Microfluidic self-assembly of tumor spheroids for anticancer drug discovery

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of in vivo tumors. Moreover, continuous dynamic perfusion allows the establishment of physiologically relevant drug profiles to exposed spheroids. Here we present a physiologically inspired design allowing microfluidic self-assembly of spheroids, formation of uniform spheroid arrays, and characterizations of spheroid dynamics all in one platform. Our microfluidic device is based on hydrodynamic trapping of cancer cells in controlled geometries and the formation of spheroids is enhanced by maintaining compact groups of the trapped cells due to continuous perfusion. It was found that spheroid formation speed (average of 7 h) and size uniformity increased with increased flow rate (up to 10 μl min⁻¹). A large amount of tumor spheroids (7,500 spheroids per square centimeter) with a narrow size distribution (10 ± 1 cells per spheroid) can be formed in the device to provide a good platform for anticancer drug assays. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Liz Y. Wu and Dino Di Carlo contributed equally to this work.     

Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids

The niche microenvironment in which cancer cells reside plays a prominent role in the growth of cancer. It is therefore imperative to mimic the in vivo tumor niche in vitro to better understand cancer and enhance development of therapeutics. Here, we engineer a 3D metastatic prostate cancer model that includes the types of surrounding cells in the bone microenvironment that the metastatic prostate cancer cells reside in. Specifically, we used a two-layer microfluidic system to culture 3D multi-cell type spheroids of fluorescently labeled metastatic prostate cancer cells (PC-3 cell line), osteoblasts and endothelial cells. This method ensures uniform incorporation of all co-culture cell types into each spheroid and keeps the spheroids stationary for easy tracking of individual spheroids and the PC-3's residing inside them over the course of at least a week. This culture system greatly decreased the proliferation rate of PC-3 cells without reducing viability and may more faithfully recapitulate the in vivo growth behavior of malignant cancer cells within the bone metastatic prostate cancer microenvironment.     

Ovarian carcinoma spheroids disaggregate on type I collagen and invade live human mesothelial cell monolayers

Ovarian carcinoma patients frequently develop malignant ascites containing single and aggregated tumor cells, or spheroids. Spheroids have been shown to be resistant to many therapies, but their contribution to ovarian cancer dissemination remains undetermined. We have previously shown that ascites spheroids adhere to extracellular matrix (ECM) proteins and live human mesothelial cells via beta1 integrin subunits. Here, we assessed the ability of spheroids that were generated from the human ovarian carcinoma cell line NIH:OVCAR5 to disseminate and invade in vitro. Spheroids were seeded on ECM proteins for 24 h. While laminin and type IV collagen stimulated some cell migration, spheroids completely disaggregated on type I collagen substrates. A monoclonal antibody against the beta1 integrin subunit significantly inhibited disaggregation on all proteins tested. To test their invasive ability, spheroids were added to monolayers of live human LP9 mesothelial cells. Within 24 h, the spheroids adhered and disaggregated on top of the monolayers, and within a week had established foci of invasion encompassing a 200-fold larger surface area. Addition of a monoclonal antibody against the beta1 integrin subunit drastically reduced spheroid invasion into the mesothelial cell monolayers. GM 6001, a broad-scale matrix metalloproteinase inhibitor, also significantly blocked spheroid invasion into the mesothelial cell monolayers. Epsilon-amino-N-caproic acid, a serine protease inhibitor, partially inhibited spheroid invasion. Based on their ability to attach to, disaggregate on, and invade into live human mesothelial cell monolayers, spheroids should thus be regarded as potential contributors to the dissemination of ovarian cancer.     

Mesothelial cells interact with tumor cells for the formation of ovarian cancer multicellular spheroids in peritoneal effusions

Epithelial ovarian cancer (EOC) dissemination is primarily mediated by the shedding of tumor cells from the primary site into ascites where they form multicellular spheroids that rapidly lead to peritoneal carcinomatosis. While the clinical importance and fundamental role of multicellular spheroids in EOC is increasingly appreciated, the mechanisms that regulate their formation and dictate their cellular composition remain poorly characterized. To investigate these important questions, we characterized spheroids isolated from ascites of women with EOC. We found that in these spheroids, a core of mesothelial cells was encased in a shell of tumor cells. Analysis further revealed that EOC spheroids are dynamic structures of proliferating, non-proliferating and hypoxic regions. To recapitulate these in vivo findings, we developed a three-dimensional co-culture model of primary EOC and mesothelial cells. Our analysis indicated that, compared to the OVCAR3 cell line, primary EOC cells isolated from ascites as well as mesothelial cells formed compact spheroids. Analysis of heterotypic spheroid microarchitecture revealed a structure that grossly resembles the structure of spheroids isolated from ascites. Cells that formed compact spheroids had elevated expression of β1 integrin and low expression of E-cadherin. Addition of β1 integrin blocking antibody or siRNA-mediated downregulation of β1 integrin resulted in reduced tightness of the spheroids. Interestingly, the loss of MUC16 and E-cadherin expression resulted in the formation of more compact spheroids. Therefore, our findings support the heterotypic nature of spheroids from malignant EOC ascites. In addition, our data describe an unusual link between E-cadherin expression and less compact spheroids. Our data also emphasize the role of MUC16 and β1 integrin in EOC spheroid formation.     

A cell-instructive hydrogel to regulate malignancy of 3D tumor spheroids with matrix rigidity

Three dimensional (3D) tumor spheroid models are becoming important biomedical tools for both fundamental and applied cancer studies, but current models do not account for different levels of cancer malignancy. Several studies have reported that the mechanical rigidity of a hydrogel plays a significant role in regulating the phenotypes of cancer cells adhered to the gel surface. This finding suggests that matrix rigidity should also modulate the malignancy of 3D tumor spheroids. However, the role of matrix stiffness is often confounded by concurrent changes in 3D matrix permeability. This study reports an advanced strategy to assemble 3D liver tumor spheroids with controlled intercellular organization, phenotypes, and angiogenic activities using hydrogels with controlled stiffness and minimal differences in molecular diffusivity. The elastic moduli of cell-encapsulated collagen gels were increased by stiffening interconnected collagen fibers with varied amounts of poly(ethylene glycol) di-(succinic acid N-hydroxysuccinimidyl ester). Interestingly, hepatocellular carcinoma cells encapsulated in a fat-like, softer hydrogel formed malignant cancer spheroids, while cells cultured in a liver-like, stiffer gel formed compact hepatoids with suppressed malignancy. Overall, both the hydrogel and the 3D tumor spheroids developed in this study will be greatly useful to better understand and regulate the emergent behaviors of various cancer cells.     

Ovarian carcinoma spheroids disaggregate on type I collagen and invade live human mesothelial cell monolayers

Ovarian carcinoma patients frequently develop malignant ascites containing single and aggregated tumor cells, or spheroids. Spheroids have been shown to be resistant to many therapies, but their contribution to ovarian cancer dissemination remains undetermined. We have previously shown that ascites spheroids adhere to extracellular matrix (ECM) proteins and live human mesothelial cells via β1 integrin subunits. Here, we assessed the ability of spheroids that were generated from the human ovarian carcinoma cell line NIH: OVCAR5 to disseminate and invade in vitro. Spheroids were seeded on ECM proteins for 24 h. While laminin and type IV collagen stimulated some cell migration, spheroids completely disaggregated on type I collagen substrates. A monoclonal antibody against the β1 integrin subunit significantly inhibited disaggregation on all proteins tested. To test their invasive ability, spheroids were added to monolayers of live human LP9 mesothelial cells. Within 24 h, the spheroids adhered and disaggregated on top of the monolayers, and within a week had established foci of invasion encompassing a 200-fold larger surface area. Addition of a monoclonal antibody against the β1 integrin subunit drastically reduced spheroid invasion into the mesothelial cell monolayers. GM 6001, a broad-scale matrix metalloproteinase inhibitor, also significantly blocked spheroid invasion into the mesothelial cell monolayers. ɛ-amino-N-caproic acid, a serine protease inhibitor, partially inhibited spheroid invasion. Based on their ability to attach to, disaggregate on, and invade into live human mesothelial cell monolayers, spheroids should thus be regarded as potential contributors to the dissemination of ovarian cancer.This revised version was published online in August 2005 with a corrected cover date.     

Three-dimensional modeling of transport of nutrients for multicellular tumor spheroid culture in a microchannel

The growth dynamics of avascular tumors in a microchannel bioreactor is investigated. A three-dimensional flow and nutrient transport model, incorporating the multicellular tumor spheroid (MTS) growth model, has been developed to study the influence of nutrients (oxygen and glucose) supply and distribution on the MTS growth. Numerical simulations based on the EMT6/Ro tumor cells show that the continuous-flow perfusion is more efficient to deliver nutrients to the MTS than the diffusion-only static culture. It is further demonstrated that as long as there is bulk flow, the growth of a single tumor spheroid at the early stage is insensitive to the flow velocity and the channel size. For multiple tumor spheroids in the same microchannel, however, increasing the perfusion velocity can improve the nutrient environment for the disadvantageous downstream tumor spheroid. The flow shear stress exerting on the MTSs in the current microchannel bioreactor is estimated to be far below the critical value to affect the MTS growth, which means that there is still much room for increasing perfusion velocity to satisfy the higher nutrient requirement by the growing tumor spheroids.     

Spheroid culture of LuCaP 136 patient-derived xenograft enables versatile preclinical models of prostate cancer

LuCaP serially transplantable patient-derived xenografts (PDXs) are valuable preclinical models of locally advanced or metastatic prostate cancer. Using spheroid culture methodology, we recently established cell lines from several LuCaP PDXs. Here, we characterized in depth the features of xenografts derived from LuCaP 136 spheroid cultures and found faithful retention of the phenotype of the original PDX. In vitro culture enabled luciferase transfection into LuCaP 136 spheroids, facilitating in vivo imaging. We showed that LuCaP 136 spheroids formed intratibial, orthotopic, and subcutaneous tumors when re-introduced into mice. Intratibial tumors responded to castration and were highly osteosclerotic. LuCaP 136 is a realistic in vitro–in vivo preclinical model of a subtype of bone metastatic prostate cancer.     

Precise glioma targeting of and penetration by aptamer and peptide dual-functioned nanoparticles

The treatment of a brain glioma is still one of the most difficult challenges in oncology. To effectively treat brain glioma and reduce the side effects, drugs must be transported across the blood brain barrier (BBB) and then targeted to the brain cancer cells because most anti-tumor drugs are highly toxic to the normal brain tissue. A cascade delivery strategy was developed to perform these two aims and to achieve enhanced and precisely targeted delivery. Herein, we utilize a phage-displayed TGN peptide and an AS1411 aptamer, which are specific targeting ligands of the BBB and cancer cells, respectively and we conjugate them with nanoparticles to establish the brain glioma cascade delivery system (AsTNP). In vitro cell uptake and three-dimensional tumor spheroid penetration studies demonstrated that the system could not only target endothelial and tumor cells but also penetrate the endothelial monolayers and tumor cells to reach the core of the tumor spheroids, which was extremely important but mostly ignored in glioma therapy. In vivo imaging further demonstrated that the AsTNP provided the highest tumor distribution and tumor/normal brain ratio. The distribution was also reconfirmed by fluorescent images of the brain slides. As a result, the docetaxel-loaded AsTNP presents the best anti-glioma effect with improved glioma bearing survival. In conclusion, the AsTNP could precisely target to the brain glioma, which was a valuable target for glioma imaging and therapy.     

Engineered 3D tumour model for study of glioblastoma aggressiveness and drug evaluation on a detachably assembled microfluidic device

3D models of tumours have emerged as an advanced technique in pharmacology and tumour cell biology, in particular for studying malignant tumours such as glioblastoma multiforme (GBM). Herein, we developed a 3D GBM model on a detachably assembled microfluidic device, which could be used to study GBM aggressiveness and for anti-GBM drug testing. Fundamental characteristics of the GBM microenvironment in terms of 3D tissue organisation, extracellular matrices and blood flow were reproduced in vitro by serial manipulations in the integrated microfluidic device, including GBM spheroid self-assembly, embedding in a collagen matrix, and continuous perfusion culture, respectively. We could realize multiple spheroids parallel manipulation, whilst, compartmentalized culture, in a highly flexible manner. This method facilitated investigations into the viability, proliferation, invasiveness and phenotype transition of GBM in a 3D microenvironment and under continuous stimulation by drugs. Anti-invasion effect of resveratrol, a naturally isolated polyphenol, was innovatively evaluated using this in vitro 3D GBM model. Temozolomide and the combination of resveratrol and temozolomide were also evaluated as control. This scalable model enables research into GBM in a more physiologically relevant microenvironment, which renders it promising for use in translational or personalised medicine to examine the impact of, or identify combinations of, therapeutic agents.     

Alginate-based microfluidic system for tumor spheroid formation and anticancer agent screening

We demonstrate a microfluidic system for long-term tumor cell culture and drug testing. Three-dimensional cell culture is critical in characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Breast tumor cells were encapsulated within alginate which was gelled in situ within the microchannels. Tumor spheroid formation was observed several days after cell seeding, and various concentrations of doxorubicin were applied to the encapsulated cell aggregates. Drug effects on cell viability and proliferation were measured. In future, hydrogel-based microfluidic devices can comprise part of systems which replace labor intensive screening platforms currently implemented in the laboratory, and they address a need for improving preclinical testing of cancer cell sensitivity to anti-cancer drugs.     

Embryonic body culturing in an all-glass microfluidic device with laser-processed 4 μm thick ultra-thin glass sheet filter

In this paper, we report the development and demonstration of a method to fabricate an all-glass microfluidic cell culturing device without circulation flow. On-chip microfluidic cell culturing is an indispensable technique for cellular replacement therapies and experimental cell biology. Polydimethylsiloxane (PDMS) have become a popular material for fabricating microfluidic cell culture devices because it is a transparent, biocompatible, deformable, easy-to-mold, and gas-permeable. However, PDMS is also a chemically and physically unstable material. For example, PDMS undergoes aging easily even in room temperature conditions. Therefore, it is difficult to control long term experimental culturing conditions. On the other hand, glass is expected to be stable not only in physically but also chemically even in the presence of organic solvents. However, cell culturing still requires substance exchanges such as gases and nutrients, and so on, which cannot be done in a closed space of a glass device without circulation flow that may influence cell behavior. Thus, we introduce a filter structure with micropores onto a glass device to improve permeability to the cell culture space. Normally, it is extremely difficult to fabricate a filter structure on a normal glass plate by using a conventional fabrication method. Here, we demonstrated a method for fabricating an all-glass microfluidic cell culturing device having filters structure. The function of this all-glass culturing device was confirmed by culturing HeLa, fibroblast and ES cells. Compared with the closed glass devices without a filter structure, the numbers of cells in our device increased and embryonic bodies (EBs) were formed. This method offers a new tool in microfluidic cell culture technology for biological analysis and it expands the field of microfluidic cell culture.     

In situ formation and collagen-alginate composite encapsulation of pancreatic islet spheroids

In this study, we suggest in situ islet spheroid formation and encapsulation on a single platform without replating as a method for producing mono-disperse spheroids and minimizing damage to spheroids during encapsulation. Using this approach, the size of spheroid can be controlled by modulating the size of the concave well. Here, we used 300 μm concave wells to reduce spheroid size and thereby eliminating the central necrosis caused by large volume. As the encapsulation material, we used alginate and collagen-alginate composite (CAC), and evaluated their suitability through diverse in vitro tests, including measurements of viability, oxygen consumption rate (OCR), hypoxic damage to encapsulated spheroids, and insulin secretion. For in situ encapsulation, alginate or CAC was spread over a concave microwell array containing spheroids, and CaCl₂ solution was diffused through a nano-porous dialysis membrane to achieve uniform polymerization, forming convex structures. By this process, the formation of uniform-size islet spheroids and their encapsulation without an intervening replating step was successfully performed. As a control, intact islets were evaluated concurrently. The in vitro test demonstrated excellent performance of CAC-encapsulated spheroids, and on the basis of these results, we transplanted the islet spheroids-encapsulated with CAC into the intraperitoneal cavity of mice with induced diabetes for 4 weeks, and evaluated subsequent glucose control. Intact islets were also transplanted as control to investigate the effect of encapsulation. Transplanted CAC-encapsulated islet spheroids maintained glucose levels below 200 mg/dL for 4 weeks, at which they were still active. At the end of the implantation experiment, we carried out intraperitoneal glucose tolerance test (IPGTT) in mice to investigate whether the implanted islets remained responsive to glucose. The glucose level in mice with CAC-encapsulated islet spheroids dropped below 200 mg/dL 60 min after glucose injection and was stably maintained. In conclusion, the proposed encapsulation method enhances the viability and function of islet spheroids, and protects these spheroids from immune attack.     

Acquisition of epithelial–mesenchymal transition and cancer stem-like phenotypes within chitosan-hyaluronan membrane-derived 3D tumor spheroids

Cancer drug development has to go through rigorous testing and evaluation processes during pre-clinical in vitro studies. However, the conventional two-dimensional (2D) in vitro culture is often discounted by the insufficiency to present a more typical tumor microenvironment. The multicellular tumor spheroids have been a valuable model to provide more comprehensive assessment of tumor in response to therapeutic strategies. Here, we applied chitosan-hyaluronan (HA) membranes as a platform to promote three-dimensional (3D) tumor spheroid formation. The biological features of tumor spheroids of human non-small cell lung cancer (NSCLC) cells on chitosan-HA membranes were compared to those of 2D cultured cells in vitro. The cells in tumor spheroids cultured on chitosan-HA membranes showed higher levels of stem-like properties and epithelial–mesenchymal transition (EMT) markers, such as NANOG, SOX2, CD44, CD133, N-cadherin, and vimentin, than 2D cultured cells. Moreover, they exhibited enhanced invasive activities and multidrug resistance by the upregulation of MMP2, MMP9, BCRC5, BCL2, MDR1, and ABCG2 as compared with 2D cultured cells. The grafting densities of HA affected the tumor sphere size and mRNA levels of genes on the substrates. These evidences suggest that chitosan-HA membranes may offer a simple and valuable biomaterial platform for rapid generation of tumor spheroids in vitro as well as for further applications in cancer stem cell research and cancer drug screening.     

Immunological Analyses of Whole Blood via “Microfluidic Drifting” Based Flow Cytometric Chip

Cost-effective, high-performance diagnostic instruments are vital to providing the society with accessible, affordable, and high-quality healthcare. Here we present an integrated, “microfluidic drifting” based flow cytometry chip as a potential inexpensive, fast, and reliable diagnostic tool. It is capable of analyzing human blood for cell counting and diagnosis of diseases. Our device achieves a throughput of ~3754 events/s. Calibration with Flow-Check calibration beads indicated good congruency with a commercially available benchtop flow cytometer. Moreover, subjection to a stringent 8-peak rainbow calibration particle test demonstrated its ability to perform high-resolution immunological studies with separation resolution of 4.28 between the two dimmest fluorescent populations. Counting accuracy at different polystyrene bead concentrations showed strong correlation (r = 0.9991) with hemocytometer results. Finally, reliable quantification of CD4+ cells in healthy human blood via staining with monoclonal antibodies was demonstrated. These results demonstrate the potential of our microfluidic flow cytometry chip as an inexpensive yet high-performance point-of-care device for mobile medicine.     

Relationship of sialyl-Lewis(x/a) underexpression and E-cadherin overexpression in the lymphovascular embolus of inflammatory breast carcinoma

Inflammatory breast carcinoma (IBC) is characterized by florid tumor emboli within lymphovascular spaces called lymphovascular invasion. These emboli have a unique microscopic appearance of compact clumps of tumor cells retracted away from the surrounding endothelial cell layer. Using a human SCID model of IBC (MARY-X), we, in previous studies, demonstrated that the tumor cell embolus (IBC spheroid) forms on the basis of an intact and overexpressed E-cadherin/alpha,beta-catenin axis that mediates tumor cell-tumor cell adhesion. In the present study we examine the mechanism behind the apparent lack of binding of the tumor embolus to the surrounding endothelium. We find that this lack of tumor cell binding is because of markedly decreased sialyl-Lewis(x/a) (sLe(x/a)) carbohydrate ligand-binding epitopes on its overexpressed MUC1 and other surface molecules that bind endothelial E-selectin. Decreased sLe(x/a) is because of decreased alpha3/4-fucosyltransferase activity in MARY-X. The decreased sLe(x/a) fail to confer electrostatic repulsions between tumor cells, which further contributes to the compactness of the MARY-X spheroid by allowing the E-cadherin homodimeric interactions to go unopposed. MARY-X spheroids were retrovirally transfected with FucT-III cDNA, significantly raising their levels of fucosyltransferase activity and surface sLe(x/a). In parallel experiments, enzymatic transfers with a milk alpha1,3-fucosyltransferase and an alpha2,3-sialyltransferase (ST3GalIV) were performed on the MARY-X spheroids and increased surface sLe(x/a). The addition of sLe(x/a) by either manipulation caused disadherence of the MARY-X spheroids and the disruption of the E-cadherin homodimers mediating cell adhesion. Our findings support the cooperative relationship of sLe(x/a) underexpression and E-cadherin overexpression in the genesis of the lymphovascular embolus of IBC.     

BMP signalling controls the malignant potential of ascites-derived human epithelial ovarian cancer spheroids via AKT kinase activation

Epithelial ovarian cancer (EOC) cells have the ability to form multi-cellular aggregates in malignant ascites which dramatically alters cell signalling, survival, and metastatic potential. Herein, we demonstrate that patient ascites-derived EOC cells down-regulate endogenous bone morphogenetic protein (BMP) signalling by decreasing BMP ligand expression when grown in suspension culture to form spheroids. Enforced BMP signalling in these cells via constitutively-active BMP type I ALK3^QD receptor expression causes the formation of smaller, more loosely-aggregated spheroids. Additionally, ALK3^QD-expressing spheroids have an increased rate of adhesion and dispersion upon reattachment to substratum. Inhibition of endogenous BMP signalling using recombinant Noggin or small molecule inhibitor LDN-193189, on the other hand, opposed these phenotypic changes. To identify potential targets that impact the phenotype of EOC spheroids due to activated BMP signalling, we performed genome-wide expression analyses using Affymetrix arrays. Using the online Connectivity Map resource, the BMP signalling gene expression signature revealed that the AKT pathway is induced by activated BMP signalling in EOC cells; this finding was further validated by phospho-AKT immuno-blotting. In fact, treatment of EOC spheroids with an AKT inhibitor, Akti-1/2, reduced BMP-stimulated cell dispersion during reattachment as compared to controls. Thus, we have identified AKT as being one important downstream component of activated BMP signalling on EOC spheroid pathobiology, which may have important implications on the metastatic potential of this malignancy.     

Clinical significance of circulating tumor cells from lung cancer patients using microfluidic chip

Circulating tumor cells (CTCs) exist in the peripheral blood and have an important role in the disease development, tumor metastasis and clinical surveillance, especially in the process of metastasis. However, the technology of detecting CTCs still had a large challenge since they were rare in the peripheral blood. Here, we developed a size-based microfluidic chip, which contained array and filter channel array that could enrich CTCs from blood samples more quickly and conveniently. Combined with clinical specimen, we analyzed CTCs in 200 lung cancer patients by this microfluidic chip. The microfluidic device has high specificity and sensitivity in detecting CTCs (86.0% sensitivity and 98% specificity). Furthermore, the number of CTCs showed a increasing trend according to the stage of the disease (the mean number of I stage 5.0 ± 5.121 versus II stage 8.731 ± 6.36 versus III stage 16.81 ± 9.556 versus IV stage 28.72 ± 17.39 cells/mL, P < 0.05). The number of CTCs was concurrent with the condition of pathological type and metastasis patients. Compared to conventional markers like CEA, CY211, SCC, CTCs showed a higher positive rate in diagnosed patients. The advanced microfluidic device could capture tumor cells without reliance on cell surface expression markers and provide a fast, convenient, economical method in detecting CTCs, thereby offering potential to design effective and individualized cancer therapies.     

Dynamics of the self-assembly of complex cellular aggregates on micromolded nonadhesive hydrogels

The process by which cells self-assemble to form three-dimensional (3D) structures is central to morphogenesis and development of living tissues and hence is of growing interest to the field of tissue engineering. Using rapid prototyping technology we made micromolded nonadhesive hydrogels to study the dynamics of self-assembly in a low-shear environment with simple spherical geometries as well as more complex geometries such as a toroid. Aggregate size, shape, and composition were easily controlled; aggregates were easily retrieved; and the dynamics of the assembly process were readily observed by time-lapse microscopy. When two cell types, normal human fibroblasts (NHFs) and human umbilical vein endothelial cells (HUVECs), were seeded together, they self-segregated into multilayered spherical microtissues with a core of NHFs enveloped by a layer of HUVECs. Surprisingly, when a single cell suspension of NHFs was added to 7-day-old HUVEC spheroids, the HUVEC spheroid reorganized such that NHFs occupied the center and HUVECs coated the outside, demonstrating that self-assembly is not terminal and that spheroids are fluid structures that retain the ability to reassemble. We also showed that cells can self-assemble to form a complex toroid shape, and we observed several phenomena indicating that cellular contraction and tension play a significant role in the assembly process of complex shapes.     

Optimization of an endovascular magnetic filter for maximized capture of magnetic nanoparticles

To computationally optimize the design of an endovascular magnetic filtration device that binds iron oxide nanoparticles and to validate simulations with experimental results of prototype devices in physiologic flow testing. Three-dimensional computational models of different endovascular magnetic filter devices assessed magnetic particle capture. We simulated a series of cylindrical neodymium N52 magnets and capture of 1500 iron oxide nanoparticles infused in a simulated 14 mm-diameter vessel. Device parameters varied included: magnetization orientation (across the diameter, “D”, along the length, “L”, of the filter), magnet outer diameter (3, 4, 5 mm), magnet length (5, 10 mm), and spacing between magnets (1, 3 mm). Top designs were tested in vitro using ⁸⁹Zr-radiolabeled iron oxide nanoparticles and gamma counting both in continuous and multiple pass flow model. Computationally, “D” magnetized devices had greater capture than “L” magnetized devices. Increasing outer diameter of magnets increased particle capture as follows: “D” designs, 3 mm: 12.8–13.6 %, 4 mm: 16.6–17.6 %, 5 mm: 21.8–24.6 %; “L” designs, 3 mm: 5.6–10 %, 4 mm: 9.4–15.8 %, 5 mm: 14.8–21.2 %. In vitro, while there was significant capture by all device designs, with most capturing 87–93 % within the first two minutes, compared to control non-magnetic devices, there was no significant difference in particle capture with the parameters varied. The computational study predicts that endovascular magnetic filters demonstrate maximum particle capture with “D” magnetization. In vitro flow testing demonstrated no difference in capture with varied parameters. Clinically, “D” magnetized devices would be most practical, sized as large as possible without causing intravascular flow obstruction.