Long-Term Tissue Preservation at Ambient Temperature for Post-Mass Fatality Incident DNA-Based Victim Identification.

In a mass fatality incident (MFI), effective preservation of tissue samples is the cornerstone for downstream DNA-based identification of victims. This is commonly achieved through freezing of tissue samples excised from bodies/fragmented remains which may be buried or stored in refrigerated containers. This may, however, not be possible depending on the nature of the MFI; in particular, during armed conflict/war where extended periods of electrical outages would be expected. The present study compared the effectiveness of long-term tissue preservation at ambient temperatures using two commercial products (non-iodized kitchen salt and a 40% alcoholic beverage) against a chemical preservative (Allprotect™ Tissue Reagent (Qiagen, Germantown, MD, USA)) and freezing at -20 °C. Bovine muscle tissue, used as a proxy for human tissue, was treated with the four preservation methods and sampled at six different time-points over a 24-month period. All four methods were able to preserve the bovine tissue, generally yielding STR-PCR (Short Tandem Repeat-Polymerase Chain Reaction) amplicons > 200 bp in size even at the end of 24 months. Gel electrophoresis, however, indicated that salt was more effective in preserving DNA integrity with high-molecular-weight DNA clearly visible as compared to the low-molecular-weight DNA smears observed in the other methods. This study also proposes a simple process for the rapid and low-cost preservation of tissue samples for long-term storage at ambient temperatures in support of post-incident victim identification efforts.

Anatomical and Clinical Education in Physical and Occupational Therapy: Influence of Integration and Soft Preservation / Educación Anatómica y Clínica en Terapia Física y Ocupacional: Influencia de la Integración y la Preservación Blanda

SUMMARY: Anatomy and clinical skills are taught separately to physical and occupational therapy students. Formaldehyde is the primary chemical used to embalm donors which creates a challenge in integrating clinical skills into the anatomy curriculum. This study aimed to evaluate the integration of clinical skills into anatomical education using Imperial College London- Soft Preservation (ICL-SP) and formaldehyde embalming through the evaluation of student perceived learning and confidence. Students were invited to complete a survey after dissecting an ICL-SP and formaldehyde embalmed donors and perform clinical tests. It was easier to dissect and identify neurovascular structures on ICL-SP donors compared to formaldehyde. Clinical tests, like measuring range of motion and ligament tests were also more realistic on ICL-SP donors. The integration of clinical skills in the anatomical curriculum increased student perceived understanding of associated anatomy (p < 0.001), gave better understanding of how anatomy is important to their professions (p < 0.001) and increased motivation to learn anatomy (p < 0.001). The integration of clinical skills into anatomical education can facilitate student learning with higher confidence performing clinical skills and is complemented by the utilization of the new ICL-SP methodology instead of the traditional formaldehyde preservation.

Las habilidades anatómicas y clínicas se enseñan por separado a los estudiantes de terapia física y ocupacional. El formaldehído es el químico principal que se usa para embalsamar a los donantes, lo que crea un desafío para integrar las habilidades clínicas en el plan de estudios de anatomía. Este estudio tuvo como objetivo evaluar la integración de habilidades clínicas en la educación anatómica utilizando Imperial College London-Soft Preservation (ICL-SP) y embalsamamiento de formaldehído a través de la evaluación del aprendizaje y la confianza percibidos por los estudiantes. Se invitó a los estudiantes a completar una encuesta después de diseccionar un ICL-SP y donantes embalsamados formolizados y realizar pruebas clínicas. Fue más fácil diseccionar e identificar estructuras neurovasculares en donantes ICL-SP en comparación con los fijados en formaldehído. Las pruebas clínicas, como la medición del rango de movimiento y las pruebas de ligamentos, también fueron más realistas en los donantes de ICL-SP. La integración de habilidades clínicas en el plan de estudios anatómico aumentó la comprensión percibida por los estudiantes de anatomía asociada (p < 0,001), dio una mejor comprensión de cómo la anatomía es importante para sus profesiones (p < 0,001) y aumentó la motivación para aprender anatomía (p < 0,001). La integración de las habilidades clínicas en la educación anatómica puede facilitar el aprendizaje de los estudiantes con mayor confianza en el desempeño de las habilidades clínicas y se complementa con la utilización de la nueva metodología ICL-SP en lugar de la conservación tradicional con formaldehído.

Conservation of Organs (Heart, Brain and Kidney) of Canine by Cold-Temperature Silicone Plastination in an Animal Anatomy Laboratory in Ecuador / Conservación de Órganos (Corazón, Encéfalo y Riñón) de Canino Mediante la Técnica de Plastinación en Silicona al Frío Realizada en Laboratorio de Anatomía Animal en Ecuador

SUMMARY: For the purposes of teaching anatomy, the use of cadaver preparations is considered the most efficient way of ensuring that students retain knowledge. Nevertheless, in Ecuador the use of animal specimens in universities must comply with the internationally accepted principles of replacement, reduction and refinement (3Rs). Plastination is an alternative technique which allows organs to be conserved in the long term and complies with the 3Rs. The object of the present work was to use cold-temperature silicone plastination with Biodur® products to obtain long-lasting, easy-to-handle canine organs for use as tools for the teaching of animal anatomy. Six canine cadavers were obtained from local animal protection charities. The hearts, brains and kidneys of the cadavers were dissected and fixed with formaldehyde 10 %. They were then dehydrated with acetone at -20 °C. The specimens were impregnated with Biodur® S10:S3 (-20 °C) and finally cured with Biodur® S6. We plastinated six hearts, twelve kidneys, four brains and one encephalic slice of canine. The application of cold-temperature plastination to canine organs followed the parameters established for the conventional protocol, enabling us to obtain organs of brilliant appearance, free of odours, in which the anatomical form was preserved. Thus the technique helped us to comply with the 3Rs, as we obtained easy-to-handle teaching models to replace fresh or formaldehyde-fixed samples for the teaching-learning of the canine anatomy.

En la enseñanza de la Anatomía, el uso de preparaciones cadavéricas se considera el método que permite a los estudiantes retener el conocimiento de una forma más eficiente. No obstante, en Ecuador, el uso de especímenes animales en las universidades se debe realizar bajo el principio internacional de reemplazo, reducción y refinamiento (3Rs). La técnica de plastinación es una técnica alternativa que permite preservar órganos a largo plazo y que se adapta al principio de las 3Rs. El objetivo del trabajo fue utilizar la técnica de plastinación en silicona al frío con productos Biodur® para obtener órganos caninos duraderos y manejables útiles como herramienta para la enseñanza de la anatomía animal. Se obtuvieron seis cadáveres de caninos de fundaciones locales para la protección animal. Se realizaron disecciones de corazones, cerebros y riñones de los cadáveres caninos. Los órganos se fijaron con formalina al 10 %. A continuación, se llevó a cabo la deshidratación con acetona a -20 °C. Los especímenes fueron impregnados con S10:S3 Biodur® (-20 °C) y al final fueron curados con Biodur® S6. Se lograron plastinar seis corazones, doce riñones, cinco encéfalos y un tallo encefálico de canino. La técnica de plastinación al frío utilizada para obtener órganos de canino conservó los parámetros empleados en el protocolo convencional y permitió obtener órganos que presentaron aspecto brillante, ausencia de olores y mantuvieron la forma anatómica. Por lo que, la técnica facilitó cumplir con el principio de las 3Rs al obtenerse modelos didácticos fáciles de manipular que pueden reemplazar muestras frescas o formolizadas en el proceso de enseñanza-aprendizaje de la anatomía del canino.

A Novel Tissue Preservation and Transport Solution as a Substitute for Formalin.

Formalin, a common laboratory fixative, is a type 1 carcinogen; a biohazard with risks, environmental, disposal, and legal costs; and a chemical modifier of protein epitopes in tissues. A less-toxic tissue preservation method is therefore badly needed. We have developed a novel tissue preservation medium, Amber, composed of low-potassium dextran glucose, 10% honey, and 1% coconut oil. This study investigates Amber as compared with formalin with respect to the following aspects: (1) histologic preservation, (2) epitope integrity with immunohistochemistry (IHC) and immunofluorescence (IF), and (3) integrity of tissue RNA. Rat and human lung, liver, kidney, and heart tissues were collected and stored for 24 hours at 4 °C in Amber or formalin. The tissues were evaluated with hematoxylin and eosin; IHC: thyroid transcription factor, muscle-specific actin, hepatocyte-specific antigen, and common acute lymphoblastic leukemia antigen; and IF: VE-cadherin, vimentin, and muscle-specific actin. RNA quality upon extraction was also assessed. Amber demonstrated superior and/or noninferior performance in rat and human tissue evaluation with respect to standard techniques of histology, IHC, IF, and extracted RNA quality. Amber maintains high-quality morphology without compromising the ability to perform IHC and nucleic acid extraction. As such, Amber could be a safer and superior substitute to formalin for clinical tissue preservation for contemporary pathological examination.

Variability in the Processing of Fresh Osteochondral Allografts.

The indications for fresh osteochondral allograft continue to increase. As a result, variations in graft processing and preservation methods have emerged. An understanding of these techniques is important when evaluating the optimal protocol for processing fresh osteochondral allografts prior to surgical implantation. The aim of this study is to review the literature and understand various tissue processing protocols of four leading tissue banks in the United States. Donor procurement, serological and microbiological testing, and storage procedures were compared among companies of interest. Similarities between the major tissue banks include donor screening, aseptic processing, and testing for microorganisms. Variability exists between these companies with relation to choice of storage media, antibiotic usage, storage temperature, and graft expiration dates. Potential exists for increased chondrocyte viability and lengthened time-to-expiration of the graft through a protocol of delicate tissue handling, proper choice of storage medium, adding hormones and growth factors like insulin growth factor-1 (IGF-1) to serum-free nutrient media, and storing these grafts closer to physiologic temperatures.

Preservation of donor corneal epithelium in McCarey-Kaufman medium.

Purpose: To evaluate the role of McCarey-Kaufman (MK) medium in maintaining the integrity of donor corneal epithelium. Methods: Nineteen corneal buttons were harvested and stored in MK media at 2°C-8°C for four days. Serial photographs were done every day till the 3rd day, and images were then analyzed with ImageJ software (LOCI, University of Wisconsin, USA). The area of exposure and epithelial defect (ED) was calculated every day for each corneal button. Results: The average age of the donors was 56.5 ± 22.7 years and mean time from death to preservation of the corneal buttons was 7.7 ± 3.1 hours. The average corneal area was 145.6 ± 18.8 mm2. The total mean area of exposure was 3.6 ± 4.8, 7.2 ± 9.2, and 9.0 ± 11.9 mm2, and ED was 1.7 ± 4.6, 2.8 ± 5.3, and 3.3 ± 5.9 mm2 on days 1, 2, and 3, respectively. The percentage of increase in the area of exposure and ED in MK media was 3.71% and 1.1% from day 1 to day 3, respectively. Six out of 19 corneal buttons (31.57%) were utilized for keratoplasties, of which two were utilized in house and four were distributed outside. Of the two utilized corneas, none had epithelial defect on postoperative day 1. Rest 13 corneas were either used for training and research purposes, stored in glycerol media, or discarded. Conclusion: Since the percentage change in area of exposure/ED is not much at the end of day 3, corneas stored in MK media can be safely used even after three days of storage. Hence, MK medium serves as an excellent medium in maintaining the integrity of donor corneal epithelium.

Preservation of Adrenoceptor and Endothelin Receptor Mediated Vasoconstriction and of Endothelium-Dependent Relaxation after Cold Storage of Explanted Blood Vessels for ex vivo Analyses.

INTRODUCTION: Adrenoceptor and endothelin (ET) receptor-mediated vasoconstriction as well as endothelium-dependent vasodilation of human saphenous veins were compared before and after 20 h of cold storage. METHODS: Contractile responses to potassium chloride (KCl), norepinephrine (NE), and ET-1 as well as vasodilator responses to acetylcholine (ACh) were evaluated. RESULTS: Storage in HEPES-supplemented Dulbecco's modified Eagle's medium (HDMEM) diminished KCl induced contractile forces to 71% (p = 0.002) and NE induced contractions to 80% (p = 0.037), in contrast to HEPES-supplemented Krebs-Henseleit solution (HKH) and TiProtec solution. KCl-normalized NE contractions were not affected by storage. NE EC50 values were slightly lower (7.1E-8 vs. 7.5E-8, p = 0.019) after storage in HKH, with no changes after storage in the other solutions. Endothelium-dependent responses to ACh were not affected by storage. ET-1 induced contractions were attenuated after storage in HDMEM (77%, p = 0.002), HKH (75%, p = 0.020), and TiProtec (73%, p = 0.010) with no changes in normalized constrictions. ET-1 EC50 values were not affected by storage. CONCLUSION: Loss of contractility after storage in HDMEM may reflect the lower content of dextrose. There was no specific attenuation of adrenoceptor, ET-receptor, or ACh receptor mediated signal transduction after storage in any of the media. HKH or TiProtec are equally suitable cold storage solutions for ex vivo measurements.

Comparación de resultados entre soluciones con y sin formol como medios de fijación y conservación en corazones de Gallus gallus domesticus / Comparison of results between solutions with and without formaldehyde as means of fixation and conservation in Gallus gallus domesticus hearts

RESUMEN: La solución de formol es utilizada en las Escuelas de medicina como medio de fijación y conservación de cadáveres para el estudio de la Anatomía, a la que están expuestos estudiantes, técnicos y personal docente; es alergénica e irritante a las mucosas, y reconocida carcinogénica en humanos por International Agency for Research on Cancer (2006). El objetivo del presente estudio fue comparar resultados cuantitativos y cualitativos entre corazones de Gallus gallus domesticus, luego de aplicarles soluciones con y sin formol. Se formaron dos grupos al azar, a uno se le aplicó solución de formol al 10 %, y al otro solución libre de formol. Se realizaron medidas antropométricas, organolépticas, y de fotografía (Pretest, durante y Postest). Se elaboró base datos en Microsoft Excel (2019), y su procesamiento en SPSS Statistics 2017 Versión 25. Para variables cuantitativas se aplicó la prueba de Shapiro-Wilk, y t-Student pareada. Para variables cualitativas el test Alfa de Cronbach, Chi cuadrado (X2) y los correspondientes coeficientes de asociación (D de Somers y Tau b de Kendal). Los resultados obtenidos de las variables peso, largo, y altura presentaron diferencia estadística significativa (p-valor <0,05), siendo diferente para el ancho y grosor de la pared del ventrículo izquierdo. Las variables color y consistencia presentaron diferencias significativa (p-valor <0,05). El olor irritante a las mucosas estuvo presente durante todo el estudio con la solución con formol. A la inspección, ninguno de los dos grupos presento colonización - descomposición. Se concluye que, los órganos en experimentación que se les aplicó solución libre de formol, presentaron mejores resultados con respecto a los que se les aplico formol al 10 %.

SUMMARY: The formaldehyde solution is used in medical schools as a means of fixing and preserving corpses for the study of Anatomy, to which students, technicians and teaching personnel are exposed; it is allergenic and irritant to the mucosa, and recognized as a human carcinogen by the International Agency for Research on Cancer (2006). The objective of the present study was to compare quantitative and qualitative results between Gallus gallus domesticus hearts, after applying solutions with and without formaldehyde. Two groups were formed at random, to one a 10 % formaldehyde solution was applied, and to the other formaldehyde- free solution. Anthropometric, organoleptic, and photographic measurements were carried out (Pretest, during and Posttest). A database was prepared in Microsoft Excel (2019), and its processing in SPSS Statistics 2017 Version 25. For quantitative variables, the Shapiro-Wilk test and t-Student paired were applied. For qualitative variables the Cronbach's Alpha test, Chi square (X2) and the corresponding association coefficients (Somers D and Kendal's Tau b). The results obtained from the variables weight, length, and height presented a statistically significant difference (p-value <0.05), being different for the width and thickness of the left ventricular wall. The variables color and consistency showed significant differences (p-value <0.05). The irritating smell to the mucous membranes was present throughout the study with the formaldehyde solution. Upon inspection, neither group showed colonization - decomposition. It is concluded that the organs in experimentation that were applied formaldehyde-free solution presented better results compared to those that were applied 10 % formaldehyde.

A new reliable method for tissue preservation.

Studying and developing preanalytical tools and technologies for preserving high-quality tissues for histological assays is a constantly growing field because preserving biological samples always poses a challenge. Formalin fixation and paraffin embedding (FFPE) remains a key component of long-term tissue preservation in the histopathology and pathology laboratory, despite its known carcinogenic effect and poor preservation of nucleic acids. Over the years, some other methods have been described to preserve biological tissues such as plastination. However, the current techniques do not consent further dissection or histopathologically based studies. The present study describes the applicability of a new method for tissues preservation after surgery or endoscopic removal, to preserve specimens for subsequent histopathological examination and tissue banking as well as for immunohistochemistry, preserving the nucleic acids and protein integrity. This method, protected by patent (EP 18185364.9A) is able to restore tissues to the state prior to drying, thereby allowing them to be further processed for histologic, cytological, immunologic, and/or genetic analyses. This new technique offers an alternative to classic tissue preservation, featuring an innovative and cost-effective process, available locally, to obtain fully preserved samples for longer periods without toxic procedures. In conclusion this new method can be successfully applied in any pathological institute, responding to the increasing demands for reliable tissue preservation in the expanding field of histological, molecular diagnostic and forensic medicine.

Comparison of the Effects of Temperature and Dehydration Mode on Glycerin-Based Approaches to SMILE-Derived Lenticule Preservation.

PURPOSE: The aim of this study was to explore the optimal method of small-incision lenticule extraction (SMILE)-derived lenticules, subjected to long-term preservation using glycerol, under a range of temperatures, and using an array of dehydration agents. METHODS: In total, 108 myopic lenticules were collected from patients undergoing the SMILE procedure. Fresh lenticules served as a control group for this study, whereas all other lenticules were separated into 8 groups, which were preserved at 4 different temperatures (room temperature [RT], 4, -20, and -80°C) with or without silica gel in anhydrous glycerol. Evaluated parameters included thickness, transmittance, hematoxylin and eosin staining, transmission electron microscopy, and immunohistochemistry analyses. RESULTS: After a 3-month preservation period, lenticular thickness in these different groups was significantly increased, particularly for samples stored at RT. The mean percentage transmittance of lenticules stored at -80°C with or without silica gel was closest to that of fresh lenticules. Hematoxylin and eosin staining revealed sparsely arranged collagen fibers that were more scattered in preserved lenticules relative to fresh lenticules, particularly in RT samples. Transmission electron microscopy revealed that the fibril bundles densities in lenticules stored at RT were significantly less than those stored at other temperatures. Immunohistochemistry analyses revealed reductions in or loss of CD45 and human leukocyte antigens in all preserved lenticules relative to control samples. CONCLUSIONS: Of the tested approaches, the preservation of SMILE-derived lenticules over a 3-month period was optimal at -80°C with or without silica gel in anhydrous glycerol.

Establishing a Standardized Method for the Effective Intraoperative Collection and Biological Preservation of Brain Tumor Tissue Samples Using a Novel Tissue Preservation System: A Pilot Study.

BACKGROUND: Glioblastoma (GB) is an aggressive tumor showing extensive intertumoral and intratumoral heterogeneity. Several possible reasons contribute to the historical inability to develop effective therapeutic strategies for treatment of GB. One such challenge is the inability to consistently procure high-quality biologically preserved specimens for use in molecular research and patient-derived xenograft model development. No scientifically derived standardized method exists for intraoperative tissue collection specifically designed with the fragility of RNA in mind. METHODS: In this investigation, we set out to characterize matched specimens from 6 GB patients comparing the traditional handling and collection processes of intraoperative tissue used in most neurosurgical operating rooms versus an automated resection, collection, and biological preservation system (APS) which captures, preserves, and biologically maintains tissue in a prescribed and controlled microenvironment. Matched specimens were processed in parallel at various time points and temperatures, evaluating viability, RNA and protein concentrations, and isolation of GB cell lines. RESULTS: We found that APS-derived GB slices stored in an APS modified medium remained viable and maintained high-quality RNA and protein concentration for up to 24 hours. CONCLUSIONS: Our results showed that primary GB cell cultures derived in this manner had improved growth over widely used collection and preservation methods. By implementing an automated intraoperative system, we also eliminated inconsistencies in methodology of tissue collection, handling and biological preservation, establishing a repeatable and standardized practice that does not require additional staff or a laboratory technician to manage it.

The cost-effectiveness of maintaining an in-house glycerol-preserved skin bank.

INTRODUCTION: Glycerol-preserved skin allograft (GPA) plays a vital role, especially in the management of burns injury. Where it is utilized as temporary wound closure, the considerably cost-effective, simpler handling and storage of GPA makes it preferable in almost all clinical indications. The GPA was first introduced to the Hospital Universiti Sains Malaysia in 2001. The supply was imported from Euro Skin Bank, Beverwijk, The Netherlands. In the year 2013, our center had started maintaining an in-house glycerolized skin bank. METHOD: We preserved donor skin grafts from patients who underwent plastic surgery-related procedures in 85% glycerol and stored them at +2 °C to +10 °C. Cost estimation of the GPA per cm2 was calculated to analyze the effectiveness of its preservation technique. RESULTS: The cost of GPA from our skin bank is estimated to be almost 90% reduction in cost as compared to the supply from Euro Skin Bank. CONCLUSION: The selective and strategic use of the GPA in major burn patients assure effective advantages in the treatment of burns. The clinical significance of skin allograft usage is very high. The cost-effectiveness of maintaining an in-house skin bank made it possible for various centers for skin allograft usage.

Ovarian tissue features assessed in bovine fetuses after vitrification and xenotransplantation procedures.

Cryopreservation and transplantation of ovarian tissue are proposed methods for the restoration of endocrine function and reproductive potential. Therefore, this study aimed to evaluate the effects of vitrification and xenotransplantation on follicle viability, activation, stromal cell integrity, vascularization, and micronuclei formation. Bovine fetal ovaries were fragmented and assigned to the following groups: Fresh control (FC), ovarian fragments immediately fixed; Vitrified control (VC), ovarian fragments vitrified; Vitrified xenotransplanted (VX), ovarian fragments vitrified and xenotransplanted; and Fresh xenotransplanted (FX), ovarian fragments xenotransplanted. Ovarian fragments were grafted in female BALB/c mice and recovered after 14 days. Follicular viability was preserved (P > 0.05) in VC group. The rate of developing follicles was greater (P < 0.05) in the FX group compared to other groups. Follicular density was higher (P < 0.05) in the VC group than the FC, VX, and FX groups. A decrease (P < 0.05) of stromal cell density was recorded after vitrification (VC vs. FX). Blood vessel density decreased in VC, VX, and FX groups compared with the FC group, and blood vessel density was correlated with follicular viability (positively; P = 0.07) and developing follicles (negatively; P < 0.001). Both vitrification and xenotransplantation groups (VC, VX, and FX) had a greater (P < 0.05) number of cells with one MN compared to the FC group. In summary, our findings showed that both vitrification and xenotransplantation modified blood vessel, follicular and stromal cell densities, follicular viability and activation, and micronuclei formation in ovarian tissue.

Optimization of long-term cold storage of rat precision-cut lung slices with a tissue preservation solution.

Precision-cut lung slices (PCLS) are used as ex vivo model of the lung to fill the gap between in vitro and in vivo experiments. To allow optimal utilization of PCLS, possibilities to prolong slice viability via cold storage using optimized storage solutions were evaluated. Rat PCLS were cold stored in DMEM/F-12 or two different preservation solutions for up to 28 days at 4°C. After rewarming in DMEM/F-12, metabolic activity, live/dead staining, and mitochondrial membrane potential was assessed to analyze overall tissue viability. Single-cell suspensions were prepared and proportions of CD45+, EpCAM+, CD31+, and CD90+ cells were analyzed. As functional parameters, TNF-α expression was analyzed to detect inflammatory activity and bronchoconstriction was evaluated after acetylcholine stimulus. After 14 days of cold storage, viability and mitochondrial membrane potential were significantly better preserved after storage in solution 1 (potassium chloride rich) and solution 2 (potassium- and lactobionate-rich analog) compared with DMEM/F-12. Analysis of cell populations revealed efficient preservation of EpCAM+, CD31+, and CD90+ cells. Proportion of CD45+ cells decreased during cold storage but was better preserved by both modified solutions than by DMEM/F-12. PCLS stored in solution 1 responded substantially longer to inflammatory stimulation than those stored in DMEM/F-12 or solution 2. Analysis of bronchoconstriction revealed total loss of function after 14 days of storage in DMEM/F-12 but, in contrast, a good response in PCLS stored in the optimized solutions. An improved base solution with a high potassium chloride concentration optimizes cold storage of PCLS and allows shipment between laboratories and stockpiling of tissue samples.

Placental extracellular vesicles retain biological activity after short-term storage (14 days) at 4 °C or room temperature.

INTRODUCTION: To investigate the role of placental extracellular vesicles (EVs), especially in pathological pregnancy, the use of freshly isolated EVs is often limited due to the sporadic and unpredictable availability of placental samples. Therefore, it is important to understand and use optimised storage conditions for placental EVs. In this study, we investigated different conditions for the short-term storage of placental micro- and nano-EVs and examined their biological activity. METHODS: Placental EVs were collected from first trimester placentae. EVs were suspended in PBS and aliquoted, and then stored for up to 14 days at room temperature, 4 °C or -20 °C. Total protein and DNA levels were measured at various time points. The ability of stored placental EVs to alter endothelial cell activation was quantified by monocyte adhesion assays. RESULTS: There was no difference in the concentration of placental micro- or nano-EVs between each time point, when stored at either room temperature or 4 °C. However, there was a significant loss of placental EVs after storage at -20 °C. There was no difference in protein or DNA levels of placental EVs when stored at either room temperature or 4 °C. Biological activity of placental EVs was retained for up to 14 days at either room temperature or 4 °C measured by monocyte adhesion assays. DISCUSSION: We have shown that placental micro- and nano-EVs are stable and retain biological activities following storage in PBS or media for 14 days at either room temperature or 4 °C.

The Japanese Society of Pathology Practical Guidelines on the handling of pathological tissue samples for cancer genomic medicine.

Clinical cancer genomic testing based on next-generation sequencing can help select genotype-matched therapy and provide diagnostic and prognostic information. Pathological tissue from malignant tumors obtained during routine practice are frequently used for genomic testing. This article is aimed to standardize the proper handling of pathological specimens in practice for genomic medicine based on the findings established in "Guidelines on the handling of pathological tissue samples for genomic medicine (in Japanese)" published by The Japanese Society of Pathology (JSP) in 2018. The two-part practical guidelines are based on empirical data analyses; Part 1 describes the standard preanalytic operating procedures for tissue collection, processing, and storage of formalin-fixed paraffin-embedded (FFPE) samples, while Part 2 describes the assessment and selection of FFPE samples appropriate for genomic testing, typically conducted by a pathologist. The guidelines recommend that FFPE sample blocks be used within 3 years from preparation, and the tumor content should be ≥30% (minimum 20%). The empirical data were obtained from clinical studies performed by the JSP in collaboration with leading Japanese cancer genome research projects. The Japanese Ministry of Health, Labour, and Welfare (MHLW) recommended to comply with the JSP practical guidelines in implementing cancer genomic testing under the national health insurance system in over 200 MHLW-designated core and cooperative cancer genome medicine hospitals in Japan.

Isochoric supercooled preservation and revival of human cardiac microtissues.

Low-temperature biopreservation and 3D tissue engineering present two differing routes towards eventual on-demand access to transplantable biologics, but recent advances in both fields present critical new opportunities for crossover between them. In this work, we demonstrate sub-zero centigrade preservation and revival of autonomously beating three-dimensional human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues via isochoric supercooling, without the use of chemical cryoprotectants. We show that these tissues can cease autonomous beating during preservation and resume it after warming, that the supercooling process does not affect sarcomere structural integrity, and that the tissues maintain responsiveness to drug exposure following revival. Our work suggests both that functional three dimensional (3D) engineered tissues may provide an excellent high-content, low-risk testbed to study complex tissue biopreservation in a genetically human context, and that isochoric supercooling may provide a robust method for preserving and reviving engineered tissues themselves.

Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs.

The standard histological processing procedure, which produces excellent staining of sections for most tissues, fails to yield satisfactory results in adult mouse orbits or eyeballs. Here, we show that a protocol using tissue block staining and domestic adhesive tapes resulted in qualified integral serial cryo-sections of whole orbits or eyeballs, and the fine structures were well preserved. The histological processing protocol comprises paraformaldehyde fixation, ethylenediaminetetraacetic acid decalcification, tissue block staining with hematoxylin and eosin, embedding, adhesive tape aided sectioning, and water-soluble mounting. This protocol was proved to be the best in comparison with seven other related existing histological traditional or non-traditional processing methods, according to the staining slice quality. We observed a hundred percent success rate in sectioning, collection, and mounting with this method. The reproducibility tested on qualified section success rates and slice quality scores confirmed that the technique is reliable. The feasibility of the method to detect target molecules in orbits was verified by successful trial tests on block immunostaining and adhesive tape-aided sectioning. Application of this protocol in joints, brains, and so on,-the challenging integral sectioning tissues, also generated high-quality histological staining sections.

Advanced technologies for the preservation of mammalian biospecimens.

The three classical core technologies for the preservation of live mammalian biospecimens-slow freezing, vitrification and hypothermic storage-limit the biomedical applications of biospecimens. In this Review, we summarize the principles and procedures of these three technologies, highlight how their limitations are being addressed via the combination of microfabrication and nanofabrication, materials science and thermal-fluid engineering and discuss the remaining challenges.

Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells.

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin ß4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.