Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Summary

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.

Abstract

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 26−28 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.

Introduction

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 k....

Protocol

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

1. Maintain human MSCs transfected with GFP and Fluc in complete media (minimum essential .......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
6-0 silk	Ethicon	1916G
Alcohol Prep Pad	CSD	3032
BD Stemflow hMSC Analysis Kit	BD Biosciences	562245	CD markers
CMV-Luciferase-EF1α-copGFP BLIV 2.0 Lentivector for In Vivo Imaging	SBI	BLIV511PA-1
CryoStor10	BioLife Solutions	640222
Human MSCs	Meridigen Biotech Co., Ltd. Taipei, Taiwan
Infrared light	JING SHANG	JS300T
Isoflurane	Halocarbon	26675-46-7
IVIS-200 small animal imaging system	Caliper LifeSciences, Hopkinton, MA
Luciferin potassium salt	Promega, Madison, WI
Micro-scissors, straight	Vannas	H4240
Normal saline	TAIWAN BIOTECH CO., LTD.	113531	Isotonic Sodium Chloride Solution
Small Hub RN Needle, 30 gauge	Hamilton Company, Reno, NV	7799-06
Syringe (100 µl)	Hamilton Company, Reno, NV	81065
Xenogen Living Image 2.5 software	Caliper LifeSciences, Hopkinton, MA	N/A