label-free-high-resolution-3d-imaging-machine-learning-analysis

Label-free, High-Resolution 3D Imaging and Machine Learning Analysis of Intestinal Organoids via Low-Coherence Holotomography

Accurate, label-free imaging of intestinal organoids is crucial for studying their morphology, growth dynamics, and responses to environmental stimuli. ...

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One major challenge associated with lung cancer organoids (LCOs) is their predominant derivation from surgical specimens of patients with early-stage lung cancer. However, patients with advanced lung cancer, who are in need of chemotherapy, often cannot undergo surgery. Therefore, there is an urgent need to successfully generate LCOs from biopsy specimens. Conventional lung biopsy techniques, such as transthoracic needle biopsy and forceps biopsy, only yield small amounts of lung tissue, resulting in a low success rate for culturing LCOs from biopsy samples. Furthermore, potential complications, like bleeding and pneumothorax, make it difficult to obtain sufficient tissue. Another critical issue is the overgrowth of normal lung cells in later passages of LCO culture, and the optimal culture conditions for LCOs are yet to be determined. To address these limitations, we attempted to create LCOs from cryobiopsy specimens obtained from patients with lung cancer ( = 113). Overall, the initial success rate of establishing LCOs from cryobiopsy samples was 40.7% ( = 46). Transbronchial cryobiopsy enables the retrieval of significantly larger amounts of lung tissue than bronchoscopic forceps biopsy. Additionally, cryobiopsy can be employed for peripheral lesions, and it is aided via radial endobronchial ultrasonography. This study significantly improved the success rate of LCO culture and demonstrated that the LCOs retained characteristics that resembled the primary tumors. Single-cell RNA sequencing confirmed high cancer cell purity in early passages of LCOs derived from patients with advanced lung cancer. Furthermore, the three-dimensional structure and intracellular components of LCOs were characterized using three-dimensional holotomography. Finally, drug screening was performed using a specialized micropillar culture system with cryobiopsy-derived LCOs. LCOs derived from cryobiopsy specimens offer a promising solution to the critical limitations of conventional LCOs. Cryobiopsy can be applied to patients with lung cancer at all stages, including those with peripheral lesions, and can provide sufficient cells for LCO generation. Therefore, we anticipate that cryobiopsy will serve as a breakthrough strategy for the clinical application of LCOs in all stages of lung cancer.

Cryobiopsy: A Breakthrough Strategy for Clinical Utilization of Lung Cancer Organoids.

Organoids, which are miniature in vitro versions of organs, possess significant potential for studying human diseases and elucidating their underlying mechanisms. Live imaging techniques play a crucial role in organoid research and contribute to elucidating the complex structure and dynamic biological phenomena of organoids. However, live, unlabeled high-resolution imaging of native organoids is challenging, primarily owing to the complexities of sample handling and optical scattering inherent in three-dimensional (3D) structures. Additionally, conventional imaging methods fail to capture the real-time dynamic processes of growing organoids. In this study, we introduce low-coherence holotomography as an advanced, label-free, quantitative imaging modality designed to overcome several technical obstacles for long-term live imaging of 3D organoids. We demonstrate the efficacy of low-coherence holotomography by capturing high-resolution morphological details and dynamic activities within mouse small intestinal organoids at subcellular resolution. Moreover, our approach facilitates the distinction between viable and nonviable organoids, significantly enhancing its utility in organoid-based research. This advancement underscores the critical role of live imaging in organoid studies, offering a more comprehensive understanding of these complex systems.

Long-term three-dimensional high-resolution imaging of live unlabeled small intestinal organoids via low-coherence holotomography.

Adipogenesis involves complex changes in gene expression, morphology, and cytoskeletal organization. However, the quantitative analysis of live cell images to identify their stages through morphological markers is limited. Distinct adipogenesis markers on human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were identified through holotomography, a label-free live cell imaging technique. In the MSC-to-preadipocyte transition, the nucleus-to-cytoplasm ratio (0.080 vs. 0.052) and lipid droplet (LD) refractive index variation decreased (0.149 % vs. 0.061 %), whereas the LD number (20 vs. 65) increased. This event was also accompanied by the downregulation and upregulation of THY1 and Preadipocyte Factor-1 (PREF-1), respectively. In the preadipocyte to immature adipocyte shift, cell sphericity (0.20 vs. 0.43) and LD number (65 vs. 200) surged, large LDs (>10 μm) appeared, and the major axis of the cell was reduced (143.7 μm vs. 83.12 μm). These findings indicate features of preadipocyte and immature adipocyte stages, alongside the downregulation of PREF-1 and upregulation of Peroxisome Proliferator-Activated Receptor gamma (PPARγ). In adipocyte maturation, along with PPARγ and Fatty Acid-Binding Protein 4 upregulation, cell compactness (0.15 vs. 0.29) and sphericity (0.43 vs. 0.59) increased, and larger LDs (>30 μm) formed, marking immature and mature adipocyte stages. The study highlights the distinct adipogenic morphological biomarkers of adipogenesis stages in UC-MSCs, providing potential applications in biomedical and clinical settings, such as fostering innovative medical strategies for treating metabolic disease.

Article	Year
Cryobiopsy: A Breakthrough Strategy for Clinical Utilization of Lung Cancer Organoids. Cells\| PubMed ID: 37508518	2023
Long-term three-dimensional high-resolution imaging of live unlabeled small intestinal organoids via low-coherence holotomography. Experimental & molecular medicine\| PubMed ID: 39349827	2024
Determination of adipogenesis stages of human umbilical cord-derived mesenchymal stem cells using three-dimensional label-free holotomography. Methods (San Diego, Calif.)\| PubMed ID: 39395684	2024

Jaehyeok Lee

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