Peyer's patch follicle-associated epithelium (FAE) regulates intestinal antigen access to the immune system in part through the action of microfold (M) cells which mediate transcytosis of antigens and microorganisms. Studies on M cells have been limited by the difficulties in isolating purified cells, so we applied TOGA mRNA expression profiling to identify genes associated with the in vitro induction of M cell-like features in Caco-2 cells and tested them against normal Peyer's patch tissue for their expression in FAE. Among the genes identified by this method, laminin beta3, a matrix metalloproteinase and a tetraspan family member, showed enriched expression in FAE of mouse Peyer's patches. Moreover, the C. perfringens enterotoxin receptor (CPE-R) appeared to be expressed more strongly by UEA-1(+) M cells relative to neighboring FAE. Expression of the tetraspan TM4SF3 gene and CPE-R was also confirmed in human Peyer's patch FAE. Our results suggest that while the Caco-2 differentiation model is associated with some functional features of M cells, the genes induced may instead reflect the acquisition of a more general FAE phenotype, sharing only select features with the M cell subset.

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We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus’s S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip’s surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent.

Objective: To evaluate a microfluidics-based positive selection technology for isolating circulating trophoblasts (CTs) from peripheral blood of women whose pregnancies are affected by aneuploidy and to evaluate fetal karyotype using fluorescence in situ hybridization (FISH).Method: Ten 18-ml samples of peripheral blood were collected consecutively from pregnant women whose fetus was affected by aneuploidy. A preservation buffer was added, and the specimens were shipped overnight to the testing laboratory at ambient temperature. The specimen was infused into the fully automated microfluidics-based LiquidScan ® instrument without pre-processing. This instrument contains microfluidic chips, which are coated with antibodies (anti-huEpCAM and a proprietary antibody mixture) specific to CT surface epitopes. FISH analysis was performed on the enriched cells.Results: Fetal aneuploidy evaluated included trisomy 21 (n = 3), trisomy 18 (n = 1), trisomy 13 (n = 1), monosomy X (n = 3), and triploidy (n = 1). CTs for analysis by FISH were identified in all samples. The average number of mononucleate cells per 1 ml of whole blood was 2.11 (range 0.38-4.63) overall and was 2.67 (range 1.13-4.63) using the proprietary combination of antibodies. FISH results were concordant with the aneuploidy based on other testing in all cases. Multinucleate cells were searched for and identified in the last seven samples (average number: 0.84/1 ml). Conclusions:Our study demonstrates that the LiquidScan ® , a high-sensitivity microfluidic platform, can enrich circulating trophoblasts (mononucleate and multinucleate). FISH can then be used to detect fetal aneuploidy. Key pointsWhat is already known about this topic? � Fetal trophoblasts are found in maternal circulation during pregnancy � Existing methods used to enrich circulating trophoblasts are time-intensive and expensive

Circulating tumor cells (CTCs) are rare cells found in peripheral blood or other body fluids of cancer patients. Single-cell multi-omics analysis of CTCs can provide critical information and insights for tumor heterogeneity, early detection, residual disease, recurrence, and response and resistance to therapies. However, the adoption of single cell analysis in a clinical setting has been challenging due to complicated and lengthy workflows, lack of automation, low throughput, cell loss, inefficient cell picking, and nucleic acid degradation. To overcome these challenges, we have developed the fully automated LiquidScan liquid biopsy platform as a solution for CTC enrichment and single cell isolation. Whole blood samples were collected from breast, prostate, and lung cancer patients using BioFluidica blood collection tubes. CTCs were enriched with LiquidScan platform coupled with microfluidic affinity selection of rare cells with antibodies against cancer specific surface markers. Cell eluates released from microfluidic chips were further sorted and individual cells were placed into individual wells of PCR plates. The isolated single cells were processed with either whole genome amplification for DNA analysis or pre-amplification of RNA for transcriptome profiling. Cell viability and RNA integrity were assessed for enriched cells post LiquidScan processing. Whole blood samples were directly loaded to microfluidic chips and processed (capture, wash, release and elute) using Hamilton robot for automated processing with no require to red blood cell lysis. The sample processing time was approximately 3 hours with up to 8 samples processed simultaneously. Enriched cells were 70% viable on average post LiquidScan sample processing. The RNA RIN score for the enriched cells was 6 on average. Success rate of DNA amplifications and RNA pre-Amplifications were both over 90% across all samples processed. The LiquidScan platform provides a solution for fast, automated single viable cell isolation making downstream molecular analysis possible in a clinical setting. Protocols for RNA pre-amplification and DNA sequencing including copy number analysis and targeted resequencing have been developed. Single cell DNA methylation and proteomics assay development is in progress. Citation Format: Janet Dickerson, Dylan Dufek, Robbie Huff, Stephenie Jones, Jennifer Barber-Singh, Christopher Brandt, Judy Muller-Cohn, Rolf Muller, Yipeng Wang. Automated viable circulating tumor cell (CTC) isolation enables efficient single-cell multi-omics analysis in a clinical setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB179.