Unraveling radiation-induced skeletal muscle damage: Insights from a 3D human skeletal muscle organoid model.

BACKGROUND: Three-dimensional (3D) organoids derived from human pluripotent stem cells (hPSCs) have revolutionized in vitro tissue modeling, offering a unique opportunity to replicate physiological tissue organization and functionality. This study investigates the impact of radiation on skeletal muscle response using an innovative in vitro human 3D skeletal muscle organoids (hSMOs) model derived from hPSCs. METHODS: The hSMOs model was established through a differentiation protocol faithfully recapitulating embryonic myogenesis and maturation via paraxial mesodermal differentiation of hPSCs. Key skeletal muscle characteristics were confirmed using immunofluorescent staining and RT-qPCR. Subsequently, the hSMOs were exposed to a clinically relevant dose of 2 Gy of radiation, and their response was analyzed using immunofluorescent staining and RNA-seq. RESULTS: The hSMO model faithfully recapitulated embryonic myogenesis and maturation, maintaining key skeletal muscle characteristics. Following exposure to 2 Gy of radiation, histopathological analysis revealed deficits in hSMOs expansion, differentiation, and repair response across various cell types at early (30 min) and intermediate (18 h) time points post-radiation. Immunofluorescent staining targeting γH2AX and 53BP1 demonstrated elevated levels of foci per cell, particularly in PAX7+ cells, during early and intermediate time points, with a distinct kinetic pattern showing a decrease at 72 h. RNA-seq data provided comprehensive insights into the DNA damage response within the hSMOs. CONCLUSIONS: Our findings highlight deficits in expansion, differentiation, and repair response in hSMOs following radiation exposure, enhancing our understanding of radiation effects on skeletal muscle and contributing to strategies for mitigating radiation-induced damage in this context.

Applications and perspectives of tumor organoids in radiobiology (Review).

Radiotherapy exhibits significant versatility and efficacy in cancer treatment, thereby playing a crucial role in the field of oncology. However, there remains an urgent need for extensive research on various aspects of radiotherapy, including target selection, damage repair and its combination with immunotherapy. Particularly, the development of in vitro models to replicate in vivo tumor lesion responses is vital. The present study provides a thorough review of the establishment and application of tumor organoids in radiotherapy, aiming to explore their potential impact on cancer treatment.

Modeling Radiation-Induced Epithelial Cell Injury in Murine Three-Dimensional Esophageal Organoids.

Esophageal squamous cell carcinoma (ESCC) is a deadly consequence of radiation exposure to the esophagus. ESCC arises from esophageal epithelial cells that undergo malignant transformation and features a perturbed squamous cell differentiation program. Understanding the dose- and radiation quality-dependence of the esophageal epithelium response to radiation may provide insights into the ability of radiation to promote ESCC. We have explored factors that may play a role in esophageal epithelial radiosensitivity and their potential relationship to ESCC risk. We have utilized a murine three-dimensional (3D) organoid model that recapitulates the morphology and functions of the stratified squamous epithelium of the esophagus to study persistent dose- and radiation quality-dependent changes. Interestingly, although high-linear energy transfer (LET) Fe ion exposure induced a more intense and persistent alteration of squamous differentiation and 53BP1 DNA damage foci levels as compared to Cs, the MAPK/SAPK stress pathway signaling showed similar altered levels for most phospho-proteins with both radiation qualities. In addition, the lower dose of high-LET exposure also revealed nearly the same degree of morphological changes, even though only ~36% of the cells were predicted to be hit at the lower 0.1 Gy dose, suggesting that a bystander effect may be induced. Although p38 and ERK/MAPK revealed the highest levels following high-LET exposure, the findings reveal that even a low dose (0.1 Gy) of both radiation qualities can elicit a persistent stress signaling response that may critically impact the differentiation gradient of the esophageal epithelium, providing novel insights into the pathogenesis of radiation-induced esophageal injury and early stage esophageal carcinogenesis.

LPA5-Dependent signaling regulates regeneration of the intestinal epithelium following irradiation.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via activation of cognate receptors. The LPA5 receptor is highly expressed in the intestinal epithelium, but its function in restoring intestinal epithelial integrity following injury has not been examined. Here, we use a radiation-induced injury model to study the role of LPA5 in regulating intestinal epithelial regeneration. Control mice (Lpar5f/f) and mice with an inducible, epithelial cell-specific deletion of Lpar5 in the small intestine (Lpar5IECKO) were subjected to 10 Gy total body X-ray irradiation and analyzed during recovery. Repair of the intestinal mucosa was delayed in Lpar5IECKO mice with reduced epithelial proliferation and increased crypt cell apoptosis. These effects were accompanied by reduced numbers of OLFM4+ intestinal stem cells (ISCs). The effects of LPA5 on ISCs were corroborated by studies using organoids derived from Lgr5-lineage tracking reporter mice with deletion of Lpar5 in Lgr5+-stem cells (Lgr5Cont or Lgr5ΔLpar5). Irradiation of organoids resulted in fewer numbers of Lgr5ΔLpar5 organoids retaining Lgr5+-derived progenitor cells compared with Lgr5Cont organoids. Finally, we observed that impaired regeneration in Lpar5IECKO mice was associated with reduced numbers of Paneth cells and decreased expression of Yes-associated protein (YAP), a critical factor for intestinal epithelial repair. Our study highlights a novel role for LPA5 in regeneration of the intestinal epithelium following irradiation and its effect on the maintenance of Paneth cells that support the stem cell niche.NEW & NOTEWORTHY We used mice lacking expression of the lysophosphatidic acid receptor 5 (LPA5) in intestinal epithelial cells and intestinal organoids to show that the LPA5 receptor protects intestinal stem cells and progenitors from radiation-induced injury. We show that LPA5 induces YAP signaling and regulates Paneth cells.

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Spatiotemporally resolved colorectal oncogenesis in mini-colons ex vivo.

Three-dimensional organoid culture technologies have revolutionized cancer research by allowing for more realistic and scalable reproductions of both tumour and microenvironmental structures1-3. This has enabled better modelling of low-complexity cancer cell behaviours that occur over relatively short periods of time4. However, available organoid systems do not capture the intricate evolutionary process of cancer development in terms of tissue architecture, cell diversity, homeostasis and lifespan. As a consequence, oncogenesis and tumour formation studies are not possible in vitro and instead require the extensive use of animal models, which provide limited spatiotemporal resolution of cellular dynamics and come at a considerable cost in terms of resources and animal lives. Here we developed topobiologically complex mini-colons that are able to undergo tumorigenesis ex vivo by integrating microfabrication, optogenetic and tissue engineering approaches. With this system, tumorigenic transformation can be spatiotemporally controlled by directing oncogenic activation through blue-light exposure, and emergent colon tumours can be tracked in real-time at the single-cell resolution for several weeks without breaking the culture. These induced mini-colons display rich intratumoural and intertumoural diversity and recapitulate key pathophysiological hallmarks displayed by colorectal tumours in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, mini-colons can be used to identify tumorigenic determinants and pharmacological opportunities. As a whole, our study paves the way for cancer initiation research outside living organisms.

Assessment of Normal Tissue Radiosensitivity by Evaluating DNA Damage and Repair Kinetics in Human Brain Organoids.

DNA-double strand break (DSB), detected by immunostaining of key proteins orchestrating repair, like γH2AX and 53BP1, is well established as a surrogate for tissue radiosensitivity. We hypothesized that the generation of normal brain 3D organoids ("mini-brains") from human induced pluripotent stem cells (hiPSC) combined with detection of DNA damage repair (DDR) may hold the promise towards developing personalized models for the determination of normal tissue radiosensitivity. In this study, cerebral organoids, an in vitro model that stands in its complexity between 2D cellular system and an organ, have been used. To quantify radiation-induced response, immunofluorescent staining with γH2AX and 53BP1 were applied at early (30 min, initial damage), and late time points (18 and 72 h, residual damage), following clinical standard 2 Gy irradiation. Based on our findings, assessment of DDR kinetics as a surrogate for radiosensitivity in hiPSC derived cerebral organoids is feasible. Further development of mini-brains recapitulating mature adult neuronal tissue and implementation of additional signaling and toxicity surrogates may pave the way towards development of next-generation personalized assessment of radiosensitivity in healthy neuronal tissue.

Bio-modulated mice epithelial endometrial organoids by low-level laser therapy serves as an invitro model for endometrial regeneration.

Endometrial regeneration is a dynamic process that is not well understood. The destruction of the endometrium with the formation of intrauterine adhesions is known as Asherman's syndrome. The lesions range from minor to severe adhesions and their impact on pregnancy is well documented. Operative hysteroscopy is the mainstay of diagnosis and treatment of intrauterine adhesions. Nevertheless, the recurrence rates remain high. It was recorded that low-level laser therapy in low doses has a stimulatory effect on different tissues while the high dose produces a suppressive effect. Organoid is a three-dimensional assembly that displays architectures and functionalities similar to in vivo organs that are being developed from human or animal stem cells or organ-specific progenitors through a self-organization process. Our prospective was to study the effect of Low-Level Laser Therapy (LLLT) on mouse epithelial endometrial organoids regarding cell proliferation and endometrial regeneration as a new modality of treatment. An in vitro clinical trial to generate mouse epithelial organoid model and testing LLLT using He:Ne 632.8 nm device on organoids proliferation, function, and their response to ovarian hormones was performed. Trying endometrial regeneration by culturing organoids with decellularized uterine matrix (DUM) and studying the LLLT effect on the regeneration process. LLLT produced a proliferative effect on the epithelial mouse organoids confirmed by Ki67 and PCNA IHC. The organoids could regenerate the epithelial layer of the endometrium in vitro on DUM and LLLT could help in this process. In conclusion, organoids whether control or bio-stimulated proved a new modality to regenerate the endometrium.

Stem Cells and Irradiation.

The main difficulty of radiotherapy is to destroy cancer cells without depletion of healthy tissue [...].

Establishment of intestinal organoid cultures modeling injury-associated epithelial regeneration.

The capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro. However, conventional organoid cultures primarily represent the homeostasis of self-organizing stem cells and their derivatives. Here, we established a novel intestinal organoid culture system composed of 8 components, mainly including VPA, EPZ6438, LDN193189, and R-Spondin 1 conditioned medium, which mimics the gut epithelium regeneration that produces hyperplastic crypts following injury; therefore, these organoids were designated hyperplastic intestinal organoids (Hyper-organoids). Single-cell RNA sequencing identified different regenerative stem cell populations in our Hyper-organoids that shared molecular features with in vivo injury-responsive Lgr5+ stem cells or Clu+ revival stem cells. Further analysis revealed that VPA and EPZ6438 were indispensable for epigenome reprogramming and regeneration in Hyper-organoids, which functioned through epigenetically regulating YAP signaling. Furthermore, VPA and EPZ6438 synergistically promoted regenerative response in gut upon damage in vivo. In summary, our results demonstrated a new in vitro organoid model to study epithelial regeneration, highlighting the importance of epigenetic reprogramming that pioneers tissue repair.

Current and Future Perspectives of the Use of Organoids in Radiobiology.

The majority of cancer patients will be treated with radiotherapy, either alone or together with chemotherapy and/or surgery. Optimising the balance between tumour control and the probability of normal tissue side effects is the primary goal of radiation treatment. Therefore, it is imperative to understand the effects that irradiation will have on both normal and cancer tissue. The more classical lab models of immortal cell lines and in vivo animal models have been fundamental to radiobiological studies to date. However, each of these comes with their own limitations and new complementary models are required to fill the gaps left by these traditional models. In this review, we discuss how organoids, three-dimensional tissue-resembling structures derived from tissue-resident, embryonic or induced pluripotent stem cells, overcome the limitations of these models and thus have a growing importance in the field of radiation biology research. The roles of organoids in understanding radiation-induced tissue responses and in moving towards precision medicine are examined. Finally, the limitations of organoids in radiobiology and the steps being made to overcome these limitations are considered.

Targeting Acid Ceramidase to Improve the Radiosensitivity of Rectal Cancer.

Previous work utilizing proteomic and immunohistochemical analyses has identified that high levels of acid ceramidase (AC) expression confers a poorer response to neoadjuvant treatment in locally advanced rectal cancer. We aimed to assess the radiosensitising effect of biological and pharmacological manipulation of AC and elucidate the underlying mechanism. AC manipulation in three colorectal cancer cell lines (HT29, HCT116 and LIM1215) was achieved using siRNA and plasmid overexpression. Carmofur and a novel small molecular inhibitor (LCL521) were used as pharmacological AC inhibitors. Using clonogenic assays, we demonstrate that an siRNA knockdown of AC enhanced X-ray radiosensitivity across all colorectal cancer cell lines compared to a non-targeting control siRNA, and conversely, AC protein overexpression increased radioresistance. Using CRISPR gene editing, we also generated AC knockout HCT116 cells that were significantly more radiosensitive compared to AC-expressing cells. Similarly, two patient-derived organoid models containing relatively low AC expression were found to be comparatively more radiosensitive than three other models containing higher levels of AC. Additionally, AC inhibition using carmofur and LCL521 in three colorectal cancer cell lines increased cellular radiosensitivity. Decreased AC protein led to significant poly-ADP ribose polymerase-1 (PARP-1) cleavage and apoptosis post-irradiation, which was shown to be executed through a p53-dependent process. Our study demonstrates that expression of AC within colorectal cancer cell lines modulates the cellular response to radiation, and particularly that AC inhibition leads to significantly enhanced radiosensitivity through an elevation in apoptosis. This work further solidifies AC as a target for improving radiotherapy treatment of locally advanced rectal cancer.

Small subset of Wnt-activated cells is an initiator of regrowth in colorectal cancer organoids after irradiation.

Most colorectal cancers (CRCs) are differentiated adenocarcinomas, which maintain expression of both stemness and differentiation markers. This observation suggests that CRC cells could retain a regeneration system of normal cells upon injury. However, the role of stemness in cancer cell regeneration after irradiation is poorly understood. Here, we examined the effect of radiation on growth, stemness, and differentiation in organoids derived from differentiated adenocarcinomas. Following a sublethal dose of irradiation, proliferation and stemness markers, including Wnt target genes, were drastically reduced, but differentiation markers remained. After a static growth phase after high dose of radiation, regrowth foci appeared; these consisted of highly proliferating cells that expressed stem cell markers. Radiosensitivity and the ability to form foci differed among the cancer tissue-originated spheroid (CTOS) lines examined and showed good correlation with in vivo radiation sensitivity. Pre-treating organoids with histone deacetylase inhibitors increased radiation sensitivity; this increase was accompanied by the suppression of Wnt signal-related gene expression. Accordingly, Wnt inhibitors increased organoid radiosensitivity. These results suggested that only a small subset of, but not all, cancer cells with high Wnt activity at the time of irradiation could give rise to foci formation. In conclusion, we established a radiation sensitivity assay using CRC organoids that could provide a novel platform for evaluating the effects of radiosensitizers on differentiated adenocarcinomas in CRC.

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

Exposure of Human Skin Organoids to Low Genotoxic Stress Can Promote Epithelial-to-Mesenchymal Transition in Regenerating Keratinocyte Precursor Cells.

For the general population, medical diagnosis is a major cause of exposure to low genotoxic stress, as various imaging techniques deliver low doses of ionizing radiation. Our study investigated the consequences of low genotoxic stress on a keratinocyte precursor fraction that includes stem and progenitor cells, which are at risk for carcinoma development. Human skin organoids were bioengineered according to a clinically-relevant model, exposed to a single 50 mGy dose of γ rays, and then xeno-transplanted in nude mice to follow full epidermis generation in an in vivo context. Twenty days post-xenografting, mature skin grafts were sampled and analyzed by semi-quantitative immuno-histochemical methods. Pre-transplantation exposure to 50 mGy of immature human skin organoids did not compromise engraftment, but half of xenografts generated from irradiated precursors exhibited areas displaying focal dysplasia, originating from the basal layer of the epidermis. Characteristics of epithelial-to-mesenchymal transition (EMT) were documented in these dysplastic areas, including loss of basal cell polarity and cohesiveness, epithelial marker decreases, ectopic expression of the mesenchymal marker α-SMA and expression of the EMT promoter ZEB1. Taken together, these data show that a very low level of radiative stress in regenerating keratinocyte stem and precursor cells can induce a micro-environment that may constitute a favorable context for long-term carcinogenesis.

Human Forebrain Organoids from Induced Pluripotent Stem Cells: A Novel Approach to Model Repair of Ionizing Radiation-Induced DNA Damage in Human Neurons.

Human induced pluripotent stem cells (iPSCs) can generate virtually any cell type and therefore are applied to studies of organ development, disease modeling, drug screening and cell replacement therapy. Under proper culture conditions in vitro induced pluripotent stem cells (iPSCs) can be differentiated to form organ-like tissues, also known as "organoids", which resemble organs more closely than cells, in vivo. We hypothesized that human brain organoids can be used as an experimental model to study mechanisms underlying DNA repair in human neurons and their progenitors after radiation-induced DNA double-strand breaks (DSBs), the most severe form of DNA damage. To this end, we customized a protocol for brain organoid generation that is time efficient. These organoids recapitulate key features of human cortical neuron development, including a subventricular zone containing neural progenitors that mature to postmitotic cortical neurons. Using immunofluorescence to measure DNA DSB markers, such as γ-H2AX and 53BP1, we quantified the kinetics of DSB repair in neural progenitors within the subventricular zone for up to 24 h after a single 2 Gy dose of ionizing radiation. Our data on DNA repair in progenitor versus mature neurons indicate a similar timeline: both repair DNA DSBs which is mostly resolved by 18 h postirradiation. However, repair kinetics are more acute in progenitors than mature neurons in the mature organoid. Overall, this study supports the use of 3D organoid culture technology as a novel platform to study DNA damage responses in developing or mature neurons, which has been previously difficult to study.

LIF is essential for ISC function and protects against radiation-induced gastrointestinal syndrome.

Leukemia inhibitory factor (LIF) is a cytokine essential for maintaining pluripotency of mouse embryonic stem cells. However, its role in adult intestinal stem cells (ISCs) is unclear. The adult intestinal epithelium has a high self-renewal rate driven by ISCs in crypts. Here, we find that LIF is present in the ISC niche in crypts and critical for the function of ISCs in maintaining the intestinal epithelial homeostasis and regeneration. Mechanistically, LIF maintains ß-catenin activity through the AKT/GSK3ß signaling to regulate ISC functions. LIF deficiency in mice impairs the renewal of the intestinal epithelium under the physiological condition. Further, LIF deficiency in mice impairs the regeneration of intestinal epithelium in response to radiation and shortens the lifespan of mice after high doses of radiation due to gastrointestinal (GI) syndrome, which can be rescued by administering recombinant LIF (rLIF). Importantly, LIF exhibits a radioprotective role in wild-type (WT) mice by protecting mice from lethal radiation-induced GI syndrome; administering rLIF promotes intestinal epithelial regeneration and prolongs survival in WT mice after radiation. These results reveal a previously unidentified and a crucial role of LIF in ensuring ISC function, promoting regeneration of the intestinal epithelium in response to radiation and protecting against radiation-induced GI syndrome.

Room temperature shipment does not affect the biological activity of pluripotent stem cell-derived retinal organoids.

The generation of laminated and light responsive retinal organoids from induced pluripotent stem cells (iPSCs) provides a powerful tool for the study of retinal diseases and drug discovery and a robust platform for cell-based therapies. The aim of this study is to investigate whether retinal organoids can retain their morphological and functional characteristics upon storage at room temperature (RT) conditions and shipment by air using a commercially available container that maintains the environment at ambient temperature. Morphological analysis and measurements of neuroepithelial thickness revealed no differences between control, RT incubated and shipped organoids. Similarly immunohistochemical analysis showed no differences in cell type composition and position within the laminated retinal structure. All groups showed a similar response to light, suggesting that the biological function of retinal organoids was not affected by RT storage or shipment. These findings provide an advance in transport of ready-made retinal organoids, increasing their availability to many research and pharma labs worldwide and facilitating cross-collaborative research.

Interactive Effect of Melatonin and UV-C on Phenylpropanoid Metabolite Production and Antioxidant Potential in Callus Cultures of Purple Basil (Ocimum basilicum L. var.s purpurascens).

The present study evaluated the interactive effect of melatonin and UV-C on phenylpropanoid metabolites profile and antioxidant potential of Ocimum basilicum L. Callus was treated with varying concentrations of melatonin and UV-C radiations for different time durations, either alone and/or in combination. Individual treatments of both UV-C and melatonin proved to be more effective than combine treatments. Results indicated that UV-C (10 min) exposure increased rosmarinic acid (134.5 mg/g dry weight (DW)), which was 2.3-fold greater than control. Chichoric acid (51.52 mg/g DW) and anthocyanin (cyanide 0.50 mg/g DW) were almost 4.1-fold, while peonidin was found 2.7-fold higher in UV-C (50 min) exposure. In the case of melatonin, 1.0 mg/L concentrations showed maximum rosmarinic acid (79.4 mg/g DW) accumulation; i.e., 1.4-fold more, as compared to the control. However, 2 mg/L melatonin accumulate chichoric acid (39.99 mg/g DW) and anthocyanin (cyanide: 0.45 mg/g DW and peonidin: 0.22 mg/g DW); i.e., 3.2, 3.7 and 2.0-fold increase, as compared to the control, respectively. On the other hand, melatonin-combined treatment (melatonin (Mel) (4 mg/L) + UV-C (20 min)) was proved to be effective in caffeic acid elicitation, which was 1.9-fold greater than the control. Furthermore, antioxidant potential was evaluated by both in vitro (DPPH, ABTS and FRAP assays) and in cellulo methods. Maximum in vitro antioxidant activity (DPPH: 90.6% and ABTS: 1909.5 µM) was observed for UV-C (50 min)-treated cultures. The highest in vitro antioxidant activity measured with the ABTS assay as compared to the FRAP assay, suggesting the main contribution of antioxidants from basil callus extracts acting through a hydrogen atom transfer (HAT) over an electron transfer (ET)-based mechanism. Cellular antioxidant assay was evaluated by production of ROS/RNS species using yeast cell cultures and further confirmed the protective action of the corresponding callus extracts against oxidative stress. Overall, both melatonin and UV-C are here proved to be effective elicitors since a positive correlation between the induced production of phenolic compounds, and in cellulo antioxidant action of basil callus extracts were observed.

Standardization of organoid culture for evaluation of melanogenesis induced by UVB, UVA and visible light

Abstract Background: Organoid cultures are primary cultures that maintain architectural characteristics and the relationships between cells, as well as the extracellular matrix. They are alternatives for pathophysiological or therapeutic investigation rather than animal and in vitro tests. Objective: Development of a cutaneous organoid culture model, aiming at the study of radiation-induced melanogenesis. Method: A validation study, which involved biopsies of the skin of the back of the adult ear. One sample was irradiated with different doses of UVB, UVA, or visible light (VL); the other was maintained in the dark for 72 h. The viability of the tissues was evaluated from the morphological and architectural parameters of the histology, and the expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, by real-time polymerase chain reaction (PCR). The radiation-induced melanin pigmentation was standardized according to the doses of each radiation and evaluated by digital image analysis (Fontana-Masson). Results: The primary skin culture was standardized at room temperature using DMEM medium. The doses of UVB, UVA, and VL (blue light) that induced differential melanogenesis were: 166 mJ/cm2, 1.524 J/cm2, and 40 J/cm2. The expression of the GAPHD constitutional gene did not differ between the sample of skin processed immediately after tissue collection and the sample cultured for 72 h in the standardized protocol. Study limitations: This was a preliminary study that evaluated only the viability and integrity of the melanogenic system, and the effect of the radiation alone. Conclusions: The standardized model maintained viable melanocytic function for 72 h at room temperature, allowing the investigation of melanogenesis induced by different forms of radiation.