Novel Method of Laser Direct Writing for Precise Patterning of Human Dermal Fibroblasts

Abstract

The ability to control a cell’s location, pattern geometry, and proximity to neighboring cells, in vitro, is highly desired to gain insight into cell-cell interactions, such as the modes of cellular signaling (direct cell contact, paracrine, or endocrine). A laser-based cell patterning technique, laser direct write, enables the precise spatial placement of living cells, with all the advantages of CAD/CAM control [1]. However, this technique is limited in usefulness due to the dependence on Matrigel® (BD Biosciences, Bedford, MA). The growth factor constituents of Matrigel® may interfere with many cellular processes under investigation and may preclude or greatly limit the utility of laser direct writing for precise cell cultures [2]. Therefore, to address this limitation, the objective of this study was to develop a Matrigel®-free laser direct writing method. Through the use of customized gelatin coatings on both the ribbon and receiving substrate, we effectively adapted the direct write technique to precisely pattern cells without the use of Matrigel®, as demonstrated with human dermal fibroblasts. The gelatin partially encapsulates the trypsinized cells on the ribbon, providing a volitization zone to protect the cells, and on the receiving substrate cushions the impact of transfer while maintaining moisture. Gelatin liquefies at 37°C, which allows it to be removed from the growth surface ensuring cellular proliferation, uninhibited by growth surface treatments. This represents a fundamental change from the original direct write technique in which cells must first form initial attachments to the ribbon via Matrigel® and then are written to a Matrigel® coated receiving substrate for their sustained growth. Additionally, we have developed a method to monitor the location of the patterned cells post-transfer to show that a gelatin coated-receiving substrate is effective as a patterning surface and ensures the registry of the pattern until cell attachment, even after the gelatin has been removed with the first growth medium application. This precise patterning technique can now be used in many biomedical applications, including those that involve cell types highly sensitive to growth factors, such as stem cells and cancer cells.