Securing high-quality cell sources is important in regenerative medicine. In this study, we developed a device that can accumulate autologous stem cells in the body. When small wire-assembled molds were embedded in the dorsal subcutaneous pouches of beagles for several weeks, collagen-based tissues with minimal inflammation formed inside the molds. At 3 weeks of embedding, the outer areas of the tissues were composed of immature type III collagen with large amounts of cells expressing SSEA3 or SSEA4 markers, in addition to growth factors such as HGF or VEGF. When separated from the tissues by collagenase treatment, approximately four million cells with a proportion of 70% CD90-positive and 20% SSEA3- or SSEA4-positive cells were recovered from the single mold. The cells could differentiate into bone or cartilage cells. The obtained cell-containing tissues are expected to have potential as therapeutic materials or cell sources in regenerative medicine.

The construction of versatile functional hydrogel interfaces holds promising prospects in biosensing and bioengineering. Herein, we introduced a light-induced protein conjugation strategy for on-demand surface modification of hydrogel interface based on the photoclick cyclization between primary amine and o-nitrobenzyl alcohol. We achieved the on-demand protein conjugation by grafting the molecular plugin, 4-(hydroxymethyl)-3-nitrobenzoic acid (HNBA), onto the hydrogel surface, followed by the mask-aided photoclick reaction with the protein of interest. This method enables the creation of protein patterns on hydrogel interface with a lower limit of pattern width at ∼70 μm. With this method, we demonstrated the surface engineering of epidermal growth factor (EGF) on hydrogel interface for selective capture of EGF receptor-positive cancer cells with an efficiency over 80%. Moreover, we applied the mask-aided photoclick conjugation method for antigen capture and developed a photoclickable hydrogel interface-based dot blotting assay. Due to the high-efficient antigen capture of photoclick conjugation, the photoclickable hydrogel interface-based dot blotting assay shows improved sensitivity for antigen detection with a limit of detection as 0.065 ng. We believed that this light-induced protein conjugation method holds the potential as a robust strategy for the construction of bioactive hydrogel interfaces for various bio-related applications.

Liquid biopsy has progressed to its current use to diagnose and monitor cancer. Despite the recent advances in investigating cancer detection and diagnosis strategies, there is still a room for improvements in capturing CTCs. We developed an efficient CTC detection system by integrating gold nanoparticles with a microfluidic platform, which can achieve CTC capture within 120 min. Here, we report our development of a simple and effective way to isolate CTCs using antibodies attached on gold nanoparticles to the surface of a lateral filter array (LFA) microdevice. Our method was optimized using three pancreatic tumor cell lines, enabling the capture with high efficiency (90% ± 3.2%). The platform was further demonstrated for isolating CTCs from patients with metastatic pancreatic cancer. Our method and platform enables the production of functionalized, patterned surfaces that interact with tumor cells, enhancing the selective capture of CTCs for biological assays.

Circulating tumor cells (CTCs) have now emerged as a type of promising circulating biomarkers in liquid biopsy and can predict the occurrence and development of cancers. In this work, an integrated and renewable interface is fabricated for the capture, release and quantitative analysis of CTCs. As designed, folate receptor-positive CTCs are captured by folic acid-modified DNA probes at the interface through the receptor-ligand interaction, and are efficiently released from the interface with the aid of bleomycin-ferrous complex-regulated cleavage. Taking MCF-7 cells as the model, the functional interface demonstrates high efficiency to selectively capture the folate receptor-positive tumor cells, and the bleomycin-ferrous complex-regulated cleavage not only easily releases the captured cells with well-maintained viability and proliferation ability, but also releases silver nanoparticles that are labeled at the cell surface for highly sensitive quantification by adopting electrochemical techniques with a detection limit of 6 cells/mL. At the meanwhile, the interface is proved to be regenerated through a simple cleavage-hybridization event and reused with high stability. Therefore, our work may provide a new idea for the collection and downstream researches of circulating tumor cells in the future.

Mycobacterium tuberculosis whole-genome sequencing (WGS) is a powerful tool as it can provide data on population diversity, drug resistance, disease transmission, and mixed infections. Successful WGS is still reliant on high concentrations of DNA obtained through M. tuberculosis culture. Microfluidics technology plays a valuable role in single-cell research but has not yet been assessed as a bacterial enrichment strategy for culture-free WGS of M. tuberculosis. In a proof-of-principle study, we evaluated the use of Capture-XT, a microfluidic lab-on-chip cleanup and pathogen concentration platform to enrich M. tuberculosis bacilli from clinical sputum specimens for downstream DNA extraction and WGS. Three of the four (75%) samples processed by the microfluidics application passed the library preparation quality control, compared to only one of the four (25%) samples not enriched by the microfluidics M. tuberculosis capture application. WGS data were of sufficient quality, with mapping depth of ≥25× and 9 to 27% of reads mapping to the reference genome. These results suggest that microfluidics-based M. tuberculosis cell capture might be a promising method for M. tuberculosis enrichment in clinical sputum samples, which could facilitate culture-free M. tuberculosis WGS. IMPORTANCE Diagnosis of tuberculosis is effective using molecular methods; however, a comprehensive characterization of the resistance profile of Mycobacterium tuberculosis often requires culturing and phenotypic drug susceptibility testing or culturing followed by whole-genome sequencing (WGS). The phenotypic route can take anywhere from 1 to >3 months to result, by which point the patient may have acquired additional drug resistance. The WGS route is a very attractive option; however, culturing is the rate-limiting step. In this original article, we provide proof-of-principle evidence that microfluidics-based cell capture can be used on high-bacillary-load clinical samples for culture-free WGS.

The development of specific and sensitive immunomagnetic cell separation nanotechnologies is central to enhancing the diagnostic relevance of circulating tumor cells (CTCs) and improving cancer patient outcomes. The limited number of specific biomarkers used to enrich a phenotypically diverse set of CTCs from liquid biopsies has limited CTC yields and purity. The ultra-high molecular weight mucin, mucin16 (MUC16) is shown to physically shield key membrane proteins responsible for activating immune responses against ovarian cancer cells and may interfere with the binding of magnetic nanoparticles to popular immunomagnetic cell capture antigens. MUC16 is expressed in ≈90% of ovarian cancers and is almost universal in High Grade Serous Epithelial Ovarian Cancer. This work demonstrates that cell bound MUC16 is an effective target for rapid immunomagnetic extraction of expressor cells with near quantitative yield, high purity and viability from serum. The results provide a mechanistic insight into the effects of nanoparticle physical properties and immunomagnetic labeling on the efficiency of immunomagnetic cell isolation. The growth of these cells has also been studied after separation, demonstrating that nanoparticle size impacts cell-particle behavior and growth rate. These results present the successful isolation of \"masked\" CTCs enabling new strategies for the detection of cancer recurrence and select and monitor chemotherapy.

Stent thrombosis remains one of the main causes that lead to vascular stent failure in patients undergoing percutaneous coronary intervention (PCI). Type 2 diabetes mellitus is accompanied by endothelial dysfunction and platelet hyperactivity and is associated with suboptimal outcomes following PCI, and an increase in the incidence of late stent thrombosis. Evidence suggests that late stent thrombosis is caused by the delayed and impaired endothelialization of the lumen of the stent. The endothelium has a key role in modulating inflammation and thrombosis and maintaining homeostasis, thus restoring a functional endothelial cell layer is an important target for the prevention of stent thrombosis. Modifications using specific molecules to induce endothelial cell adhesion, proliferation and function can improve stents endothelialization and prevent thrombosis. Blood endothelial progenitor cells (EPCs) represent a potential cell source for the in situ-endothelialization of vascular conduits and stents. We aim in this review to summarize the main biofunctionalization strategies to induce the in-situ endothelialization of coronary artery stents using circulating endothelial stem cells.

With circulating tumor cells (CTCs) playing a critical role in cancer metastasis, the quantitation and characterization of CTCs promise to provide precise diagnostic and prognostic information in service of personalized therapies. However, as CTCs are extremely rare, high yield, high purity strategies are required to target and isolate CTCs from patient samples. Recently, we demonstrated the selective capture of CTCs upon antibody-functionalized polyethylene glycol diacrylate (PEGDA) hydrogels photopolymerized within polydimethylsiloxane (PDMS) microfluidic molds. Isolated CTC purity was subsequently enriched by selectively releasing desired cells from photodegradable hydrogel capture surfaces. However, the fabrication of these acrylate-based hydrogels by photopolymerization is subject to oxygen inhibition, which dramatically affects the physical and chemical properties of hydrogel interfaces formed in proximity to PDMS boundaries. To evaluate how antibody conjugation density and cell capture is impacted by fabrication parameters affected by oxygen inhibition, PEGDA hydrogel features were polymerized within PDMS micromolds under different UV exposure conditions and linker (acrylate-PEG-biotin) concentrations. Predictions of acrylate conversion throughout the hydrogel feature were performed using a 1D reaction-diffusion model that describes oxygen-inhibited photopolymerization. The functional consequences of photopolymerization parameters and solution stoichiometry on CTC capture were experimentally quantified and evaluated. Results show that hydrogel surfaces polymerized under shorter exposure times and with higher linker concentrations display superior functionalization and higher CTC capture efficiency. Conversely, highly cross-linked hydrogel surfaces polymerized under longer exposure times are insensitive to functionalization and display poor capture, regardless of linker concentration. By highlighting the importance of oxygen-inhibited photopolymerization, these findings provide guidelines to design micromolded hydrogels with controlled ligand expression. In addition to enhancing the selective cell capture capacity of immunofunctional hydrogels, the ability to quantifiably design hydrogel interfaces described here will improve the sensitivity of hydrogel biosensors, provide a platform to finely screen cell-matrix interactions, and generally enhance the fidelity of micromolded hydrogel features.

Biofilms initiate when bacteria encounter and are retained on surfaces. The surface orchestrates biofilm growth through direct physico-chemical and mechanical interactions with different structures on bacterial cells and, in turn, through its influence on cell-cell interactions. Individual cells respond directly to a surface through mechanical or chemical means, initiating \"surface sensing\" pathways that regulate gene expression, for instance producing extra cellular matrix or altering phenotypes. The surface can also physically direct the evolving colony morphology as cells divide and grow. In either case, the physico-chemistry of the surface influences cells and cell communities through mechanisms that involve additional factors. For instance the numbers of cells arriving on a surface from solution relative to the generation of new cells by division depends on adhesion and transport kinetics, affecting early colony density and composition. Separately, the forces experienced by adhering cells depend on hydrodynamics, gravity, and the relative stiffnesses and viscoelasticity of the cells and substrate materials, affecting mechanosensing pathways. Physical chemistry and surface functionality, along with interfacial mechanics also influence cell-surface friction and control colony morphology, in particular 2D and 3D shape. This review focuses on the current understanding of the mechanisms in which physico-chemical interactions, deriving from surface functionality, impact individual cells and cell community behavior through their coupling with other interfacial processes.

Selective isolation of individual target cells from a heterogeneous population is technically challenging; however, the ability to retrieve single cells can have high significance in various aspects of biological research. Here, we present a new photoelectrochemical surface based on a transparent electrode that is compatible with high-resolution fluorescence microscopy for isolating individual rare cells from complex biological samples. This is underpinned by two important factors: (i) careful design of the electrode by patterning discrete Au disks of micron dimension on amorphous silicon-indium tin oxide films and (ii) orthogonal surface chemistry, which modifies the patterned electrode with self-assembly layers of different functionalities, to selectively capture target cells on the Au disks and resist cell binding to the amorphous silicon surface. The co-stimulation of the surface using light from a microscope and an electric potential triggers the reductive desorption of the alkanethiol monolayer from the Au disks to release the single cells of interest from the illuminated regions only. Using circulating tumor cells as a model, we demonstrate the capture of cancer cells on an antibody-coated surface and selective release of single cancer cells with low expression of epithelial cell adhesion molecules.