Fabrication of Decellularized Spleen Matrix Derived from Rats

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.

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.
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.
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. 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.

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 relatively intact, with visible vascular structures (Figure 2B).

Hematoxylin-eosin staining confirmed the removal of cellular components, revealing an intact ECM within the DSM. This is in stark contrast to the native spleen, as depicted in Figure 3A, where cellular nuclei and cytoplasmic elements are visible. The absence of cells was further confirmed through 4',6-diamidino-2-phenylindole (DAPI) staining and measurement of residual DNA (DSM: 10.1 ± 4 ng/mg dry weight, native spleen: 6,200 ± 300 ng/mg dry weight). Scanning electron microscopic images revealed the ultrastructural characterization of the DSM, which showed a honeycomb structure after the complete removal of lymphocytes from the spleen (Figure 3C). This indicated that the decellularization of the spleen preserved the normal spleen's ultrastructure and architecture. For a more comprehensive evaluation of the crucial ECM proteins present within the DSM, Masson trichrome (Figure 3B) and immunofluorescence staining were employed. Specifically, collagen I (Figure 3D) and fibronectin (Figure 3E) were targeted for analysis. The results indicated that both structural and basement membrane components of the ECM were retained similarly to the native spleen.

Figure 1: The experimental setup. Please click here to view a larger version of this figure.

Figure 2: The workflow of decellularization and gross morphological changes. (A) The decellularized spleen matrix preparation workflow. The complete decellularization of the spleen was achieved in approximately 11 h. (B) Gross morphological changes of the spleen during the preparation of DSM. During this process, the spleen's color gradually transitioned from deep red to a mottled, light red, and eventually, a white translucent appearance. (B) 1. After repeated freeze-thaw cycles and before decellularization. 2. After rinsing with deionized water for 1 h. 3. After perfusion with SDS for 4 h. 4. After perfusion with Triton for 2 h. Abbreviations: DSM = decellularized spleen matrix; SDS = sodium dodecyl sulfate; PBS = phosphate-buffered saline; PS = penicillin-streptomycin. Please click here to view a larger version of this figure.

Figure 3: Morphological observations of the decellularized spleen matrix. (A) H&E staining; (B) Masson trichrome staining; (C) SEM; (D,E) Immunofluorescence staining of collagen I and fibronectin; (F) DAPI staining. Scale bars = 50 µm (A, B, D-F), 5 µm (C). Abbreviations: DSM = decellularized spleen matrix; H&E = hematoxylin-eosin; SEM = scanning electron microscopy; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

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 challenge, we propose a novel strategy that involves employing a decellularized spleen matrix (DSM) as an alternative scaffold for BALs^8,9,10,11. Spleens are more readily accessible in various clinical scenarios and exhibit similar characteristics to livers. In this work, we present detailed methods of harvesting rat spleens and preparing DSM that preserve the microstructures and components of the ECM.

An ideal decellularization method would remove cellular components while keeping the original structure, composition, and mechanical properties of the ECM^12,13,14. Decellularization methods encompass physical, chemical, and enzymatic treatments, each with its distinct advantages and drawbacks¹⁵. While these methods can partially remove cellular components, they may also compromise the composition, structure, and functionality of the remaining ECM. The quality of the decellularization can be influenced by variations in cell density, matrix thickness, and tissue morphology across different tissue sources.

To date, there is no gold standard for the decellularization process. Typically, simply employing any of these methods is inadequate for minimizing adverse effects on the ECM and maximizing the removal of cellular content. Consequently, the most effective approach relies on the tissue characteristics, necessitating a combination of these methods. In this study, we utilized a protocol that combined physical methods (freeze-thaw cycles and perfusion) with chemical methods (SDS and TritonX-100) to decellularize rat spleens.

The freeze-thaw cycles promote cell lysis and the rapid detachment of cells from the ECM¹⁶. Simultaneously, perfusion through the native vasculature significantly enhances decellularization efficiency and preserves the original vascular network¹⁷. Sodium dodecyl sulfate (SDS), functioning as an ionic detergent, proficiently dissolves both cell and nuclear membranes, leading to a more comprehensive removal of cytoplasmic and nuclear components. However, this process also inflicts damage on the ECM ultrastructure due to the depletion of glycosaminoglycans (GAGs) and collagen.

Elevated concentrations of SDS correlated with diminished residual DNA content and reduced mechanical strength within the ECM scaffold. Conversely, lower concentrations of SDS preserved a greater amount of collagen and induced less denaturation of ECM proteins. In contrast, Triton X-100, serving as a non-ionic detergent, effectively disrupts lipid-lipid, lipid-protein, and DNA-protein interactions, offering a milder approach to cell membrane dissolution. Nevertheless, it proves inadequate for the complete removal of cell nuclei and DNA. Therefore, it needs to be combined with low concentrations of SDS and physical treatments to ensure the complete removal of cellular components while preserving the original structure, composition, and performance of the ECM. It is important to note that residual detergents can have certain cytotoxicity, so posttreatment rinsing with sterile PBS or distilled water is necessary before storage.

One limitation of this protocol is the absence of quantification for residual SDS and Triton X-100. This decision is informed by both our team's experience and corroborating reports, which suggest that a 4 h PBS wash is sufficient to remove these substances effectively. Furthermore, our prior cell culture experiments employing this protocol have not demonstrated any signs of cytotoxicity. To minimize the protocol's expenses, a deliberate choice was made to forego the quantification of residual detergents.

In conclusion, this protocol presents a feasible method for the preparation of DSMs, demonstrating efficiency, stability, and minimal invasiveness. The DSMs prepared using this protocol maintain the spleen's inherent architecture, composition, and natural vascular network. Moreover, it offers a scaffold for cell implantation and three-dimensional dynamic culture, thereby establishing a basis for advancing investigations in tissue-engineered liver.