Endometriotic tissue fragments are viable after cryopreservation in an ex vivo tissue model recapitulating the fibrotic microenvironment.

STUDY QUESTION: Is it possible to establish an ex vivo endometriosis model using cryopreserved endometriotic tissue fragments? SUMMARY ANSWER: Cryopreserved endometriotic tissue fragments remain viable after thawing and during at least 3 days of culture and can therefore be used to establish an ex vivo endometriosis model to efficiently test potential therapeutic agents. WHAT IS KNOWN ALREADY: Endometriosis is the most prevalent benign gynecologic disease with an enormous societal burden; however, curative therapies are still lacking. To efficiently test potential new therapies, an ex vivo model based on previously cryopreserved endometriotic tissue that recapitulates the different endometriosis subtypes and their microenvironment is highly desirable. STUDY DESIGN, SIZE, DURATION: Endometriotic tissue fragments of three different subtypes were obtained from 28 patients by surgical resection. After cryopreservation and thawing, viability and metabolic activity of these tissue fragments were assessed. Viability was compared with fresh fragments from 11 patients directly after surgical removal. Experimental intervention studies were performed in cryopreserved and thawed tissue fragments from two patients to confirm the usability of these tissues for ex vivo intervention studies. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometriotic tissue fragments (n = 45) were cryopreserved according to three different protocols. After thawing, fragments were cultured for 24 h. A resazurin-based assay was performed to assess the metabolic activity of the tissue fragments. In addition, cell type-specific viability was analyzed by VivaFix, Hoechst 33342, and α-smooth muscle actin immunofluorescence staining and confocal microscopy. The presence of endometriosis was histologically confirmed based on hematoxylin-eosin staining. Cryopreserved and thawed tissue fragments were treated for 72 h with pirfenidone or metformin and COL1A1 and CEMIP gene expressions were assessed using RT-PCR and RT-qPCR, either in the whole tissue fragments or in myofibroblasts isolated by laser capture microdissection. MAIN RESULTS AND THE ROLE OF CHANCE: Metabolic activity of endometriotic tissue fragments obtained from peritoneal (PER), ovarian (OMA), and deep (DE) endometriotic lesions was well preserved after cryopreservation in a dimethyl sulfoxide-based medium and was comparable with fresh tissue fragments. Relative metabolic activity compared to fresh tissue was 70% (CI: 92-47%) in PER, 43% (CI: 53-15%) in OMA and 94% (CI: 186-3%) in DE lesions. In fragments from PE lesions 92% (CI: 87-96%), from OMA lesions 95% (CI: 91-98%), and from DE lesions 88% (CI: 78-98%) of cells were viable after cryopreservation and thawing followed by a 24-h culture period. Differences in gene expression of fibrotic markers COL1A1 and CEMIP after 72-h treatment with pirfenidone or metformin could be detected in whole tissue fragments and in isolated myofibroblasts, indicating that cryopreserved and thawed endometriotic tissue fragments are suitable for testing anti-fibrotic interventions. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Viability and metabolic activity of the endometriotic tissue fragments may have been partially compromised by damage sustained during the surgical procedure, contributing to inter-sample variance. WIDER IMPLICATIONS OF THE FINDINGS: The storage of viable endometriotic tissue fragments for later usage in an ex vivo model creates the possibility to efficiently test potential new therapeutic strategies and facilitates the exchange of viable endometriotic tissue between different research laboratories. STUDY FUNDING/COMPETING INTEREST(S): This study was not financially supported by external funding. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

Organ-on-a-chip models for development of cancer immunotherapies.

Cancer immunotherapy has emerged as a promising approach in the treatment of diverse cancer types. However, the development of novel immunotherapeutic agents faces persistent challenges due to poor translation from preclinical to clinical stages. To address these challenges, the integration of microfluidic models in research efforts has recently gained traction, bridging the gap between in vitro and in vivo systems. This approach enables modeling of the complex human tumor microenvironment and interrogation of cancer-immune interactions. In this review, we analyze the current and potential applications of microfluidic tumor models in cancer immunotherapy development. We will first highlight current trends in the immunooncology landscape. Subsequently, we will discuss recent examples of microfluidic models applied to investigate mechanisms of immune-cancer interactions and for developing and screening cancer immunotherapies in vitro. First steps toward their validation for predicting human in vivo outcomes are discussed. Finally, promising opportunities that microfluidic tumor models offer are highlighted considering their advantages and current limitations, and we suggest possible next steps toward their implementation and integration into the immunooncology drug development process.

The Application of Organs-on-a-Chip in Dental, Oral, and Craniofacial Research.

The current development of microfluidics-based microphysiological systems (MPSs) will rapidly lead to a paradigm shift from traditional static 2-dimensional cell cultivation towards organized tissue culture within a dynamic cellular milieu. Especially organs-on-a-chip (OoCs) can very precisely re-create the mechanical and unique anatomical structures of the oral environment. This review provides an introduction to such technology, from commonly used chip materials and fabrication methods to the application of OoC in in vitro culture. OoCs are advantageous because of their small-scaled culture environment, the highly controlled dynamic experimental conditions, and the likeness to the in vivo structure. We specifically focus on current chip designs in dental, oral, and craniofacial (DOC) research. Also, future perspectives are discussed, like model standardization and the development of integrated platforms with advanced read-out functionality. By doing so, it will be possible for OoCs to serve as an alternative for animal testing and to develop highly predictive human models for clinical experiments and even personalized medicine.

Unravelling Receptor and RGD Motif Dependence of Retargeted Adenoviral Vectors using Advanced Tumor Model Systems.

Recent advances in engineering adenoviruses are paving the way for new therapeutic gene delivery approaches in cancer. However, there is limited knowledge regarding the impact of adenoviral retargeting on transduction efficiency in more complex tumor architectures, and the role of the RGD loop at the penton base in retargeting is unclear. To address this gap, we used tumor models of increasing complexity to study the role of the receptor and the RGD motif. Employing tumor-fibroblast co-culture models, we demonstrate the importance of the RGD motif for efficient transduction in 2D through the epithelial cell adhesion molecule (EpCAM), but not the epidermal growth factor receptor (EGFR). Via optical clearing of co-culture spheroids, we show that the RGD motif is required for transduction via both receptors in 3D tumor architectures. We subsequently employed a custom-designed microfluidic model containing collagen-embedded tumor spheroids, mimicking the interplay between interstitial flow, extracellular matrix and adenoviral transduction. Image analysis of on-chip cleared spheroids indicated the importance of the RGD motif for on-chip adenoviral transduction. Together, our results show the interrelationship between receptor characteristics, the RGD motif, the 3D tumor architecture and retargeted adenoviral transduction efficiency. The findings are important for the rational design of next-generation therapeutic adenoviruses.

Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure.

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane-cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane-cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.