Ramila Joshi

Interfacial Tension Effect on Cell Partition in Aqueous Two-Phase Systems.

Aqueous two-phase systems (ATPS) provide a mild environment for the partition and separation of cells. We report a combined experimental and theoretical study on the effect of interfacial tension of polymeric ATPS on the partitioning of cells between two phases and their interface. Two-phase systems are generated using polyethylene glycol and dextran of specific properties as phase-forming polymers and culture media as the solvent component. Ultralow interfacial tensions of the solutions are precisely measured using an axisymmetric drop shape analysis method. Partition experiments show that two-phase systems with an interfacial tension of 30 μJ/m(2) result in distribution of majority of cells to the bottom dextran phase. An increase in the interfacial tension results in a distribution of cells toward the interface. An independent cancer cell spheroid formation assay confirms these observations: a drop of the dextran phase containing cancer cells is dispensed into the immersion polyethylene glycol phase to form a cell-containing drop. Only at very small interfacial tensions do cells remain within the drop to aggregate into a spheroid. We perform a thermodynamic modeling of cell partition to determine variations of free energy associated with displacement of cells in ATPS with respect to the ultralow interfacial tensions. This modeling corroborates with the experimental results and demonstrates that at the smallest interfacial tension of 30 μJ/m(2), the free energy is a minimum with cells in the bottom phase. Increasing the interfacial tension shifts the minimum energy and partition of cells toward the interfacial region of the two aqueous phases. Examining differences in the partition behavior and minimum free energy modeling of A431.H9 cancer cells and mouse embryonic stem cells shows that the surface properties of cells further modulate partition in ATPS. This combined approach provides a fundamental understanding of interfacial tension role on cell partition in ATPS and a framework for future studies.

Liquid-based three-dimensional tumor models for cancer research and drug discovery.

Tumors are three-dimensional tissues where close contacts between cancer cells, intercellular interactions between cancer and stromal cells, adhesion of cancer cells to the extracellular matrix, and signaling of soluble factors modulate functions of cancer cells and their response to therapeutics. Three-dimensional cultures of cancer cells overcome limitations of traditionally used monolayer cultures and recreate essential characteristics of tumors such as spatial gradients of oxygen, growth factors, and metabolites and presence of necrotic, hypoxic, quiescent, and proliferative cells. As such, three-dimensional tumor models provide a valuable tool for cancer research and oncology drug discovery. Here, we describe different tumor models and primarily focus on a model known as tumor spheroid. We summarize different technologies of spheroid formation, and discuss the use of spheroids to address the influence of stromal fibroblasts and immune cells on cancer cells in tumor microenvironment, study cancer stem cells, and facilitate compound screening in the drug discovery process. We review major techniques for quantification of cellular responses to drugs and discuss challenges ahead to enable broad utility of tumor spheroids in research laboratories, integrate spheroid models into drug development and discovery pipeline, and use primary tumor cells for drug screening studies to realize personalized cancer treatment.

Engineered Breast Cancer Cell Spheroids Reproduce Biologic Properties of Solid Tumors.

Solid tumors develop as 3D tissue constructs. As tumors grow larger, spatial gradients of nutrients and oxygen and inadequate diffusive supply to cells distant from vasculature develops. Hypoxia initiates signaling and transcriptional alterations to promote survival of cancer cells and generation of cancer stem cells (CSCs) that have self-renewal and tumor-initiation capabilities. Both hypoxia and CSCs are associated with resistance to therapies and tumor relapse. This study demonstrates that 3D cancer cell models, known as tumor spheroids, generated with a polymeric aqueous two-phase system (ATPS) technology capture these important biological processes. Similar to solid tumors, spheroids of triple negative breast cancer cells deposit major extracellular matrix proteins. The molecular analysis establishes presence of hypoxic cells in the core region and expression of CSC gene and protein markers including CD24, CD133, and Nanog. Importantly, these spheroids resist treatment with chemotherapy drugs. A combination treatment approach using a hypoxia-activated prodrug, TH-302, and a chemotherapy drug, doxorubicin, successfully targets drug resistant spheroids. This study demonstrates that ATPS spheroids recapitulate important biological and functional properties of solid tumors and provide a unique model for studies in cancer research.

Molecular analysis of stromal cells-induced neural differentiation of mouse embryonic stem cells

Deriving specific neural cells from embryonic stem cells (ESCs) is a promising approach for cell replacement therapies of neurodegenerative diseases. When co-cultured with certain stromal cells, mouse ESCs (mESCs) differentiate efficiently to neural cells. In this study, a comprehensive gene and protein expression analysis of differentiating mESCs is performed over a two-week culture period to track temporal progression of cells from a pluripotent state to specific terminally-differentiated neural cells such as neurons, astrocytes, and oligodendrocytes. Expression levels of 26 genes consisting of marker genes for pluripotency, neural progenitors, and specific neuronal, astroglial, and oligodendrocytic cells are tracked using real time q-PCR. The time-course gene expression analysis of differentiating mESCs is combined with the hierarchal clustering and functional principal component analysis (FPCA) to elucidate the evolution of specific neural cells from mESCs at a molecular level. These statistical analyses identify three major gene clusters representing distinct phases of transition of stem cells from a pluripotent state to a terminally-differentiated neuronal or glial state. Temporal protein expression studies using immunohistochemistry demonstrate the generation of neural stem/progenitor cells and specific neural lineages and show a close agreement with the gene expression profiles of selected markers. Importantly, parallel gene and protein expression analysis elucidates long-term stability of certain proteins compared to those with a quick turnover. Describing the molecular regulation of neural cells commitment of mESCs due to stromal signaling will help identify major promoters of differentiation into specific cell types for use in cell replacement therapy applications.

Microprinted Stem Cell Niches Reveal Compounding Effect of Colony Size on Stromal Cells-Mediated Neural Differentiation

Microenvironmental factors have a major impact on differentiation of embryonic stem cells (ESCs). Here, a novel phenomenon that size of ESC colonies has a significant regulatory role on stromal cells induced differentiation of ESCs to neural cells is reported. Using a robotic cell microprinting technology, defined densities of ESCs are confined within aqueous nanodrops over a layer of supporting stromal cells immersed in a second, immiscible aqueous phase to generate ESC colonies of defined sizes. Temporal protein and gene expression studies demonstrate that larger ESC colonies generate disproportionally more neural cells and longer neurite processes. Unlike previous studies that attribute neural differentiation of ESCs solely to interactions with stromal cells, it is found that increased intercellular signaling of ESCs significantly enhances neural differentiation. This study offers an approach to generate neural cells with improved efficiency for potential use in translational research.

Stem cell colony interspacing effect on differentiation to neural cells

Efforts to enhance the efficiency of neural differentiation of stem cells are primarily focused on exogenous modulation of physical niche parameters such as surface topography and extracellular matrix proteins, or addition of certain growth factors or small molecules to culture media. We report a novel neurogenic niche to enhance the neural differentiation of embryonic stem cells (ESCs) without any external intervention by micropatterning ESCs into spatially organized colonies of controlled size and interspacing. Using an aqueous two‐phase system cell microprinting technology, we generated pairs of uniformly sized isolated ESC colonies at defined interspacing distances over a layer of differentiation‐inducing stromal cells. Our comprehensive analysis of temporal expression of neural genes and proteins of cells in colony pairs showed that interspacing two colonies at approximately 0.66 times the colony diameter (0.66D) significantly enhanced neural differentiation of ESCs. Cells in these colonies displayed higher expression of neural genes and proteins and formed thick neurite bundles between the two colonies. A computational model of spatial distribution of soluble factors of cells in interspaced colony pairs showed that the enhanced neural differentiation is due to the presence of stable concentration gradients of soluble signalling factors between the two colonies. Our results indicate that culturing ESCs in colony pairs with defined interspacing is a promising approach to efficiently derive neural cells. Additionally, this approach provides a platform for quantitative studies of molecular mechanisms that regulate neurogenesis of stem cells.

Colony size effect on neural differentiation of embryonic stem cells microprinted on stromal cells

Controlling cellular microenvironment to induce neural differentiation of embryonic stem cells (ESCs) remains a major challenge. We address this need by introducing a micro-engineered co-culture system that resembles embryonic development in terms of direct intercellular interactions and induces neural differentiation of ESCs. A polymeric aqueous two-phase system (ATPS)-mediated robotic microprinting technology allows precise localization of mouse ESCs (mESCs) over a layer of supporting stromal cells. mESCs proliferate over a 2-week culture period into a single colony of defined size. Physical and chemical cues from the stromal cells guide mESCs to differentiate toward specific neural lineages. We generated mESC colonies of three different sizes from 100, 250 and 500 single cells and showed that size of mESC colonies is an important factor determining the yield of neural cells. Expression of early neural cell markers nestin denoting neural stem cells, NCAM specifying neural progenitors, and β-III tubulin (TuJ) indicating post mitotic neurons escalated from day 4. Differentiation into specific neural cells astrocytes marked by GFAP, oligodendrocytes indicated by CNPase, and TH-positive dopaminergic neurons was observed during the second week of culture. Unexpectedly, analysis of protein expression revealed a disproportionate increase in neural differentiation of mESCs by increase in the colony size. For the first time, our study establishes colony size as an important regulator of fate of ESCs in this heterocellular niche. This approach of deriving neural cells may make a major impact on stem cell research for treating neurodegenerative diseases.