Determining The Optimal Protocol Of Decellularizaiton To Prepare Decellular Lung Scaffolds

Lung transplantation is the only effective treatment for end-stage lung disease.However,shortage of donor lung restrict the development of clinical lung transplantation.Artificial lungs that used by engineering technology could be a feasible way to solve the above problem.Decellularized lung scaffold(DCLS)is an ideal scaffold for artificial tissue engineering lung.Lung is characterized by loose structure,airway and vascular two-tube systems.What kind of acellular method can be used to obtain ideal effects has been an important research topic in the project of lung organ engineering.There are a variety of methods to prepare organ decellularized scaffolds.The preparation of DCLS usually adopts the solution of descaling agent.For example,SDS、CHAPS、or Triton-X 100 and so on.The reagent,CHAPS,represented by zwitterionic detergent,has good effect of decellularization,and can preserve collagen and maintain the performance of ECM.However,but it tends to destroy collagen fibers and has poor preservation effect on GAG.SDS,as an ionic decellular agent,can enhance the dissolution of cell membranes and nuclear membranes and cause protein denaturation.The nuclear material and cytoplasmic protein can be effectively removed from the dense tissue,resulting in ultrastructure damage,removal of GAG and growth factors,and injury of collagen.EDTA-2Na,represented by chelating agent,can disrupt connections between cells and ECM,and the effect is poor when used alone,but it is often used in combination with enzymes or with other agents.As a physical cell removal method,froze/melted method can effectively dissolve tissue or organ cells,but the cell membrane and cell contents still exist,and will not cause the loss of ECM protein.The experiment first compared the HE staining of the lavage lung tissues in the experimental group with detergent and the control group without detergent at first.Then,four methods including SDS,CHAPS,EDTA-2Na and froze/melted Plus SDS were used to prepare DCLS in the present study.The effect of these four agents on the removal of all cellular and nuclear material was compared.And the effect on the retaining extracellular matrix structure and composition was compared too.At last,The effect of concentration on the decellularization of SDS and CHAPS was explored.[Objective] 1、HE staining of lung tissue was compared between the experimental group and the control group.2、To compare the decellularizaiton effect and the integrity of DCLS prepared by four different methods: SDS,CHAPS,EDTA-2Na and froze/melted Plus SDS.3、To explore the effect of concentration on the decellularization of SDS and CHAPS.[Methods] 1、Lung tissue were lavaged with reagents from the experimental group(containing detergent)and the control group(no detergent)respectively,and the lung tissue after lavage were compared by HE staining.2、Different concentrations of SDS、CHAPS、EDTA-2Na perfusion solutions were prepared.The slices of DCLS were stained using Hematoxylin-Eosin staining 、 Masson staining 、 Elastic Van Gieson staining and immunochemistry,Determining which reagent has the best decellurization effect by comparing the result of various staining methods.3、Three different concentrations of SDS(0.1%SDS、0.2%SDS、0.3%SDS)and CHAPS(4mmol/LCHAPS、8 mmol/LCHAPS、12 mmol/LCHAPS)were used to perfuse lung tissues respectively,and the optimal concentration and reagent were selected to prepare the extracellular lung scaffold by comparing the immunofluorescence staining of collagen 3 and fibronectin in the lung tissues after lavage and determing the residual nucleic acid content in the acellular lung tissues of each group.[Results] 1、Both HE staining of the acellular lung tissue of the control group and the experimental group were intact after lavage,while the nuclei of the experimental group were almost completely exfoliated,and the nuclei of the control group were intact.2、All the four methods,including SDS,CHAPS,EDTA-2Na and froze/melted Plus SDS,can achieve better acellular effect.The results from Hematoxylin-Eosin staining,Masson staining,Elastic Van Gieson staining and immunochemistry showed that the structure integrity and composition of DCLS prepared with CHAPS perfusion is the best.3、The result of immunofluorescence staining and determination of nucleic acid residue in the acellular lung tissue showed that the 4mmmol/L CHAPS group is the optimal group of acelllar lung tissue group.[Conclusions] Perfusion with 4mmmol/L CHAPS solution is the optimal protocol to prepare DCLS in our present condition.