Harvesting, Decellularization, and Pepsin Digestion of Bovine Lung Tissues for Decellularized Lung Extracellular Matrix Hydrogel Formation

1 To begin, remove the frozen bovine lung tissues2 from 80 degrees Celsius 3 and allow them to thaw at room temperature. 4 Dissect the tissue using sterile scalpels and scissors 5 to remove the trachea and cartilaginous airways, 6 then mint the tissue into small pieces. 7 Wash the tissue three times 8 with ultrapure water 9 containing 2%penicillin and streptomycin.

10 Immerse the minced tissue sample 11 in 2%iodine solution for one minute, 12 then perform two consecutive washes 13 in sterile distilled water. 14 Transfer the tissue pieces into a 50 milliliter tube 15 up to the 15 milliliter mark. 16 Fill the tube to the top with distilled water 17 and freeze it in liquid nitrogen for two minutes.

18 Then immediately transfer the tube to a beaker 19 containing 37 degrees Celsius water for 10 minutes. 20 After five freeze thaw cycles, 21 incubate the sample in 30 milliliters 22 of DNase solution for one hour 23 at 37 degrees Celsius with constant stirring. 24 Freeze the wet decellularized extracellular matrix samples 25 at 80 degrees Celsius for 24 hours.

26 Transfer frozen samples into a lyophilizer 27 and operate in drying mode under a vacuum 28 for three to four days until fully dried. 29 Following freeze drying, 30 mill the samples into a fine powder 31 in a grinding apparatus containing dry ice. 32 Treat the lung tissues with 1%Triton-X-100 33 for three days under gentle rotation 34 at four degrees Celsius, 35 changing the solution every 24 hours.

36 Incubate the sample in DNase solution 37 for one hour at 37 degrees Celsius with constant stirring. 38 Wash the tissue with distilled water 39 for three days under constant rolling, 40 replenishing the water every 24 hours. 41 After the last wash, perform lyophilization and cryo milling 42 as demonstrated previously.

43 Digest 15 milligrams per milliliter 44 powdered decellularized extracellular matrix samples 45 in one milligram per milliliter pepsin solution 46 at room temperature under constant stirring for 48 hours. 47 Maintain the solution pH at two for effective digestion. 48 Centrifuge the tissue digest at 5, 000 g for 10 minutes 49 and remove the supernatant.