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A microfluidic cell concentrator.

ABSTRACT: Cell concentration via centrifugation is a ubiquitous step in many cell culture procedures. At the macroscale, centrifugation suffers from a number of limitations, particularly when dealing with small numbers of cells (e.g., less than 50,000). On the other hand, typical microscale methods for cell concentration can affect cell physiology and bias readouts of cell behavior and function. In this paper, we present a microfluidic concentrator device that utilizes the effects of gravity to allow cells to gently settle out of a suspension into a collection region without the use of specific adhesion ligands. Dimensional analysis was performed to compare different device designs and was verified with flow modeling to optimize operational parameters. We are able to concentrate low-density cell suspensions in a microfluidic chamber, achieving a cell loss of only 1.1 ± 0.6% (SD, n = 7) with no observed loss during a subsequent cell staining protocol which incorporates ?36 complete device volume replacements. This method provides a much needed interface between rare cell samples and microfluidic culture assays.

SUBMITTER: Warrick J

PROVIDER: S-EPMC3074536 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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A microfluidic cell concentrator.

Warrick Jay J Casavant Ben B Frisk Megan M Beebe David D

Analytical chemistry 20101001 19

Cell concentration via centrifugation is a ubiquitous step in many cell culture procedures. At the macroscale, centrifugation suffers from a number of limitations, particularly when dealing with small numbers of cells (e.g., less than 50,000). On the other hand, typical microscale methods for cell concentration can affect cell physiology and bias readouts of cell behavior and function. In this paper, we present a microfluidic concentrator device that utilizes the effects of gravity to allow cell ...[more]

PMID: 20843010

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Project description:Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next-generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis. We investigated gene expression in mouse embryonic cells using microfluidic-facilitated RNA-Seq to analyze 56 single mouse ES cell (mESC) transcriptomes and 6 single mouse embryonic fibroblast (MEF) transcriptomes. To quantitatively evaluate the sensitivity and precision of our technique, we also sequenced 23 libraries from extracted mESC RNA, representing three sets of technical replicates with varying starting amounts of material.

Dataset Information

A microfluidic cell concentrator.

Publications

A microfluidic cell concentrator.

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