Continuous-flow sorting of stem cells and differentiation products based on dielectrophoresis.

This paper presents a continuous-flow microfluidic device for sorting stem cells and their differentiation progenies. The principle of the device is based on the accumulation of multiple dielectrophoresis (DEP) forces to deflect cells laterally in conjunction with the alternating on/off electric field to manipulate the cell trajectories. The microfluidic device containing a large array of oblique interdigitated electrodes was fabricated using a combination of standard and soft lithography techniques to generate a PDMS-gold electrode construct. Experimental testing with human mesenchymal stem cells (hMSC) and their differentiation progenies (osteoblasts) was carried out at different flow rates, and clear separation of the two populations was achieved. Most of the osteoblasts experiencing stronger DEP forces were deflected laterally and continuously, following zig-zag trajectories, and moved towards the desired collection outlet, whereas most of the hMSCs remained on the original trajectory due to weaker DEP forces. The experimental measurements were characterized and evaluated quantitatively, and consistent performance was demonstrated. Collection efficiency up to 92% and 67% for hMSCs and osteoblasts, respectively, along with purity up to 84% and 87% was obtained. The experimental results established the feasibility of our microfluidic DEP sorting device for continuous, label-free sorting of stem cells and their differentiation progenies.

A microfluidic impedance flow cytometer for identification of differentiation state of stem cells.

This paper presents a microfluidic electrical impedance flow cytometer (FC) for identifying the differentiation state of single stem cells. This device is comprised of a novel dual micropore design, which not only enhances the processing throughput, but also allows the associated electrodes to be used as a reference for one another. A signal processing algorithm, based on the support vector machine (SVM) theory, and a data classification method were developed to automate the identification of sample types and cell differentiation state based on measured impedance values. The device itself was fabricated using a combination of standard and soft lithography techniques to generate a PDMS-gold electrode construct. Experimental testing with non-biological particles and mouse embryonic carcinoma cells (P19, undifferentiated and differentiated) was carried out using a range of excitation frequencies. The effects of the frequency and the interrogation parameters on sample identification performance were investigated. It was found that the real and imaginary part of the detected impedance signal were adequate for distinguishing the undifferentiated P19 cells from non-biological polystyrene beads at all tested frequencies. A higher frequency and an opacity index were required to resolve the undifferentiated and differentiated P19 cells by capturing capacitive changes in electrophysiological properties arising from differentiation. The experimental results demonstrated salient accuracy of the device and algorithm, and established its feasibility for non-invasive, label-free identification of the differentiation state of the stem cells.

Targeted delivery of VEGF to treat myocardial infarction.

Noninvasive injection of pro-angiogenic compounds such as vascular endothelial growth factor (VEGF) has shown promising results in regenerating cardiac microvasculature. However, these results have failed to translate into successful clinical trials in part due to the short half-life of VEGF in circulation. Increasing the dose of VEGF may increase its availability to the target tissue, but harmful side-effects remain a concern. Encapsulating and selectively targeting VEGF to the MI border zone may circumvent these problems. Anti-P-selectin conjugated immunoliposomes containing VEGF were developed to target the infarct border zone in a rat MI model. Targeted VEGF therapy significantly improves vascularization and cardiac function after an infarction.

Targeted Delivery of VEGF after a Myocardial Infarction Reduces Collagen Deposition and Improves Cardiac Function.

The development of adjunctive therapies which attenuate adverse remodeling and improve LV function post myocardial infarction (MI) is of significant clinical interest. Previously, we have shown that targeted delivery of therapeutic vascular endothelial growth factor (VEGF) to the infarct border zone significantly increases vascular perfusion and results in improvements in LV function. In this study, we tested the hypothesis that improvements in cardiac function observed with this novel targeted drug delivery system strongly correlate with reductions in collagen deposition in the scar tissue after an MI. Rats received anti-P-selectin conjugated immunoliposomes containing VEGF immediately post-MI. Over 4 weeks, evolutionary changes in LV geometry and function were correlated with collagen deposition and infarct size quantified by Gomori's trichrome and picrosirius red staining. Targeted VEGF treated hearts showed a 37% decrease in collagen deposition in the anterior wall, as well as significant improvements in LV filling pressures. Multi-regression analysis showed that the extent of collagen deposition post MI can be predicted by a linear combination of normalized LV mass and ejection fraction. Targeted delivery of VEGF post-MI results in significant decreases in collagen deposition and adverse remodeling. Improvements in cardiac function in this model are related to degree of collagen deposition and extent of scar formation.

Fourier transform infrared spectroscopic imaging of cardiac tissue to detect collagen deposition after myocardial infarction.

Myocardial infarction often leads to an increase in deposition of fibrillar collagen. Detection and characterization of this cardiac fibrosis is of great interest to investigators and clinicians. Motivated by the significant limitations of conventional staining techniques to visualize collagen deposition in cardiac tissue sections, we have developed a Fourier transform infrared imaging spectroscopy (FT-IRIS) methodology for collagen assessment. The infrared absorbance band centered at 1338 cm(-1), which arises from collagen amino acid side chain vibrations, was used to map collagen deposition across heart tissue sections of a rat model of myocardial infarction, and was compared to conventional staining techniques. Comparison of the size of the collagen scar in heart tissue sections as measured with this methodology and that of trichrome staining showed a strong correlation (R=0.93). A Pearson correlation model between local intensity values in FT-IRIS and immuno-histochemical staining of collagen type I also showed a strong correlation (R=0.86). We demonstrate that FT-IRIS methodology can be utilized to visualize cardiac collagen deposition. In addition, given that vibrational spectroscopic data on proteins reflect molecular features, it also has the potential to provide additional information about the molecular structure of cardiac extracellular matrix proteins and their alterations.

Towards a targeted multi-drug delivery approach to improve therapeutic efficacy in breast cancer.

IMPORTANCE OF THE FIELD: Significant improvements in breast cancer treatments have resulted in a significant decrease in mortality. However, current breast cancer therapies, for example, chemotherapy, often result in high toxicity and nonspecific side effects. Other treatments, such as hormonal and antiangiogenic therapies, often have low treatment efficacy if used alone. In addition, acquired drug resistance decreases further the treatment efficacy of these therapies. Intra-tumor heterogeneity of the tumor tissue may be a major reason for the low treatment efficacy and the development of chemoresistance. Therefore, targeted multi-drug therapy is a valuable option for addressing the multiple mechanisms that may be responsible for reduced efficacy of current therapies. AREAS COVERED IN THIS REVIEW: In this article, different classes of drugs for treating breast cancer, the possible reasons for the drug resistance in breast cancer, as well as different targeted drug delivery systems are summarized. The current targeting strategies used in cancer treatment are discussed. WHAT THE READER WILL GAIN: This article considers the current state of breast cancer therapy and the possible future directions in targeted multi-drug delivery for treating breast cancer. TAKE HOME MESSAGE: A better understanding of tumor biology and physiological responses to nanoparticles, as well as advanced nanoparticle design, are needed to improve the therapeutic outcomes for treating breast cancer using nanoparticle-based targeted drug delivery systems. Moreover, selective delivery of multi-drugs to tumor tissue using targeted drug delivery systems may reduce systemic toxicity further, overcome drug resistances, and improve therapeutic efficacy in treating breast cancer.

Targeting VEGF-encapsulated immunoliposomes to MI heart improves vascularity and cardiac function.

Recent attempts at rebuilding the myocardium using stem cells have yielded disappointing results. The lack of a supporting vasculature may, in part, explain these disappointing findings. However, concerns over possible side effects have hampered attempts at revascularizing the infarcted myocardium using systemic delivery of proangiogenic compounds. In this study, we develop the technology to enhance the morphology and function of postinfarct neovasculature. Previously, we have shown that the up-regulated expression of endothelial cell adhesion molecules in the myocardial infarction (MI) region provides a potential avenue for selectively targeting drugs to infarcted tissue. After treatment with anti-P-selectin-conjugated liposomes containing vascular endothelial growth factor (VEGF), changes in cardiac function and vasculature post-MI were quantified in a rat MI model. Targeted delivery of VEGF to post-MI tissue resulted in significant increase in fractional shortening and improved systolic function. These functional improvements were accompanied by a 21% increase in the number of anatomical vessels and a 74% increase in the number of perfused vessels in the MI region of treated animals. No significant improvements in cardiac function were observed in untreated, systemic VEGF-treated, nontargeted liposome-treated, or blank immunoliposome-treated animals. Targeted delivery of low doses of proangiogenic compounds to post-MI tissue results in significant improvements in cardiac function and vascular structure.

A physiologically realistic in vitro model of microvascular networks.

Existing microfluidic devices, e.g. parallel plate flow chambers, do not accurately depict the geometry of microvascular networks in vivo. We have developed a synthetic microvascular network (SMN) on a polydimethalsiloxane (PDMS) chip that can serve as an in vitro model of the bifurcations, tortuosities, and cross-sectional changes found in microvascular networks in vivo. Microvascular networks from a cremaster muscle were mapped using a modified Geographical Information System, and then used to manufacture the SMNs on a PDMS chip. The networks were cultured with bovine aortic endothelial cells (BAEC), which reached confluency 3-4 days after seeding. Propidium iodide staining indicated viable and healthy cells showing normal behavior in these networks. Anti-ICAM-1 conjugated 2-mum microspheres adhered to BAEC cells activated with TNF-alpha in significantly larger numbers compared to control IgG conjugated microspheres. This preferential adhesion suggests that cultured cells retain an intact cytokine response in the SMN. This microfluidic system can provide novel insight into characterization of drug delivery particles and dynamic flow conditions in microvascular networks.