原发性肾组织段的显微解剖与新型无支架结构技术的结合

The overall goal of this microscopic dissection procedure is to isolate discrete segments of living renal tissue with intact renal architecture for tissue engineering applications. This method can help answer key questions in the field of tissue engineering such as can whole segments of isolated primary organ tissue be harvested sterilely and be kept alive in culture? The main advantage of this technique is the segments contain intact renal architecture as opposed to using cell lines to recreate a complex organ with over 26 different cell types.

The implications of this technique extend towards therapy of end-stage renal disease because these constructs will improve filtration and may help patients stay off dialysis. During the surgery, wear proper surgical gear including a mask and a cap to minimize the risk of contamination. Likewise, make every effort to maintain sterility throughout.

Start preparations by draping the operating table. Next, open the pack of autoclaved instruments onto the sterile drapes. They must include three small hemostats, fine forceps with teeth, fine forceps without teeth, and straight iris scissors.

Cover the center of the operating table with a second non-fenestrated surgical drape. Prior to setting up the operating room, prepare 50 milliliters of antibiotic supplemented DBPS and aliquot five milliliters into a 15 milliliter tube. Now anesthetize an eight to 12-week-old C57 black six mouse and periodically monitor the level of anesthesia by assessing its pedal reflex with a firm toe pinch.

Next, use a hair removal cream to remove all of the hair from the ventral torso area and rinse off the cream and hair with distilled water. Then transfer the mouse to the operating table and place it supine under the non-fenestrated drape. Now cut out a two centimeter square fenestration in the drape over the mouse's abdomen.

Then scrub the exposed skin with povidone-iodine followed by 70%ethanol three times to sterilize the skin and complete the preparations for the surgery. Begin the surgery by using fine forceps with teeth and iris scissors. Make a three to four centimeter skin incision parallel to the midline one-half centimeter to the left of the midline.

Next, grasp the peritoneum using fine forceps without teeth and incise with iris scissors to enter the abdominal cavity. Then apply hemostats to the superior and inferior lateral edges of skin and peritoneum to place tension laterally and enhance the exposure. Now delicately shift the intestinal contents to the right of the abdomen to expose the left kidney.

Then gently place traction on the kidney to elevate it out of the abdomen. Now place a hemostat across the hilum of the kidney and use iris scissors to transect the ureter and renal vessels. Then transfer the kidney to the tube of 1%pen-strep DBPS on ice.

Take this tube to a sterile culture hood for processing. There transfer the kidney to a 60 millimeter Petri dish and rinse it twice with fresh five milliliters of DPBS containing calcium and magnesium. After the second wash, suspend the kidney in fresh five milliliters of DBPS in a new 60 millimeter dish.

Then prepare an additional Petri dish containing only five milliliters of DBPS for later use. Continue using sterile technique including sterile gloves, surgical mask, and bouffant to minimize contamination risks. Then drape the stage area and area surrounding the stereo microscope.

Next, place plastic adhesive sheets on the microscope focus knobs and spray them with 70%ethanol. Then turn on the dual gooseneck illuminator to light the stage. Now onto the sterile drapes, open a package of sterilized instruments containing fine forceps without teeth, a scalpel handle, a number 15 scalpel blade, two straight hemostats, and two 30.5 gauge hollow-bore needles.

Place the number 15 blade onto the scalpel handle and attach one hemostat to each needle adapter hub to create needle dissection instruments. Now transfer the two dishes, one with the kidney in DPBS and the other only DPBS, to the microscope area. Remove the lid on the kidney dish and focus the microscope on the anterior or posterior surface of the kidney at 3.2 to 4X magnification.

Next, with the non-dominant hand, use fine forceps without teeth to pierce and pin the kidney against the dish at the inferior and superior poles of the kidney. Then with the dominant hand, use the number 15 blade to remove the translucent fibrous capsular layer from the exposed surface. Shave the most superficial 0.5 millimeters from the exposed surface of the kidney surface to remove the remainder of the capsule.

Continue by using the number 15 blade to dissect an inverted pyramid of tissue from the de-capsulated area that is approximately two square millimeters. Then transfer the tissue into the clean dish of DBPS and discard the remainder of the kidney. Now focus on the dissected tissue and decrease the magnification to 1.5 to 2.0X.

Using the two hemostat needle instruments as cutting tools, slice the tissue fragment into progressively smaller pieces until the tissue segments are the size of the needle tips or smaller. About 50 segments should be produced. This is a critical portion of the procedure.

Each segment needs to closely approximate the diameter of the 30-1/2 gauge needle tip to avoid large segments which could hinder diffusion. Return the tissue to the tissue culture hood and remove the DPBS using a P1000 micropipette. Then add back five millimeters of DMEM supplemented with 10%fetal bovine serum and 1%pen-strep.

Then culture the tissue or implant it within cellular constructs immediately. The viability of the intact renal segments produced by the described procedure was examined over three days in culture using a viability assay. Green fluorescent calcium AM is present with intracellular esterase activity indicative of living cells.

Red fluorescent ethidium homodimer-1 is seen with loss of integrity of the plasma membrane. When the renal segments were embedded in scaffold-free endothelial fibroblast constructs, by day three they were incorporated with the cellular constructs to form intact structures. The constructs maintained their pre-vascular endothelial network shown by labeling with von Willebrand factor.

Renal epithelial cells can be seen in green labeled by cytokertain-18. To test in-vitro renal functionality, the constructs were incubated with FITC-labeled albumin. While there was residual albumin found away from the segments of embedded renal tissue, there were also hotspots in the renal tubular epithelial cells.

These hotspots are albumin that traverses intraluminally which is thought to represent albumin reuptake in the renal segment cellular construct. After watching this video, you should have a good understanding of how to isolate renal segments from murine kidneys for tissue engineering applications. Once mastered, this procedure can be done in one to 1-1/2 hours.

Following this procedure, other methods including seeding cellularized constructs can be performed in order to answer other questions regarding renal construct fabrication and characterization. Don't forget that working with surgical instruments and pressurized oxygen cylinders can be extremely hazardous. Be careful when working with sharp objects and remember to anchor oxygen tanks to a lab bench.