Name: intratracheal instillation of stem cells in term neonatal rats
Uploaded: 2020-05-04T14:30:00
Description: Watch this Scientific Journal Video about Intratracheal Instillation of Stem Cells in Term Neonatal Rats at JoVE.com

This study was partly supported by a grant from Meridigen Biotech Co., Ltd. Taipei, Taiwan (A-109-008).

This procedure was approved by the Animal Care and Use Committee at Taipei Medical University.
NOTE: Human MSCs stably transfected with green fluorescent protein (GFP) and firefly luciferase genes (Fluc) were obtained from a commercial company (Table of Materials).
1. Characterization of human MSCs with firefly luciferase and green fluorescent protein
<ol>
	<li>Maintain human MSCs transfected with GFP and Fluc in complete media (minimum essential ...

<ol>
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Newborn infants with respiratory distress commonly require intratracheal surfactant and/or corticosteroid treatment19. Human phase I clinical trials have demonstrated the safety of intratracheal MSCs in preterm infants8. These studies suggest that intratracheal administration of drugs is an important option for newborn infants with respiratory distress. Animal model studies are most helpful if the model features are directly pertinent to humans. Term...

Supplemental oxygen is often required to treat newborn infants with respiratory distress1. However, hyperoxia therapy in infants has adverse long-term effects. Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD)2. BPD is a major complication of hyperoxia treatment that can occur in spite of early surfactant therapy, optimal ventilation procedures, and increased use of noninvasive positive pressure ventilation in premature infants. While many treatment strategies have been reported for BPD3, no known therapy can reduce this complication.
Corticosteroid use is one potential treatment to prevent BPD, because pulmonary inflammation plays a crucial role in its pathogenesis. However, systemic corticosteroid therapy is not usually recommended for preterm infants due to long-term adverse effects4,5.
Mesenchymal stromal cells (MSCs) have pluripotent characteristics and can differentiate into various cell types, including bone, cartilage, adipose tissue, muscle, and tendons6. MSCs have immunomodulatory, anti-inflammatory, and regenerative effects7, and animal studies show the therapeutic benefits of MSCs and their secreted components in hyperoxia-induced lung injury in rodents8,9. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and corticosteroid therapy might be a potential treatment strategy to treat newborns with respiratory disorders. Preclinical studies have used intratracheal administration of stem cells and adeno-associated virus in newborn rats10,11,12. However, a step-by-step presentation of the technique and in vivo tracking of the transplanted stem cells is not available. The newborn rat is appropriate for studying the effects of intratracheal administration on preterm infants with respiratory distress because the saccular stage of the rat lung at birth is equivalent to that of the human lung at 26&#8722;28 week of gestation13. An effective method for administration into the rat trachea is crucial for successful pulmonary distribution. The technique presented here allows for the study of intratracheal administration of cells and/or drugs for treatment of neonatal pulmonary diseases using rats as a model for humans.

<table>
	<tbody>
		<tr>
			<td>6-0 silk</td>
			<td>Ethicon</td>
			<td>1916G</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep Pad</td>
			<td>CSD</td>
			<td>3032</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Stemflow hMSC Analysis Kit</td>
			<td>BD Biosciences</td>
			<td>562245</td>
			<td>CD markers</td>
		</tr>
		<tr>
			<td>CMV-Luciferase-EF1&#945;-copGFP BLIV 2.0 Lentivector for In Vivo Imaging</td>
			<td>SBI</td>
			<td>BLIV511PA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor10</td>
			<td>BioLife Solutions</td>
			<td>640222</td>
			<td></td>
		</tr>
		<tr>
			<td>Human MSCs</td>
			<td>Meridigen Biotech Co., Ltd. Taipei, Taiwan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared light</td>
			<td>JING SHANG</td>
			<td>JS300T</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Halocarbon</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS-200 small animal imaging system</td>
			<td>Caliper LifeSciences, Hopkinton, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferin potassium salt</td>
			<td>Promega, Madison, WI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-scissors, straight</td>
			<td>Vannas</td>
			<td>H4240</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>TAIWAN BIOTECH CO., LTD.</td>
			<td>113531</td>
			<td>Isotonic Sodium Chloride Solution</td>
		</tr>
		<tr>
			<td>Small Hub RN Needle, 30 gauge</td>
			<td>Hamilton Company, Reno, NV</td>
			<td>7799-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (100 &#181;l)</td>
			<td>Hamilton Company, Reno, NV</td>
			<td>81065</td>
			<td></td>
		</tr>
		<tr>
			<td>Xenogen Living Image 2.5 software</td>
			<td>Caliper LifeSciences, Hopkinton, MA</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

intratracheal instillation of stem cells in term neonatal rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

The pulmonary distribution of intratracheal instillation of stem cells in the term neonatal rats was determined by firefly luciferase (Fluc)-labeled stem cells. MSCs were labeled with Fluc and tagged with green fluorescent protein through lentiviral transduction. Figure 1A demonstrates a high level of GFP expression in human MSCs, and 93.7% of the population showed GFP positive expression detected by flow cytometry. MSCs were characterized by analyzing the expression of CD markers (i.e., CD ...

Watch this Scientific Journal Video about Intratracheal Instillation of Stem Cells in Term Neonatal Rats at JoVE.com

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.

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

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.

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