Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2–100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) generated using directed differentiation lack some cellular populations found in the native organ, including vasculature. Using single cell RNA sequencing (scRNAseq), we have identified a transient population of endothelial cells (ECs) present early in HIO differentiation that are lost over time in culture. Here, we have developed a method to enhance co-differentiation and maintenance of ECs within HIOs (vHIOs). Given that ECs are known to possess organ specific gene expression, morphology and function, we used bulk RNAseq and scRNAseq to interrogate the developing human intestine, lung, and kidney in order to identify organ-enriched EC-gene signatures in these organ systems. By comparing organ-specific gene signatures along with markers validated by fluorescent in situ hybridization to HIO ECs, we find that HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and lung. Together, these data show that HIOs can co-differentiate a native EC population that are properly patterned with an intestine-specific EC transcriptional signature in vitro..

22Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) generated using directed 23 differentiation lack some cellular populations found in the native organ, including vasculature. Using 24 single cell RNA sequencing (scRNAseq), we have identified a transient population of endothelial cells 25 (ECs) present early in HIO differentiation that are lost over time in culture. Here, we have developed a 26 method to enhance co-differentiation and maintenance of ECs within HIOs (vHIOs). Given that ECs are 27 known to possess organ specific gene expression, morphology and function, we used bulk RNAseq 28 and scRNAseq to interrogate the developing human intestine, lung, and kidney in order to identify 29 organ-enriched EC-gene signatures in these organ systems. By comparing organ-specific gene 30 signatures along with markers validated by fluorescent in situ hybridization to HIO ECs, we find that 31 HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and 32 lung. Together, these data show that HIOs can co-differentiate a native EC population that are properly 33 patterned with an intestine-specific EC transcriptional signature in vitro. 34 35 36 37 38 39 40 41 42 43 44 65highly vascularized regions of immunocompromised mice (Cortez et al., 2018; Watson et al., 2014). In 66 these environments, HIOs undergo extensive vascularization by the murine host tissue, and increase in 67 complexity to resemble mature intestinal tissue (Finkbeiner et al., 2015b; Watson et al., 2014). 68However, co-culture approaches or co-differentiating HIOs with a native vasculature prior to in vivo 69 engraftment has not yet been achieved. 71Here, we performed single cell RNA sequencing (scRNAseq) at various timepoints across HIO 72 differentiation in vitro and observed a transient population of endothelial-like cells (ECs) present within 73 HIOs early during differentiation; however, these cells are not maintained during prolonged culture 74 under standard growth conditions. This suggested that early during HIO differentiation, cells within the 75 culture are capable of giving rise to EC-like cells. Based on these observations, we hypothesized that a 76 modified directed differentiation approach would allow the induction and maintenance of a more robust 77 EC population within HIOs (termed vHIO). Our findings demonstrate that modified culture conditions 78 allow a ~13-fold increase in the induction of EC-like cells within HIOs without impacting the other HIO 79 cell populations present (i.e. epithelium, mesenchyme), and support the survival of this population of 80 ECs within HIOs in culture for months. 82Since organ-specific morphology in vascular beds has long been appreciated (Aird, 2007), and 83 organ-specific transcriptional signatures and functions have been described in mouse (Ding et al., 84 2011; Kalucka et al., 2020; Lee et al., 2014; Nolan et al., 2013) and human tissues (Chi et al., 2003; 85 Marcu et al., 2018), we further sought to determine if HIO ECs were properly...

Cryopreservation is of significance in areas including tissue engineering, regenerative medicine, and organ transplantation. We investigated endothelial cell attachment and membrane integrity in a microvasculature model at high subzero temperatures in the presence of extracellular ice. The results show that in the presence of heterogeneous extracellular ice formation induced by ice nucleating bacteria, endothelial cells showed improved attachment at temperature minimums of −6 °C. However, as temperatures decreased below −6 °C, endothelial cells required additional cryoprotectants. The glucose analog, 3-O-methyl-D-glucose (3-OMG), rescued cell attachment optimally at 100 mM (cells/lane was 34, as compared to 36 for controls), while 2% and 5% polyethylene glycol (PEG) were equally effective at −10 °C (88% and 86.4% intact membranes). Finally, endothelialized microchannels were stored for 72 h at −10 °C in a preservation solution consisting of the University of Wisconsin (UW) solution, Snomax, 3-OMG, PEG, glycerol, and trehalose, whereby cell attachment was not significantly different from unfrozen controls, although membrane integrity was compromised. These findings enrich our knowledge about the direct impact of extracellular ice on endothelial cells. Specifically, we show that, by controlling the ice nucleation temperature and uniformity, we can preserve cell attachment and membrane integrity. Further, we demonstrate the strength of leveraging endothelialized microchannels to fuel discoveries in cryopreservation of thick tissues and solid organs.