rat spleen decellularization to create a spleen matrix for liver engineering

肝臓組織工学のための脱細胞化ラット脾臓マトリックス調製

Liver transplantation remains the only definitive treatment for end-stage liver disease1,2,3. 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 solutions5,6,7. 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 BALs8,9,10,11. 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.

著者スポットライト:バイオ人工肝臓のための脱細胞化脾臓マトリックスの利用

ラット由来の脱細胞化脾臓マトリックスの作製

We purpose a novel strategy that involves employing a decellularized spleen matrix as an alternative scaffold to bioartificial livers. We showed the detailed procedure of harvesting rat spleen and preparing decellularized spleen matrix that preserves the macrostructures and the components of the extracellular matrix. Our protocol provides a readily available spleen matrix as an alternative to scarce donor livers.Our protocol present a method for the preparation of decellularized spleen matrices. Demonstrating efficiency, stability, and minimal invasiveness, this method maintains the spleen inherent architecture, composition, and the natural vascular network. It offers the scaffold for cell implantation and three-dimensional dynamic culture, thereby establishing a basis for advancing investigations in tissue-engineered liver.

ラットの脾臓脱細胞化による肝臓工学のための脾臓マトリックスの作成

まず、凍結したラットの脾臓を3回凍結して解凍し、脾臓細胞を溶解します。クリーンベンチに、蠕動ポンプ、2リットルのリザーバー、内径2.4ミリメートルのシリコンチューブ、およびバブルトラップを設置します。灌流システムに脱イオン水を入れます。10分間実行し続けます。採取した脾臓を脱イオン水で満たされた容器に注意深く移します。次に、脾臓動脈に挿入した静脈カテーテルにシリコンチューブを接続します。脱イオン水、0.1%SDS、1%Triton X-100、PBS溶液で灌流を行います。50ミリリットルの遠心分離チューブに、10%ペニシリンストレプトマイシンを含むPBSを入れます。脱細胞化した脾臓マトリックスをPBSに移します。さらなる実験まで摂氏マイナス20度で保管してください。脾臓の完全な脱細胞化は約11時間で達成されました。色は徐々に深い赤からモデル化された明るい赤に変化し、最終的には白い半透明の外観になり、全体的な形態は損なわれませんでした。

rat-spleen-decellularization-to-create-spleen-matrix-for-liver

Research

JoVE Journal

Bioengineering

Rat Spleen Decellularization to Create a Spleen Matrix for Liver Engineering

64 ビュー.  The First Affiliated Hospital of Xi'an Jiaotong University. まず、凍結したラットの脾臓を3回凍結して解凍し、脾臓細胞を溶解します。クリーンベンチに、蠕動ポンプ、2リットルのリザーバー、内径2.4ミリメートルのシリコンチューブ、およびバブルトラップを設置します。灌流システムに脱イオン水を入れます。10分間実行し続けます。採取した脾臓を脱イオン水で満たされた容器に注意深く移します。次に、脾臓動脈に...

ビデオ: ラットの脾臓脱細胞化による肝臓工学のための脾臓マトリックスの作成

Fabrication of Decellularized Spleen Matrix Derived from Rats

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.

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.

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the ...