Noninvasive and <i>in situ</i> identification of the phenotypes and differentiation stages of individual living cells entrapped within hydrogels

Microscale screening platforms that allow cells to interact in three dimensions (3D) with their microenviroment have been developed as a tool for identifying the extrinsic cues that might stimulate stem cells to replicate without differentiating within artificial cultures. Though these platforms reduce the number of valuable stem cells that must be used for screening, analyzing the fate decisions of cells in these platforms can be challenging. New noninvasive approaches for identifying the lineage-specific differentiation stages of cells while they are entrapped in the hydrogels used for these 3D cultures are especially needed. Here we used Raman spectra acquired from individual, living cells entrapped within a hydrogel matrix and multivariate analysis to identify cell phenotype noninvasively and <i>in situ</i>. We collected a single Raman spectrum from each cell of interest while it was entrapped within a hydrogel matrix and used partial least-squares discriminant analysis (PLS-DA) of the spectra for cell phenotype identification. We first demonstrate that this approach enables identifying the lineages of individual, living cells from different laboratory lines entrapped within two different hydrogels that are used for 3D culture, collagen and gelatin methacrylate (gelMA). Then we use a hematopoietic progenitor cell line that differentiates into different types of macrophages to show that the lineage-specific differentiation stages of individual, living hematopoietic cells entrapped inside of gelMA scaffolds may be identified by PLS-DA of Raman spectra. This ability to noninvasively identify the lineage-specific differentiation stages of cells without removing them from a 3D culture could enable tracking the differentiation of the same cell over time.