三維 (Three-dimensional, 3D) 細胞培養近年來已普遍應用於研究中，因為其可以更好地模擬體內細胞行為的特性。在細胞球培養過程中，細胞數量是一項關鍵的影響因素，然而，很少有研究提供準確量化細胞數量的方法。因此，我們開發了一個工作流程，以使用高度可及的設備準確計算細胞球體的細胞數量。在我們提出的工作流程中，細胞以超低附著 (ULA) 圓底 96 孔盤培養成球，並在微流體裝置進行 FUnGI 光學透明化後用雷射掃描共軛焦顯微鏡 (CLSM) 成像，最後將影像使用細胞球內核定量演算法 (ASNQ) 進行分析。微流道裝置和FUnGI透明化試劑的結合提供了近乎兩倍的成像深度以及更高的影像品質，並且比較ASNQ與Imaris的細胞核切割以及數量計算， ASNQ演算法是具備可競爭性的。從 4T1、LF 和 HepaRG 球體的分析結果來看，細胞數與細胞球直徑以及球體微環境並沒有直接相關。我們的研究提供了一種高度可及的方法來準確量化細胞球中的細胞數量，從而進一步了解細胞球之研究。

Three-dimensional (3D) cell culture has been generally implemented in studies in recent years for it can better mimic the in vivo cell behaviors. During the process of spheroid culture, one of the key influences is the cell number. However, little research provides an approach to quantify the cell number accurately. Hence, we developed a workflow to accurately calculate the cell number of spheroids with highly approachable equipment. In our proposed workflow, the spheroids were cultured with the ultra-low attachment (ULA) round-bottom 96-well plate. They were imaged with confocal laser scanning microscopy (CLSM) after the FUnGI optical clearing in the microfluidic device, and the images were finally analyzed with the algorithm of spheroid nuclear quantification (ASNQ). The combination of the microfluidic device and FUnGI clearing reagent provided almost two times the imaging depth, and higher image quality than the non-clear group; the nuclei segmentation of the ASNQ showed a competitive result compared with the result from Imaris. From the analysis result of 4T1, LF, and HepaRG spheroids, the cell number was not directly correlated to the spheroid diameter and microenvironment. Our study provided a highly accessible method to accurately quantify the cell number in spheroids, enabling further insights into the studies of spheroids.

[1] E. Krausz et al., “Translation of a tumor microenvironment mimicking 3D tumor growth co-culture assay platform to high-content screening,” J. Biomol. Screen., 2013
[2] E. C. Costa et al., “3D tumor spheroids: an overview on the tools and techniques used for their analysis,” Biotechnol. Adv., vol. 34, no. 8, pp. 1427–1441, 2016
[3] W. Sun et al., “Interaction study of cancer cells and fibroblasts on a spatially confined oxygen gradient microfluidic chip to investigate the tumor microenvironment,” Analyst, vol. 143, no. 22, pp. 5431–5437, 2018
[4] F. Fontana et al., “Three-dimensional cell cultures as an in vitro tool for prostate cancer modeling and drug discovery,” Int. J. Mol. Sci., vol. 21, no. 18, pp. 1–18, 2020
[5] Shawn P. Carey, “in a Three-Dimensional Heterotypic Tumor Spheroid Model,” Clin. Exp. Metastasis., vol. 30, no. 5, pp. 615–630, 2014
[6] S. Gunti et al., “Organoid and spheroid tumor models: Techniques and applications,” Cancers (Basel)., vol. 13, no. 4, pp. 1–18, 2021
[7] Y. Li et al., “Hydrogel microenvironments for cancer spheroid growth and drug screening,” Sci. Adv., vol. 4, no. 4, pp. 1–11, 2018
[8] Y. Tan et al., “Cell number per spheroid and electrical conductivity of nanowires influence the function of silicon nanowired human cardiac spheroids,” Acta Biomater., vol. 51, pp. 495–504, 2017
[9] C.-L. Lin et al., “Development of an In Vitro 3D Model for Investigating Ligamentum Flavum Hypertrophy,” Biol. Proced. Online, 2020
[10] S. A. DuPré et al., “The mouse mammary carcinoma 4T1: Characterization of the cellular landscape of primary tumours and metastatic tumour foci,” Int. J. Exp. Pathol., vol. 88, no. 5, pp. 351–360, 2007
[11] L. Li et al., “Optimizing a 3D culture system to study the interaction between epithelial breast cancer and its surrounding fibroblasts,” J. Cancer, vol. 2, no. 1, pp. 458–466, 2011
[12] A. C. Gilmore et al., “An in vitro tumorigenesis model based on live-cell-generated oxygen and nutrient gradients,” Commun. Biol., vol. 4, no. 1, pp. 1–13, 2021
[13] C. Sun et al., “Ligamentum flavum fibrosis and hypertrophy: Molecular pathways, cellular mechanisms, and future directions,” FASEB J., vol. 34, no. 8, pp. 9854–9868, 2020
[14] N. Bailly et al., “Numerical investigation of the relative effect of disc bulging and ligamentum flavum hypertrophy on the mechanism of central cord syndrome,” Clin. Biomech., vol. 74, no. October 2019, pp. 58–65, 2020
[15] L. Yu et al., “The Relationship between Dural Ossification and Spinal Stenosis in Thoracic Ossification of the Ligamentum Flavum,” J. Bone Jt. Surg. - Am. Vol., vol. 101, no. 7, pp. 606–612, 2019
[16] M. Mandon et al., “Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay,” Sci. Rep., vol. 9, no. 1, pp. 1–9, 2019
[17] Z. Y. Wang et al., “A DMSO-free hepatocyte maturation medium accelerates hepatic differentiation of HepaRG cells in vitro,” Biomed. Pharmacother., vol. 116, no. March, p. 109010, 2019
[18] F.-C. Liao et al., “A Preliminary Investigation of Embedding In Vitro HepaRG Spheroids into Recombinant Human Collagen Type I for the Promotion of Liver Differentiation,” Polymers (Basel)., vol. 14, no. 9, p. 1923, 2022
[19] U. Grässer et al., “Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis,” Ann. Anat., 2018
[20] W. Wang et al., “3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells,” Biomaterials, vol. 30, no. 14, pp. 2705–2715, 2009
[21] Z. Berahim et al., “Characterization of periodontal fibroblast spheroids in vitro,” Chiang Mai J. Sci., vol. 40, no. 6 SPEC. ISSUE 2, pp. 1020–1029, 2013.
[22] I. Smyrek et al., “Quantitative three-dimensional evaluation of immunofluorescence staining for large whole mount spheroids with light sheet microscopy,” Biomed. Opt. Express, vol. 8, no. 2, p. 484, 2017
[23] J. F. Dekkers et al., “High-resolution 3D imaging of fixed and cleared organoids,” Nat. Protoc., vol. 14, no. 6, pp. 1756–1771, 2019
[24] B. editing: precision chemistry on the genome And et al., “乳鼠心肌提取 HHS Public Access,” Physiol. Behav., vol. 176, no. 3, pp. 139–148, 2016
[25] Y. Zhan et al., “Organic solvent-based tissue clearing techniques and their applications,” J. Biophotonics, vol. 14, no. 6, pp. 1–16, 2021
[26] E. Nürnberg et al., “Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward,” Front. Mol. Biosci., 2020
[27] M. P. I. Williams et al., “A novel optical tissue clearing protocol for mouse skeletal muscle to visualize endplates in their tissue context,” Front. Cell. Neurosci., vol. 13, no. February, pp. 1–12, 2019
[28] M. E. Boutin et al., “A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models,” Sci. Rep., 2018
[29] R. L. van Ineveld et al., “Single-cell resolution three-dimensional imaging of intact organoids,” J. Vis. Exp., vol. 2020, no. 160, pp. 1–8, 2020
[30] C. L. Roberge et al., “Non-Destructive Tumor Aggregate Morphology and Viability Quantification at Cellular Resolution, During Development and in Response to Drug,” Acta Biomater., vol. 117, pp. 322–334, 2020
[31] P. Järvinen et al., “Simultaneous Culturing of Cell Monolayers and Spheroids on a Single Microfluidic Device for Bridging the Gap between 2D and 3D Cell Assays in Drug Research,” Adv. Funct. Mater., vol. 30, no. 19, 2020
[32] R. Achanta et al., “SLIC superpixels compared to state-of-the-art superpixel methods,” IEEE Trans. Pattern Anal. Mach. Intell., vol. 34, no. 11, pp. 2274–2281, 2012
[33] R. L. van Ineveld et al., “Single-cell resolution three-dimensional imaging of intact organoids,” J. Vis. Exp., 2020
[34] E. Leary et al., “Quantitative Live-Cell Confocal Imaging of 3D Spheroids in a High-Throughput Format,” SLAS Technol., vol. 23, no. 3, pp. 231–242, 2018
[35] E. C. Costa et al., “ClearT immersion optical clearing method for intact 3D spheroids imaging through confocal laser scanning microscopy,” Opt. Laser Technol., vol. 106, pp. 94–99, 2018
[36] A. G. Pershina et al., “Variation in tumor pH affects pH-triggered delivery of peptide-modified magnetic nanoparticles,” Nanomedicine Nanotechnology, Biol. Med., vol. 32, no. 19, p. 102317, 2021
[37] Z. Huang et al., “Characterization of triple-negative breast cancer MDA-MB-231 cell spheroid model,” Onco. Targets. Ther., vol. 13, pp. 5395–5405, 2020