Our aim is to develop an organotypic lung tissue model. Therefore, we have optimized the decellularization of bovine lung tissue and reconstitution of lung extracellular matrix hydrogels. In this study, we seek to understand the effect of different decellularization methods on the biochemical and mechanical characteristics of reconstituted lung hydrogels.
One of the main challenges of decellularization protocols is achieving mechanical stability in reconstitute hydrogels, which requires an understanding of the effects of decellularization on mechanical properties. These properties, such as stiffness and viscoelasticity, have crucial effects on cellular behaviors. We have established methodologies that effectively decellularize the bovine lung, yielding reproducible lung hydrogels that demonstrate noteworthy analogies with the extracellular matrix of the human lung.
Therefore, there is substantial promise in utilizing native lung dECM hydrogels for the purpose of disease modeling in the context of the lung. We engineered tissues to model homeostatic and disease conditions with a particular focus on cell matrix interactions. Therefore, we're interested in understanding the role of organotypic extracellular matrices, both in terms of unique content and mechanical aspects on cell behavior.
Our current research focuses on building biomimetic, patient-specific organoid models of cancer. To begin, remove the frozen bovine lung tissues from 80 degrees Celsius and allow them to thaw at room temperature. Dissect the tissue using sterile scalpels and scissors to remove the trachea and cartilaginous airways.
Then mince the tissue into small pieces. Wash the tissue three times with ultrapure water containing 2%penicillin and streptomycin. Immerse the minced tissue sample in 2%iodine solution for one minute.
Then perform two consecutive washes in sterile distilled water. Transfer the tissue pieces into a 50-milliliter tube up to the 15-milliliter mark. Fill the tube to the top with distilled water and freeze it in liquid nitrogen for two minutes.
Then immediately transfer the tube to a beaker containing 37 degrees Celsius water for 10 minutes. After five freeze thaw cycles, incubate the sample in 30 milliliters of DNA solution for one hour at 37 degrees Celsius with constant stirring. Freeze the wet decellularized extracellular matrix samples at 80 degrees Celsius for 24 hours.
Transfer frozen samples into a lyophilizer and operate in drying mode under a vacuum for three to four days until fully dried. Following freeze drying, mill the samples into a fine powder in a grinding apparatus containing dry ice. Treat the lung tissues with 1%Triton X-100 for three days under gentle rotation at 4 degrees Celsius, changing the solution every 24 hours.
Incubate the sample in DNA solution for one hour at 37 degrees Celsius with constant stirring. Wash the tissue with distilled water for three days under constant rolling, replenishing the water every 24 hours. After the last wash, perform lyophilization and cryomilling as demonstrated previously.
Digest 15 milligrams per milliliter powdered decellularized extracellular matrix samples in one milligram per milliliter pepsin solution at room temperature under constant stirring for 48 hours. Maintain the solution pH at 2 for effective digestion. Centrifuge the tissue digest at 5000g for 10 minutes and remove the supernatant.
Turn on the discovery HR-2 rheometer to implement oscillatory rheology measurements. Pour 250 microliters of pre-gel digest solution placed on ice onto a pre-cooled lower plate to avoid rapid gelation. Lower the 20-millimeter parallel plate until the pre-gel solution forms a disc with a one-millimeter gap width between the two plates.
Set the strain at 0.1%and frequency to 0.5 hertz. Measure storage and loss moduli over time while heating the lower plate to 37 degrees Celsius for 30 minutes to observe dilation kinetics. After the storage modulus value stops increasing and reaches a plateau, perform a creep recovery test to assess the stress relaxation behavior of hydrogels.
Apply one pascal sheer stress to the hydrogel for 15 minutes and measure the strain. Then unload the sample from the stress and record the change in strain values for 15 minutes. Finally, draw a strain versus time graph to show the stress relaxing behavior.
The average storage modulus and loss modulus of hydrogels produced by the freeze thaw method demonstrated significantly higher stiffness compared to hydrogels produced using the Triton X-100 method. Creep recovery tests revealed that hydrogels obtained from both methods exhibited distinct stress responses, indicating different viscoelastic properties.
