rat spleen harvest for the preparation of decellularized spleen matrix

Decellulariseret rotte milt matrix forberedelse til levervævsteknik

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.

Forfatter Spotlight: Udnyttelse af decellulariseret miltmatrix til biokunstige lever

Fremstilling af decellulariseret miltmatrix afledt af rotter

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.

Milthøst af rotter til fremstilling af decellulariseret miltmatrix

Til at begynde med skal du placere en bedøvet barberet rotte i liggende stilling på et kirurgisk bord. Injicer to milliliter hepariniseret saltvand i penis dorsale vene til systemisk antikoagulation. Desinficer den barberede mavehud med povidon jodopløsning og dæk med en steril drapering klud.Lav et korsformet snit i maven med en kirurgisk saks. Strækning af bukhulen med hæmostatiske tang, vend leveren mod membranen, eksteriør mave-tarmkanalen til højre side af maven og dække den med fugtigt gasbind. Adskil forsigtigt og skær det splenogastriske ledbånd for at udsætte milthilumet.Disseker gradvist langs milthilum for at adskille og udsætte den fælles leverarterie, gastroduodenalarterie og miltarterie. Ligate og skære gastroduodenal arterie og fælles leverarterie, mens gradvist dissociere det omgivende væv. Vend milten mod højre side for at udsætte abdominal aorta.Udfør forsigtigt stump dissektion med vatpinde for at udsætte abdominal aorta og cøliakistammen. Placer en 3-0 silkesutur cirka tre centimeter lang over og under grene af cøliakistammen. Placer derefter en 10 centimeter lang 6-0 silkesutur ved grenen af cøliakistammen.Ligate abdominal aorta under og over grene af cøliakistammen. Lav derefter et lille snit i arteriegrenen. Løft forsigtigt 6-0 silkesuturen og indsæt et 24-gauge venekateter i miltarterien langs cøliakistammen.Ligate kateteret for at fastgøre det på plads. Brug en sprøjtepumpe til at infundere 50 ml hepariniseret normalt saltvand med en hastighed på fire ml pr. Minut. Afbryd samtidig portalvenen som en udstrømningskanal for at tillade den infunderede væske at strømme ud af milten.Dissekere forsigtigt miltens omgivende væv, undgå beskadigelse af bugspytkirtlen, samtidig med at de store tilbehørsbeholdere bevares. Kontroller for lækage omkring milten. Fjern derefter milten og bugspytkirtlen og skyl dem i normalt saltvand.Overfør milten til et 50 ml centrifugerør fyldt med normalt saltvand. Og opbevar den i en 80 grader Celsius fryser.

Research

JoVE Journal

Bioengineering

Rat Spleen Harvest for the Preparation of Decellularized Spleen Matrix

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 ...

Rat Spleen Decellularization to Create a Spleen Matrix for Liver Engineering

Rotte milt decellularisering for at skabe en miltmatrix til leverteknik