A kind of decellularized submandibular gland matrix material and its preparation method
A matrix material and submandibular gland technology, applied in the field of decellularized submandibular gland matrix material and its preparation, can solve the problems of extracellular matrix biotoxicity, damage to the structural integrity and biological activity of cell matrix, unfavorable three-dimensional structure reconstruction, etc., and achieve good cell differentiation The effect of proliferation environment, simple method and good mechanical properties
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Embodiment 1
[0040] Example 1 Production of mouse submandibular gland ECM
[0041] (1) Material collection: the submandibular gland tissues of healthy mice were selected, cut into small pieces of 1 cm square, and washed twice with PBS. As for the EP tube, store it in a -80°C refrigerator for later use.
[0042] (2) Broken cells: Take out the tissue block from the -80°C refrigerator, thaw it in a 37°C water bath, and then put it back into the -80°C refrigerator for 30 minutes, and repeat the freezing and thawing process 3 times.
[0043] (3) Disinfection and preparation: Place the tissue block in 75% alcohol, suspend the submandibular gland tissue block with sutures, hang it in a 250ml jar, and place a 1cm magnetic stirring bar at the bottom of the bottle.
[0044] (4) Pre-cleaning and crushing: add 250ml of sterile third-grade water, and treat on a magnetic stirrer (gear 2) at 4°C for 6 hours.
[0045] (5) Clean and enhance nuclease enzyme activity: replace the submandibular gland tissue...
Embodiment 2
[0056] Embodiment 2, the making of rat submandibular gland ECM
[0057] (1) Material collection: select submandibular gland tissues of healthy rats, decompose them into small pieces of 1 cm square, and wash them twice with PBS. As for the EP tube, store it in a -80°C refrigerator for later use.
[0058] (2) Broken cells: Take out the tissue block from the -80°C refrigerator, thaw it in a 37°C water bath, and then put it back into the -80°C refrigerator for 30 minutes, and repeat the freezing and thawing process 3 times.
[0059] (3) Disinfection and preparation: Place the tissue block in 75% alcohol, suspend the submandibular gland tissue block with sutures, hang it in a 250ml jar, and place a 1cm magnetic stirring bar at the bottom of the bottle.
[0060] (4) Pre-cleaning and crushing: add 250ml of sterile third-grade water, and treat on a magnetic stirrer (gear 2) at 4°C for 12 hours.
[0061] (5) Clean and enhance nuclease enzyme activity: replace the submandibular gland ...
Embodiment 3
[0068] Example 3, the application of mouse submandibular gland ECM in the study of submandibular gland tissue remodeling
[0069] (1) Prepare cells: take two C57 mouse salivary glands and soak them in 75% ethanol for 15 seconds, rinse twice with pre-cooled 1×PBS, and cut them into 2mm pieces 3 sized pieces. Add 3ml dispase and digest in a 37°C water bath for 60min. Rinse twice with pre-cooled 1×PBS (centrifuge at 1000rpm for 4min, add 10ml 1×PBS and stir at 4°C for 10min). The submandibular gland cells were collected by filtration with a 40um filter membrane.
[0070] (2) Cell injection: the mouse submandibular gland ECM prepared in Example 1 was fixed on a new wax plate with a sterile pin in a clean bench. Use an alcohol lamp to pull the Pasteur tube into a glass needle with a diameter of about 100 μm, and draw the primary cells of the submandibular gland isolated in step (1) and inject them into the ECM tissue of the submandibular gland.
[0071] (2) Culture: Put the sub...
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