Label-free enrichment of human adipose-derived stem cells using a continuous microfluidic sorting cascade.

Human adipose tissue is a rich source of mesenchymal stem cells (MSCs). Human adipose-derived stem cells (ADSCs) are first prepared by tissue digestion of lipoaspirate. The remaining constituent contains a mixture of ADSCs, other cell types and lysed fragments. We have developed a scalable microfluidic sorter cascade which enabled high-throughput and label-free enrichment of ADSCs prepared from tissue-digested human adipose samples to improve the quality of purified stem cell product. The continuous microfluidic sorter cascade was composed of spiral-shaped inertial and deterministic lateral displacement (DLD) sorters which separated cells based on size difference. The cell count characterization results showed >90% separation efficiency. We also demonstrated that the enriched ADSC sub-population by the microfluidic sorter cascade yielded 6× enhancement of expansion capacity in tissue culture. The incorporation of this microfluidic sorter cascade into ADSC preparation workflow facilitates the generation of transplantation-scale stem cell product. We anticipate our stem cell microfluidic sorter cascade will find a variety of research and clinical applications in tissue engineering and regeneration medicine.

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.

The U.S. Army Telemedicine and m-Health Program: making a difference at home and abroad.

This article highlights the deployment of telemedicine by the U.S. Army through the various echelons of care and in overseas locations, including range and scope of health services provided by telemedicine in a challenging environment. This is followed by a discussion of technological developments advances in mobile communications likely to change the practice of telemedicine in the military from limited fixed-point access to a highly mobile individual with handheld communication devices.

Real time monitoring of lipoplex molar mass, size and density.

Time-resolved multiangle laser light scattering (TR-MALLS) is used to monitor the temporal variation of DNA/cationic liposome lipoplex molar masses and geometric sizes throughout the complexation process. The measured molar masses and geometric sizes are in turn used to estimate lipoplex density. The DNA/cationic lipid charge ratio is found to be the primary factor governing lipoplex formation kinetics and the final lipoplex molar mass, geometric size and density. Charge ratios near unity lead to a growing kinetic regime in which initially formed primary lipoplexes undergo further aggregation eventually forming large molar mass lipoplexes of high density, while charge ratios very far from unity yield low molar mass lipoplexes of lower density. It is also noted that solvent composition can play a significant role in the lipoplex formation process with lipoplexes formed in ion-containing media being larger and denser than those formed in dextrose solution.

Evidence of lipoplex dissociation in liquid formulations.

Aggregation is typically cited as the root cause of lipoplex transfection efficacy loss in liquid formulations. Typically, this conclusion is based on observed increases in lipoplex hydrodynamic size. A more detailed physical characterization of the lipoplex transfection efficacy diminution has previously never been conducted. As a result, most research has focused on either methods of forming more stable lipoplex formulations or preservation methods such as lyophilization. These studies typically consider the use of stabilizing additives, such as polymer and sugar molecules, to enhance the efficacy of lipoplex dispersions. This report details a recent multiangle laser light scattering study of the temporal evolution of lipoplex geometric size (i.e., radius of gyration) and molar mass in liquid-based formulations over several months. The results indicate that for the lipoplex systems considered, the primary factor underlying the long-term loss of lipoplex transfection efficiency is actually lipoplex dissociation caused by a decrease in the observed molar mass of some lipoplex formulations. The increasing geometric sizes observed are actually the result of lipoplex dissociation combined with an increase in volume. That is, the lipoplexes lose mass and expand in volume, leading to a less dense lipoplex structure over time.