Neutrophils are the most abundant white blood cell, with 1 × 10¹¹ new cells produced in the bone marrow daily with a short lifespan of about ∼7–24 hours in the blood (1). Neutrophils, also known as polymorphonuclear cells (PMNs) are terminally differentiated leukocytes with typical lobulated nuclei and antimicrobial cytoplasmic granules. Neutrophils are essential immune cells, crucial for the body’s innate immune response, and are often the first line of defense against invading pathogens (2). Neutrophils are involved in various disease processes, including pathogen infection (3), pulmonary diseases (4), cardiovascular diseases (5), inflammatory disorders (6) and cancer (7). Importantly, modifying or reducing neutrophil influx by pharmacological treatment can serve as a potential therapy. For example, during infection, enhancing neutrophil numbers and function by G-CSF treatment has been shown to be beneficial to treat severe neutropenia, particularly in the case of chemotherapy-induced neutropenia (8, 9). Furthermore, in pulmonary diseases and atherosclerosis, inhibition of neutrophil functions such as blocking neutrophil recruitment and targeting NETs has been demonstrated to be helpful (10–12). Neutrophils are challenging to study because they are short-lived effector cells of the innate immune system with a complex and diverse role in fighting pathogens, acute inflammation and they actively participate in several diseases including cancer (13).

Reverse migration has been described as a process whereby neutrophils migrate away from sites of tissue damage or infection once the infection has been resolved (14). The resolution of inflammation is generally thought to be driven by macrophages by efferocytosis of dead neutrophils at the wound site (15). However, following the identification of retrotaxis as an alternative path to resolution of neutrophilic inflammation in zebrafish (14), the functional role of neutrophil reverse migration during an immune response is still unclear as it is challenging to study in a human model. Evidence that suggests that neutrophil reverse migration can be a protective response, but it can also be detrimental and lead to systemic inflammation (16, 17). The protective response is attained by facilitating an efficient immune response resolution once the infection has been cleared, and this has been demonstrated using both mouse and zebrafish models (18, 19). Animal models have provided strong evidence of reverse migration in neutrophils and its impact on disease, but these findings do not always translate to human neutrophil activity.

Microfluidic approaches represent an integrative technology that enables customizable studies of primary leukocytes ex vivo. Microfluidic devices have contributed to improved understanding of the interaction and behavior of neutrophils ex vivo in 2D and 3D microenvironments. To better understand forward and reverse migration in neutrophils and the regulatory interactions between neutrophils and other cells, factors and molecular mechanisms driving this process, there is a need to identify appropriate in vitro models to study this process with human cells. In addition, these in vitro or ex vivo systems should account for relevant geometries, cell-cell interactions, and cell-matrix interactions. This review will provide a summary of the current applications of microfluidic devices to investigate neutrophil behavior and interactions with other immune cells with a focus on forward and reverse migration in neutrophils. The current state of knowledge regarding the mechanisms that control reverse migration are also reviewed and the potential for microfluidic devices to continue to help unravel the underlying biology that drives these phenomena is described. Finally, the use of microfluidic devices to improve our understanding of neutrophil behavior in other contexts (e.g. cancer, pathogen interaction) is discussed.

Neutrophils act as first responders during an innate immune response and are often the first cells to arrive at a site of infection or injury. After neutrophils execute their antimicrobial function at the site of infection, timely clearance of neutrophils is crucial to maintain homeostasis (27, 28). Macrophages carry out clearance of dead neutrophils at the site of infection via apoptosis or necrosis and subsequent phagocytosis (17), however recent evidence suggests that live neutrophils recirculate away from the site of infection and back into the blood vessels. Reverse migration in neutrophils was first suggested by Hughes et al. The authors used a rat model that describes the tracking and migration of radiolabeled neutrophils from an inflamed glomerular capillary back to the main circulation (29).

The mechanism involved in reverse migration and its visualization in vivo was reported by Mathias et al. The authors demonstrated that most neutrophils at the site of tissue damage reverse migrated away from the wound back to blood circulation (14). In agreement with Mathias et al. study, Buckley and colleagues reported that human neutrophils undergo reverse transendothelial migration (rTEM) through a tumor necrosis factor-α (TNF-α)-activated endothelial monolayer (25). Interestingly, Tauzin and colleagues also showed that macrophages could drive reverse migration in neutrophils using the zebrafish model. The authors found that reverse migration of neutrophils from a wound site was mediated by ROS-Src family kinase signaling in macrophages, providing a mechanism by which macrophages modulate resolution of neutrophil-mediated inflammation (30, 31).

Multiple phenotypic markers of reverse migrated neutrophils have been identified. Neutrophils that undergo reverse migration have a high expression level of intercellular adhesion molecule-1 (ICAM-1^high) and a low expression level of C-X-C motif chemokine receptor 1 (CXCR1^low) compared to neutrophils in blood circulation which are ICAM-1^low and CXCR1^high (32, 33). Interestingly, ICAM-1^high/CXCR1^low neutrophils were found to be increased in patients with systemic inflammation (25). Wang and colleagues visualized and imaged neutrophils that reverse migrated from an inflammatory site in the liver to the lungs and bone marrow (34). While these studies ( Table 1 ) indicated that neutrophils reverse migrate, the fate of reverse-migrated neutrophils and the underlying mechanism of reverse migration remains unclear.

The mechanisms that mediate reverse migration in neutrophils from sterile injury remain unclear. However, factors like chemotactic repellents, neutrophil-endothelial interaction, and chemokine receptors have been identified as contributing to reverse migration in neutrophils (35) ( Figure 1 ). Neutrophils migrate from the circulation to the infection site by breaching the endothelium (36). Compromise in the structural and functional integrity of the endothelial junction and permeability could play a crucial role in the mechanism of reverse migration in neutrophils (37). During inflammation, structural damage occurs at endothelial junctions, thus increasing endothelial permeability. There can also be diffusion of chemokines from damage sites that influence vascular permeability. It is possible that these factors may affect neutrophil reverse migration and transmigration across vessel walls (38, 39).

Junctional adhesion molecules are members of an immunoglobulin subfamily, consisting of JAM-A, -B, -C, -4, endothelial cell selective adhesion molecule (ESAM), and coxsackievirus and adenovirus receptor (CAR) that are specifically enriched at the tight junctions of cell-cell contacts (40). Woodfin et al. demonstrated that neutrophil reverse transmigration is regulated by the junctional adhesion molecule pathway in mouse models (26). Accordingly, inhibiting the junctional adhesion molecule pathway reduces reverse migration in neutrophils in septic mice (41). Furthermore, Colom and colleagues reported that CD11b and ICAM-1 interaction lead to junctional adhesion molecule cleavage by neutrophil elastase, promoting reverse transmigration in neutrophils (42) ( Figure 1 ). The study further showed that the leukotriene B-neutrophil elastase pathway may contribute to neutrophil reverse transmigration in mice (42). Li et al. demonstrated that blocking LTB₄ receptors could decrease reverse transmigration of neutrophils in mice (43). In another study by Hirano and colleagues, it was demonstrated that inhibition of JAM-C degradation significantly decreases neutrophil reverse transmigration in septic mice (41).

Studies have begun to identify some chemokines and chemokine receptors that are involved in neutrophil reverse migration. Chemokine receptors such as CXCR1 and CXCR2 are crucial for neutrophils to sense chemokines during cell migration. Interestingly, a study showed that reverse migrated neutrophils had decreased expression of CXCR1 (25). Therefore, it was speculated that those neutrophils might lose their ability to sense chemokine cues and migrate in a reverse direction. However, the actual evidence and mechanism remain unclear. Another study using zebrafish showed that CXCL8a and CXCR2 (human equivalent of CXCR1) are required for neutrophils to undergo reverse migration. The authors further showed that a CXCR2 knockout in zebrafish had impaired reverse migration of neutrophils (44).

Wang and colleagues, in their study, used advanced intravital imaging techniques in mice to visualize neutrophil dynamics within a thermal hepatic injury site. The authors reported that neutrophils migrate away from the injury site following tissue repair back into the vasculature. They further demonstrated that the reversed migrating neutrophils traffic to the lung and eventually to the bone marrow, where they die via apoptosis (34). Some studies have molecular mechanisms that regulate the reverse migration in neutrophils. For example, Elk and colleagues showed the role of hypoxia-inducible factor 1 subunit alpha (HIF1α) in suppressing reverse migration of neutrophils in zebrafish (45). The authors also noted that the activation of HIF1α reduced reverse migration of neutrophils and delayed inflammation resolution in zebrafish. A more recent study showed that the paracrine factor myeloid derived growth factor (MYDGF) affected neutrophil reverse migration through a HIF1a dependent pathway (46).

Chemotactic repellency in the inflammatory site is another mechanistic hypothesis for reverse migration in neutrophils (17). CXCL8 is a potent neutrophil chemoattractant in human inflammation (47). CXCL8 binds to the G protein-coupled receptors CXCR1 and CXCR2 (48). Tharp et al. reported that CXCL8 functions as a chemoattractant and chemorepellent in lower and higher concentrations, respectively. CXCL8 as a chemorepellent has been shown to drive reverse migration in neutrophils (49). Other studies have reported eicosanoid prostaglandin E₂ (PGE2), and macrophages as chemorepellents that drive reverse migration in zebrafish neutrophils (50). These studies ( Table 2 ) collectively showed the complexity and diversity of the suggested mechanisms driving reverse migration in neutrophils. Thus, we need to improve our knowledge about neutrophil reverse migration and its effect on immune response and inflammation.

What is the final fate of reverse migrated neutrophils? Several studies have suggested several outcomes. For example, a study showed that neutrophils could be found in circulation for at least 48 hours after migrating away from the wound site using the zebrafish model (51). Imbalance in reverse migration could lead to multiple organ failure (52). For example, neutrophils expressing ICAM-1^high/CXCR1^low markers were found in the circulation of patients with acute pancreatitis that developed acute lung injury (53). The same authors reported a similar observation in a mouse model of acute pancreatitis that an increased level of neutrophils expressing ICAM-1^high/CXCR1^low markers were found in circulation (53). Another study showed that following ischemia–reperfusion injury in mice, ICAM-1^high/CXCR1^low neutrophils were found to be resident in the lungs (26). Colom and colleagues, in agreement, showed that experimentally induced rTEM resulted in increased organ damage in the lungs, liver, and heart (42). Furthermore, neutrophils have also been demonstrated to migrate away from the site of infection and re-localize to the lymph nodes (54) or bone marrow (55) to affect host defense. The clinical implication of this re-localization is that neutrophils can shuttle live pathogens to lymph nodes (56) ( Table 3 ).

The functional role of reverse migration in neutrophils during inflammation needs further studies. However, current evidence suggests that it could be context dependent. Reverse migration in neutrophils can be both a protective response by facilitating an efficient resolution to an innate immune reaction and also a tissue-damaging event (17, 34, 42, 57, 58). Migration of activated neutrophils away from the infection sites through reverse migration may resolve an inflammatory reaction. However, reverse migrating neutrophils re-entering the vasculature can spread infections (26, 59). Few studies have shown strong in vivo evidence that neutrophils can reverse migrate into circulation (26, 42). For example, Elks et al. and Tharp et al. demonstrated that reverse migration in neutrophils could promote inflammation resolution using a zebrafish model. These studies also showed that inhibition of reverse migration in neutrophils by macrophage depletion and activation of HIF1α resulted in more wound damage due to infiltration of neutrophils (45, 49).

Pathologically, several studies have shown that reverse migrated neutrophils contribute to the spreading of infection and distant organ dysfunction. For example, Colom et al. and Woodfin et al. demonstrated that inhibition of reverse migration in neutrophils protected the host from distant organ damage using a murine model of ischemia-reperfusion (26, 42). Though the underlying mechanism of how reverse migrated neutrophils drive distant organ damage remains unclear, they speculate that the mechanism could result from cellular interaction between reverse migrated neutrophils and circulating cells. In a recent study by Bernut et al. the authors demonstrated that the loss of cystic fibrosis transmembrane conductance regulator (CFTR) delays resolution of inflammation by reducing neutrophil reverse migration in the context of sterile inflammation using a zebrafish model of cystic fibrosis (CF). The authors further reported that the pathogenic mechanisms leading to persistent neutrophilic inflammation in CF involve CFTR-related defect in reverse migration of neutrophils (16).

Another study by Hirano and colleagues showed that neutrophils with a similar phenotype to reverse migrated neutrophils modulate T cell functions by suppressing their proliferation in a CD18/CD11b dependent manner (53). Furthermore, inflammatory conditions that drive reverse migration in neutrophils are linked with a high expression level of ICAM and ROS generating neutrophils in the pulmonary vasculature, a response that is also associated with lung inflammation (26). Interestingly, in chronic inflammatory conditions like atherosclerosis and rheumatoid arthritis, there has been a report of high numbers of ICAM-1^high neutrophils in circulation (25), suggesting that reverse migrated neutrophils may be associated with the pathogenesis of these inflammatory diseases. In conclusion, most reverse migration studies have been demonstrated using animal models. However, studies of reverse migration in humans are limited and can be challenging to investigate. Thus, the use of microfluidic platforms to study reverse migration in human neutrophils could be helpful.

During an inflammatory response, neutrophils must interact and communicate with other immune cells in a variety of ways that are crucial for the immune response. After transmigration, neutrophils must also navigate complex blood vessel networks and ECM to reach the infection site. To achieve this, neutrophils must migrate toward the chemokine gradient produced by other immune cells and pathogens at the site of infection ( Figure 3 ). During an immune response, neutrophils signal to other migrating neutrophils. The interaction is through a signal amplification mechanism known as swarming. Neutrophil swarming has been described as an essential tissue response that is orchestrated to protect healthy tissue from unnecessary inflammation, limiting neutrophil migration to the pathogen-infected tissue (94). The role of neutrophil swarming during infection has been seen to be context dependent. It is essential for arresting the growth and spread of various pathogens (82, 83, 95–97).

Interestingly, it has been shown that response to swarming signals depends on LTB₄ (98). It varies in magnitude depending on the pathogen type, thus indicating a role for the pathogen in modulating neutrophil function (73). In addition, swarming has been reported in a cell line model of neutrophils. A study by Babatunde et al. demonstrated that differentiated human leukemia- neutrophil-like cells (dHL-60) could exhibit swarming ability toward a cluster of zymosan particles using a swarming microfluidic device. The authors demonstrated that swarming in dHL-60 cells is comparable to primary neutrophils through quantitative but not qualitative data. The authors also showed that swarming in dHL-60 cells depends on the expression and secretion of LTB₄ during coordinated migration toward the zymosan particles cluster (80) ( Table 4 ).

Monocytes are also known to migrate from the blood vessels into the surrounding tissue. Once in the tissue, they differentiate into either macrophages or dendritic cells (99). Neutrophil-monocyte interaction has been reported using LumeNEXT. For example, a study demonstrated that monocytes significantly increase chemotactic response in neutrophils to A. fumigatus infection. The authors also showed that the observed increase in chemotactic response was macrophage inflammatory protein (MIP-1) dependent (93). Though few studies use microfluidic devices to study neutrophil-monocyte interactions, simple in vitro models, have also been used to demonstrate cellular crosstalk between monocytes and neutrophils. Zhang et al. show that macrophage-derived exosomes activate ROS production and NETosis in neutrophils (93).

The interaction of neutrophils and dendritic cells has not been studied using microfluidic platforms that mimic the in vivo microenvironment. However, simple in vitro approaches have been used to study the crosstalk between these cell types. For example, a study demonstrated that Aspergillus fumigatus-infected dendritic cells induce a chemotactic response in neutrophils by activating the IL-8 receptor (100). Other immune cells like T cells, mast cells, and natural killer cells (NK cells) have also been reported to interact with neutrophils. Pelletier et al. demonstrated that neutrophils induce chemotaxis in T-helper 17 cells by releasing CCL2 and CCL20 in vitro (101). In another report by Tamassia et al., the authors reported that neutrophil-secreted IL-23 induces naive CD4 T cells to differentiate into Th17 cells (102).

Neutrophil and Natural killer (NK) cells interactions have been shown to result in changes in the expression level of neutrophil receptors and survival. For example, a study showed that activated NK cells secrete factors and signals necessary for neutrophil survival and increase the expression of receptors including CD64, CD11b, and CD69 on neutrophils (103). In contrast, a study demonstrated that NK cells could induce apoptosis of neutrophils in a caspase-dependent manner (104). In addition, NK cells have also been reported to drive the apoptotic process in neutrophils following ROS-induced NK interaction (105). Neutrophil interactions with other immune cells like NK cells, dendritic cells, and macrophages play a crucial role in their immune responses. Overall, the study of neutrophil interactions with other immune cells is active field where microfluidic models could offer a versatile tool to decipher the cellular and molecular mechanisms underlying such processes.

Tumor-associated Neutrophils (TANs) are neutrophils that have infiltrated into the tumor microenvironment. TANs are functionally classified as a tumor-suppressing N1 or tumor-promoting N2 phenotype although it is likely that there is a spectrum of phenotypes between N1 and N2. Each subpopulation of TANs has a distinct role in the tumor. N1 neutrophils have potent anti-tumor activity mainly due to their release of pro-inflammatory or immunostimulatory cytokines, such as interleukin (IL)-12, tumor necrosis factor (TNF)-α, CCL3, CXCL9, CXCL10 (32, 106). In contrast, N2 neutrophils have strong immunosuppressive and tumor-promoting activity, including stimulation of tumor angiogenesis, invasion and metastases via various factors (107). The role of neutrophils in cancer and the tumor microenvironment is multifactorial, and still unclear.

Few studies have demonstrated using a microfluidic device to investigate neutrophil behavior in the tumor microenvironment. For example, a recent study by Surendran et al. designed a microfluidic device to study the role of neutrophils in the tumor microenvironment. The 3D microfluidic device is known as the tumor immune microenvironment (TIME)-on-Chip device and serves both as a neutrophil migration and 3D tumor invasion platform. Briefly, a tumor spheroid was formed and embedded within the collagen matrix. The tumor spheroid setup was then hybrid-integrated with 3D bioprinting-enabled microfluidic channels. The TIME-on-Chip mimics the in vivo tumor microenvironment with key physiological components like extracellular collagen matrix compared to 2D models. The authors reported that the tumor spheroid recruited neutrophils by a chemotactic process and led to NETosis. However, the released NETs stimulated the invasion of the tumor cells into the surrounding collagen matrix, in a manner comparable to transforming growth factor-beta (TGF-β) and IL-8 effects on tumor cells. Furthermore, they reported that the tumor invasion was reversed by a drug that inhibits the NET formation pathway in neutrophils (108).

Another study by Chen and colleagues described and characterized a microfluidics-based, in vitro assay featuring 3D perfusable microvascular networks for studying tumor cell extravasation dynamics. The device is a self-organized human microvascular network formed by human umbilical vein endothelial cells (HUVECs) in fibrin gels, through which tumor cells can be perfused and extravasation events can be tracked via microscopy. The authors described a dynamic interaction between intravascular tumor cells and neutrophils at high spatiotemporal resolutions. They showed that neutrophil clusters were formed around the tumor cells. The clusters aggregation was chemotactically driven by neutrophil secreted IL-8 and tumor-derived CXCL1. However, the localization of the neutrophils around the tumor cells and secreted factors imply that it increases tumor extravasation potential by modulating the endothelial barrier (109).

Microfluidic devices could play an important role in improving our knowledge of how neutrophil related processes like ROS production and NETosis reduces NK and T cell tumor cytotoxicity and drives tumor progression ( Figure 3 ). As such, a novel device has to be designed to allow the incorporation of these different immune cell populations to allow real-time monitoring and analyses of these interactions in a microenvironment that mimics the TME in vivo. Furthermore, the factors that drive neutrophil phenotype into pro-tumorigenic N2 neutrophils and anti-tumorigenic N1 cells remain unclear; microfluidic platforms could be useful in characterizing and identifying these factors.

Coronavirus disease 2019 (COVID-19) is a novel, viral-induced respiratory disease that in ∼15% of patients progresses to acute respiratory distress syndrome (ARDS) and its thought to be triggered by a cytokine storm (110). Neutrophils are present in many lung diseases associated with ARDS, including infections with influenza virus and COVID-19 (111). Increases in neutrophils has been identified as an indicator of severe respiratory symptoms and poor outcome in patients with COVID-19 (112–116). Elevated neutrophil numbers have also been reported in the nasopharyngeal epithelium of individuals infected with COVID-19 (117) as well as in the inferior lobe of the lung (118). Furthermore, plasma levels of neutrophil associated to factors such as resistin (RETN), lipocalin-2 (LCN2), and hepatocyte growth factor (HGF), were recently proposed as biomarkers for critical illness and mortality during COVID-19 (115).

Viral infection can also induce the release of NETs by neutrophils (119) NETs are known to immobilize and degrade pathogens such as bacteria, fungi, viruses, being a critical effector mechanism to contain infections (120). Several clinical and experimental studies have demonstrated an elevated level of NETs in individuals infected with COVID-19 (121, 122), and an increase in NETs in plasma is correlated with increased COVID-19 severity (123). NET formation in the lungs may have been triggered by direct contact of COVID-19 infected neutrophils. This has been shown in vitro as COVID-19 induced NET formation by neutrophils (123–125). In addition, the interaction between neutrophils and platelets has been implicated in the mechanism involved in increased NET levels during severe COVID-19 (126). The process of immunothrombosis can lead to blockage of hepatic micro-vessels causing hepatic cell death, thereby contributing to impaired functions of the lung. During severe COVID-19, the risk of immunothrombosis is increased by vasoconstriction driven by excessive cytokine release (127). In addition, a study by Wang and colleagues demonstrated at the transcriptional level the activation of several NETs-associated genes such as myeloperoxidase (MPO) and neutrophil elastase (NE) in individuals infected with COVID-19. The authors further hypothesized that the observed molecular changes could be as a result of negative regulation of anti-viral immune cells such as NK and T cells (128). This cascade of events associated with neutrophils triggered in COVID-19 infection undoubtedly contributes to the disease severity, thus there is a need to have a better understanding of the role of neutrophil in COVID-19.

There are limited studies using microfluidic devices to understand the role of neutrophils in COVID-19. Most studies to date are in vitro (123), in vivo using mouse models (129) and patients samples (129). However, microfluidics can provide a platform to help understand the neutrophil interactions with COVID-19 and how these interactions drive the pathophysiology of COVID-19 ex vivo. Reverse migrated neutrophils may become mechanically entrapped in the microvasculature of major organs such as lungs, thus contributing to its damage and failure as observed in COVID-19 (130). We postulate that the observed increased number of neutrophils during COVID-19 infection may be due to the tissue damage induced by the virus. Importantly, the presence of neutrophils in the lungs and nasopharyngeal epithelium during COVID-19 infection may be due to reverse migration of neutrophils back to the lungs from infection sites. If this is the case, microfluidic devices can provide a platform to study the interaction of COVID-19 and neutrophils ex vivo in a micro-environment that incorporates other immune cells such as endothelial cells, macrophages, NK and T cells in an environment that closely mimics the hepatic environment in vivo.

Neutrophils are crucial to the body’s innate immune response as they are responsible for fighting against invading pathogens as well as cancer. During infection, neutrophils must process several inflammatory signals from their microenvironment in other to elicit their immune response. Several studies have tried to understand how neutrophils process and respond to these signals using various experimental systems that include in vitro and in vivo models.

Neutrophil phenotypes can exist as either pro-inflammatory, anti-tumor “N1” neutrophils or anti-inflammatory, pro-tumor “N2” neutrophils. Currently, it’s not clear if these different phenotypes of the neutrophil have an active role in reverse migration. It will be interesting to know if reverse migration specifically of N1 neutrophils can result in systemic inflammation or if reverse migration in the “N2” neutrophil phenotype could serve as a means of cancer immunotherapy. We speculate that increasing reverse migration in N2 neutrophil phenotype from TME and driving forward migration of N1 phenotype toward TME could limit tumor progression. There are limited studies demonstrating the use of microfluidic devices to understand the role of neutrophils in cancer. To understand how reverse migration in the N2 neutrophil phenotype could reduce tumor progression in the TME, we suggest that a novel microfluidic platform that allows the in vitro polarization of the N2 neutrophil phenotype towards the N1 neutrophil phenotype using suggested/known factors responsible for driving these phenotypes. Furthermore, this microfluidic platform should allow the monitoring and investigation of the interaction of neutrophils and tumor cells in single-cell resolution and in real-time in a microenvironment that mimics the TME in vivo.

Retrotaxis in neutrophils has been observed using both in vitro and in vivo models. It has been identified as a way to resolve inflammation, though it could also result in the spread of infection into the blood circulation. So far, in vitro and in vivo models have improved our understanding of the mechanisms involved in the neutrophil immune response. However, how these signals result in an effective neutrophil response following an infection remains unclear. Future studies should then focus on developing and designing devices that will include cell populations known to influence reverse migration in neutrophils, like macrophages. This will facilitate investigation of the factors driving the yet to be understood reverse migration of neutrophils in a microenvironment that closely recapitulates tissues in vivo. However, challenges remain as different immune cell populations need different culture periods, nutrients/media, and conditions in vitro, which poses additional hurdles to decipher the complex immune interactions during human disease. We suggest that different cell populations that are involved in the multicellular signaling cascades that drive reverse migration in neutrophils should be incorporated into the design of these microfluidic devices.

KB drafted the article. KB prepared the figures. JA, SK, AH, and DB provided critical feedback. All authors contributed to the article and approved the submitted version.

This work supported by the University of Wisconsin Carbone Cancer Center, Cancer Center Support Grant NIH P30CA014520. This work was also supported by the National Institutes of Health NIH R01AI134749 to AH and DB, the National Institutes of Health NIH U24AI152177 to DB, and the Swiss National funding (P500PB_203002) to KB.

DB holds equity in Bellbrook Labs LLC, Tasso Inc., Salus Discovery LLC, Lynx Biosciences Inc., Stacks to the Future LLC, Turba LLC, Flambeau Diagnostics LLC, and Onexio Biosystems LLC. DB is also a consultant for Abbott Laboratories.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.