Microfluidic Co-culture System Research Articles

Establishment of a novel microfluidic co-culture system for simultaneous analysis of multiple indicators of gefitinib sensitivity in colorectal cancer cells.

The therapeutic effect of gefitinib on colorectal cancer (CRC) is unclear, but it has been reported that stromal cells in the tumor microenvironment may have an impact on drug sensitivity. Herein, we established a microfluidic co-culture system and explored the sensitivity of CRC cells co-cultured with cancer-associated fibroblasts (CAFs) to gefitinib. The system consisted of a multichannel chip and a Petri dish. The chambers in the chip and dish were designed to continuously supply nutrients for long-term cell survival and create chemokine gradients for driving cell invasion without any external equipment. Using this system, the proliferation and invasiveness of cells were simultaneously evaluated by quantifying the area of cells and the migration distance of cells. In addition, the system combined with live cell workstation could evaluate the dynamic drug response of co-cultured cells and track individual cell trajectories in real-time. When CRC cells were co-cultured with CAFs, CAFs promoted CRC cell proliferation and invasion and reduced the sensitivity of cells to gefitinib through the exosomes secreted by CAFs. Furthermore, the cells that migrated out of the chip were collected, and EMT-related markers were determined by immunofluorescent and western blot assays. The results demonstrated that CAFs affected the response of CRC cells to gefitinib by inducing EMT, providing new ideas for further research on the resistance mechanism of gefitinib. This suggests that targeting CAFs or exosomes might be a new approach to enhance CRC sensitivity to gefitinib, and our system could be a novel platform for investigating the crosstalk between tumor cells and CAFs and understanding multiple biological changes of the tumor cells in the tumor microenvironment.

Guiding Hepatic Differentiation of Pluripotent Stem Cells Using 3D Microfluidic Co-Cultures with Human Hepatocytes.

Human pluripotent stem cells (hPSCs) are capable of unlimited proliferation and can undergo differentiation to give rise to cells and tissues of the three primary germ layers. While directing lineage selection of hPSCs has been an active area of research, improving the efficiency of differentiation remains an important objective. In this study, we describe a two-compartment microfluidic device for co-cultivation of adult human hepatocytes and stem cells. Both cell types were cultured in a 3D or spheroid format. Adult hepatocytes remained highly functional in the microfluidic device over the course of 4 weeks and served as a source of instructive paracrine cues to drive hepatic differentiation of stem cells cultured in the neighboring compartment. The differentiation of stem cells was more pronounced in microfluidic co-cultures compared to a standard hepatic differentiation protocol. In addition to improving stem cell differentiation outcomes, the microfluidic co-culture system described here may be used for parsing signals and mechanisms controlling hepatic cell fate.

Modeling of three-dimensional innervated epidermal like-layer in a microfluidic chip-based coculture system

Reconstruction of skin equivalents with physiologically relevant cellular and matrix architecture is indispensable for basic research and industrial applications. As skin-nerve crosstalk is increasingly recognized as a major element of skin physiological pathology, the development of reliable in vitro models to evaluate the selective communication between epidermal keratinocytes and sensory neurons is being demanded. In this study, we present a three-dimensional innervated epidermal keratinocyte layer as a sensory neuron-epidermal keratinocyte co-culture model on a microfluidic chip using the slope-based air-liquid interfacing culture and spatial compartmentalization. Our co-culture model recapitulates a more organized basal-suprabasal stratification, enhanced barrier function, and physiologically relevant anatomical innervation and demonstrated the feasibility of in situ imaging and functional analysis in a cell-type-specific manner, thereby improving the structural and functional limitations of previous coculture models. This system has the potential as an improved surrogate model and platform for biomedical and pharmaceutical research.

Analysis of metabolites and metabolism-mediated biological activity assessment of ginsenosides on microfluidic co-culture system.

In vivo, the complex process of drugs metabolism alters the change in drug composition and determines the final pharmacological properties of oral drugs. Ginsenosides are primary constituents of ginseng, whose pharmacological activities are greatly affected by liver metabolism. However, the predictive power of existing in vitro models is poor due to their inability to mimic the complexity of drug metabolism in vivo. The advance of organs-on-chip-based microfluidics system could provide a new in vitro drug screening platform by recapitulating the metabolic process and pharmacological activity of natural product. In this study, an improved microfluidic device was employed to establish an in vitro co-culture model by culturing multiple cell types in compartmentalized microchambers. Different cell lines were seeded on the device to examine the metabolites of ginsenosides from the hepatocytes in top layer and its resulting efficacy on the tumors in bottom layer. Metabolism dependent drug efficacy of Capecitabine in this system demonstrated the model is validated and controllable. High concentrations of CK, Rh2 (S), and Rg3 (S) ginsenosides showed significant inhibitory effects on two types of tumor cells. In addition, apoptosis detection showed that Rg3 (S) through liver metabolism promoted early apoptosis of tumor cells and displayed better anticancer activity than prodrug. The detected ginsenoside metabolites indicated that some protopanaxadiol saponins were converted into other anticancer aglycones in varying degrees due to orderly de-sugar and oxidation. Ginsenosides exhibited different efficacy on target cells by impacting their viabilities, indicating hepatic metabolism plays an important role in determining ginsenosides efficacy. In conclusion, this microfluidic co-culture system is simple, scalable, and possibly widely applicable in evaluating anticancer activity and metabolism of drug during the early developmental phases of natural product.

A Microfluidic Cell Co-Culture Chip for the Monitoring of Interactions between Macrophages and Fibroblasts

Macrophages and fibroblasts are two types of important cells in wound healing. The development of novel platforms for studying the interrelationship between these two cells is crucial for the exploration of wound-healing mechanisms and drug development. In this study, a microfluidic chip composed of two layers was designed for the co-culturing of these two cells. An air valve was employed to isolate fibroblasts to simulate the wound-healing microenvironment. The confluence rate of fibroblasts in the co-culture system with different macrophages was explored to reflect the role of different macrophages in wound healing. It was demonstrated that M2-type macrophages could promote the activation and migration of fibroblasts and it can be inferred that they could promote the wound-healing process. The proposed microfluidic co-culture system was designed for non-contact cell-cell interactions, which has potential significance for the study of cell-cell interactions in biological processes such as wound healing, tumor microenvironment, and embryonic development.

PDMS 마이크로 스텐실이 내재된 세포공동배양 미세유체 장치 제작

In this paper, a microfluidic co-culture system comprising an embedded polydimethylsiloxane (PDMS) microstencil was fabricated. The fabricated co-culture system has two micro-channels separated with a PDMS microstencil membrane. Master molds for microchannels and stencil membranes were fabricated by photolithography, then used for casting of PDMS devices. The stencil membrane was 10 thick, with holes 10-μm large in diameter. The fabricated system co-cultured two types of cells (HepG2, NIH-3T3 Cells) successfully for seven days. The viability and stability of the cells were verified through LIVE/DEAD® staining and analysis. Additionally, albumin secretion of HepG2 cells was measured for seven days, using an HSA ELISA kit. The measured data were analyzed, to compare the activity of HepG2 cells. Results confirmed that cells can be co-cultured in the fabricated microfluidic system.

Modelling T-cell immunity against hepatitis C virus with liver organoids in a microfluidic coculture system.

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here, we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV non-structural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells.

Microfluidic Neuromuscular Co-culture System for Tracking Cell-to-Cell Transfer and Axonal Transport of Labeled Proteins.

The molecular communication mechanisms within the Motor Neurons (MN) distant axon and its soma, as well as between MN and their neighboring cells and extracellular environment are of keen interest for our understanding of neurodevelopment and neurodegenerative diseases. One tool that has significantly improved our ability to study such processes with high spatiotemporal resolution is microfluidic devices. Here we describe a step-by-step guide to the neuromuscular co-culturing procedure and demonstrate how to track trophic factors transmission from muscle-to-neuron and their transport along the axons.

Nitric Oxide and a Conditioned Medium Affect the Hematopoietic Development in a Microfluidic Mouse Embryonic Stem Cell/OP9 Co-Cultivation System.

A microfluidic co-culture system, consisting of mouse embryonic stem cells (mESCs)/OP9 cells, was evaluated as a platform for studying hematopoietic differentiation mechanisms in vitro. mESC differentiation into blood cells was achieved in a microchannel that had the minimum size necessary to culture cells. The number of generated blood cells increased or decreased based on the nitric oxide (NO) donor or inhibitor used. Conditioned medium from OP9 cell cultures also promoted an increase in the number of blood cells. The number of generated blood cells under normal medium flow conditions was lower than that observed under the static condition. However, when using a conditioned medium, the number of generated blood cells under flow conditions was the same as that observed under the static condition. We conclude that secreted molecules from OP9 cells have a large influence on the differentiation of mESCs into blood cells. This is the first report of a microfluidic mESC/OP9 co-culture system that can contribute to highly detailed hematopoietic research studies by mimicking the cellular environment.

Human dental pulp stem cells exhibit enhanced properties in comparison to human bone marrow stem cells on neurites outgrowth.

Mesenchymal stem cells (MSCs) have the capacity to self-renew and differentiate into specific cell types and are, therefore, key players during tissue repair and regeneration. The use of MSCs for the regeneration of tissues in vivo is increasingly being explored and already constitutes a promising alternative to existing clinical treatments. MSCs also exert paracrine and trophic functions, including the promotion of innervation that plays fundamental roles in regeneration and in restoration of the function of organs. Human bone marrow stem cells (hBMSCs) and human dental pulp stem cells (hDPSCs) have been used in studies that aimed at the repair and/or regeneration of bone or other tissues of the craniofacial complex. However, the capabilities of hBMSCs and hDPSCs to elicit the growth of specific axons in order to reestablish functional innervation of the healing tissues are not known. Here, we compared the neurotrophic effects of hDPSCs and hBMSCs on trigeminal and dorsal root ganglia neurons using microfluidic organs-on-chips devices. We found that hDPSCs express significantly higher levels of neurotrophins than hBMSCs and consequently neurons cocultured with hDPSCs develop longer axons in the microfluidic co-culture system when compared to neurons cocultured with hBMSCs. Moreover, hDPSCs elicited the formation of extensive axonal networks and established close contacts with neurons, a phenomenon not observed in presence of hBMSCs. Taken together, these findings indicate that hDPSCs constitute a superior option for restoring the functionality of damaged craniofacial tissues, as they are able to support and promote extensive trigeminal innervation.

A microfluidic mammary gland coculture model using parallel 3D lumens for studying epithelial-endothelial migration in breast cancer.

In breast cancer development, crosstalk between mammary epithelial cells and neighboring vascular endothelial cells is critical to understanding tumor progression and metastasis, but the mechanisms of this dynamic interplay are not fully understood. Current cell culture platforms do not accurately recapitulate the 3D luminal architecture of mammary gland elements. Here, we present the development of an accessible and scalable microfluidic coculture system that incorporates two parallel 3D luminal structures that mimic vascular endothelial and mammary epithelial cell layers, respectively. This parallel 3D lumen configuration allows investigation of endothelial-epithelial crosstalk and its effects of the comigration of endothelial and epithelial cells into microscale migration ports located between the parallel lumens. We describe the development and application of our platform, demonstrate generation of 3D luminal cell layers for endothelial cells and three different breast cancer cell lines, and quantify their migration profiles based on number of migrated cells, area coverage by migrated cells, and distance traveled by individual migrating cells into the migration ports. Our system enables analysis at the single-cell level, allows simultaneous monitoring of endothelial and epithelial cell migration within a 3D extracellular matrix, and has potential for applications in basic research on cellular crosstalk as well as drug development.

Abstract 958: Tumor-vascular interactions promote STING-driven inflammation in the tumor microenvironment

Abstract The recruitment of T cells following intratumoral administration of Stimulation of Interferon Genes (STING) agonists in the tumor microenvironment (TME) is a critical event in the STING-driven antitumor immune response, a pathway with great relevance in the context of cancer immunotherapy. We have previously demonstrated that LKB1 mutation is associated with suppression of tumor cell STING levels and reduced production of T-cell chemoattractants such as CXCL10 in KRAS-driven non-small cell lung cancer (NSCLC). Consistent with this, immunohistochemical staining of patient samples showed poor infiltration of CD3, CD4, and CD8 T cells into LKB1 negative versus LKB1 intact cancer epithelium, and instead, retention of T-cells in stroma. To examine how LKB1 alters immune cell recruitment in a STING-dependent manner, we used a 3-D microfluidic co-culture system to study interactions between vasculature and tumor spheroids derived from a KRAS/LKB1 mutated (KL) cell line with LKB1 reconstitution +/- STING deletion. To form the vasculature, we co-cultured tumor spheroids with fibroblasts and endothelial cells for 7 days, and identified changes in morphology, cytokine production, and gene expression that occur in co-culture. We first observed that co-culture induced synergistic production of multiple immune cell chemo-attractants such as CXCL10, CCL2, CCL5, and G-CSF. Interestingly, this more physiologic ex vivo tumor model of LKB1 reconstitution revealed particularly strong cooperative production of STING-dependent cytokines such as CXCL10 in the vasculature. Moreover, STING depletion in LKB1 reconstituted tumor cells did not significantly attenuate production of CXCL10 and other cytokines in co-culture, suggesting that tumor/vessel interaction may promote STING activation in the vasculature regardless of cancer cell-intrinsic STING function. Furthermore, although there was no appreciable response after treatment of KL cancer cells with cGAMP based STING agonists, treatment of isolated 3-D vascular networks with cGAMP enhanced vascular permeability and increased production of CXCL10 and CCL5, possibly contributing to defective chemokine gradients that retain T cells near the vasculature. Thus, developing these more complex models that incorporate the vasculature may elucidate important aspects of STING biology and may ultimately aid further development of effective immunotherapies targeting this signaling axis. Citation Format: Marco Campisi, Shriram K. Sundararaman, Shunsuke Kitajima, Valeria Chiono, Roger D. Kamm, David A. Barbie. Tumor-vascular interactions promote STING-driven inflammation in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 958.

Adipose-on-a-chip: a dynamic microphysiological in vitro model of the human adipose for immune-metabolic analysis in type II diabetes.

Infiltration of immune cells into adipose tissue is associated with chronic low-grade inflammation in obese individuals. To better understand the crosstalk between immune cells and adipocytes, in vivo-like in vitro models are required. Conventionally transwell culture plates are used for studying the adipocyte-immune cell interaction; however, the static culture nature of this approach falls short of closely recapitulating the physiological environment. Here we present a compartmentalized microfluidic co-culture system which provides a constant-rate of nutrient supply as well as waste removal, resembling the microvascular networks of the in vivo environment. Human adipocytes and U937 cells were co-cultured in close proximity in an enclosed system. The porous barrier between the adjacent compartments comprises an array of microchannels, which enables paracrine interaction between cells in adjacent compartments and improved perfusion-based long term cell feeding. Human pre-adipocytes were fully differentiated into adipocytes on the chip and remained viable for several weeks. Upon co-culturing with immune cells, adipocytes showed a tendency to develop insulin resistance. The immune-metabolic correlation has been studied by monitoring adiponectin and IL-6 expression, as well as glucose uptake upon treatment with insulin. Our microfluidic system can be potentially used to develop physiologically relevant adipose tissue models to study obesity-associated diseases such as insulin resistance and type 2 diabetes and therefore, facilitate drug development to treat these diseases.

Microfluidic Coculture Device for Monitoring of Inflammation-Induced Myocardial Injury Dynamics.

Emerging awareness of cardiac macrophages' role in inflammation after myocardial infarction indicates that overabundant proinflammatory macrophages induce accentuated myocardial injury. The investigation of the macrophages-cardiomyocytes interaction and inflammation-induced dynamic damage in myocardial infarction, especially in a spatiotemporally controlled manner, remains a huge challenge. Here, we developed an in vitro model using a microfluidic coculture system to mimic inflammatory cardiac injury. To our knowledge, on-chip pathological models focused on inflammation-induced myocardial injury have not been reported. The device consists of two sets of thin interconnecting grooves that isolate heterogeneous cells spatially but maintain their soluble factors communication. The mass transportation is visually characterized, and the complete diffusion reaches equilibrium within 100 s. We investigate the dynamic interaction between the macrophages and the cardiomyocytes in the spatiotemporal controlled microenvironment, mimicking a key aspect of the in vivo pathophysiological process. The results show that the activated macrophages induce time-lapsed apoptotic responses of the cardiac cells and damage mitochondria membrane integrity. The anti-inflammatory and cardio-protective effects of quercetin were explored on the chip. The extent of caspase-3 activation is asynchronous in the individual cardiac cells, suggesting the different apoptosis dynamics. We further demonstrate that the mechanism of activated inflammation is associated with the upregulation of several inflammatory cytokines and NF-κB pathway. Thus, the developed microfluidic coculture device provides a useful tool for real-time monitoring of inflammatory response for myocardial disease and holds potential for anti-inflammatory drug screening.

Dorsal root ganglion neurons regulate the transcriptional and translational programs of osteoblast differentiation in a microfluidic platform

Innervation by the sensory nervous system plays a key role in skeletal development and in orchestration of bone remodeling and regeneration. However, it is unclear how and in which bone cells can sensory nerves act to control these processes. Here, we show a microfluidic coculture system comprising dorsal root ganglion (DRG) neurons and mesenchymal stem cells (MSCs) that more faithfully represents the in vivo scenario of bone sensory innervation. We report that DRG neurons promote the osteogenic differentiation capacity of MSCs, by mediating the increase of alkaline phosphatase activity and the upregulation of osteoblast-specific genes. Furthermore, we show that DRG neurons have a positive impact on Cx43 levels in MSCs during osteoblastogenesis, especially at an early stage of this process. Conversely, we described a negative impact of DRG neurons on MSCs N-cadherin expression at a later stage. Finally, we demonstrate a cytoplasmic accumulation of β-catenin translocation into the nucleus, and subsequently Lymphoid Enhancer Binding Factor 1—responsive transcriptional activation of downstream genes in cocultured MSCs. Together, our study provides a robust body of evidence that the direct interaction of DRG neurons with MSCs in a bone-like microenvironment leads to an enhancement of osteoblast differentiation potential of MSCs. The osteogenic effect of DRG neurons on MSCs is mediated through the regulation of Cx43 and N-cadherin expression and activation of the canonical/β-catenin Wnt signaling pathway.

Ductular reaction-on-a-chip: Microfluidic co-cultures to study stem cell fate selection during liver injury.

Liver injury modulates local microenvironment, triggering production of signals that instruct stem cell fate choices. In this study, we employed a microfluidic co-culture system to recreate important interactions in the liver stem cell niche, those between adult hepatocytes and liver progenitor cells (LPCs). We demonstrate that pluripotent stem cell-derived LPCs choose hepatic fate when cultured next to healthy hepatocytes but begin biliary differentiation program when co-cultured with injured hepatocytes. We connect this fate selection to skewing in production of hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β1 caused by injury. Significantly, biliary fate selection of LPCs was not observed in the absence of hepatocytes nor did it happen in the presence of TGF-β inhibitors. Our study demonstrates that microfluidic culture systems may offer an interesting new tool for dissecting cellular interactions leading to aberrant stem cell differentiation during injury.

Microfluidic co-culture system for cancer migratory analysis and anti-metastatic drugs screening.

Tumour metastasis is an important reason for cancer death, and cancer cell migration is an important step in the process of tumour metastasis. Studying cancer cell migration is of great significance. Here, we present a novel microfluidic co-culture system and establish mild, moderate and severe cancer models by using HMEpiC and MDA-MB–231 cells to study cancer cell migration and anti-cancer drug screening. Using this device, we achieved high cell viability (over 90%) and a stable analysis of the migration ability of cancer cells. We observed that the density of the cancer cells determined the probability of the occurrence of metastatic cells and that the induction of normal cells affected the metastatic velocity of each cancer cell. We verified that the increase in the migration ability of MDA-MB-231 cells co-cultured with HMEpiC cells was relative to the increased secretion of IL-6 and that this was verified by an IL-6 inhibitor assay. This co-culture also led to decreased CK-14 secretion and morphological changes in HMEpiC cells. Finally, significant inhibition of paclitaxel and tamoxifen on cancer migration was observed. Taken together, our microfluidic device could be a useful tool for the quantitation of the migratory capability and anti-metastatic drug screening.

Microfluidic co-culture system model to study the interplay between sensory neurons and mesenchymal stem cells: new clues towards osteogenesis

Event Abstract Back to Event Microfluidic co-culture system model to study the interplay between sensory neurons and mesenchymal stem cells: new clues towards osteogenesis Hugo Oliveira1*, Bruno P. Dos Santos1*, Diana I. Silva1*, Jacques Leng2* and Joelle Amedee1* 1 Inserm U1026, Université Bordeaux Segalen, BioIngénierie Tissulaire, France 2 UMR 5258 CNRS/SOLVAY/Univ. BORDEAUX, LaboratoireduFutur, France Introduction: The existence of nerve fibers in bone and the expression of neural factors in close vicinity to bone cells have been reported. Neuron-derived molecules have demonstrated to play a role on bone turnover [1] and accumulating evidence has revealed the importance of the sensory nervous system in the orchestration of bone repair [2]. The aim of this work is to establish a co-culture system between sensorial neurons and mesenchymal stem cells (MSC) and to study the cellular interplay, between these cells, in view of osteogenesis. Materials and Methods: In order to allow the establishment of co-culture between rat dorsal root ganglia (DRG) sensory neurons and rat bone marrow MSCs we fabricated PDMS microfluidic chambers, using standard photolithography techniques. This approach allows to physically separate cellular bodies from the distinct cell types, to use two different cell culture medias and to some extent to mimic the in vivo physiological environment. The primary cultures were co-cultured during 7 and 14 days in microfluidics system using DMEM (high glucose) with 2% B27 medium or DMEM with 10%FBS medium for DRGs and MSCs, respectively. At designated time points (i.e. 7 and 14 days), metabolic activity, immunostaining and gene expression profiles (qPCR) were performed. Results: DRGs were able to spread neurites through microchannels toward MSCs (Figure 1). Figure 1. Co-culture microfluidic system. DRG neurons and MSCs are cultured separately but cells can interact through channels. Immunocytochemistry of DRG neurons (anti-beta III tubulin, green) and MSCs (actin, red) co-cultures at 7 and 14 days of culture. MSCs metabolic activity was not influenced by the co-culture with DRGs. MSCs in co-culture conditions shown an upregulation of the osteogenic markers Runx2, Col1 and Sp7 (Osx). Additionally, in view of further evaluate the mechanism involved in this regulation we assessed the gene expression of SMAD1 and Ctnnb1 (beta-catenin) (Figure 2). Figure 2. Quantitative gene expression of Runx2, collagen type I (Col1), osterix (osx), beta cathenin (Bcath) and Smad 1 of MSCs in mono and co-culture with sensory neurons, at 7 and 14 days of culture (Aver±SD, n=3, * denotes p <0.05). Discussion: We show that that sensorial neurons can closely interact with MSCs and modulate their phenotype toward the osteogenic lineage. Further studies are underway in order to further understand the mechanism underlying this cellular communication. Conclusion: These co-culture systems will allow the broadening of current knowledge regarding the impact of the sensorial nervous system in bone remodeling and repair, and can open new avenues for the establishment of new strategies for bone tissue regeneration.

A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions.

ABSTRACTBidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF – facilitating growth and muscle innervation at axon terminals and survival pathways in the soma.

Automated co-culture system for spatiotemporal analysis of cell-to-cell communication.

We present a microfluidic co-culture system that generates localized and precisely formulated immune signals among a population of cells, enabling spatiotemporal analysis of paracrine signal transmission between different cell types. The automated system allows us to create temporally modulated chemical inputs that can be delivered to single signal-transmitting and receiving cells in a highly controlled way. Using this system we stimulated a single macrophage with brief pulses of bacterial LPS and observed the macrophage transmitted TNF signal propagating in a population of fibroblasts via NF-κB activation. The signal receiving fibroblasts transformed the TNF signal into a spatiotemporally distributed NF-κB output, recapitulating the initiation of immune response to bacterial infection.