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.

Progressive Circuit Hyperexcitability in Mouse Neocortical Slice Cultures with Increasing Duration of Activity Silencing.

Forebrain neurons deprived of activity become hyperactive when activity is restored. Rebound activity has been linked to spontaneous seizures in vivo following prolonged activity blockade. Here, we measured the time course of rebound activity and the contributing circuit mechanisms using calcium imaging, synaptic staining, and whole-cell patch clamp in organotypic slice cultures of mouse neocortex. Calcium imaging revealed hypersynchronous activity increasing in intensity with longer periods of deprivation. While activity partially recovered 3 d after slices were released from 5 d of deprivation, they were less able to recover after 10 d of deprivation. However, even after the longer period of deprivation, activity patterns eventually returned to baseline levels. The degree of deprivation-induced rebound was age-dependent, with the greatest effects occurring when silencing began in the second week. Pharmacological blockade of NMDA receptors indicated that hypersynchronous rebound activity did not require activation of Hebbian plasticity. In single-neuron recordings, input resistance roughly doubled with a concomitant increase in intrinsic excitability. Synaptic imaging of pre- and postsynaptic proteins revealed dramatic reductions in the number of presumptive synapses with a larger effect on inhibitory than excitatory synapses. Putative excitatory synapses colocalizing PSD-95 and Bassoon declined by 39 and 56% following 5 and 10 d of deprivation, but presumptive inhibitory synapses colocalizing gephyrin and VGAT declined by 55 and 73%, respectively. The results suggest that with prolonged deprivation, a progressive reduction in synapse number is accompanied by a shift in the balance between excitation and inhibition and increased cellular excitability.

Editorial for special issue-human precision cut lung slices: an Ex vivo platform for therapeutic target discovery and drug testing in lung disease.

Publications utilizing precision cut lung slices (PCLS) steadily increased from the 1970's, with a significant increase in 2010, to tripling by 2023. PCLS have been used to study a vast array of pulmonary diseases and exposures to pathogens and toxicants to understand pathogenesis of disease but also to examine basic cellular mechanisms that underly lung biology. This Special Issue will highlight new, exciting, and novel research using PCLS, while acknowledging the substantial fund of knowledge that has been gained using this platform.

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.

Organotypic 3D Cell Culture of the Embryonic Lacrimal Gland.

Ectodermal organ development, including lacrimal gland, is characterized by an interaction between an epithelium and a mesenchyme. Murine lacrimal gland is a good model to study non-stereotypical branching morphogenesis. In vitro cultures allow the study of morphogenesis events with easy access to high-resolution imaging. Particularly, embryonic lacrimal gland organotypic 3D cell cultures enable the follow-up of branching morphogenesis thanks to the analysis of territories organization by immunohistochemistry. In this chapter, we describe a method to culture primary epithelial fragments together with primary mesenchymal cells, isolated from embryonic day 17 lacrimal glands.

Review of state-of-the-art micro and macro-bioreactors for the intervertebral disc.

Lower back pain continues to be a global epidemic, limiting quality of life and ability to work, due in large part to symptomatic disc degeneration. Development of more effective and less invasive biological strategies are needed to treat disc degeneration. In vitro models such as macro- or micro-bioreactors or mechanically active organ-chips hold great promise in reducing the need for animal studies that may have limited clinical translatability, due to harsher and more complex mechanical loading environments in human discs than in most animal models. This review highlights the complex loading conditions of the disc in situ, evaluates state-of-the-art designs for applying such complex loads across multiple length scales, from macro-bioreactors that load whole discs to organ-chips that aim to replicate cellular or engineered tissue loading. Emphasis was placed on the rapidly evolving more customizable organ-chips, given their greater potential for studying the progression and treatment of symptomatic disc degeneration. Lastly, this review identifies new trends and challenges for using organ-chips to assess therapeutic strategies.

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.

Establishment of an organ culture system to maintain the structure of mouse Müllerian ducts during development.

We previously showed that the anti-Müllerian hormone (AMH), infiltrating from the testis to the mesonephros reaches the cranial and middle regions of the Müllerian duct (MD) and induces their regression using an organ culture in mice. However, it is difficult to maintain structural integrity, such as the length and diameter and normal direction of elongation of the caudal region of the MD, in conventional organ culture systems. Therefore, the pathway of AMH to the caudal MD region remains uncharted. In this study, we established an organ culture method that can maintain the morphology of the caudal region of the MD. The gonad-mesonephros complex, metanephros, and urinary bladder of mouse fetuses at 12.5 dpc attached to the body trunk were cultured on agarose gels for 72 hr. The cultured caudal region of the mesonephros was elongated along the body trunk, and the course of the mesonephros was maintained in many individuals. In males, mesenchymal cells aggregated around the MD after culture. Moreover, the male MD diameter was significantly smaller than the female. Based on these results, it was concluded that the development of the MD was maintained in the present organ culture system. Using this culture system, AMH infiltration to the caudal region of the MD can be examined without the influence of AMH in the blood. This culture system is useful for clarifying the regression mechanism of the caudal region of the MD.

Direct diffusion of anti-Müllerian hormone from both the cranial and caudal regions of the testis during early gonadal development in mice.

BACKGROUND: The Müllerian duct (MD), the primordium of the female reproductive tract, is also formed in males during the early stage of development, then regresses due to the anti-Müllerian hormone (AMH) secreted from the testes. However, the detailed diffusion pathway of AMH remains unclear. We herein investigated the mechanism by which AMH reaches the middle region of the MD using an organ culture system. RESULTS: Injection of recombinant human AMH into the testis around the start of MD regression induced diffuse immunoreactivity in the mesonephros near the injection site. When the testis and mesonephros were cultured separately, the diameters of both cranial and middle MDs were significantly increased compared to the control. In the testis-mesonephros complex cultured by inhibiting the diffusion of AMH through the cranial region, the cranial MD diameter was significantly increased compared to the control, and there was no difference in middle MD diameter. CONCLUSIONS: These results indicate that AMH, which infiltrates from the testis through the cranial region at physiological concentrations, induces regression of the cranial MD at the start of MD regression. They also indicate that AMH infiltrating through the caudal regions induces regression of the middle MD.

'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.

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.

Injectable hydrogel induces regeneration of naturally degenerate human intervertebral discs in a loaded organ culture model.

Low back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc (IVD). This study investigates the ability of an injectable hydrogel (NPgel), to inhibit catabolic protein expression and promote matrix expression in human nucleus pulposus (NP) cells within a tissue explant culture model isolated from degenerate discs. Furthermore, the injection capacity of NPgel into naturally degenerate whole human discs, effects on mechanical function, and resistance to extrusion during loading were investigated. Finally, the induction of potential regenerative effects in a physiologically loaded human organ culture system was investigated following injection of NPgel with or without bone marrow progenitor cells. Injection of NPgel into naturally degenerate human IVDs increased disc height and Young's modulus, and was retained during extrusion testing. Injection into cadaveric discs followed by culture under physiological loading increased MRI signal intensity, restored natural biomechanical properties and showed evidence of increased anabolism and decreased catabolism with tissue integration observed. These results provide essential proof of concept data supporting the use of NPgel as an injectable therapy for disc regeneration. STATEMENT OF SIGNIFICANCE: Low back pain resulting from disc degeneration is a leading cause of disability worldwide. However, to date few therapies target the cause and fail to repair the intervertebral disc. This study investigated the potential regenerative properties of an injectable hydrogel system (NPgel) within human tissue samples. To mimic the human in vivo conditions and the unique IVD niche, a dynamically loaded intact human disc culture system was utilised. NPgel improved the biomechanical properties, increased MRI intensity and decreased degree of degeneration. Furthermore, NPgel induced matrix production and decreased catabolic factors by the native cells of the disc. This manuscript provides evidence for the potential use of NPgel as a regenerative biomaterial for intervertebral disc degeneration.

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.

Organ-Chips Enhance the Maturation of Human iPSC-Derived Dopamine Neurons.

While cells in the human body function in an environment where the blood supply constantly delivers nutrients and removes waste, cells in conventional tissue culture well platforms are grown with a static pool of media above them and often lack maturity, limiting their utility to study cell biology in health and disease. In contrast, organ-chip microfluidic systems allow the growth of cells under constant flow, more akin to the in vivo situation. Here, we differentiated human induced pluripotent stem cells into dopamine neurons and assessed cellular properties in conventional multi-well cultures and organ-chips. We show that organ-chip cultures, compared to multi-well cultures, provide an overall greater proportion and homogeneity of dopaminergic neurons as well as increased levels of maturation markers. These organ-chips are an ideal platform to study mature dopamine neurons to better understand their biology in health and ultimately in neurological disorders.

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.

P05-A152†Human platelet lysate for cornea organ culture.

PURPOSE: Comprehensible concerns have been raised regarding the safety of FBS-based culture media. In this talk we discuss the benefits of using human platelet lysate (HPL) for the xeno-free culture of human donor corneas, isolated corneal stromal keratocytes (CSK) and stromal fibroblasts (SF). METHODS: 32 human corneas unsuitable for transplantation from 16 human donors were cultured for 25-days in either 2%FBS or 2%HPL medium and compared by phase contrast microscopy (ECD and morphology), and next generation sequencing (NGS). Effects of 0.5%FBS, 5%FBS, 0.5%HPL, 2%HPL and 10%hPL on cultured human CSK and SF were evaluated. RESULTS: Differential cornea culture showed lower endothelial cell loss in the 2%HPL vs. 2%FBS group (ECL hPL=-0.7% vs. FBS=-3.8%; p=0.01). 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (e.g. HMOX1, SERPINE1, ANGPTL4, LEFTY2) and the downregulation of pro-inflammatory/apoptotic genes (e.g. CXCL14, SIK1B, PLK5, PPP2R3B). CSK/SF cell viability remained high in all groups (98-100%). Cell numbers and proliferation rates increased (p=0.024-0.001), CSK marker expression decreased with higher fractions of HPL and FBS (p<0.001). SMA1 increased with higher amounts of FBS (p=0.003) but decreased with incremental HPL substitution in both cell types (p=0.014). HPL contained more TGF-ß1 (100%hPL 1.861±0.231ng/ml vs. 100%FBS 0.015±0.010ng/ml, p<0.001). bFGF and HGF were only detectable in 100% hPL (bFGF 0.067±0.017ng/ml, HGF 1.074±0.050ng/ml). CONCLUSION: 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and potentially beneficial alterations in gene expression. CSK and SF can be cultured with xeno-free hPL. To maintain CSK characteristics substitution must remain minimal (0.5% hPL/FBS). hPL contains the antifibrotic HGF und bFGF, suppressing myofibroblast conversion.

Krüppel-like factor 12 regulates aging ovarian granulosa cell apoptosis by repressing SPHK1 transcription and sphingosine-1-phosphate (S1P) production.

Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1-phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide-treated GCs and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress-induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress-induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide-KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid-induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback.