Control of vascular network location in millimeter-sized 3D-tissues by micrometer-sized collagen coated cells

Abstract

Engineering three-dimensional (3D) vascularized constructs remains a central challenge because capillary network structures are important for sufficient oxygen and nutrient exchange to sustain the viability of engineered constructs. However, construction of 3D-tissues at single cell level has yet to be reported. Previously, we established a collagen coating method for fabricating a micrometer-sized collagen matrix on cell surfaces to control cell distance or cell densities inside tissues. In this study, a simple fabrication method is presented for constructing vascular networks in 3D-tissues over micrometer-sized or even millimeter-sized with controlled cell densities. From the results, well vascularized 3D network structures can be observed with a fluorescence label method mixing collagen coated cells and endothelia cells, indicating that constructed ECM rich tissues have the potential for vascularization, which opens up the possibility for various applications in pharmaceutical or tissue engineering fields.

An in vitro 3D diabetic human skin model from diabetic primary cells

“…First of all, type 2 diabetic human dermal fibroblasts (4.5 × 10 6 cells ml −1 ) and HUVECs (2.25 × 10 6 cells ml −1 ) at passages 2-4 were incubated in DMEM-F12 medium containing 0.3 mg ml −1 atelocollagen for 20 min at 37 • C to prevent excess water loss from the cells during photocrosslinking [18]. After the incubation, the cells were centrifuged at 1500 rpm for 5 min and the pellet was resuspended in GelMA prehydrogel solution (8% w/v) which was then transferred to 12-well culture inserts (3 µm pore size, Millipore) and exposed to UV A for 22 min at a distance of 5.8 cm.…”

“…Current commercially available in vitro systems such as EpiSkin TM , T-Skin TM (L'Orèal, Paris, France), Epiderm TM , and EpiDermFT TM (MatTek, Ashland, MA, United States) have been used as full thickness or epidermal models for the analysis of drug delivery, sensitization and wound healing [17], but they are still far from a realization of physiologically faithful reproduction of the human skin. One of the main shortcomings of such aforementioned skin models is that they do not recapitulate the dynamic transport of molecules such as nutrients, growth factors, and migrating cells because of the absence of a capillary network system which is required for tissues thicker than 200 µm [18]. Many in vitro 3D skin models which consist of various cell types of skin such as fibroblasts and keratinocytes [13,[19][20][21][22]; fibroblasts and human umbilical vein endothelial cells (HUVECs) [18]; keratinocytes, fibroblasts and HUVECs [23]; keratinocytes, fibroblasts, adipocytes, endothelial cells, smooth muscle cells and neural cells [16] have been developed for research purposes.…”

“…One of the main shortcomings of such aforementioned skin models is that they do not recapitulate the dynamic transport of molecules such as nutrients, growth factors, and migrating cells because of the absence of a capillary network system which is required for tissues thicker than 200 µm [18]. Many in vitro 3D skin models which consist of various cell types of skin such as fibroblasts and keratinocytes [13,[19][20][21][22]; fibroblasts and human umbilical vein endothelial cells (HUVECs) [18]; keratinocytes, fibroblasts and HUVECs [23]; keratinocytes, fibroblasts, adipocytes, endothelial cells, smooth muscle cells and neural cells [16] have been developed for research purposes. The cells of these skin models are either cell lines or have a normoglycemic human skin origin.…”

“…When separate cell-laden scaffolds are formed as the layers of the skin and are finally going to be assembled together, this approach may prevent the formation of a continuous model. Another limitation of current in vitro 3D skin models is that greater than 100-2000 µm thick 3D skin models are still not constructed [18]. To our knowledge, there is no commercially available or experimentally developed in vitro 3D skin model which hosts primary cells derived from type 2 diabetic humans.…”

Collagen/Cellulose Nanofiber Blend Scaffolds Prepared at Various pH Conditions

“…The native ECMs including collagen, gelatin, elastin, and fibronectin are important for cell adhesion, proliferation, and differentiation in native tissues. Among these ECNs, type I collagen (COL) is one of the most abundant proteins that is widely employed in biomedical or tissue engineering fields − and is sensitively denatured by environmental factors . COL molecules have rod-like triple-helical structures composed of three polypeptide chains.…”