In vitro 3D model for human vascularized adipose tissue - PubMed (original) (raw)

In vitro 3D model for human vascularized adipose tissue

Jennifer H Kang et al. Tissue Eng Part A. 2009 Aug.

Abstract

The clinical need for both three-dimensional (3D) soft tissue replacements and in vitro adipose tissue models continues to grow. In this study, we evaluated structural and functional characteristics of an in vitro 3D coculture model of vascularized adipose tissue. Tomato red-infected human adipose tissue-derived mesenchymal stem cells (hASCs) and green fluorescence protein-infected human umbilical vein endothelial cells were cocultured on 3D aqueous-derived silk scaffolds for 2 weeks. Confocal microscopy images demonstrated viability of cocultures and organization of both cell types over time. Endothelial cells aligned with time, and further histological analyses revealed continuous endothelial lumen formation in both differentiated and undifferentiated cocultures. Differentiated adipose cocultures secreted significantly higher levels of leptin than undifferentiated cocultures at 1 and 2 weeks. Additionally, lipid accumulation was demonstrated with Oil Red O staining, where positive staining was higher in the differentiated cocultures. A promising in vitro approach for the vascularization of tissue-engineered adipose tissue, and the ability to vascularize a construct containing hASCs was demonstrated. The strategy outlined provides a basis for the formation of other in vitro vascularized tissues as well as a path forward for the sustainable formation of soft tissue due to the use of slowly degrading silk scaffolds.

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Figures

FIG. 1.

FIG. 1.

Research design for cocultivation of HUVECs and hASCs/differentiated adipocytes on silk scaffolds.

FIG. 2.

FIG. 2.

DNA content results for optimization of coculture media. hASCs and HUVECs were grown in three types of media: EGM:GM, EGM:Diff, and EGM:MM in 1:1, 1:2, and 1:3 medium ratios. EGM, endothelial growth medium; GM, growth medium; Diff, differentiation medium; MM, maintenance medium. hASCs exhibited significantly greater proliferation in 1:1 EGM:Diff than 1:2 or 1:3 (+p < 0.05) and in EGM:GM and EGM:MM at 1:2 and 1:3 ratios (*p < 0.05). Additionally, HUVECs exhibited significantly less proliferation in EGM:Diff media at all three ratios (*p < 0.05), and 1:1 EGM:MM showed significantly more proliferation than 1:3 EGM:MM (+p < 0.05).

FIG. 3.

FIG. 3.

Confocal microscopy images for hASC and HUVEC cocultures on day 4 (A) and day 8 (B). hASCs and HUVECs were coseeded on day 0. HUVECs, GFP labeled; hASCs, tomato red labeled. Scale bar = 375 μm. Color images available online at

www.liebertonline.com/ten

.

FIG. 4.

FIG. 4.

Confocal images of induced coculture on days 2 (A, B), 7 (C, D), and 14 (E–H). HUVECs, GFP labeled; hASCs, tomato red labeled. Scale bars as marked. Color images available online at

www.liebertonline.com/ten

.

FIG. 5.

FIG. 5.

CD31 ICC with hematoxylin counterstain for uninduced (A, B) and induced (C, D) cocultures after 14 days. Lumen organization evident in both uninduced and induced cocultures as marked by both aligned endothelial cells (arrows) and open lumens (*). Scale bar = 50 μm. Original magnification, × 320. Color images available online at

www.liebertonline.com/ten

.

FIG. 6.

FIG. 6.

Lumen quantification of uninduced and induced cocultures after 2 weeks of culture. Uninduced cocultures did not exhibit statistically greater number of lumens per scaffold cross-sectional area than induced cocultures.

FIG. 7.

FIG. 7.

Continuity of lumen structures through consecutive sections of scaffolds. Both uninduced (A–C) and induced (G–I) cocultures show lumen continuity through consecutive sections. Additionally, converging of lumen, as shown in this representative uninduced coculture sample (D–F), and branching of lumen, as shown by this representative induced coculture sample (J–L), have both been observed. Scale bars: (A–C, G–I) 100 μm (original magnification, ×100); (D–F, J–L) 50 μm (original magnification, × 320). Color images available online at

www.liebertonline.com/ten

.

FIG. 8.

FIG. 8.

Secreted leptin levels in both uninduced and induced cocultures. Induced cocultures secreted significantly more leptin than uninduced at day 14 (*p < 0.05) and increased secretion at days 7 and 14 (+p < 0.05), indicative of the presence of more mature adipocytes. Uninduced cocultures increased leptin section at day 14 as well (++p < 0.05).

FIG. 9.

FIG. 9.

Oil Red O–stained frozen sections after 2 weeks of coculture. Uninduced cocultures (A) exhibited less frequent Oil Red O stain, whereas all induced cocultures (B) were positive for lipid. (C) Frequency of adipogenesis was determined, and induced cocultures contained higher percentages of lipid-accumulating cells than uninduced cocultures (*p < 0.0001). Scale bars = 50 μm. Original magnification, × 40. Color images available online at

www.liebertonline.com/ten

.

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