Guiding cell cultures by engineering their extracellular environment is an area which has gathered a lot of attention because of its potential to repair, maintain and develop tissues and organs. However, current biofabrication approaches struggle to construct 3D networks because of the precision and complexity required.
A team of Researchers from Hong Kong have now developed an optical µ-printing technology that can fabricate 3D scaffold arrays for an optical µ-printing technology to study the interactions between 3D cell cultures and cell-scaffolds on a single chip.
The topographies of a cell on the micro and nanoscale have been revealed to have an essential influence on the regulation of cell behavior and ultimately its fate. This is particularly relevant for stem cells, where their self-renewal and differentiation are significantly dependent upon the surrounding microenvironment, also known as the stem cell niche.
However, the study of stem cells, particularly human mesenchymal stem cells (hMSCs), in three-dimensions has been rarely achieved. The lack of studies on this scale have been attributed to the lack of efficient approaches towards fabricating large 3D micro-scaffolds with tuneable structural and surface properties.
The Researchers have now created a printing technology that utilizes dynamic optical projection stereolithography (DOPsL) with machine vision metrology and new biomaterial processing protocols for the production of 3D cubic micro-scaffold arrays. The cubic arrays produced were of the order of tens of micrometers, thus, they mimicked the natural structure of bone lacunae.
In-situ printing of gelatin methacrylate (GelMA) onto a suspended beam of an SU-8 photoresist polymer was utilized by the Researchers. The GelMA acted as an active coating for the beam to enhance and guide cell adhesion and spreading. To help bind the GelMA to the beam, polydopamine (PDA) was first coated on the scaffolds. hMSCs were grown to a constant density of 20 000 cells/cm2 onto the micro-scaffolds and incubated in a growth medium for 48 hours.
The µ-printer itself was composed of six parts: a UV light source (L10561, Hamamatsu Photonics K.K), a DMD chip (DLP9500, Digital Light Innovations), projection optics (Thorlabs Inc), a digital camera, a motorized stage (ANT130-XY, Aerotech Inc) and a computer for sliced images (Techplot Inc). All of which contributed to the formation of an automated optical μ-printing process.
To study the interaction of the 3D scaffolds within the hMSC cell cultures, the Researchers employed fluorescence staining and image analysis approaches. Amongst washing steps, the cells were fixed with 4% paraformaldehyde solution, 2% Triton-X100, 1% BSA and were then stained using primary antibodies against a molecular sensor known as yes-associated protein (YAP).
Secondary antibody staining was performed upon incubation in goat anti-mouse IgG containing Alexa488. The cytoskeleton was also stained with phalloidin-TRITC, whilst the cell nuclei were stained using 6-diamidino-2-phenylindole (DAPI). The fluorescent images were taken using a Nikon C2+ confocal microscope and analyzed using Image J (NIH) software. Statistical analyzes were also performed using one-way ANOVA and Tukey’s post hoc testing methods.
The Researchers produced an array of 3D cubic microscaffolds which matched the size of a single cell. This approach was found to aid the cell spreading across the microbeams and exposed both the optical and basal cell membranes. The printing process first allowed the cells to adhere to the microbeams and then spread across the scaffold.
The Researchers also demonstrated that by increasing the cubical size of the scaffold, they could enhance the spreading of hMSc and activate mechanosensing signaling which promoted osteogenesis.
The Researchers also showed that by selectively modifying the morphology (shape and area) of the coated beam surface, they could produce a process which was tailorable in terms of cell adhesion and spreading capabilities across the micro-scaffolds. The modification of the morphology was also found to be dependent upon the geometry of the micro-scaffold.
The dual-material based printing protocol was found to produce a biomimetic 3D microplatform with chemically and structurally tuneable properties for cell and culture and migration studies. The information obtained will provide a new pathway for tailorable 3D cell cultures, downstream cell studies and will provide a platform for the investigation of hMSC behaviors in bone research.
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“Optical µ-Printing of Cellular-Scale Microscaffold Arrays for 3D Cell Culture”- Ouyang X., et al, Scientific Reports, 2017, DOI:10.1038/s41598-017-08598-3