rat spleen decellularization to create a spleen matrix for liver engineering

הכנת מטריצת טחול חולדה דה-צלולרית להנדסת רקמת כבד

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.

מחבר זרקור: ניצול מטריצת טחול דה-צלולרית עבור כבדים ביו-מלאכותיים

ייצור מטריצת טחול דה-צלולרית שמקורה בחולדות

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.

דה-צלולריזציה של טחול חולדה ליצירת מטריצת טחול להנדסת כבד

כדי להתחיל, להקפיא ולהפשיר טחול חולדה קפוא שלוש פעמים כדי לשכב את תאי הטחול. על ספסל נקי הציבו משאבה פריסטלטית, מאגר של שני ליטרים, צינור סיליקון בקוטר פנימי של 2.4 מילימטרים ומלכודת בועות. מלאו את מערכת הזילוח במים נטולי יונים.השאר אותו פועל במשך 10 דקות. בזהירות להעביר את הטחול שנקטף למיכל מלא מים deionized. לאחר מכן, חבר את צינור הסיליקון לצנתר ורידי שהוחדר לעורק הטחול.בצע זילוח עם מים נטולי יונים, 0.1% SDS, 1% Triton X-100 ופתרונות PBS. מלאו צינור צנטריפוגה של 50 מיליליטר ב-PBS המכיל 10% פניצילין סטרפטומיצין. העבר את מטריצת הטחול decellularized לתוך PBS.אחסנו אותו בטמפרטורה של מינוס 20 מעלות צלזיוס עד לניסויים נוספים. דה-צלולריזציה מלאה של הטחול הושגה תוך כ-11 שעות. הצבע עבר בהדרגה מאדום עמוק לאדום בהיר מודל, ובסופו של דבר מראה לבן שקוף, כאשר המורפולוגיה הכללית נותרה שלמה.

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 Views.  The First Affiliated Hospital of Xi'an Jiaotong University. כדי להתחיל, להקפיא ולהפשיר טחול חולדה קפוא שלוש פעמים כדי לשכב את תאי הטחול. על ספסל נקי הציבו משאבה פריסטלטית, מאגר של שני ליטרים, צינור סיליקון בקוטר פנימי של 2.4 מילימטרים ומלכודת בועות. מלאו את מערכת הזילוח במים נטולי יונים.השאר אותו פועל במשך 10 דקות. בזהירות להעביר את ...

Video: דה-צלולריזציה של טחול חולדה ליצירת מטריצת טחול להנדסת כבד

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 ...