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. 2015 Jun;3(6):821-32.
doi: 10.1039/c5bm00034c. Epub 2015 Mar 27.

In Vitro Model Alveoli From Photodegradable Microsphere Templates

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Free PMC article

In Vitro Model Alveoli From Photodegradable Microsphere Templates

Katherine J R Lewis et al. Biomater Sci. .
Free PMC article

Abstract

Recreating the 3D cyst-like architecture of the alveolar epithelium in vitro has been challenging to achieve in a controlled fashion with primary lung epithelial cells. Here, we demonstrate model alveoli formed within a tunable synthetic biomaterial platform using photodegradable microspheres as templates to create physiologically relevant, cyst structures. Poly(ethylene glycol) (PEG)-based hydrogels were polymerized in suspension to form microspheres on the order of 120 μm in diameter. The gel chemistry was designed to allow erosion of the microspheres with cytocompatible light doses (≤15 min exposure to 10 mW cm(-2) of 365 nm light) via cleavage of a photolabile nitrobenzyl ether crosslinker. Epithelial cells were incubated with intact microspheres, modified with adhesive peptide sequences to facilitate cellular attachment to and proliferation on the surface. A tumor-derived alveolar epithelial cell line, A549, completely covered the microspheres after only 24 hours, whereas primary mouse alveolar epithelial type II (ATII) cells took ∼3 days. The cell-laden microsphere structures were embedded within a second hydrogel formulation at user defined densities; the microsphere templates were subsequently removed with light to render hollow epithelial cysts that were cultured for an additional 6 days. The resulting primary cysts stained positive for cell-cell junction proteins (β-catenin and ZO-1), indicating the formation of a functional epithelial layer. Typically, primary ATII cells differentiated in culture to the alveolar epithelial type I (ATI) phenotype; however, each cyst contained ∼1-5 cells that stained positive for an ATII marker (surfactant protein C), which is consistent with ATII cell numbers in native mouse alveoli. This biomaterial-templated alveoli culture system should be useful for future experiments to study lung development and disease progression, and is ideally suited for co-culture experiments where pulmonary fibroblasts or endothelial cells could be presented in the hydrogel surrounding the epithelial cysts.

Figures

Fig. 1
Fig. 1
Schematic of the overall cyst-forming procedure, cross-sectional view. (i) Bioadhesive, photodegradable microspheres (orange) were (ii) incubated with epithelial cells ( purple) to coat the surface of the microspheres with cells. The cell-microsphere pre-cyst structures were (iii) encapsulated in a second bioadhesive hydrogel (blue), followed by (iv) erosion of the microsphere template with 365 nm light, leaving a hollow epithelial cyst inside the encapsulating hydrogel.
Fig. 2
Fig. 2
Microsphere synthesis and erosion. (A) The microsphere hydrogel network was composed of poly(ethylene glycol) tetrathiol (PEG4SH; Mn ~ 5 kDa) and poly(ethylene glycol) di-photodegradable acrylate (PEGdiPDA; Mn ~ 4.1 kDa), with the bioadhesive peptide CRGDS included at 1.5 mM. The monomers were reacted via an inverse suspension polymerization using a base-catalyzed Michael addition to form the microspheres. (B) Microspheres labeled with a green fluorescent dye were imaged and analyzed to determine the size distribution. Histogram shows distribution of microsphere diameters, and the black line indicates the cumulative percentage of the population. Inset shows representative fluorescent image of microspheres used for diameter measurement. n = 3087 (C) The chemical structure indicates the cleavage of the nitrobenzyl ether moiety with light. The absorbance spectrum is for the PEGdiPDA crosslinker (0.8 mM) in phosphate-buffered saline, where the blue line indicates 365 nm. The molar extinction coefficient at 365 nm for the nitrobenzyl ether moiety was calculated to be ~5000 L mol−1 cm−1. (D) Microspheres containing entrapped 2 μm diameter polystyrene beads were encapsulated in a second hydrogel and exposed to 365 nm light at an intensity of ~10 mW cm−2 for 15 minutes to erode the microspheres. Images are of polystyrene bead tracks over 15 minutes before and after light exposure illustrating increased Brownian motion after microsphere erosion. The plot gives the ensemble mean squared displacement for the liquid and solid cases.
Fig. 3
Fig. 3
Encapsulating hydrogel formulation. (A) The encapsulating gel was composed of 8-arm poly(ethylene glycol) functionalized with norbornene end groups (Mn ~ 40 kDa) and an enzymatically-cleavable di-cysteine peptide crosslinker (KCGPQG↓IWGQCK), with the adhesive peptide CRGDS included at 1 mM. The arrow indicates the enzymatic cleavage site. Thiol groups (red) react with the -ene functionalities on the 8-arm PEG through a radical-initiated thiol–ene polymerization. (B) Single cell suspension of A549 cells encapsulated in the thiol–ene gel. The image is a z-projection of a 250 μm confocal stack showing healthy actin cytoskeleton (red) and cell nuclei (blue). (C) Single cell suspension of A549 cells encapsulated in thiol–ene gels. The images are z-projections of a 500 μm confocal stack with live cells stained green and dead cells stained red. The first is a representative image of a gel left in the dark and stained on day 1 after encapsulation (93 ± 3% live). The second is a representative image of a gel exposed to 15 minutes of 365 nm light at ~10 mW cm−2 on day 1 after encapsulation and stained 1 hour later (90 ± 4% live). The third is a representative image of a gel exposed to 15 minutes of 365 nm light at ~10 mW cm−2 on day 1 after encapsulation and stained 1 day later (90 ± 4% live).
Fig. 4
Fig. 4
Schematic illustration of the three conditions that epithelial cells experience during the cyst-forming procedure, cross-sectional view. (i) First, cells form integrin binding sites with the CRGDS peptide in the microsphere network. (ii) Then, pre-cysts are encapsulated within the thiol–ene gel and cells form attachments to the CRGDS in the encapsulating gel. (iii) Finally, microspheres are eroded with light and with fast integrin turnover on the apical side only the integrins on the outside of the cyst retain their connections to the gel network.
Fig. 5
Fig. 5
A549 cysts. (A) Bright field images of A549 cells progressively covering fibronectin-loaded microspheres. (B) Cross-section schematic illustrating the hollow cyst that remains in the hydrogel after erosion of the microsphere template. (C) Bright field image of an encapsulated A549 cyst after template erosion. (D) Maximum intensity projection of a confocal image stack of an A549 cyst, fixed 3 days after template erosion. Red is actin; blue is the cell nucleus. (E) Single confocal image through the center of the same A549 cyst demonstrating the hollow interior. Red is actin; blue is the cell nucleus.
Fig. 6
Fig. 6
Primary cysts. (A) Bright field images of primary cells proliferating to cover the microspheres. (B) Bright field image of an encapsulated primary cell cyst after template erosion. (C) Maximum intensity projection of a confocal image stack of a primary cell cyst, fixed 3 days after template erosion. Green is T1α, a marker of the ATI cell phenotype; blue is the cell nucleus. (D) Single confocal image through the center of the same primary cell cyst demonstrating the hollow interior. Green is T1α, a marker of ATI cell phenotype; blue is the cell nucleus. (E) Maximum intensity projections of three 50 μm cryosections, documenting the absence of cells in the central lumen. Red is SPC, a marker of the ATII cell phenotype; green is T1α, a marker of the ATI cell phenotype; blue is the cell nucleus.
Fig. 7
Fig. 7
Immunostaining of primary cysts. (A) Maximum intensity projections of a confocal image stack of a primary cell cyst, fixed 3 days after template erosion. Green is β-catenin, a marker of adherens junctions; red is ZO-1, a marker of tight junctions; blue is the cell nucleus. (B) Single confocal image through the center of the same primary cell cyst. Colors are the same as in A. (C) Maximum intensity projections of a confocal image stack of a primary cell cyst, fixed 6 days after template erosion. Green is T1α, a marker of the ATI cell phenotype; red is SPC, a marker of the ATII cell phenotype; blue is the cell nucleus. (D) Single confocal image through the center of the same primary cell cyst. Colors are the same as in C. (E) 3D surface projection of a 50 μm mouse lung tissue section showing multiple cysts, with on average 2 ATII cells per cyst. Colors are the same as in C.

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