This method allows investigation of living human tissue in it's natural 3D structure and composition for the translation of key laboratory findings into potential therapies. This technique specifically enables the generation of tissue cultures from surgically resected or explanted human lung tissue for the visualization of individual deceased patient lung tissue samples. Disease-free lung tissue can be used as 3D tissue cultures to model pulmonary diseases ex vivo enabling the analysis of early pathomechanisms in a high spatiotemporal resolution.
Begin by placing the resected lung sample into 15 milliliters of cultivation medium in a sterile 15 centimeter culture dish and sterile metal tray covered in tissue paper, and fill a 30 milliliter syringe with 42 degrees Celsius low melting-point agarose. Remove the obturator from a peripheral venous catheter and attach the catheter to the 30 milliliter syringe. Identify a 0.5 to 3 millimeter diameter bronchus in the ventilating tissue in an intact section of the tissue, and gently insert the canula into bronchus as far as possible.
Use forceps to compress the bronchiole wall around the canula, ideally clamping any adjacent pulmonary artery at the same time, to seal the bronchus around the canula and use a surgical clamp to occlude any other additional airways, to prevent agarose leakage. Next, use the forceps to lift the lung tissue from the culture dish, and use the syringe to manually deliver the agarose through the canula, no faster than 0.3 milliliters per second. When the lung tissue is filled completely without over-inflating the tissue, immediately remove the canula and clamp the bronchus.
Submerge the tissue in culture medium at four degrees Celsius for 30 minutes to facilitate agarose solidification, and repeat the agarose filling procedure for any additional bronchiole openings. Then, store the agarose-filled lung tissue sections in four degrees Celsius medium until slicing. For precision cut lung slicing, identify the solidly agarose-filled regions within the lung tissue, that do not collapse when gently pressed with tweezers against the bottom of the cell culture dish.
Excise a 1 to 1.5 cubic centimeter block of solidly filled region with one side still covered with pleura, and use cyanoacrylate glue to attach the plural side of each tissue block to the vibratome holder. Slice the whole way through the long tissue block, until only two to three milometers of tissue are left unsliced. Using forceps to transfer each 500 micrometer section into one well of a twelve-well plate containing cultivation medium, as they are obtained.
When all the tissue has been sectioned, transfer the samples from each well into individual ten centimeter culture dishes and position a four millimeter biopsy puncher orthogonally to the upper surface of the precision-cut lung slice. Then, moving the puncher in clockwise and counterclockwise rotations, obtain tissue punches of the lung sample. Placing each punch in fresh cell culture medium in individual wells of a 96-well plate as they are obtained.
When all of the tissue punches have been obtained, place the plate in the cell culture incubator for up to five days. Immunolabeling of fibronectin in cell nuclei using Immunofluorescence in human 3-D lung tissue cultures allows imaging of the preserved alveolar structure ex vivo. Treatment of the human precision cut lung slice punches with a profibrotic cytokine cocktail for 48 hours results in fibrosis-like changes in human lung 3-D tissue cultures, including the significant induction of the fibrosis relevant extracellular matrix components, collagen Type One, and fibronectin genes.
Additionally, protein levels of the mesenchymal marker vimentin are up regulated in three out of four patients after treatment of 3-D lung tissue culture punches. Following this procedure, other methods like RNA analysis by quantitative real-time PCR, or protein analysis by western blotting, can be performed to evaluate gene and protein expression respectively within the tissue. After it's development, this technique paved the way for researches in the field of experimental pulmonology, to explant lung biology as well as to perform toxicological and pharmacological studies in human tissue samples.
Remember that live human tissue is potentially infective and that precautionary measures such as using personal protective equipment and proper tissue storage, transport and waste handling, should always be taken while performing this procedure
