Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.
Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.
1. Coating of plates with ECM proteins
2. Isolation of fetal rodent lung type II epithelial cells
The day before isolation have the screen cups with different size nylon meshes autoclaved and ready.
3. Isolation of fetal rodent lung fibroblasts
4. Experimental system to provide mechanical stimulation to lung cells
5. Representative Results
Figure 1 and Figure 2 show representative phase-contrast photographs of E18 fetal mouse type II cells isolated using the technique described in this manuscript.
Figure 3 demonstrates that mechanical strain induces differentiation of fetal type II epithelial cells using surfactant protein-C as a marker.
Figure 1. Representative phase-contrast photograph taken right after isolation showing the clumped appearance of fetal type II cells.
Figure 2. E18 fetal type II epithelial cells were isolated as described here and plated on bioflex plates coated with laminin. Photograph was taken the following day after non-stretched cells were fixed in paraformaldehyde. The purity of the cells was determined to be 90 ± 5% by microscopic analysis of epithelial cell morphology and immunostaining for SP-C.
Figure 3. Fetal type II epithelial cells were exposed to 5% cyclic strain at 40 cycles/min for 16h. A) Northern blot of surfactant protein C (SP-C) mRNA expression showing that strain induces type II cell differentiation using different ECM substrates. +/- signs represent exposure or not to strain, respectively. Data are presented as mean +/- SEM, n=3; * P<0.05. B) Fluorescence immunocytochemistry images demonstrating SP-C protein levels (green) in fetal type II cells exposed or not (control) to mechanical strain . Nuclei were counterstained with dapi (blue). Bar, 10 μm. C) Western blot results from three experiments showing that mechanical stretch increases SP-C protein. N=3; * P<0.05.
In this manuscript, we describe a method to isolate fetal type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using the Flexcell Strain Apparatus. We have used this technique to assess differentiation of epithelial cells 1,2 and to study receptor and signaling pathways activated by stretch 3-9. In addition, this method can also be used to investigate cell responses induced by mechanical injury 10,11. The procedure is based on digestion of the lung tissue with collagenase. It takes advantage of the tendency of type II cells to clump together after digestion. Important steps while performing this technique are mincing the tissue well to facilitate digestion of the lung and washing thoroughly the non-filtered cells on 30 and 15 micron meshes to facilitate the filtration of non-epithelial cells and therefore to achieve greater purity of type II cells.
Our laboratory uses membranes coated with collagen or laminin to simulate the ECM composition of the basement membranes. However, when cells are exposed to 20% elongation, cells might detach from the substrate during stretch and fibronectin should be considered as an alternative substrate.
Another important consideration is to avoid 100% confluence of cell monolayers prior to stretch, given that this may promote cell-to-cell contact rather than cell-to-matrix contact and therefore cells might not “sense” the percentage of elongation set up by the protocol.
The Flexcell Strain Unit uses a vacuum to deform a flexible bottom culture plate. Application of vacuum stretches each membrane over the central cylinder post, creating uniform radial and circumferential strain across the membrane surface and therefore providing equibiaxial distension, similar to in vivo conditions (see cartoon, schematic overview video, with permission, Dr. A.J. Banes Flexcell International Corporation, www.flexcellint.com). This system provides a defined, controlled static or cyclic deformation by the specified strain regimen. To mimic fetal breathing movements a cyclic stretch regimen between 2.5-5% stretch at 40-60 cycles/minute is applied. There is not agreement on the percentage of elongation that fetal lung senses during fetal breathing. Some authors believe that 5% distension is appropriate 12 whereas other investigators argue that 2.5% is more representative of the in vivo conditions 13,14. To simulate constant distension pressure, a 2.5% continuous stretch protocol is used. To mimic lung injury, 20% cyclic stretch at 40 cycles/min is used. Potential limitations of this technique include the increase of deformation of cells seeded in the area of the membrane peripheral to the loading post. Another limitation is the inability to provide elongation greater than 15-20% if a high cycle rate is used (above 40 cycles/min).
The authors have nothing to disclose.
Supported by NIH grant HD052670.
Name of the reagent | Company | Catalogue number | Comments |
DMEM | Sigma | D5648 | |
HEPES | Sigma | H3375 | |
Collagenase 1 | Sigma | C0130 | |
Collagenase 1A | Sigma | C9891 | |
Chicken serum | Sigma | C5405 | |
Screen cups | Sigma | CD1-1KT | |
Syringe filters | Fisher Scientific | 09-754-25 | |
100 micron nylon mesh | Small Parts, INC | CMN-100-D | |
30 micron mesh | Small Parts, INC | CMN-30-D | |
15 micron mesh | Dynamic Aqua-Supply Ltd. | NTX15 | |
Laminin | Sigma | L2020 | |
Collagen-1 | Collagen Biomed | PC0701 | |
Fibronectin | Sigma | F1141 | |
Vitronectin | Sigma | V-0132 | |
Elastin | Sigma | E-6402 | |
Bioflex plate | Flexcell International | BF-3001U | Uncoated |
Flexcell Strain Unit | Flexcell International | FX-5000 |