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The long-term function of transplanted therapeutic cells typically requires systemic immune suppression. Here, we show that a retrievable implant comprising a silicone reservoir and a porous polymeric membrane protects human cells encapsulated in it after implant transplantation in the intraperitoneal space of immunocompetent mice. Membranes with pores 1 µm in diameter allowed host macrophages to migrate into the device without the loss of transplanted cells, whereas membranes with pore sizes 130 days, the device supported human cells engineered to secrete erythropoietin in immunocompetent mice, as well as transgenic human cells carrying an inducible gene circuit for the on-demand secretion of erythropoietin. Pancreatic islets from rats encapsulated in the device and implanted in diabetic mice restored normoglycaemia in the mice for over 75 days. The biocompatible device provides a retrievable solution for the transplantation of engineered cells in the absence of immunosuppression. A retrievable, porous and anti-fibrotic macrodevice for the encapsulation of cells provides long-term protection to human cells expressing therapeutic proteins after device implantation in the intraperitoneal space of immunocompetent mice.

Naïve helper (CD4+) T-cells can differentiate into distinct functional subsets including Th1, Th2, and Th17 phenotypes. Each of these phenotypes has a ‘master regulator’ – T-bet (Th1), GATA3 (Th2) and RORγT (Th17) – that inhibits the other two master regulators. Such mutual repression among them at a transcriptional level can enable multistability, giving rise to six experimentally observed phenotypes – Th1, Th2, Th17, hybrid Th/Th2, hybrid Th2/Th17 and hybrid Th1/Th17. However, the dynamics of switching among these phenotypes, particularly in the case of epigenetic influence, remains unclear. Here, through mathematical modeling, we investigated the coupled transcription-epigenetic dynamics in a three-node mutually repressing network to elucidate how epigenetic changes mediated by any ‘master regulator’ can influence the transition rates among different cellular phenotypes. We show that the degree of plasticity exhibited by one phenotype depends on relative strength and duration of mutual epigenetic repression mediated among the master regulators in a three-node network. Further, our model predictions can offer putative mechanisms underlying relatively higher plasticity of Th17 phenotype as observed in vitro and in vivo. Together, our modeling framework characterizes phenotypic plasticity and heterogeneity as an outcome of emergent dynamics of a three-node regulatory network, such as the one mediated by T-bet/GATA3/RORγT.

Humans are exposed to numerous synthetic foreign particulates in the form of environmental pollutants and diagnostic or therapeutic agents. Specialized immune cells (phagocytes) clear these particulates by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particulates may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize E. coli faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and S. epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable particulates.

Neutrophils play a crucial role in establishing inflammation in response to an infection or injury, but their production rates, as well as blood and tissue residence times, remain poorly characterized under these conditions. Herein, using a biomaterial implant model to establish inflammation followed byin vivotracking of newly formed neutrophils, we determine neutrophil kinetics under inflammatory conditions. To obtain quantifiable information from our experimental observations, we develop an ordinary differential equation-based mathematical model to extract kinetic parameters. Our data show that in the presence of inflammation resulting in emergency granulopoiesis-like conditions, neutrophil maturation time in the bone marrow and half-life in the blood reduces by about 40%, compared to non-inflammatory conditions. Additionally, neutrophil residence time at the inflammatory site increases by two-fold. Together, these data improve our understanding of neutrophil kinetics under inflammatory conditions, which could pave the way for therapies that focus on modulating in vivo neutrophil dynamics.

Identifying the design principles of complex regulatory networks driving cellular decision-making remains essential to decode embryonic development as well as enhance cellular reprogramming. A well-studied network motif involved in cellular decision-making is a toggle switch—a set of two opposing transcription factors A and B, each of which is a master regulator of a specific cell fate and can inhibit the activity of the other. A toggle switch can lead to two possible states—(high A, low B) and (low A, high B)—and drives the ‘either-or' choice between these two cell fates for a common progenitor cell. However, the principles of coupled toggle switches remain unclear. Here, we investigate the dynamics of three master regulators A, B and C inhibiting each other, thus forming three-coupled toggle switches to form a toggle triad. Our simulations show that this toggle triad can lead to co-existence of cells into three differentiated ‘single positive' phenotypes—(high A, low B, low C), (low A, high B, low C) and (low A, low B, high C). Moreover, the hybrid or ‘double positive' phenotypes—(high A, high B, low C), (low A, high B, high C) and (high A, low B, high C)—can coexist together with ‘single positive' phenotypes. Including self-activation loops on A, B and C can increase the frequency of ‘double positive' states. Finally, we apply our results to understand cellular decision-making in terms of differentiation of naive CD4 + T cells into Th1, Th2 and Th17 states, where hybrid Th1/Th2 and hybrid Th1/Th17 cells have been reported in addition to the Th1, Th2 and Th17 ones. Our results offer novel insights into the design principles of a multi-stable network topology and provide a framework for synthetic biology to design tristable systems.

The effects of senescence and aging on geriatric diseases has been well explored but how these influence infections in the elderly have been scarcely addressed. Here, we show that several innate immune responses are elevated in senescent cells and old mice, allowing them to promptly respond to bacterial infections. We have identified higher levels of iNOS as a crucial host response and show that p38 MAPK in senescent cells acts as a negative regulator of iNOS transcription. In old mice, however the ability to impede bacterial proliferation does not correlate with increased survival as elevated immune responses persist unabated eventually affecting the host. The use of anti-inflammatory drugs that could consequently be recommended also decreases iNOS disarming the host of a critical innate immune response. Overall, our study highlights that infection associated mortality in the elderly is not merely an outcome of pathogen load but is also influenced by the host’s ability to resolve inflammation induced damage. Summary statement Using cellular models and old mice we demonstrate the effect of aging on host response to bacterial infections. Aged systems mount a more effective anti-bacterial innate immune response but its persistence results in mortality of the host.

Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression. By studying the immune responses of animals to different types of biomaterial implants, colony stimulating factor-1 receptor is revealed as an important mediator of the foreign body reaction and a possible target for fibrosis inhibition.

Neutrophils are constantly generated from hematopoietic stem and progenitor cells in the bone marrow to maintain high numbers in circulation. A considerable number of neutrophils and their progenitors have been shown to be present in the spleen too; however, their exact role in this organ remains unclear. Herein, we sought to study the function of splenic neutrophils and their progenitors using a mouse model for sterile, peritoneal inflammation. In this microcapsule device implantation model, we show chronic neutrophil presence at implant sites, with recruitment from circulation as the primary mechanism for their prevalence in the peritoneal exudate. Furthermore, we demonstrate that progenitor populations in the spleen play a key role in maintaining elevated neutrophil numbers. Our results provide new insight into the role for splenic neutrophils and their progenitors and establish a model to study neutrophil function during sterile inflammation.

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. Although several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate mitochondrial fission or fusion remain largely unknown. In fission yeast (Schizosaccharomyces pombe), mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics and used high-resolution and time-lapse fluorescence microscopy, revealing that mitochondrial lengths mimic microtubule lengths. Furthermore, we determined that compared with WT cells, mutant cells with long microtubules exhibit fewer mitochondria, and mutant cells with short microtubules have an increased number of mitochondria because of reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we observed increased mitochondrial fission. We found that the consequent rise in the mitochondrial copy number is necessary to reduce partitioning errors during independent segregation of mitochondria between daughter cells. We also discovered that the association of mitochondria with microtubules physically impedes the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a mechanism for the regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton. © 2019 Mehta et al.

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. While several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate fission or fusion remain largely unknown. In fission yeast, mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics, and determined that cells with long microtubules exhibited long, but fewer mitochondria, whereas cells with short microtubules exhibited short, but several mitochondria due to reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we measured increased mitochondrial fission. We determined that the consequent rise in the mitochondrial copy number was necessary to reduce partitioning errors while stochastically partitioning mitochondria between daughter cells. We discovered that the association of mitochondria with microtubules physically impeded the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a novel mechanism for regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.

Tregs play important roles in maintaining immune homeostasis, and thus, therapies based on Treg are promising candidates for the treatment for a variety of immune-mediated disorders. These therapies, however, face the significant challenge of obtaining adequate numbers of Tregs from peripheral blood that maintains suppressive function following extensive expansion. Inducing Tregs from non-Tregs offers a viable alternative. Different methods to induce Tregs have been proposed and involve mainly treating cells with TGF-β-iTreg. However, use of TGF-β alone is not sufficient to induce stable Tregs. ATRA or rapa has been shown to synergize with TGF-β to induce stable Tregs. Whereas TGF-β plus RA-iTregs have been well-described in the literature, the phenotype, function, and migratory characteristics of TGF-β plus rapa-iTreg have yet to be elucidated. Herein, we describe the phenotype and function of mouse rapa-iTreg and reveal that these cells differ in their in vivo homing capacity when compared with mouse RA-iTreg and mouse TGF-β-iTreg. This difference in migratory activity significantly affects the therapeutic capacity of each subset in a mouse model of colitis. We also describe the characteristics of iTreg generated in the presence of TGF-β, RA, and rapa.

Cell penetrating peptides have demonstrated potential to facilitate the cellular delivery of therapeutic molecules. Here we develop a set of 50 cell penetrating peptide based formulations with potential to deliver small interfering RNAs intercellularly. The transfection efficacy of siRNA containing lipid-like nanoparticles decorated with different peptides was evaluated both in vitro and in vivo and correlated with the peptide physical and chemical properties. In vitro, these particles were internalized primarily through macropinocytosis. When the peptides were presented to bone marrow-derived dendritic cells, they induce low immunoactivation relative to control cell penetrating peptides including the antennapedia homeodomain and TAT, as quantified by the expression of activation specific surface proteins like CD80, CD86, and major histocompatibility complex class II. In vivo, peptide decorated nanoparticles primarily accumulated in the lungs and the liver. Three human peptides derived from surfactant protein B (a lung surfactant protein), orexin (a neuropeptide hormone, and lactoferricin (a globular glycoprotein) that exist in many physiological fluids facilitated the in vivo delivery of siRNA and induce significant knock down (90%) of a hepatocyte expressed protein, coagulation Factor VII.

Deficiency of regulatory T cells (Treg), a type of lymphocyte that promotes immunological homeostasis in the healthy steady state, [1–3] is a causative factor for destructive inflammation and autoimmune disease. [2,4] Accordingly, increasing the presence of Treg at the site of autoimmunity has been suggested as a potential method to treat these types of disorders. [3,4] However, to our knowledge, techniques to increase the local presence of specific immune cell types in vivo do not yet exist. Herein, we develop and test a microscale controlled-release system for the recruitment of Treg that draws inspiration from a specific biological system: tumors. Specifically, a wide variety of tumors release the chemokine CCL22, [5,6] which is responsible for tumor-directed migration of Treg and corresponding tumor-specific immune evasion. Our hypothesis was that if a steady CCL22 release could be maintained, by tuning the material properties of a controlled-release system, Treg may be preferentially recruited to a local site in vivo. The first step towards testing this hypothesis was to design a release vehicle suitable for the steady release of CCL22. Although polymeric-microparticle-based controlled-release systems for proteins (including chemokines) have previously been developed, [7,8] the release of hydrophilic proteins from these particles typically follows a triphasic release profile. [9,10] Our goal was to achieve release of CCL22 without any periods of lag in order to produce a corresponding steady gradient of chemokine originating from a point source. To this end, new mechanistic descriptions of how controlled release from degradable materials [9,11] suggest that one of the major factors affecting the rate of release is the erosion of a dense polymer matrix into a porous macrostructure that allows for protein egress. The timing of this process and, in turn, the period of lag is determined by the type of polymer, its molecular weight (inherent viscosity) and the degree of porosity in the matrix. Accordingly, we hypothesized that addition of contiguous pores to the microparticles during fabrication would pre-establish pathways for diffusive protein egress and should bypass the need for erosion as a requisite for release. Thus, we used the polymer poly(lactic-co-glycolic acid) (PLGA) of a specific inherent viscosity (0.16–0.24 dl/g in 0.1% chloroform; in order to achieve release within a 3–8 week period) and modified the surface porosity using an osmotic gradient between the inner aqueous emulsion and the outer bulk aqueous phase (Supporting Information, Figure S1). We observed that porous microparticles prepared using a specific osmotic gradient (corresponding to 15 × 10−3 m NaCl in inner aqueous emulsion), in contrast to unmodified degradable particles ( Figure 1a), did not display the standard lag phase that would be expected with release of proteins and released CCL22 continuously over a 4-week period (Figure 1b). As an additional design parameter, the particles were made to be large enough to avoid uptake by phagocytic cells and to prohibit their movement across vascular endothelium (Supporting Information, Figure S2). As a consequence, particles would remain immobilized at the site of injection. Figure 1 CCL22MP Characteristics. a) Scanning electron micrographs of nonporous (left; top and bottom) and porous (right; top and bottom) CCL22 microparticles (CCL22MP). The top images were taken at 500× magnification, while the bottom images were taken ... In order to test the ability of rationally designed CCL22MP to attract Treg, an in vivo adoptive transfer model coupled with non-invasive live-animal imaging was used. Specifically, fluorescently labeled CCL22MP were injected into the triceps surae of normal FVB mice followed by intravenous (i.v.) infusion of ex vivo-alloactivated Treg [12] (AATreg) that constitutively expresses the luciferase gene. The hind limb muscles were chosen as the site for MP injections as these distal sites are not expected to produce large quantities of CCL22. The migration pattern of these bioluminescent AATreg was studied following the injection of non-labeled mature allogeneic dendritic cells (DC), which provide an activation stimulus (Supporting Information, Figure S3). Soon after DC stimulation, a significantly greater number of AATreg was recruited to the site of CCL22MP injection compared to an internal control of microparticles lacking CCL22 ( Figure 2a and Supporting Information Figure S4). The adoptively transferred Treg are expected to translocate to sites in the body (e.g., lungs, gut etc.) that secrete CCL22, as observed in Figure S4, but not sites such as the hind limb (as confirmed by our preliminary experiments (data not shown)). However, a significant number of Treg do migrate towards the hind limb muscles upon injection of CCL22MP but not upon addition of blank particles ( p < 0.05), suggesting that the migration must be due to the release of CCL22 from these particles. Importantly, several reports suggest that small changes in Treg numbers at local sites (leading to a change in the ratio of Treg to effector T cells) is sufficient to dramatically alter local immune responses. [13] In line with these reports, and as a test for the ability of CCL22MP to replace the function of CCL22 secreting cells in vivo (e.g., site-specific recruitment of regulatory cells), we also demonstrate that CCL22MP are effectively able to delay the rejection of transplanted allogeneic cells. Specifically, allogeneic luciferase-expressing Lewis lung carcinoma cells (which do not endogenously produce CCL22) were implanted subcutaneously into mice at the site of CCL22MP injections (or for comparison BlankMP or Bolus CCL22 as controls), and the time to rejection was recorded using non-invasive live imaging (Figure S3). We observed that the rejection rates were significantly slower in the CCL22MP group when compared to both bolus CCL22 and BlankMP controls (Figure 2b and Figure S5), supporting the hypothesis that establishing a CCL22 gradient in vivo using synthetic systems can help modulate local immune respons1es. Figure 2 CCL22MP in vivo. a) Representative fluorescence (red-gold) and luminescence images (blue-yellow) showing localization of particles and Treg, respectively; fluorescence images were used to outline (red-line) areas of particle localization (Igor Pro Living ... Potential therapeutic implications of such a bioinspired degradable controlled-release formulation capable of recruiting Treg in vivo are manifold. One obvious application is the use of CCL22MP in combination with an infusion of Treg expanded ex vivo. Current pre-clinical data suggest that freshly-isolated or ex vivo-expanded Treg infusion can prevent organ transplant rejection or suppress autoimmune diseases. [2,14,15] However, challenges such as obtaining adequate numbers and highly-purified populations of Treg have hindered progress into clinical trials. [14,15] Using formulations that release CCL22, it may be possible to lower the numbers of injected Treg, or potentially use populations with lower purity. Another use of CCL22MP would be to attract endogenous Treg populations, wherein these formulations have the potential to function as “off-the-shelf” therapeutics for the treatment of a wide variety of disorders associated with unrestrained immune reactivity. A potential drawback of using a CCL22 sustained release vehicle is that the receptor for this chemokine (CCR4) is expressed on both activated Treg and activated effector T cells, [16] suggesting that both these cell populations would be attracted towards CCL22MP. Yet in vivo studies show that CCL22 production associated with tumors [5,17] or long-surviving allografts [18] result in preferential recruitment of Treg leading to local immunosuppression. One possible explanation for these results is that Treg could express significantly more CCR4 than effector T cells [17] (Supporting Information, Figure S6). Further, it has been suggested that an optimal ratio of Treg to effector T cells, and not a complete absence of effector T cells, is necessary for effective local suppression of immunity. [14,19] Regardless, if local effector T cells abrogate a suppressive environment, CCL22MP can easily be modified to simultaneously release immunosuppressive agents (such as rapamycin) [20] to inhibit these effector T cells in situ, thereby assisting Treg to control adverse immune responses. In conclusion, we demonstrate that site-specific attraction of Treg leading to local immunomodulation can be achieved in vivo using CCL22MP. These bioinspired controlled-release formulations are particularly attractive as modular platforms for therapeutic development, as well as tools to study Treg-dependent modulation of immune responses in situ.

Degradable microparticles have the potential to protect and release drugs over extended periods and, if sized appropriately, can be passively targeted to phagocytic cells in vivo. Dendritic cells (DC) are a class of phagocytic cells known to play important roles in transplant rejection. Previously, we have demonstrated that DC treated with an immunosuppressive drug, rapamycin, have the ability to suppress transplant rejection. Herein, we describe a strategy to deliver an intracellular depot of rapamycin to DC. To achieve this, rapamycin was encapsulated into ∼3.4 μm sized poly(lactic-co-glycolic)acid (PLGA) microparticles (rapaMPs), and release behavior was examined under intra-phagosomal (pH = 5) and extracellular (pH = 7.4) conditions. It was observed that 4 days following phagocytosis of rapaMP, DC have significantly reduced ability to activate T cells, in comparison to DC treated with soluble rapamycin. Hence, we conclude that DC-specific intracellular delivery of rapamycin results in better efficacy of the drug, with respect to its ability to modulate DC function, when compared to treating DC with extracellular rapamycin.

Intracellular delivery of materials is a challenge in research and therapeutic applications. Physical methods of plasma membrane disruption have recently emerged as an approach to facilitate the delivery of a variety of macromolecules to a range of cell types. We use the microfluidic CellSqueeze delivery platform to examine the kinetics of plasma membrane recovery after disruption and its dependence on the calcium content of the surrounding buffer (recovery time ~5 min without calcium vs. ~30 s with calcium). Moreover, we illustrate that manipulation of the membrane repair kinetics can yield up to 5× improvement in delivery efficiency without significantly impacting cell viability. Membrane repair characteristics initially observed in HeLa cells are shown to translate to primary naïve murine T cells. Subsequent manipulation of membrane repair kinetics also enables the delivery of larger materials, such as antibodies, to these difficult to manipulate cells. This work provides insight into the membrane repair process in response to mechanical delivery and could potentially enable the development of improved delivery methods., National Institutes of Health (U.S.) (Grant RC1 EB011187-02), National Institutes of Health (U.S.) (Grant R01GN101420-01A1), Kathy and Curt Marble Cancer Research Fund, National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051), National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant MPP-09Call-Langer-60)

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Therapeutic gene replacement of a dystrophin cDNA into dystrophic muscle can provide functional dystrophin protein to the tissue. However, vector-mediated gene transfer is limited by anti-vector and anti-transgene host immunity that causes rejection of the therapeutic protein. We hypothesized that rapamycin (RAPA) would diminish immunity due to vector-delivered recombinant dystrophin in the adult mdx mouse model for DMD. To test this hypothesis, we injected limb muscle of mdx mice with RAPA-containing, poly-lactic-co-glycolic acid (PLGA) microparticles prior to dystrophin gene transfer and analyzed treated tissue after 6 weeks. RAPA decreased host immunity against vector-mediated dystrophin protein, as demonstrated by decreased cellular infiltrates and decreased anti-dystrophin antibody production. The interpretation of the effect of RAPA on recombinant dystrophin expression was complex because of an effect of PLGA microparticles., National Institutes of Health (U.S.) (F31-NS056780-01A2), National Center for Research Resources (U.S.) (KL2 RR024154), United States. Army Medical Research and Materiel Command (grant W81XWH-05-1-0334)

Intracellular delivery of biomolecules, such as proteins and siRNAs, into primary immune cells, especially resting lymphocytes, is a challenge. Here we describe the design and testing of microfluidic intracellular delivery systems that cause temporary membrane disruption by rapid mechanical deformation of human and mouse immune cells. Dextran, antibody and siRNA delivery performance is measured in multiple immune cell types and the approach’s potential to engineer cell function is demonstrated in HIV infection studies., National Institutes of Health (U.S.) (Grant R01GM101420-01A1), Kathy and Curt Marble Cancer Research Fund, Ragon Institute of MGH, MIT and Harvard, National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)