A novel scale‐down cell culture and imaging design for the mechanistic insight of cell colonisation within porous substrate

Abstract

SummaryAt the core of translational challenges in tissue engineering is the mechanistic understanding of the underpinning biological processes and the complex relationships among components at different levels, which is a challenging task due to the limitations of current tissue culture and assessment methodologies. Therefore, we proposed a novel scale-down strategy to deconstruct complex biomatrices into elementary building blocks, which were resembled by thin modular substrate and then evaluated separately in miniaturised bioreactors using various conventional microscopes. In order to investigate cell colonisation within porous substrate in this proof-of-concept study, TEM specimen supporters (10-30 μm thick) with fine controlled open pores (100ß600 μm) were selected as the modular porous substrate and suspended in 3D printed bioreactor systems. Noninvasive imaging of human dermal fibroblasts cultured on these free-standing substrate using optical microscopes illustrated the complicated dynamic processes used by both individual and coordinated cells to bridge and segment porous structures. Further in situ analysis via SEM and TEM provided high-quality micrographs of cell-cell and cell-scaffold interactions at microscale, depicted cytoskeletal structures in stretched and relaxed areas at nanoscale. Thus this novel scaled-down design was able to improve our mechanistic understanding of tissue formation not only at singleand multiple-cell levels, but also at micro-and nanoscales, which could be difficult to obtain using other methods.

Comparison of Human Dermal Fibroblasts and HaCat Cells Cultured in Medium with or without Serum via a Generic Tissue Engineering Research Platform

“…The traditional 2D cell culture is simple and cost-effective, but it is usually considered as a poor proxy [ 18 , 19 ]. Complex 3D tissue models have been developed to mimic the complicated in vivo microenvironments [ 20 ], but it has remained difficult to distinguish the regulatory functions of each individual architectural feature at different scales, as well as other biochemical and biomechanical properties during 3D tissue cultures [ 21 , 22 ]. Moreover, the data from the distinct 2D cell and 3D tissue systems are usually not comparable [ 18 , 19 , 20 ].…”

Mechanistic Insights of Cells in Porous Scaffolds via Integrated Culture Technologies

“…Commercial TEM (transmission electron microscopy) nickel specimen supporters (Diameter: 3.05 mm, thickness: 10-30 µm, strut bar width: 40-80 µm, Agar Scientific, Stansted, UK) with finely controlled square meshes of different sizes (125, 200, 280 and 420 µm) were utilized as the modular substrates in the 3D CCIS. After washed thoroughly using distilled water, dried and autoclaved, the thin modular substrates were then suspended in the 3D CCISs for cell culture as previously described [15].…”

“…The cells were also processed for SEM (scanning electron microscopy), or labelled with Cell Tracker TM Red (CellTracker™ CM-DiI Dye) or Green (CellTracker™ Green CMFDA Dye) for live cell fluorescent microscopy as previously described [15]. To examine cell nuclei, the cells were fixed in IC (Intracellular) Fixation Buffer (Fisher Scientific) for 10 minutes, washed gently using PBS (×3), stained with 7-AAD (7-Aminoactinomycin D, sigma) for 45 minutes, further washed using PBS (×3) and then imaged at λ ex = 546 nm, λ em = 647 nm (for TRITC/cell nuclei visualisation).…”

“…However, the introduction of these in vitro models are a trade-off between the value of obtaining physiologically relevant data against their through-put as they are usually more labour intensive [11]. Furthermore, it is problematic to utilise tissue models for thorough investigation of the aforementioned complex relationships during tissue regeneration, as it is difficult to distinguish the regulatory functions of each individual architectural features at nano-and micro-scales, as well as other biochemical and biomechanical properties in the 3D culture environments [12][13][14][15]. To bridge the gap between the 2D and 3D culture technologies, we have recently developed a 3D CCIS (3D cell culture and imaging system) [15].…”

“…Furthermore, it is problematic to utilise tissue models for thorough investigation of the aforementioned complex relationships during tissue regeneration, as it is difficult to distinguish the regulatory functions of each individual architectural features at nano-and micro-scales, as well as other biochemical and biomechanical properties in the 3D culture environments [12][13][14][15]. To bridge the gap between the 2D and 3D culture technologies, we have recently developed a 3D CCIS (3D cell culture and imaging system) [15]. In this scale-down design, cells are cultivated on thin modular substrates with finely controlled open pores, and monitored non-invasively at single cell level using optical microscopes, thus it can be utilised as a high through-put platform to yield mechanistic insights of cell-cell and cell-scaffold interactions.…”