Human Anterior Segment Perfusion Organ Culture.

The human anterior segment perfusion organ culture is an ex vivo model system for studying the human conventional outflow pathway with reference to pressure regulation. In this model, anterior segments dissected from human donor eyes can be fixed to a modified petri dish and perfused with media along with various study agents at the physiological flow rate of 2.5 µL/min. The model mimics the one-way flow of aqueous humor in human eyes and can be used to evaluate the effects of various drugs on eye pressure in real time. Using this model, cells and tissues of the anterior segment can be maintained for up to 28 days, enabling histological and molecular evaluations.

Human Corneal Rim Perfusion Culture.

Human anterior segment perfusion cultures are frequently used for trabecular meshwork research. However, this model requires the use of whole eye globes which are expensive. Here, we describe a method using human corneal rims as an alternative to anterior segments for perfusion culture. In this method, the human corneal rim is glued to a three-dimensional-printed perfusion plates instead of using mechanical compression in traditional perfusion culture setup. The corneal rim and perfusion plate are placed onto a special holder for perfusion culture. This method is affordable and easy to set up. The instruments for traditional perfusion culture studies can be used for this model without modification.

Segmental Aqueous Humor Outflow.

Of the known risk factors for glaucoma, elevated intraocular pressure (IOP), is the primary one. The conventional aqueous humor outflow pathway contains the key source of IOP regulation, which is predominantly the trabecular meshwork (TM). Studies of outflow have demonstrated that the outflow pathway is not uniform around the circumference of the eye but highly segmental with regions of relative high flow (HF) and intermediate or medium flow (IF) and regions of low or no flow (LF). Herein we present protocols that we use to study outflow segmentation through the conventional outflow pathway, mostly focusing on human eyes. These methods are quite similar for nonhuman primates and other species. These studies are mostly conducted using ex vivo intact globes or perfused anterior segment organ culture. One potential therapy for IOP reduction in those with elevated IOP to reduce progression of glaucomatous optic nerve damage would be to increase HF or IF and reduce LF proportions.

Neonatal exposure to high D-galactose affects germ cell development in neonatal testes organ culture.

Excess exogenous supplementation of D-galactose (D-gal), a monosaccharide and reducing sugar, generates reactive oxygen species (ROS), leading to cell damage and death. ROS accumulation is critical in aging. Therefore, D-gal-induced aging mouse models are used in aging studies. Herein, we evaluated D-gal's effect on neonatal testis development using an in vitro organ culture method. Mouse testicular fragments (MTFs) derived from neonatal testes (postnatal day 5) were cultured with 500 mM D-gal for 5 days. D-gal-treated MTFs showed a significantly increased and decreased expression of undifferentiated and differentiated germ cell markers, respectively, with a substantial reduction in meiotic cells. In D-gal-exposed MTFs, expression levels of Sertoli cell markers (Sox9 and Wt1) increased, while those of StAR and 17ß-HSD3, whose expressions are abundant in D-Gal treated adult Leydig cells, decreased. Additionally, the enzyme 3 ß-HSD1, essential for steroidogenesis in Leydig cells, was significantly reduced in D-gal-exposed MTFs compared to that in controls.D-gal significantly increased the expression of Bad, Bax, and cleaved caspase-3 and -8. Via oxidative stress in MTF. Overall, D-gal negatively regulates germ cell and Leydig cell development in neonatal testes through pro-apoptotic mechanisms and ROS.

Application of testicular organ culture system for the evaluation of spermatogenesis impairment.

Recently, it was reported that a testicular organ culture system (TOCS) using polydimethylsiloxane (PDMS) chips with excellent oxygen permeability and biocompatibility, called the PDMS-chip ceiling (PC) method, enables improved spermatogenesis efficiency. We investigated whether this PC method is useful for detecting impaired spermatogenesis caused by busulfan (Bu), a typical testicular toxicant. In this study, testicular tissue fragments from Acro3-EGFP mice, which express the green fluorescent protein (GFP) and reflect the progression of spermatogenesis, were subjected to the PC method. When treated with Bu, cultured tissues shrank in volume, and their GFP-expressing area decreased or disappeared. Histological examination confirmed the regression of spermatogenesis. In addition, immunohistochemical examination revealed that spermatogonia, including spermatogonial stem cells (SSCs), were the primary targets of Bu toxicity. Time-course analysis demonstrated that the recovery of spermatogenesis, dependent on Bu concentration, correlated closely with the severity of damage to these target cells. These results suggest that the PC method is a useful approach for detecting spermatogenesis impairment accurately through faithful recapitulation of spermatogenesis in vivo.

The Organotypic Culture of Mouse Seminiferous Tubules as a Reliable Methodology for the Study of Meiosis In Vitro.

Male mouse meiosis has been traditionally studied using descriptive methods like histological sections and spreading or squashing techniques, which allow the observation of fixed meiocytes in either wildtype or genetically modified mice. For these studies, the sacrifice of the males and the extraction of the testicles are required to obtain the material of study. Other functional in vivo studies include the administration of intravenous or intraperitoneal drugs, or the exposure to mutagenic agents or generators of DNA damage, in order to study their impact on meiosis progression. However, in these studies, the exposure times or drug concentration are important limitations to consider when acknowledging animal welfare. Recently, several approaches have been proposed to offer alternative methodologies that allow the in vitro study of spermatocytes with a considerable reduction in the use of animals. Here we revisit and validate an optimal technique of organotypic culture of fragments of seminiferous tubules for meiotic studies. This technique is a trustable methodology to develop functional studies that preserve the histological configuration of the seminiferous tubule, aim homogeneity of the procedures (the use of the same animal for different study conditions), and allow procedures that would compromise the animal welfare. Therefore, this methodology is highly recommendable for the study of meiosis and spermatogenesis, while it supports the principle of 3R's for animal research.

Protocol to study early lung developmental branching in mouse embryos using explant culture.

Mouse lung branching morphogenesis creates epithelial tree structures required for respiration. Here, we present a protocol for studying mouse lung developmental branching using lung explant cultures. We describe steps for isolating lungs with a video at embryonic day 12.5 (E12.5) and culturing as an explant for 2 days. We also detail procedures for microscopic imaging on days 0-2 and analysis of peripheral lung buds. This technique has the potential to investigate lung development in various conditions. For complete details on the use and execution of this protocol, please refer to Talvi et al.1.

A Novel Use of Embryonic Gut Organoid Culture to Investigate Duodenal Atresia.

BACKGROUND: The cause of duodenal atresia (DA) is not known. Tandler's "solid cord" hypothesis conflicts with current biological evidence. In humans, a genetic aetiology is supported by the association with Trisomy 21. Interruption of Fgf10 is the strongest genetic link to DA in mice, demonstrating an increased incidence and severity as embryos mature. This project aimed to develop an organoid model to facilitate ex vivo DA research on the FGF10/FGFR2b signalling pathway. We hypothesised that DA morphology represents an evolving spectrum of disease and that Fgf10 knockout organoids would vary in growth pattern compared to wild-type. METHODS: Organoids were cultured from the duodenum of E12.5 Fgf10 knockout, heterozygous and wild-type embryos, using an air-liquid interface with Growth Factor reduced Matrigel. Organoids were photographed every 48 h to observe growth. Organoids were isolated and fixed after 14 days, then stained with DAPI, KI-67, and cytokeratin to demonstrate proliferation and differentiation. RESULTS: Wild-type duodenum developed into crypt-forming organoids. Fgf10 heterozygous duodenum failed to progress beyond the development stage of spheroids. Fgf10 knockout duodenum failed to demonstrate any growth. Wholemount staining showed the greatest cell proliferation and differentiation in wild-type tissue. CONCLUSION: This research presents a novel concept for the growth of embryonic gastrointestinal tissue to inform normal biology. The small sample numbers and restricted culture duration limit longer-term growth analysis. While this model serves as a potential ex vivo setting for future research, that research should consider organoid models with greater standardisation and other gastrointestinal regions. LEVEL OF EVIDENCE: Animal/laboratory study.

Tebuconazole Induces Mouse Fetal Testes Damage via ROS Generation in an Organ Culture Method.

The fungicide tebuconazole (TEB) poses risks to human and animal health via various exposure routes. It induces toxicity in multiple organs and disrupts reproductive health by affecting steroid hormone synthesis and fetal development. In this study, we investigated the impact of TEB on fetal testes using in vitro models, focusing on germ, Sertoli, and Leydig cells, and explored the mechanisms underlying cellular damage. The results revealed significant damage to germ cells and disruption of Leydig cell development. TEB exposure led to a decrease in germ cell numbers, as indicated by histological and immunostaining analyses. TEB induced the up- and down-regulation of the expression of fetal and adult Leydig cell markers, respectively. Additionally, TEB-treated fetal testes exhibited increased expression of oxidative-stress-related genes and proteins. However, co-treatment with the antioxidant N-acetylcysteine mitigated TEB-induced germ cell damage and prevented abnormal Leydig cell development. These findings suggest that administration of antioxidants can prevent the intratesticular damage typically caused by TEB exposure.

Comparison of the structural integrity and quality of corneal endothelium stored in organ culture storage medium versus Eusol-C.

In this experimental study, we compared the structural integrity and cell quality of corneal endothelium stored in organ culture medium (OCS) and Eusol-C. The experiment included rabbit and human cornea experiments in vitro. Thirty rabbit corneas and thirty-two human corneas were collected and divided into two groups. All right corneas were allocated in experiment group and left corneas were placed in control group. The corneas in experimental group were stored in OCS at 34 °C, and the corneas in control group were stored in Eusol-C at 4 °C for 7, 14, 21, 28, and 35 days, respectively. Endothelial cell morphology, cell count, and trypan blue staining for viability were assessed before storage (Day 0) and at days 7, 14, 21, 28 and 35. The structural integrity of human corneal endothelial cell was analyzed using immunohistochemistry. The samples of storage solution for microbial culture were collected on the third day and at the end of storage. The results show that no bacterial and fungal infections were found in both groups. After 14 days of storage, the morphology of endothelial cell was better in the experimental group than in the control group. The endothelial cell stored in OCS were better than those stored in Eusol-C at the end of storage times, except human cornea 14 days storage group. The ZO-1 protein staining showed the typical polygonal morphology of endothelial cell stored in the OCS. Corneal endothelial cells stored in the OCS had better quality up to 28 days. It can be applied to Chinese eye banks as a method of corneal preservation.

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury.

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs. Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (≥30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

An untargeted metabolomics approach to study changes of the medium during human cornea culture.

INTRODUCTION: Two main approaches (organ culture and hypothermia) for the preservation and storage of human donor corneas are globally adopted for corneal preservation before the transplant. Hypothermia is a hypothermic storage which slows down cellular metabolism while organ culture, a corneal culture performed at 28-37 °C, maintains an active corneal metabolism. Researchers, till now, have just studied the impact of organ culture on human cornea after manipulating and disrupting tissues. OBJECTIVES: The aim of the current work was to optimize an analytical procedure which can be useful for discovering biomarkers capable of predicting tissue health status. For the first time, this research proposed a preliminary metabolomics study on medium for organ culture without manipulating and disrupting the valuable human tissues which could be still used for transplantation. METHODS: In particular, the present research proposed a method for investigating changes in the medium, over a storage period of 20 days, in presence and absence of a human donor cornea. An untargeted metabolomics approach using UHPLC-QTOF was developed to deeply investigate the differences on metabolites and metabolic pathways and the influence of the presence of the cornea inside the medium. RESULTS: Differences in the expression of some compounds emerged from this preliminary metabolomics approach, in particular in medium maintained for 10 and 20 days in presence but also in the absence of cornea. A total of 173 metabolites have been annotated and 36 pathways were enriched by pathway analysis. CONCLUSION: The results revealed a valuable untargeted metabolomics approach which can be applied in organ culture metabolomics.

3D Approaches to Culturing Bovine Skin: Explant Culture versus Organotypic Skin Model.

INTRODUCTION: Digital dermatitis (DD) in cattle appears with high prevalence; nevertheless, the knowledge on its pathogenesis is still limited. In this context, in vitro skin models represent a valuable tool to facilitate the study of DD. METHODS: Two in vitro skin models were established using bovine distal limb skin: a skin explant model and an organotypic skin model. For the skin explant model, skin samples were cultured with an air-liquid interface for up to 7 days. Besides routine histopathological examination, readout parameters were Ki-67 and cleaved Caspase-3 stainings. For the organotypic model, primary keratinocytes were layered on top of a dermal equivalent containing mainly mitotically inactive fibroblasts and maintained for up to 21 days. At regular intervals (days 7, 14, and 21), cultured skin samples were taken for (immuno)histological analysis. RESULTS: Both cultures could be maintained for the entire duration of the intended culture period. In the histopathological assessment, explant skin cultures showed ballooning degeneration of keratinocytes and segmental necrosis starting at day 5 of culturing. Initially, basal keratinocytes in the organotypic model differentiated as demonstrated by positive Keratin 14, Desmoglein-1, Loricrin, and Involucrin immunofluorescent stainings. Ki-67 was observed occasionally and suprabasally still after 21 days of culture. CONCLUSION: Both in vitro models proved dependable and constitute a viable option for replacing experiments on live animals, each with its own benefits. Whereas skin explants include all cell types available in vivo and can therefore reflect realistic cell-cell interactions and signaling pathways, the organotypic model offers a higher standardization and reproducibility. Depending on the focus of future studies, both models can be used for specific experimental purposes of bovine dermatological research in general or specialized questions concerning (infectious) claw diseases as, e.g., DD.

New uses for an old technique: live imaging on the slice organ culture to study reproductive processes†.

Reproductive processes are dynamic and involve extensive morphological remodeling and cell-cell interactions. Live imaging of organs enhances our understanding of how biological processes occur in real time. Slice culture is a type of organ culture where thick slices are collected from an organ and cultured for several days. Slice culture is a useful and easy-to-implement technique for live imaging of reproductive events at cellular resolution. Here we describe a pipeline of live imaging on slice culture to visualize the process of urethra closure in mouse embryonic penis as a proof of principle. In combination with genetic reporter mice, nuclear stains, and exposure experiments, we demonstrate the feasibility of slice culture on a reproductive organ. We also provide a step-by-step protocol and troubleshooting guide to facilitate the adoption of slice culture with live imaging in other reproductive organs. Lastly, we discuss potential utilities and experiments that could be implemented with slice culture in reproductive sciences.

Human organotypic brain slice cultures: a detailed and improved protocol for preparation and long-term maintenance.

The investigation of the human brain at cellular and microcircuit level remains challenging due to the fragile viability of neuronal tissue, inter- and intra-variability of the samples and limited availability of human brain material. Especially brain slices have proven to be an excellent source to investigate brain physiology and disease at cellular and small network level, overcoming the temporal limits of acute slices. Here we provide a revised, detailed protocol of the production and in-depth knowledge on long-term culturing of such human organotypic brain slice cultures for research purposes. We highlight the critical pitfalls of the culturing process of the human brain tissue and present exemplary results on viral expression, single-cell Patch-Clamp recordings, as well as multi-electrode array recordings as readouts for culture viability, enabling the use of organotypic brain slice cultures of these valuable tissue samples for basic neuroscience and disease modeling (Fig. 1).

A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

In Vitro Porcine (Explant) Colon Culture.

Models have been extensively used to investigate disease pathogenesis. Animal models are costly and require extensive logistics for animal care, and samples are not always suitable for different analytical techniques or to answer the research question. In vitro cell culture models are generally focused on recreating a specific characteristic of an organ and are limited to a single cell population that does not display the characteristic tissue architecture of the source organ. In addition, such models do not account for the many interactions between pathogens and the diverse cell subsets that are normally present in a given organ. Conclusions based on conventional 2D cell culture methods are limited, requiring extrapolation from a reductionist model to understand in vivo events. In vitro organ culture (IVOC) offers a way to overcome some of these limitations. Explants conserve important in vivo characteristics, such as different cell types and complex tissue architecture. This in vitro (ex vivo) organ culture protocol of the swine large intestine aims at maintaining viable colonic mucosa for up to 5 days. The protocol described herein applies a combination of methods used for immortalized cell culture and stem cell stimulation to support the physiological cellular flow inherent of the intestinal mucosa. Required equipment includes a hyperoxic chamber and culture at the air-liquid interface.

Generation of Human Lung Organoid Cultures from Healthy and Tumor Tissue to Study Infectious Diseases.

Respiratory viruses cause mild to severe diseases in humans every year, constituting a major public health problem. Characterizing the pathogenesis in physiologically relevant models is crucial for developing efficient vaccines and therapeutics. Here, we show that lung organoids derived from human primary or lung tumor tissue maintain the cellular composition and characteristics of the original tissue. Moreover, we show that these organoids sustain viral replication with particular infection foci formation, and they activate the expression of interferon-associated and proinflammatory genes responsible for mediating a robust innate immune response. All together, we show that three-dimensional (3D) lung organoids constitute a relevant platform to model diseases and enable the development of drug screenings. IMPORTANCE Three-dimensional (3D) human lung organoids reflect the native cell composition of the lung as well as its physiological properties. Human 3D lung organoids offer ideal conditions, such as timely availability in large quantities and high physiological relevance for reassessment and prediction of disease outbreaks of respiratory pathogens and pathogens that use the lung as a primary entry portal. Human lung organoids can be used in basic research and diagnostic settings as early warning cell culture systems and also serve as a relevant platform for modeling infectious diseases and drug development. They can be used to characterize pathogens and analyze the influence of infection on, for example, immunological parameters, such as the expression of interferon-associated and proinflammatory genes in the context of cancer. In our study, we found that cancer-derived lung organoids were more sensitive to influenza A virus infection than those derived from healthy tissue and demonstrated a decreased innate immune response.

Engineering brain assembloids to interrogate human neural circuits.

The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region-specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell-cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3-4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.

A Microfluidic Cancer-on-Chip Platform Predicts Drug Response Using Organotypic Tumor Slice Culture.

Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies. SIGNIFICANCE: The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.