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In the second reconstructive phase of the breast after mastectomy, lipofilling is often necessary. Currently, lipofilling occurs immediately after autologous adipose tissue harvesting procedure, but most of the patients, usually, require multiple sessions to obtain a satisfactory result. Therefore, the need of repeated surgical harvesting outputs implies high risk of patients’ morbidity and discomfort as well as increasing medical time and costs. The aim of our pilot study was to find out a feasible method to cryopreserve adipose tissue, in order to avoid reiterated liposuctions. Lipoaspirates samples have been harvested from 10 women and preserved by three methods: (1) the first one, using 10% Me2SO and 20% human albumin from human plasma as cryoprotective agents; (2) the second one, adding 5% Me2SO as cryoprotective agent; 3) the last one, without any cryoprotective agent. Fresh and cryopreserved fat samples, obtained through the aforementioned processes, have been analyzed ex vivo. The efficiency of the cryopreservation methods used was determined by adipocyte viability and the expression of adipocytes surface markers. Lipoaspirates stored at −196 °C for 3 months, after thawing, retained comparable adipocyte viability and histology to fresh tissue and no significant differences were found between the three methods used. Although the current results, differences between the methodologies in terms of viability may not become evident until breast lipofilling using frozen-thawed cryopreserved tissue.

Objective To test the applicability of a fresh tissue model for teaching facial plastic techniques and approaches to the eyelids and orbit. Design Observational animal experiments. Subjects Ten prepubescent sheep heads harvested following humane euthanasia at the completion of unrelated live animal research. Methods Young sheep were saline perfused at the end of an in vivo protocol. Head and neck tissues were harvested and refrigerated for 3–7 days. An experienced oculoplastic surgeon and an otolaryngologist explored the feasibility of common oculoplastic procedures in the ovine model. Results The model has potential for teaching basic principles in eyelid surgery including upper lid blepharoplasty, aponeurotic ptosis repair, upper lid gold weight lid loading for facial paralysis, lateral canthotomy and inferior limb cantholysis, lower lid tightening, and transconjunctival approach to the orbital floor. Eye muscle advancement, optic nerve sheath fenestration, and enucleation also accurately simulated human surgery. Anatomic variations limit the sheep model for orbital floor reconstruction and lacrimal drainage procedures. Conclusions The sheep head and neck provide an inexpensive, safe model for developing skills in several oculoplastic procedures. Formal simulation testing is needed to confirm these expert opinions.

Anatomi biliminde, ölümden sonra hayvan veya insan bedenlerinde meydana gelen değişikliklerin (koku, renk, elastikiyet) en aza indirilmesini sağlamak için kullanılan birçok koruma tekniği vardır. Bu teknikler arasında, bilinen en modern anatomik örnek hazırlama yöntemlerinden biri, plastinasyon tekniğidir. Bu sebeple bu çalışmada kas dokusuna temel plastinasyon aşamaları uygulandı. Çalışmada, standart silikon plastinasyon tekniğinde son ürün elde edilene kadar geçen her aşamada kas dokusunda meydana gelen renk değişimlerinin kolorimetre cihazıyla sayısal olarak ortaya konulması amaçlandı. Plastinasyonun her aşamasında, kas dokusuna renk analizi yapıldı. İlk aşamadan son aşamaya kadar olan tüm süreç boyunca renk değişimi değerlendirildiğinde; elde edilen son üründeki yeşilden kırmızıya renk değişiminin taze dokuya en yakın olduğu aşamanın; 1. aseton banyosu olduğu gözlemlendi. Maviden sarıya değişimin taze dokuya en yakın olduğu aşamanın ise gaz kürleme ve sertleştirme aşaması olduğu görüldü. Ayrıca plastinatların parlaklık açısından değişimi ele alındığında; taze dokuya en yakın değerleri, vakumla gömme aşamasından sonra kaydedildi. Yapılan bu çalışmayla, bugüne kadar doğal renge yakın olarak tanımlanan plastinatlardaki renk değişimi sayısal verilerle belirlendi. Bununla birlikte bu tez çalışması sonucunda kolorimetre cihazının, sahip olduğu avantajlar sebebiyle, anatomi alanında oldukça etkili bir şekilde kullanılabilir olduğu ortaya konuldu., There are many preservation techniques that are used to ensure that the changes (odor, color, and elasticity) in the characteristics of the animal or human bodies after death are minimized in the field of anatomy. One of the most modern anatomic specimen preparation methods is the plastination technique. Therefore, primary plastination stages were applied to the muscle tissue in this study. The aim of the current study is to present color changes in muscle tissue quantitatively by using a colorimeter device in every stage of the standard silicone plastination technique until the last product is obtained. Color analyses were performed on the muscle tissue after each stage of the plastination. Throughout the whole process, it was observed that the stage when the value of color change from green to red in the product was the closest to the fresh tissue was the 1st acetone bath. The value of color change from blue to yellow was closest to the fresh tissue at the gas curing and hardening stage. Furthermore, the closest values to the fresh tissue were recorded after the impregnation stage when the variations in plastinates were evaluated in terms of brightness. Color changes in plastinates, which have been described as close to the natural color up to today, were determined through statistical data in this study. Moreover, as a result of this dissertation, it was asserted that colorimeter can be effectively used in the field of anatomy due to the advantages it holds.

Among women, ovarian cancer (OC) is one of the most severe forms of malignancy, accounting for a low 5-year survival rate, of approximately 52%. Early symptoms are unspecific and hence hard to detect. The origin of OC and its subtypes are still unclear, underlying the need for efficient diagnostic biomarkers. In that regard, epigenetics studies are emerging in cancer diagnostics, with encouraging outcomes. Among them, DNA methylation profiling has shown that the origins of the cancer epigenome are associated with molecular factors that are crucial to carcinogenesis, such as regulation of oncogenes and tumor suppressors. Furthermore, those events have been detected in abnormal cell morphology before neoplastic formation, indicating its potential crucial use in the OC diagnostics in the future. Nonetheless, studies are limited, and whether methylation analysis can be performed optimally in formalin-fixed paraffin-embedded (FFPE) preparations of OC cases is still elusive. In the present report, we investigated the performance of DNA methylation analysis in FFPE samples, compared to their matched fresh frozen tissue in a small cohort of OC samples. We found that the overall DNA methylation profile in FFPE tissue showed high concordance to that found in fresh frozen tissue, and accounting for the small cohort size, the differentially methylated sites found primarily in frozen tissue, compared to benign samples, were also reproducible in FFPE. Overall, by using samples from our current clinical setting of tissue preservation, these preliminary observations might provide insights into the clinical use of FFPE tissues in methylation studies without critically compromising the outcome. Supplementary Information The online version contains supplementary material available at 10.1007/s43032-021-00589-0.

One of the most widely used solutions to fix and preserve organic tissues is formaldehyde, despite reservations regarding its toxicity and the fact that formaldehyde-embalmed bodies lose their original characteristics. Anatomy laboratories have been replacing formaldehyde with solutions that retain the characteristics of fresh tissue. For this purpose, alternative solutions with a very low concentration of formaldehyde or without any formaldehyde have been analyzed. The objective of this study was to compare biometry, coloration, haptic properties, and bacterial load on animal specimens (pig kidneys) embalmed with formaldehyde, and with Chilean Conservative Fixative Solution with and without formaldehyde (formaldehyde chCFS and formaldehyde-free chCFS). Also, the perception of health and biological science students towards specimens treated with different solutions was assessed. The results indicated that there were no significant differences in specimens' retraction, or bacterial load. Students showed a preference for organs embalmed in formaldehyde chCFS and formaldehyde-free chCFS; indicating that with these treatments they could better visualize structures and that the prosections had greater flexibility and the colors were more similar to those of fresh tissue. Additionally, students recommended the material embalmed in formaldehyde chCFS and formaldehyde-free chCFS for anatomy learning. In contrast, students indicated that formaldehyde-fixation negatively affected their practical experience. In conclusion, embalming with formaldehyde chCFS or formaldehyde-free chCFS provides an advantageous practical experience over the use of formaldehyde and may be an alternative to replace the use of formaldehyde in anatomy laboratories.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. “biobanking” of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-020-01952-z.

The practical application of genome-scale technologies to precision oncology research requires flexible tissue processing strategies that can be used to differentially select both tumour and normal cell populations from formalin-fixed, paraffin-embedded tissues. As tumour sequencing scales towards clinical implementation, practical difficulties in scheduling and obtaining fresh tissue biopsies at scale, including blood samples as surrogates for matched 'normal' DNA, have focused attention on the use of formalin-preserved clinical samples collected routinely for diagnostic purposes. In practice, such samples often contain both tumour and normal cells which, if correctly partitioned, could be used to profile both tumour and normal genomes, thus identifying somatic alterations. Here we report a semi-automated method for laser microdissecting entire slide-mounted tissue sections to enrich for cells of interest with sufficient yield for whole genome and transcriptome sequencing. Using this method, we demonstrated enrichment of tumour material from mixed tumour-normal samples by up to 67%. Leveraging new methods that allow for the extraction of high-quality nucleic acids from small amounts of formalin-fixed tissues, we further showed that the method was successful in yielding sequence data of sufficient quality for use in BC Cancer's Personalized OncoGenomics (POG) program. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Successful reproductive cloning depends on obtaining intact donor nuclei from viable cells, ideally isolated by tissue biopsy of a living donor. However, owners and veterinarians often freeze deceased animals, which eventually causes damage to cellular micro-organelles due to the formation of intracellular water crystals. In the present study, we have reported the production of viable cloned puppies using donor nuclei of cells obtained from frozen carcasses. Five cases of deceased and frozen canine specimens were presented to be cloned. Skin fibroblast cell lines were successfully established for four specimens. Significant longer time was needed for the cell growth from frozen tissues (4 days) to reach 80% confluency compared to fresh tissue and frozen tissues frozen for 1- or 2-days. Similarly, SA-βgal positive cells (death cells) were significantly higher in frozen cells for 2- or 4- days compared to samples from fresh or frozen (1 day) sources. The cloning efficiency (CE) and the pregnancy rates (PR) of frozen cells were lower than those obtained from fresh or living donors (CE 2.4 ± 1.8% vs. 0.6 ± 0.3%, PR 21.7 ± 16.1% vs. 7.7 ± 5.3% for fresh vs. frozen, respectively). Here we demonstrate is the possibility to produce healthy offspring from cell lines obtained from frozen tissue collected post-mortem.

Flow cytometry is an effective and widely used tool for determination of ploidy in fish, but it is not always possible to access the fresh samples for analysis. We investigated the potential for extended storage of fish tissue with sterlet and tench as representative species of Chondrostei and Teleostei, using blood and fin of subadult/adult specimens and tail of larvae. Thirteen procedures for extending storage, selected for rapidity and simplicity in both field and laboratory conditions, were tested for each tissue sample. Flow cytometry was applied to fresh tissue immediately after sampling and to tissue subjected to experimental protocols, always along with species‐specific standard, after 1, 5, and 10 days storage at 0–4°C or freezing at −80°C. The fluorochrome 4′,6‐diamidine‐2′‐phenylindole dihydrochloride was used with excitation/emission maximum 358/461 nm. Based on the measurability of stored samples, evaluation of directly measured coefficients of variation of their DNA peaks and the changes in fluorescence intensity compared to fresh tissue, optimal procedures for extended storage of the selected tissue types of the model species are suggested. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Histological visualizations are critical to clinical disease management and are fundamental to biological understanding. However, current approaches that rely on bright-field microscopy require extensive tissue preparation prior to imaging. These processes are labor intensive and contribute to delays in clinical feedback that can extend to two to three weeks for standard paraffin-embedded tissue preparation and interpretation. Here, we present a label-free reflection-mode imaging modality that reveals cellular-scale morphology by detecting intrinsic endogenous contrast. We accomplish this with the novel photoacoustic remote sensing (PARS) detection system that permits non-contact optical absorption contrast to be extracted from thick and opaque biological targets with optical resolution. PARS was examined both as a rapid assessment tool that is capable of managing large samples (>1 cm2) in under 10 minutes, and as a high contrast imaging modality capable of extracting specific biological contrast to simulate conventional histological stains such as hematoxylin and eosin (H&E). The capabilities of the proposed method are demonstrated in a variety of human tissue preparations including formalin-fixed paraffin-embedded tissue blocks and unstained slides sectioned from these blocks, including normal and neoplastic human brain, and breast epithelium involved with breast cancer. Similarly, PARS images of human skin prepared by frozen section clearly demonstrated basal cell carcinoma and normal human skin tissue. Finally, we imaged unprocessed murine kidney and achieved histologically relevant subcellular morphology in fresh tissue. This represents a vital step towards an effective real-time clinical microscope that overcomes the limitations of standard histopathologic tissue preparations and enables real-time pathology assessment., 32 pages, 11 figures, includes Supplementary Information section

This study evaluated the effect of formalin fixation for near-infrared (NIR) fluorescence imaging of an antibody-dye complex (panitumumab-IRDye800CW) that was intravenously administered to patients with head and neck squamous cell carcinoma (HNSCC) scheduled to undergo surgery of curative intent. HNSCC patients were infused with 25 or 50 mg of panitumumab-IRDye800CW followed by surgery 1–5 days later. Following resection, primary tumor specimens were imaged in a closed-field fluorescence imaging device, before and after formalin fixation. The fluorescence images of formalin-fixed specimens were compared with images prior to formalin fixation. Regions of interest were drawn on the primary tumor and on the adjacent normal tissue on the fluorescence images. The mean fluorescence intensity (MFI) and tumor-to-background ratios (TBRs) of the fresh and formalin-fixed tissues were compared. Of the 30 enrolled patients, 20 tissue specimens were eligible for this study. Formalin fixation led to an average of 10 % shrinkage in tumor specimen size (p

Background aims Umbilical cord (UC) tissue is recognized as an advantageous source of mesenchymal stromal cells (MSCs), whose therapeutic properties are being actively evaluated in pre-clinical and clinical trials. In recognition of its potential value, storage of UC tissue or cells from UC tissue in newborn stem cell banks is now commonplace; however, strategies for isolating UC-derived MSCs (UCMSCs) from UC tissue have not been standardized. The majority of newborn stem cell banks take one of two approaches to cord tissue processing and cryopreservation: enzymatic digestion of the fresh tissue with cryopreservation of the subsequent cell suspension or cryopreservation of the tissue as a composite whole with later, post-thaw isolation of cells by explantation. Evaluation of UCMSCs derived by these two principal preparation and cryopreservation strategies is important to understanding whether the methods currently employed by newborn stem cell banks retain the desirable clinical attributes of UC cells. Methods UCMSCs were isolated from 10 UC tissue samples by both explantation and enzymatic digestion methods to allow for comparison of cells from the same donor. Cell isolates from both methods were compared pre- and post-cryopreservation as well as after serial passaging. Cell viability, morphology, growth kinetics, immunophenotype, cytokine secretion and differentiation capacity were evaluated. Results UCMSCs could be derived from fresh UC tissue by both explantation and digestion methods and from thawed UC tissue by explantation. Initial cell populations isolated by digestion were heterogeneous and took longer to enrich for UCMSCs in culture than populations obtained by explantation. However, once isolated and enriched, UCMSCs obtained by either method showed no significant difference in viability, morphology, rate of proliferation, surface marker expression, levels of cytokine secretion or differentiation capacity. Conclusions Derivation of UCMSCs by explantation after thawing UC cryopreserved as a composite tissue may be favorable in terms of initial purity and number of cells achievable by a specific passage. However, we observed no evidence of functional difference between UCMSCs derived by explanation or digestion, suggesting that cells isolated from cryopreserved material obtained by either method maintain their therapeutic properties.

Modified agarose cell-block (CB) technique can be effectively used to have the CB embedded in the OCT compound for the preparation of frozen CB (F-CB) sections in the same way as in the preparation of frozen sections from cryoembedded fresh tissue samples for the intraoperative consultation. In this report, we demonstrate the amenability of F-CB sections to the diagnostic immunocytochemistry. The pelleted cytologic material was at first compactly embedded in ultralow-gelling temperature agarose gel and then re-embedded in conventional agarose gel. The resulting agarose-embedded cell-pellet was cut in halves so that one half is embedded in OCT compound for cryosectioning on a cryostat, while the other half is reserved for the preparation of paraffin-embedded CB (P-CB). The F-CB sections were comparable to sections cut from the paraffin-embedded CB in terms of the quality of H&E-staining and immunocytochemistry. We suppose this method can also facilitate a rapid quantitative and qualitative assessment of the future P-CB. We have extended this technique to the cryo-embedded cell-block method, in which the compact agarose cell pellet is directly embedded in the OCT compound so that the frozen sections can be cut from the cryo-embedded cell block in the same way as in intraoperative frozen section analysis. In this technical report, we illustrate how the frozen cell blocks (F-CBs) can be effectively prepared not only from liquid-based cytology samples but also from conventional fine-needle aspiration cytology slides.

Senescence-associated beta-galactosidase (SA-β-gal) activity assay is commonly used to evaluate the increased beta-galactosidase (β-gal) activity in senescent cells related to enhanced lysosomal activity. Although the optimal pH for β-gal is 4.0, this enzymatic activity has been most commonly investigated at a suboptimal pH by using histochemical reaction on fresh tissue material. In the current study, we optimized a SA-β-gal activity histochemistry protocol that can also be applied on cryopreserved hepatic tissue. This protocol was developed on livers obtained from control rats and after bile duct resection (BDR). A significant increase in β-gal liver activity was observed in BDR rats vs controls after 2 hr of staining at physiological pH 4.0 (6.98 ± 1.19% of stained/total area vs 0.38 ± 0.22; p

The aim of this study was to develop a new chitosan derivative and investigate its effects on fresh tissue healing in rats. A chitosan-fructose Schiff based quaternary ammonium salt (CS = Fru-DEAE) was synthesized for the first time and characterized using FT-IR and

BackgroundOvarian tissue cryopreservation (OTC) is important for fertility preservation and conservation. Delay in OTC may be required for transport or workflow management, however little is understood about the effect of processing delay on the tissue.ObjectiveTo determine whether a delay of 24-48 hours to OTC affects primordial follicle (PF) health.MethodsOvaries (n=6 sheep) were processed immediately or after storage at 4°C (24h, 48h). Tissue was fixed fresh, after cryopreservation or 10-day xenotransplantation. Morphological assessment of follicle health and development was performed.FindingsA total of 1541 follicles were analysed. A 24h processing delay did not impact PF health in fresh or cryopreserved tissue. In fresh tissue a 48h delay had an adverse effect on follicle health (OR=2.47, 95% CI 1.29-4.71). Interestingly, a 48h delay resulted in cryopreserved tissue being less likely to be graded as unhealthy compared to control (OR=0.56, 95% CI 0.36-0.87). There was no difference in PF health or development across groups following xenotransplantation.ConclusionOvarian tissue can be stored for up to 48 hours prior to cryopreservation with no net impact on PF health.

Post-operative adhesions, a common complication of surgery, cause pain, impair organ functionality, and often require additional surgical interventions. Control of inflammation, protection of injured tissue, and rapid tissue repair are critical for adhesion prevention. Adhesion barriers are biomaterials used to prevent adhesions by physical separation of opposing injured tissues. Current adhesion barriers have poor anti-inflammatory and tissue regenerative properties. Umbilical cord tissue (UT), a part of the placenta, is inherently soft, conforming, biocompatible, and biodegradable, with antimicrobial, anti-inflammatory, and antifibrotic properties, making it an attractive alternative to currently available adhesion barriers. While use of fresh tissue is preferable, availability and short storage time limit its clinical use. A viable cryopreserved UT (vCUT) “point of care” allograft has recently become available. vCUT retains the extracellular matrix, growth factors, and native viable cells with the added advantage of a long shelf life at −80 °C. In this study, vCUT's anti-adhesion property was evaluated in a rabbit abdominal adhesion model. The cecum was abraded on two opposing sides, and vCUT was sutured to the abdominal wall on the treatment side; whereas the contralateral side of the abdomen served as an internal untreated control. Gross and histological evaluation was performed at 7, 28, and 67 days post-surgery. No adhesions were detectable on the vCUT treated side at all time points. Histological scores for adhesion, inflammation, and fibrosis were lower on the vCUT treated side as compared to the control side. In conclusion, the data supports the use of vCUT as an adhesion barrier in surgical procedures., Highlights • Umbilical tissue (UT) has properties desirable for an adhesion barrier biomaterial. • Study shows viable cryopreserved umbilical tissue (vCUT) retains components of fresh UT in their native state. • vCUT prevents post-operative adhesions in vivo, suggesting vCUT could be used as an adhesion-barrier in surgical procedures.

Ex vivo fluorescence confocal microscopy (FCM) is a developing tool providing rapid digital imaging of fresh tissue utilizing high-resolution optical sectioning that highly corresponds with conventional hmatoxylin and eosin (HE)-stained slides. A very little data on oral mucosa lesions exist currently. The present work aimed to create an image atlas of benign and malignant oral tissues and compare them to the corresponding histopathology. Furthermore, we aimed to evaluate the learning curve for confocal image interpretation. From 50 samples obtained from the oral mucosa, including oral squamous cell carcinoma (OSCC), dysplasia, and healthy oral tissue, ex vivo FCM images and corresponding HE slides were created and collected into a tissue atlas. Additionally, two experts were asked to analyze the images to assess the learning curve. Ex vivo FCM images revealed high comparability with histopathological images. Tissues including OSCC, dysplasia, and normal oral mucosa were implemented in the image atlas to provide the diagnostic fundament for pathologists and surgeons; the learning curve was short. Future studies on this topic will be advantageous for the development of artificial intelligence-based diagnostic approaches. The current work provides a novel set of data that are structured as an atlas of common pathologies of the mucosa to enhance the existing knowledge and material on confocal images.

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer (BU). Rapid detection of M. ulcerans in clinical specimens is essential for early diagnosis so that patients can be treated appropriately as soon as possible. This chapter describes suitable methods for the extraction of M. ulcerans DNA from the most common specimens submitted to the laboratory for confirmation of BU: swabs, fresh tissue biopsies, and fixed tissue sections. The resulting DNA extracts may be used for downstream procedures including standard gel-based PCR and real-time PCR assays. Protocols for direct detection of M. ulcerans DNA by real-time PCR are described in Chapter 8 .

Histological images can reveal rich cellular information of tissue sections, which are widely used by pathologists in disease diagnosis. However, the gold standard for histopathological examination is based on thin sections on slides, which involves inevitable time-consuming and labor-intensive tissue processing steps, hindering the possibility of intraoperative pathological assessment of the precious patient specimens. Here, by incorporating ultraviolet photoacoustic microscopy (UV-PAM) with deep learning, we show a rapid and label-free histological imaging method that can generate virtually stained histological images (termed Deep-PAM) for both thin sections and thick fresh tissue specimens. With the tissue non-destructive nature of UV-PAM, the imaged intact specimens can be reused for other ancillary tests. We demonstrated Deep-PAM on various tissue preparation protocols, including formalin-fixation and paraffin-embedding sections (7-µm thick) and frozen sections (7-µm thick) in traditional histology, and rapid assessment of intact fresh tissue (~ 2-mm thick, within 15 min for a tissue with a surface area of 5 mm × 5 mm). Deep-PAM potentially serves as a comprehensive histological imaging method that can be simultaneously applied in preoperative, intraoperative, and postoperative disease diagnosis.

Considerable recent research has indicated the presence of bacteria in a variety of human tumours and matched normal tissue. Rather than focusing on further identification of bacteria within tumour samples, we reversed the hypothesis to query if establishing the bacterial profile of a tissue biopsy could reveal its histology / malignancy status. The aim of the present study was therefore to differentiate between malignant and non-malignant fresh breast biopsy specimens, collected specifically for this purpose, based on bacterial sequence data alone. Fresh tissue biopsies were obtained from breast cancer patients and subjected to 16S rRNA gene sequencing. Progressive microbiological and bioinformatic contamination control practices were imparted at all points of specimen handling and bioinformatic manipulation. Differences in breast tumour and matched normal tissues were probed using a variety of statistical and machine-learning-based strategies. Breast tumour and matched normal tissue microbiome profiles proved sufficiently different to indicate that a classification strategy using bacterial biomarkers could be effective. Leave-one-out cross-validation of the predictive model confirmed the ability to identify malignant breast tissue from its bacterial signature with 84.78% accuracy, with a corresponding area under the receiver operating characteristic curve of 0.888. This study provides proof-of-concept data, from fit-for-purpose study material, on the potential to use the bacterial signature of tissue biopsies to identify their malignancy status.

Temporary storage of nasal tissues and nasal cell sheets, which entails transportation between hospitals and cell culture facilities, is an important issue in regenerative medicine. Herein, we investigated the preservation of chilled and frozen nasal tissues and expiry dates of ready-to-use nasal cell sheets. Although the cell number in preserved tissues was lower than that in fresh tissue, nasal cell sheets could be fabricated from tissues that had been refrigerated for 5 days and frozen–thawed over 5 days. Moreover, the nasal mucosal cell sheets were preserved in a non-hazardous buffer. The cell number, viability, and structure were not maintained in saline containing E-cadherin for 2 days; however, these were maintained in Hank’s balanced salt solution for 2 days, but not for 5 days. To assess the proliferation capacity of cells in the stored cell sheets, we performed cell sheet grafting assays in vitro. Cell sheets stored in Hank’s balanced salt solution for 2 days adhered to collagen gel and expanded normally. Our results show that nasal tissues can be stored temporarily in refrigerators or deep freezers, and Hank’s balanced salt solution can be used for preservation of ready-to-use cell sheets for a few days.

Three-dimensional (3D) organoid culture systems are emerging as potential reliable tools to investigate basic developmental processes of human disease, especially cancer. The present study used established and modified culture conditions to report successful generation and characterization of patient-derived organoids from fresh primary tissue specimens of patients with treatment-naive prostate cancer (PCa). Fresh tissue specimens were collected, digested enzymatically and the resulting cell suspensions were plated in a 3D environment using Matrigel as an extracellular matrix. Previously established 12-factor medium for organoid culturing was modified to create a minimal 5-factor medium. Organoids and corresponding tissue specimens were characterized using transcriptomic analysis, immunofluorescent analysis, and immunohistochemistry. Furthermore, patient-derived organoids were used to assess the drug response. Treatment-naive patient-derived PCa organoids were obtained from fresh radical prostatectomy specimens. These PCa organoids mimicked the heterogeneity of corresponding parental tumor tissue. Histopathological analysis demonstrated similar tissue architecture and cellular morphology, as well as consistent immunohistochemical marker expression. Also, the results confirmed the potential of organoids as an in vitro model to assess potential personalized treatment responses as there was a differential drug response between different patient samples. In conclusion, the present study investigated patient-derived organoids from a cohort of treatment-naive patients. Derived organoids mimicked the histological features and prostate lineage profiles of their corresponding parental tissue and may present a potential model to predict patient-specific treatment response in a pre-clinical setting.

The aim of this work was to inspect the presence of asbestos fibers in colon tissue from a patient, with history of indirect exposure to asbestos and affected by colon cancer, who underwent surgery. Variable pressure scanning electron microscopy, coupled with energy dispersive spectroscopy (VP-SEM/EDS), was used for identification of inorganic fibers and for their morphological—chemical characterization. Fresh tissue samples from both, healthy area close to the neoplasia and from the neoplastic regions, were separately digested to eliminate the biological matrix. The precipitate was analyzed by VP-SEM/EDS, identifying in samples from healthy tissue asbestos bodies and small asbestos fibers, and in samples from neoplastic tissue long fibers of asbestos, free from covering. A quantification of the asbestos bodies and the free fibers in the two type of specimens is proposed. Moreover, to locate the fibers in the biological medium, histological sections from the colon of the same patient were also examined. Free asbestos fibers appeared concentrated in the tissue bridge between the healthy and the neoplastic areas. Immuno-histochemical investigation performed on the neoplasia seems to exclude a role of microsatellite instability in the carcinogenesis process, suggesting an influence of the fibers.

Minimally invasive core needle biopsies for medical diagnoses have become increasingly common for many diseases. Although tissue cores can yield more diagnostic information than fine needle biopsies and cytologic evaluation, there is no rapid evaluation at the point-of-care for intact tissue cores that is low-cost and non-destructive to the biopsy. We have developed a proof-of-concept 3D printed millifluidic histopathology lab-on-a-chip device to automatically handle, process, and image fresh core needle biopsies. This device, named CoreView, includes modules for biopsy removal from the acquisition tool, transport, staining and rinsing, imaging, segmentation, and multiplexed storage. Reliable removal from side-cutting needles and bidirectional fluid transport of core needle biopsies from five tissue types has been demonstrated with 0.5-mm positioning accuracy. Automation is aided by a MATLAB-based biopsy tracking algorithm that can detect the location of tissue and air bubbles in the channels of the millifluidic chip. With current and emerging optical imaging technologies, CoreView can be used for a rapid adequacy test at the point-of-care for tissue identification and glomeruli counting from renal core needle biopsies, and has the potential to be used for breast cancer diagnosis, phenotyping, and mapping of enriched tumorous regions for downstream analyses in global health applications.

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.