Dataset Information

Isolating single cells in a neurosphere assay using inertial microfluidics.

ABSTRACT: 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.

SUBMITTER: Nathamgari SS

PROVIDER: S-EPMC4665643 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Isolating single cells in a neurosphere assay using inertial microfluidics.

Nathamgari S Shiva P SS Dong Biqin B Zhou Fan F Kang Wonmo W Giraldo-Vela Juan P JP McGuire Tammy T McNaughton Rebecca L RL Sun Cheng C Kessler John A JA Espinosa Horacio D HD

Lab on a chip 20151029 24

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, w ...[more]

PMID: 26511875

Similar Datasets

Project description:Simple Summary Prostate cancer (PCa) is notoriously difficult to diagnose owing to the lack of reliable biomarkers and the invasiveness of obtaining a tissue biopsy from the prostate. As an alternative, we developed a liquid biopsy technique, based on isolating tumor cells from semen samples via a microfluidic device. To optimize the device, we first attempted to recover PCa cells from semen samples spiked with PCa cell lines, achieving an average efficiency of >87% cell recovery at the chosen flow rate. We then transitioned to a clinical setting using semen samples from PCa patients. The yield of isolated clinical PCa cells varied between 67 and 307 cells per mL of semen (in 15 cancer patients). These cells were stained and compared to the standard prognostic parameters such as Gleason score and PSA serum level. This study presents a potential liquid biopsy technique to augment the existing diagnosis and prognosis of PCa. Abstract Prostate cancer (PCa) diagnosis is primarily based on prostate-specific antigen (PSA) testing and prostate tissue biopsies. However, PSA testing has relatively low specificity, while tissue biopsies are highly invasive and have relatively low sensitivity at early stages of PCa. As an alternative, we developed a technique of liquid biopsy, based on isolation of circulating tumor cells (CTCs) from seminal fluid (SF). The recovery of PCa cells from SF was demonstrated using PCa cell lines, achieving an efficiency and throughput as high as 89% (±3.8%) and 1.7 mL min−1, respectively, while 99% (±0.7%) of sperm cells were disposed of. The introduced approach was further tested in a clinical setting by collecting and processing SF samples of PCa patients. The yield of isolated CTCs measured as high as 613 cells per SF sample in comparison with that of 6 cells from SF of healthy donors, holding significant promise for PCa diagnosis. The correlation analysis of the isolated CTC numbers with the standard prognostic parameters such as Gleason score and PSA serum level showed correlation coefficient values at 0.40 and 0.73, respectively. Taken together, our results show promise in the developed liquid biopsy technique to augment the existing diagnosis and prognosis of PCa.

Dataset Information

Isolating single cells in a neurosphere assay using inertial microfluidics.

Publications

Isolating single cells in a neurosphere assay using inertial microfluidics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets