自组装类人心类中的缺氧-再氧合损伤模型
2026年3月17日
自组装型人类心脏类器官（hCOs）是一种新兴的系统，用于模拟人体心脏发育和疾病。本文提供了通过缺氧再氧化生成和损伤hCO的方法，以及损伤后预期的组织学和功能结果细节。
我们的研究重点是寻找帮助心脏在受伤后再生的新方法。为此，我们生成自组装的心脏类器官并进行缺氧再氧化研究。该方案可用于研究损伤反应、促进再生的治疗干预，并可用于患者特异性疾病建模。
首先，抽取D1心脏中胚层指定培养基，并加入足够的细胞脱离液，以完全覆盖人为诱导的多能干细胞来源心脏中胚层细胞。然后将盘子放入37摄氏度的培养箱中，浸泡五分钟。使用移液器，向每个孔槽加入等量的D2心脏中胚层培养基，轻轻上下移液以释放粘附细胞，将细胞悬浮液转移到15毫升锥形管中。
用离心机在室温下以300克沉淀细胞三分钟。将沉淀物重新悬浮于一毫升D2心脏中胚层规格培养基中，含5微摩尔IWP-2和50微克/毫升抗坏血酸，浓度为RB-。将10微升的锥蓝与10微升细胞悬浮液混合在微管中。
使用首选计数方法计数细胞，然后将细胞稀释至每毫升100,000个细胞浓度，加入D2心脏中胚层规格培养基，并补充两微摩尔噻佐维文。将细胞悬浮液转移到适当大小的试剂储液槽中。然后，使用多通道移液器，向超低附着的96孔板的每个孔注入100微升，达到每孔10,000个细胞的最终浓度。
在室温下，500克旋转该板五分钟，诱导悬浮培养中人类心脏类器官的形成。观察每个孔中形成一个大型且边界良好的细胞颗粒。如果颗粒没有被限制，重复离心。
24小时后，制备10毫升含5微摩尔IWP-2和50微克/毫升抗坏血酸的D3培养基，每片板均为RB-。使用多通道移液器，在不去除任何现有培养基的情况下，向每个孔添加100微升的D3培养基。第二天，从每个孔中取出100微升的D3培养基。
为降低吸入风险，将板倾斜至45度角，并将多通道移液器与板侧壁呈90度角。将移液管牢牢放在井壁上，缓慢吸气。去除培养基后，换成100微升含5微摩尔IWP-2的D4培养基，含量为RB减。
使用1000微升移液管，去除吸头，将人体心脏类器官转移到1.7毫升微离心管中。让类器官沉入管底，轻柔地去除多余的培养基。添加含磷盐的生理盐水清洗类器官一次。
等几秒钟让植物沉下去，然后轻轻去除多余的PBS。接着加入4%的对甲醛固定类器官，并将管子置于室温下30分钟以固定。然后将所需数量的固定类器官转移到1.7毫升微离心管中。
去除固定液，用含0.1%Tween 20的PBS清洗类器官。通过将抗体稀释至染色溶液中的所需浓度，制备初级抗体溶液。使用移液管向每个微离心管加入50至100微升的初级抗体溶液，确保类器官完全覆盖。
培养和后续清洗后，将样本与稀释的二级抗体在染色溶液中以4摄氏度在摇晃平台上培养24小时。孵化后，按照之前描述的进行洗涤。接着，使用切过的移液器尖端，将类器官转移到带正电的玻璃玻片上。
用Tween 20轻轻去除多余的PBS，并滴几滴装载介质覆盖类器官。小心地将盖片覆盖在类器官上使其平整。然后进行荧光显微镜检查。
在将固定类器官浸泡在30%蔗糖、四摄氏度中孵育一夜后，转移到塑料模具中，加入一小滴最佳切割温度化合物，刚好覆盖类器官。用细镊子或移液头轻轻将类器官在化合物中重新定位，使其不结块，远离模具边缘。将类器官尽可能靠近模具底部，以最大化单次冷冻切割中的数量。
将模具放入含有干冰层的泡沫冷却器中，冻结最佳切割温度的初始层。等几分钟让模具硬化，然后把模具移到工作台上。在模具填充前，添加额外的最佳切割温度配方。
让第一层完全融化后，再将模具放回干冰中完全冷冻。冷冻后，将模具存放在零下80摄氏度，或进行冷冻切割以获得六微米切片。使用移液器，将每96孔人体心脏类器官板中5毫升缺氧培养基转移到细胞培养瓶中。
将烧瓶放入缺氧舱，用含5%二氧化碳和95%氮气的缺氧气体冲洗三分钟。在开始缺氧前，将舱体放入37摄氏度的保温箱中至少一小时。使用多通道移液器，从人体心脏类器官板的所有孔中提取约150微升培养基。
然后，使用200微升的移液器，小心地从每个孔中取出剩余培养基，同时不扰动类器官。每口井加入50微升缺氧介质。将人体心脏类器官板放入缺氧舱。
并用含5%二氧化碳和95%氮气的缺氧气体冲洗舱室三分钟。将缺氧舱放入37摄氏度的保温箱中，浸泡六小时。要启动再氧化，将人体心脏类器官从缺氧舱中取出。
使用多通道移液器，向每个孔加入150微升预热的RB+培养基。最后，将平板放入37摄氏度的培养箱，进行至少三小时的再氧化后再进行下游检测。在缺氧6小时和再灌注24小时后，受伤人类心脏类器官的细胞外乳酸脱氢酶活性显著增加，相较于未受伤对照组。
缺氧处理的心脏类器官表现出TUNEL阳性心肌细胞核，受伤类器官中TUNEL阳性心肌细胞核的比例显著高于未受伤对照组。在重新氧合前缺氧持续时间增加时，瘢痕形成时间依赖性增加。再灌注后24小时，69%的未受伤人类心脏类器官跳动，而受伤类器官均未跳动。
未受伤的人类心脏类器官在五个关注区域表现出同步的钙瞬变，峰值同时出现且波形相似。受伤的人类心脏类器官不表现出协调收缩，反而在关注区域表现出不规则的自发钙瞬变。该方案使研究人员能够模拟iPSC来源的人类心脏类器官中的心肌梗死。
确保伤害可复现的最大技术挑战是完全去除现有介质。我们未来的研究旨在识别能够促进人类心脏类器官模型组织再生的因素。
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Overview
This protocol details the generation, maintenance, and injury modeling of human induced pluripotent stem cell (iPSC)-derived cardiac organoids (hCOs). It provides a step-by-step guide for creating self-assembling hCOs, inducing hypoxia-reoxygenation injury to mimic ischemia/reperfusion, and performing histological and functional analyses. The approach enables researchers to study cardiac injury responses and test therapeutic interventions in a human tissue context.
Key Study Components
Area of Science
- Cardiac biology
- Stem cell research
- Organoid technology
- Ischemia-reperfusion injury modeling
Background
- Ischemic heart disease can cause irreversible cardiomyocyte loss and impaired cardiac function if not rapidly treated.
- There is a need for human tissue models to study hypoxia-reoxygenation injury and evaluate therapies.
- Human cardiac organoids (hCOs) offer a promising model but present technical challenges due to their small size and culture requirements.
- Existing animal models do not fully recapitulate human cardiac injury and regeneration.
Purpose of Study
- To provide a user-friendly protocol for generating and maintaining iPSC-derived hCOs.
- To establish a reproducible method for inducing hypoxia-reoxygenation injury in hCOs.
- To enable histological and functional analysis of injury and recovery in human cardiac tissue models.
Methods Used
- Differentiation of human iPSCs into cardiac mesoderm and formation of self-assembling organoids in ultra-low attachment plates.
- Suspension culture and stepwise media changes to promote cardiac organoid maturation.
- Fixation, immunostaining, and cryosectioning for histological analysis.
- Induction of hypoxia-reoxygenation injury using a hypoxia chamber and controlled gas environment.
- Assessment of injury via lactate dehydrogenase release, TUNEL staining, contractility, and calcium imaging.
Main Results
- Hypoxia-reoxygenation injury in hCOs led to increased extracellular lactate dehydrogenase activity, indicating cell damage.
- Injured organoids showed a higher percentage of TUNEL-positive cardiomyocyte nuclei, reflecting increased apoptosis.
- Scar formation increased with longer hypoxia duration before reoxygenation.
- Beating activity and synchronized calcium transients were lost in injured organoids, while uninjured controls maintained coordinated contractions.
Conclusions
- This protocol enables reproducible modeling of myocardial infarction-like injury in human cardiac organoids.
- It provides a platform for studying injury responses and testing regenerative therapies in a human context.
- Future work will focus on identifying factors that promote tissue regeneration using this model.
Frequently Asked Questions
Executive Industry Relevance
Modeling hypoxia-reoxygenation injury in self-assembling human cardiac organoids enables predictive interrogation of human myocardial injury and recovery mechanisms. This system provides a translationally relevant platform for evaluating therapeutic interventions and de-risking targets in ischemic heart disease. The approach supports early-stage portfolio decisions by bridging the gap between in vitro discovery and preclinical validation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables mechanistic interrogation of cardiomyocyte injury and regeneration pathways in a human context.
- Supports functional target validation by quantifying apoptosis, scar formation, and contractility loss after injury.
- Facilitates predictive confidence in candidate targets by modeling clinically relevant injury phenotypes.
Screening & Assay Development
- Provides a reproducible, scalable platform for compound screening in human cardiac tissue models.
- Delivers quantitative outputs such as lactate dehydrogenase release and TUNEL positivity for assay standardization.
- Enables assessment of synchronized calcium transients and contractility for functional screening endpoints.
Translational & Preclinical Research
- Aligns with disease-relevant human myocardial injury, supporting translational biomarker development.
- Offers continuity from discovery through preclinical validation by complementing animal models.
- De-risks advancement decisions by providing human-specific injury and recovery data.
Pipeline & Workflow Integration
This model integrates into the discovery-to-preclinical continuum, enabling hypothesis testing, target validation, and translational assessment of cardiac injury interventions.
- Discovery Biology: Supports hypothesis-driven testing of regenerative and protective factors in human cardiac tissue.
- Screening: Provides standardized, quantitative injury readouts for compound evaluation.
- Analytics: Enables statistical comparison of injury markers and functional outputs across experimental conditions.
- Translational Research: Bridges in vitro findings with preclinical and clinical relevance in ischemic heart disease.
- Enterprise Reuse: Establishes a reusable platform for diverse cardiac injury and regeneration studies.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in cardiac injury research.
- Operational Value: Standardizes injury modeling and readouts for reproducibility and scalability.
- Strategic Value: Informs go/no-go decisions and optimizes resource allocation in early cardiac portfolio programs.
- Portfolio Impact: Enables risk-adjusted prioritization of regenerative and protective cardiac therapies.
Implementation Considerations
- Requires expertise in iPSC culture, cardiac differentiation, and organoid handling.
- Needs access to hypoxia chambers, fluorescence microscopy, and quantitative assay platforms.
- Demands rigorous cross-team standardization for reproducible injury induction and analysis.
- Adaptation across iPSC lines and disease models may require protocol optimization.
- Technical reproducibility hinges on precise media removal and injury timing.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:22
Differentiation of iPSC Derived Cardiac Mesoderm Cells to Human Cardiac Organoids (hCOs)
3:31
Fixation and Whole-Mount Immunostaining of Human Cardiac Organoids
5:37
Embedding the Cardiac Organoids for Cryosectioning
6:55
Inducing Hypoxia-Reoxygenation Injury in Human Cardiac Organoids
8:53
Results
10:13
Conclusion
