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In vitro reconstruction of branched tubular structures from lung epithelial cells in high cell concentration gradient environment.

Hagiwara M, Peng F, Ho CM - Sci Rep (2015)

Bottom Line: However, homogeneous high cell concentration does not make a branching structure.Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation.This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.

View Article: PubMed Central - PubMed

Affiliation: 1] Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan [2] Mechanical and Aerospace Engineering Department, University of California Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.

ABSTRACT
We have succeeded in developing hollow branching structure in vitro commonly observed in lung airway using primary lung airway epithelial cells. Cell concentration gradient is the key factor that determines production of the branching cellular structures, as optimization of this component removes the need for heterotypic culture. The higher cell concentration leads to the more production of morphogens and increases the growth rate of cells. However, homogeneous high cell concentration does not make a branching structure. Branching requires sufficient space in which cells can grow from a high concentration toward a low concentration. Simulation performed using a reaction-diffusion model revealed that long-range inhibition prevents cells from branching when they are homogeneously spread in culture environments, while short-range activation from neighboring cells leads to positive feedback. Thus, a high cell concentration gradient is required to make branching structures. Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation. This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.

No MeSH data available.


Comparison of lung airway morphogenesis in 3D culture.(A) Phase contrast image of monocultured NHBE cells at day 8. NHBE cells formed spherical colonies, but no branching morphogenesis occurred. (B) Phase contrast image of NHBE cells co-cultured with HUVECs. No branching morphogenesis occurred, but the growth rate of NHBE cells was slightly higher than controls. (C) Phase contrast image of NHBE cells co-cultured with MSCs. No branching morphogenesis occurred. The size of the NHBE cells is double that of the control case at day 8. (D) Phase contrast image of NHBE cell clots in Matrigel at day 1, day 4, and day 8. The clot consisted of 2 × 105 cells at day 1. The branches grew out from the clot at day 4, and the growth rate was much faster than the cultures that were homogeneously distributed. At day 8, the maximum length of branches reached 700 μm and number of branches from the clot was increased. (E) Higher magnification image of branches at day 8. Secondary branching was observed. (F) Quantitation of cell growth rate. Co-culture of NHBE cells increased the growth rate, but the cells grew much faster in NHBE cell clots. The size of branches from cell clots at day 3 were 3 times higher than the cell sizes cultured homogeneously (p = 0.027). Error bars indicate standard deviation (n = 20). Scale bars 100 μm (A–E).
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f1: Comparison of lung airway morphogenesis in 3D culture.(A) Phase contrast image of monocultured NHBE cells at day 8. NHBE cells formed spherical colonies, but no branching morphogenesis occurred. (B) Phase contrast image of NHBE cells co-cultured with HUVECs. No branching morphogenesis occurred, but the growth rate of NHBE cells was slightly higher than controls. (C) Phase contrast image of NHBE cells co-cultured with MSCs. No branching morphogenesis occurred. The size of the NHBE cells is double that of the control case at day 8. (D) Phase contrast image of NHBE cell clots in Matrigel at day 1, day 4, and day 8. The clot consisted of 2 × 105 cells at day 1. The branches grew out from the clot at day 4, and the growth rate was much faster than the cultures that were homogeneously distributed. At day 8, the maximum length of branches reached 700 μm and number of branches from the clot was increased. (E) Higher magnification image of branches at day 8. Secondary branching was observed. (F) Quantitation of cell growth rate. Co-culture of NHBE cells increased the growth rate, but the cells grew much faster in NHBE cell clots. The size of branches from cell clots at day 3 were 3 times higher than the cell sizes cultured homogeneously (p = 0.027). Error bars indicate standard deviation (n = 20). Scale bars 100 μm (A–E).

Mentions: To evaluate the lung airway morphogenesis, we cultured NHBE cells in reconstituted basement membrane (rBM). Matrigel was used as rBM in the following 3D culture experiments. It was reported that co-culture of HUVECs or mesenchymal stem cells (MSCs) is required to make a branching structure of lung epithelium524252627. Thus, we examined the effects of both heterotypic co-culture of NHBE with HUVECs or MSCs as well as NHBE clotting monoculture on NHBE morphogenesis. When NHBE was homogeneously distributed in Matrigel, cells formed spherical colonies, but no branching was observed (Fig. 1A). On the other hand, when NHBE cells were cultured with HUVECs or MSCs, they formed larger spherical colonies in comparison with homogeneously distributed NHBE monocultures (Fig. 1B and C). Despite this, NHBE cell branching morphogenesis was absent in both cases. In order to observe the effects of cell concentration gradient, we then established NHBE cell clots using fibrin produced from the reaction between fibrinogen and thrombin (Fig. 1D). This procedure causes radial elongation of cells from the clot, and subsequent formation of branching structures. After 5 d, some of the initial branches also began to form secondary branches (Fig. 1E).


In vitro reconstruction of branched tubular structures from lung epithelial cells in high cell concentration gradient environment.

Hagiwara M, Peng F, Ho CM - Sci Rep (2015)

Comparison of lung airway morphogenesis in 3D culture.(A) Phase contrast image of monocultured NHBE cells at day 8. NHBE cells formed spherical colonies, but no branching morphogenesis occurred. (B) Phase contrast image of NHBE cells co-cultured with HUVECs. No branching morphogenesis occurred, but the growth rate of NHBE cells was slightly higher than controls. (C) Phase contrast image of NHBE cells co-cultured with MSCs. No branching morphogenesis occurred. The size of the NHBE cells is double that of the control case at day 8. (D) Phase contrast image of NHBE cell clots in Matrigel at day 1, day 4, and day 8. The clot consisted of 2 × 105 cells at day 1. The branches grew out from the clot at day 4, and the growth rate was much faster than the cultures that were homogeneously distributed. At day 8, the maximum length of branches reached 700 μm and number of branches from the clot was increased. (E) Higher magnification image of branches at day 8. Secondary branching was observed. (F) Quantitation of cell growth rate. Co-culture of NHBE cells increased the growth rate, but the cells grew much faster in NHBE cell clots. The size of branches from cell clots at day 3 were 3 times higher than the cell sizes cultured homogeneously (p = 0.027). Error bars indicate standard deviation (n = 20). Scale bars 100 μm (A–E).
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Related In: Results  -  Collection

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f1: Comparison of lung airway morphogenesis in 3D culture.(A) Phase contrast image of monocultured NHBE cells at day 8. NHBE cells formed spherical colonies, but no branching morphogenesis occurred. (B) Phase contrast image of NHBE cells co-cultured with HUVECs. No branching morphogenesis occurred, but the growth rate of NHBE cells was slightly higher than controls. (C) Phase contrast image of NHBE cells co-cultured with MSCs. No branching morphogenesis occurred. The size of the NHBE cells is double that of the control case at day 8. (D) Phase contrast image of NHBE cell clots in Matrigel at day 1, day 4, and day 8. The clot consisted of 2 × 105 cells at day 1. The branches grew out from the clot at day 4, and the growth rate was much faster than the cultures that were homogeneously distributed. At day 8, the maximum length of branches reached 700 μm and number of branches from the clot was increased. (E) Higher magnification image of branches at day 8. Secondary branching was observed. (F) Quantitation of cell growth rate. Co-culture of NHBE cells increased the growth rate, but the cells grew much faster in NHBE cell clots. The size of branches from cell clots at day 3 were 3 times higher than the cell sizes cultured homogeneously (p = 0.027). Error bars indicate standard deviation (n = 20). Scale bars 100 μm (A–E).
Mentions: To evaluate the lung airway morphogenesis, we cultured NHBE cells in reconstituted basement membrane (rBM). Matrigel was used as rBM in the following 3D culture experiments. It was reported that co-culture of HUVECs or mesenchymal stem cells (MSCs) is required to make a branching structure of lung epithelium524252627. Thus, we examined the effects of both heterotypic co-culture of NHBE with HUVECs or MSCs as well as NHBE clotting monoculture on NHBE morphogenesis. When NHBE was homogeneously distributed in Matrigel, cells formed spherical colonies, but no branching was observed (Fig. 1A). On the other hand, when NHBE cells were cultured with HUVECs or MSCs, they formed larger spherical colonies in comparison with homogeneously distributed NHBE monocultures (Fig. 1B and C). Despite this, NHBE cell branching morphogenesis was absent in both cases. In order to observe the effects of cell concentration gradient, we then established NHBE cell clots using fibrin produced from the reaction between fibrinogen and thrombin (Fig. 1D). This procedure causes radial elongation of cells from the clot, and subsequent formation of branching structures. After 5 d, some of the initial branches also began to form secondary branches (Fig. 1E).

Bottom Line: However, homogeneous high cell concentration does not make a branching structure.Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation.This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.

View Article: PubMed Central - PubMed

Affiliation: 1] Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan [2] Mechanical and Aerospace Engineering Department, University of California Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.

ABSTRACT
We have succeeded in developing hollow branching structure in vitro commonly observed in lung airway using primary lung airway epithelial cells. Cell concentration gradient is the key factor that determines production of the branching cellular structures, as optimization of this component removes the need for heterotypic culture. The higher cell concentration leads to the more production of morphogens and increases the growth rate of cells. However, homogeneous high cell concentration does not make a branching structure. Branching requires sufficient space in which cells can grow from a high concentration toward a low concentration. Simulation performed using a reaction-diffusion model revealed that long-range inhibition prevents cells from branching when they are homogeneously spread in culture environments, while short-range activation from neighboring cells leads to positive feedback. Thus, a high cell concentration gradient is required to make branching structures. Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation. This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.

No MeSH data available.