In situ laser micropatterning of proteins for dynamically arranging living cells

Abstract

This study shows the modification of the surface of polymer-layered glass substrates to form biofunctional micropatterns through femtosecond laser ablation in an aqueous solution. Domains of micrometer size on a substrate can be selectively converted from proteinphobic (resistant to protein adsorption) to proteinphilic, allowing patterning of protein features under physiological aqueous conditions. When femtosecond laser pulses (800 nm, 1 kHz, 200-500 nJ per pulse) were focused on and scanned on the substrate, which was glass covered with the proteinphobic polymer 2-methacryloyloxyethylphosphorylcholine (MPC), the surface became proteinphilic. Surface analysis by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) reveals that the laser ablates the MPC polymer. Extracellular matrix (ECM) proteins were bound to the laser-ablated surface by physisorption. Since femtosecond laser ablation is induced under physiological aqueous conditions, this approach can form micropatterns of functional ECM proteins with minimal damage. This method was applied to pattern collagen, laminin, and gelatin on the substrate. Removal of an ECM protein from the substrate followed by replacement with another ECM protein was achieved on demand at a specific location and time by the same laser ablation method. Living cells adhered to the fabricated domains where ECM proteins were arranged. The modification of patterning during cell culture was used to control cell migration and form arrays of different cells.

Selective induction of targeted cell death and elimination by near-infrared femtosecond laser ablation

“…Plasma-cleaned glass substrates (Borosilicate, 24 mmφ, 0.12–0.17 mm thickness) were coated with cytophobic copolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) followed by micropatterning to form cell adhesion domains by NIR fs laser scanning as previously reported [ 25 ]. In detail, NIR fs laser (1 kHz, 150 μW) was focused with a water immersion objective (20×, NA.…”

High-throughput screening for integrative biomaterials design: exploring advances and new trends

“…Techniques such as contact, inkjet, and laser printing allowed patterning biomaterials and biomaterial/cell mixtures with diameters ranging from 50 mm to 500 mm [13,[26][27][28][29][30][31][32][33] attaining, mainly in the case of cell-laden hydrogels, lower values than indirect writing techniques (Table 2). However, the spreading of the biomaterials in such platforms is dependent on their chemical/physical interactions with the surface.…”

“…Piezoelectric stimuli or heat may be applied in order to separate the liquid from the tip of the nozzle; the use of the heating strategy allowed cell encapsulation with viability in the range of 90% [29]. Alternatively, laser printing (Table 2), as firstly suggested by Guillemot et al [30], was used to print microarrays of cells -avoiding DNA fragmentation [31,32] -ceramic/polymeric biomaterials, and proteins [33].…”

Guided Axon Outgrowth of Neurons by Real-Time Femtosecond Laser Penetration on a 4 μm Thick Thin-Glass Sheet

“…Cell transplantation is the current gold standard for treating brain injuries, especially those identified by large lesions, such as traumatic method for direct axon outgrowth in real-time conditions to attain robust axon regeneration and promote functional recovery after brain injury. Femtosecond (fs) laser is a powerful tool for precisely manipulating cells and scaffolds (Hosokawa et al, 2009;Kaji et al, 2007;Okano et al, 2016Okano et al, , 2013Okano et al, , 2011Rukmana et al, 2019;Yamamoto et al, 2011). Fs laser can be positioned in sophisticated optical arrangements that allow the placement of other controllers (e.g., a motorized stage and an environmental chamber), resulting in reliable and reproducible experiments.…”

“…Compared to other lasers, fs laser processing produces low heat which can minimize thermalinduced cell death (Kasaai et al, 2003;Le Harzic et al, 2002). Based on these favorable characteristics, our group has successfully developed guided neurite outgrowth by ablating the cytophobic area to expose the cytophilic area of the scaffold, resulting in neurites grow precisely along with the linear shape of the ablated area (Okano et al, 2016(Okano et al, , 2013Yamamoto et al, 2011). However, these studies have limitations for transplantation purposes in donating molecules to control axon outgrowth of neurons in real-time conditions.…”