Laser Photofabrication of Cell-Containing Hydrogel Constructs

Abstract

The two-photon polymerization (2PP) of photosensitive gelatin in the presence of living cells is reported. The 2PP technique is based on the localized cross-linking of photopolymers induced by femtosecond laser pulses. The availability of water-soluble photoinitiators (PI) suitable for 2PP is crucial for applying this method to cell-containing materials. Novel PIs developed by our group allow 2PP of formulations with up to 80% cell culture medium. The cytocompatibility of these PIs was evaluated by an MTT assay. The results of cell encapsulation by 2PP show the occurrence of cell damage within the laser-exposed regions. However, some cells located in the immediate vicinity and even within the 2PP-produced structures remain viable and can further proliferate. The control experiments demonstrate that the laser radiation itself does not damage the cells at the parameters used for 2PP. On the basis of these findings and the reports by other groups, we conclude that such localized cell damage is of a chemical origin and can be attributed to reactive species generated during 2PP. The viable cells trapped within the 2PP structures but not exposed to laser radiation continued to proliferate. The live/dead staining after 3 weeks revealed viable cells occupying most of the space available within the 3D hydrogel constructs. While some of the questions raised by this study remain open, the presented results indicate the general practicability of 2PP for 3D processing of cell-containing materials. The potential applications of this highly versatile approach span from precise engineering of 3D tissue models to the fabrication of cellular microarrays.

3D Microporous Scaffolds Manufactured via Combination of Fused Filament Fabrication and Direct Laser Writing Ablation

“…Composite material structures of various geometries and periodicities are of great interest and already technologically realized [16,17]. Centimeter-scale sample scaffolds are already producible overnight [18,19], with the ability to incorporate living cells [20]. It is noteworthy that many of the laser cross-linkable materials are biocompatible in vivo have shown in ex vivo tests of implantation into rabbit tissues [21].…”

Two-photon polymerization of hydrogels – versatile solutions to fabricate well-defined 3D structures

“…97 In a similar manner, GelMOD was processed in formulations with up to 80% water in the presence of living cells. 86 In this case, the benzylidenecycloketones (G2CK and P2CK) were used as photoinitiators ( Table 1) that allowed the fabrication of well-dened microstructures. Human osteosarcoma cells (MG63) incorporated in the cross-linked GelMOD hydrogel were affected during the 2PP fabrication process, whereas the ones found in the immediate proximity or within the 2PP structures survived the fabrication process.…”

Laser 3D Printing with Sub‐Microscale Resolution of Porous Elastomeric Scaffolds for Supporting Human Bone Stem Cells

“…For example, freeze-drying of collagen can be used to create a variety of structures with microscale porosities [ 21 ] ; unlike 3D printing, freezedrying of collagen does not enable a wide variety of geometries to be created. [ 22 ] Recent advances in microelectronics fabrication have allowed for surface geometry manipulation on the micrometer and sub-micrometer scales. Although some techniques such as photolithography, nanoimprinting, dip-pen lithography, and others have been available for a decade or longer, [ 23 ] they have only recently begun to gain the attention of the medical device industry as a platform for understanding fundamental cell responses.…”

“…It was recently demonstrated that 3D structures can be prepared from gelatin-based photopolymer formulations with high water content; live cells can be trapped within the gelatin-based photopolymer structures during 2PP processing. [ 22 ] 2PP laser printing has been used successfully with a variety of polymeric materials. [ 22,31 ] The present study expands the use of 2PP to an elastomer material, which allows for the creation of large, durable scaffolds that resist tearing and have appropriate stiffness for use with a variety of cell and tissue types.…”

“…[ 22 ] 2PP laser printing has been used successfully with a variety of polymeric materials. [ 22,31 ] The present study expands the use of 2PP to an elastomer material, which allows for the creation of large, durable scaffolds that resist tearing and have appropriate stiffness for use with a variety of cell and tissue types. The resulting scaffolds are fabricated relatively rapidly and allow for facile handling with standardized biological assays.…”