Tissue Culture Model Research Articles

Epac activation reduces trans-endothelial migration of undifferentiated neuroblastoma cells and cellular differentiation with a CDK inhibitor further enhances Epac effect.

Neuroblastoma (NB) is the most common solid extracranial neoplasm found in children and is derived from primitive sympathoadrenal neural precursor. The disease accounts for 15% of all cancer deaths in children. The mortality rate is high in patients presenting with a metastatic tumour even with extensive treatments. This signifies the need for further research towards the development of new additional therapies that can combat not only tumour growth but metastasis, especially amongst the high-risk groups. During metastasis, primary tumour cells become migratory and travel towards a capillary within the tumour. They then degrade the matrix surrounding the pericytes and endothelial cells traversing the endothelial barrier twice to establish a secondary. This led to the hypothesis that modulation of the endothelial cell junctional stability could have an influence on tumour metastasis. To test this hypothesis, agents that modulate endothelial permeability on NB cell line migration and invasion were assessed in vitro in a tissue culture model. The cAMP agonist and its antagonists were found to have no obvious effect on both SK-N-BE2C and SK-N-AS migration, invasion and proliferation. Next, NB cells were cocultured with HDMEC cells and live cell imaging was used to assess the effect of an Epac agonist on trans-endothelial cell migration of NB cells. Epac1 agonist remarkably reduced the trans-endothelial migration of both SK-N-BE2C and SK-N-AS cells. These results demonstrate that an Epac1 agonist may perhaps serve as an adjuvant to currently existing therapies for the high-risk NB patients.

In Vitro Models of Cardiovascular Calcification.

Cardiovascular calcification, characterized by hydroxyapatite deposition in the arterial wall and heart valves, is associated with high cardiovascular morbidity and mortality. Cardiovascular calcification is a hallmark of aging but is frequently seen in association with chronic diseases, such as chronic kidney disease (CKD), diabetes, dyslipidemia, and hypertension in the younger population as well. Currently, there is no therapeutic approach to prevent or cure cardiovascular calcification. The pathophysiology of cardiovascular calcification is highly complex and involves osteogenic differentiation of various cell types of the cardiovascular system, such as vascular smooth muscle cells and valve interstitial cells. In vitro cellular and ex vivo tissue culture models are simple and useful tools in cardiovascular calcification research. These models contributed largely to the discoveries of the numerous calcification inducers, inhibitors, and molecular mechanisms. In this review, we provide an overview of the in vitro cell culture and the ex vivo tissue culture models applied in the research of cardiovascular calcification.

Reduction of RAD23A extends lifespan and mitigates pathology in TDP-43 mice.

Protein misfolding and aggregation are cardinal features of neurodegenerative disease (NDD) and they contribute to pathophysiology by both loss-of-function (LOF) and gain-of-function (GOF) mechanisms. This is well exemplified by TDP-43 which aggregates and mislocalizes in several NDDs. The depletion of nuclear TDP-43 leads to reduction in its normal function in RNA metabolism and the cytoplasmic accumulation of TDP-43 leads to aberrant protein homeostasis. A modifier screen found that loss of rad23 suppressed TDP-43 pathology in invertebrate and tissue culture models. Here we show in a mouse model of TDP-43 pathology that genetic or antisense oligonucleotide (ASO)-mediated reduction in rad23a confers benefits on survival and behavior, histological hallmarks of disease and reduction of mislocalized and aggregated TDP-43. This results in improved function of the ubiquitin-proteasome system (UPS) and correction of transcriptomic alterations evoked by pathologic TDP-43. RAD23A-dependent remodeling of the insoluble proteome appears to be a key event driving pathology in this model. As TDP-43 pathology is prevalent in both familial and sporadic NDD, targeting RAD23A may have therapeutic potential.

Characterization of drusen formation in a primary porcine tissue culture model of dry AMD

Age-related macular degeneration (AMD) affects millions of individuals worldwide and is a leading cause of blindness in the elderly. In dry AMD, lipoproteinaceous deposits called drusen accumulate between the retinal pigment epithelium (RPE) and Bruch's membrane, leading to impairment of oxygen and nutrient trafficking to the neural retina, and degeneration of the overlying photoreceptor cells. Owing to key differences in human and animal ocular anatomy and the slowly progressing nature of the disease, AMD is not easily modeled invivo. In this study, we further characterize a "drusen-in-a-dish" primary porcine RPE model system by employing vital lipid staining to monitor sub-RPE deposition over time in monolayers of cells cultured on porous transwell membranes. We demonstrate for the first time using a semi-automated image analysis pipeline that the number and size of sub-RPE deposits increases gradually but significantly over time and confirm that sub-RPE deposits grown in culture immunostain positive for multiple known components found in human drusen. As a result, we propose that drusen-in-a-dish cell culture models represent a high-throughput and cost-scalable alternative to animal models in which to study the pathobiology of drusen accumulation and may serve as useful tools for screening novel therapeutics aimed at treating dry AMD.

Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood-Brain Barrier.

The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood-brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood-brain barrier and is primarily responsible for preventing activated T-cells from entering the brain.

Monitoring of three-dimensional live-cell cultures using a multimode, multiscale imaging system combining confocal fluorescence microscopy and optical coherence microscopy

Live-cell monitoring involves long-term observation and analysis of living cells in tissue cultures, which develop cells or tissues in an artificial environment and are essential for tissue engineering. Fluorescence microscopy (FM) is widely used as a monitoring tool owing to its powerful ability to visualize protein distribution within cells and tissues, thereby providing information on their functions in biological processes. To reduce the photobleaching effect, which is a major limiting factor in FM, FM is generally combined with other imaging modalities, such as phase contrast and differential interference contrast microscopy, which are nondestructive to fluorochromes, to selectively use fluorescence signals only in specific areas of interest within a sample. However, these methods are restricted to thin samples and cannot produce depth-resolved images. Here, we propose a method to determine three-dimensional (3D) positions in volumetric samples for application in high-resolution 3D fluorescence imaging using a multimode and multiscale imaging system that combines optical coherence microscopy (OCM) and line confocal FM (LC–FM). We also demonstrate the benefits of multimodal imaging in 3D cell culture monitoring. To evaluate the performance of the proposed system and method, we rapidly and accurately located the regions of interest in 3D tissue culture models, such as tumor spheroids and microvascular bed, and instantaneously acquired volumetric high-resolution fluorescence images. We also used an integrated system to monitor tumor spheroids mixed with extracellular matrix (ECM) over five days of the culture process, and the FM–OCM integrated technique successfully imaged the overall 3D structures, such as the distribution and boundaries of the spheroids and ECM as well as stained tumor cells. This complementary information is useful for understanding the relationship between cellular behaviors, such as the proliferation and migration of tumor cells, and microenvironments, such as the ECM.

Evaluation of bacteriophages for the alleviation of potential bacterial contamination risks in developmental engineering.

This research aimed to address the potential bacterial contamination risks in developmental engineering (DE) using bacteriophages. To compare and contrast the exemplar Escherichia coli T4 and M13 bacteriophages, human dermal fibroblasts cultivated on culture plates, natural cellulosic scaffolds, and poly(methyl methacrylate) (PMMA) particles were utilized as two-dimensional (2D) cell, three-dimensional (3D) tissue, and modular tissue culture models, respectively. When directly introduced into these distinct culture systems, both phages survived, exhibited no significant effects on the cultured cells or tissues, yet displayed their potentials to alleviate the infections caused by corresponding bacterial host cells. Apart from direct addition into the culture medium, both phages were also coated on PMMA, polystyrene, poly(lactic acid) particles with different diameters (5, 10, 30, and 100 µm) and cellulosic scaffolds. The coated phages endured the coating processes and demonstrated their viabilities in plaque assays. Further testing indicated that the phages coated on the PMMA particles tolerated multiple deliberate rinses and centrifugations, but not thermal treatment at 60-80°C. In summary, T4 and M13 bacteriophages not only manifested their antibacterial functions in diverse 2D cell, 3D tissue, and modular tissue culture systems, but also demonstrated their potentials of coating modular scaffolds to alleviate the bacterial contamination risks in DE.

Steven A. Brown and the synchronization of circadian rhythms by body temperature cycles.

The mammalian circadian timing system has a hierarchical architecture, with a central pacemaker located in the brain's suprachiasmatic nucleus orchestrating rhythms in behaviour and physiology. In cooperation with environmental cycles, it synchronizes the phases of peripheral oscillators operating in most cells of the body. Even cells kept in tissue culture harbour self-sustained and cell-autonomous circadian clocks that keep ticking throughout their lifespan. The master pacemaker in the suprachiasmatic nucleus is synchronized primarily by light-dark cycles, whereas peripheral oscillators are phase entrained by a multitude of systemic signalling pathways. These include pathways depending on feeding-fasting cycles, cellular actin polymerization dynamics, endocrine rhythms and, surprisingly, body temperature oscillations. Using tissue culture and murine models, Steve Brown was the first one to demonstrate that shallow rhythms of mammalian body temperature are timing cues (zeitgebers) for peripheral circadian clocks.

Vaginal irritation testing-prospects of human organotypic vaginal tissue culture models.

Personal lubricants intended for local or systemic delivery via the vaginal route can induce vaginal irritation, damage the vaginal epithelial barrier which can enhance microbial entry, induce inflammation, and alter the microbiome of the vaginal ecosystem. Therefore, manufacturers of personal lubricants and medical devices are required to show biocompatibility and safety assessment data to support regulatory decision-making within a specified context of use. Furthermore, due to ethical concerns and the introduction of the 7th amendment of the European Council Directive which bans animal testing for cosmetic ingredients and products coupled with the Food and Drug Administration modernization Act 2.0 guidelines, there is a wave of drive to develop alternative test methods to predict human responses to chemical or formulation exposure. In this framework, there is a potential to use three-dimensional organotypic human vaginal-ectocervical tissue models as a screening tool to predict the vaginal irritation potential of personal lubricants and medicaments. To be physiologically relevant, the in vitro tissue models need to be reconstructed using primary epithelial cells of the specific organ or tissue and produce organ-like structure and functionality that recapitulate the in vivo-like responses. Through the years, progress has been made and vaginal tissue models are manufactured under controlled conditions with a specified performance criterion, which leads to a high level of reproducibility and reliability. The utility of vaginal tissue models has been accelerated in the last 20years with an expanded portfolio of applications ranging from toxicity, inflammation, infection to drug safety, and efficacy studies. This article provides an overview of the state of the art of diversified applications of reconstructed vaginal tissue models and highlights their utility as a tool to predict vaginal irritation potential of feminine care products.

The influence of viscosity of hydrogels on the spreading and migration of cells in 3D bioprinted skin cancer models.

Various in vitro three-dimensional (3D) tissue culture models of human and diseased skin exist. Nevertheless, there is still room for the development and improvement of 3D bioprinted skin cancer models. The need for reproducible bioprinting methods, cell samples, biomaterial inks, and bioinks is becoming increasingly important. The influence of the viscosity of hydrogels on the spreading and migration of most types of cancer cells is well studied. There are however limited studies on the influence of viscosity on the spreading and migration of cells in 3D bioprinted skin cancer models. In this review, we will outline the importance of studying the various types of skin cancers by using 3D cell culture models. We will provide an overview of the advantages and disadvantages of the various 3D bioprinting technologies. We will emphasize how the viscosity of hydrogels relates to the spreading and migration of cancer cells. Lastly, we will give an overview of the specific studies on cell migration and spreading in 3D bioprinted skin cancer models.

An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro.

In the field of biomaterials for prosthetic reconstructive surgery, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials before in vivo tests. Despite the complex osteointegration process being difficult to recreate in vitro, this study proposes an advanced in vitro tissue culture model of osteointegration using human bone. Cubic samples of trabecular bone were harvested, as waste material, from hip arthroplasty; inner cylindrical defects were created and assigned to the following groups: (1) empty defects (CTRneg); (2) defects implanted with a cytotoxic copper pin (CTRpos); (3) defects implanted with standard titanium pins (Ti). Tissues were dynamically cultured in mini rotating bioreactors and assessed weekly for viability and sterility. After 8 weeks, immunoenzymatic, microtomographic, histological, and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, while Ti appears to have a trophic effect on bone. MicroCT and a histological analysis supported the results, with signs of matrix and bone deposition at the Ti implant site. Data suggest the reliability of the tested model in recreating the osteointegration process in vitro with the aim of reducing and refining in vivo preclinical models.

Regenerative Medicine in Gynecology.

The female reproductive tract undergoes dynamic changes across the life span. Congenital abnormalities, life events, and medical interventions can negatively affect the structure and function of reproductive tract organs, resulting in lifelong sequelae. The objective of regenerative gynecology is to discover and promote endogenous mechanisms by which a healthy tissue maintains overall tissue integrity after injury, after disease, or with age. In this review, we discuss some of the key state-of-the-art cell-based and scaffolding therapies that have been applied to regenerate gynecologic tissues and organs primarily in animal and tissue culture models. We further discuss the limitations of current technologies, problems of implementation and scalability, and future outlook of the field.

P141 The Roles and Therapeutic Implications of Macrophages in Inducing Calcinosis Via Activin-A Pathway and CD206 Expression in Patients with Systemic Sclerosis

Abstract Background/Aims Calcinosis may result from localised trans-differentiation of tissue resident stem cells in the subcutaneous layer of affected skin in systemic sclerosis (SSc), as a severe disabling manifestation linked to ischaemia and local trauma. Polarised macrophages synergistically promote osteogenesis: M1 macrophages recruit mesenchymal stem cells leading to osteogenic differentiation, while M2 are responsible for osteoblast formation. Previous studies have implicated two pathways-Activin-A signalling and CD206 expression by macrophages - in heterotopic osteogenesis. We investigated the pathogenesis of calcinosis through macrophages, using a novel tissue culture model created by stimulation of adipose derived mesenchymal stem cells (ASCs) with SSc macrophages and explored the relevance of the Activin-A pathway. Methods Activin-A was measured by ELISA in diffuse cutaneous systemic sclerosis (dcSSc) patients' (n = 72), and healthy control (HC) plasma samples (n = 42). Macrophages were derived from peripheral blood monocytes of dcSSc patients (n = 4) and cultured under basal and BzATP-stimulated conditions. dcSSc macrophages were maintained in co-culture with ASCs in osteogenic media ±anti-activin-A neutralising antibody (anti-AA). Osteogenesis was assessed using Alizarin Red. Results Activin-A was increased in dcSSc patients’ plasma when compared to HC, most notably in anti-centromere antibody (ACA) subgroup (plasma activin-A 521±199pg/ml vs 255 ± 170pg/ml [p = 0.000065]) (Figure 1A). Moreover, there was synthesis of Activin A by SSc macrophages, enhanced by stimulation with BzATP (0.1µM) (basal Activin A 3.3 (0-14.5), BzATP stimulated 21.7 (7.7-418) pg/ml p NS) (Figure 1B). In co-cultures, the addition of SSc macrophages to ASCs led to significantly enhanced synthesis of Activin A, which was reduced by the addition of anti-AA (ASC monoculture 987+/-87, SSc macrophages+AScs 1599+/- 122, p < 0.0065, SSc macrophages+ASCs+anti-AA 1034+/-108 pg/ml, p < 0.013) (Figure 1C). Moreover, adding SSc macrophages to ASC cultures significantly enhanced calcinosis, as measured by Alizarin red stain, but anti-AA did not fully supress the Alizarin staining (ASC: 0.5, 0.4-0.7; ASC+M: 13.0, 5.8-17.2 p = 0.0022; ASC+M+anti-AA p NS median, IQR, red pixels per field) (Figure1D). Conclusion Elevated plasma Activin-A, most notable in ACA subgroup, is consistent with systemic upregulation of the Activin-A pathway in SSc. The interaction of activated macrophages with ASCs represents a potential model for calcinosis. Targeting Activin-A might benefit in this severe complication. Disclosure K. Liang: None. S. Lopez Garces: None. T. Searle: None. H. Lopez: Corporate appointments; CEO of Murigenics. G. Martin: Corporate appointments; Senior Scientific Officer Riptide. C. Denton: None. D. Abraham: None. R.J. Stratton: None.

A lentivirus-vectored feline erythropoietin gene therapy strategy in tissue culture and rodent models for the potential treatment of chronic renal disease-associated anemia.

The aim of this study was to assess the efficacy and safety of a third-generation lentivirus-based vector encoding the feline erythropoietin (EPO) (feEPO) gene in vitro and in rodent models in vivo. This vector incorporates a genetic mechanism to facilitate the termination of the therapeutic effect in the event of supraphysiologic polycythemia, the herpes simplex virus thymidine kinase (HSV-TK) "suicide gene." CFRK cells and replication-defective lentiviral vectors encoding feEPO were used for in vitro experiments. Eight Fischer rats were enrolled in the pilot in vivo study, 24 EPO-deficient mice were used in the initial mouse study, and 15 EPO-deficient mice were enrolled in the final mouse study. Efficacy of a third-generation lentivirus encoding feEPO was determined in vitro using western blot assays. Subsequently, in a series of rodent experiments, animals were administered the viral vector in progressively increasing inoculation doses with serial measurements of blood packed cell volume (PCV) over time. We documented production of feEPO protein in transduced CRFK cells with subsequent cessation of production when treated with the HSV-TK substrate ganciclovir. In vivo, we demonstrated variably persistent elevated PCV values in treated rats and mice with eventual return to baseline values over time. These results provide justification for a lentiviral gene therapy approach to the treatment of nonregenerative anemia associated with chronic renal disease in cats.

Validation of Human Bone Marrow-derived Mesenchymal Stem Cells and MCF-7 Breast Cancer Cells Co-culture Using a 3D Perfused Biomimetic Microfluidic Platform.

Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time. MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks. VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment. Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs.

Abstract 5253: Epigenetic drugs partially restored anti-cholangiocarcinoma activity of dysfunctional CD3+CD56+ cells

Abstract The cholangiocarcinoma cell-exposed cytokine-induced killer (CIK) cells or their most efficacious CD3+CD56+ subset displayed weaken anti-tumor cytotoxicity upon subsequent exposure. RNAseq analysis of the resistant phenotype gave rise to a list of drugs that could potentially counter the activity of genes associated with resistance to the anti-tumor activity of CIK cells. The top-ranking drugs appeared to be epigenetic drugs. Sublethal concentration of these drugs were assayed for their synergistic activity with CIK cell or the CD3+CD56+ subset for cytolysis of resistant CCA cells in tissue culture model. These drugs, although in low concentration, contained anti-tumor activity. The tumor cytolysis was greater after the combination with either CIK cells or the CD3+CD56+ subset. An in vivo model of CCA-implanted mice exhibited the synergistic activity of subtherapeutic dose of these drugs and CD3+CD56+ subset in suppressing tumor growth. It is concluded that epigenetic drugs in subtherapeutic dose could prevent the dysfunction of CCA-exposed CIK cells. Citation Format: Adisak Wongkajornsilp, Porncheera Chusorn, Kriengkrai Phongkitkarun, Khin Su Su Htwe, Sunisa Duangsa-ard, Kanda Kasetsinsombat, Nathawadee Sawatpiboon. Epigenetic drugs partially restored anti-cholangiocarcinoma activity of dysfunctional CD3+CD56+ cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5253.

Abstract 237: Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and there is a critical need for more effective treatments. The development of new therapies, especially those aiming to modulate the tumor microenvironment (TME), is limited by the lack of in vitro and in vivo models that recapitulate the complexity of the TME and spatial architecture of HCC. Here, we aimed to establish an HCC model to assess the treatment response and hepatotoxicity of selected drugs by ex vivo culturing fresh human liver tumor tissue and adjacent normal tissue, respectively. Materials and Methods: Fresh human liver tissues, including 5 malignant and 20 adjacent-normal, were obtained from surgical resections. PDX tumors were generated from patient-derived organoids. Tissue resections were cut into ultra-thin slices (from 150µm to 350µm) using a vibratome. Related culture conditions such as high oxygen partial pressure, air-liquid interface (ALI) culture, artificial extracellular matrix (AEM), and delay duration were evaluated to optimize the protocol. As a readout, cell viability was quantified by an expert pathologist using Hematoxylin/Eosin staining. The preservation and complexity of the TME were evaluated by immunohistochemistry or flow cytometry using specific antibodies for each cell population (T cells, anti-CD3; stroma cells, a-SMA; endothelial cells, CD34; Kupffer cells, CD68). Liver cell function was tested by measuring ALT/AST activity, albumin release, and CYP340 activity. Results: The highest tissue viability was observed in normal and tumor tissue with a thickness of 150µm and 250µm, respectively. Compared to standard conditions, the combination of ALI and high-oxygen partial pressure conditions dramatically improved tissue viability, with most of the samples having 70% and 50% cell viability at days 3 and 5 of ex vivo culture, respectively (p<0.05). AEM could prevent the efflux of cellular components (p=0.016). The impact of the time delay between the liver resection and the start of slicing was minimal when the slicing occurred within two hours after hepatectomy (p<0.05). Key cellular components including hepatocytes, T cells, fibroblasts, and Kupffer cells also remained better preserved throughout the 5-day culture period in optimized conditions. Conclusion: Our study has successfully optimized the ex vivo tissue culture model of liver tissue, capable of maintaining the tissue for several days while preserving the unique TME. These results highlight the potential of using the ex vivo tissue culture model in the preclinical setting to further investigate and evaluate TME-related treatments and their hepatotoxicity in HCC tissues. Citation Format: Huiche Feng, Miriam Cieri, Cinzia Esposito, Karolina Guja-Jarosz, Daniela Liberati, Lukas Bubendorf, Savas Deniz Soysal, Mairene Coto-Llerena, Luigi Maria Terracciano, Otto Kollmar, Salvatore Piscuoglio. Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 237.

ADVANCED IN VITRO 3D TISSUE CULTURE MODEL FOR THE ASSESSMENT OF TITANIUM IMPLANT OSTEOINTEGRATION

The use of implant biomaterials for prosthetic reconstructive surgery and osteosynthesis is consolidated in the orthopaedic field, improving the quality of life of patients and allowing for healthy and better ageing. However, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials, particularly for the study of local effects of implant and osteointegration. Despite the complex process of osseointegration is difficult to recreate in vitro, the growing challenges in developing alternative models require to set-up and validate new approaches. Aim of the present study is to evaluate an advanced in vitro tissue culture model of osteointegration of titanium implants in human trabecular bone. Cubic samples (1.5×1.5 cm) of trabecular bone were harvested as waste material from hip arthroplasty surgery (CE AVEC 829/2019/Sper/IOR); cylindrical defects (2 mm Ø, 6 mm length) were created, and tissue specimens assigned to the following groups: 1) empty defects- CTR-; 2) defects implanted with a cytotoxic copper pin (Merck cod. 326429)- CTR+; 3) defects implanted with standard titanium pins of 6 µm-rough (ZARE S.r.l) -Ti6. Tissue specimens were cultured in mini rotating bioreactors in standard conditions, weekly assessing viability. At the 8-week-timepoint, immunoenzymatic, microtomographic, histological and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, differently from Ti6 which appears to have a trophic effect on the bone. MicroCT and histological analysis supported the results, with lower BV/TV and Tb.Th values observed in CTR- compared to CTR+ and Ti6 and signs of matrix and bone deposition at the implant site. The collected data suggest the reliability of the tested model which can recreate the osseointegration process in vitro and can therefore be used for preliminary evaluations to reduce and refine in vivo preclinical models.Acknowledgment: This work was supported by Emilia-Romagna Region for the project “Sviluppo di modelli biologici in vitro ed in silico per la valutazione e predizione dell'osteointegrazione di dispositivi medici da impianto nel tessuto osseo”

Feasibility of engineered Bacillus subtilis for use as a microbiome-based topical drug delivery platform.

Non-adherence to medication is a major challenge in healthcare that results in worsened treatment outcomes for patients. Reducing the frequency of required administrations could improve adherence but is challenging for topical drug delivery due to the generally short residence time of topical formulations on the skin. In this study, we sought to determine the feasibility of developing a microbiome-based, long-acting, topical delivery platform using Bacillus subtilis for drug production and delivery on the skin, which was assessed using green fluorescent protein as a model heterologous protein for delivery. We developed a computational model of bacteria population dynamics on the skin and used its qualitative predictions to guide experimental design choices. Using an ex vivo pig skin model and a human skin tissue culture model, we saw persistence of delivered bacteria for multiple days and observed little evidence of cytotoxicity to human keratinocyte cells in vitro. Finally, using an in vivo mouse model, we found that the delivered bacteria persisted on the skin for at least 1 day during every-other-day application and did not appear to present safety concerns. Taken together, our results support the feasibility of using engineered B. subtilis for topical drug delivery.

Visualizing Lipophagy as a New Mechanism of the Synthesis of Sex Steroids in Human Ovary and Testis Using Immunofluorescence Staining Method.

Immunofluorescence, a transformative tool in cellular biology, is employed to dissect the intricate mechanisms of cholesterol trafficking in human reproductive tissues. Autophagy, a key player in cellular homeostasis, particularly lipophagy, emerges as a free cholesterol source for steroidogenesis. In this chapter, we describe a comprehensive immunofluorescence staining protocol, with details provided for the precise visualization of subcellular dynamics of mitochondria, lysosomes, and lipid droplets in ex vivo testicular tissue and primary luteal granulosa cell culture models, pivotal components in sex steroid biosynthesis. Here, we detail the culture, treatment, and immunofluorescence protocols, providing a comprehensive guide for researchers. The provided immunofluorescence toolkit serves as a valuable resource for researchers, paving way for advancements in human reproductive health to investigate the intricate interplay between autophagy, lipophagy, and cholesterol trafficking.