Keith H. K. Wong

Three-Dimensional Blood-Brain Barrier Model for in vitro Studies of Neurovascular Pathology

Blood–brain barrier (BBB) pathology leads to neurovascular disorders and is an important target for therapies. However, the study of BBB pathology is difficult in the absence of models that are simple and relevant. In vivo animal models are highly relevant, however they are hampered by complex, multi-cellular interactions that are difficult to decouple. In vitro models of BBB are simpler, however they have limited functionality and relevance to disease processes. To address these limitations, we developed a 3-dimensional (3D) model of BBB on a microfluidic platform. We verified the tightness of the BBB by showing its ability to reduce the leakage of dyes and to block the transmigration of immune cells towards chemoattractants. Moreover, we verified the localization at endothelial cell boundaries of ZO-1 and VE-Cadherin, two components of tight and adherens junctions. To validate the functionality of the BBB model, we probed its disruption by neuro-inflammation mediators and ischemic conditions and measured the protective function of antioxidant and ROCK-inhibitor treatments. Overall, our 3D BBB model provides a robust platform, adequate for detailed functional studies of BBB and for the screening of BBB-targeting drugs in neurological diseases.

Enhanced Isolation and Release of Circulating Tumor Cells Using Nanoparticle Binding and Ligand Exchange in a Microfluidic Chip

The detection of rare circulating tumor cells (CTCs) in the blood of cancer patients has the potential to be a powerful and noninvasive method for examining metastasis, evaluating prognosis, assessing tumor sensitivity to drugs, and monitoring therapeutic outcomes. In this study, we have developed an efficient strategy to isolate CTCs from the blood of breast cancer patients using a microfluidic immune-affinity approach. Additionally, to gain further access to these rare cells for downstream characterization, our strategy allows for easy detachment of the captured CTCs from the substrate without compromising cell viability or the ability to employ next generation RNA sequencing for the identification of specific breast cancer genes. To achieve this, a chemical ligand-exchange reaction was engineered to release cells attached to a gold nanoparticle coating bound to the surface of a herringbone microfluidic chip (NP-HBCTC-Chip). Compared to the use of the unmodified HBCTC-Chip, our approach provides several advantages, including enhanced capture efficiency and recovery of isolated CTCs.

Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry

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.

The Role of Physical Stabilization in Whole Blood Preservation

The rapid degradation of blood ex vivo imposes logistical limitations on the utilization of blood-borne cells in medical diagnostics and scientific investigations. A fundamental but overlooked aspect in the storage of this fluid tissue is blood settling, which induces physical stress and compaction, aggregates blood cells, and causes collateral damage due to leukocyte activation. Here we show that the polymer Ficoll 70 kDa stabilized blood samples and prevented blood settling over the course of 72 hours, primarily by inhibiting depletion-mediated red blood cell aggregation. Physical stabilization decreased echinocyte formation, improved leukocyte viability, and inhibited the release of neutrophil elastase—a marker of neutrophil extracellular trap formation. In addition, Ficoll-stabilized blood was compatible with common leukocyte enrichment techniques including red blood cell lysis and immunomagnetic purification. This study showed for the first time that blood settling can be prevented using polymers and has implications in diagnostics.

Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells.

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.The current FDA-approved whole blood stabilization method for circulating tumor cell (CTC) isolation suffers from RNA degradation. Here the authors combine hypothermic preservation and antiplatelet strategies to stabilize whole blood up to 72 h without compromising CTC yield and RNA integrity.

Microengineering in cardiovascular research: new developments and translational applications

Microfluidic, cellular co-cultures that approximate macro-scale biology are important tools for refining the in vitro study of organ-level function and disease. In recent years, advances in technical fabrication and biological integration have provided new insights into biological phenomena, improved diagnostic measurements, and made major steps towards de novo tissue creation. Here we review applications of these technologies specific to the cardiovascular field, emphasizing three general categories of use: reductionist vascular models, tissue-engineered vascular models, and point-of-care diagnostics. With continued progress in the ability to purposefully control microscale environments, the detailed study of both primary and cultured cells may find new relevance in the general cardiovascular research community.

Ultra-fast vitrification of patient-derived circulating tumor cell lines

Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.

Trapped Chromatin Fibers Damage Flowing Red Blood Cells

Neutrophils are the most abundant white blood cells in the circulation and serve antimicrobial functions. One of their antimicrobial mechanisms involves the release of neutrophil extracellular traps (NETs), long chromatin fibers decorated with antimicrobial granular proteins that contribute to the elimination of pathogens. However, the release of NETs has also been associated with disease processes. While recent research has focused on biochemical reactions catalyzed by NETs, significantly less is known about the mechanical effect of NETs in circulation. Here, microfluidic devices and biophysical models are employed to study the consequences of the interactions between NETs trapped in channels and red blood cells (RBCs) flowing in blood over the NETs. It has been found that the RBCs can be deformed and ruptured after interactions with NETs, generating RBC fragments. Significant increases in the number of RBC fragments have also been found in the circulation of patients with conditions in which NETs have been demonstrated to be present in circulation, including sepsis and kidney transplant. Further studies will probe the potential utility of RBC fragments in the diagnostic, monitoring, and treatment of diseases associated with the presence of NETs in circulation.

Neutrophil extracellular traps are increased in cancer patients but does not associate with venous thrombosis

Background A single center, prospective tissue-based study was conducted to investigate an association between neutrophil extracellular traps (NETs) and venous thromboembolic disease in patients with malignancy. Methods Plasma was collected from 65 patients in which 27 were cancer patients and 38 were age-matched non-cancer patients. Plasma NETs, circulating free DNA (cfDNA), DNase-1, endonuclease-G, endonuclease activity and thrombin-antithrombin III (TAT) complex levels was quantified. Laboratory values were also compared. Additionally, NETs detection and quantification was performed with fluorescent immunohistochemistry (IHC) in tissue-banked tumor sections and fresh human venous thrombus derived from cancer patients. Results Plasma samples from cancer patients contained higher levels of nucleosomes (P=0.0009) and cfDNA (P=0.0008) compared to the non-cancer group. Western blot analysis revealed significantly lower DNase-1 protein levels (P=0.016) that paralleled lower nuclease activity (P=0.03) in plasma samples from cancer patients compared to non-cancer patients. Thrombus tissue from cancer patients and tumor tissue from liver and lung cancer also showed marked levels of NETs. However, increased levels of NETs in cancer patients did not correlate with TAT complex activation or prevalence of venous thrombosis in cancer patients. Conclusions Further studies are warranted to determine the role of NETs as a procoagulant in human thrombosis.