Biomedical researchers have become increasingly aware of the limitations of the conventional 2-D tissue cell cultures where most tissue cell studies including cancer and tumor cells have been carried out. They are now searching and testing 3-D cell culture systems, something between a petri dish and a mouse. The important implications of 3-D tissue cell cultures for basic cell biology, tumor biology, high-content drug screening, and regenerative medicine and beyond are far-reaching. How can nanobiotechnology truly advance the traditional cell, tumor, and cancer biology? Why nano is important in biomedical research and medical science? A nanometer is 1000 times smaller than a micrometer, but why it matters in biology? This chapter addresses these questions. It has become more and more apparent that 3-D cell culture offers a more realistic local environment through the nanofiber scaffolds where the functional properties of cells can be observed and manipulated. A new class of designer self-assembling peptide nanofiber scaffolds now provides an ideal alternative system. Time has come to address the 3-D questions because quantitative biology requires in vitro culture systems that more authentically represent the cellular microenvironment in a living organism. In doing so, in vitro experimentation can become truly more predictive of in vivo systems.