These methods can help address key questions in the biomaterials field such as the development of a scaffold with excellent fluid transfer capabilities with sophisticated ultra-structures and scalable manufacturing. The main advantage of these techniques is that they can be applied to most plants for stem tissues without the need for specialized equipment. First, collect fresh or frozen samples of F.hispida leaves.
Store any remaining fresh sample at minus 20 degrees Celsius for future use. Let the leaf remain emersed in deionized water at 20 to 25 degrees Celsius. Use a clean biopsy punch to cut the leaf into eight millimeter diameter discs.
Next, to wash the leaf samples, incubate them on a shake plate at low speed for five to 10 minutes, emersed in deionized water. Then prepare 10%weight by volume of SDS in deionized water. After preparing the SDS solution, place the leaf samples on a plastic dish.
Then add the SDS solution to completely cover the samples. Place the SDS emersed leaf samples, covered, on a shake plate to prevent evaporation. Then incubate the leaf sample at room temperature for five days.
After five days of incubation, replace the SDS solution with deionized water. Incubate the samples soaked in deionized water for 10 to 15 minutes on a shake plate. Next, mix five milliliters of non-ionic surfactant with 50 milliliters of bleach.
Add this mixture to 445 milliliters of deionized water. Then emerse the samples in the freshly prepared non-ionic surfactant bleach solution. Continue changing the non-ionic surfactant bleach solution every 24 hours till the samples are cleared.
Then leave the cleared samples emersed in deionized water for two minutes on a shake plate. For detergent free decellularization, prepare five percent volume by volume bleach in 3%weight by volume sodium bicarbonate solution. Next, warm the solution to 60 to 70 degrees Celsius on a hot plate within a fume hood.
As soon as the solution reaches the desired temperature, soak the leaf samples. Then reduce the speed of the stir plate to prevent damaging the samples and look for the visibly cleared samples and transfer them from the bath carefully. Finally, incubate the samples in deionized water for one to two minutes.
As all the samples are intrinsically different, it's recommended to check on the progress of decellularization approximately every 15 minutes to ensure the samples are not being damaged. MTM, SAF, and UTS parameters are first studied to investigate the unique decellularized scaffold properties of both P.aquatica and F.hispida species. In fact, all three parameters display similar mechanical properties using both the detergent based and detergent free decellularization methods.
However, the UTS mechanical property of F.hispida shows a significantly higher average value using SDS rather than bleach for decellularization. The genomic DNA content of the cleared plant species studied is also significantly reduced upon decellularization when compared to the non-decellularized samples. Next, the metabolic activity of human dermal thyroblasts is also measured in the presence of decellularized P.aquatica or Garcinia scaffolds.
Interestingly, detergent free based scaffolds elicited a reduced impact on cellular metabolic activity, but washing with Trishydrochloricde buffer, followed by serum-free medium after SDS clearing decreases the impact on cellular metabolic activity When compared to washing with deionized water alone. Finally, growth of the mesenchymal stem cells stained with calcine on the surface of the F.hispida leaf structure is shown here. Once mastered, these techniques can be completed from around seven days to as little as 15 minutes when performed properly.
When performing these procedures, it's important to remember to monitor the samples as they decellularize because the samples can vary from batch to batch which can result in different clearing times. Following this procedure, other methods such as dequantification or mechanical testing can be applied to answer additional questions such as to what extent are the samples cleared and to what other tissues the samples are mechanically similar to. After its development, this technique has helped pave the way for researchers in the field of tissue engineering to manufacture perfusable scaffolds.
After watching this video you should have a good understanding of how to decellularize plant tissues for use as scaffolds. Don't forget that working with detergents and bleach fumes can be hazardous and precautions such as using proper protective equipment and using a fume hood should always be taken while performing these procedures.
