Factors Affecting the Evaluation of Collagen Deposition and Fibrosis In Vitro.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Intersection of Immunity, Metabolism, and Muscle Regeneration in an Autoimmune-Prone MRL Mouse Model.

Due to the limited capacity of mammals to regenerate complex tissues, researchers have worked to understand the mechanisms of tissue regeneration in organisms that maintain that capacity. One example is the MRL/MpJ mouse strain with unique regenerative capacity in ear pinnae that is absent from other strains, such as the common C57BL/6 strain. The MRL/MpJ mouse has also been associated with an autoimmune phenotype even in the absence of the mutant Fas gene described in its parent strain MRL/lpr. Due to these findings, the differences between the responses of MRL/MpJ versus C57BL/6 strain are evaluated in volumetric muscle injury and subsequent material implantation. One salient feature of the MRL/MpJ response to injury is robust adipogenesis within the muscle. This is associated with a decrease in M2-like polarization in response to biologically derived extracellular matrix scaffolds. In pro-fibrotic materials, such as polyethylene, there are fewer foreign body giant cells in the MRL/MpJ mice. As there are reports of both positive and negative influences of adipose tissue and adipogenesis on wound healing, this model can provide an important lens to investigate the interplay between stem cells, adipose tissue, and immune responses in trauma and material implantation.

Pro-regenerative biomaterials recruit immunoregulatory dendritic cells after traumatic injury.

During wound healing and surgical implantation, the body establishes a delicate balance between immune activation to fight off infection and clear debris and immune tolerance to control reactivity against self-tissue. Nonetheless, how such a balance is achieved is not well understood. Here we describe that pro-regenerative biomaterials for muscle injury treatment promote the proliferation of a BATF3-dependent CD103+XCR1+CD206+CD301b+ dendritic cell population associated with cross-presentation and self-tolerance. Upregulation of E-cadherin, the ligand for CD103, and XCL-1 in injured tissue suggests a mechanism for cell recruitment to trauma. Muscle injury recruited natural killer cells that produced Xcl1 when stimulated with fragmented extracellular matrix. Without cross-presenting cells, T-cell activation increases, pro-regenerative macrophage polarization decreases and there are alterations in myogenesis, adipogenesis, fibrosis and increased muscle calcification. These results, previously observed in cancer progression, suggest a fundamental mechanism of immune regulation in trauma and material implantation with implications for both short- and long-term injury recovery.

Ectopic adipogenesis in response to injury and material implantation in an autoimmune mouse model.

Due to the limited capacity of mammals to regenerate complex tissues, researchers have worked to understand the mechanisms of tissue regeneration in organisms that maintain that capacity. One example is the MRL/MpJ mouse strain with unique regenerative capacity in ear pinnae that is absent from other strains, such as the common C57BL/6 strain. The MRL/MpJ mouse has also been associated with an autoimmune phenotype even in the absence of the mutant Fas gene described in its parent strain MRL/lpr. Due to these findings, we evaluated the differences between the responses of MRL/MpJ versus C57BL/6 strain in traumatic muscle injury and subsequent material implantation. One salient feature of the MRL/MpJ response to injury was a robust adipogenesis within the muscle. This was associated with a decrease in M2-like polarization in response to biologically derived extracellular matrix scaffolds. In pro-fibrotic materials, such as polyethylene, there were fewer foreign body giant cells in the MRL/MpJ mice. As there are reports of both positive and negative influences of adipose tissue and adipogenesis on wound healing, this model could provide an important lens to investigate the interplay between stem cells, adipose tissue, and immune responses in trauma and materials implantation.

Conserved and tissue-specific immune responses to biologic scaffold implantation.

Upon implantation into a patient, any biomaterial induces a cascade of immune responses that influences the outcome of that device. This cascade depends upon several factors, including the composition of the material itself and the location in which the material is implanted. There is still significant uncertainty around the role of different tissue microenvironments in the immune response to biomaterials and how that may alter downstream scaffold remodeling and integration. In this study, we present a study evaluating the immune response to decellularized extracellular matrix materials within the intraperitoneal cavity, the subcutaneous space, and in a traumatic skeletal muscle injury microenvironment. All different locations induced robust cellular recruitment, specifically of macrophages and eosinophils. The latter was most prominent in the subcutaneous space. Intraperitoneal implants uniquely recruited B cells that may alter downstream reactivity as adaptive immunity has been strongly implicated in the outcome of scaffold remodeling. These data suggest that the location of tissue implants should be taken together with the composition of the material itself when designing devices for downline therapeutics.

Dynamics of SARS-CoV-2 Seroprevalence in a Large US population Over a Period of 12 Months.

Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.

Characterization of the antispike IgG immune response to COVID-19 vaccines in people with a wide variety of immunodeficiencies.

Research on coronavirus disease 2019 vaccination in immune-deficient/disordered people (IDP) has focused on cancer and organ transplantation populations. In a prospective cohort of 195 IDP and 35 healthy volunteers (HV), antispike immunoglobulin G (IgG) was detected in 88% of IDP after dose 2, increasing to 93% by 6 months after dose 3. Despite high seroconversion, median IgG levels for IDP never surpassed one-third that of HV. IgG binding to Omicron BA.1 was lowest among variants. Angiotensin-converting enzyme 2 pseudo-neutralization only modestly correlated with antispike IgG concentration. IgG levels were not significantly altered by receipt of different messenger RNA-based vaccines, immunomodulating treatments, and prior severe acute respiratory syndrome coronavirus 2 infections. While our data show that three doses of coronavirus disease 2019 vaccinations induce antispike IgG in most IDP, additional doses are needed to increase protection. Because of the notably reduced IgG response to Omicron BA.1, the efficacy of additional vaccinations, including bivalent vaccines, should be studied in this population.

Label-free cleared tissue microscopy and machine learning for 3D histopathology of biomaterial implants.

Tissue clearing of whole intact organs has enhanced imaging by enabling the exploration of tissue structure at a subcellular level in three-dimensional space. Although clearing and imaging of the whole organ have been used to study tissue biology, the microenvironment in which cells evolve to adapt to biomaterial implants or allografts in the body is poorly understood. Obtaining high-resolution information from complex cell-biomaterial interactions with volumetric landscapes represents a key challenge in the fields of biomaterials and regenerative medicine. To provide a new approach to examine how tissue responds to biomaterial implants, we apply cleared tissue light-sheet microscopy and three-dimensional reconstruction to utilize the wealth of autofluorescence information for visualizing and contrasting anatomical structures. This study demonstrates the adaptability of the clearing and imaging technique to provide sub-cellular resolution (0.6 µm isotropic) 3D maps of various tissue types, using samples from fully intact peritoneal organs to volumetric muscle loss injury specimens. Specifically, in the volumetric muscle loss injury model, we provide 3D visualization of the implanted extracellular matrix biomaterial in the wound bed of the quadricep muscle groups and further apply computational-driven image classification to analyze the autofluorescence spectrum at multiple emission wavelengths to categorize tissue types at the injured site interacting with the biomaterial scaffolds.

Leveraging CTSA hubs for rapid, large-scale, high-impact research: A case study during a global public health emergency.

As the COVID-19 pandemic took hold in the USA in early 2020, it became clear that knowledge of the prevalence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among asymptomatic individuals could inform public health policy decisions and provide insight into the impact of the infection on vulnerable populations. Two Clinical and Translational Science Award (CTSA) Hubs and the National Institutes of Health (NIH) set forth to conduct a national seroprevalence survey to assess the infection's rate of spread. This partnership was able to quickly design and launch the project by leveraging established research capacities, prior experiences in large-scale, multisite studies and a highly skilled workforce of CTSA hubs and unique experimental capabilities at the NIH to conduct a diverse prospective, longitudinal observational cohort of 11,382 participants who provided biospecimens and participant-reported health and behavior data. The study was completed in 16 months and benefitted from transdisciplinary teamwork, information technology innovations, multimodal communication strategies, and scientific partnership for rigor in design and analytic methods. The lessons learned by the rapid implementation and dissemination of this national study is valuable in guiding future multisite projects as well as preparation for other public health emergencies and pandemics.

Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

Development of a High-Color Flow Cytometry Panel for Immunologic Analysis of Tissue Injury and Reconstruction in a Rat Model.

The rat model is an important resource in biomedical research due to its similarities to the human immune system and its use for functional studies. However, because of the preponderance of mouse models in foundational and mechanistic immunological studies, there is a relative lack of diverse, commercially available flow cytometry antibodies for immunological profiling in the rat model. Available antibodies are often conjugated to common fluorophores with similar peak emission wavelengths, making them hard to distinguish on conventional flow cytometers and restricting more comprehensive immune analysis. This can become a limitation when designing immunological studies in rat injury models to investigate the immune response to tissue injury. In addition, this lack of available antibodies limits the number of studies that can be done on the immune populations in lymphoid organs in other research areas. To address this critical unmet need, we designed a spectral flow cytometry panel for rat models. Spectral cytometry distinguishes between different fluorophores by capturing their full emission spectra instead of their peak emission wavelengths. This flow cytometry panel includes 24 distinct immune cell markers to analyze the innate and adaptive immune response. Importantly, this panel identifies different immune phenotypes, including tolerogenic, Type 1, and Type 2 immune responses. We show that this panel can identify unique immune populations and phenotypes in a rat muscle trauma model. We further validated that the panel can identify distinct adaptive and innate immune populations and their unique phenotypes in lymphoid organs. This panel expands the scope of previous rat panels providing a tool for scientists to examine the immune system in homeostasis and injury while pairing mechanistic immunological studies with functional studies.

Examining multi-level immune response to determine prevalence of COVID-19 in pediatric tonsillectomy.

OBJECTIVE: To determine the prevalence of COVID-19 in a cohort of children undergoing tonsillectomy through assessment of B cell immune responses to SARS-CoV-2 in both peripheral blood and tonsil tissue. METHODS: In this cohort study at a tertiary pediatric hospital (Children's National Hospital) in Washington, DC, we recruited 100 children undergoing tonsillectomy from late September 2020 to January 2021. Serum, peripheral blood cells, and tonsil tissue were collected and examined for immune reactivity to SARS-CoV-2. Parent-reported clinical histories were compared to antibody and B-cell responses. RESULTS: Among 100 children undergoing tonsillectomy, 19% had evidence of immune responses to SARS-CoV-2 (CoV2+), indicating prior COVID-19. In all seropositive participants, we detected SARS-CoV-2 specific B cells in both peripheral blood mononuclear cells and tonsils, providing evidence for tissue-specific immunity in these children. Of the 19, 63% reported no known history of COVID-19, and an additional 3 were asymptomatic or unaware of an acute infection when detected on pre-surgery screen. Hispanic children represented 74% of CoV2+ subjects compared to 37% of the full cohort. 100% of CoV2+ children lived in a zip code with poverty level >10%. CONCLUSIONS: Nearly one-fifth of children undergoing tonsillectomy at an urban U.S. hospital had evidence of prior COVID-19 during the early pandemic, with the majority unaware of prior infection. Our results underscore the ethnic and socio-economic disparities of COVID-19. We found concordant evidence of humoral immune responses in children in both blood and tonsil tissue, providing evidence of local immune responses in the upper respiratory tract. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:1993-1999, 2023.

Comprehensive profiling of the human viral exposome in households containing an at-risk child with mitochondrial disease during the 2020-2021 COVID-19 pandemic.

BACKGROUND: Viral infection is a major cause of morbidity in children with mitochondrial disease (MtD). As a result, families with children with MtD are highly adherent to risk mitigation behaviours (RMBs) advised by the Centers for Disease Control and Prevention during the COVID-19 pandemic that can modulate infection risk. METHODS: Deep serologic phenotyping of viral infections was performed via home-based sampling by combining SARS-CoV-2 serologic testing and phage display immunoprecipitation and sequencing. Samples were collected approximately 1 year apart (October 2020 to April 2021 and October 2021 to March 2022) on households containing a child with MtD. RESULTS: In contrast to our first collection in 2020-2021, SARS-CoV-2 antibody profiles for all participants in 2021-2022 were marked by greater isotype diversity and the appearance of neutralizing antibodies. Besides SARS-CoV-2, households (N = 15) were exposed to >38 different respiratory and gastrointestinal viruses during the study, averaging five viral infections per child with MtD. Regarding clinical outcomes, children with MtD (N = 17) experienced 34 episodes of illness resulting in 6 hospitalizations, with some children experiencing multiple episodes. Neurologic events following illness were recorded in five patients. Infections were identified via clinical testing in only seven cases. Viral exposome profiles were consistent with clinical testing and even identified infections not captured by clinical testing. CONCLUSIONS: Despite reported adherence to RMBs during the COVID-19 pandemic by families with a child with MtD, viral infection was pervasive. Not all infections resulted in illness in the child with MtD, suggesting that some were subclinical or asymptomatic. However, selected children with MtD did experience neurologic events. Our studies emphasize that viral infections are inexorable, emphasizing the need for further understanding of host-pathogen interactions through broad serologic surveillance.

Undiagnosed COVID-19 in households with a child with mitochondrial disease.

Background: The impact of the COVID-19 pandemic on medically fragile populations, who are at higher risk of severe illness and sequelae, has not been well characterized. Viral infection is a major cause of morbidity in children with mitochondrial disease (MtD), and the COVID-19 pandemic represents an opportunity to study this vulnerable population. Methods: A convenience sampling cross-sectional serology study was conducted (October 2020 to June 2021) in households (N = 20) containing a child with MtD (N = 22). Samples (N = 83) were collected in the home using a microsampling apparatus and shipped to investigators. Antibodies against SARS-CoV-2 nucleocapsid (IgG), spike protein (IgG, IgM, IgA), and receptor binding domain (IgG, IgM, IgA) were determined by enzyme linked immunosorbent assay. Results: While only 4.8% of participants were clinically diagnosed for SARS-CoV-2 infection, 75.9% of study participants were seropositive for SARS-CoV-2 antibodies. Most samples were IgM positive for spike or RBD (70%), indicating that infection was recent. This translated to all 20 families showing evidence of infection in at least one household member. For the children with MtD, 91% had antibodies against SARS-CoV-2 and had not experienced any adverse outcomes at the time of assessment. For children with recent infections (IgM+ only), serologic data suggest household members as a source. Conclusions: COVID-19 was highly prevalent and undiagnosed in households with a child with MtD through the 2020-2021 winter wave of the pandemic. In this first major wave, children with MtD tolerated SARS-CoV-2 infection well, potentially due to household adherence to CDC recommendations for risk mitigation.

Robust, persistent adaptive immune responses to SARS-CoV-2 in the oropharyngeal lymphoid tissue of children.

SARS-CoV-2 infection triggers adaptive immune responses from both T and B cells. However, most studies focus on peripheral blood, which may not fully reflect immune responses in lymphoid tissues at the site of infection. To evaluate both local and systemic adaptive immune responses to SARS-CoV-2, we collected peripheral blood, tonsils, and adenoids from 110 children undergoing tonsillectomy/adenoidectomy during the COVID-19 pandemic and found 24 with evidence of prior SARS-CoV-2 infection, including detectable neutralizing antibodies against multiple viral variants. We identified SARS-CoV-2-specific germinal center (GC) and memory B cells; single cell BCR sequencing showed that these virus-specific B cells were class-switched and somatically hypermutated, with overlapping clones in the adenoids and tonsils. Oropharyngeal tissues from COVID-19-convalescent children showed persistent expansion of GC and anti-viral lymphocyte populations associated with an IFN-γ-type response, with particularly prominent changes in the adenoids, as well as evidence of persistent viral RNA in both tonsil and adenoid tissues of many participants. Our results show robust, tissue-specific adaptive immune responses to SARS-CoV-2 in the upper respiratory tract of children weeks to months after acute infection, providing evidence of persistent localized immunity to this respiratory virus.

SARS-CoV-2 Cross-Reactivity in Prepandemic Serum from Rural Malaria-Infected Persons, Cambodia.

Inhabitants of the Greater Mekong Subregion in Cambodia are exposed to pathogens that might influence serologic cross-reactivity with severe acute respiratory syndrome coronavirus 2. A prepandemic serosurvey of 528 malaria-infected persons demonstrated higher-than-expected positivity of nonneutralizing IgG to spike and receptor-binding domain antigens. These findings could affect interpretation of large-scale serosurveys.

Rapidly Increasing Severe Acute Respiratory Syndrome Coronavirus 2 Seroprevalence and Limited Clinical Disease in 3 Malian Communities: A Prospective Cohort Study.

BACKGROUND: The extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure and transmission in Mali and the surrounding region is not well understood. We aimed to estimate the cumulative incidence of SARS-CoV-2 in 3 communities and understand factors associated with infection. METHODS: Between July 2020 and January 2021, we collected blood samples and demographic, social, medical, and self-reported symptoms information from residents aged 6 months and older over 2 study visits. SARS-CoV-2 antibodies were measured using a highly specific 2-antigen enzyme-linked immunosorbent assay optimized for use in Mali. We calculated cumulative adjusted seroprevalence for each community and evaluated factors associated with serostatus at each visit by univariate and multivariate analysis. RESULTS: Overall, 94.8% (2533/2672) of participants completed both study visits. A total of 31.3% (837/2672) were aged <10 years, 27.6% (737/2672) were aged 10-17 years, and 41.1% (1098/2572) were aged ≥18 years. The cumulative SARS-CoV-2 exposure rate was 58.5% (95% confidence interval, 47.5-69.4). This varied between sites and was 73.4% in the urban community of Sotuba, 53.2% in the rural town of Bancoumana, and 37.1% in the rural village of Donéguébougou. Study site and increased age were associated with serostatus at both study visits. There was minimal difference in reported symptoms based on serostatus. CONCLUSIONS: The true extent of SARS-CoV-2 exposure in Mali is greater than previously reported and may now approach hypothetical "herd immunity" in urban areas. The epidemiology of the pandemic in the region may be primarily subclinical and within background illness rates.

Mobilizing Endogenous Repair Through Understanding Immune Reaction With Biomaterials.

With few exceptions, humans are incapable of fully recovering from severe physical trauma. Due to these limitations, the field of regenerative medicine seeks to find clinically viable ways to repair permanently damaged tissue. There are two main approaches to regenerative medicine: promoting endogenous repair of the wound, or transplanting a material to replace the injured tissue. In recent years, these two methods have fused with the development of biomaterials that act as a scaffold and mobilize the body's natural healing capabilities. This process involves not only promoting stem cell behavior, but by also inducing activity of the immune system. Through understanding the immune interactions with biomaterials, we can understand how the immune system participates in regeneration and wound healing. In this review, we will focus on biomaterials that promote endogenous tissue repair, with discussion on their interactions with the immune system.

Severe Acute Respiratory Syndrome Coronavirus 2 Seroassay Performance and Optimization in a Population With High Background Reactivity in Mali.

BACKGROUND: False positivity may hinder the utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests in sub-Saharan Africa. METHODS: From 312 Malian samples collected before 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and 4 other betacoronaviruses by enzyme-linked immunosorbent assay (ELISA). In a subset of samples, we assessed antibodies to a panel of Plasmodium falciparum antigens by suspension bead array and functional antiviral activity by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA using SARS-CoV-2 spike protein and receptor-binding domain developed in the United States using Malian positive and negative control samples. To optimize test performance, we compared single- and 2-antigen approaches using existing assay cutoffs and population-specific cutoffs. RESULTS: Background reactivity to SARS-CoV-2 antigens was common in prepandemic Malian samples. The SARS-CoV-2 reactivity varied between communities, increased with age, and correlated negligibly/weakly with other betacoronavirus and P falciparum antibodies. No prepandemic samples demonstrated functional activity. Regardless of the cutoffs applied, test specificity improved using a 2-antigen approach. Test performance was optimal using a 2-antigen assay with population-specific cutoffs (sensitivity, 73.9% [95% confidence interval {CI}, 51.6-89.8]; specificity, 99.4% [95% CI, 97.7-99.9]). CONCLUSIONS: We have addressed the problem of SARS-CoV-2 seroassay performance in Africa by using a 2-antigen assay with cutoffs defined by performance in the target population.

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.