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>
	<li>Ramanathan, R., Bhatia, J. J., Sekar, K., Ernst, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+in+preterm+infants+with+respiratory+distress+syndrome+treated+with+poractant+alfa,+calfactant+or+beractant:+a+retrospective+study.">Mortality in preterm infants with respiratory distress syndrome treated with poractant alfa, calfactant or beractant: a retrospective study.</a> Journal of Perinatology. 33, 119-125 (2013).</li><li>Gien, J., Kinsella, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+treatment+of+bronchopulmonary+dysplasia.">Pathogenesis and treatment of bronchopulmonary dysplasia.</a> Current Opinion in Pediatrics. 23, 305-313 (2011).</li><li>Pasha, A. B., Chen, X. Q., Zhou, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchopulmonary+dysplasia:+Pathogenesis+and+treatment.">Bronchopulmonary dysplasia: Pathogenesis and treatment.</a> Experimental and Therapeutic. 16, 4315-4321 (2018).</li><li>Committee on Fetus and Newborn. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+corticosteroids+to+treat+or+prevent+chronic+lung+disease+in+preterm+infants.">Postnatal corticosteroids to treat or prevent chronic lung disease in preterm infants.</a> Pediatrics. 109, 330-338 (2002).</li><li>Watterberg, K. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+stromal+cells+as+stem+cells+for+nonhematopoietic+tissues.">Marrow stromal cells as stem cells for nonhematopoietic tissues.</a> Science. 276, 71-74 (1997).</li><li>Nemeth, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+attenuate+sepsis+via+prostaglandin+E(2)-+dependent+reprogramming+of+host+macrophages+to+increase+their+interleukin-10+production.">Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)- dependent reprogramming of host macrophages to increase their interleukin-10 production.</a> Nature Medicine. 15 (2), 42-49 (2009).</li><li>Chou, H. C., Li, Y. T., Chen, C. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+transplantation+of+mesenchymal+stem+cells+simultaneously+attenuates+both+lung+and+brain+injuries+in+hyperoxic+newborn+rats.">Intratracheal transplantation of mesenchymal stem cells simultaneously attenuates both lung and brain injuries in hyperoxic newborn rats.</a> Pediatric Research. 80, 415-424 (2016).</li><li>Fleurence, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+efficacy+of+intratracheal+adeno-associated+virus+administration+to+newborn+rats.">Comparative efficacy of intratracheal adeno-associated virus administration to newborn rats.</a> Human Gene Therapy. 16, 1298-1306 (2005).</li><li>Waszak, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+intratracheal+adenoviral+vector+administration+on+lung+development+in+newborn+rats.">Effect of intratracheal adenoviral vector administration on lung development in newborn rats.</a> Human Gene Therapy. 13, 1873-1885 (2002).</li><li>O'Reilly, M., Th&#233;baud, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+bronchopulmonary+dysplasia.+The+term+rat+models.">Animal models of bronchopulmonary dysplasia. The term rat models.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 307, 948 (2014).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+labeling+and+hepatic+differentiation+potential+of+human+placenta-derived+mesenchymal+stem+cells.">GFP labeling and hepatic differentiation potential of human placenta-derived mesenchymal stem cells.</a> Cellular Physiology and Biochemistry. 35, 2299-2308 (2015).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 315-317 (2006).</li><li>Zhang, Z. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+osteogenic+capacity+for+bone+tissue+engineering+of+fetal+compared+with+perinatal+and+adult+mesenchymal+stem+cells.">Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells.</a> Stem Cells. 27, 126-137 (2009).</li><li>Huang, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+surfactant+and+budesonide+on+pulmonary+distribution+of+fluorescent+dye+in+mice.">Effect of surfactant and budesonide on pulmonary distribution of fluorescent dye in mice.</a> Pediatric and Neonatology. 56, 19-24 (2015).</li><li>Keyaerts, M., Caveliers, V., Lahoutte, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging:+looking+beyond+the+light.">Bioluminescence imaging: looking beyond the light.</a> Trends in Molecular Medicine. 18, 164-172 (2012).</li><li>Yeh, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-tracheal+administration+of+budesonide/surfactant+to+prevent+bronchopulmonary+dysplasia.">Intra-tracheal administration of budesonide/surfactant to prevent bronchopulmonary dysplasia.</a> American Journal of Respiratory and Critical Care Medicine. 193, 86-95 (2016).</li><li>Nguyen, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+inoculation+of+Fischer+344+rats+with+Francisella+tularensis.">Intratracheal inoculation of Fischer 344 rats with Francisella tularensis.</a> Journal of Visualized Experiments. (127), e56123 (2017).</li><li>de Almeida, P. E., van Rappard, J. R., Wu, J. 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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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5.5K 次观看.  Taipei Medical University. 内切注射是一种临床生物技术，用于将干细胞和药物移植到新生儿肺部，以评估Chilman的疗效。这种技术在新生儿大鼠的各种初级应用中可能非常适用，是一种相对容易且具有成本效益的肺病治疗措施。产后第五天，在60度角的插管站上，对斯普拉格-道利大鼠幼崽进行麻醉。并在每只小狗的所有四肢上贴上实验室标签胶带，以确保它们就位。在每个鼻子下面涂抹胶带来固...