Integration and application of vitrified collagen in multilayered microfluidic devices for corneal microtissue culture

Abstract

This paper describes the fabrication and application of microfluidic devices containing collagen vitrigel (CV) used as both a functional and sacrificial cell growth substrate for the development of corneal microtissue patches. Within the device, vacuum fixation of the CV in a dehydrated state enables quick integration with standard multilayer soft lithographic techniques, while on-chip rehydration results in a gel-like collagen substrate for microfluidic cell culture. Fluidic connectivity to both the apical and basal side of the CV permits bilayered culture of epithelium and supporting stromal cell layers. In addition, microfluidic introduction of a collagenase etching media enables sacrificial degradation of the supporting CV membrane for development of barrier tissue constructs containing minimal synthetic substrate. The utility of this platform was evaluated by miniaturizing the standard transepithelial permeability (TEP) assay in order to measure the integrity of an array of corneal tissue micropatches.

Membrane microchannel made of collagen with self-assembled micro fibril structures for tissue engineering

“…Even after introducing culture medium in the capillary, the 3D membrane structure was self-sustained, and the swelling of the membrane was so small that the whole microchannels were kept opened (Fig.3d). This result shows enhanced mechanical properties of the vitrified collagen compared to conventional collagen hydrogel as previously reported [11,12]. Although vitrified collagen has attracted attention in tissue engineering field, the usage has been limited to 2-D simple structures due to lack of processing methods.…”

Integrated Micro/Nanoengineered Functional Biomaterials for Cell Mechanics and Mechanobiology: A Materials Perspective

“…It suggests that integration of different micro/nanoengineered extracellular cues within an in vitro microenvironment may be a prerequisite for engineered physiological biomimicry. [33,34] Recently, the greatly advanced and widely adapted micro/nanofabrication techniques and biomaterials research have culminated in successful developments of integrated on-chip cell culture systems reconstituting in vivo-like organ-level functions of lungs [306,307] , intestines [308] and kidneys [309–311] , as well as many other “organ-on-a-chip” models that succeed in mimicking tissue-level functions of corneal [312] , cardiac muscle [313] , neural network [314] , bile canaliculi [315] and vascular vessels [48] .…”

Microfluidic approaches for epithelial cell layer culture and characterisation

“…This approach leads to another interesting set of microfluidic models in which structured ECM gels are utilised as substrate for epithelial cell layers. Puleo et al manufactured a chip in which a collagen vitrigel layer serves as separator between apical and basolateral fluid channel networks, thus replacing a porous membrane as permeable carrier of the epithelial cell layer 40 (Figure 2C). The collagen vitrigel layer has the advantage that, once a corneal epithelial cell layer is grown on top, the gel can be removed by enzymatic degradation, and a supporting stromal cell layer can be grown underneath 40 .…”

“…Puleo et al manufactured a chip in which a collagen vitrigel layer serves as separator between apical and basolateral fluid channel networks, thus replacing a porous membrane as permeable carrier of the epithelial cell layer 40 (Figure 2C). The collagen vitrigel layer has the advantage that, once a corneal epithelial cell layer is grown on top, the gel can be removed by enzymatic degradation, and a supporting stromal cell layer can be grown underneath 40 . Furthermore, traditional ECM gel-based culture systems, in which epithelial cells form self-organised three-dimensional cysts, can be equipped with a microfluidic perfusion system 74 .…”