High-purity isolation of rare single cells from blood using a tiered microchip system

PLOS ONE RESEARCH ARTICLE High-purity isolation of rare single cells from blood using a tiered microchip system Onur Gur1,2, Chun-Li Chang2,3, Rohil Jain2,3, Yuan Zhong2,3, Cagri A. Savran2,3* 1 School of Electrical Engineering, Purdue University, West Lafayette, IN, United States of America, 2 Birck Nanotechnology Center, Purdue University, West Lafayette, IN, United States of America, 3 School of Mechanical Engineering, Purdue University, West Lafayette, IN, United States of America * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Gur O, Chang C-L, Jain R, Zhong Y, Savran CA (2020) High-purity isolation of rare single cells from blood using a tiered microchip system. PLoS ONE 15(3): e0229949. https://doi. org/10.1371/journal.pone.0229949 Abstract We present a two-tiered microchip system to capture and retrieve rare cells from blood samples with high purity. The first module of the system is a high throughput microfluidic interface that is used to immunomagnetically isolate targeted rare cells from whole blood, and discard > 99.999% of the unwanted leukocytes. The second module is a microwell array that furthers the purification by magnetically guiding each cell into a separate well concurrently, and allows individual retrieval of each cell. We demonstrate the design of the system as well as its characterization by experiments using model cell lines that represent circulating fetal trophoblasts. Our results show that single cells can be retrieved with efficiencies and purities as high as 100% within 145 mins. Editor: Juan Carlos del Alamo, University of California San Diego, UNITED STATES Received: August 9, 2019 Accepted: February 18, 2020 Introduction Published: March 17, 2020 Chromosomal abnormalities, including aneuploidy, translocations, dislocations and deletions occur in 1 in every 150 live births [1]. Current methods to diagnose these abnormalities include amniocentesis and chorionic villus sampling (CVS). These invasive procedures come with a risk of miscarriage; around 1% for amniocentesis and 2% for CVS [2–6]. To alleviate these difficulties, non-invasive prenatal diagnostics methods are being developed. One commercially available method involves retrieval of cell free fetal DNA (cffDNA) from the blood plasma of the mother and analyzing it to detect genetic anomalies. While this method is effective in detecting a few conditions that include Trisomies 13, 18, and 21; the fragmented nature of the fetal DNA, and the contamination from maternal DNA makes it difficult to diagnose many other genetic disorders stemming from conditions such as mosaicism, small deletions, duplications or expansions [1,7]. An alternate non-invasive diagnostics method involves circulating fetal cells (CFCs). CFCs can be found as early as 6–8 weeks into pregnancy and can be retrieved from maternal blood without risking the fetus or the mother [8]. They are more effective in diagnosing chromosomal abnormalities in fetuses compared to cffDNA due to their intact fetal genome, and lack of contamination from maternal DNA [9]. The major challenge regarding CFCs is that they are extremely rare, ranging from 1–2 cells per milliliter of blood [10,11]. This has led to the development of several isolation methods for CFCs over the years. We provide below a Copyright: © 2020 Gur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: CAS: National Science Foundation (Award 1509097), https://www.nsf.gov Donations from McKinley Educational Foundation, and Tom Hurvis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: C.S. and C-L.C. are PLOS ONE | https://doi.org/10.1371/journal.pone.0229949 March 17, 2020 1 / 20 PLOS ONE share holders of Savran Technologies Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials. High-purity isolation of rare single cells from blood using a tiered microchip system detailed summary of existing methods, recent developments as well as the ensuing opportunities for improvement. Conventional methods for CFC enrichment include fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), and methods based on the size of the cell such as density gradient centrifugation and filtration [2,12]. FACS and MACS are methods that rely on specific biomarkers that target cells express to separate them from a sample fluid. They both result in relatively low purity, i.e. a great number of unwanted cells which could necessitate additional enrichment steps. For example, a study by Bianchi et al., where 20 ml of maternal blood was enriched for cells that express the transferrin receptor (TfR), yielded between 46,000 to 673,000 TfR+ cells; of which an average of only 150 were determined to be the targeted cells by subsequent PCR and Southern blot analyses [13]. Experiments performed by Chen et al. where 20 target cells were spiked into 5 ml blood showed that negative enrichment by MACS result in recovery rates of around 35% with a total number of 27900 cells [9]. Hatt et al. used MACS by targeting the marker set CD105 and CD141 which resulted in 500,000 cells, only 0 to 18 of which were classified as candidate fetal cells after fluorescent labeling and manual scanning of the cells [14]. Density gradient separation, where cells are suspended in a solution with density gradient also have purity levels that are generally low. Two studies by Calabrese et al. in 2011 and 2016 on fetuses with aneuploidy yielded a total of 50,000–100,000 cells of which only 4–9 were target cells per 25 ml blood, and 160,000–220,000 cells of which only 4–34 were target cells per 24 ml blood respectively [15,16]. Multiple groups used size-based detection to target CFCs. Vona et al. used polycarbonate filters with 8 μm to target CFCs and Mohamed et al. used successively narrowing channels to separate CFCs based on their size and deformation characteristics [17,18]. These filtration methods rely on the assumption that there are significant size and deformity differences between the targeted cells and other cells, which is not necessarily the case at all times. Recently, various microfluidic devices were developed to further advance CFC isolation. Byeon et al. used a 2-step enrichment process to increase the purity of retrieved CFCs. A red blood cell hyperagregation step was used to facilitate the removal of white blood cells (WBCs) remaining in the supernatant ; followed by further purification by negative (...truncated)