Effect of surface acoustic waves on the viability, proliferation and differentiation of primary osteoblast-like cells

Surface acoustic waves (SAWs) have been used as a rapid and efficient technique for driving microparticles into a three-dimensional scaffold matrix, raising the possibility that SAW may be effective in seeding live cells into scaffolds, that is, if the cells were able to survive the infusion process. Primary osteoblast-like cells were used to specifically address this issue: To investigate the effects of SAW on the cells’ viability, proliferation, and differentiation. Fluorescence-labeled osteoblast-like cells were seeded into polycaprolactone scaffolds using the SAW method with a static method as a control. The cell distribution in the scaffold was assessed through image analysis. The cells were far more uniformly driven into the scaffold with the SAW method compared to the control, and the seeding process with SAW was also significantly faster: Cells were delivered into the scaffold in seconds compared to the hour-long process of static seeding. Over 80% of the osteoblast-like cells were found to be viable after being treated with SAW at 20 MHz for 10–30 s with an applied power of 380 mW over a wide range of cell suspension volumes (10–100 μℓ) and cell densities (1000–8000 cells∕μℓ). After determining the optimal cell seeding parameters, we further found that the treated cells offered the same functionality as untreated cells. Taken together, these results show that the SAW method has significant potential as a practical scaffold cell seeding method for tissue and orthopedic engineering.     

Microstructured multi-well plate for three-dimensional packed cell seeding and hepatocyte cell culture

In this article, we present a microstructured multi-well plate for enabling three-dimensional (3D) high density seeding and culture of cells through the use of a standard laboratory centrifuge to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro without the addition of animal derived or synthetic matrices or coagulants. Each well has microfeatures on the bottom that are comprised of a series of ditches/open microchannels. The dimensions of the microchannels promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro. After cell seeding with a standard pipette, the microstructured multi-well plates were centrifuged to tightly pack cells inside the ditches in order to enhance cell-cell interactions and induce formation of 3D cellular structures during cell culture. Cell-cell interactions were optimized based on cell packing by considering dimensions of the ditches/open microchannels, orientation of the microstructured multi-well plate during centrifugation, cell seeding density, and the centrifugal force and time. With the optimized cell packing conditions, we demonstrated that after 7 days of cell culture, primary human hepatocytes adhered tightly together to form cord-like structures that resembled 3D tissue-like cellular architecture. Importantly, cell membrane polarity was restored without the addition of animal derived or synthetic matrices or coagulants.     

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.     

In situ phase transitional polymeric vaccines for improved immunotherapy

Cancer vaccines have exhibited immense potential in cancer treatment. Through activating the host''s immune system, vaccines stimulate extensive functional T cells to eliminate cancer. However, the therapeutic efficacy of cancer vaccines is limited by their inferior lymph node delivery and inadequate uptake of dendritic cells. Herein, we propose an in situ phase transitional strategy on vaccine manufacturing to maximally enhance lymph node drainage while ensuring adequate dendritic cell uptake. The phase transitional vaccines, with dynamic size modulation property, retain a small size (24.4 ± 3.1 nm) during lymph node draining then transform into larger particles (483.0 ± 41.6 nm) on-site by external signal input. Results show that this strategy induced rapid and robust immune response in a mouse melanoma tumor model. Furthermore, a stronger humoral immune response was observed in mice when immunized with MHC-II restricted antigen, which demonstrated that lymph node-targeted cancer vaccine delivery could be effectively manipulated through dynamic size modulation.     

Microfluidic cell fragmentation for mechanical phenotyping of cancer cells

Circulating tumor cells (CTCs) shed from the primary tumor undergo significant fragmentation in the microvasculature, and very few escape to instigate metastases. Inspired by this in vivo behavior of CTCs, we report a microfluidic method to phenotype cancer cells based on their ability to arrest and fragment at a micropillar-based bifurcation. We find that in addition to cancer cell size, mechanical properties determine fragmentability. We observe that highly metastatic prostate cancer cells are more resistant to fragmentation than weakly metastatic cells, providing the first indication that metastatic CTCs can escape rupture and potentially initiate secondary tumors. Our method may thus be useful in identifying phenotypes that succumb to or escape mechanical trauma in microcirculation.     

Variation in the life history strategy underlies functional diversity of tumors

Classical r- vs. K-selection theory describes the trade-offs between high reproductive output and competitiveness and guides research in evolutionary ecology. While its impact has waned in the recent past, cancer evolution may rekindle it. Herein, we impose r- or K-selection on cancer cell lines to obtain strongly proliferative r cells and highly competitive K cells to test ideas on life-history strategy evolution. RNA-seq indicates that the trade-offs are associated with distinct expression of genes involved in the cell cycle, adhesion, apoptosis, and contact inhibition. Both empirical observations and simulations based on an ecological competition model show that the trade-off between cell proliferation and competitiveness can evolve adaptively. When the r and K cells are mixed, they exhibit strikingly different spatial and temporal distributions. Due to this niche separation, the fitness of the entire tumor increases. The contrasting selective pressure may operate in a realistic ecological setting of actual tumors.     

Administering the Optimum Dose of l-Arginine in Regional Tumor Therapy

The purpose of this study is optimizing the l-arginine (l-Arg) doses on the basis of chemical structure in regional accessible tumor therapy to settle down a new protocol for the treatment of cancer. ³H-thymidine-based cell proliferation assay was performed in vitro on tumor cell lines of fibrosarcoma (FS), lymphosarcoma-ascitic and on normal cell line of NIH 3T3 after treatment with different concentrations of l-Arg in phosphate buffered saline (PBS). The cultures were harvested after 22 h and the incorporated radioactivity was counted to identify their histologic grades as described in earlier studies. In vivo therapy of murine tumors was conducted where FS cells injected subcutaneously at ventro-lateral position of mice. Various drug delivery schedules were injected into the centre of tumor base, once a day for 4 days. Tumor diameter and survivals were monitored where the day of sacrifice was considered for monitoring the survival period. By identifying the histologic grades of the treated cultures in vitro and in vivo by different concentrations of l-Arg, the corresponding energy of such concentrations were determined. An efficient model with a good fit (R² = 0.98) was established to describe the energy yield by l-Arg dose. The equivalence between the tumor histologic grade and energy of the l-Arg dose delivered in saline (PBS) environment is the optimum condition for regional tumor therapy achieves higher survival rate. The selective cytotoxicity to tumor cells with minimal damage to normal cells by l-Arg due to its chemical structure suggests to be considered the most promising drug for regional therapy of the accessible tumors like breast cancers of early stage with no distant metastasis.     

A microfluidic model for organ-specific extravasation of circulating tumor cells

Circulating tumor cells (CTCs) are the principal vehicle for the spread of non-hematologic cancer disease from a primary tumor, involving extravasation of CTCs across blood vessel walls, to form secondary tumors in remote organs. Herein, a polydimethylsiloxane-based microfluidic system is developed and characterized for in vitro systematic studies of organ-specific extravasation of CTCs. The system recapitulates the two major aspects of the in vivo extravasation microenvironment: local signaling chemokine gradients in a vessel with an endothelial monolayer. The parameters controlling the locally stable chemokine gradients, flow rate, and initial chemokine concentration are investigated experimentally and numerically. The microchannel surface treatment effect on the confluency and adhesion of the endothelial monolayer under applied shear flow has also been characterized experimentally. Further, the conditions for driving a suspension of CTCs through the microfluidic system are discussed while simultaneously maintaining both the local chemokine gradients and the confluent endothelial monolayer. Finally, the microfluidic system is utilized to demonstrate extravasation of MDA-MB-231 cancer cells in the presence of CXCL12 chemokine gradients. Consistent with the hypothesis of organ-specific extravasation, control experiments are presented to substantiate the observation that the MDA-MB-231 cell migration is attributed to chemotaxis rather than a random process.     

Effect of vimentin on cell migration in collagen-coated microchannels: A mimetic physiological confined environment

Cancer cell migration through tissue pores and tracks into the bloodstream is a critical biological step for cancer metastasis. Although in vivo studies have shown that expression of vimentin can induce invasive cell lines, its role in cell cytoskeleton reorganization and cell motility under in vitro physical confinement remains unknown. Here, a microfluidic device with cell culture chamber and collagen-coated microchannels was developed as an in vitro model for physiological confinement environments. Using this microchannel assay, we demonstrated that the knockdown of vimentin decreases 3T3 fibroblast cell directional migration speed in confined microchannels. Additionally, as cells form dynamic membranes that define the leading edge of motile cells, different leading edge morphologies of 3T3 fibroblast and 3T3 vimentin knockdown cells were observed. The leading edge morphology change under confinement can be explained by the effect of vimentin on cytoskeletal organization and focal adhesion. The microfluidic device integrated with a time-lapse microscope provided a new approach to study the effect of vimentin on cell adhesion, migration, and invasiveness.     

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.     

A green,efficient and precise hydrogen therapy of cancer based on in vivo electrochemistry

By combined use of traditional Chinese acupuncture Fe needle electrode and in vivo electrochemistry, we achieved in vivo H₂ generation in tumors in a controllable manner and exploited it for effective and green therapy of tumors for the first time. The cathodic acupuncture electrodes working under an applied voltage of ∼3 V (with minimal damage to the living body) undergo effective electrochemical reactions in the acidic tumor area that produce sufficient H₂ locally to cause cancer cells to burst and die. Due to puncture positioning, the acidic tumor microenvironment and gas diffusion effect, the developed H₂ generation electrochemotherapy (H₂-ECT) strategy enables precise and large-scale tumor therapy, as demonstrated by in vivo treatment of diseased mice (glioma and breast cancers). Such green H₂-ECT is simple, highly efficient and minimally invasive, requiring no expensive medical equipment or nano materials and medication, and is therefore very promising for potential clinical applications.     

Photogenerated-hole-induced rapid elimination of solid tumors by the supramolecular porphyrin photocatalyst

The rapid, complete, targeted and safe treatment for tumors remains a key issue in cancer therapy. A novel treatment of solid tumors by supramolecular photocatalyst Nano-SA-TCPP with the irradiation of 600–700 nm wavelength is established. Solid tumors (100 mm³) can be eliminated within 10 min. The 50-day mouse survival rate was increased from 0% to 100% after the photocatalytic therapy. The breakthrough was owing to the cell membrane rupture and the cytoplasmic loss caused by photogenerated holes inside cancer cells. The porphyrin-based photocatalysts can be internalized in a targeted manner by cancer cells due to the size selection effect, without entering the normal cells. The therapy has no toxicity or side effects for normal cells and organisms. Moreover, the photocatalytic therapy is effective for a variety of cancer cell lines. Because of its high efficiency, safety and universality, the photocatalytic therapy provides us with a new lancet to conquer the tumor.     

Engineered three-dimensional microfluidic device for interrogating cell-cell interactions in the tumor microenvironment

Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-μm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the “single file” pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12.     

Biodegradable magnesium alloy with eddy thermal effect for effective and accurate magnetic hyperthermia ablation of tumors

Magnetic hyperthermia therapy (MHT) is able to ablate tumors using an alternating magnetic field (AMF) to heat up magnetocaloric agents (e.g. magnetic nanoparticles) administered into the tumors. For clinical applications, there is still a demand to find new magnetocaloric agents with strong AMF-induced heating performance and excellent biocompatibility. As a kind of biocompatible and biodegradable material, magnesium (Mg) and its alloys have been extensively used in the clinic as an implant metal. Herein, we discovered that the eddy thermal effect of the magnesium alloy (MgA) could be employed for MHT to effectively ablate tumors. Under low-field-intensity AMFs, MgA rods could be rapidly heated, resulting in a temperature increase in nearby tissues. Such AMF-induced eddy thermal heating of MgA could not only be used to kill tumor cells in vitro, but also be employed for effective and accurate ablation of tumors in vivo. In addition to killing tumors in mice, we further demonstrated that VX₂ tumors of much larger sizes growing in rabbits after implantation of MgA rods could also be eliminated after exposure to an AMF, illustrating the ability of MgA-based MHT to kill large-sized tumors. Moreover, the implanted MgA rods showed excellent biocompatibility and ∼20% of their mass was degraded within three months. Our work thus discovered for the first time that non-magnetic biodegradable MgA, an extensively used implant metal in clinic, could be used for effective magnetic thermal ablation of tumors under a low-field-intensity AMF. Such a strategy could be readily translated into clinical use.     

Integrated carbon fiber electrodes within hollow polymer microneedles for transdermal electrochemical sensing

In this study, carbon fiber electrodes were incorporated within a hollow microneedle array, which was fabricated using a digital micromirror device-based stereolithography instrument. Cell proliferation on the acrylate-based polymer used in microneedle fabrication was examined with human dermal fibroblasts and neonatal human epidermal keratinocytes. Studies involving full-thickness cadaveric porcine skin and trypan blue dye demonstrated that the hollow microneedles remained intact after puncturing the outermost layer of cadaveric porcine skin. The carbon fibers underwent chemical modification in order to enable detection of hydrogen peroxide and ascorbic acid; electrochemical measurements were demonstrated using integrated electrode-hollow microneedle devices.     

On-chip three-dimensional tumor spheroid formation and pump-less perfusion culture using gravity-driven cell aggregation and balanced droplet dispensing

This paper presents a spheroid chip in which three-dimensional (3D) tumor spheroids are not only formed by gravity-driven cell aggregation but also cultured at the perfusion rates controlled by balanced droplet dispensing without fluidic pumps. The previous spheroid chips require additional off-chip processes of spheroid formation and extraction as well as bulky components of fluidic pumps. However, the present spheroid chip, where autonomous medium droplet dispensers are integrated on a well array, achieves the on-chip 3D tumor spheroid formation and perfusion culture using simple structure without bulky fluidic pumps. In the experimental study, we demonstrated that the spheroid chip successfully forms 3D tumor spheroids in the wide diameter range of 220 μm–3.2 mm (uniformity > 90%) using H358, H23, and A549 non-small cell lung cancer cells. At the pump-less perfusion culture (Q = 0.1–0.3 μl/min) of spheroids, the number of H358 cells in the spheroid increased up to 50% from the static culture (Q = 0 μl/min) and the viability of the cultured cells also increased about 10%. Therefore, we experimentally verified that the perfusion environment created by the spheroid chip offers a favourable condition to the spheroids with high increase rate and viability. The present chip achieves on-chip 3D tumor spheroid formation and pump-less perfusion culture with simple structure, thereby exhibiting potential for use in integrated in-vivo-like cell culture systems.     

Dielectrophoresis has broad applicability to marker-free isolation of tumor cells from blood by microfluidic systems

The number of circulating tumor cells (CTCs) found in blood is known to be a prognostic marker for recurrence of primary tumors, however, most current methods for isolating CTCs rely on cell surface markers that are not universally expressed by CTCs. Dielectrophoresis (DEP) can discriminate and manipulate cancer cells in microfluidic systems and has been proposed as a molecular marker-independent approach for isolating CTCs from blood. To investigate the potential applicability of DEP to different cancer types, the dielectric and density properties of the NCI-60 panel of tumor cell types have been measured by dielectrophoretic field-flow fractionation (DEP-FFF) and compared with like properties of the subpopulations of normal peripheral blood cells. We show that all of the NCI-60 cell types, regardless of tissue of origin, exhibit dielectric properties that facilitate their isolation from blood by DEP. Cell types derived from solid tumors that grew in adherent cultures exhibited dielectric properties that were strikingly different from those of peripheral blood cell subpopulations while leukemia-derived lines that grew in non-adherent cultures exhibited dielectric properties that were closer to those of peripheral blood cell types. Our results suggest that DEP methods have wide applicability for the surface-marker independent isolation of viable CTCs from blood as well as for the concentration of leukemia cells from blood.     

Cytotoxicity and Apoptosis Inducing Activities of 2-Amino-4H-chromene-3-carbonitrile Derivatives Loaded on Gold Nanoparticles Against Human Breast Cancer Cell Line T47D

Chemotherapy drugs, used for prevention of uncontrolled cell proliferation in certain tissues as well as inducing apoptosis in tumor cells, are important candidates for treatment of cancer. The synthesized 2-amino-4H-chromene-3-carbonitrile derivatives effective on cancerous cells resistant to other drugs such as Paclitaxel were used due to their ability in induction of apoptosis. The growth inhibitory and inducing apoptosis activities were determined. In order to make it target-oriented, the best compound was conjugated with gold nanoparticles (NPs) by aspartic acid with chemical reduction method. Cytotoxicity effect of 2-amino-4H-chromene-3-carbonitrile derivatives against the T47D breast cancer cell line was determined by MTT assay. The synthesis of gold NPs was confirmed by transmission electron microscopy, UV–Vis and dynamic light scattering. To assess the effects of compounds on the process of apoptosis, staining methods with acridine orange–ethidium bromide and Hoechst staining by fluorescence microscopy and DNA fragmentation by the diphenylamine method were used. The synthesized compounds containing two NH₂ groups on benzene rings, demonstrated more cytotoxicity effect. The effect of conjugation with gold NPs and the induction of apoptosis were studied with the best compound. The cytotoxicity effects of the synthesized 2-amino-4H-chromene-3-carbonitrile compounds were changed by replacement of NO₂ group on thiol ring with different chemical groups on the benzene ring. Analyses of treated cell lines by conjugated and non-conjugated forms of compounds verified their ability in inducing apoptosis while conjugated form demonstrated higher apoptosis.     

Red blood cell membrane-camouflaged nanoparticles loaded with AIEgen and Poly(I : C) for enhanced tumoral photodynamic-immunotherapy

Red blood cell (RBC)-mimicking nanoparticles (NPs) offer a promising platform for drug delivery because of their prolonged circulation time, reduced immunogenicity and specific targeting ability. Herein, we report the design and preparation of RBC membrane-bound NPs (M@AP), for tumoral photodynamic-immunotherapy. The M@AP is formed by self-assembly of the positively charged aggregation-induced emission luminogen (AIEgen) (named P2-PPh3) and the negatively charged polyinosinic : polycytidylic acid (Poly(I : C)), followed by RBC membrane encapsulation. P2-PPh3 is an AIE-active conjugated polyelectrolyte with additional photosensitizing ability for photodynamic therapy (PDT), while Poly(I : C) serves as an immune-stimulant to stimulate both tumor and immune cells to activate immunity, and thus reduces tumor cell viability. When applied in tumor-bearing mice, the M@AP NPs are enriched in both the tumor region as a result of an enhanced permeability and retention (EPR) effect, and the spleen because of the homing effect of the RBC-mimicking shell. Upon light irradiation, P2-PPh3 promotes strong ROS generation in tumor cells, inducing the release of tumor antigens (TA). The anti-tumor immunity is further enhanced by the presence of Poly(I : C) in M@AP. Thus, this strategy combines the PDT properties of the AIE-active polyelectrolyte and immunotherapy properties of Poly(I : C) to achieve synergistic activation of the immune system for anti-tumor activity, providing a novel strategy for tumor treatment.