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.

Organotypic 3D Ex Vivo Co-culture Model of the Macro-metastasis/Organ Parenchyma Interface.

The macro-metastasis/organ parenchyma interface (MMPI) is gaining increasing significance due to its prognostic relevance for cancer (brain) metastasis. We have developed an organotypic 3D ex vivo co-culture model that mimics the MMPI and allows us to evaluate the histopathological growth pattern (HGP) and infiltration grade of the tumor cells into the neighboring brain tissue and to study the interactions of cancer and glial cells ex vivo. This system consists of a murine brain slice and a 3D tumor plug that can be co-cultured for several days. After slicing the brain of 5- to 8-day-old mice, a Matrigel plug containing fluorescent-labelled tumor cells is placed next to it, so that tumor cells in the 3D plug and glial cells in the brain slice can interact at the interface for up to 96 h. To facilitate the positioning of the co-culture and increase the reproducibility of the model, a brain spacer can be used. The HGP and infiltration of the tumor cells into the brain slice as well as the activation of the glial cells can be assessed by live and/or confocal microscopy after immunofluorescence staining of microglia and/or astrocytes. Alternatively, the co-culture can also be used for other purposes, such as RNA analysis. This organotypic 3D ex vivo co-culture offers a perfect tool for preliminary screenings before in vivo experiments and reduces the number of animals, thus contributing to the 3R concept as a central precept in preclinical research.

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.

'Speed-ageing' of human skin in serum-free organ culture ex vivo: An instructive novel assay for preclinical human skin ageing research demonstrates senolytic effects of caffeine and 2,5-dimethylpyrazine.

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ-cultured in a defined serum-free medium. Quantitative (immuno-)histomorphometry documented this unexpected ex vivo phenotype on the basis of ageing-associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin-1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA-damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT-PCR of whole skin RNA extracts also showed up-regulated mRNA levels of several (inflamm-) ageing biomarkers (MMP-1, -2, -3, -9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti-ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up-regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti-ageing effects of topical 2,5-dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant "speed-ageing" assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well-suited to identify novel anti-ageing actives directly in the human target organ.

Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche.

Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell-matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.

Human immature testicular tissue organ culture: a step towards fertility preservation and restoration.

Background: Cryopreservation of immature testicular tissue (ITT) is currently the only option to preserve fertility of prepubertal patients. Autologous transplantation of ITT may not be safe or appropriate for all patients. Therefore, methods to mature ITT ex vivo are needed. Objectives: Aim to investigate the feasibility of inducing in vitro spermatogenesis from ITT cryopreserved for pediatric patients prior to initiation of gonadotoxic therapy. Materials and methods: Cryopreserved-thawed ITT from prepubertal and peripubertal patients were cultured for 7, 16, and 32 days in medium with no hormones or supplemented with 5 IU/L FSH, 1 IU/L hCG, or 5IU/L FSH+1 IU/L hCG. Samples were evaluated histologically to assess tissue integrity, and immunofluorescence staining was performed to identify VASA (DDX4)+ germ cells, UCHL1+ spermatogonia, SYCP3+ spermatocytes, CREM+ spermatids, SOX9+ Sertoli cells. Proliferation (KI67) and apoptosis (CASPASE3) of germ cells and Sertoli cells were also analyzed. Sertoli and Leydig cell maturation was evaluated by AR and INSL3 expression as well as expression of the blood testis barrier protein, CLAUDIN11, and testosterone secretion in the culture medium. Results: Integrity of seminiferous tubules, VASA+ germ cells and SOX9+ Sertoli cells were maintained up to 32 days. The number of VASA+ germ cells was consistently higher in the peripubertal groups. UCHL1+ undifferentiated spermatogonia and SOX9+ Sertoli cell proliferation was confirmed in most samples. SYCP3+ primary spermatocytes began to appear by day 16 in both age groups. Sertoli cell maturation was demonstrated by AR expression but the expression of CLAUDIN11 was disorganized. Presence of mature and functional Leydig cells was verified by INSL3 expression and secretion of testosterone. Gonadotropin treatments did not consistently impact the number or proliferation of germ cells or somatic cells, but FSH was necessary to increase testosterone secretion over time in prepubertal samples. Conclusion: ITT were maintained in organotypic culture for up to 32 days and spermatogonia differentiated to produce primary spermatocytes in both pre- and peripubertal age groups. However, complete spermatogenesis was not observed in either group.

P17-A140†Clinical outcomes of the first organ-cultured preloaded DMEK-a single centre studie.

The use of ready-to-use grafts from specialized eye banks might provide a number of benefits, including graft quality control, assured availability at a certain operation time, a decreased likelihood of case cancellation, and a shorter and less sophisticated DMEK surgery, with a resulting lower risk of graft damage. However, it is critical to thoroughly establish the clinical safety of employing these preloaded tissues. Especially since most of the studies were prepared and stored under hypothermic conditions. There are only a few studies on preprepared tissues in organ culture, which are partly controversial.In this prospective study we included patients who received DMEK surgery at the Knappschaft Eye Clinic Sulzbach. Patients received either a preloaded DMEK (pDMEK), prepared five days before surgery in the eye bank, or a conventional, directly before surgery, surgeon-prepared DMEK (sDMEK).The preliminary data show a trend towards more frequent need for rebubbing in the pDMEK group and a statistically non-significant lower postoperative endothelial cell count compared to the sDMEK group. However, the development of visual acuity and decrease in corneal thickness is comparable in both groups.Therefore, we investigated the clinical outcomes of the first organ-cultured preloaded DMEK cases and compared these outcomes with those from our very last cases with surgically loaded tissues from a single centre.

Oestrogen and Vibration Improve Intervertebral Disc Cell Viability and Decrease Catabolism in Bovine Organ Cultures.

Postmenopausal women are at an increased risk for intervertebral disc degeneration, possibly due to the decrease in oestrogen levels. Low-magnitude, high-frequency vibration (LMHFV) is applied as a therapeutic approach for postmenopausal osteoporosis; however, less is known regarding possible effects on the intervertebral disc (IVD) and whether these may be oestrogen-dependent. The present study investigated the effect of 17ß-oestradiol (E2) and LMHFV in an IVD organ culture model. Bovine IVDs (n = 6 IVDs/group) were treated with either (i) E2, (ii) LMHFV or (iii) the combination of E2 + LMHFV for 2 or 14 days. Minor changes in gene expression, cellularity and matrix metabolism were observed after E2 treatment, except for a significant increase in matrix metalloproteinase (MMP)-3 and interleukin (IL)-6 production. Interestingly, LMHFV alone induced cell loss and increased IL-6 production compared to the control. The combination of E2 + LMHFV induced a protective effect against cell loss and decreased IL-6 production compared to the LMHFV group. This indicates possible benefits of oestrogen therapy for the IVDs of postmenopausal women undergoing LMHFV exercises.

Establishing a Microfluidic Tumor Slice Culture Platform to Study Drug Response.

Accurate models for tumor biology and prediction of drug responses of individual tumors require novel technology to grow tumor tissue ex vivo to maintain tumor growth characteristics in situ. Models containing only tumor cells, without the stromal components of the tumor, are suboptimal for many purposes and are generally problematic because the cells are passed through extensive culture and selection. Therefore, direct culture of (human) tumors is of considerable interest for basic tumor biology and diagnostic purposes. Microfluidic technologies have been proposed to accurately mimic physiological conditions for tissue growth. Most published systems build tissues from individual cell types in so-called Organ-on-Chip (OoC) cultures. We here describe a novel OoC device for growing tumor specimens. Thin tumor slices are grown in a microfluidic 'chip' that allows precisely controlled in vitro culture conditions. The performance of the OoC device was extensively validated for predicting therapeutic responses in human breast cancer patient-derived xenograft (PDX) tumor material. The system is amenable to primary tumor material from surgery or biopsies. In addition to using the model to predict and evaluate therapeutic responses, the model can also be used for mechanistic studies of human cancers, such as clonal evolution or immune responses, or to validate new or repurposed (cancer) drugs. The Bi/ond Cancer-on-Chip (CoC) device is designed to culture tumor slices and investigate aspects of tumor growth and drug responses. Here, we describe the step-by-step process of setting up tumor slice cultures using a Bi/ond CoC device and performing in vitro drug response evaluation. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of breast cancer tumor slice culture using a microfluidic cancer-on-chip platform for chemotherapy testing ex vivo Basic Protocol 2: Histology and immunohistochemistry-based analysis of tumor tissue architecture, cell proliferation, and cell death.

Cisplatin Induces Apoptosis in Mouse Neonatal Testes Organ Culture.

Chemotherapy is used for childhood cancer but may lead to infertility in patients. Spermatogonia stem cells are present in the testes of prepubertal boys, although they do not produce sperm at this age. Herein, we evaluated the toxicity of cisplatin, a known medicine for cancer treatment, in neonatal mouse testes using in vitro organ culture. Mouse testicular fragments (MTFs) derived from 5.5-d postpartum mouse testes were exposed to 1-10 µg/mL cisplatin. The results showed that cisplatin significantly downregulated the expression of germ cell marker genes, including differentiated and undifferentiated, in a dose-dependent manner. In particular, a high dose of cisplatin (10 µg/mL) led to germ cell depletion. In addition, the expression levels of the Sertoli cell marker gene, the number of SOX9+ Sertoli cells, and the levels of SOX9 protein were markedly decreased in cisplatin-treated MTFs compared to controls. The mRNA expression of steroidogenic enzyme-related genes significantly increased in cisplatin-treated MTFs, except for estrogen receptor 1 (Esr1). Consistently, 3ß-hydroxysteroid dehydrogenase protein was also observed in the interstitial regions of cisplatin-treated MTFs. Altogether, our findings showed a significant impairment in germ cell development, Sertoli cell survival, and steroidogenesis in the MTFs of cisplatin-treated mice.

Recent advances in organotypic tissue slice cultures for anticancer drug development.

Organotypic tissue slice culture is established from animal or patient tissues and cultivated in an in vitro ecosystem. This technique has made countless contributions to anticancer drug development due to the vast number of advantages, such as the preservation of the cell repertoire and immune components, identification of invasive ability of tumors, toxicity determination of compounds, quick assessment of therapeutic efficacy, and high predictive performance of drug responses. Importantly, it serves as a reliable tool to stratify therapeutic responders from nonresponders and select the optimal standard-of-care treatment regimens for personalized medicine, which is expected to become a potent platform and even the gold standard for anticancer drug screening of individualization in the near future.

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1.

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Small hairpin inhibitory RNA delivery in the metanephric organ culture identifies long noncoding RNA Pvt1 as a modulator of cyst growth.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder characterized by the formation of kidney cysts that originate from the epithelial tubules of the nephron and primarily results from mutations in polycystin-1 (PKD1) and polycystin-2 (PKD2). The metanephric organ culture (MOC) is an ex vivo system in which explanted embryonic kidneys undergo tubular differentiation and kidney development. MOC has been previously used to study polycystic kidney disease as treatment with 8-bromo-cAMP induces the formation of kidney cysts. However, the inefficiency of manipulating gene expression in MOC has limited its utility for identifying genes and pathways that are involved in cystogenesis. Here, we used a lentivirus and three serotypes of self-complementary adeno-associated viral (scAAV) plasmids that express green fluorescent protein and found that scAAV serotype D/J transduces the epithelial compartment of MOC at an efficiency of 68%. We used scAAV/DJ to deliver shRNA to knockdown Pvt1, a long noncoding RNA, which was upregulated in kidneys from Pkd1 and Pkd2 mutant mice and humans with ADPKD. shRNA delivery by scAAV/DJ downregulated expression of Pvt1 by 45% and reduced the cyst index by 53% in wild-type MOCs and 32% in Pkd1-null MOCs. Knockdown of Pvt1 decreased the level of c-MYC protein by 60% without affecting Myc mRNA, indicating that Pvt1 regulation of c-MYC was posttranscriptional. These results identify Pvt1 as a long noncoding RNA that modulates cyst progression in MOC.NEW & NOTEWORTHY This study identified scAAV/DJ as effective in transducing epithelial cells of the metanephric organ culture (MOC). We used scAAV/DJ shRNA to knockdown Pvt1 in cystic MOCs derived from Pkd1-null embryos. Downregulation of Pvt1 reduced cyst growth and decreased levels of c-MYC protein. These data suggest that suppression of Pvt1 activity in autosomal dominant polycystic kidney disease might reduce cyst growth.

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.

Effect of Descemet Membrane Endothelial Keratoplasty Graft Storage Time on Graft Elasticity.

PURPOSE: The purpose of this study was to evaluate the effect of Descemet membrane endothelial keratoplasty (DMEK) graft storage time on its elastic properties, measured using atomic force microscopy (AFM). METHODS: Twenty human corneas (from 10 donors), unsuitable for transplantation, were obtained from the eye bank (S. Fyodorov Eye Microsurgery State Institution, Moscow). Ten DMEK grafts were prepared and stored in the corneal storage medium, Optisol-GS at 4°C after preparation, and AFM analysis was performed within 12 hours after preparation (group A). Ten paired corneas from the respective donors were stored in Optisol-GS at 4°C for 1 week after preparation before AFM analysis (group B). Data were analyzed using the Hertz model for the evaluation of the Young modulus of elasticity. RESULTS: Force-distance curve analysis showed an increase in the Young modulus of elasticity in group B in comparison with that in group A, and the mean values were 10.4 ± 1.8 kPa and 6.77 ± 2.25 kPa, respectively (P < 0.001). There was no correlation between the Young modulus of elasticity and donor age (r = 0.110, P = 0.644), endothelial cell count (r = -0.145, P = 0.541), and procurement interval (r = 0.14, P = 0.755). CONCLUSIONS: A longer graft storage time in cold storage medium was found to significantly reduce the elasticity of the DMEK graft. Clinically, this could potentially influence the unfolding of the DMEK graft within the anterior chamber during surgery and the postoperative detachment rate.

An ex vivo organ culture screening model revealed that low temperature conditions prevent side effects of anticancer drugs.

Development of chemotherapy has led to a high survival rate of cancer patients; however, the severe side effects of anticancer drugs, including organ hypoplasia, persist. To assume the side effect of anticancer drugs, we established a new ex vivo screening model and described a method for suppressing side effects. Cyclophosphamide (CPA) is a commonly used anticancer drug and causes severe side effects in developing organs with intensive proliferation, including the teeth and hair. Using the organ culture model, we found that treatment with CPA disturbed the growth of tooth germs by inducing DNA damage, apoptosis and suppressing cellular proliferation and differentiation. Furthermore, low temperature suppressed CPA-mediated inhibition of organ development. Our ex vivo and in vitro analysis revealed that low temperature impeded Rb phosphorylation and caused cell cycle arrest at the G1 phase during CPA treatment. This can prevent the CPA-mediated cell damage of DNA replication caused by the cross-linking reaction of CPA. Our findings suggest that the side effects of anticancer drugs on organ development can be avoided by maintaining the internal environment under low temperature.

Biotechnological production of bacosides from cell and organ cultures of Bacopa monnieri.

Bacopa monnieri (L.) Wettst. (BM), also known as 'Brahmi' or 'Water Hyssop', has been utilized as a brain tonic, memory enhancer, sensory organ revitalizer, cardiotonic, anti-anxiety, antidepressant and anticonvulsant agent in the Indian system of medicine Ayurveda for centuries. BM is beneficial in the treatment of Parkinson's disease, Alzheimer's disease, epileptic seizures and schizophrenia in recent pharmacological research. Dammarane-type triterpenoid saponins containing jujubogenin and pseudojujubogenin as aglycones, also known as bacosides, are the principal chemical ingredients identified and described from BM. Bacosides have been shown to have anti-ageing, anticancer, anticonvulsant, antidepressant, anti-emetic, anti-inflammatory and antibacterial properties in a variety of pre-clinical and clinical studies. The pharmaceutical industry's raw material comes from wild sources; nevertheless, the concentration of bacosides varies in different regions of the plants, as well as seasonal and genotypic variation. Cell and tissue cultures are appealing alternatives for the long-term manufacture of bioactive chemicals, and attempts to produce bacosides using in vitro cultures have been made. This review discusses the biotechnological approaches used to produce bacosides, as well as the limitations and future potential. KEY POINTS: • Bacosides extracted from Bacopa monnieri are important pharmaceutical compounds. • The current review provides insight into biotechnological interventions for the production of bacosides using in vitro cultures. • Highlights the prospects improvement of bacoside production through metabolic engineering.

Protocol for Studying Embryonic Mammary Gland Branching Morphogenesis Ex Vivo.

Mammary gland development starts during embryogenesis, and the process continues after birth. During development, the mammary gland undergoes massive morphological and physiological alterations including growth, invasion, and branching morphogenesis providing an ideal model for stem cell and cancer biology studies. Great efforts have been made in understanding mammary gland development during puberty and adulthood; however, the process during embryogenesis is still elusive. One reason is that the tools to study tissue dynamics during development are limited, which is partially due to the lack of an ex vivo culture method. Here we describe an updated organ culture protocol of the murine embryonic mammary gland. This powerful tool allows monitoring of growth and branching morphogenesis of mammary gland ex vivo by live imaging. In addition, we introduce a novel method for culturing intact, stroma-free mammary rudiments from late gestation mouse embryos in 3D in Matrigel. This approach can be used to identify the direct stromal cues for branching morphogenesis.

TrkB agonist antibody ameliorates fertility deficits in aged and cyclophosphamide-induced premature ovarian failure model mice.

Premature ovarian failure (POF) is a leading cause of women's infertility without effective treatment. Here we show that intravenous injection of Ab4B19, an agonistic antibody for the BDNF receptor TrkB, penetrates into ovarian follicles, activates TrkB signaling, and promotes ovary development. In both natural aging and cyclophosphamide-induced POF models, treatment with Ab4B19 completely reverses the reduction of pre-antral and antral follicles, and normalizes gonadal hormone. Ab4B19 also attenuates gonadotoxicity and inhibits apoptosis in cyclophosphamide-induced POF ovaries. Further, treatment with Ab4B19, but not BDNF, restores the number and quality of oocytes and enhances fertility. In human, BDNF levels are high in granulosa cells and TrkB levels increase in oocytes as they mature. Moreover, BDNF expression is down-regulated in follicles of aged women, and Ab4B19 activates TrkB signaling in human ovary tissue ex vivo. These results identify TrkB as a potential target for POF with differentiated mechanisms, and confirms superiority of TrkB activating antibody over BDNF as therapeutic agents.

Trypsin-induced elevated contractile responses in a rat model of interstitial cystitis/bladder pain syndrome: Involvement of PAR2 and intracellular Ca2+ release pathways.

AIMS: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disease with unclear etiology. Different receptors play a role in the pathophysiology including protease activated receptors (PARs). The present study aimed to investigate the subtypes and the effects of PARs on contractility using permeabilized detrusor smooth muscle strips in IC/BPS. MAIN METHODS: IC/BPS was induced by cyclophosphamide injection. Histopathological analysis, PCR for detecting PAR proteins, western blotting for indicating PAR2 protein expression levels and myograph recording for measuring contractile force were used. KEY FINDINGS: The present study reveals that in rat bladder PAR1 and PAR2 but not PAR4 were found to be expressed. The first evidence was revealed where trypsin-induced contractions in rat permeabilized detrusor were potentiated in CYP-induced cystitis. Moreover, the functional inhibition of trypsin-induced contractions by selective PAR2 antagonist (ENMD-1068) and the supporting immunoblotting results emphasized that the main PAR subtype involved in IC/BPS model in rat bladder is PAR2. Our data emphasize the prominent role of IP3 in cystitis pathology besides ryanodine channels. Trypsin-induced Ca2+sensitization contractions were also higher in cystitis. Both Rho kinase and protein kinase C played a role in this increased Ca2+sensitization situation. SIGNIFICANCE: The present paper highlights the intracellular pathways that are involved in trypsin-induced contractions mainly via PAR2 in permeabilized bladder detrusor smooth muscle in a rat model of IC/BPS.