Daniel Irimia | Harvard University (original) (raw)
Papers by Daniel Irimia
Proceedings of the National Academy of Sciences of the United States of America, Jan 13, 2016
Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations ... more Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations associated with low chemical concentrations and dynamically changing gradients as well as high saturating chemical concentrations. Although a number of reports have identified cellular behavior consistent with a directional memory that could account for behavior in these complex environments, the quantitative and molecular details of such a memory process remain unknown. Using microfluidics to confine cellular motion to a 1D channel and control chemoattractant exposure, we observed directional memory in chemotactic neutrophil-like cells. We modeled this directional memory as a long-lived intracellular asymmetry that decays slower than observed membrane phospholipid signaling. Measurements of intracellular dynamics revealed that moesin at the cell rear is a long-lived element that when inhibited, results in a reduction of memory. Inhibition of ROCK (Rho-associated protein kinase), downstr...
Langmuir : the ACS journal of surfaces and colloids, Jan 6, 2015
Cell-adhesive particles are of significant interest in the biotechnologies, bioengineering of com... more Cell-adhesive particles are of significant interest in the biotechnologies, bioengineering of complex tissues, and biomedical research. Their applications range from platforms to increase the efficiency of anchorage dependent cell culture to building blocks to load cells in heterogeneous structures and from clonal-population growth monitoring to cell sorting. Although useful, currently available cell-adhesive particles can only accommodate homogenous cell culture. Here, we report the design of anisotropic hydrogel microparticles with tunable cell-adhesive regions, as first step towards micropatterned cell cultures on particles. We employed stop flow lithography (SFL), coupling reaction between amine and N-hyroxysuccinimide (NHS), and streptavidin-biotin chemistry, to adjust the localization of conjugated collagen and poly-L-lysine on the surface of microscale particles. Using the new particles, we demonstrate the attachment and formation of tight junctions between brain endothelial ...
Pediatric nephrology (Berlin, Germany), Jan 24, 2015
Advances in therapeutics have dramatically improved short-term graft survival, but the incidence ... more Advances in therapeutics have dramatically improved short-term graft survival, but the incidence of chronic rejection has not changed in the past 20 years. New insights into mechanism are sorely needed at this time and it is hoped that the development of predictive biomarkers will pave the way for the emergence of preventative therapeutics. In this review, we discuss a paradigm suggesting that sequential changes within graft endothelial cells (EC) lead to an intragraft microenvironment that favors the development of chronic rejection. Key initial events include EC injury, activation and uncontrolled leukocyte-induced angiogenesis. We propose that all of these early changes in the microvasculature lead to abnormal blood flow patterns, local tissue hypoxia, and an associated overexpression of HIF-1α-inducible genes, including vascular endothelial growth factor. We also discuss how cell intrinsic regulators of mTOR-mediated signaling within EC are of critical importance in microvascula...
Pnas, 2010
Gene expression signatures are used in the clinic as prognostic tools to determine the risk of in... more Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different "poor-outcome" cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by "poor-prognosis" signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.
Scientific reports, 2015
Blood-brain barrier (BBB) pathology leads to neurovascular disorders and is an important target f... more Blood-brain barrier (BBB) pathology leads to neurovascular disorders and is an important target for therapies. However, the study of BBB pathology is difficult in the absence of models that are simple and relevant. In vivo animal models are highly relevant, however they are hampered by complex, multi-cellular interactions that are difficult to decouple. In vitro models of BBB are simpler, however they have limited functionality and relevance to disease processes. To address these limitations, we developed a 3-dimensional (3D) model of BBB on a microfluidic platform. We verified the tightness of the BBB by showing its ability to reduce the leakage of dyes and to block the transmigration of immune cells towards chemoattractants. Moreover, we verified the localization at endothelial cell boundaries of ZO-1 and VE-Cadherin, two components of tight and adherens junctions. To validate the functionality of the BBB model, we probed its disruption by neuro-inflammation mediators and ischemic...
Nature communications, 2015
Tau pathology is known to spread in a hierarchical pattern in Alzheimer's disease (AD) brain ... more Tau pathology is known to spread in a hierarchical pattern in Alzheimer's disease (AD) brain during disease progression, likely by trans-synaptic tau transfer between neurons. However, the tau species involved in inter-neuron propagation remains unclear. To identify tau species responsible for propagation, we examined uptake and propagation properties of different tau species derived from postmortem cortical extracts and brain interstitial fluid of tau-transgenic mice, as well as human AD cortices. Here we show that PBS-soluble phosphorylated high-molecular-weight (HMW) tau, though very low in abundance, is taken up, axonally transported, and passed on to synaptically connected neurons. Our findings suggest that a rare species of soluble phosphorylated HMW tau is the endogenous form of tau involved in propagation and could be a target for therapeutic intervention and biomarker development.
The Journal of infectious diseases, Jan 12, 2015
The contribution of human neutrophils to the protection against fungal infections by Aspergillus ... more The contribution of human neutrophils to the protection against fungal infections by Aspergillus fumigatus is essential but not fully understood. Whereas healthy people can inhale spores of A. fumigatus without developing disease, neutropenic patients and those taking immunosuppressive drugs have a higher incidence of invasive fungal infections. To study the role of neutrophils in protection against A. fumigatus infections, we developed an in vitro assay where the interactions between human neutrophils and A. fumigatus were observed in real time, at single cell resolution, in precisely controlled conditions. We measured the outcomes of neutrophil-fungus interactions and found that human neutrophils have limited ability to migrate towards A. fumigatus and block the growth of 69% of A. fumigatus conidia. Human neutrophil blocking ability increased to 85.1% when stimulated by uniform concentrations of fMLP, and was enhanced further, to 99.4% , in the presence of chemoattractant gradien...
Neutrophils play a key role in the inflammatory response and the defense of the host against inva... more Neutrophils play a key role in the inflammatory response and the defense of the host against invading pathogens like bacteria and fungi. They possess the remarkable ability to undergo directional migration in response to spatial and temporal changes in gradients of chemokinetic agents in their environment, which allows them to localize to site of tissue injury. We were interested in characterizing primary human neutrophil population responses and quantifying response times of these cells to changes in chemokine levels in vitro. Traditional tools such as the Boyden chamber do not fully capture the complexity of neutrophil responses because they generate unstable chemoattractant gradients and cannot be manipulated to create precise temporal changes. We employed a novel microfluidics system in conjunction with time-lapse video-microscopy to study neutrophil migratory responses to rapid changes between two linear gradients of the chemokine, interleukin-8 (IL-8). We described the behavio...
Lab on a chip, 2015
Appropriate inflammatory responses to wounds and infections require adequate numbers of neutrophi... more Appropriate inflammatory responses to wounds and infections require adequate numbers of neutrophils arriving at injury sites. Both insufficient and excessive neutrophil recruitment can be detrimental, favouring systemic spread of microbes or triggering severe tissue damage. Despite its importance in health and disease, the trafficking of neutrophils through tissues remains difficult to control and the mechanisms regulating it are insufficiently understood. These mechanisms are also complex and difficult to isolate using traditional in vivo models. Here we designed a microfluidic model of tissue infection/inflammation, in which human neutrophils emerge from a droplet-size samples of whole blood and display bi-directional traffic between this and micro-chambers containing chemoattractant and microbe-like particles. Two geometrical barriers restrict the entrance of red blood cells from the blood to the micro-chambers and simulate the mechanical function of the endothelial barrier separ...
A simple technique for controlling cell adhesion on glass substrates by surface modi®cation using... more A simple technique for controlling cell adhesion on glass substrates by surface modi®cation using a commercially available poly(ethylene glycol) (PEG) disilane, which can bind directly to glass in a single step, in combination with photolithographic micropatterning, was developed, characterized, and evaluated for patterning of HepG2 hepatoma cells and 3T3 ®broblasts. The optimal concentration of PEG-disilane for surface modi®cation was 5 mM, and patterning of strongly adherent cells such as HepG2 required the chelation of divalent metal cations in order to inhibit nonspeci®c binding and cell aggregation. Whereas the average thickness of the PEG-disilane layer was 18 + 3.5 nm, the perimeters of patterned areas of exposed glass exhibited ridges of average height 857 + 50 nm, which may have aided in constraining cell spreading and migration. Although unpatterned PEG-treated substrates were hydrophilic (contact angle 46 + 1 ), micropatterned surfaces behaved as if they were somewhat hydrophobic (contact angle * 90 ), necessitating special protocols for preventing deleterious dewetting of cells. For optimized protocols, the probability of adhesion of HepG2 cells to a patterned area of exposed glass was almost 15 times higher than the probability of adhesion to a PEG-treated background region of equal area. Our technique is useful for short-to intermediate-term patterning of cell or tissue morphology, e.g., for investigation of the effects of cell±cell interactions or cell geometry.
TECHNOLOGY, 2014
We combined microfluidic tools and molecular probes to monitor the migration speed of successive ... more We combined microfluidic tools and molecular probes to monitor the migration speed of successive generations of cancer cells. We found that the migratory speed of individual cells changes stochastically from parent cells to their descendants, while the average speed of successive generations of cells remains constant. Further studies of the interrelations between cell migration and division processes may help identify the molecular determinants of cell speed and lead to new therapies to slow the invasion of cancer cells and delay metastases.
Methods in Cell Biology, 2014
We describe a protocol for measuring the speed of human neutrophils migrating through small chann... more We describe a protocol for measuring the speed of human neutrophils migrating through small channels, in conditions of mechanical confinement comparable to those experienced by neutrophils migrating through tissues. In such conditions, we find that neutrophils move persistently, at constant speed for tens of minutes, enabling precise measurements at single cells resolution, for large number of cells. The protocol relies on microfluidic devices with small channels in which a solution of chemoattractant and a suspension of isolated neutrophils are loaded in sequence. The migration of neutrophils can be observed for several hours, starting within minutes after loading the neutrophils in the devices. The protocol is divided into four main steps: the fabrication of the microfluidic devices, the separation of neutrophils from whole blood, the preparation of the assay and cell loading, and the analysis of data. We discuss the practical steps for the implementation of the migration assays in biology labs, the adaptation of the protocols to various cell types, including cancer cells, and the supplementary device features required for precise measurements of directionality and persistence during migration.
Advances in Bioengineering, 2002
ABSTRACT
Encyclopedia of Microfluidics and Nanofluidics, 2013
Proceedings of the National Academy of Sciences of the United States of America, Jan 13, 2016
Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations ... more Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations associated with low chemical concentrations and dynamically changing gradients as well as high saturating chemical concentrations. Although a number of reports have identified cellular behavior consistent with a directional memory that could account for behavior in these complex environments, the quantitative and molecular details of such a memory process remain unknown. Using microfluidics to confine cellular motion to a 1D channel and control chemoattractant exposure, we observed directional memory in chemotactic neutrophil-like cells. We modeled this directional memory as a long-lived intracellular asymmetry that decays slower than observed membrane phospholipid signaling. Measurements of intracellular dynamics revealed that moesin at the cell rear is a long-lived element that when inhibited, results in a reduction of memory. Inhibition of ROCK (Rho-associated protein kinase), downstr...
Langmuir : the ACS journal of surfaces and colloids, Jan 6, 2015
Cell-adhesive particles are of significant interest in the biotechnologies, bioengineering of com... more Cell-adhesive particles are of significant interest in the biotechnologies, bioengineering of complex tissues, and biomedical research. Their applications range from platforms to increase the efficiency of anchorage dependent cell culture to building blocks to load cells in heterogeneous structures and from clonal-population growth monitoring to cell sorting. Although useful, currently available cell-adhesive particles can only accommodate homogenous cell culture. Here, we report the design of anisotropic hydrogel microparticles with tunable cell-adhesive regions, as first step towards micropatterned cell cultures on particles. We employed stop flow lithography (SFL), coupling reaction between amine and N-hyroxysuccinimide (NHS), and streptavidin-biotin chemistry, to adjust the localization of conjugated collagen and poly-L-lysine on the surface of microscale particles. Using the new particles, we demonstrate the attachment and formation of tight junctions between brain endothelial ...
Pediatric nephrology (Berlin, Germany), Jan 24, 2015
Advances in therapeutics have dramatically improved short-term graft survival, but the incidence ... more Advances in therapeutics have dramatically improved short-term graft survival, but the incidence of chronic rejection has not changed in the past 20 years. New insights into mechanism are sorely needed at this time and it is hoped that the development of predictive biomarkers will pave the way for the emergence of preventative therapeutics. In this review, we discuss a paradigm suggesting that sequential changes within graft endothelial cells (EC) lead to an intragraft microenvironment that favors the development of chronic rejection. Key initial events include EC injury, activation and uncontrolled leukocyte-induced angiogenesis. We propose that all of these early changes in the microvasculature lead to abnormal blood flow patterns, local tissue hypoxia, and an associated overexpression of HIF-1α-inducible genes, including vascular endothelial growth factor. We also discuss how cell intrinsic regulators of mTOR-mediated signaling within EC are of critical importance in microvascula...
Pnas, 2010
Gene expression signatures are used in the clinic as prognostic tools to determine the risk of in... more Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different "poor-outcome" cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by "poor-prognosis" signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.
Scientific reports, 2015
Blood-brain barrier (BBB) pathology leads to neurovascular disorders and is an important target f... more Blood-brain barrier (BBB) pathology leads to neurovascular disorders and is an important target for therapies. However, the study of BBB pathology is difficult in the absence of models that are simple and relevant. In vivo animal models are highly relevant, however they are hampered by complex, multi-cellular interactions that are difficult to decouple. In vitro models of BBB are simpler, however they have limited functionality and relevance to disease processes. To address these limitations, we developed a 3-dimensional (3D) model of BBB on a microfluidic platform. We verified the tightness of the BBB by showing its ability to reduce the leakage of dyes and to block the transmigration of immune cells towards chemoattractants. Moreover, we verified the localization at endothelial cell boundaries of ZO-1 and VE-Cadherin, two components of tight and adherens junctions. To validate the functionality of the BBB model, we probed its disruption by neuro-inflammation mediators and ischemic...
Nature communications, 2015
Tau pathology is known to spread in a hierarchical pattern in Alzheimer's disease (AD) brain ... more Tau pathology is known to spread in a hierarchical pattern in Alzheimer's disease (AD) brain during disease progression, likely by trans-synaptic tau transfer between neurons. However, the tau species involved in inter-neuron propagation remains unclear. To identify tau species responsible for propagation, we examined uptake and propagation properties of different tau species derived from postmortem cortical extracts and brain interstitial fluid of tau-transgenic mice, as well as human AD cortices. Here we show that PBS-soluble phosphorylated high-molecular-weight (HMW) tau, though very low in abundance, is taken up, axonally transported, and passed on to synaptically connected neurons. Our findings suggest that a rare species of soluble phosphorylated HMW tau is the endogenous form of tau involved in propagation and could be a target for therapeutic intervention and biomarker development.
The Journal of infectious diseases, Jan 12, 2015
The contribution of human neutrophils to the protection against fungal infections by Aspergillus ... more The contribution of human neutrophils to the protection against fungal infections by Aspergillus fumigatus is essential but not fully understood. Whereas healthy people can inhale spores of A. fumigatus without developing disease, neutropenic patients and those taking immunosuppressive drugs have a higher incidence of invasive fungal infections. To study the role of neutrophils in protection against A. fumigatus infections, we developed an in vitro assay where the interactions between human neutrophils and A. fumigatus were observed in real time, at single cell resolution, in precisely controlled conditions. We measured the outcomes of neutrophil-fungus interactions and found that human neutrophils have limited ability to migrate towards A. fumigatus and block the growth of 69% of A. fumigatus conidia. Human neutrophil blocking ability increased to 85.1% when stimulated by uniform concentrations of fMLP, and was enhanced further, to 99.4% , in the presence of chemoattractant gradien...
Neutrophils play a key role in the inflammatory response and the defense of the host against inva... more Neutrophils play a key role in the inflammatory response and the defense of the host against invading pathogens like bacteria and fungi. They possess the remarkable ability to undergo directional migration in response to spatial and temporal changes in gradients of chemokinetic agents in their environment, which allows them to localize to site of tissue injury. We were interested in characterizing primary human neutrophil population responses and quantifying response times of these cells to changes in chemokine levels in vitro. Traditional tools such as the Boyden chamber do not fully capture the complexity of neutrophil responses because they generate unstable chemoattractant gradients and cannot be manipulated to create precise temporal changes. We employed a novel microfluidics system in conjunction with time-lapse video-microscopy to study neutrophil migratory responses to rapid changes between two linear gradients of the chemokine, interleukin-8 (IL-8). We described the behavio...
Lab on a chip, 2015
Appropriate inflammatory responses to wounds and infections require adequate numbers of neutrophi... more Appropriate inflammatory responses to wounds and infections require adequate numbers of neutrophils arriving at injury sites. Both insufficient and excessive neutrophil recruitment can be detrimental, favouring systemic spread of microbes or triggering severe tissue damage. Despite its importance in health and disease, the trafficking of neutrophils through tissues remains difficult to control and the mechanisms regulating it are insufficiently understood. These mechanisms are also complex and difficult to isolate using traditional in vivo models. Here we designed a microfluidic model of tissue infection/inflammation, in which human neutrophils emerge from a droplet-size samples of whole blood and display bi-directional traffic between this and micro-chambers containing chemoattractant and microbe-like particles. Two geometrical barriers restrict the entrance of red blood cells from the blood to the micro-chambers and simulate the mechanical function of the endothelial barrier separ...
A simple technique for controlling cell adhesion on glass substrates by surface modi®cation using... more A simple technique for controlling cell adhesion on glass substrates by surface modi®cation using a commercially available poly(ethylene glycol) (PEG) disilane, which can bind directly to glass in a single step, in combination with photolithographic micropatterning, was developed, characterized, and evaluated for patterning of HepG2 hepatoma cells and 3T3 ®broblasts. The optimal concentration of PEG-disilane for surface modi®cation was 5 mM, and patterning of strongly adherent cells such as HepG2 required the chelation of divalent metal cations in order to inhibit nonspeci®c binding and cell aggregation. Whereas the average thickness of the PEG-disilane layer was 18 + 3.5 nm, the perimeters of patterned areas of exposed glass exhibited ridges of average height 857 + 50 nm, which may have aided in constraining cell spreading and migration. Although unpatterned PEG-treated substrates were hydrophilic (contact angle 46 + 1 ), micropatterned surfaces behaved as if they were somewhat hydrophobic (contact angle * 90 ), necessitating special protocols for preventing deleterious dewetting of cells. For optimized protocols, the probability of adhesion of HepG2 cells to a patterned area of exposed glass was almost 15 times higher than the probability of adhesion to a PEG-treated background region of equal area. Our technique is useful for short-to intermediate-term patterning of cell or tissue morphology, e.g., for investigation of the effects of cell±cell interactions or cell geometry.
TECHNOLOGY, 2014
We combined microfluidic tools and molecular probes to monitor the migration speed of successive ... more We combined microfluidic tools and molecular probes to monitor the migration speed of successive generations of cancer cells. We found that the migratory speed of individual cells changes stochastically from parent cells to their descendants, while the average speed of successive generations of cells remains constant. Further studies of the interrelations between cell migration and division processes may help identify the molecular determinants of cell speed and lead to new therapies to slow the invasion of cancer cells and delay metastases.
Methods in Cell Biology, 2014
We describe a protocol for measuring the speed of human neutrophils migrating through small chann... more We describe a protocol for measuring the speed of human neutrophils migrating through small channels, in conditions of mechanical confinement comparable to those experienced by neutrophils migrating through tissues. In such conditions, we find that neutrophils move persistently, at constant speed for tens of minutes, enabling precise measurements at single cells resolution, for large number of cells. The protocol relies on microfluidic devices with small channels in which a solution of chemoattractant and a suspension of isolated neutrophils are loaded in sequence. The migration of neutrophils can be observed for several hours, starting within minutes after loading the neutrophils in the devices. The protocol is divided into four main steps: the fabrication of the microfluidic devices, the separation of neutrophils from whole blood, the preparation of the assay and cell loading, and the analysis of data. We discuss the practical steps for the implementation of the migration assays in biology labs, the adaptation of the protocols to various cell types, including cancer cells, and the supplementary device features required for precise measurements of directionality and persistence during migration.
Advances in Bioengineering, 2002
ABSTRACT
Encyclopedia of Microfluidics and Nanofluidics, 2013