Three-dimensional encapsulation of cells within nanostructured silica gels or matrices enables applications as diverse as biosensors, microbial fuel cells, artificial organs, and vaccines. It also allows study of individual cell behaviors. Recent progress has improved the performance and flexibility of cellular encapsulation, yet there remains a need for robust scalable processes for large format production of cell-encapsulating materials. Here, we detail two novel techniques, that enable the large-scale production of functional Nano-Bio-Composites (NBCs) containing living cells within ordered 3-D lipid/silica nanostructures: 1) thick-casting and 2) spray drying. Furthermore, we detail a third technique for material scaling in which aqueous, silicate-based gel monoliths encapsulate biofunctional yeast or bacteria. Both dry processes are demonstrated to work with multiple cell types and result in dry powders exhibiting a unique combination of properties including: highly ordered 3-D nanostructure, extended lipid fluidity, tunable macro-morphologies and aerodynamic diameters, and unexpectedly high physical strength. Nanoindentation of the encasing nanostructure revealed Young’s modulus and hardness of 13 and 1.4 GPa respectively, which was unexpected considering the low processing conditions.
We hypothesized and confirmed that NBC-encapsulated cells would remain viable for extended periods of time under elevated aging conditions. We attribute this due to the high material strength as observed with nanoindentation, which would prevent cell growth and force bacteria into viable but not culturable (VBNC) states. In concordance with the VBNC state, cellular ATP levels remained elevated even over eight months confirming temperature stable, viable cells. However, their ability to undergo resuscitation and enter growth phase greatly decreased with time in the VBNC state. A quantitative method of determining resuscitation frequencies was developed and showed that, after 36 weeks in an NBC-induced VBNC state, less than 1 in 10,000 cells underwent resuscitation. We verify the VBNC phenotype in gel-encapsulated cells by studying cellular RNA expression levels. These latent behaviors are further demonstrated with an in-vivo immunological study in which mice, immunized with NBCs containing the vaccine Bacillus Calmette-Guérin, were observed to be immunized against a latent form of Tuberculosis. This finding is, in our understanding, the first demonstration of a latent disease-specific live cell immunotherapy. The NBC platform production of industrially scalable quantities of VBNC cells is of interest for research in bacterial persistence and screening of drugs targeting such cells. NBC’s may also enable long-term preservation of living cells for applications in cell-based sensing and the packaging and delivery of live-cell vaccines. Moreover, our methodology represents a novel process for preparing formulations of latent cells in-silico, which could find application in basic cellular research and for the development of a latent-specific vaccine.
Viable but not culturable, cell encapsulation, latent vaccine, biopreservation
Nanoscience and Microsystems
Level of Degree
Nanoscience and Microsystems
First Committee Member (Chair)
C. Jeffrey Brinker
Second Committee Member
Third Committee Member
Fourth Committee Member
Johnson, Patrick E.; C. Jeffrey Brinker; Graham Timmins; Jacob Agola; and Jason Harper. "Scalable, biofunctional, ultra-stable nano- bio- composite materials containing living cells." (2018). https://digitalrepository.unm.edu/nsms_etds/48