隨著老年化社會來臨，老年人及退化性疾病日益增多。腰椎椎管狹窄症(LSS) 常見於老年人脊椎退化性疾病，往往造成慢性下背痛和下肢疼痛、神經性跛行或馬尾症候群等脊椎神經病變。造成腰椎椎管狹窄症的因素與黃韌帶肥厚、小面關節退化性關節炎以及椎間盤突出相關，在這些因素之中黃韌帶肥厚是造成腰椎椎管狹窄的一個重要因素。黃韌帶外觀呈現黃色，主要由膠原蛋白及彈性纖維組成，位於上下脊椎骨骨椎板(laminae)之間，其功能為提供脊椎穩定度及保護椎管內神經組織。雖然已經有大量研究針對黃韌帶肥厚進行研究，但其確切發病機制尚不清楚。這部分是由於大多數研究是在傳統的2D細胞培養中進行的，缺乏關鍵的生理學面向，例如在三維（3D）組織結構中發現的細胞之間信號傳導，阻礙了2D培養模擬體內條件，相比之下，3D細胞培養系統已顯示出比2D更好地反映體內條件。透過3D細胞球(spheroid)的培養，不僅透過細胞結構改善，細胞因子梯度、營養物和氣體交換，增加細胞與細胞間相互作用，獲得更多生理相關性。目前還沒有關於3D培養應用於黃韌帶細胞的研究。因此本篇研究目的是想建立體外培養原代黃韌帶細胞的3D培養平台，並且將常規細胞培養和不同的3D培養條件做進一步的比較。
在本研究中，我們首先利用蘇木精-伊紅（H&E）染色來觀察細胞結構。我們使用低貼附的圓底96孔盤培養原代黃韌帶細胞，並且通過免疫螢光染色證實培養的細胞具有典型的黃韌帶表型，根據活死細胞的染色顯示，細胞在平台上生長良好。我們的結果發現不同細胞數與細胞球的大小變化相關，此外我們還證明了3D球體可以維持長期培養。透過細胞週期分析、qRT-PCR及Western blot 進一步比較2D培養與3D培養。我們的實驗指出2D和3D培養在細胞週期分析上沒有顯著差異，3D培養中collagen mRNA表現顯著高於2D培養，但兩種培養方法之間蛋白質表達無顯著差異。Elastin mRNA表現2D培養較多，蛋白的表現3D培養則隨著時間增多，此外，在3D培養的細胞中，Fibronectin蛋白表現量在第7天和第14天顯著高於2D培養的細胞。黃韌帶三維模型發展具有潛力，因黃韌帶細胞可在三維條件下穩定培養，這有助於我們應用在未來的研究，進一步了解黃韌帶肥厚的發病機制。

With the aging society, the number of diseases observed among elderly patients as well as the number of degenerative diseases are increasing. Lumbar spinal stenosis (LSS) is a degenerative disease of the spine in elderly patients that may cause lower extremity pain, back pain, cauda equina syndrome and neurogenic claudication. Hypertrophy of the ligamentum flavum (LF), facet joints and herniated disc are important contributing factors in degenerative LSS. LF hypertrophy (LFH) is known to be the main cause of LSS. LF is a yellow ligament containing collagen and elastin fibers that connects the lam-inae of adjacent vertebrae and provides protection and stability to the spinal column. The exact patho-genesis of LF hypertrophy is not well understood, although it has been evaluated in numerous studies. Most studies have been carried out using conventional 2D cell cultures that do not resemble in vivo conditions, as they lack crucial physiological aspects such as cell-cell signaling that are found in three-dimensional (3D) tissue structures. By comparison, 3D cell culture systems have been shown to reflect in vivo conditions better than 2D culture systems. By using 3D cultures, including spheroids, cell-cell interactions can be immensely improved to ensure greater physiological relevance through not only structural improvements but also cytokine gradients and nutrient and gas exchange. There is currently no research on the application of 3D culture to ligamentum flavum cells. Therefore, the aims of this study are to build an in vitro 3D culture platform for culturing primary LF cells and to compare conventional cell culture with 3D culture under different conditions.
In this study, we first utilized hematoxylin and eosin (H&E) staining to observe cell structure. We used ultralow attachment (ULA) 96-well plates to perform 3D LF primary cell culture and confirmed that the cultured cells had a typical LF phenotype according to immunofluorescence staining. The cells exhibited good viability and growth on the platform according to live/dead staining. Our results show that different cell seeding numbers (100, 1000 and 5000 cells) correlate with the growth of LF spheroids based on size. We also demonstrated that 3D spheroids can be maintained in long-term culture. 2D culture and 3D culture were further compared by cell cycle, qRT-PCR and Western blot analyses. There was no significant difference between 2D and 3D cultures in the cell cycle analysis. Collagen mRNA expression was significantly higher in 3D culture than in 2D culture, but there was no significant difference in protein expression between the two culture methods. Elastin mRNA expression was more abundant in 2D culture, while at the protein level, elastin expression increased with time in 3D culture. Fibronectin protein expression was significantly higher at 7 and 14 days in 3D-cultured cells than in 2D-cultured cells. The development of the LF 3D spheroid model has great potential, as LF cells could be stably cultured in 3D, which could further our understanding of the pathogenesis of LFH and have applications in future studies.

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