生成基于小鼠前列腺类器官的模型以研究宿主-病原体相互作用
February 27th, 2026
该协议描述了生成基于二维小鼠前列腺类器官模型的过程。包括类器官解离、细胞播种、分化、上皮完整性评估以及体 外 感染尿路病原 性大肠杆菌。该模型提供了顶端可及性，适合研究宿主-病原体相互作用，克服了传统三维类器官系统的局限。
本研究重点是了解细菌病原体如何感染前列腺上皮，以及哪些宿主病原体相互作用决定了感染结果。现有体外模型未能很好地捕捉前列腺上皮的多样性。该模型提供了一个生理学相关的系统，反映了组织中存在的不同细胞类型。
首先，获取在24孔板中细胞外基质滴液培养的小鼠前列腺类器官。从所需数量的孔中抽取培养基，并向每个井加入500微升新鲜培养基。使用P1000移液器机械地破坏每个孔中的胞外基质滴落。
将悬浮液收集在15毫升的锥形管中，每管最多收集两孔。加入新鲜培养基，使体积达到10毫升。轻轻倒置管子三到四次，混合后以450克、摄氏四度离心五分钟。
抽吸上清液。将颗粒重新悬浮在两毫升酶溶液中，预热至37摄氏度，并在37摄氏度的水浴中孵育15分钟。在培养期间，使用P-1000移液器每五分钟混合一次细胞悬浮液。
孵育后加入新鲜培养基，达到最终体积10毫升。轻轻将管子倒转三到四次以混合。离心机在450克，四摄氏度，离心五分钟。
在不扰动沉淀的情况下抽取上清液。将沉淀重新悬浮在300微升培养基中，辅以10微摩尔岩石抑制剂。使用新鲍尔计数室，计数懸浮液中的细胞数。
将悬浮液稀释至每毫升62,500个细胞，培养基中添加10微摩尔岩石抑制剂。然后每孔为48孔板投放200微升悬浮液，为腔室微载玻片每孔注入300微升。将平板放入细胞培养箱，温度为37摄氏度，二氧化碳为5%。
一天后，检查大多数单细胞是否已附着在二维培养表面。每两到三天更换培养基，并持续进行，直到七天内达到汇合点。为了诱导分化以富集腔腔细胞，在播种时向细胞悬浮液中加入10纳摩尔的五α二氢睾酮（DHT）。
将泌尿病性大肠杆菌株（UPEC）划线到补充100微克每毫升氨苄青霉素的LB琼脂平板上。在37摄氏度下孵育16至24小时。使用无菌环接种：3毫升LB汤，辅以100微克/毫升氨苄青霉素，单个细菌菌落。
在37摄氏度下静止孵育12至16小时。Vortex 一夜间细菌培养。测量培养物的光学密度为600纳米。
然后计算在三毫升汤中达到0.05起始光学密度所需的培养体积。将计算出的细菌培养液加入新鲜氨苄青霉素补充的LB汤中。静置于37摄氏度静止孵育24小时。
孵育后，对细菌培养物进行漩涡处理。测量600纳米的光学密度。计算达到1的光学密度所需的培养体积。
在室温下以12,000克离心所需体积两分钟。抽吸并丢弃上清液。将细菌沉淀重新悬浮于一毫升Primus和无DHT的游离类器官培养基中。
然后用小鼠前列腺类器官培养基稀释悬浮液，达到感染倍数为100。从制备的类器官模型中抽吸培养基。在48孔板的每个孔中加入200微升含细菌培养基。
在37摄氏度、5%二氧化碳的细胞培养箱中孵育一小时。从孔中抽取含菌基，并用新鲜培养基清洗三次，以去除细胞外细菌。接着加入含有50微克/毫升的庆他霉素培养基，培养30分钟。
吸入介质。加入新鲜培养基，补充10微克每毫升的庆他霉素，以应对剩余感染时间。使用共焦显微镜在荧光染色后成像感染细胞，并使用流式细胞仪定量感染细胞。
在无二氢睾酮的对照组中，大多数细胞为细胞角蛋白，基底细胞有五个阳性，只有少数表现出低CD 24 8腔表表达。补充一纳摩尔DHT产生混合基底腔族群，尽管CD 24 A保持较低水平。将DHT增加到10纳摩尔促进分化到腔腔细胞，同时在培养七天后保留部分基底细胞。
这与定量研究显示细胞角蛋白减少、5个阳性基底细胞、CD 24A阳性腔腔细胞增加、DHT浓度增加相关。感染后零小时，UPEC大量附着于上皮细胞。在接受一小时的优大霉素治疗后，UPEC阳性细胞数量急剧下降。
24小时时，感染细胞数与1小时时相似。24小时后的共焦点成像显示，细胞内细菌继续复制，形成群落状簇。该方案使研究人员能够在一个保留上皮细胞多样性的模型中研究细菌感染动态及病原体相互作用。
未来的研究可以利用该模型研究多种前列腺疾病，包括感染、癌症生物学，并测试潜在的治疗化合物。
Overview
This article presents a step-by-step protocol for generating a two-dimensional (2D) culture model derived from mouse prostate organoids. The model maintains key epithelial characteristics and allows direct access to the apical surface, enabling detailed studies of host-pathogen interactions, particularly with uropathogenic Escherichia coli (UPEC). The protocol includes organoid dissociation, cell seeding, differentiation, and infection procedures, validated by immunofluorescence and functional assays.
Key Study Components
Area of Science
- Prostate epithelial biology
- Host-pathogen interactions
- Organoid and cell culture models
Background
- Prostate diseases such as benign prostatic hyperplasia, prostatitis, and prostate cancer are prevalent in aging men.
- Organoid models replicate epithelial structure and function but limit access to the apical surface.
- Existing in vitro models do not fully capture the diversity of prostatic epithelial cells.
- Improved models are needed to study infection dynamics and disease mechanisms.
Purpose of Study
- To develop a physiologically relevant 2D culture system from mouse prostate organoids.
- To enable direct experimental access to the apical surface for infection studies.
- To validate the model's epithelial diversity and suitability for host-pathogen interaction research.
Methods Used
- Dissociation of mature mouse prostate organoids cultured in extracellular matrix.
- Seeding of dissociated cells onto 2D culture surfaces with optimized conditions for attachment and proliferation.
- Differentiation induction using dihydrotestosterone (DHT) supplementation.
- Infection of the 2D model with UPEC and analysis via immunofluorescence microscopy, TEER measurements, and flow cytometry.
Main Results
- The 2D model retained essential epithelial characteristics and cellular diversity.
- DHT supplementation modulated the balance between basal and luminal cell populations.
- UPEC adhered to and invaded epithelial cells, forming intracellular clusters observed by confocal microscopy.
- Gentamycin treatment effectively reduced extracellular bacteria, allowing study of intracellular infection dynamics.
Conclusions
- The 2D organoid-derived model provides a robust platform for studying prostate epithelial biology and host-pathogen interactions.
- It enables detailed investigation of infection dynamics and cellular responses.
- The model is suitable for future studies on prostate diseases and therapeutic testing.
Frequently Asked Questions
Executive Industry Relevance
Prostate organoid-derived 2D models address a critical gap in preclinical research by enabling direct interrogation of host-pathogen interactions at the epithelial barrier. This system preserves cellular diversity and barrier function, supporting predictive confidence in early-stage target validation and mechanistic de-risking for infection and prostate disease research. The platform's translational relevance positions it as a reusable asset for portfolio-wide evaluation of disease mechanisms and therapeutic strategies.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables mechanistic interrogation of epithelial responses to uropathogenic Escherichia coli infection.
- Supports functional validation of host-pathogen interaction targets in a physiologically relevant context.
- Facilitates biological de-risking by modeling both basal and luminal cell populations.
- Improves predictive confidence for downstream infection biology studies.
Screening & Assay Development
- Provides a standardized, reproducible 2D epithelial system for quantitative infection assays.
- Enables direct access to the apical surface for compound or pathogen application.
- Supports TEER and immunofluorescence-based readouts for barrier integrity and cell differentiation.
- Allows scalable preparation for high-content screening of infection-modulating agents.
Translational & Preclinical Research
- Aligns with disease-relevant epithelial biology for translational infection and prostate disease models.
- Maintains continuity from discovery through preclinical validation of host-pathogen mechanisms.
- Enables risk-adjusted advancement of therapeutic hypotheses targeting epithelial infection pathways.
- Supports evaluation of candidate interventions in a system reflecting in vivo cellular diversity.
Pipeline & Workflow Integration
This organoid-based 2D model bridges early discovery and preclinical infection research, enabling hypothesis testing, target validation, and quantitative readouts within a unified workflow.
- Discovery Biology: Supports null hypothesis testing of infection mechanisms and epithelial responses.
- Screening: Delivers reproducible, quantitative infection and barrier function assays for compound evaluation.
- Analytics: Provides immunofluorescence, TEER, and flow cytometry outputs for robust condition comparison.
- Translational Research: Maintains disease-relevant cellular organization for preclinical continuity.
- Enterprise Reuse: Functions as a reusable platform for diverse prostate disease and infection studies.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in infection biology.
- Operational Value: Standardizes epithelial infection assays with scalable, reproducible workflows.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient portfolio triage.
- Portfolio Impact: Supports risk-adjusted prioritization of infection and prostate disease programs.
Implementation Considerations
- Requires expertise in organoid culture, epithelial differentiation, and infection assays.
- Needs access to immunofluorescence microscopy, TEER instrumentation, and flow cytometry.
- Demands cross-team standardization of cell seeding, differentiation, and infection protocols.
- Adaptable to other epithelial models with protocol optimization for tissue specificity.
- Barrier function and cellular diversity must be validated for each experimental batch.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:27
3D Prostate Organoid Disruption into Single Cells
1:58
Plating Single-Cell Suspension onto a 2D Culture Surface
3:17
Preparation of Uropathogenic Escherichia coli (UPEC) for Infecting the Organoids
5:12
Infecting the Prepared Prostate-Organoid Model with Uropathogenic Escherichia coli (UPEC)
6:16
Results
7:38
Conclusion
