following+microencapsulation+with+a+clinically+suitable+alginate%5BTitle%5D)+AND+%22World+J.+Surg%22%5BJournal%5D)">Parathyroid xenotransplantation without immunosuppression in experimental hypoparathyroidism: long-term in vivo function following microencapsulation with a clinically suitable alginate.</a> World J. Surg. 24, 1361-1366 (2000).</li>
<li>Oster, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+and+reliable+dissection+of+porcine+parathyroid+glands+-+A+protocol+for+molecular+and+histological+analyses%5BTitle%5D)+AND+%22Ann.+Anat.+-+Anat.+Anz%22%5BJournal%5D)">Fast and reliable dissection of porcine parathyroid glands - A protocol for molecular and histological analyses.</a> Ann. Anat. - Anat. Anz. 219, 76-81 (2018).</li>
<li>Hyun, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure-inherent+targeting+of+near-infrared+fluorophores+for+parathyroid+and+thyroid+gland+imaging%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">Structure-inherent targeting of near-infrared fluorophores for parathyroid and thyroid gland imaging.</a> Nat. Med. 21, 192-197 (2015).</li>
<li>Klute, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Contribution+to+the+topography+and+histology+of+porcine+parathyroid+glands%22">Contribution to the topography and histology of porcine parathyroid glands</a>. , Justus Liebig University of Giessen. Beitrag zur Topographie und Histologie der EpithelkÃ¶rperchen des Schweines.Â Ph.D. thesis. Justus Liebig-UniversitÃ¤t zu Giessen (1959).</li>
<li>Arndt, H. On the topography of epithelial cells in pigs with special consideration of the suitability of pigs for the production of organo-therapeutic epithelial cell preparations. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Z.+Anat%22">Z. Anat</a>. 81, (Zur Topographie der Epithelkörperchen beim Schwein mitbesonderer Berücksichtigung der Eignung des Schweines für die Gewinnungorgano-therapeutischer Epithelkörperchenpräparate) 191-197 (1926).</li>
<li>Butylina, M., Föger-Samwald, U., Gelles, K., Pietschmann, P., Sipos, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+in+establishing+animal+models+for+studying+osteoimmunology+of+hypoparathyroidism%5BTitle%5D)+AND+%22Front.+Vet.+Sci%22%5BJournal%5D)">Challenges in establishing animal models for studying osteoimmunology of hypoparathyroidism.</a> Front. Vet. Sci. 10, 1163903(2023).</li>
</ol>

This protocol presents a rapid and non-lethal method for parathyroid gland procurement in pigs. The key step within the protocol involves localizing the porcine thymus to ensure complete retrieval for further ex vivo investigation. Once the thymus is extracted, the parathyroid gland can be easily identified using transillumination.
One of the main challenges of this technique is the need to avoid relying on human surgical habits associated with parathyroidectomy, as our protocol circu...

This protocol presents a rapid and non-lethal method for parathyroid gland procurement from pigs. Hypoparathyroidism, characterized by insufficient parathormone secretion, critically disrupts calcium homeostasis1. This condition, predominantly a surgical complication following parathyroidectomy or thyroidectomy, manifests through symptoms like asthenia, chronic pain, tachycardia, seizures, and even end-stage renal disease2. Occasionally, it arises from rare autoimmune or genetic disorders. Current treatments, mainly calcium and vitamin D oral supplementation or recombinant parathormone, provide only partial relief from systemic symptoms like fatigue and depression3,4. The growing necessity for parathyroid allotransplantation as a potential remedy for severe hypoparathyroidism underscores the necessity for the establishment of uniform transplant protocols5,6,7,8,9. However, preclinical studies on parathyroid transplantation remain limited, and there is a lack of standardized animal models. Furthermore, the recent boost in the xenotransplantation field10,11, increases the interest in clinical applications of xenotransplantation, especially in the field of endocrine cell therapy12,13. However, porcine models of parathyroid xenotransplantation are challenging due to the complexity of identifying parathyroid tissue in pigs. There is, thus, a need to provide a reproducible, sustainable, and ethical technique to retrieve parathyroid glands from pigs to further develop parathyroid xenotransplantation.
Unlike human beings, who typically have four parathyroid glands located behind the thyroid, pigs possess, for most, two parathyroid glands, each located in the cranial part of the thymus. Previous studies that aimed to locate parathyroid glands in pigs either relied on lethal methods for ex vivo identification or fluorophore-based techniques, which may be difficult to implement in xenotransplantation14,15,16. We propose here a simple and non-lethal technique, suitable for xenotransplantation, based on two key steps: porcine thymus retrieval and ex vivo identification of the parathyroid gland using transillumination. This technique might be useful for research on parathyroid physiology, as well as transplantation.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>22G Short Peripheral Catheter</td><td>Smiths Medical</td><td>5060</td><td></td></tr><tr><td>Basic set II, crepe</td><td>Hartmann</td><td>9389253</td><td></td></tr><tr><td>Bupaq (bupr&amp;eacute;norphine)</td><td>Virbac</td><td></td><td></td></tr><tr><td>Clippers Favorita II</td><td>Aesculap Schermaschinen</td><td>GT104</td><td></td></tr><tr><td>Disposable Scalpels</td><td>Swann-Morton</td><td>0510</td><td></td></tr><tr><td>Electrode spatula</td><td>Erbe</td><td>21191-002</td><td></td></tr><tr><td>Electrosurgery Generator BE VIO 200p</td><td>Erbe</td><td>10140-400</td><td></td></tr><tr><td>Euroklav 23 VS+</td><td>MELAG</td><td>10056</td><td></td></tr><tr><td>Gravity infusion 3-way valve with mobile Luer Lock tip</td><td>Care Fusion</td><td>A64-3V</td><td></td></tr><tr><td>Handle with two buttons for electrodes Slim-Line&amp;nbsp;</td><td>Erbe</td><td>20190-065</td><td></td></tr><tr><td>Isoflurane Funnel Fill Vaporizer</td><td>Supera Anesthesia Innovations</td><td>VAP3000</td><td></td></tr><tr><td>Isorane (isoflurane)</td><td>Axience</td><td></td><td></td></tr><tr><td>Ketamidor (k&amp;eacute;tamine)</td><td>Axience</td><td></td><td></td></tr><tr><td>Light Ceiling Double</td><td>RIMSA</td><td>PENTA81+30E</td><td></td></tr><tr><td>Medisorb Carbon Dioxide Absorbent</td><td>Vyaire</td><td>8570043</td><td></td></tr><tr><td>Mersutures 0</td><td>Ethicon</td><td>F2520</td><td></td></tr><tr><td>Microtome</td><td>LEICA RM2255</td><td></td><td></td></tr><tr><td>Miller Blade</td><td>Opticlar</td><td>600.020</td><td></td></tr><tr><td>Oxygen Concentrator</td><td>DeVilbiss</td><td>525KS</td><td></td></tr><tr><td>Paxman (xylazine)</td><td>Virbac</td><td></td><td></td></tr><tr><td>Portable Multi-Parameter Veterinary Monitor</td><td>Mindray</td><td>IPM 10</td><td></td></tr><tr><td>Reusable Laryngoscope Handles</td><td>Opticlar</td><td>600.080</td><td></td></tr><tr><td>Small Animal Anesthesia Ventilator</td><td>Midmark</td><td>91806001</td><td></td></tr><tr><td>Sodium Chloride Injection Viaflo</td><td>Baxter</td><td></td><td></td></tr><tr><td>Surgical Instruments</td><td>Kohler medizintechnik</td><td></td><td></td></tr><tr><td>Surgical Table</td><td>Vetinox</td><td>TA700005</td><td></td></tr><tr><td>Tracheal Tube</td><td>Smiths Medical</td><td>100/179/070</td><td></td></tr><tr><td>Vicryl 2-0</td><td>Ethicon</td><td>JV453</td><td></td></tr><tr><td>VMS</td><td>Midmark</td><td>91800003</td><td></td></tr><tr><td>&lt;strong&gt;Materials and Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM F-12 medium</td><td>ThermoFischer</td><td>11320033</td><td></td></tr><tr><td>Parathormone antibody</td><td>SantaCruz</td><td>sc-398856</td><td></td></tr><tr><td>Rapid intraoperative pth assay</td><td>Beckman Coulter</td><td>A16972</td><td></td></tr></tbody></table>

procurement of parathyroid glands from living donor pigs and ex vivo identification

Procurement of Parathyroid Glands from Living Donor Pigs and Ex Vivo Identification

Hypoparathyroidism, often resulting from surgical complications, severely disrupts calcium homeostasis, leading to disabling symptoms. Current treatments offer limited relief, highlighting the need for alternative therapies like parathyroid transplantation. This study introduces a simple method for living-donor parathyroid procurement from pigs, with potential applications in transplantation research, especially for the field of xenotransplantation. The procedure involves three key steps: porcine preparation with anesthesia, unilateral thymus retrieval, and ex vivo identification and extraction of the intrathymic parathyroid gland. The protocol utilizes Landrace pigs, with procedures ensuring ethical treatment and recovery post-operation. We successfully extracted parathyroid glands, confirming their viability and integrity through subsequent analysis. PTH levels in tissue supernatant exceeded 1,000 pg/mL, and immunofluorescence staining distinguished parathyroid glands from thymic tissue, validating the procedure's reliability. This protocol offers a rapid, non-lethal, and ethically sound method for parathyroid gland procurement from pigs, addressing anatomical differences between pigs and humans. It offers a sustainable approach with significant implications for hypoparathyroidism research and xenotransplantation.

This study's primary outcomes include the successful extraction of parathyroid glands from Landrace pigs and subsequent analysis to confirm their viability and integrity. The results are summarized as follows. Identification of parathyroid tissue was confirmed intraoperatively via transillumination (Figure 1A). After extraction, two small samples of the tissue were collected (approximately 10% each). One sample was minced in a dish-plate in 1 mL of DMEM-F12 culture medium. After min...

This protocol presents a rapid and non-lethal method for procuring parathyroid glands from pigs. The procedure consists of three main steps: porcine preparation with anesthesia, unilateral thymus retrieval, and ex vivo identification and extraction of the intrathymic parathyroid gland.

Hypoparathyroidism, often resulting from surgical complications, severely disrupts calcium homeostasis, leading to disabling symptoms. Current treatments ...

procurement-parathyroid-glands-from-living-donor-pigs-ex-vivo

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Learn a non-lethal method for parathyroid gland procurement in pigs using thymus retrieval and transillumination. This technique supports ethical xenotransplantation research for treating hypoparathyroidism.

Procurement of Parathyroid Glands from Living Donor Pigs and Ex Vivo Identification

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