Systematic development of ionizable lipid nanoparticles for placental mRNA delivery using a design of experiments approach.

Ionizable lipid nanoparticles (LNPs) have gained attention as mRNA delivery platforms for vaccination against COVID-19 and for protein replacement therapies. LNPs enhance mRNA stability, circulation time, cellular uptake, and preferential delivery to specific tissues compared to mRNA with no carrier platform. However, LNPs are only in the beginning stages of development for safe and effective mRNA delivery to the placenta to treat placental dysfunction. Here, we develop LNPs that enable high levels of mRNA delivery to trophoblasts in vitro and to the placenta in vivo with no toxicity. We conducted a Design of Experiments to explore how LNP composition, including the type and molar ratio of each lipid component, drives trophoblast and placental delivery. Our data revealed that utilizing C12-200 as the ionizable lipid and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as the phospholipid in the LNP design yields high transfection efficiency in vitro. Analysis of lipid molar composition as a design parameter in LNPs displayed a strong correlation between apparent pKa and poly (ethylene) glycol (PEG) content, as a reduction in PEG molar amount increases apparent pKa. Further, we present one LNP platform that exhibits the highest delivery of placental growth factor mRNA to the placenta in pregnant mice, resulting in synthesis and secretion of a potentially therapeutic protein. Lastly, our high-performing LNPs have no toxicity to both the pregnant mice and fetuses. Our results demonstrate the feasibility of LNPs as a platform for mRNA delivery to the placenta, and our top LNP formulations may provide a therapeutic platform to treat diseases that originate from placental dysfunction during pregnancy.

Sex-related external factors influence pulmonary vascular angiogenesis in a sex-dependent manner.

Bronchopulmonary dysplasia (BPD) is a disease with a significant sexual dimorphism where males have a disadvantage compared with their female counterparts. Although mechanisms behind this sexual dimorphism are poorly understood, sex differences in angiogenesis have been identified as one possible source of the male disadvantage in BPD. Pulmonary angiogenesis was assessed in vitro using a bead sprouting assay with pooled male or female human pulmonary microvascular endothelial cells (HPMECs, 18-19 wk gestation, canalicular stage of human lung development) in standard (sex-hormone containing) and hormone-stripped medium. We identified sex-specific phenotypes in angiogenesis where male HPMECs produce fewer but longer sprouts compared with female HPMECs. The presence of sex hormones from standard culture medium modifies the male HPMEC phenotype with shorter and fewer sprouts but does not influence the female phenotype. Using a conditioned medium model, we further characterized the influence of the sex-specific secretome. Male and female HPMECs secrete factors that increase the maximum length of sprouts in female, but not male HPMECs. The presence of sex hormones abolishes this response. The male HPMEC secretome inhibits angiogenic sprouting in male HPMECs in the absence of sex hormones. Taken together, these results demonstrate that the pulmonary endothelial cell phenotypes are influenced by sex hormones and sex-specific secreted factors in a sex-dependent manner.NEW & NOTEWORTHY We identified a sex-specific phenotype wherein male HPMECs produce fewer but longer sprouts than females. Surprisingly, the presence of sex hormones only modifies the male phenotype, resulting in shorter and even fewer sprouts. Furthermore, we found the sex-specific secretome has a sex-dependent influence on angiogenesis that is also sex-hormone sensitive. These new and surprising findings point to the unappreciated role of sex and sex-related exogenous factors in early developmental angiogenesis.

Artificial engineering of the protein corona at bio-nano interfaces for improved cancer-targeted nanotherapy.

Nanoparticles (NPs) have been used in numerous applications as anticancer, antibacterial and antioxidant agents. Artificial engineering of protein interactions with NPs in biological systems is crucial to develop potential NPs for drug delivery and cancer nanotherapy. The protein corona (PC) on the NP surface, displays an interface between biomacromolecules and NPs, governing their pharmacokinetics and pharmacodynamics. Upon interaction of proteins with the NPs, their surface features are modified and they can easily be removed from the circulation by the mononuclear phagocytic system (MPS). PC properties heavily depend on the biological microenvironment and NP physicochemical parameters. Based on this context, we have surveyed different approaches that have been used for artificial engineering of the PC composition on NP surfaces. We discussed the effects of NP size, shape, surface modifications (PEGylation, self-peptide, other polymers), and protein pre-coating on the PC properties. Additionally, other factors including protein source and structure, intravenous injection and the subsequent shear flow, plasma protein gradients, temperature and local heat transfer, and washing media were considered in the context of their effects on the PC properties and overall target cellular effects. Moreover, the effects of NP-PC complexes on cancer cells based on cellular interactions, organization of intracellular PC (IPC), targeted drug delivery (TDD) and regulation of burst drug release profile of nanoplatforms, enhanced biocompatibility, and clinical applications were discussed followed by challenges and future perspective of the field. In conclusion, this paper can provide useful information to manipulate PC properties on the NP surface, thus trying to provide a literature survey to shorten their shipping from preclinical to clinical trials and to lay the basis for a personalized PC.

An Updated Review on EPR-Based Solid Tumor Targeting Nanocarriers for Cancer Treatment.

The enhanced permeability and retention (EPR) effect in cancer treatment is one of the key mechanisms that enables drug accumulation at the tumor site. However, despite a plethora of virus/inorganic/organic-based nanocarriers designed to rely on the EPR effect to effectively target tumors, most have failed in the clinic. It seems that the non-compliance of research activities with clinical trials, goals unrelated to the EPR effect, and lack of awareness of the impact of solid tumor structure and interactions on the performance of drug nanocarriers have intensified this dissatisfaction. As such, the asymmetric growth and structural complexity of solid tumors, physicochemical properties of drug nanocarriers, EPR analytical combination tools, and EPR description goals should be considered to improve EPR-based cancer therapeutics. This review provides valuable insights into the limitations of the EPR effect in therapeutic efficacy and reports crucial perspectives on how the EPR effect can be modulated to improve the therapeutic effects of nanomedicine.

Secretion of the disulfide bond generating catalyst QSOX1 from pancreatic tumor cells into the extracellular matrix: association with extracellular vesicles and matrix proteins.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a disulfide bond generating catalyst that is overexpressed in solid tumors. Expression of QSOX1 is linked to cancer cell invasion, tumor grade, and extracellular matrix (ECM) protein deposition. While the secreted version of QSOX1 is known to be present in various fluids and secretory tissues, its presence in the ECM of cancer is less understood. To characterize secreted QSOX1, we separated conditioned media based on size and density. We discovered that the majority of secreted QSOX1 resides in the EV-depleted fraction and in the soluble protein fraction. Very little QSOX1 could be detected in the EVP fraction. We used immunofluorescence to image subpopulations of EVs and found QSOX1 in Golgi-derived vesicles and medium/large vesicles, but in general, most extracellular QSOX1 was not attributed to these vesicles. Next, we quantified QSOX1 co-localization with the EV marker Alix. For the medium/large EVs, ~98% contained QSOX1 when fibronectin was used as a coating. However, on collagen coatings, only ~60% of these vesicles contained QSOX1, suggesting differences in EV cargo based on ECM coated surfaces. About 10% of small EVs co-localized with QSOX1 on every ECM protein surface except for collagen (0.64%). We next investigated adhesion of QSOX1 to ECM proteins in vitro and in situ and found that QSOX1 preferentially adheres to fibronectin, laminins, and Matrigel compared to gelatin and collagen. This mechanism was found to be, in part, mediated by the formation of mixed disulfides between QSOX1 and cysteine-rich ECM proteins. In summary, we found that QSOX1 (1) is in subpopulations of medium/large EVs, (2) seems to interact with small Alix+ EVs, and (3) adheres to cysteine-rich ECM proteins, potentially through the formation of intermediate disulfides. These observations offer significant insight into how enzymes, such as QSOX1, can facilitate matrix remodeling events in solid tumor progression.

Lipid Nanoparticle Composition Drives mRNA Delivery to the Placenta.

Ionizable lipid nanoparticles (LNPs) have gained attention as mRNA delivery platforms for vaccination against COVID-19 and for protein replacement therapies. LNPs enhance mRNA stability, circulation time, cellular uptake, and preferential delivery to specific tissues compared to mRNA with no carrier platform. However, LNPs have yet to be developed for safe and effective mRNA delivery to the placenta as a method to treat placental dysfunction. Here, we develop LNPs that enable high levels of mRNA delivery to trophoblasts in vitro and to the placenta in vivo with no toxicity. We conducted a Design of Experiments to explore how LNP composition, including the type and molar ratio of each lipid component, drives trophoblast and placental delivery. Our data revealed that a specific combination of ionizable lipid and phospholipid in the LNP design yields high transfection efficiency in vitro . Further, we present one LNP platform that exhibits highest delivery of placental growth factor mRNA to the placenta in pregnant mice, which demonstrates induced protein synthesis and secretion of a therapeutic protein. Lastly, our high-performing LNPs have no toxicity to both the pregnant mice and fetuses. Our results demonstrate the feasibility of LNPs as a platform for mRNA delivery to the placenta. Our top LNPs may provide a therapeutic platform to treat diseases that originate from placental dysfunction during pregnancy.

Mask Reuse in the COVID-19 Pandemic: Creating an Inexpensive and Scalable Ultraviolet System for Filtering Facepiece Respirator Decontamination.

As the current COVID-19 pandemic illustrates, not all hospitals and other patient care facilities are equipped with enough personal protective equipment to meet the demand in a crisis. Health care workers around the world use filtering facepiece respirators to protect themselves and their patients, yet during this global pandemic they are forced to reuse what are intended to be single-use masks. This poses a significant risk to these health care workers along with the people they are trying to protect. Ultraviolet germicidal irradiation (UVGI) has been validated previously as a method to effectively decontaminate these masks between use. However, not all facilities have access to the expensive commercial ultraviolet type C (UV-C) lamp decontamination equipment required for UVGI. UV-C bulbs are sitting idle in biosafety cabinets at universities and research facilities around the world that have been shuttered to slow the spread of COVID-19. These bulbs may also be available in existing medical centers where infectious diseases are commonly treated. We developed a method to modify existing light fixtures or create custom light fixtures that are compatible with new or existing UV-C bulbs. This system is scalable; can be created for less than US$50, on site and at the point of need; and leverages resources that are currently untapped and sitting unused in public and private research facilities during the pandemic. The freely accessible design can be easily modified for use around the world. Health care facilities can obtain this potentially lifesaving UVGI resource with minimal funds by collaborating with research facilities to obtain the UV-C meters and UV-C bulbs if they are unavailable from other sources. Although mask reuse is not ideal, we must do what we can in emergency situations to protect our health care workers responding to the pandemic and the communities they serve.

A Microfluidic System to Measure Neonatal Lung Compliance Over Late Stage Development as a Functional Measure of Lung Tissue Mechanics.

Premature birth interrupts the development of the lung, resulting in functional deficiencies and the onset of complex pathologies, like bronchopulmonary dysplasia (BPD), that further decrease the functional capabilities of the immature lung. The dysregulation of molecular targets has been implicated in the presentation of BPD, but there is currently no method to correlate resultant morphological changes observed in tissue histology with these perturbations to differences in function throughout saccular and alveolar lung development. Lung compliance is an aggregate measure of the lung's mechanical properties that is highly sensitive to a number of molecular, cellular, and architectural characteristics, but little is known about compliance in the neonatal mouse lung due to measurement challenges. We have developed a novel method to quantify changes in lung volume and pressure to determine inspiratory and expiratory compliance throughout neonatal mouse lung development. The compliance measurements obtained were validated against compliance values from published studies using mature lungs following enzymatic degradation of the extracellular matrix (ECM). The system was then used to quantify changes in compliance that occurred over the entire span of neonatal mouse lung development. These methods fill a critically important gap connecting powerful mouse models of development and disease to measures of functional lung mechanics critical to respiration and enable insights into the genetic, molecular, and cellular underpinnings of BPD pathology to improve lung function in premature infants.

Osmotic Gradients in Epithelial Acini Increase Mechanical Tension across E-cadherin, Drive Morphogenesis, and Maintain Homeostasis.

Epithelial cells spontaneously form acini (also known as cysts or spheroids) with a single, fluid-filled central lumen when grown in 3D matrices. The size of the lumen is dependent on apical secretion of chloride ions, most notably by the CFTR channel, which has been suggested to establish pressure in the lumen due to water influx. To study the cellular biomechanics of acini morphogenesis and homeostasis, we used MDCK-2 cells. Using FRET-force biosensors for E-cadherin, we observed significant increases in the average tension per molecule for each protein in mature 3D acini as compared to 2D monolayers. Increases in CFTR activity resulted in increased E-cadherin forces, indicating that ionic gradients affect cellular tension. Direct measurements of pressure revealed that mature acini experience significant internal hydrostatic pressure (37 ± 10.9 Pa). Changes in CFTR activity resulted in pressure and/or volume changes, both of which affect E-cadherin tension. Increases in CFTR chloride secretion also induced YAP signaling and cellular proliferation. In order to recapitulate disruption of acinar homeostasis, we induced epithelial-to-mesenchymal transition (EMT). During the initial stages of EMT, there was a gradual decrease in E-cadherin force and lumen pressure that correlated with lumen infilling. Strikingly, increasing CFTR activity was sufficient to block EMT. Our results show that ion secretion is an important regulator of morphogenesis and homeostasis in epithelial acini. Furthermore, this work demonstrates that, for closed 3D cellular systems, ion gradients can generate osmotic pressure or volume changes, both of which result in increased cellular tension.

MicroRNA-30a as a candidate underlying sex-specific differences in neonatal hyperoxic lung injury: implications for BPD.

Premature male neonates are at a greater risk of developing bronchopulmonary dysplasia (BPD). The reasons underlying sexually dimorphic outcomes in premature neonates are not known. The role of miRNAs in mediating sex biases in BPD is understudied. Analysis of the pulmonary transcriptome revealed that a large percentage of angiogenesis-related differentially expressed genes are miR-30a targets. We tested the hypothesis that there is differential expression of miR-30a in vivo and in vitro in neonatal human pulmonary microvascular endothelial cells (HPMECs) upon exposure to hyperoxia. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia [95% fraction of inspired oxygen (FiO2), postnatal day ( PND) 1-5] and euthanized on PND 7 and 21. HPMECs (18-24-wk gestation donors) were subjected to hyperoxia (95% O2 and 5% CO2) or normoxia (air and 5% CO2) up to 72 h. miR-30a expression was increased in both males and females in the acute phase ( PND 7) after hyperoxia exposure. However, at PND 21 (recovery phase), female mice showed significantly higher miR-30a expression in the lungs compared with male mice. Female HPMECs showed greater expression of miR-30a in vitro upon exposure to hyperoxia. Delta-like ligand 4 (Dll4) was an miR-30a target in HPMECs and showed sex-specific differential expression. miR-30a increased angiogenic sprouting in vitro in female HPMECs. Lastly, we show decreased expression of miR-30a and increased expression of DLL4 in human BPD lung samples compared with controls. These results support the hypothesis that miR-30a could, in part, contribute to the sex-specific molecular mechanisms in play that lead to the sexual dimorphism in BPD.

Pulmonary endothelial cells exhibit sexual dimorphism in their response to hyperoxia.

Abnormal pulmonary vascular development is a critical factor in the pathogenesis of bronchopulmonary dysplasia (BPD). Despite the well-established sex-specific differences in the incidence of BPD, the molecular mechanism(s) behind these are not completely understood. Exposure to a high concentration of oxygen (hyperoxia) contributes to BPD and creates a profibrotic environment in the lung. Our objective was to elucidate the sex-specific differences in neonatal human pulmonary microvascular endothelial cells (HPMECs) in normoxic and hyperoxic conditions, including the propensity for endothelial-to-mesenchymal transition. HPMECs (18- to 24-wk gestation donors, 6 male donors and 5 female donors) were subjected to hyperoxia (95% O2 and 5% CO2) or normoxia (air and 5% CO2) up to 72 h. We assessed cell migration and angiogenesis at baseline. Cell proliferation, viability, and expression of endothelial (CD31) and fibroblast markers (α-smooth muscle actin) were measured upon exposure to hyperoxia. Female HPMECs had significantly higher cell migration when assessed by the wound healing assay (40.99 ± 4.4%) compared with male HPMECs (14.76 ± 3.7%) and showed greater sprouting (1710 ± 962 µm in female cells vs. 789 ± 324 in male cells) compared with male endothelial cells in normoxia. Hyperoxia exposure decreased cell viability (by 9.8% at 48 h and 11.7% at 72 h) and proliferation (by 26.7% at 72 h) markedly in male HPMECs, whereas viability was sustained in female endothelial cells. There was greater expression of α-smooth muscle actin (2.5-fold) and decreased expression (5-fold) of CD31 in male HPMECs upon exposure to hyperoxia. The results indicate that cellular sex affects response in HPMECs in normoxia and hyperoxia. NEW & NOTEWORTHY Cellular sex affects response in human neonatal pulmonary microvascular endothelial cells in normoxia and hyperoxia. Under normoxic conditions, female human neonatal pulmonary microvascular endothelial cells display greater migration and angiogenic sprouting compared with male endothelial cells. Compared with female endothelial cells, hyperoxia exposure decreased cell viability and proliferation and increased α-smooth muscle actin and decreased CD31 expression in male endothelial cells, indicating an increased endothelial-mesenchymal transition.

Investigating the role of Hedgehog/GLI1 signaling in glioblastoma cell response to temozolomide.

Resistance to chemotherapy substantially hinders successful glioblastoma (GBM) treatment, contributing to an almost 100% mortality rate. Resistance to the frontline chemotherapy, temozolomide (TMZ), arises from numerous signaling pathways that are deregulated in GBM, including Hedgehog (Hh) signaling. Here, we investigate suppression of Hh signaling as an adjuvant to TMZ using U87-MG and T98G cell lines as in vitro models of GBM. We found that silencing GLI1 with siRNA reduces cell metabolic activity by up to 30% in combination with TMZ and reduces multidrug efflux activity by 2.5-fold. Additionally, pharmacological GLI inhibition modulates nuclear p53 levels and decreases MGMT expression in combination with TMZ. While we surprisingly found that silencing GLI1 does not induce apoptosis in the absence of TMZ co-treatment, we discovered silencing GLI1 without TMZ co-treatment induces senescence as evidenced by a significant 2.3-fold increase in senescence associated ß-galactosidase staining, and this occurs in a loss of PTEN-dependent manner. Finally, we show that GLI inhibition increases apoptosis in glioma stem-like cells by up to 6.8-fold in combination with TMZ, and this reduces the size and number of neurospheres grown from glioma stem-like cells. In aggregate, our data warrant the continued investigation of Hh pathway inhibitors as adjuvants to TMZ chemotherapy and highlight the importance of identifying signaling pathways that determine whether co-treatment will be successful.

Lymphatic function is required prenatally for lung inflation at birth.

Mammals must inflate their lungs and breathe within minutes of birth to survive. A key regulator of neonatal lung inflation is pulmonary surfactant, a lipoprotein complex which increases lung compliance by reducing alveolar surface tension (Morgan, 1971). Whether other developmental processes also alter lung mechanics in preparation for birth is unknown. We identify prenatal lymphatic function as an unexpected requirement for neonatal lung inflation and respiration. Mice lacking lymphatic vessels, due either to loss of the lymphangiogenic factor CCBE1 or VEGFR3 function, appear cyanotic and die shortly after birth due to failure of lung inflation. Failure of lung inflation is not due to reduced surfactant levels or altered development of the lung but is associated with an elevated wet/dry ratio consistent with edema. Embryonic studies reveal active lymphatic function in the late gestation lung, and significantly reduced total lung compliance in late gestation embryos that lack lymphatics. These findings reveal that lymphatic vascular function plays a previously unrecognized mechanical role in the developing lung that prepares it for inflation at birth. They explain respiratory failure in infants with congenital pulmonary lymphangiectasia, and suggest that inadequate late gestation lymphatic function may also contribute to respiratory failure in premature infants.

Host epithelial geometry regulates breast cancer cell invasiveness.

Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment.

Engineered tissues to quantify collective cell migration during morphogenesis.

Renal development is a complex process involving the dynamic interplay of over 25 different cell types. One distinct step in this process is the formation of the ureteric tree, which develops from the repeated branching of the ureteric bud. During branching of the ureteric bud, cells migrate collectively in unison to form the complex structure of the tree. Here, we present a microlithography-based 3D culture model in which multiple identical kidney epithelial tissues are used to quantify collective cell migration during the process of branching morphogenesis.

Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device.

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45- cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2-400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.

Immunocapture of prostate cancer cells by use of anti-PSMA antibodies in microdevices.

Patients suffering from cancer can shed tumor cells into the bloodstream, leading to one of the most important mechanisms of metastasis. As such, the capture of these cells is of great interest. Circulating tumor cells are typically extracted from circulation through positive selection with the epithelial cell-adhesion molecule (EpCAM), leading to currently unknown biases when cells are undergoing epithelial-to-mesenchymal transition. For prostate cancer, prostate-specific membrane antigen (PSMA) presents a compelling target for immunocapture, as PSMA levels increase in higher-grade cancers and metastatic disease and are specific to the prostate epithelium. This study uses monoclonal antibodies J591 and J415-antibodies that are highly specific for intact extracellular domains of PSMA on live cells-in microfluidic devices for the capture of LNCaPs, a PSMA-expressing immortalized prostate cancer cell line, over a range of concentrations and shear stresses relevant to immunocapture. Our results show that J591 outperforms J415 and a mix of the two for prostate cancer capture, and that capture performance saturates following incubation with antibody concentrations of 10 micrograms per milliliter.

Rare Cell Capture in Microfluidic Devices.

This article reviews existing methods for the isolation, fractionation, or capture of rare cells in microfluidic devices. Rare cell capture devices face the challenge of maintaining the efficiency standard of traditional bulk separation methods such as flow cytometers and immunomagnetic separators while requiring very high purity of the target cell population, which is typically already at very low starting concentrations. Two major classifications of rare cell capture approaches are covered: (1) non-electrokinetic methods (e.g., immobilization via antibody or aptamer chemistry, size-based sorting, and sheath flow and streamline sorting) are discussed for applications using blood cells, cancer cells, and other mammalian cells, and (2) electrokinetic (primarily dielectrophoretic) methods using both electrode-based and insulative geometries are presented with a view towards pathogen detection, blood fractionation, and cancer cell isolation. The included methods were evaluated based on performance criteria including cell type modeled and used, number of steps/stages, cell viability, and enrichment, efficiency, and/or purity. Major areas for improvement are increasing viability and capture efficiency/purity of directly processed biological samples, as a majority of current studies only process spiked cell lines or pre-diluted/lysed samples. Despite these current challenges, multiple advances have been made in the development of devices for rare cell capture and the subsequent elucidation of new biological phenomena; this article serves to highlight this progress as well as the electrokinetic and non-electrokinetic methods that can potentially be combined to improve performance in future studies.

Stiffness of photocrosslinked RGD-alginate gels regulates adipose progenitor cell behavior.

Adipose progenitor cells (APCs) are widely investigated for soft tissue reconstruction following tumor resection; however, the long-term success of current approaches is still limited. In order to develop clinically relevant therapies, a better understanding of the role of cell-microenvironment interactions in adipose tissue regeneration is essential. In particular, the effect of extracellular matrix (ECM) mechanics on the regenerative capability of APCs remains to be clarified. We have used artificial ECMs based on photocrosslinkable RGD-alginate to investigate the adipogenic and pro-angiogenic potential of 3T3-L1 preadipocytes as a function of matrix stiffness. These hydrogels allowed us to decouple matrix stiffness from changes in adhesion peptide density or extracellular Ca(2+) concentration and provided a physiologically relevant 3D culture context. Our findings suggest that increased matrix rigidity promotes APC self-renewal and angiogenic capacity, whereas, it inhibits adipose differentiation. Collectively, this study advances our understanding of the role of ECM mechanics in adipose tissue formation and vascularization and will aid in the design of efficacious biomaterial scaffolds for adipose tissue engineering applications.

Capture of circulating tumor cells from whole blood of prostate cancer patients using geometrically enhanced differential immunocapture (GEDI) and a prostate-specific antibody.

Geometrically enhanced differential immunocapture (GEDI) and an antibody for prostate-specific membrane antigen (PSMA) are used for high-efficiency and high-purity capture of prostate circulating tumor cells from peripheral whole blood samples of castrate-resistant prostate cancer patients.