Micro- and Nanoscale Technologies for Delivery into Adherent Cells

Abstract

Several recent micro- and nano-technologies have provided novel methods for biological studies of adherent cells because the small features of these new biotools provide unique capabilities for accessing cells without the need for suspension or lysis. These novel approaches have enabled gentle, yet effective delivery of molecules into specific adhered target cells, with unprecedented spatial resolution. Here we review recent progress in the development of these technologies with an emphasis on in vitro delivery into adherent cells utilizing mechanical penetration or electroporation. We discuss major advantages and limitations of these approaches and propose possible strategies for improvements. Finally, we discuss the impact of these technologies on biological research concerning cell-specific temporal studies, e.g., non-destructive sampling and analysis of intracellular molecules. Need For Techniques To Study Adherent Cells A mechanistic understanding of cell biology is often limited by both the complexity of the processes and limitations of commonly available research tools that lack temporal or spatial resolution. The lack of tools capable of providing cell-specific, non-destructive biomolecular delivery and analysis is a particular barrier for advancing fundamental discoveries of cell heterogeneity, single-cell behavior within a complex environment, and the mechanisms that govern disease states, responses to drugs or other stimuli, and differentiation of stem cells. To gain new mechanistic understanding, advances in methods for precise intracellular delivery and non-destructive biochemical analyses of non-secretory molecules (e.g., mRNA and proteins) are greatly needed so that individual cells can be experimentally controlled and repeatedly analyzed over time and/or within a particular location of the cell. For example, developing neurons must undergo a series of sequential changes in gene expression to achieve a mature phenotype; hence, understanding the process will require the ability to accurately monitor the sequence of intracellular events, within individual cells, in a non-destructive manner. In addition, neuronal maturation is influenced by interactions with surrounding cells and with extracellular matrix, so it is necessary to be able to simultaneously monitor events occurring in multiple cells that are interacting with each other and with the matrix. While the requirements are challenging, these experimental capabilities would provide unprecedented insight into the determinants of both the timing of cellular processes and their phenotype, the principles of cell heterogeneity, and the role of cell-cell communication in homogeneous cell populations and co-cultures. Because most cells adhere to a substrate or to other cells during their growth or differentiation [1], it is advantageous for new technologies to be capable of accessing adhered cells to avoid the need to disrupt cell processes by suspension and replating. Several technologies for studying adhered cells are currently being developed, and ...

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