近年來，全球感音性聽力損失（sensorineural hearing loss， SNHL）患者人數持續增加，然由於哺乳類聽毛細胞於受損後幾乎不具再生能力，且現行臨床治療仍以輔具補償為主，缺乏可促進功能修復之有效策略，使聽損研究長期面臨臨床轉譯瓶頸。傳統臨床前研究高度依賴動物模型，尤以小鼠為主要系統，然而其在耳蝸發育歷程、離子微環境組成及藥物反應上與人類存在本質差異，限制研究成果向臨床應用延伸的可靠性。隨著 FDA Modernization Act 3.0 對新方法學（New Approach Methodologies， NAMs）與動物實驗替代策略的推動，建立具人源相關性且可重現耳蝸發育微環境的體外模型，已成為聽覺醫療研發的重要方向。本研究建構於本實驗室既有之小鼠耳蝸類器官培養與離子交換微環境調控技術基礎，進一步導入人類誘導多能幹細胞（induced pluripotent stem cells， iPSCs），結合3D 生物列印工程與離子微環境設計，建立一套以微環境重建為核心的體外耳蝸類器官新方法學平台。本研究之關鍵並非單一細胞分化流程之優化，而在於回應既有文獻所指出的核心限制：耳前驅細胞（Otic Progenitor Cells， OPCs）中之上皮前驅細胞與神經前驅細胞於體外條件下通常需分別誘導，難以在同一培養系統中穩定共存與協同發育。為突破此限制，本研究以胚胎發育階段之耳蝸微環境為設計藍本，透過三維結構支撐、離子梯度調控與動態培養條件之整合，重建可支持多譜系前驅細胞共存、互動與組織化的培養系統，促進具層次結構之耳蝸類器官形成。預期本研究所建立之人源體外耳蝸類器官平台，將可更貼近人類耳蝸發育與微環境特性，作為探討聽毛細胞分化機制、評估聽損再生策略，以及進行聽力相關藥物與醫材臨床前測試的關鍵研究工具。整體而言，本研究不僅延續既有動物模型成果，更透過人源化與微環境工程導向之方法學設計，展現作為動物實驗替代方案的潛力，並為未來聽覺醫療產品開發與臨床轉譯提供具系統性與前瞻性的研究基礎。;In recent years， the prevalence of sensorineural hearing loss (SNHL) has continued to increase worldwide. However， because mammalian cochlear hair cells possess little regenerative capacity after damage， current clinical treatments remain largely dependent on hearing aids and cochlear implants， which compensate for hearing function rather than restore it. Consequently， the development of regenerative therapies has become a major challenge in hearing research. Conventional preclinical studies rely heavily on animal models， particularly mice. Nevertheless， fundamental differences in cochlear development， ionic microenvironment， and drug responses between mice and humans limit the translational value of these models. With the implementation of the FDA Modernization Act 3.0， which promotes New Approach Methodologies (NAMs) and alternatives to animal testing， the development of physiologically relevant human in vitro models has become increasingly important. Building upon our laboratory’s previous work on mouse cochlear organoids and ionic microenvironment engineering， this study integrates induced pluripotent stem cells (iPSCs) with 3D bioprinting and microenvironmental engineering to establish a human cochlear organoid platform that better recapitulates cochlear development. Rather than focusing solely on optimizing differentiation protocols， this study addresses a critical limitation reported in previous studies: epithelial and neural otic progenitor cells are typically induced separately in vitro， making it difficult to achieve their stable coexistence and coordinated development within a single culture system. Inspired by the embryonic cochlear microenvironment， we reconstructed a biomimetic culture platform by integrating a three-dimensional structural scaffold， ionic microenvironment regulation， and dynamic culture conditions. This engineered microenvironment is designed to support the coexistence， interaction， and spatial organization of multiple otic progenitor lineages， thereby promoting the formation of structurally organized cochlear organoids. The proposed human cochlear organoid platform is expected to more faithfully mimic human cochlear development and provide a valuable in vitro model for investigating hair cell differentiation， evaluating regenerative strategies for hearing loss， and conducting preclinical testing of hearing-related drugs and medical devices. Overall， this study extends previous animal-based research by introducing a humanized， microenvironment-oriented engineering strategy， demonstrating its potential as an alternative to animal models and providing a promising foundation for future translational research and hearing healthcare innovation.
