Fabrication of Decellularized Spleen Matrix Derived from Rats

Summary

The decellularized spleen matrix (DSM) holds promising applications in the field of liver tissue engineering. This protocol outlines the procedure for preparing rat DSM, which includes harvesting rat spleens, decellularizing them through perfusion, and evaluating the resulting DSM to confirm its characteristics.

Abstract

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the development of alternative therapies. Bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions. However, sourcing suitable DLMs remains challenging. The use of a decellularized spleen matrix (DSM) has been explored as a foundation for BALs, offering a readily available alternative. In this study, rat spleens were harvested and decellularized using a combination of freeze-thaw cycles and perfusion with decellularization reagents. The protocol preserved the microstructures and components of the extracellular matrix (ECM) within the DSM. The complete decellularization process took approximately 11 h, resulting in an intact ECM within the DSM. Histological analysis confirmed the removal of cellular components while retaining the ECM's structure and composition. The presented protocol provides a comprehensive method for obtaining DSM, offering potential applications in liver tissue engineering and cell therapy. These findings contribute to the development of alternative approaches for the treatment of end-stage liver disease.

Introduction

Liver transplantation remains the only definitive treatment for end-stage liver disease^1,2,3. However, the critical shortage and declining quality of donor organs have heightened the need for alternative treatments⁴. In the realm of regenerative medicine, bioartificial livers (BALs) utilizing decellularized liver matrix (DLM) have emerged as promising solutions^5,6,7. The DLM preserves the original liver structure, including its intricate microvascular network and components of the ECM, offering a scaffold for creating transplantable BALs that could potentially alleviate liver diseases.

Despite the promise, the adoption of this technology faces challenges, particularly in sourcing suitable DLMs. Human-derived DLMs are in short supply, while those from animal sources carry the risks of disease transmission and immune rejection. In an innovative approach, our research has explored the use of a decellularized spleen matrix (DSM) as a foundation for BALs^8,9,10,11. Spleens are more readily available in various medical situations, such as portal hypertension, traumatic rupture, idiopathic thrombocytopenic purpura, and donation after cardiac death. Therefore, spleens are more widely available than livers for research purposes. Patients who have undergone splenectomies do not suffer from severe conditions, further confirming the dispensability of the spleen. The microenvironment of the spleen, particularly the extracellular matrix and sinusoids, is similar to that of the liver. This makes the spleen a suitable organ for cell adhesion and proliferation in hepatocyte transplantation research. Based on these findings, our previous investigations have demonstrated that DSMs share comparable microstructures and components with DLMs and can support the survival and function of hepatocytes, including albumin and urea production. Furthermore, DSMs have been shown to enhance the hepatic differentiation of bone marrow mesenchymal stem cells, leading to improved and consistent functionality.

By employing DSMs treated with heparin, we have engineered functional BALs capable of demonstrating effective short-term anticoagulation and partial liver function compensation¹¹. Consequently, this three-dimensional DSM holds significant promise for the advancement of liver tissue engineering and cell therapy. In this work, we present the detailed methods of harvesting rat spleens and preparing DSM that preserve the microstructures and components of the ECM.

Protocol

This study was approved by the Committee on the Ethics of Animal Experiments of Xi'an Jiaotong University and carried out in accordance with the guidelines for the Care and Use of Laboratory Animals.

1. Spleen harvesting

1. Use male Sprague Dawley rats weighing 250-280 g. House the rats in rooms with controlled temperature and humidity, and provide them with food and water ad libitum, except for fasting before surgery.
2. Subcutaneously inject buprenorphine (0.05 mL/kg) as an analgesic 1 h before operation. Anesthetize the rat by isoflurane inhalation. Use a flow rate of 1.5 L/min of 5% isoflurane for induction anesthesia in a plexiglass box and maintain anesthesia with a flow rate of 0.6-0.8 L/min of 2% isoflurane through a mask. Confirm the depth of anesthesia by pinching the toes.
3. Use an electric shaver to shave the skin over the entire abdomen. Secure the rat in a supine position on the surgical table. Inject 2 mL of heparinized saline (1,000 U of heparin) via the penile dorsal vein to achieve systemic anticoagulation. Disinfect the shaved skin with a povidone-iodine solution and cover with a sterile draping cloth.
4. Make a cruciform incision using surgical scissors in the abdomen, expose the abdominal cavity by stretching with the hemostatic forceps, and flip the liver towards the diaphragm. Exteriorize the gastrointestinal tract to the right side of the abdomen and cover it with moist gauze.
5. Carefully separate and cut the splenogastric ligament to expose the splenic hilum.
   NOTE: The spleen, which appears as a reddish elongated structure, approximately 3.0 cm x 0.6 cm x 0.6 cm in size, can be identified in the left abdomen.
6. Gradually separate and expose the common hepatic artery, gastroduodenal artery, and splenic artery by dissecting along the splenic hilum. Ligate and cut the gastroduodenal artery and common hepatic artery while progressively dissociating the surrounding tissue.
7. Flip the spleen towards the right side to expose the abdominal aorta. Gently perform blunt dissection and expose the abdominal aorta and celiac trunk using cotton swabs. Place a 3-0 silk suture, approximately 3 cm in length, above and below the branches of the celiac trunk, and place a 6-0 silk suture, approximately 10 cm in length, at the branch of the celiac trunk.
8. Ligature the abdominal aorta below and above the branches of the celiac trunk. Make a small incision at the arterial branch. Gently lift the 6-0 silk suture, insert a 24 G venous catheter into the splenic artery along the celiac trunk, and ligate and secure it.
9. Using a syringe pump at a rate of 4 mL/min, infuse heparinized normal saline (25 U/mL) at a volume of 50 mL. At the same time, sever the portal vein as an outflow channel to allow the infused fluid to flow out of the spleen. The animal is euthanized by exsanguination.
10. Carefully dissect the surrounding tissue of the spleen, avoiding damage to the pancreas, while preserving the major accessory vessels.
11. Check for any leakage around the spleen, then remove the spleen and pancreas and rinse them in normal saline.
    NOTE: The spleen and pancreas of rats are closely connected, with the pancreas wrapping around the splenic artery. If the spleen is removed separately, it can be challenging to ligate numerous small blood vessels. In this procedure, the spleen is removed together with the pancreas. After decellularization, the spleen and pancreas become transparent and the microvasculature is visible, which facilitates the preservation of the spleen with intact blood vessels.
12. Transfer the spleen into a 50 mL centrifuge tube filled with normal saline and store it in a -80 °C freezer.
    NOTE: The spleen and the venous catheter inserted into the splenic artery will be cryopreserved together for convenient connection during the perfusion experiments.

2. Spleen decellularization

1. Repeat the freeze-thaw cycle 3x in a sterile container to lyse the spleen cells.
   NOTE: The freeze-thaw cycle is a physical method used for scaffold decellularization. The spleen tissue is placed in a -80 °C freezer overnight for freezing, then removed from the low-temperature environment and placed in a room temperature or 37 °C water bath for thawing. This freezing and thawing process is repeated 3-6 times, which disrupts cell membranes and leads to cell lysis.
2. Set up a perfusion system within a clean bench comprising a peristaltic pump, a 2 L reservoir, a silicone tube with an inner diameter of 2.4 mm, and a bubble trap (Figure 1).
3. Fill the perfusion system with deionized water (ddH₂O) and keep it running for 10 min.
4. Carefully transfer the harvested spleen to the ddH₂O-filled container.
5. Connect the silicone tube to the venous catheter that has been inserted into the splenic artery.
6. Start perfusion with ddH₂O at a rate of 2 mL/min for 30 min.
7. Continue perfusion with ddH₂O at a rate of 4 mL/min for 30 min.
8. Perfuse with 0.1% (w/v) SDS solution for 4 h.
9. Perfuse with 1% (v/v) Triton X-100 solution for 2 h.
10. Perfuse with PBS at a rate of 4 mL/min for 4 h to wash the DSM.
    NOTE: Using the peristaltic pump for unidirectional perfusion of all liquids.
11. After the perfusion is completed, ligate and transect the vascular branches between the pancreas and the spleen, and then remove the pancreas. Store the DSM in a clean and sealed 50 mL centrifuge tube soaked in PBS containing 10% penicillin-streptomycin at -20 °C until ready to use for future experiments.

Results

This protocol utilized a combination of repeated freeze-thaw cycles and perfusion with decellularization reagents for the decellularization of rat spleen. The complete decellularization of the spleen was achieved in approximately 11 h (Figure 2A). Throughout the decellularization process, the spleen's color gradually transitioned from deep red to a mottled, light red, and eventually, a white translucent appearance (Figure 2B). The overall morphology remained...

Discussion

The BALs represent an effective approach for the treatment of end-stage liver disease, particularly in cases where liver transplantation is hindered by the current shortage of donor organs⁶. A promising option for creating BALs is the utilization of DLM, which preserves the native liver's natural ECM and vascular structure. However, the scarcity of human DLM and the potential risks of infection and immunogenicity associated with animal DLM pose significant limitations. To address this challeng...

Materials

Name	Company	Catalog Number	Comments
Anesthesia Machine	Harvard Apparatus	tabletop	animal anesthesia
bubble trap	Shandong Weigao Group Medical Polymer Co., Ltd.	pore diameter: 5 μm	prevent air bubbles
Buprenorphine	TIPR Pharmaceutical Responsible Co.,Ltd		an analgesic
Hemostatic Forceps	Shanghai Medical Instruments  Co., Ltd	J31020	surgical tool
Heparinized Saline	SPH No.1 Biochemical & Pharmaceutical Co., LTD		prevent the formation of thrombosis
Isoflurane	RWD life Science Co.		anesthetic:for the induction and maintenanceof anesthesia
Penicillin-Streptomycin	Beyotime Biotechnology Co., Ltd.	C0222	antibiotics in vitro to prevent microbial contamination
Peristaltic Pump	Baoding Longer Precision Pump Co., Ltd.	BT100-1L
Phosphate-Buffered Saline	Shanghai Titan Scientific Co., Ltd.	4481228	phosphoric acid buffer salt solution
Silicone Tube	Baoding Longer Precision Pump Co., Ltd.	2.4×0.8mm
Silk Suture	Yangzhou Jinhuan Medical Instrument Factory	6-0 and 3-0	ligate blood vessels
Sodium Dodecyl Sulfate	Shanghai Titan Scientific Co., Ltd.	151-21-3	ionic detergent, dissolves both cell and nuclear membranes
Syringe Pump	Shenzhen Mindray Bio-Medical Electronics Co., Ltd	BeneFusion SP5	intravenous infusion
Triton X-100	Shanghai Titan Scientific Co., Ltd.	9002-93-1	non-ionic detergent, disrupts lipid-lipid, lipid-protein, and DNA-protein interactions
Venous Catheter	B. Braun Company	24G	inserting the spleen artery