Both hard material photolithography and soft lithography are widely used for patterned cell culture. Soft lithography techniques enable bioactive molecules incorporation, however complex surface modifications are required to introduce specific ligands or proteins in conventional photolithography. We have developed a novel substrate by patterning titanium diboride (TiB2) layers deposited on silicon (Si) by electron-beam evaporation and micropatterned using photolithography. Micropatterned cell growth specificity on geometric shapes of circle and/or lines is achieved via differential heparin bindinggrowth factors adsorption in the presence of heparin. Specifically, the deposited films of TiB2 show increased stiffness, hardness, hydrophilicity and less negative surface charges when compared to background Si. The substrates are biocompatible based on human umbilical vein endothelial cell (HUVEC) and mesenchymal stem cell (MSC) viability, biomarker expression and RNA-sequence transcriptome analysis. Cell-type dependent selective growth patterns and behaviours such as contact guidance, alignment, and durotaxis based preferential growth on micropatterns were observed. In addition, cell clustering into three-dimensional (3D) aggregates was achieved for MSCs enabling a 3D microenvironment during culture. The microfabricated Si-TiB2 substrate enables patterned cell culture in vitro, independent of any additional surface modification, and can be used for culture and co-culture of a variety of cells of epithelial and mesodermal lineage.