Organoids in the Development of Pharmacokinetics, Pharmacodynamics, and Potency Assays

What are organoids?

Organoids are three-dimensional (3D) tissue culture systems, that mimic the architecture and functionality of human organs, generated from pluripotent stem cells (PSCs), adult stem cells (ASCs), or primary cells. They are cultured in extracellular matrix scaffolds (e.g., Matrigel) and exposed to organ-specific growth factors, enabling self-organization into structures that mirror in vivo tissues such as the intestine, brain, liver, and lung. These organoid models offer unparalleled advantages over traditional two-dimensional (2D) cell cultures and animal models by providing a physiologically relevant microenvironment, better genetic fidelity, and functional complexity (Xu, et al (2018)). As such, organoids are rapidly becoming integral to preclinical drug development, disease modeling, toxicity testing, and the implementation.

How Organoids are Made

The generation and maintenance of organoids involve several key components. Organoids can be derived from various cell sources, including pluripotent stem cells (PSCs), adult stem cells (ASCs), or primary cells (Landcaster et al (2013), Boonekamp, et al (2020), Huch et al (2015)). To support the three-dimensional structure and development of these cells, a 3D matrix such as Matrigel is typically used (Poudel et al (2021)). Differentiation into organ-specific tissue types is guided by specialized growth factor cocktails that often include Wnt, FGF, and EGF (Boonekamp, et al (2020), Cala et al (2023)) . The culture process usually spans 30 to 60 days to allow for maturation, and passaging is employed to support long-term maintenance and expansion of the organoids.

Advantages of Organoids Over 2-D Cell Cultures

Organoids offer significant advantages over traditional 2D cell cultures by providing enhanced physiological relevance and greater predictive accuracy in biomedical research and drug development (Yang et al (2023), Liu et al (2025)). Unlike flat monolayer cultures, organoids replicate the three-dimensional architecture of tissues and preserve complex cell-to-cell interactions, closely mimicking in vivo environments such as oxygen gradients and extracellular matrix composition. This 3D structure supports more realistic cell signaling and tissue-specific functions, including neural activity and contractility, and allows for the compartmentalization of multiple cell types, improving the modeling of organ-specific diseases. Hydrogels like Matrigel further facilitate accurate cell-matrix interactions, contributing to their biological fidelity. The differences between 2D and 3D (organoid) cell cultures systems are summarized in Table 1.

Table 1: Differences between 2D and 3D (organoid) cell cultures systems

Applications of Organoids in Drug Discovery and Development

Organoids have emerged as versatile tools in drug discovery, offering a range of applications that enhance the development and evaluation of new therapeutics. They enable high-throughput drug screening to identify promising drug candidates and provide a physiologically relevant in vitro environment for assessing drug efficacy. Additionally, organoids facilitate drug repurposing by uncovering new indications for existing drugs and play a critical role in toxicity evaluation by predicting potential adverse effects. They also support biomarker validation to identify markers that predict drug response, and importantly, contribute to personalized medicine by allowing treatments to be tailored to individual patients based on their unique biological profiles. Different types of organoids have been developed and are used for specific, specialized applications (Table 2).

Table 2: Types of organoids used in drug discovery and development

Can Organoids Replace Animal Models

Organoids are emerging as powerful human-specific models that offer major advantages over animal studies in the context of disease modeling, drug screening, and personalized medicine. Grown from human stem cells, primary cell, or patient-derived tissues, they replicate key features of human biology, including architecture, signaling, and genetic specificity, that animal models simply can’t capture. For instance, human-only cell types like BEST4⁺/OTOP2⁺ intestinal cells or patient-specific responses to chemotherapy can be accurately studied in organoids but not in mice (Malonga et al (2024)) Their scalability, compatibility with high-throughput platforms, and quick gene editing using CRISPR make them especially valuable in early-stage drug development. Organoids also meet ethical goals by reducing animal use and supporting the 3Rs (the guiding framework for ethically responsible animal research and testing such as replacement, reduction, and refinement) all while enabling real-time imaging of disease processes in a 3D system. As a result, they’ve already begun to replace animal models in areas like liver toxicity testing, neurodevelopmental disorders, and individualized cancer therapy.

That said, organoids aren’t a full replacement for animal models, at least not yet. They still lack the systemic complexity of a living organism, such as integrated immune responses, vascular flow, and multi-organ signaling, all of which are necessary for studying conditions like diabetes, autoimmunity, or cancer metastasis (Park et al (2024)) Brain organoids can’t simulate memory or behavioral outcomes, and their developmental immaturity makes it difficult to model chronic or aging-related diseases. For now, organoids are best viewed as complementary tools: ideal for the early, human-relevant phases of research, while animal models remain necessary for whole-body validation and long-term safety studies. Looking forward, the most effective approach will likely combine organoids with organ-on-chip technologies, humanized animal models, and AI-enabled analytics, reducing the need for animals while improving predictive power in biomedical research (Wang et al (2024)).

Pharmacokinetic Assay Development with Organoids

Organoids are revolutionizing pharmacokinetic (PK) and pharmacodynamic (PD) assay development by providing complex, human-relevant in vitro models that better recapitulate tissue architecture, cellular heterogeneity, and functionality than traditional 2D cell lines.

The development of PK assays using organoids has demonstrated remarkable progress, particularly in modeling drug absorption, distribution, metabolism, and excretion (ADME) processes. Human intestinal organoids have proven especially valuable for studying drug absorption mechanisms (Tanaka et al (2025)). Researchers have successfully generated pharmacokinetically functional intestinal organoids from human induced pluripotent stem cells that express critical drug transporters and demonstrate efflux transport activity through ABCB1/MDR1, as well as inducible drug-metabolizing enzyme CYP3A4 expression (Tanaka et al (2025)). These organoids exhibit physiologically relevant features including microvilli, tight junctions, and various intestinal cell types including enterocytes, goblet cells, and Paneth cells. A significant breakthrough was achieved in kidney organoids, where transplanted human kidney organoids were used for preclinical PD studies with the investigational drug GFB-887 (Westerling-Bui et al (2022)) The study demonstrated that orally administered GFB-887 reached human podocytes in transplanted organoids at therapeutically meaningful concentrations, successfully performing both PK evaluation (what the organism does to a drug) and PD evaluation (how the drug affects the organism) (Westerling-Bui et al (2022)).  This approach represents a paradigm shift by enabling human-specific pharmacokinetic evaluation using organoids transplanted into animal models.

The liver organoid systems have been particularly instrumental in ADME-Tox studies, with human liver organoids demonstrating superior drug-metabolizing enzyme expression and nuclear receptor activity compared to traditional 2D cell cultures (Brooks et al (2021), Li et al (2022)) . These organoids maintain bile acid transport function and can be scaled for high-throughput screening applications (Shinozawa et al (2020), Kim et al (2025)). The development of standardized protocols has enabled the generation of hepatic organoids with functional characteristics that closely mirror primary hepatocytes, including the expression of cytochrome P450 enzymes essential for drug metabolism6. Recent advances have incorporated microfluidic “organ-on-chip” systems that enhance organoid function and provide more controlled environments for PK studies (Zhang et al (2024)).

Development of Pharmacodynamics (PD) Assays with Organoids

The development of PD and potency assays using organoids has revolutionized drug efficacy evaluation by providing human-relevant models that capture target engagement and therapeutic response. Patient-derived organoids (PDOs) have emerged as powerful tools for predicting individual drug responses, with studies demonstrating remarkable concordance between organoid drug sensitivity and clinical patient outcomes (Xiang et al (2024), Qu et al (2024)). In colorectal cancer, organoid response testing has achieved 88% positive predictive value for effective drugs and 100% negative predictive value for ineffective treatments (Qu et al (2024)). This high predictive accur(acy stems from organoids’ ability to maintain the genetic, molecular, and cellular characteristics of the original patient tissue (Qu et al (2024), Vlachogiannis et al (2018)).

Development of Potency Assays with Organoids

Potency assay development in the context of organoids refers to the process of designing, optimizing, and validating laboratory tests that measure the biological activity of a therapeutic product using three-dimensional, stem cell–derived organ-like structures. Organoids provide distinct advantages for potency assay development by closely mimicking the structural and functional characteristics of human tissues. As three-dimensional, self-organizing cell cultures derived from stem cells, organoids bridge the gap between conventional two-dimensional cell cultures and animal models, offering a more physiologically relevant cellular architecture and tissue-like organization.

Strategies for developing potency assays with organoids

Developing robust potency assays using organoids requires strategic planning to capture the relevant biological activity of a therapeutic product (cell therapy, biologic, or drug) in a physiologically relevant yet standardized system. Effective potency assays with organoids combine human relevance, biological complexity, and quantitative rigor to predict how a drug will perform in humans more accurately than traditional cell lines or animal models alone.

Here are key strategies:

Defining the Mechanism of Action (MoA) and Critical Quality Attributes (CQAs): A well-designed potency assay must measure a biological function that directly reflects the product’s intended therapeutic mechanism of action (MoA). Achieving this demands a deep understanding of the underlying biology.  For example, a stem cell therapy for lung repair might measure epithelial barrier restoration in organoids or secretion of healing factors. Key quality attributes (CQAs) like cell differentiation, secretion levels, or gene expression changes then become the assay’s readouts. Controls such as inactive cells help prove the response is specific to the MoA. However, the FDA does not always require a fully defined MoA. If the MoA is unclear, surrogate measures must show the product’s activity relates to its intended effect. In such cases, a mix of biological, analytical, or functional tests can together show potency, as long as they align with clinical benefit and ensure consistent quality (Klinker, M (2023), FDA Draft Guidance (2023)). 

Selecting and Standardizing the Organoid System: Choosing the right organoid system is fundamental to developing a relevant potency assay. The selected organoid type—whether liver, kidney, intestinal, or tumor—must accurately model the target tissue and disease context of the therapeutic product. To ensure reproducibility, organoid sources should be well-characterized and consistent, such as defined induced pluripotent stem cell (iPSC) lines or standardized primary cell protocols. For patient-derived organoids (PDOs) used in personalized applications, clear acceptance criteria for organoid quality must be established. It is also crucial to confirm that organoids are consistently at the appropriate stage of maturation and functional relevance. Rigorous quality control should be in place to monitor morphology, expression of key markers, and baseline function across batches. Additionally, protocols must be scalable to generate sufficient numbers of uniform organoids for assay development, validation, and routine use, with automation considered to streamline organoid handling and dosing.

Choosing the Optimal Readout Technology: Choosing the right readouts is key to measuring potency well. Functional outputs that show organ-level activity should come first. For example, barrier strength can be tested by TEER for gut or lung models; liver function by albumin or urea levels; pancreatic or brain models by hormone or neurotransmitter release. Contractility can be checked by force tests in heart or muscle organoids, while brain activity can be tracked with multi-electrode arrays. Shape and structure changes are measured with high-content imaging to capture cysts, lumen size, cell ratios, or tubule growth, which needs advanced image analysis. Molecular markers from qPCR, RNA-seq, or proteomics add proof of MoA effects and back up functional data. Basic viability tests like ATP or resazurin are good controls but must be paired with MoA-linked readouts to prove potency.

Assay Design, Optimization, and Validation: A strong potency assay must show a clear link between product dose (or cell count) and the biological response, often as a sigmoidal dose-response curve that sets assay sensitivity and range. To ensure reliable results, the assay should be tested for variability within runs, between runs, and between operators, with factors like organoid size, dosing time, media, and endpoints tightly controlled. Good reference standards, like proven positive and negative controls, help calibrate and track performance. Solid stats are essential, with clear acceptance limits, valid analysis methods like EC50, and checks for run validity. The assay must also meet FDA and EMA standards for specificity, accuracy, precision, range, and robustness from the start.

Key Considerations and Challenges in Potency Assay Development with Organoids: Balancing biological relevance with practical limits is a key challenge in organoid-based potency assays. High-content functional assays give strong insights but are often complex and slower than simple molecular tests, so trade-offs are needed. Organoids naturally vary, so solid protocols, enough samples, and good data analysis are critical. Costs can be high for imaging, electrophysiology, or ‘omics tools. Finally, turning complex organoid data into a clear potency measure needs sound science tied to the MoA to meet scientific and regulatory demands.

Phase-Appropriate Potency Assay Development Strategies:  The potency assays generally progress through three critical stages. The first stage is fit-for-purpose demonstration, conducted during preclinical and Phase 1 studies to verify that the assay reliably measures the potency of the test drug in organoids. The second stage is assay qualification in Phase 2, which confirms the assay’s robustness and suitability for process development, dose selection, and broader clinical evaluation. The final stage is full assay validation, performed during Phase 3 and for commercial release, to ensure the assay meets regulatory standards for accuracy, precision, specificity, linearity, and robustness. The stages of potency assay development with organoids across clinical phases are shown in Table 3.

Table 3: Stages of Potency Assay Development with Organoids Across Clinical Phases*

*Read our white paper “Selecting Bioanalytical Assays for FDA Approved Clinical Trials and Commercialization” for detailed information.

Conclusion

Organoids bridge the gap between traditional cell cultures and animal models by providing human-relevant, 3D systems that better predict real-world drug behavior. Their adoption for PK, PD, and potency assays enhances early-stage drug evaluation, reduces reliance on animals, and supports personalized medicine. Although they are not yet a complete substitute for animal models, their integration with organ-on-chip systems and AI analytics holds promise for safer, faster, and more ethical drug development pipelines. Continued advances in standardization, scalability, and functional complexity will expand their impact in shaping the future of translational research.

Disclaimer: This blog post is intended solely for educational and scientific informational purposes. Any mention of therapeutic drug names, including FDA-approved medications, is for the purpose of accurate reporting and discussion of biomedical research and does not constitute medical advice, endorsement, or promotion. Readers should not interpret the content as a recommendation for any specific treatment. Always consult a qualified healthcare professional for medical advice or treatment decisions.

About Marin Biologic Laboratories

Our Recent Publication/Meeting Presentation on Gene Therapy

1.  Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody
bioRxiv 2025.03.19.644217; doi: https://doi.org/10.1101/2025.03.19.644217
2. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. bioRxiv 2025.04.15.648836v1 https://www.biorxiv.org/content/10.1101/2025.04.15.648836v1
3.  American Society of Hematology (ASH) Annual Meeting 2024.
Abstract link: Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity  Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager.

Comprehensive Assay Solutions for In Vitro and Cell Based Potency Assays and Pharmacokinetics Studies- Our Expertise

With 30 years of expertise in cell culture, cell-based assays, and preclinical/clinical PK/PD analysis, we specialize in offering assay services essential for a wide variety of therapeutic drug development programs, preclinical studies, IND/BLA applications, and commercialization. Our comprehensive services include both preclinical non-GLP and GLP assays, as well as non-GMP and GMP assays, providing critical support throughout the entire development pipeline.

Watch the following video and explore our latest presentation on the development and validation of potency and pharmacokinetic (PK) assays for AAV vectors, highlighting innovative methodologies and industry-leading expertise.

Download the full presentation: Development of Custom Cell Based and In vitro Potency and Pharmacokinetics (PK) Assays for AAV vectors- Marin biologic Laboratories

Development of Cell-Based Potency Assays: Case Studies and Blogs from Marin Biologic Laboratories (MarinBio)

Drug Discovery & Development Assays Offered by Marin Biologic Laboratories (MarinBio)

• ADME/Tox
• Molecular Biology
• The Art of Cell Culture
• Exosomes
• Stability Services
• Potency Assay
• Gene Therapy Assays
• Immunotherapy Assays
• Antiviral Therapy Assays
• cGMP
• MLR
• Cell Based Assays
• ELISA
• Flow Cytometry
• Protein
• PCR-qPCR
• Immunoassay
• Radioimmunoassay
• GLP
• Transfection
• Cell Therapy Assays
• Targeted Protein Degradation
• Research to Commercialization