Single-Cell Microfluidic Separation and Analytical Platforms Based on Biophysical Phenotypes

The complex functional and structural organization of biosystems leads to a degree of
heterogeneity of cellular phenotypes. To parse through this heterogeneity, there is the need for
platforms for separation and analysis, with single-cell sensitivity, to associate biological function
and disease with particular cellular markers. The current state-of-the-art method for this purpose
is based on single-cell analysis by flow cytometry, after fluorescent staining for their characteristic
cell surface proteins, which is then used to identify and separate cells based on biochemical
characteristics. However, there is an increasing recognition that biological processes, such as
prediction of cancer metastasis, stem cell differentiation lineage, or different immune cells
activation levels that cannot be linked solely to biochemical traits. Hence, there is emerging
interest in identifying cells, cellular aggregates, and subcellular bodies based on biophysical
properties for separation and quantification. These biophysical properties can include cell size
distribution, shape, deformability, and electrophysiology-based characteristics.
Biomechanical metrics, such as deformability of cells and cellular aggregates, caused by
microfluidic constrictions or post structures can allow for stratification of biosystems based on cell
size, rigidity, and its extracellular matrix properties. Devices for separation and analysis using
biomechanical metrics were developed for two distinct applications. The first focused on
monitoring biophysical heterogeneity of the integration of pancreatic islets with adipose-derived
stems cells (ADSCs), which alter the basement membrane, and angiogenic factors in the islet, which
are important for its transplantation to treat diabetic conditions. Given the heterogeneity in size,
shape, and extracellular matrix of human islets, we seek to determine if biomechanical metrics of
single islets can be used as a marker to indicate the completion of their integration with ADSCs. The
second application focused on microfluidic deterministic lateral displacement (DLD) for size-selective separation of macrophages to enrich the activated fraction from heterogeneous samples.
This can be used to measure the immunomodulation conditions during macrophage interactions
with tissues to reduce inflammations.
Electrophysiology can serve as a subcellular marker to stratify and separate cells by
dielectrophoresis (DEP), based on membrane capacitance or interior conductivity characteristics.
We seek to integrate on-chip sample preparation and phenotypic assessment functionalities on
DEP separation platforms. Specifically, to reduce cumbersome off-chip operations, we propose to
integrate the capability for on-chip swapping of cells from culture media to a low conductivity
buffer prior to DEP manipulation, and then back to the culture media following DEP separation.
This will be demonstrated using red blood cells.