This method can help answer essential questions in the skin tissue regeneration field which is how to replace the diverse cell types and structures that are contained in natural skin. The main advantage of this technique is that autologous full-thickness skin can be collected with minimal donor site morbidity. Demonstrating the procedure will be Bill Farinelli, who is a research associate in our lab.
Begin this procedure with setup of the production stage as described in the text protocol. Install two cutoff wheels concentrically onto the rotary tool. Use a smaller lower grid diamond cutoff wheel over a larger higher grid stone cutoff wheel.
Position an overhead light source with an adjustable arm over the rotary tool with the light aimed at the cutting wheels. To enhance visualization, position a dissecting microscope over the production setup so that the eyepiece is focused on the cutting discs. When reshaping the needle tip, wear protective eyewear and a surgical mask to prevent fine metal particles from entering the eyes or airways.
Choose hypodermic needles of the appropriate gauge size based on experimental requirements. Mark off the intended length on each harvesting needle. Lower the needle perpendicularly to the rotary tool with the power on using the edge of the outer cutting disc to cut off the excess length of the needle at the point marked.
Connect the shortened blunt needle to the female Luer lock connector on the production stage. Adjust the horizontal rotation stage so that the needle is at a 12 degree angle parallel to the cutting discs on the rotary tool. Turn on the overhead light and adjust its position while observing the needle under magnification until the light is reflected off the midline of the needle.
Power on the rotary tool. Then use the translation stage to advance the needle toward the inner cutting disc. Keep advancing the needle slowly against the cutting disc until the cutting disc reaches approximately the midline of the needle.
Slowly move the cut needle surface from the inner diamond wheel onto the outer stone wheel to finish the cut needle surface with a finer polish. Retract the needle away from the cutting disc. Using the vertical rotation stage, rotate the needle 180 degrees.
Repeat these steps to reshape the other side of the needle. Now remove the needle from the production stage. Clean the inside bore with a metal wire that is slightly smaller than the needle's inner diameter.
Using a sharp wooden stick, remove any burrs that may still be attached to the edges of the newly formed needle. When a high volume of microcolumns is needed, remove the plunger from a 20 milliliter syringe. Then attach the syringe to a suction adapter.
Complete the assembly by attaching a harvesting needle to the syringe. Use a piece of sterile suction tubing to connect the suction adapter to a sterile suction canister. Make sure the harvesting apparatus is connected to a canister input that allows fluid to flow into the canister unimpeded.
Now connect the apparatus to a negative pressure source. Harvest the microcolumns by inserting the harvesting needle into the skin. Intermittently dip the harvesting needle into a container of sterile saline during the harvesting procedure to flush the system.
Keep on hand a metal wire or a needle that is slightly smaller than the harvesting needle's inner diameter. If the needle becomes clogged, it can be cleared by inserting the metal wire into the needle bore. When the desired amount of microcolumns have accumulated in the canister, disconnect the device from suction.
Then pour the contents of the suction canister out through a filter to collect the microcolumns. Shown here is a finished harvesting needle viewed from the front and from the side. Note the two cutting points at the tip.
The harvesting needles should be able to collect microcolumns from full-thickness skin tissue with an approximate 80 to 90%success rate. In this figure, check marks represent one millimeter. Each microcolumn should contain epidermis, dermis, and some subcutaneous fat.
Microcolumns can be applied directly to wound healing or they may be combined with different matrix materials to produce combination constructs. Microcolumns can also be maintained in culture for in vitro studies. While performing high-volume harvesting, it's important to remember to incorporate an intermittent saline flush.
Though this method was designed for skin wound repair, a similar principle could be applied to other organ systems such as muscles where autologous tissue grafting is needed.
