The ability to precisely deliver molecules into single cells is of great interest to biotechnology researchers for advancing applications in therapeutics, diagnostics, and drug delivery toward the promise of personalized medicine. The use of bulk electroporation techniques for cell transfection has increased significantly in the last decade, but the technique is nonspecific and requires high voltage, resulting in variable efficiency and low cell viability. We have developed a new tool for electroporation using nanofountain probe (NFP) technology, which can deliver molecules into cells in a manner that is highly efficient and gentler to cells than bulk electroporation or microinjection. Here we demonstrate NFP electroporation (NFP-E) of single HeLa cells within a population by transfecting them with fluorescently labeled dextran and imaging the cells to evaluate the transfection efficiency and cell viability. Our theoretical analysis of the mechanism of NFP-E reveals that application of the voltage creates a localized electric field between the NFP cantilever tip and the region of the cell membrane in contact with the tip. Therefore, NFP-E can deliver molecules to a target cell with minimal effect of the electric potential on the cell. Our experiments on HeLa cells confirm that NFP-E offers single cell selectivity, high transfection efficiency (>95%), qualitative dosage control, and very high viability (92%) of transfected cells.

The precise immunological role played by CD4 ؉ T cells in retroviral infections is poorly defined. Here, we describe a new function of these cells, the elimination of retrovirus-infected macrophages. After experimental CD8 ؉ cell depletion, elite controlling macaques with set-point viral loads <500 viral RNA copies/mL mounted robust Gagand Nef-specific CD4 ؉ T cell responses during reestablishment of control with >54% of all virus-specific CD4 ؉ T cells targeting these 2 proteins. Ex vivo, these simian immunodeficiency virus (SIV)-specific CD4 ؉ T cells neither recognized nor suppressed viral replication in SIV-infected CD4 ؉ T cells. In contrast, they recognized SIV-infected macrophages as early as 2 h postinfection because of presentation of epitopes derived from virion-associated Gag and Nef proteins. Furthermore, virus-specific CD4 ؉ T cells displayed direct effector function and eliminated SIV-infected macrophages. These results suggest that retrovirus-specific CD4 ؉ T cells may contribute directly to elite control by inhibiting viral replication in macrophages.antigen processing and presentation ͉ HIV

New techniques to deliver of nucleic acids and other molecules for gene editing and gene expression profiling, which can be performed with minimal perturbation to cell growth or differentiation, are essential for advancing biological research. Studying cells in their natural state, with temporal control, is particularly important for primary cells that are derived by differentiation from stem cells and are adherent, e.g., neurons. Existing high-throughput transfection methods either require cells to be in suspension or are highly toxic and limited to a single transfection per experiment. Here we present a microfluidic device that couples on-chip culture of adherent cells and transfection by localized electroporation. Integrated microchannels allow long-term cell culture on the device and repeated temporal transfection. The microfluidic device was validated by first performing electroporation of HeLa and HT1080 cells, with transfection efficiencies of ~95% for propidium iodide and up to 50% for plasmids. Application to primary cells was demonstrated by on-chip differentiation of neural stem cells and transfection of postmitotic neurons with a green fluorescent protein plasmid.

The role of CD4 ؉ T cells in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication is not well understood. Even though strong HIV-and SIV-specific CD4؉ T-cell responses have been detected in individuals that control viral replication, major histocompatibility complex class II (MHC-II) molecules have not been definitively linked with slow disease progression. In a cohort of 196 SIVmac239-infected Indian rhesus macaques, a group of macaques controlled viral replication to less than 1,000 viral RNA copies/ml. These elite controllers (ECs) mounted a broad SIV-specific CD4 ؉ T-cell response. Here, we describe five macaque MHC-II alleles (Mamu-DRB*w606, -DRB*w2104, -DRB1*0306, -DRB1*1003, and -DPB1*06) that restricted six SIV-specific CD4 ؉ T-cell epitopes in ECs and report the first association between specific MHC-II alleles and elite control. Interestingly, the macaque MHC-II alleles, Mamu-DRB1*1003 and -DRB1*0306, were enriched in this EC group (P values of 0.02 and 0.05, respectively). Additionally, Mamu-B*17-positive SIV-infected rhesus macaques that also expressed these two MHC-II alleles had significantly lower viral loads than Mamu-B*17-positive animals that did not express Mamu-DRB1*1003 and -DRB1*0306 (P value of <0.0001). The study of MHC-II alleles in macaques that control viral replication could improve our understanding of the role of CD4 ؉ T cells in suppressing HIV/SIV replication and further our understanding of HIV vaccine design.

Sphere forming assays are routinely used for in vitro propagation and differentiation of stem cells. Because the stem cell clusters can become heterogeneous and polyclonal, they must first be dissociated into a single cell suspension for further clonal analysis or differentiation studies. The dissociated population is marred by the presence of doublets, triplets and semi-cleaved/intact clusters which makes identification and further analysis of differentiation pathways difficult. In this work, we use inertial microfluidics to separate the single cells and clusters in a population of chemically dissociated neurospheres. In contrast to previous microfluidic sorting technologies which operated at high flow rates, we implement the spiral microfluidic channel in a novel focusing regime that occurs at lower flow rates. In this regime, the curvature-induced Dean’s force focuses the smaller, single cells towards the inner wall and the larger clusters towards the center. We further demonstrate that sorting in this low flow rate (and hence low shear stress) regime yields a high percentage (> 90%) of viable cells and preserves multipotency by differentiating the sorted neural stem cell population into neurons and astrocytes. The modularity of the device allows easy integration with other lab-on-a-chip devices for upstream mechanical dissociation and downstream high-throughput clonal analysis, localized electroporation and sampling. Although demonstrated in the case of the neurosphere assay, the method is equally applicable to other sphere forming assays.