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 ....

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 bupr.......

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