Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Intratracheal instillation is a clinical biotechnical for delivering stem cells and drugs into a neonatal lung to evaluate Chilman efficacy. This technique may be bona fide for use with a variety of primary application in a neonatal rat and is a relative easy and cost-effective measure for pulmonary disease treatment. On postnatal day five, restrain anesthetized Sprague-Dawley rat pups, on a 60 degree-angled intubation stand.

And affix laboratory labeling tape to all four limbs of each pup to secure them in place. Apply tape below each nose to fix the heads. And pinpoint the first neck for puncture tracheotomy.

Use a 75%alcohol prep pad to disinfect the incision area. And make a vertical 3 centimeter midline incision above the trachea to avoid damaging the carotid arteries. Use curved tapered tweezers without a hook to dissociate away the fat and muscle layers to locate the trachea.

When the trachea can be observed, use the tweezers to grasp the trachea and use a 100 microliter syringe equipped with a 30 gauge needle to inject one times ten of the fifth firefly luciferase GFP labeled mesenchymal stromal cells in 30 microliters of saline into the trachea, during the inspiratory phase. When all of the cells have been injected, close the incision with 160 silk stitch tying the knot as tightly as possible. And cutting the ends of the suture as short as possible.

Then allow the pup to recover from the anesthesia, in a warm spot with monitoring until it is warm, pink, and capable of spontaneous movement before returning the animal to its cage. To track the transplanted human mesenchymal stromal cells 15 minutes after the rats have recovered from the surgery, intraperitoneally inject the re-anesthetized pups with 125 milligrams per kilogram of Luciferian potassium salt in PBS. 10 minutes after the injection, use a small animal imaging system to acquire sequential images at 5-15 second intervals with a medium binning, a 1 f-stop, and a 26 centimeter field of view.

Then use the imaging software to quantify the luminescence activity from the lungs based on the automatic regions of interest. A high level of GFP expression is observed in luciferase firefly labeled human mesenchymal stem cells. Mesenchymal stem cells can be further characterized by their cell surface marker expression and their ability to differentiate into osteocytes, chondrocytes, and adipocytes.

Luminescence imaging of the transplanted human mesenchymal cells in VIVO, reveals an absence of luminescent signaling in the lung regions of rats treated with normal saline. In rats treated with mesenchymal stem cells, luminescence is observed within the trachea and central lung regions of those animals. Indeed, in this representative analysis, quantification of the luminescence intensity revealed that rats treated with mesenchymal stem cells exhibited an approximately 13 fold increase in luminescence activity compared to rats treated with normal saline.

When attempting this procedure, it is important to dissect and isolate the trachea without limiting the surrounding arteries. This technique allows study of the effects of intratracheal administration of cells and drugs of neonatal Palmer disease using rays with the mode of human patient.