Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Summary

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146⁺ magnetic bead selection.

Abstract

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O₂, 5% CO₂ conditions. To deplete fibroblasts (CD146^-) and to ensure a population of only L-MSCs (CD146⁺), positive selection for CD146⁺ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Introduction

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. During development, including in the lung, the mesenchyme is an important source of developmental cues, and resident MSCs are a likely candidate to be at the center of this. Moreover, evidence is emerging that resident MSCs are perturbed in adult diseases, including cancer^1,2 and fibrosis³. The main hurdle for the study of....

Protocol

All procedures were approved by the Animal Care Committee of the University of Ottawa (animal ethics protocol OHRI-1696). Animal care was performed in accordance with institutional guidelines.

1. Isolation of Lung Mesenchymal Stromal Cells

1. Prepare the enzyme mix in a 50 ml tube: weigh in 30 U Neutral Protease, 2,500 U Collagenase I and 500 U DNAse I. These amounts suffice for the lungs of an adult mouse or rat pup. Prepare on day of isolation and store at 4 °C until use.
2. Sacrifice rat pups at day 13 by an intra-peritoneal injection of pentobarbital sodium (0.2 ml, 65 mg/ml). Use the toe pinch reflex to establish u....

Results

Two of the most reliable physical characteristics of MSCs, density and plastic adherence, are used in the first part of this protocol to obtain the mesenchymal cell fraction of the lung that contains L-MSCs. Although the density gradient interphase will include monocytes and macrophages in addition to lung mesenchymal cells, the plastic adherence followed by 3-5 days of culture ensures that only the lung mesenchymal cells remain. Indeed this cell population expresses the classic MSC surfa.......

Discussion

The isolation and culture of primary L-MSCs presents an opportunity to better understand their function and their interaction with other cell populations at a cellular level, and their role in lung development, health and disease. This is especially important as the lack of a specific single marker of these cells makes it nearly impossible to study these cells in situ. As with all primary cell populations, one should keep in mind that these cells are more likely to change their character the longer they are kept.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neutral Protease	Worthington Biochemical Corporation	LS02104	Prepare on day of isolation and store at 4 °C until use
Collagenase I	Worthington Biochemical Corporation	LS004196	Prepare on day of isolation and store at 4 °C until use
DNAse I	Sigma-Aldrich	D5025	Prepare on day of isolation and store at 4 °C until use
Pentobarbital sodium (Euthanyl)	Bimeda-MTC Animal Health Inc, Dublin, Ireland	-	Use 0.2 ml for rat pups between 20-30 g; larger animals may need more.
Dulbecco’s PBS + Sodium-pyruvate + Glucose	Life Technologies	14287-072	Very crucial to use this type of D-PBS, as the calcium and magnesium that are present in this D-PBS facilitate facilitate the enzymatic digestion
Ficoll-Paque PREMIUM (1.073 g/cm3)	GE Healthcare	17-5446-52	Density gradient media. To obtain a good interphase, it is crucial that the layering of the single cell suspension occurs at a >45° angle at very low speed, and that the subsequent centrifugation is done at 19 °C.
αMEM	Sigma-Aldrich	M8042	Warm in 37 °C water bath before use
200 mM L-Glutamine	Life Technologies	25030-164
100x Antibiotic-Antimycotic	Life Technologies	15240-062	Penicillin/Streptomycin/Fungizone
M-280 Dynabeads	Life Technologies	11205D	Magnetic beads
biotinylated polyclonal rabbit-anti-mouse IgG	Dako, Agilent Technologies	E0464	Biotinylated secondary antibody that matches with the anti-rat CD146 antibody described below
DynaMag5-magnet	Life Technologies	12303D	Magnet that is recommended for use with Dynabeads. NOTE: magnet should be chosen based on the manufacturer’s instructions of the magnet beads of choice.
TrypLE express	Life Technologies	12605-028	Gentle non-trypsin alternative, use 1 ml of TrypLE express/25 cm2 surface area. After detachment, 10 ml L-MSC culture medium is sufficient to inactivate 3 ml TrypLE
anti-rat CD146 antibody	Lifespan Biosciences Inc.	C35841	12.5 μl per 0.5 x 106 cells
Pentaspan (pentastarch solution)	Bristol-Myers Squibb Canada	-	Can be obtained through local blood donation services or hematology departments. Alternatively, one could use the PSI 20% Pentastarch solution (Preservation Solutions, PST001) diluted 1:1 with 0.9% NaCl.
Mr. Frosty freezing container	ThermoFisher Scientific	5100-0001	Improves viability when freezing L-MSCs overnight at -80 °C