Spatial Transcriptomics Resolve an Emphysema-Specific Lymphoid Follicle B Cell Signature in Chronic Obstructive Pulmonary Disease.

Rationale: Within chronic obstructive pulmonary disease (COPD), emphysema is characterized by a significant yet partially understood B cell immune component. Objectives: To characterize the transcriptomic signatures from lymphoid follicles (LFs) in ever-smokers without COPD and patients with COPD with varying degrees of emphysema. Methods: Lung sections from 40 patients with COPD and ever-smokers were used for LF proteomic and transcriptomic spatial profiling. Formalin- and O.C.T.-fixed lung samples obtained from biopsies or lung explants were assessed for LF presence. Emphysema measurements were obtained from clinical chest computed tomographic scans. High-confidence transcriptional target intersection analyses were conducted to resolve emphysema-induced transcriptional networks. Measurements and Main Results: Overall, 115 LFs from ever-smokers and Global Initiative for Chronic Obstructive Lung Disease (GOLD) 1-2 and GOLD 3-4 patients were analyzed. No LFs were found in never-smokers. Differential gene expression analysis revealed significantly increased expression of LF assembly and B cell marker genes in subjects with severe emphysema. High-confidence transcriptional analysis revealed activation of an abnormal B cell activity signature in LFs (q-value = 2.56E-111). LFs from patients with GOLD 1-2 COPD with emphysema showed significantly increased expression of genes associated with antigen presentation, inflammation, and B cell activation and proliferation. LFs from patients with GOLD 1-2 COPD without emphysema showed an antiinflammatory profile. The extent of centrilobular emphysema was significantly associated with genes involved in B cell maturation and antibody production. Protein-RNA network analysis showed that LFs in emphysema have a unique signature skewed toward chronic B cell activation. Conclusions: An off-targeted B cell activation within LFs is associated with autoimmune-mediated emphysema pathogenesis.

Spatial Transcriptomics Resolve an Emphysema-specific Lymphoid Follicle B Cell Signature in COPD.

RATIONALE: Within chronic obstructive pulmonary disease (COPD), emphysema is characterized by a significant yet partially understood B cell immune component. OBJECTIVE: To characterize the transcriptomic signatures from lymphoid follicles (LFs) in ever-smokers without COPD and COPD patients with varying degrees of emphysema. METHODS: Lung sections from 40 COPD patients and ever-smokers were used for LF proteomic and transcriptomic spatial profiling. Formalin and OCT-fixed lung samples obtained from biopsies or lung explants, were assessed for LF presence. Emphysema measurements were obtained from clinical chest CT scans. High confidence transcriptional (HCT) target intersection analyses were conducted to resolve emphysema-induced transcriptional networks. MEASUREMENTS AND MAIN RESULTS: Overall, 115 LFs from ever-smokers and GOLD 1-2 and GOLD 3-4 patients were analyzed. No LFs were found in never-smokers. Differential gene expression analysis revealed significantly increased expression of LF assembly and B cell markers genes in subjects with severe emphysema. HCT analysis revealed activation of abnormal B cell activity signature in LFs (q-value: 2.56E-111). LFs from GOLD 1-2 COPD patients with emphysema showed significantly increased expression of genes associated with antigen presentation, inflammation, and B cell activation and proliferation. LFs from GOLD 1-2 COPD patients without emphysema showed an anti-inflammatory profile. The extent of centrilobular emphysema was significantly associated with genes involved in B cell maturation and antibody production. Protein-RNA network analysis showed that LFs in emphysema have a unique signature skewed towards chronic B cell activation. CONCLUSIONS: An off-targeted B cell activation within LFs is associated with autoimmune-mediated emphysema pathogenesis.

Digital spatial profiling of melanoma shows CD95 expression in immune cells is associated with resistance to immunotherapy.

Although immune checkpoint inhibitor (ICI) therapy has dramatically improved outcome for metastatic melanoma patients, many patients do not benefit. Since adverse events may be severe, biomarkers for resistance would be valuable, especially in the adjuvant setting. We performed high-plex digital spatial profiling (DSP) using the NanoString GeoMx® on 53 pre-treatment specimens from ICI-treated metastatic melanoma cases. We interrogated 77 targets simultaneously in four molecular compartments defined by S100B for tumor, CD68 for macrophages, CD45 for leukocytes, and nonimmune stromal cells defined as regions negative for all three compartment markers but positive for SYTO 13. For DSP validation, we confirmed the results obtained for some immune markers, such as CD8, CD4, CD20, CD68, CD45, and PD-L1, by quantitative immunofluorescence (QIF). In the univariable analysis, 38 variables were associated with outcome, 14 of which remained significant after multivariable adjustment. Among them, CD95 was further validated using multiplex immunofluorescence in the Discovery immunotherapy (ITX) Cohort and an independent validation cohort with similar characteristics, showing an association between high levels of CD95 and shorter progression-free survival. We found that CD95 in stroma was associated with resistance to ICI. With further validation, this biomarker could have value to select patients that will not benefit from immunotherapy.

Tensile properties of freeze-cast collagen scaffolds: How processing conditions affect structure and performance in the dry and fully hydrated states.

Tensile properties of directionally freeze-cast biopolymer scaffolds are rarely reported, even though they are of interest from a fundamental science perspective and critical in applications such as scaffolds for the regeneration of nerves or when used as ureteral stents. The focus of this study is on collagen scaffolds freeze-cast with two different applied cooling rates (10 °C/min and 1 °C/min) in two freezing directions (longitudinal and radial). Reported are the results of a systematic structural characterization of dry scaffolds by scanning electron microscopy and the mechanical characterization in tension of both dry and fully hydrated scaffolds. Systematic structure-property-processing correlations are obtained for a comparison of the tensile performance of longitudinally and radially freeze-cast collagen scaffolds with their performance in compression. Collated, the correlations, obtained both in tension in this study and in compression for collagen and chitosan in two earlier reports, not only enable the custom-design of freeze-cast biopolymer scaffolds for biomedical applications but also provide new insights into similarities and differences of scaffold and cell-wall structure formation during the directional solidification of "smooth" and "fibrillar" biopolymers.

A Knock-In Mouse Model of Thymoma With the GTF2I L424H Mutation.

INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.

Single-nucleus and spatial transcriptome profiling of pancreatic cancer identifies multicellular dynamics associated with neoadjuvant treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment-refractory cancer. Molecular stratification in pancreatic cancer remains rudimentary and does not yet inform clinical management or therapeutic development. Here, we construct a high-resolution molecular landscape of the cellular subtypes and spatial communities that compose PDAC using single-nucleus RNA sequencing and whole-transcriptome digital spatial profiling (DSP) of 43 primary PDAC tumor specimens that either received neoadjuvant therapy or were treatment naive. We uncovered recurrent expression programs across malignant cells and fibroblasts, including a newly identified neural-like progenitor malignant cell program that was enriched after chemotherapy and radiotherapy and associated with poor prognosis in independent cohorts. Integrating spatial and cellular profiles revealed three multicellular communities with distinct contributions from malignant, fibroblast and immune subtypes: classical, squamoid-basaloid and treatment enriched. Our refined molecular and cellular taxonomy can provide a framework for stratification in clinical trials and serve as a roadmap for therapeutic targeting of specific cellular phenotypes and multicellular interactions.

Spatial meta-transcriptomics reveal associations of intratumor bacteria burden with lung cancer cells showing a distinct oncogenic signature.

BACKGROUND: The lung intratumor microbiome influences lung cancer tumorigenesis and treatment responses, but detailed data on the extent, location, and effects of microbes within lung tumors are missing, information needed for improved prognosis and treatment. METHODS: To address this gap, we developed a novel spatial meta-transcriptomic method simultaneously detecting the expression level of 1,811 host genes and 3 microbe targets (bacteria, fungi, and cytomegalovirus). After rigorous validation, we analyzed the spatial meta-transcriptomic profiles of tumor cells, T cells, macrophages, other immune cells, and stroma in surgically resected tumor samples from 12 patients with early-stage lung cancer. RESULTS: Bacterial burden was significantly higher in tumor cells compared with T cells, macrophages, other immune cells, and stroma. This burden increased from tumor-adjacent normal lung and tertiary lymphoid structures to tumor cells to the airways, suggesting that lung intratumor bacteria derive from the latter route of entry. Expression of oncogenic ß-catenin was strongly correlated with bacterial burden, as were tumor histological subtypes and environmental factors. CONCLUSIONS: Intratumor bacteria were enriched with tumor cells and associated with multiple oncogenic pathways, supporting a rationale for reducing the local intratumor microbiome in lung cancer for patient benefit. TRIAL REGISTRATION NUMBER: NCT00242723, NCT02146170.

Discovery of Biomarkers of Resistance to Immune Checkpoint Blockade in NSCLC Using High-Plex Digital Spatial Profiling.

INTRODUCTION: Despite the clinical efficacy of immune checkpoint inhibitors (ICIs) in NSCLC, only approximately 20% of patients remain disease-free at 5 years. Here, we use digital spatial profiling to find candidate biomarker proteins associated with ICI resistance. METHODS: Pretreatment samples from 56 patients with NSCLC treated with ICI were analyzed using the NanoString GeoMx digital spatial profiling method. A panel of 71 photocleavable oligonucleotide-labeled primary antibodies was used for protein detection in four molecular compartments (tumor, leukocytes, macrophages, and immune stroma). Promising candidates were orthogonally validated with quantitative immunofluorescence. Available pretreatment samples from 39 additional patients with NSCLC who received ICI and 236 non-ICI-treated patients with operable NSCLC were analyzed to provide independent cohort validation. RESULTS: Biomarker discovery using the protein-based molecular compartmentalization strategy allows 284 protein variables to be assessed for association with ICI resistance by univariate analysis using continuous log-scaled data. Of the 71 candidate protein biomarkers, CD66b in the CD45+CD68 molecular compartment (immune stroma) predicted significantly shorter overall survival (OS) (hazard ratio [HR] 1.31, p = 0.016) and was chosen for validation. Orthogonal validation by quantitative immunofluorescence illustrated that CD66b was associated with resistance to ICI therapy but not prognostic for poor outcomes in untreated NSCLC (discovery cohort [OS HR 2.49, p = 0.026], validation cohort [OS HR 2.05, p = 0.046], non-ICI-treated cohort [OS HR 1.67, p = 0.06]). CONCLUSIONS: Using the technique, we have discovered that CD66b expression is indicative of resistance to ICI therapy in NSCLC. Given that CD66b identifies neutrophils, further studies are warranted to characterize the role of neutrophils in ICI resistance.

Intratarget Microdosing for Deep Phenotyping of Multiple Drug Effects in the Live Brain.

A main impediment to effective development of new therapeutics for central nervous system disorders, and for the in vivo testing of biological hypotheses in the brain, is the ability to rapidly measure the effect of novel agents and treatment combinations on the pathophysiology of native brain tissue. We have developed a miniaturized implantable microdevice (IMD) platform, optimized for direct stereotactic insertion into the brain, which enables the simultaneous measurement of multiple drug effects on the native brain tissue in situ. The IMD contains individual reservoirs which release microdoses of single agents or combinations into confined regions of the brain, with subsequent spatial analysis of phenotypic, transcriptomic or metabolomic effects. Using murine models of Alzheimer's disease (AD), we demonstrate that microdoses of various approved and investigational CNS drugs released from the IMD within a local brain region exhibit in situ phenotypes indicative of therapeutic responses, such as neuroprotection, reduction of hyperphosphorylation, immune cell modulation, and anti-inflammatory effects. We also show that local treatments with drugs affecting metabolism provide evidence for regulation of metabolite profiles and immune cell function in hMAPT AD mice. The platform should prove useful in facilitating the rapid testing of pharmacological or biological treatment hypotheses directly within native brain tissues (of various animal models and in patients) and help to confirm on-target effects, in situ pharmacodynamics and drug-induced microenvironment remodeling, much more efficiently than currently feasible.

High-plex expression profiling reveals that implants drive spatiotemporal protein production and innate immune activation for tissue repair.

Surprisingly little clarity exists concerning effects of biomaterial properties on spatially localized protein expression, which drives implant success. Wound healing and tissue regeneration must be optimally supported by the implant, adsorbed proteins, immune cells, and fibroblasts; cells determine repair and functional recovery through protein production and regulation. However, not yet fully understood is how implants differentially drive spatial quantities of individual proteins both within the implant interior and the tissue surrounding it. Here we apply GeoMxⓇ digital spatial profiling to site-specifically investigate protein production in porous implants. Data is collected on the location and quantity of 40+ proteins from formalin-fixed, paraffin-embedded tissue slides of anisotropic tissue scaffolds (n = 18) with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days); matching bulk gene expression data (700+ genes) is measured for identical implants. Notably, we discover fundamental spatial relationships in protein localization that in both the implant interior and the exterior are either uniquely independent or dependent of implant microstructure: dendritic cell marker CD11c and fibronectin significantly dominate the scaffold interior, while cell-to-cell adhesion marker CD34 and anti-inflammatory M2 polarization marker CD163 localize in the exterior. Lastly, collating spatial and bulk information, unique spatiotemporal expression patterns are identified for markers such as fibronectin, which are only uncoverable through spatial profiling and are otherwise hidden in bulk expression results. Together, these discoveries illustrate the critical importance of quantifying spatial expression patterns for implants, facilitating a paradigm shift in the iterative design, mechanistic understanding, and rapid assessment of biomaterials. STATEMENT OF SIGNIFICANCE: Spatial localization and expression of proteins, which determine implant success, are not fully understood because quantitative high-plex profiling is challenging. Applying GeoMxⓇ digital spatial profiling to site-specifically investigate protein production in porous implants, data is collected on the location and quantity of 40+ protein targets from tissue scaffolds with differing pore sizes (35 µm, 53 µm) and implantation durations (2, 14, 28 days). Collecting in parallel matched bulk gene expression data (700+ genes) for identical implants, we discover significant spatiotemporal expression patterns that remain otherwise hidden in differential bulk results. This new approach for the rapid assessment of biomaterials offers an enhanced mechanistic understanding and enables the tailoring of implants for superior regenerative outcomes.

Best Practices for Spatial Profiling for Breast Cancer Research with the GeoMx® Digital Spatial Profiler.

Breast cancer is a heterogenous disease with variability in tumor cells and in the surrounding tumor microenvironment (TME). Understanding the molecular diversity in breast cancer is critical for improving prediction of therapeutic response and prognostication. High-plex spatial profiling of tumors enables characterization of heterogeneity in the breast TME, which can holistically illuminate the biology of tumor growth, dissemination and, ultimately, response to therapy. The GeoMx Digital Spatial Profiler (DSP) enables researchers to spatially resolve and quantify proteins and RNA transcripts from tissue sections. The platform is compatible with both formalin-fixed paraffin-embedded and frozen tissues. RNA profiling was developed at the whole transcriptome level for human and mouse samples and protein profiling of 100-plex for human samples. Tissue can be optically segmented for analysis of regions of interest or cell populations to study biology-directed tissue characterization. The GeoMx Breast Cancer Consortium (GBCC) is composed of breast cancer researchers who are developing innovative approaches for spatial profiling to accelerate biomarker discovery. Here, the GBCC presents best practices for GeoMx profiling to promote the collection of high-quality data, optimization of data analysis and integration of datasets to advance collaboration and meta-analyses. Although the capabilities of the platform are presented in the context of breast cancer research, they can be generalized to a variety of other tumor types that are characterized by high heterogeneity.

Alpha-smooth Muscle Actin Expression in the Stroma Predicts Resistance to Trastuzumab in Patients with Early-stage HER2-positive Breast Cancer.

PURPOSE: The companion diagnostic test for trastuzumab has not changed much in the last 25 years. We used high-plex digital spatial profiling to identify biomarkers besides HER2 that can help predict response to trastuzumab in HER2-positive breast cancer. EXPERIMENTAL DESIGN: Fifty-eight protein targets were measured in three different molecularly defined compartments by the NanoString GeoMx Digital Spatial Profiler (DSP) in a tissue microarray containing 151 patients with breast cancer that received adjuvant trastuzumab as part of the Hellenic Cooperative Oncology Group 10/05 clinical trial. Promising candidate biomarkers were orthogonally validated with quantitative immunofluorescence (QIF). RNA-sequencing data from the Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimisation Study (NeoALTTO) were accessed to provide independent cohort validation. Disease-free survival (DFS) was the main outcome assessed. Statistical analyses were performed using a two-sided test (α = 0.05) and multiple testing correction (Benjamini-Hochberg method, FDR < 0.1). RESULTS: By DSP, high expression of alpha-smooth muscle actin (α-SMA), both in the leukocyte and stromal compartments, was associated with shorter DFS in univariate analysis (P = 0.002 and P = 0.023, respectively). High α-SMA expression in the stroma was validated by QIF after controlling for estrogen receptor and progesterone receptor status [HR, 3.12; 95% confidence interval (CI), 1.12-8.68; P = 0.029] showing recurrence on trastuzumab in the same cohort. In the NeoALTTO cohort, elevated levels of ACTA2 were predictive for shorter DFS in the multivariate analysis (HR, 3.21; 95% CI, 1.14-9.05; P = 0.027). CONCLUSIONS: This work identifies α-SMA as a novel, easy-to-implement biomarker of resistance to trastuzumab that may be valuable in settings where trastuzumab is combined with other therapies.

The CD155/TIGIT axis promotes and maintains immune evasion in neoantigen-expressing pancreatic cancer.

The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.

Structure-property-processing correlations of longitudinal freeze-cast chitosan scaffolds for biomedical applications.

Needed for the custom-design of longitudinally freeze-cast chitosan scaffolds for biomedical applications are systematic structure-property-processing correlations. Combining mechanical testing in compression with both scanning electron microscopy and semiautomated confocal microscopy for a quantitative structural characterization of fully hydrated chitosan scaffolds, robust correlations were determined. Decreasing the applied cooling rate from 10 °C/min to 0.1 °C/min, the short and long axes of the pore cross-sections, the pore aspect ratio, and the pore area were found to increase from 68.0 µm to 120.5 µm, from 189.2 µm to 401.2 µm, from 2.64 to 3.52, and from 8,922 µm2 to 35,596 µm2, respectively. Values for the scaffolds' modulus, yield strength, and toughness range from 1,067 kPa to 3,209 kPa, from 37.7 kPa to 75.5 kPa, and from 20.3 kJ/m3 to 35.3 kJ/m3, respectively. Because of additional structural features, such as cell wall stiffening ridges, affecting the mechanical properties, not linear but more complex correlation with modulus, yield strength, and toughness were observed. Contrasting the results of this study with those obtained in an earlier study of dry and fully hydrated collagen scaffolds, we were able to identify features that are important and peculiar to each material system. Highlighted in this study are newly determined robust structure-property-processing correlations as well as processing conditions and features that are critical for the mechanical performance of chitosan and other biopolymer scaffolds made by freeze casting for biomedical applications.

COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

Quantitative evaluation of the in vivo biocompatibility and performance of freeze-cast tissue scaffolds.

Quantitative methods are little used for the in vivo assessment of tissue scaffolds to evaluate biocompatibility. To complement current histological techniques, we introduce as a measure of biocompatibility a straightforward, geometric analysis for the quantitative assessment of encapsulation thickness, cross-sectional area, and biomaterial shape. Advantages of this new technique are that it enables, on the one hand, a more complete and objective comparison of scaffolds with differing compositions, architectures, and mechanical properties, and, on the other, a more objective approach to their selection for a given application. In this contribution, we focus on freeze-cast polymeric scaffolds for tissue regeneration and their subcutaneous implantation in mice for biocompatibility testing. Initially, seven different scaffold types are screened. Of these, three are selected for systematic biocompatibility studies based on histopathological criteria: EDC-NHS-crosslinked bovine collagen, EDC-NHS-crosslinked bovine collagen-nanocellulose, and chitin. Geometric models developed to quantify scaffold size, ovalization, and encapsulation thickness are tested, evaluated, and found to be a powerful and objective metric for the in vivo assessment of biocompatibility and performance of tissue scaffolds.

Plant-Derived Nanocellulose as Structural and Mechanical Reinforcement of Freeze-Cast Chitosan Scaffolds for Biomedical Applications.

Despite considerable recent interest in micro- and nanofibrillated cellulose as constituents of lightweight structures and scaffolds for applications that range from thermal insulation to filtration, few systematic studies have been reported to date on structure-property-processing correlations in freeze-cast chitosan-nanocellulose composite scaffolds, in general, and their application in tissue regeneration, in particular. Reported in this study are the effects of the addition of plant-derived nanocellulose fibrils (CNF), crystals (CNCs), or a blend of the two (CNB) to the biopolymer chitosan on the structure and properties of the resulting composites. Chitosan-nanocellulose composite scaffolds were freeze-cast at 10 and 1 °C/min, and their microstructures were quantified in both the dry and fully hydrated states using scanning electron and confocal microscopy, respectively. The modulus, yield strength, and toughness (work to 60% strain) were determined in compression parallel and the modulus also perpendicular to the freezing direction to quantify anisotropy. Observed were the preferential alignments of CNCs and/or fibrils parallel to the freezing direction. Additionally, observed was the self-assembly of the nanocellulose into microstruts and microbridges between adjacent cell walls (lamellae), features that affected the mechanical properties of the scaffolds. When freeze-cast at 1 °C/min, chitosan-CNF scaffolds had the highest modulus, yield strength, toughness, and smallest anisotropy ratio, followed by chitosan and the composites made with the nanocellulose blend, and that with crystalline cellulose. These results illustrate that the nanocellulose additions homogenize the mechanical properties of the scaffold through cell-wall material self-assembly, on the one hand, and add architectural features such as bridges and pillars, on the other. The latter transfer loads and enable the scaffolds to resist deformation also perpendicular to the freezing direction. The observed property profile and the materials' proven biocompatibility highlight the promise of chitosan-nanocellulose composites for a large range of applications, including those for biomedical implants and devices.

Values and property charts for anisotropic freeze-cast collagen scaffolds for tissue regeneration.

Presented in this article are systematic microstructural and mechanical property data for anisotropic collagen scaffolds made by freeze casting. Three applied cooling rates (10 °C/min, 1 °C/min, 0.1 °C/min) and two freezing directions (longitudinal and radial) were used during scaffold manufacture. Utilizing a semi-automated image analysis technique applied to confocal micrographs of fully hydrated scaffolds, pore area, long and short pore axes, and pore aspect ratio were determined. Compression testing was performed to determine scaffold modulus, yield strength, and toughness.

Anisotropic freeze-cast collagen scaffolds for tissue regeneration: How processing conditions affect structure and properties in the dry and fully hydrated states.

Few systematic structure-property-processing correlations for directionally freeze-cast biopolymer scaffolds are reported. Such correlations are critical to enable scaffold design with attractive structural and mechanical cues in vivo. This study focuses on freeze-cast collagen scaffolds with three different applied cooling rates (10, 1, and 0.1 °C/min) and two freezing directions (longitudinal and radial). A semi-automated approach for the structural characterization of fully hydrated scaffolds by confocal microscopy is developed to facilitate an objective quantification and comparison of structural features. Additionally, scanning electron microscopy and compression testing are performed longitudinally and transversely. Structural and mechanical properties are determined on dry and fully hydrated scaffolds. Longitudinally frozen scaffolds have aligned and regular pores while those in radially frozen ones exhibit greater variations in pore geometry and alignment. Lamellar spacing, pore area, and cell wall thickness increase with decreasing cooling rate: in longitudinally frozen scaffolds from 25 µm to 83.5 µm, from 814 µm2 to 8452 µm2, and from 4.21 µm to 10.4 µm, and in radially frozen ones, from 69 µm to 116 µm, from 7679 µm2 to 25,670 µm2, and from 6.18 µm to 13.6 µm, respectively. Both longitudinally and radially frozen scaffolds possess higher mechanical property values, when loaded parallel rather than perpendicular to the ice-crystal growth direction. Modulus and yield strength range from 779 kPa to 4700 kPa and from 38 kPa to 137 kPa, respectively, as a function of cooling rate and freezing direction. Collated, the correlations obtained in this study enable the custom-design of freeze-cast collagen scaffolds, which are ideally suited for a large variety of tissue regeneration applications.