Presence of tumor cells in intra-operative blood salvage autotransfusion samples from hepatocellular carcinoma liver transplantation: analysis using highly sensitive microfluidics technology.

BACKGROUND: The application of intra-operative blood salvage autotransfusion(IBSA) in liver transplantation(LT) for hepatocellular carcinoma(HCC) remains controversial due to the theoretical risk of tumour cell(TC) reintroduction. Current studies evaluating for presence of TC are limited by suboptimal detection techniques. This study aims to analyze the presence of TC in HCC LT autologous blood using microfluidics technology. METHODS: A prospective study of HCC patients who underwent LT from February 2018-April 2019 was conducted. Blood samples were collected peri-operatively. TCs were isolated using microfluidics technology and stained with antibody cocktails for confirmation. RESULTS: A total of 15 HCC LT patients were recruited. All recipients had tumour characteristics within the University of California, San Francisco(UCSF) criteria pre-operatively. TC was detected in all of the autologous blood samples collected from the surgical field. After IOCS wash, five patients had no detectable TC, while 10 patients had detectable TC; of these two remained positive for TC after Leukocyte Depletion Filter(LDF) filtration. CONCLUSION: The risk of tumour cell reintroduction using IBSA in HCC LT patients can be reduced with a single LDF. Future studies should evaluate the proliferation capacity and tumorigenicity of HCC TC in IBSA samples, and the effects of TC reintroduction in patients with pre-existing HCC TCs.

Protein covalently conjugated SU-8 surface for the enhancement of mesenchymal stem cell adhesion and proliferation.

Cell growing behavior is significantly dependent on the surface chemistry of materials. SU-8 as an epoxy-based negative photoresist is commonly used for fabricating patterned layers in lab-on-a-chip devices. As a hydrophobic material, SU-8 substrate is not favorable for cell culture, and cell attachment on native SU-8 is limited attributed to poor surface biocompatibility. Although physical adsorption of proteins could enhance the cell adhesion, the effect is not durable. In this work, SU-8 surface chemistry is modified by immobilizing fibronectin (FN) and collagen type I (COL I) covalently using (3-aminopropyl)triethoxysilane (APTES) and cross-linker glutaraldehyde (GA) to increase surface biofunctionality. The effectiveness of this surface treatment to improve the adhesion and viability of mesenchymal stem cells (MSCs) is investigated. It is found that the wettability of SU-8 surface can be significantly increased by this chemical modification. In addition, the spreading area of MSCs increases on the SU-8 surfaces with covalently conjugated matrix proteins, as compared to other unmodified SU-8 surface or those coated with proteins simply by physical adsorption. Furthermore, cell proliferation is dramatically enhanced on the SU-8 surfaces modified under the proposed scheme. Therefore, SU-8 surface modification with covalently bound matrix proteins assisted by APTES+GA provides a highly biocompatible interface for the enhanced adhesion, spreading, and proliferation of MSCs.

Point-of-care diagnostic tests for tuberculosis disease.

Rapid diagnosis is one key pillar to end tuberculosis (TB). Point-of-care tests (POCTs) facilitate early detection, immediate treatment, and reduced transmission of TB disease. This Review evaluates current diagnostic assays endorsed by the World Health Organization and identifies the gaps between existing conventional tests and the ideal POCT. We discuss the commercial development of new rapid tests and research studies on nonsputum-based diagnostic biomarkers from both pathogen and host. Last, we highlight advances in integrated microfluidics technology that may aid the development of new POCTs.

The role of bifurcation angles on collective smooth muscle cell biomechanics and the implication in atherosclerosis development.

Vascular smooth muscle cells (SMCs) are located in the middle of the tunica media and regulate the vasodilation and vasoconstriction of the blood vessels. SMCs also play a critical role during the development of atherosclerotic lesions, which are mainly found at sites of disturbed blood flow such as arterial branch points and bifurcations. Although the migratory and proliferative activities of SMCs and their phenotypic switch have been widely studied, the mechanotransduction of the SMC layer underlying atherosclerotic plaques remains unclear. In this study, bifurcate micropatterns with different angles were fabricated with polydimethylsiloxane and polyacrylamide gel for SMC culture and characterization of cell traction force. The cellular morphology, density and orientation-specific adaptation during branched cell layer formation on this platform were monitored until they became confluence. The results indicated that the characteristic cell traction forces and the von Mises stresses were dependent on bifurcation angles, which might provide important geometrical cues associated with the development of atherosclerosis. Immunofluorescence staining and gene analysis further revealed the proliferative and migratory states of SMCs in response to different bifurcation angles, which might elucidate the localization and progression of atherosclerotic lesions.

An in vitro study on the collective tumor cell migration on nanoroughened poly(dimethylsiloxane) surfaces.

The nanotopography of the cellular environment in vivo is an important factor that affects cellular phenomena such as adhesion, proliferation and migration. The capability of tumor cells to collectively migrate is critical during the process of tumor metastasis, which is significantly regulated by the nanotopography of the microenvironment such as its roughness. Herein, a simple and effective approach is developed to generate a controlled roughness contrast on the same poly(dimethylsiloxane) substrate using chemical etching and rapid molding, and a quantitative study is presented on the influence of surface roughness on cell collective migration. Specifically, the HuH7 (a human hepatocarcinoma) cell monolayer exhibits a slower migration mode on a nanoroughened substrate compared to its behaviour on a smooth substrate. Subsequent gene analyses indicate that the cell-substrate and cell-cell adhesion proteins are downregulated on the roughened substrate. This study shows the impact of substrate roughness on cell biochemical functioning, and hence on collective migration, suggesting that an engineered nanotopography could be applied in the design of biomedical devices in order to manipulate tumor cell behaviour.

Simple surface engineering of polydimethylsiloxane with polydopamine for stabilized mesenchymal stem cell adhesion and multipotency.

Polydimethylsiloxane (PDMS) has been extensively exploited to study stem cell physiology in the field of mechanobiology and microfluidic chips due to their transparency, low cost and ease of fabrication. However, its intrinsic high hydrophobicity renders a surface incompatible for prolonged cell adhesion and proliferation. Plasma-treated or protein-coated PDMS shows some improvement but these strategies are often short-lived with either cell aggregates formation or cell sheet dissociation. Recently, chemical functionalization of PDMS surfaces has proved to be able to stabilize long-term culture but the chemicals and procedures involved are not user- and eco-friendly. Herein, we aim to tailor greener and biocompatible PDMS surfaces by developing a one-step bio-inspired polydopamine coating strategy to stabilize long-term bone marrow stromal cell culture on PDMS substrates. Characterization of the polydopamine-coated PDMS surfaces has revealed changes in surface wettability and presence of hydroxyl and secondary amines as compared to uncoated surfaces. These changes in PDMS surface profile contribute to the stability in BMSCs adhesion, proliferation and multipotency. This simple methodology can significantly enhance the biocompatibility of PDMS-based microfluidic devices for long-term cell analysis or mechanobiological studies.

Microfluidic Assay To Study the Combinatorial Impact of Substrate Properties on Mesenchymal Stem Cell Migration.

As an alternative to complex and costly in vivo models, microfluidic in vitro models are being widely used to study various physiological phenomena. It is of particular interest to study cell migration in a controlled microenvironment because of its vital role in a large number of physiological processes, such as wound healing, disease progression, and tissue regeneration. Cell migration has been shown to be affected by variations in the biochemical and physical properties of the extracellular matrix (ECM). To study the combinatorial impact of the ECM physical properties on cell migration, we have developed a microfluidic assay to induce migration of human bone marrow derived mesenchymal stem cells (hBMSCs) on polydimethylsiloxane (PDMS) substrates with varying combinatorial properties (hydrophobicity, stiffness, and roughness). The results show that although the initial cell adhesion and viability appear similar on all PDMS samples, the cell spreading and migration are enhanced on PDMS samples exhibiting intermediate levels of hydrophobicity, stiffness, and roughness. This study suggests that there is a particular range of substrate properties for optimal cell spreading and migration. The influence of substrate properties on hBMSC migration can help understand the physical cues that affect cell migration, which may facilitate the development of optimized engineered scaffolds with desired properties for tissue regeneration applications.

Combinatorial effect of substratum properties on mesenchymal stem cell sheet engineering and subsequent multi-lineage differentiation.

Cell sheet engineering has been exploited as an alternative approach in tissue regeneration and the use of stem cells to generate cell sheets has further showed its potential in stem cell-mediated tissue regeneration. There exist vast interests in developing strategies to enhance the formation of stem cell sheets for downstream applications. It has been proved that stem cells are sensitive to the biophysical cues of the microenvironment. Therefore we hypothesized that the combinatorial substratum properties could be tailored to modulate the development of cell sheet formation and further influence its multipotency. For validation, polydimethylsiloxane (PDMS) of different combinatorial substratum properties (including stiffness, roughness and wettability) were created, on which the human bone marrow derived mesenchymal stem cells (BMSCs) were cultured to form cell sheets with their multipotency evaluated after induced differentiation. The results showed that different combinatorial effects of these substratum properties were able to influence BMSC behavior such as adhesion, spreading and proliferation during cell sheet development. Collagen formation within the cell sheet was enhanced on substrates with lower stiffness, higher hydrophobicity and roughness, which further assisted the induced chondrogenesis and osteogenesis, respectively. These findings suggested that combinatorial substratum properties had profound effects on BMSC cell sheet integrity and multipotency, which had significant implications for future biomaterials and scaffold designs in the field of BMSC-mediated tissue regeneration.

A microfluidic co-culture system to monitor tumor-stromal interactions on a chip.

The living cells are arranged in a complex natural environment wherein they interact with extracellular matrix and other neighboring cells. Cell-cell interactions, especially those between distinct phenotypes, have attracted particular interest due to the significant physiological relevance they can reveal for both fundamental and applied biomedical research. To study cell-cell interactions, it is necessary to develop co-culture systems, where different cell types can be cultured within the same confined space. Although the current advancement in lab-on-a-chip technology has allowed the creation of in vitro models to mimic the complexity of in vivo environment, it is still rather challenging to create such co-culture systems for easy control of different colonies of cells. In this paper, we have demonstrated a straightforward method for the development of an on-chip co-culture system. It involves a series of steps to selectively change the surface property for discriminative cell seeding and to induce cellular interaction in a co-culture region. Bone marrow stromal cells (HS5) and a liver tumor cell line (HuH7) have been used to demonstrate this co-culture model. The cell migration and cellular interaction have been analyzed using microscopy and biochemical assays. This co-culture system could be used as a disease model to obtain biological insight of pathological progression, as well as a tool to evaluate the efficacy of different drugs for pharmaceutical studies.

Surface chemical modification of poly(dimethylsiloxane) for the enhanced adhesion and proliferation of mesenchymal stem cells.

The surface chemistry of materials has an interactive influence on cell behavior. The optimal adhesion of mammalian cells is critical in determining the cell viability and proliferation on substrate surfaces. Because of the inherent high hydrophobicity of a poly(dimethylsiloxane) (PDMS) surface, cell culture on these surfaces is unfavorable, causing cells to eventually dislodge from the surface. Although physically adsorbed matrix proteins can promote initial cell adhesion, this effect is usually short-lived. Here, (3-aminopropyl)triethoxy silane (APTES) and cross-linker glutaraldehyde (GA) chemistry was employed to immobilize either fibronectin (FN) or collagen type 1 (C1) on PDMS. The efficiency of these surfaces to support the adhesion and viability of mesenchymal stem cells (MSCs) was analyzed. The hydrophobicity of the native PDMS decreased significantly with the mentioned surface functionalization. The adhesion of MSCs was mostly favorable on chemically modified PDMS surfaces with APTES + GA + protein. Additionally, the spreading area of MSCs was significantly higher on APTES + GA + C1 surfaces than on other unmodified/modified PDMS surfaces with C1 adsorption. However, there were no significant differences in the MSC spreading area on the unmodified/modified PDMS surfaces with FN adsorption. Furthermore, there was a significant increase in cell proliferation on the PDMS surface with APTES + GA + protein functionalization as compared to the PDMS surface with protein adsorption only. Therefore, the covalent surface chemical modification of PDMS with APTES + GA + protein could offer a more biocompatible platform for the enhanced adhesion and proliferation of MSCs. Similar strategies can be applied for other substrates and cell lines by appropriate combinations of self-assembly monolayers (SAMs) and extracellular matrix proteins.