Preserving Tissue Structure Research Articles

Super‐Resolution of Histopathological Frozen Sections via Deep Learning Preserving Tissue Structure

Histopathology plays a pivotal role in medical diagnostics. In contrast to preparing permanent sections for histopathology, a time‐consuming process, preparing frozen sections is significantly faster and can be performed during surgery, where the sample scanning time should be optimized. Super‐resolution techniques allow imaging of histopathalogical samples in lower magnification, thus sparing scanning time. Herein, a new approach is presented to super‐resolution of histopathological frozen sections, with focus on achieving better distortion measures, rather than pursuing photorealistic images that may compromise critical diagnostic information. Our deep‐learning architecture focuses on learning the error between interpolated images and real images; thereby generating high‐resolution images while preserving critical image details, which reduces the risk of diagnostic misinterpretation. This is done by leveraging the loss functions in the frequency domain and assigning higher weights to the reconstruction of complex, high‐frequency components. In comparison with existing methods, significant improvements are obtained in terms of distortion metrics, improving the pathologist's clinical decisions. This approach has a great potential to provide faster frozen‐section imaging, with less scanning, speeding up intraoperative decisions, while preserving the high‐resolution details in the imaged sample.

Updated techniques and evidence for endoscopic ultrasound-guided tissue acquisition from solid pancreatic lesions.

Endoscopic ultrasound-guided tissue acquisition (EUS-TA), including fine-needle aspiration (EUS-FNA) and fine-needle biopsy (EUS-FNB), has revolutionized specimen collection from intra-abdominal organs, especially the pancreas. Advances in personalized medicine and more precise treatment have increased demands to collect specimens with higher cell counts, while preserving tissue structure, leading to the development of EUS-FNB needles. EUS-FNB has generally replaced EUS-FNA as the procedure of choice for EUS-TA of pancreatic cancer. Various techniques have been tested for their ability to enhance the diagnostic performance of EUS-TA, including multiple methods of sampling at the time of puncture, on-site specimen evaluation, and specimen processing. In addition, advances in next-generation sequencing have made comprehensive genomic profiling of EUS-TA samples feasible in routine clinical practice. The present review describes updates in EUS-TA sampling techniques of pancreatic lesions, as well as methods for their evaluation.

Oxidative Stress, inflammation and metallomatrix Proteinase-Regulated hydrogels for intervertebral disc regeneration

Intervertebral disc degeneration (IVDD) is still a challenge and the efficient treatment approaches are still lacking, due to the high oxidative stress and inflammation and matrix degradation. The utilization of bioactive hydrogels as carriers for therapeutic factors offers a powerful means to enhance IVDD treatment. However, the therapeutic efficacy of existing hydrogels heavily relies on the loaded therapeutic drugs, resulting in reduced treatment efficiency. Herein, we report a dual-component polymer network hydrogel (FPFG) based on gelatin methacrylate (GelMA) and poly (ferulic acid)-F127- poly (ferulic acid) (FPF). The FPFG hydrogel exhibited injectability, antioxidative properties, good biocompatibility, and biodegradability. FPFG hydrogel could efficiently suppress the intracellular reactive oxygen species (ROS) and metallomatrix proteinase in nucleus pulposus (NP) cells, inhibit the expression of inflammatory factors in macrophages and enhance the extracellular matrix formation. The rat puncture model revealed that the FPFG hydrogel effectively restored disc height, maintained disc hydration, and preserved tissue structure. By exerting anti-inflammatory and antioxidant activities, the FPFG hydrogel successfully ameliorated the hostile microenvironment commonly found in degenerated disc tissue. These findings highlight the potential application of FPFG hydrogel in enhancing IVDD treatment.

Triple-layered platform utilizing electrospun nanofibers and 3D-printed sodium alginate-based hydrogel for effective topical treatment of rheumatoid arthritis

Rheumatoid arthritis (RA), an autoimmune disease impacting the joints, significantly diminishes the quality of life for patients. Conventional treatments predominantly rely on oral or injectable formulations, underscoring the crucial need for an effective topical remedy. The present study reports a novel triple-layered transdermal platform for efficient RA treatment. The patches are based on an electrospun/electrosprayed diclofenac (DIC)-conjugated polyvinyl alcohol (PVA) nanofibers/nanoparticles (NFs/NPs) composite layer sandwiched between an electrospun supporting layer of polycaprolactone (PCL) NFs, and a 3D-printed sodium alginate-based hydrogel (HG) layer incorporating sodium hyaluronate (HA) and rosuvastatin (ROS)-loaded core-shell lipid nanocapsules (LNCs). The ingeniously designed transdermal patches release the chemically conjugated DIC via skin-secreted esterases at the inflamed sites. The LNCs and patches were characterized using DLS, FTIR, DSC, and electron microscopy. ROS-loaded LNCs (<50 nm as per the TEM micrographs) were able to release about 97 % of ROS during 5 days. In-vitro and in-vivo evaluations definitively established the efficacy of the developed platform, showcasing a substantial reduction in IL-6 and TNF-α through sandwich ELISA measurements in cell culture and Rattus norvegicus plasma samples. Besides, the stained photomicrographs of the rats' ankle joints confirmed the alleviation of the RA symptoms via reducing cell infiltration with a preserved joint tissue structure.

New Optimized Academic Anti-BCMA CAR (CARTemis-1): How Does the Manufacturing Process Impact CAR-T Cell Features?

Introduction CAR therapy targeting BCMA (B-cell maturation antigen) is under research as treatment for multiple myeloma (MM). However, given the lack of a plateau in most studies, it becomes imperative to pursue more effective alternatives. BCMA downmodulation, the presence of soluble BCMA or the immune dysfunction observed in MM are some limitations that may affect efficacy. Next-generation CAR-T cells are being designed exploring new approaches to overcome restrictions to CAR-T efficacy in MM. We present a novel optimized anti-BCMA CAR (CARTemis-1) and its translation to the clinic. We have specifically focused on the study of the dynamics of the immunophenotype of CARTemis-1 along the manufacturing process, analyzing how these variables might impact the quality of the final product and influence the release decision. Materials and methods CARTemis-1 was evaluated in preclinical in vitro and in vivo models. CARTemis-1 generation from three healthy donors was validated under GMP-conditions in CliniMACS Prodigy with IL-7/IL-15. Flow cytometry was performed to characterize the immunophenotype along the manufacturing process. Quality controls of the final CARTemis-1 product were performed according to the criteria of the Spanish Medicine Agency. Stability of the final cryopreserved CARTemis-1 cell product was also analyzed. Results CARTemis-1 is a second generation anti-BCMA CAR that co-expresses the truncated epidermal growth factor receptor (EGFRt) as a transduction marker and suicide gene. Two different versions of CARTemis-1 were synthesized with different spacer regions (short vs long) with the long version showing significantly greater anti-myeloma function in vitro compared to the short version (p&amp;lt;0.001) (Figure 1A). During the preclinical validation, CARTemis-1 demonstrated potent anti-tumor capacity in vitro and in vivo, showing no susceptibility to the presence of soluble BCMA. Expansion with IL-2 or IL-7/IL-15 was compared (n=8), obtaining increased proliferation (mean 1.59x10 7 vs 3.07x10 7, respectively, p=0.002), increased stem cell memory and naïve population (mean 37.48% vs 43.95%, respectively, p=0.0052), and less PD1+LAG3+ levels (1.38% vs 0.55%, respectively, p=0.0338) with IL-7/IL-15. The generation of CARTemis-1 was validated under GMP-conditions closely monitoring the immunophenotype dynamics from leukapheresis to the final product. Through comprehensive evaluations, the optimal time-point for product release to achieve the best-fitness product was &amp;gt;6 (specifically, day 8-10) with reduced levels of exhaustion markers (Figure 1B) maintaining hallmarks of T cell activation and anti-tumor potency. In vivo biodistribution of CARTemis-1 cells was studied in mice models without myeloma cells, finding preserved tissue structure without relevant morphological alterations in brain, cerebellum, intestine, lung, endometrium, ovary, testicle, spleen, and liver. We performed graft-versus-host disease (GVHD) in vivo mice models and found no increase in GVHD incidence following CARTemis-1 inoculation. Additionally, we assessed the cryopreserved product stability, and it met all specifications and retained its functionality for up to 12 months after cryopreservation. With this positive outcome and having obtained the approval of the Spanish Medicine Agency, we plan to proceed with a phase I/II clinical trial using CARTemis-1 for patients with multiple myeloma who have experienced relapse following allogeneic transplantation (EudraCT 2021-001955-15). Conclusions CARTemis-1 has been rationally designed to increase anti-tumor efficacy and overcomes sBCMA inhibition. To our knowledge, this is the first study analyzing the impact of the manufacturing process in the dynamics of the CAR-T immunophenotype highlighting the importance of the immunophenotypic characterization of CAR-T cells throughout the manufacturing process to define the optimal cell culture protocol and expansion time to increase CAR-T cell product fitness.

Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Comprehensive review on endoscopic ultrasound-guided tissue acquisition techniques for solid pancreatic tumor.

Pancreatic ductal adenocarcinoma is speculated to become the second leading cause of cancer-related mortality by 2030, a high mortality rate considering the number of cases. Surgery and chemotherapy are the main treatment options, but they are burdensome for patients. A clear histological diagnosis is needed to determine a treatment plan, and endoscopic ultrasound (EUS)-guided tissue acquisition (TA) is a suitable technique that does not worsen the cancer-specific prognosis even for lesions at risk of needle tract seeding. With the development of personalized medicine and precision treatment, there has been an increasing demand to increase cell counts and collect specimens while preserving tissue structure, leading to the development of the fine-needle biopsy (FNB) needle. EUS-FNB is rapidly replacing EUS-guided fine-needle aspiration (FNA) as the procedure of choice for EUS-TA of pancreatic cancer. However, EUS-FNA is sometimes necessary where the FNB needle cannot penetrate small hard lesions, so it is important clinicians are familiar with both. Given these recent dev-elopments, we present an up-to-date review of the role of EUS-TA in pancreatic cancer. Particularly, technical aspects, such as needle caliber, negative pressure, and puncture methods, for obtaining an adequate specimen in EUS-TA are discussed.

MALDI mass spectrometry imaging workflow for the aquatic model organisms Danio rerio and Daphnia magna

Lipids play various essential roles in the physiology of animals. They are also highly dependent on cellular metabolism or status. It is therefore crucial to understand to which extent animals can stabilize their lipid composition in the presence of external stressors, such as chemicals that are released into the environment. We developed a MALDI MS imaging workflow for two important aquatic model organisms, the zebrafish (Danio rerio) and water flea (Daphnia magna). Owing to the heterogeneous structure of these organisms, developing a suitable sample preparation workflow is a highly non-trivial but crucial part of this work and needs to be established first. Relevant parameters and practical considerations in order to preserve tissue structure and composition in tissue sections are discussed for each application. All measurements were based on high mass accuracy enabling reliable identification of imaged compounds. In zebrafish we demonstrate that a detailed mapping between histology and simultaneously determined lipid composition is possible at various scales, from extended structures such as the brain or gills down to subcellular structures such as a single axon in the central nervous system. For D. magna we present for the first time a MALDI MSI workflow, that demonstrably maintains tissue integrity during cryosectioning of non-preserved samples, and allows the mapping of lipids in the entire body and the brood chamber inside the carapace. In conclusion, the lipid signatures that we were able to detect with our method provide an ideal basis to analyze changes caused by pollutants in two key aquatic model organisms.

Establishment of porcine and monkey colonic organoids for drug toxicity study

Pig and monkey are widely used models for exploration of human diseases and evaluation of drug efficiency and toxicity, but high cost limits their uses. Organoids have been shown to be promising models for drug test as they reasonably preserve tissue structure and functions. However, colonic organoids of pig and monkey are not yet established. Here, we report a culture medium to support the growth of porcine and monkey colonic organoids. Wnt signaling and PGE2 are important for long-term expansion of the organoids, and their withdrawal results in lineage differentiation to mature cells. Furthermore, we observe that porcine colonic organoids are closer to human colonic organoids in terms of drug toxicity response. Successful establishment of porcine and monkey colonic organoids would facilitate the mechanistic investigation of the homeostatic regulation of the intestine of these animals and is useful for drug development and toxicity studies.

Air impingement to reduce thawing time of chicken fingers for food service

Air impingement (AI) thawing was studied as alternative to time-consuming thawing, conventionally performed in refrigerators for food service. Chicken fingers’ bags (200 g), surrounded by Tylose to simulate a food service grid load, were thawed in an AI prototype. Nine combinations of process parameters (distance food to nozzles, air speed, air temperature, number of stages, and stage durations) identified by genetic algorithm optimization were applied. The optimization aimed at minimizing thawing time, food overheating (above 0℃), and food temperature unevenness. Samples were assessed for thawing time (to 0℃), thawing loss, water-holding capacity, firmness, microstructure, and FTIR spectra. AI allowed up to 85% thawing time reduction compared to refrigerator (4 ± 1℃), while thawing loss, water-holding capacity, and firmness were not significantly different (p > .05). Histological analysis and FTIR spectra indicated AI as an interesting technology to preserve tissue structure. Novelty impact statement In this paper, air impingement (AI) is applied for the first time to quickly thaw chicken fingers while avoiding overheating (above 0℃) and structural modification of meat. These results support air impingement as a valid alternative to refrigeration thawing, commonly applied for food preparation in food service.

Optimised tissue clearing minimises distortion and destruction during tissue delipidation.

AimsA variety of tissue clearing techniques have been developed to render intact tissue transparent. For thicker samples, additional partial tissue delipidation is required before immersion into the final refractive index (RI)‐matching solution, which alone is often inadequate to achieve full tissue transparency. However, it is difficult to determine a sufficient degree of tissue delipidation, excess of which can result in tissue distortion and protein loss. Here, we aim to develop a clearing strategy that allows better monitoring and more precise determination of delipidation progress.MethodsWe combined the detergent sodium dodecyl sulphate (SDS) with OPTIClear, a RI‐matching solution, to form a strategy termed Accurate delipidation with Optimal Clearing (Accu‐OptiClearing). Accu‐OptiClearing allows for a better preview of the final tissue transparency achieved when immersed in OPTIClear alone just before imaging. We assessed for the changes in clearing rate, protein loss, degree of tissue distortion, and preservation of antigens.ResultsPartial delipidation using Accu‐OptiClearing accelerated tissue clearing and better preserved tissue structure and antigens than delipidation with SDS alone. Despite achieving similar transparency in the final OPTIClear solution, more lipids were retained in samples cleared with Accu‐OptiClearing compared to SDS.ConclusionsCombining the RI‐matching solution OPTIClear with detergents, Accu‐OptiClearing, can avoid excessive delipidation, leading to accelerated tissue clearing, less tissue damage and better preserved antigens.

Deciphering the Immune Microenvironment on A Single Archival Formalin-Fixed Paraffin-Embedded Tissue Section by An Immediately Implementable Multiplex Fluorescence Immunostaining Protocol.

Simple SummaryTechnological breakthroughs have fundamentally changed our understanding on the complexity of tissue organization, both in healthy and diseased conditions. Characterizing the immune cell composition in relation to spatial distribution and histological changes may provide important diagnostic and therapeutic information. Immunohistochemistry remains a method of choice for these purposes, with crucial implications in clinics or in medical research. Nowadays, the widespread use of fluorophore-conjugated antibodies enables simultaneous visualization of an increasing number of proteins on a single tissue slice, with unprecedented resolution. However, advanced methods usually require modern and specific equipment, a significant amount of time for assay optimization, and highly specialized skills. This work reports on the use of a multiplex immunostaining method based on sequential immunostaining and antibody stripping, combined with digital image processing and analysis. Our aim is to provide the medical and research communities with a simple, cost-effective workflow to encompass some current limitations of multiplex immunohistochemistry.Technological breakthroughs have fundamentally changed our understanding on the complexity of the tumor microenvironment at the single-cell level. Characterizing the immune cell composition in relation to spatial distribution and histological changes may provide important diagnostic and therapeutic information. Immunostaining on formalin-fixed paraffin-embedded (FFPE) tissue samples represents a widespread and simple procedure, allowing the visualization of cellular distribution and processes, on preserved tissue structure. Recent advances in microscopy and molecular biology have made multiplexing accessible, yet technically challenging. We herein describe a novel, simple and cost-effective method for a reproducible and highly flexible multiplex immunostaining on archived FFPE tissue samples, which we optimized for solid organs (e.g., liver, intestine, lung, kidney) from mice and humans. Our protocol requires limited specific equipment and reagents, making multiplexing (>12 antibodies) immediately implementable to any histology laboratory routinely performing immunostaining. Using this method on single sections and combining it with automated whole-slide image analysis, we characterize the hepatic immune microenvironment in preclinical mouse models of liver fibrosis, steatohepatitis and hepatocellular carcinoma (HCC) and on human-patient samples with chronic liver diseases. The data provide useful insights into tissue organization and immune–parenchymal cell-to-cell interactions. It also highlights the profound macrophage heterogeneity in liver across premalignant conditions and HCC.

Age-dependent changes in glutathione metabolism pathways in the lens: New insights into therapeutic strategies to prevent cataract formation-A review.

Ocular tissues possess a robust antioxidant defence system to minimize oxidative stress and preserve tissue structure and function. Glutathione (GSH) is a powerful antioxidant and in the lens exists at unusually high concentrations. However, with advancing age, GSH levels deplete specifically in the lens centre initiating a chain of biochemical events that ultimately result in protein aggregation, light scattering and age-related nuclear cataract. However, antioxidant supplementation has been shown to be ineffective in preventing or delaying cataract indicating that a better understanding of the delivery, uptake and metabolism of GSH in the different regions of the lens is required. This information is essential for the development of scientifically informed approaches that target the delivery of GSH to the lens nucleus, the region of the lens most affected by age-related cataract.

Comparison of routine and microwave-assisted decalcification of bone with or without teeth: A histologic study

The aim of this study was to compare conventional and microwave-assisted decalcification of sheep bone with and without teeth and to detect any difference in tissue detail preservation, staining quality, and rate of decalcification. In this method analysis study, twenty-four specimens consisting of 12 blocks of mandibular molars with their surrounding bone and 12 blocks of mandibular osseous tissue were allocated into two microwave or routine decalcification groups using 5% nitric or formic acid as decalcifying agents. In addition to decalcification rate, a number of variables were used to assess staining quality and tissue detail preservation which were compared between the two groups using Mann-Whitney test (P < 0.05). Time to complete decalcification was significantly reduced in the microwave-treated samples as compared to the conventional method, regardless of the decalcifying agent (P = 0.025). For both acids, most variables related to staining quality and tissue detail preservation were similar between the techniques (P > 0.05). Patchy staining in bone samples and tissue tears in bone + teeth specimens were more common in the routine method when using nitric acid (NAc) (P = 0.046) and formic acid (FAc) (P = 0.046), respectively. In comparing acids, the performance of FAc was slightly inferior to that of NAc, especially for specimens containing both tooth and bone. The use of microwave technology can accelerate decalcification of bone and teeth of sheep mandibles and at the same time preserve tissue structure and staining quality. Further studies are required to help select the best demineralizing agent, especially in specimens containing bone and teeth.

Temporal evolution of acute multiple sclerosis lesions on serial sodium (23Na) MRI

BackgroundSeveral studies have reported the characteristics of acute multiple sclerosis (MS) lesions on diffusion-weighted magnetic resonance imaging (DWI MRI). Current publications reported a transient reduction of the apparent diffusion coefficient (ADC) delineating an early phase of lesion evolution, before increased diffusion occurs in parallel to blood-brain-barrier (BBB) breakdown. Sodium MRI might provide another perspective on lesion development, but clinical applications have been limited to high field MR systems. The objective in this study was to investigate the temporal evolution of acute MS lesions using conventional (T2-fluid-attenuated inversion recovery (T2-FLAIR) images, post-contrast T1-weighted images), diffusion and sodium MRI. MethodsInitial and follow-up MRI (23Na and 1H MRI) were performed on a 3T scanner. Quantitative assessment of total sodium concentration (TSC) and ADC was performed. The study was designed for frequent follow-up MRI examinations during 4 weeks after the initial presentation. ResultsThirty-one acute MS lesions (7 lesions with reduced diffusion) in eleven MS patients were included. On initial MRI, TSC in contrast-enhancing lesions was increased when compared to the normal-appearing white matter (NAWM), while lesions with an initial reduced diffusion showed a TSC comparable to the NAWM. On follow-up MRI, in lesions with reduced diffusion subsequent increase of ADC and TSC values occurred along with signs of the development of vasogenic edema and contrast-enhancement. After four weeks, TSC values decreased along with regression of vasogenic edema and contrast-enhancement. ConclusionsIn lesions with a reduction of the ADC sodium levels are near normal and precede signs of BBB breakdown. These findings suggest a relatively preserved tissue structure in this early phase of lesion evolution.

Peroxisome proliferator activated receptor-gamma stimulation for prevention of 5-fluorouracil-induced oral mucositis in mice.

Oral mucositis is a side effect of treatment regimens containing 5-fluorouracil (5-FU). The purpose of this study was to present our evaluation to see if rosiglitazone (RGZ) protected normal tissues from chemotherapy-induced oral mucositis. C57BL/6J mice were treated with 5-FU for 5 days, with or without RGZ. Mice were euthanized after 5, 8, 11, or 15 days, and mucosal segments were collected. The RGZ did not affect the 5-FU-induced decrease in mouse body weight. The 5-FU caused loss of epithelial architecture, collagen fiber impairment, and inflammatory infiltration. The RGZ reduced leukocyte infiltration, preserved tissue structure, and dampened the 5-FU-induced expression of p53 and matrix metalloproteinase (Mmp)-2 after 5 days, and of Mmp-2 and interleukin (Il-1β after 15 days. The RGZ inhibited the 5-FU-induced increase of transforming growth factor-beta (TGF-β) and nuclear factor-kappa B (NF-κB) proteins and restored collagen protein levels. The RGZ had a protective effect on oral mucosa damaged by chemotherapy. These data encourage the further study of RGZ for the prevention of 5-FU-induced mucositis in patients with cancer.

A novel supercritical CO2-based decellularization method for maintaining scaffold hydration and mechanical properties

Decellularized tissues are commonly utilized as tissue engineering scaffolds. Decellularization by extended exposure to aqueous detergents can damage the microstructure or deposit cytotoxic residue. Supercritical carbon dioxide (scCO2) has been proposed for decellularization, but reportedly causes dehydration and scaffold embrittlement.Presented herein is a novel decellularization method that preserves matrix hydration state and mechanical properties. Over 97% of the water in porcine aorta is maintained by presaturating scCO2 with water; however, complete decellularization was not attained by any process utilizing only scCO2. Instead, a novel hybrid method is presented that combines a brief (48h) exposure of tissue to aqueous detergent, followed by washing with scCO2 (1h). The hybrid method fully decellularized the tissue, as confirmed by histology and DNA quantification (<0.04μg DNA/mg tissue). This hybrid treatment was faster than the standard method (2days compared to 4–7days), while preserving tissue structure and mechanical properties.

Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies.

Age-related macular degeneration (AMD) is a devastating disease affecting more than 600 million people worldwide. Endothelial cells of the choriocapillaris (CECs) are among the first cell types lost in early AMD, and cell replacement therapy is currently the most promising option for restoring vision in patients with advanced AMD. In order to study CEC replacement strategies we have generated a 3D choroid scaffold using a novel decellularization method in human RPE/choroid tissue. To our knowledge, this is the first report describing decellularization of human RPE/choroid, as well as recellularization of a choroid scaffold with CECs. This work will aid in our development and optimization of cell replacement strategies using a tissue scaffold that is similar to the in vivo environment.

The Use of Mouse Models of Breast Cancer and Quantitative Image Analysis to Evaluate Hormone Receptor Antigenicity after Microwave-assisted Formalin Fixation

Microwave methods of fixation can dramatically shorten fixation times while preserving tissue structure; however, it remains unclear if adequate tissue antigenicity is preserved. To assess and validate antigenicity, robust quantitative methods and animal disease models are needed. We used two mouse mammary models of human breast cancer to evaluate microwave-assisted and standard 24-hr formalin fixation. The mouse models expressed four antigens prognostic for breast cancer outcome: estrogen receptor, progesterone receptor, Ki67, and human epidermal growth factor receptor 2. Using pathologist evaluation and novel methods of quantitative image analysis, we measured and compared the quality of antigen preservation, percentage of positive cells, and line plots of cell intensity. Visual evaluations by pathologists established that the amounts and patterns of staining were similar in tissues fixed by the different methods. The results of the quantitative image analysis provided a fine-grained evaluation, demonstrating that tissue antigenicity is preserved in tissues fixed using microwave methods. Evaluation of the results demonstrated that a 1-hr, 150-W fixation is better than a 45-min, 150-W fixation followed by a 15-min, 650-W fixation. The results demonstrated that microwave-assisted formalin fixation can standardize fixation times to 1 hr and produce immunohistochemistry that is in every way commensurate with longer conventional fixation methods.

Evaluation of Small Intestine Grafts Decellularization Methods for Corneal Tissue Engineering

Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.