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	Figure 1. Timeline of early lung development and associated milestones in blood‐gas barrier maturation. Blue trapezoids illustrate the magnitudes of morphometric changes during alveolarization and microvascular maturation in the rat. E, embryonic day. P, postnatal day. Gest wks, gestational weeks.
	Figure 2. Hematoxylin and eosin (H&E) staining of native rat lung fixed by formalin inflation at postnatal days (A) 1, (B) 4, (C) 7, (D) 14, (E) 21, and (F) 60. Scale bars, 50 μm.
	Figure 3. Scales of blood‐gas barrier organization. Top of each panel, native lung image. Bottom of each panel, schematic. Tops of panels: (A) Scanning electron micrograph of distal lung parenchyma in mouse. (B) Light micrograph of alveolar septa in a saline‐filled rabbit lung. (C) Transmission electron micrograph of an alveolar capillary in monkey. (D) Enlarged detail by transmission electron micrograph of the gas exchange surface. Scale bars in lower panels representative of human lung. (A) Reused, with permission, from Bastacky J and Goerke J, 1992 18, copyright the American Physiological Society; (B) Reused, with permission, from Gil J, et al., 1979 80, copyright the American Physiological Society; (C,D) Reused, with permission, from Weibel ER, 1970 246, with permission from Elsevier.
	Figure 4. Layers of the blood‐gas barrier.
	Figure 5. Alveolar‐capillary morphology. (A) Arteriolar “islands” and venous “lakes” in cat lung. (B) Pulmonary capillary beds in frog lung (left) and in the alveolar walls of cat lung (right). AL, alveolar sac. l, line delineating interalveolar wall. P, Intercapillary connective tissue posts. (C) Schematic of capillary network between a pulmonary arteriole (left) and venule (right). (D) Schematic of the alveolar‐capillary “sheet” with associated dimensions in cat. (E) Comparison of pulmonary capillary sheet width to the height of the Empire State Building. (A) Reprinted with slight modification from Sobin SS, et al. 1980 204, with permission from Elsevier; (B, left) Reprinted from Maloney JE and Castle BL, 1969 138, with permission from Elsevier; (B, right) Reprinted from Sobin SS, et al. 1980 204, with permission from Elsevier.
	Figure 6. Alveolar epithelial type I cell morphology. (A) Scanning electron micrograph of the alveolar surface of a human lung, with an alveolar epithelial type I cell (AEC1) highlighted in yellow. (B) Drawing of an AEC1 (in yellow, with inner and outer cell membranes in red and green, respectively) branching to either side of an alveolar septum. (C) Alternative view of the AEC1 in (B) illustrating the cell's structure of intercapillary stalks and multiple cytoplasmic plates on both sides of the septum. Ep1, type I epithelial cell, Ep2, type II epithelial cell. Reprinted from Weibel ER, 2015 250 with permission of the American Thoracic Society. Copyright © 2019 American Thoracic Society. The American Journal of Respiratory and Critical Care Medicine is an official journal of the American Thoracic Society.
	Figure 7. Lung pressure‐volume curve.
	Figure 8. Schematic of micromechanical forces acting upon the alveolar septum.
	Figure 9. Alveolar septal pleats. An alveolar septum is shown by transmission electron micrograph and by line drawing. Arrowheads, alveolar luminal surface. Dotted line, pleated epithelial basement membrane. C, capillaries. SLL, surface lining layer. EPII, alveolar epithelial type II cell. M, alveolar macrophage. Modified from image originally printed in Bachofen H, et al., 1987 11, copyright the American Physiological Society.
	Figure 10. Morphological differences of air‐ and saline‐filled lungs at different volumes. (A,B) Scanning electron micrographs of air‐filled lungs fixed by vascular perfusion at 40% and 80% TLC, respectively. (C,D) Saline‐filled lungs fixed by vascular perfusion at 40% and 80% TLC, respectively. Originally printed in Gil J, et al. 1979 80, copyright the American Physiological Society.
	Figure 11. Zonation of pulmonary blood flow. (A) Location of zones, with Zone 1 near the apex of the lung and Zone 3 near the base. (B) Relative pressures, (C) capillary profiles, and (D) capillary cross‐sections in Zones 1 to 3. PA, alveolar pressure. Pa, arterial pressure. Pv, venous pressure.
	Figure 12. Starling's law and alveolar protective mechanisms against edema. (A) Schematic of the alveolar septum in homeostasis. Block arrows at left depict typical relative magnitudes of the Starling forces Pc, Pis, πc, and πis across the alveolar‐capillary wall in homeostasis. Dashed arrow at right depicts small amount of lymph flow from the perivascular spaces. (B) Altered Starling forces in the setting of increased hydrostatic pressure Pc, and the structural and biological protective mechanisms that may limit edema.
	Figure 13. Schematic of major extracellular matrix and interstitial components in the alveolar wall.
	Figure 14. Lung stress and strain. Lung collagen and elastin have been described to behave as a “string‐spring” pair of mechanical elements. Collagen and elastin at low (A) and high (B) lung volumes. (C) Stress‐strain curve for the lung.
	Figure 15. Evolution of lung‐on‐a‐chip designs. (A) A rudimentary model of the alveolar blood‐gas barrier can be made by plating the underside of a porous Transwell membrane with endothelium and the apical side with pulmonary epithelium. This allows intermittent TEER measurements and visualization of the cultured cells but not stretch or flow. (B) A pioneering design by Huh et al. applied cyclic stretch to an elastic porous silicone membrane, allowing modeling of alveolar ventilatory mechanics. This design also allows media flow, thereby mimicking capillary and airway shear dynamics. (C) A later chip‐based design with integrated gold‐plated electrodes allowing simultaneous real‐time microscopy and TEER underflow dynamics. (D) A recent microimpedance tomography‐based lung‐on‐a‐chip design, which segregates all electrodes on one side of the modeled barrier and opens the apical side of this chip for direct visualization and/or intervention. Chips with integrated barrier‐measuring electrodes are generally capable of producing more repeatable and cross‐laboratory translatable results.
	Figure 16. Overview of decellularization‐recellularization paradigm for whole lung engineering. (A) Intact native lungs are decellularized via detergent rinsing, creating an extracellular matrix scaffold free of intact cells (B). (C) The scaffold is subsequently seeded with cells into the airway and/or vascular compartments. (D) The reseeded construct is transferred to a bioreactor for culture under physiologically relevant conditions. The resulting engineered organ undergoes (E) ex vivo characterization before final (F) orthotopic transplantation for in vivo functional assessment.
	Figure 17. Endothelial cell seeding strategy for decellularized lung. The leaky nature of the decellularized scaffold leads to a “sieving effect” whereby fluid preferentially leaks across the capillary wall. (A) Cells clump in the early capillary segments due to fluid leakage. (B) Seeding a dilute cell suspension from both ends of the vasculature, under higher pressure and with pulsatile perfusion, leads to enhanced distribution of endothelial cells within the decellularized scaffold.

Teaching Material

Katherine L. Leiby, Micha Sam Brickman Raredon, and Laura E. Niklason. Bioengineering the Blood-gas Barrier. Compr Physiol 10 : 2020, 415-452.

Didactic Synopsis

Major teaching points:

1. Key structural features of the blood-gas barrier:

a. Extreme thinness

b. Large alveolar surface area

c. High density of vessels within the pulmonary capillary "sheet"

2. Lungs have a complex pressure-volume relationship principally governed by tissue stretch and surface tension.

3. Biological aspects of blood-gas separation:

a. Cell-cell junctions, particularly epithelial tight junctions

b. Alveolar fluid clearance

c. Surfactant, which aids fluid balance across the pulmonary capillaries

4. Collagen I, elastin, and proteoglycans provide the bulk of lung parenchymal structural integrity. Collagen IV is the primary source of alveolar-capillary membrane strength.

5. Lung-on-a-chip:

a. Advances: Chips with multiple cell types, air-liquid interface, and cyclic strain; multi-organ chip systems

b. Challenges: Physiologically-appropriate membrane, resistance measurements comparable across platforms

6. Engineered whole lungs:

a. Advances: Decellularization and culture of human-sized lungs, improved endothelial recellularization

b. Challenges: Achieving blood-gas separation, developing/implementing methodologies for assessing barrier function

Didactic Legends

The following legends to the figures that appear throughout the article are written to be useful for teaching.

Figure 1. Teaching points: This figure illustrates the principle phases of early lung development, beginning in the fetal period. While aspects of blood-gas barrier development (noted in the figure) occur as early as the pseudoglandular stage, the most significant restructuring of the alveolar membrane takes place during alveolarization and microvascular maturation. These phases are characterized by a dramatic increase in alveolar surface area (which cannot be explained by an increase in lung volume alone) with simultaneous thinning of the alveolar membrane. The result is a substantial increase in lung diffusing capacity. It is still debated whether septation, or the subdivision of existing alveoli into new airspaces, occurs past early childhood in human.

Figure 2. Teaching points: These hematoxylin and eosin (H&E) images of native rat lung illustrate the dramatic changes that occur in airspace structure during later lung development, from the late saccular period (A) (postnatal day 1 (P1)), through alveolarization (B)-(E) (P4-P21), to adult (F) (P60). (A) The P1 rat lung is comprised of large saccules with thick walls. (B) Beginning at P4, the onset of alveolarization, secondary septa subdivide the airspaces into smaller alveoli, a process that continues through P7 (C), P14 (D), and P21 (E). Secondary septation leads to a significant increase in alveolar surface area. Note over this time period that there is a simultaneous thinning of the alveolar walls, due to both tissue remodeling and to a slowing of cellular proliferation near the end of alveolarization. The P21 lung has an appearance similar to that of adult (F), though airspace size may change with growth of the animal.

Figure 3. Teaching points: This figure illustrates several key features of the blood-gas barrier, as revealed by different length scales. In the top of each panel is an image of native lung taken by light or electron microscopy. In the bottom of each panel is a corresponding schematic for clarity. (A) The distal lung parenchyma is made up of alveoli packed together as if in a foam. This organization allows for an extremely large surface area for gas exchange to be packed into a relatively small volume. (B) This light micrograph and corresponding schematic illustrate the structure of individual alveolar septa. This image was taken from a saline-filled lung; it is the absence of surface tension that causes the capillaries to bulge into the airspaces. Note that the septa are rich with capillaries (open spaces bulging to either side of the septa in the top image; "blood-filled" round capillaries in the schematic). This high density of alveolar capillaries serves to create a very large capillary surface area, to facilitate gas exchange. (C) This transmission electron micrograph and accompanying schematic zooms in further, on a cross-section of a single alveolar capillary. Note that one side of the capillary (upper right) is "thin," comprising only of a thin epithelial cell process, a fused basement membrane, and a thin endothelial cell process. This thin side is the site of gas exchange. The "thick" side (lower left), on the other hand, contains additional interstitial components and provides support to the adjacent capillary and to the septum. (D) This panel provides a closer look at the thin gas-exchanging part of the blood gas barrier.

Figure 4. Teaching points: This schematic illustrates in more detail the layers comprising the blood-gas barrier, through which gas must diffuse to pass from the airspaces to the blood, or vice-versa. By Fick's law, an increase in thickness or a decrease in the oxygen permeation coefficient of any of these layers will decrease the diffusion capacity of gas across the barrier.

Figure 5. Teaching points: This figure illustrates several key features of the alveolar capillary network. (A) Different regions of capillaries are either supplied by an arteriole, or drained by a venule. Those regions drained by venules comprise a slightly larger area of continuous tissue surrounding "islands" of alveoli supplied by individual arterioles. (B,C) The alveolar capillary network is extremely dense, with many different capillary "paths" crossing the distance from arteriole to vein and forming a dense meshwork of vessels in the alveolar walls. (D,E) This dense meshwork may be modeled as a "sheet" of blood that is very short (distance between arteriole and venule) but extremely wide due to the large number of capillaries.

Figure 6. Teaching points: This figure illustrates the unique morphology of alveolar epithelial type I cells (AEC1s). (A) These cells cover the vast majority of the alveolar surface. (B,C) AEC1s can have a complex morphology that includes thin cell stalks passing between capillaries, and multiple flat plates of cytoplasm lining both sides of a single septum.

Figure 7. Teaching points: This graph shows the lung pressure-volume curve in the setting of either air or saline inflation. In the air-filled lung, due to the presence of surface tension at the air-liquid interface, there is prominent hysteresis between the inflation and deflation limbs of the curve (i.e., the volume at a given transpulmonary pressure depends on whether that volume was reached by inflation or deflation). In a saline-filled lung, which does not have surface tension, hysteresis is nearly absent. Note as well how the curve for the air-filled lung is nearly flat at low transpulmonary pressures, corresponding to very low compliance (low change in volume in response to a given change in pressure) at low lung volumes.

Figure 8. Teaching points: Several forces act upon the alveolar wall at the microscopic level, with important consequences for alveolar morphology and architecture as discussed in the text. 1) Surface tension acts tangential to the surface of the alveolar lining layer (ALL). It generally results in a net lumen-directed force, as is the case in the alveolar corner at the left of this figure; however it may also produce a compressive force on convex structures that bulge into the alveolar space such as the alveolar type II cell (AEC2) in this illustration. 2) Wall tension (a tissue force) arises due to stretching of the alveolar wall with lung inflation. 3) Circumferential wall tension in the capillary wall arises due to capillary transmural pressure. If this tension leads to stretch of neighboring septal components it may also contribute to tissue forces.

Figure 9. Teaching points: This figure, a transmission electron micrograph and accompanying line drawing of an alveolar septum, illustrates how the surface area of the alveolar lumen (arrow heads in line drawing) may be less than the surface area of the alveolar epithelial basement membrane (dotted line) due to extensive septal/basement membrane pleating. As a result, expansion of the lung from low volumes occurs primarily by unfolding of these tissue pleats, rather than by stretching.

Figure 10. Teaching points: These images, taken by scanning electron microscope (SEM), depict how alveolar architecture is affected by surface tension and by lung volume. In an air-filled lung with normal surface tension, the alveolar septa appear "crumpled" due to low tissue stretch at lower lung volumes, with redundant tissue pleating at the corners (A). Note how the alveolar surfaces appear smoother, and the capillaries flatter, at higher lung volumes in the setting of higher surface tension (B). By comparison, in a saline-filled lung with no surface tension, capillaries bulge at both low (C) and high (D) lung volumes, lending a bumpy appearance to the alveolar surfaces, and the alveolar septa are wavy.

Figure 11. Teaching points: Pulmonary capillary blood flow is affected by location within the lung, due in part to the effects of gravity. A capillary in Zone 1 (apex of the lung), compared to a capillary in Zone 3 (base of the lung), is relatively more compressed, with associated lower blood flow, due to relatively higher alveolar pressures and lower arterial and venous pressures higher up in the lung.

Figure 12. Teaching points: This figure illustrates how the alveolar septum is uniquely designed to protect against edema that may lead to thickening of the blood-gas barrier and, in severe cases, flooding of the alveoli, significantly impairing gas exchange. (A) In homeostasis, three of the Starling forces (capillary hydrostatic pressure P_c, the negative interstitial hydrostatic pressure P_is, and interstitial oncotic pressure π_is favor filtration from the capillaries, whereas capillary oncotic pressure π_c favors reabsorption; the net balance of these forces leads to a small amount of transudated fluid entering the lymphatic circulation in the space around the small vessels. (B) Several protective mechanisms act to prevent and/or limit edema. Illustrated here is the case of hydrostatic pulmonary edema in setting of increased P_c: 1) P_is increases (becomes less negative) as fluid enters the interstitium due to the low compliance of this space. 2) π_is decreases due to dilution of the interstitial space by transudated fluid. 3) The pulmonary capillaries are particularly impermeable (effective pore size approximately 5 nm), compared to systemic capillaries and to extraalveolar pulmonary vessels. 4) Alveolar epithelial tight junctions provide a very tight barrier, at 1 nm. 5) Both alveolar epithelial type I (AEC1s) and type II (AEC2s) cells participate in alveolar fluid clearance, transporting ions from apical to basolateral membrane to create a driving force for fluid movement out of the alveolar lumen. 6) Surfactant secreted by AEC2s, which forms the surface film of the alveolar lining layer (ALL), reduces surface tension, thereby decreasing the contribution of this force to the negative P_is. 7) Lymph flow may increase significantly.

Figure 13. Teaching points: This figure illustrates the major supporting structures of the alveolar wall. A thin basement membrane composed primarily of collagen IV and laminin, underlies the alveolar epithelium and endothelium. Note that a fused basement membrane is the only supporting structure on the "thin" side of the capillaries. However, in the space between the capillaries, and in the "thick" portions of the wall, additional support is provided by collagen I (illustrated here in both cross-sectional and longitudinal orientations) and elastin, and to a lesser degree by fibroblasts. Proteoglycans, not illustrated here, have important interactions with collagen and elastin to further stabilize the delicate alveolar walls.

Figure 14. Teaching points: This figure illustrates one model of the behavior of individual collagen and elastin fibers in the lung, and shows the stress/strain behavior of the lung parenchyma as a whole. According to the "string-spring" model of collagen and elastin in the lung, at low volumes (A), collagen fiber "strings" are wavy and incompletely distended, whereas the elastin "springs" bear the stress within the tissue. At high volumes (B) the collagen fibers eventually reach their stop lengths (beyond which they cannot be extended without rupture), and limit further lung expansion. This behavior of collagen may at least partially explain the non-linear behavior of the lung stress-strain curve at higher strain values (C). Note as well in (C) that there is a threshold strain (approximately 1.5) above which there is a risk for ventilator-associated lung injury.

Figure 15. Teaching points: This figure illustrates several published lung-on-a-chip platforms from the past 15 years. While all models involve the co-culture of different cell types on either side of a membrane, more recent chips have incorporated additional physiologically relevant features and/or technological advancements in barrier assessment or visualization. These improvements include cyclic stretch (B); media flow (B,C); simultaneous microscopy and resistance measurements by trans-epithelial electrical resistance (TEER) (C); and microimpedance tomography electrodes, which provide more accurate resistance measurements than TEER and are compatible with application of cyclic stretch.

Figure 16. Teaching points: This figure illustrates the overall paradigm of whole lung engineering. In (A), (B), and (D), the bottom part of the panel provides a schematic of the alveolar septum during each stage of the process. (A) A native lung (either from an animal model, or a human lung that is unsuitable for transplantation) serves as the initial starting material. (B) The lung is decellularized, typically via detergents rinsed through the vasculature, to create an acellular scaffold. This process is designed to preserve the underlying extracellular matrix and native lung architecture while removing as much cellular material as possible. This process is facilitated by cannulas providing access to the airways (via the trachea) and the vasculature (via the pulmonary artery, through the heart). (C) The decellularized scaffold is next repopulated with cells that are typically derived either from primary isolation, or from the differentiation of induced pluripotent stem cells (iPSCs). Cells may be seeded into the airways and/or the vasculature. (D) The reseeded lung is transferred to a bioreactor that provides a sterile environment for lung culture, and which is designed to mimic physiological conditions by providing stimuli such as vascular perfusion and ventilation. (E) Following culture, engineered lungs may be analyzed ex vivo, before ultimate transplantation into a recipient (F).

Figure 17. Teaching points: Successful introduction of endothelial cells into the microvasculature of decellularized lung scaffolds has been a significant challenge for the field of lung engineering. (A) Due to the leaky nature of vessels within the decellularized lung (due to removal of endothelial cells and potential damage to the underlying basement membrane), fluid preferentially leaks out, leaving cells (which are relatively large in diameter compared to the caliber of the alveolar capillaries) stuck in the initial vascular segments. (B) Recent recognition of this "sieving" effect has led to strategies to more effectively introduce cells for revascularization. Seeding a dilute cell suspension helps to counteract the leakage of fluid, delaying the time until cell clumping, while seeding from a greater initial pressure allows cells to travel further into the capillary bed. Reaching the most distal segments of the capillaries is aided by seeding from both ends (i.e. from both the pulmonary artery and the pulmonary veins). Finally, seeding under pulsatile flow aids in recruitment of collapsed capillaries, facilitating entry of cells into more vessels.