Ph.D., Rice University

The general focus of the Darling lab is to understand the relationship between the biological function of cells and tissues and their micro/nano-scale mechanical properties. Specific goals include investigating the mechanical biomarkers associated with stem cells before, during, and after differentiation along various cell lineages. It is possible that cell harvests can even be sorted via mechanical properties to enrich source populations used for tissue engineering and cell therapies. We primarily accomplish these cell-level experiments using atomic force microscopy (AFM), which allows high resolution imaging and force measurements at small scales. Biological experiments involve extended cell culture as well as molecular and biochemical assays. We are also interested in applying a wide range of AFM technologies from cellular manipulation to micropatterning. Our long-term goals are to improve cell-based therapies that can be translated to clinical applications. General areas of interest to the lab include:

1. AFM techniques for evaluating the mechanical characteristics of cells and tissues
2. Single-cell characterization of normal and diseased/damaged cells
3. Stem cell enrichment for improving tissue regeneration approaches
4. Understanding the microscale interactions that occur with respect to cell-based therapeutics and tissue engineering procedures

Project summaries:
I. Temporal changes in the biomechanical properties of stem cells during differentiation
Previous results have indicated that cells possess distinct mechanical biomarkers that are associated with lineage. Osteoblasts exhibit a larger modulus than chondrocytes, which in turn exhibit a larger modulus than adipocytes. The local environment plays a major role in the expressed phenotype of cells, as do the biochemical stimuli present in the surrounding media. This project investigates how the mechanical properties of adipose-derived stem cells (ASCs) change during differentiation along three different lineages: osteoblastic, chondrocytic, and adipocytic. Atomic force microscopy (AFM) in conjunction with fluorescent imaging will be used to evaluate the mechanical properties of cells, as well as the extent of differentiation within ASC populations.

II. Mechanical characterization of the stromal fraction of lipoaspirate
ASCs are a promising type of progenitor cell that reside within adipose (fat) tissue. Previous research has indicated that ASCs are a multipotent cell source capable of differentiating along numerous lineages, which combined with its abundance within the body (there is no shortage of fat in America), make it a desirable cell type for cell-based therapies. Harvest of ASCs is relatively simple, involving a short period of tissue dissociation followed by centrifugation. The resulting cell pellet is plated in a monolayer environment and sequentially expanded to create the "stem cell" population. The initial purity of these populations is as yet unknown so experiments utilizing these cells can have unpredictable results. This project focuses on the mechanical characterization of primary cell types present within lipoaspirate. AFM will be used to probe single cells for their elastic and viscoelastic properties. Representative cell populations from adipose-associated tissues will be used to establish a library of mechanical traits than can later be applied to the sorting of ASC harvests.

III. Investigations into the heterogeneity of adipose-derived stem cell clones
A primary goal of the laboratory is to determine whether mechanical biomarkers can be used as a reliable means of determining stem cell differentiation potential. This project will investigate whether single-cell mechanical properties are more consistent within clonal populations than across an entire cell harvest. Heterogeneous, ASC populations will be separated into clonal populations, taking care that each population initially has only a single cell. After approximately twenty doublings, the mechanical characteristics of the cells will be evaluated, as well as the differentiation potential of the population in general. The relationship between mechanical properties and multipotential will be examined across a large number of clones. An alternative (and more difficult) extension to this project is to characterize the mechanical properties of cells prior to the formation of clonal colonies.

IV. High-throughput, mechanical sorting of viscoelastic bodies
For a cell sorting technique to be successful, it has to either characterize cells very quickly or utilize a highly redundant system. This is also true for a mechanically-based sorting approach. Currently, individual cells are tested one-by-one using an AFM-based approach. While perfectly functional for small sample sizes, it would be impractical to test millions of cells using this technique. Alternative approaches range from simple (deformation-based filters) to complex (multiplexed mechanical assays in a microfluidic environment). The goal of this project is to create a prototype system that can mechanically sort viscoelastic bodies (i.e. cells) at a high enough throughput that manual characterization is surpassed. Ideally, individual cell properties will be recorded during the sorting process, which would allow further characterization of sample cell populations.