Lecture 17: Organoids and Organ-on-a-chip PDF

🥽 Lecture 17: Organoids and Organ-on-a-chip Describe the derivation of adult and iPSC organoids Summary of Systems Used in Human Biology Research 1. Animal Models Advantages:...

🥽 Lecture 17: Organoids and Organ-on-a-chip Describe the derivation of adult and iPSC organoids Summary of Systems Used in Human Biology Research 1. Animal Models Advantages: Useful for studying physiological systems in vivo. Provide insights into complex biological processes. Disadvantages: Not always reflective of human-specific characteristics (e.g., brain differences between humans and mice). Ethical concerns and species differences limit their applicability to human disease. 2. Cell Lines (2D Systems) Advantages: Derived from human tissues, easy to culture and manipulate. Lecture 17: Organoids and Organ-on-a-chip 1  Cost-effective and scalable. Disadvantages: Do not mimic the complex 3D structure and interactions of human tissues. Limited ability to replicate the true tissue environment. 3. Patient-Derived Xenograft (PDX) Models Advantages: More representative of human diseases compared to traditional animal models. Can model human-specific cancer and other diseases in vivo. Disadvantages: Still involves animal use and may not fully capture human disease complexities. 4. Spheroid Models (2D to 3D transition) Advantages: Better approximation of tissue architecture than 2D cell lines. Useful for drug screening and disease modeling. Disadvantages: Still a simplified model compared to fully developed organs or tissues. 5. Organoids/Organ-on-a-Chip Models Advantages: Accurately mimic human tissue structure and function. Can model multiple tissues and organ interactions. Suitable for high-throughput drug testing and disease modeling. Ethical advantage as they do not require animal testing for all stages. Disadvantages: Complex to develop and maintain. Lecture 17: Organoids and Organ-on-a-chip 2  Can be expensive and time-consuming. What are Organoids? Definition: miniature, 3D versions of organs, grow in three dimensions and contain multiple cell types. Key features of organoids: 3D cell culture: Organoids grow in 3D, providing a more accurate representation of the tissue they mimic. Heterogeneous population of cells: Contain more than one type of cell. Especially in adult organoids, they may mainly have epithelial cells, though they can also contain different tissues within the Lecture 17: Organoids and Organ-on-a-chip 3  cell layer. Maintains cellular hierarchy: Stem Cells: Serve as the basis for organoid cultures, "seeding" the growth of the organoid. Differentiation: Organoids contain both stem cells and differentiated cells, maintaining hierarchies like those seen in actual organs. Mimics many features of the original tissue: Organoids often replicate the gene expression and tissue organization of the organ from which they are derived. What can organoids be used for? 1. Disease Modeling Patient-Derived Organoids: Can be derived from a patient with a specific disease. Allow researchers to study the disease in the context of the patient’s unique genetic makeup. Introduction of Disease-Causing Mutations: Gene mutations can be introduced into organoid models. Useful for studying the effects of specific mutations on cells and tissues. 2. Studying Specific Cells and Diseases Human Brain and Kidney Models: Organoids can model diseases affecting specific cells in the brain or kidney, where direct study in humans may not be possible. Provides a way to observe how gene mutations affect individual cells within these organs. 3. Personalized Medicine Patient-Specific Models: Lecture 17: Organoids and Organ-on-a-chip 4  Organoids can be tailored to individual patients, allowing for the study of disease characteristics specific to them rather than general disease patterns. 4. Organ Production / Organ engineering Whole Organs in a Dish: Organoids can produce a miniature version of an entire organ, containing many different cell types. This involves engineering and growing a functioning, complex structure that mimics the organ it is derived from. 5. Fundamental Biological Research Human Development: Organoids are valuable for studying the development of organs and human biology at the cellular and tissue levels. Disease Development: Useful for researching how diseases develop in organs and tissues. How are organoids generated? 1. Adult Tissue-Derived Organoids Source: Isolated from epithelial tissues of various organs. Key Feature: These organoids are primarily epithelial models. Process: A stem cell population within the organ is isolated and used to seed the organoid culture. Result: An adult model of the tissue, representing fully formed adult tissues. 2. iPS-Derived Organoids (Induced Pluripotent Stem Cell-Derived) Source: Begin with undifferentiated stem cells (iPS cells). Process: These cells undergo a differentiation process. Lecture 17: Organoids and Organ-on-a-chip 5  Developmental signals are mimicked to guide the cells toward becoming the desired tissue type. Key Feature: Tend to produce organoids that are more fetal-like, representing earlier developmental stages of tissues. Key Differences Between the Two Types iPS-Derived Organoids: Represent fetal or pre-birth stages of tissue development. Adult Tissue-Derived Organoids: Represent fully developed adult tissues. List the key steps in generation of organoids and key features Adult-Derived Organoids (Example: Liver Organoids) 1. Organoid Type Type: Adult liver organoids (one of the first adult organoids developed), commonly grown in the lab. 2. Process of Generating Liver Organoids Cell Source: Cells are isolated from biopsies, either from animals or humans. Cell Isolation: A series of digestion steps are performed to isolate the cells, including stem cells. Seeding in Matrigel: The isolated cells are seeded into a small dome of Matrigel (a gel-like substance). Culture Medium: Medium containing essential growth factors is added. These growth factors provide the niche signals needed to support stem cells and promote organoid generation. Describe how cells are organised in intestinal organoids Lecture 17: Organoids and Organ-on-a-chip 6  1. Normal Small Intestine Structure Stem Cells: Located at the base in the crypts. Dividing Cells: Transit-amplifying cells that are actively dividing. Differentiated Cells: Non-dividing cells located in the villi. 2. Intestinal Organoid Structure Bud-like Structures: The organoid is covered by structures resembling buds. Cellular Composition: At the end of these buds, stem cells are distributed in a similar way to the stem cells in the crypts of the small intestine (in both animals and humans). Stem cells progress into differentiated cell types. 3. Mimicking Intestinal Tissue Differentiation Process: Although organoids do not form large villi-like structures (due to the lack of the body’s supporting camel structure), cells still migrate upwards. Similar to the small intestine, these cells undergo apoptosis and slough off into the center of the organoid. Functional Mimicry: The organoid mimics the tissue structure and cell turnover processes found in the small intestine. Lecture 17: Organoids and Organ-on-a-chip 7  Describe how organoids can be used to study cancer biology and test drug therapies Personalised precision medicine Introduction to Personalized Medicine Individual Differences: Humans are individuals, as evidenced by traits like hair and eye color. Tumors also have individual characteristics, making them unique to each patient. Current Treatment Approach: Most cancer therapies follow a "one-size-fits-all" approach, with common treatments applied to large groups of patients. Limitations of Current Treatments Example: Bowel Cancer: Common treatment often involves combination therapy, such as 5-FU (fluorouracil). However, only around 30% of patients show any response to this treatment. Trial and Error Approach: Current treatment often involves giving the same drug to a group of patients, knowing that only a fraction will respond. There is no effective method to predict which patients will respond to specific treatments. Precision Approach to Medicine Goal: Shift towards precision medicine, where treatments are tailored to individual patients. Select patients based on their likely response to specific treatments or drug combinations. Lecture 17: Organoids and Organ-on-a-chip 8  Benefits: More effective treatment by using drugs that are specifically tailored to the patient’s unique tumor characteristics. Ability to test different drug combinations on a patient’s individual condition, improving the chance of finding the most effective therapy. Common cancers in Australia 1 in 12 Australians develop bowel cancer 1 in 8 Australian women develop breast cancer Impact of Tumor Stage on Patient Outcome Lecture 17: Organoids and Organ-on-a-chip 9  Early vs. Late-Stage Diagnosis Early Diagnosis (Stage 1): Tumor can often be excised through surgery. Most patients survive if the tumor is caught early. Late Diagnosis (Stage 4): Survival rate drops significantly, with less than 10% chance of surviving for five years. Need for Effective Treatments Not only is it crucial to administer the right treatment, but there is also a significant need to find more effective treatments, especially for advanced-stage cancers like bowel cancer. Using Organoids for Personalised Cancer Treatment 1. Concept of Organoid Screening Objective: Determine the most effective drug for individual cancer patients. Process: A biopsy is taken from the patient. Lecture 17: Organoids and Organ-on-a-chip 10  Organoids are grown from the patient’s tissue sample. Organoids are divided into multiple wells and exposed to a panel of drugs. Example: Drug F is identified as the most effective for a specific patient. 2. Current Approach Proof of Principle: Tissue from patients is used to grow organoids. Organoids are exposed to standard chemotherapy or radiation treatments received by patients in the clinic. The organoid responses are measured and compared to the actual patient responses. 3. Goal Validate the correlation between organoid drug response and patient treatment outcomes. Demonstrate the effectiveness of organoid screening technology as a tool for personalised medicine. Establishing Tumor and Normal Organoid Cultures 1. Dual Culture Establishment Tumor Organoid Culture: Derived from the patient’s tumor tissue. Normal Organoid Culture: Lecture 17: Organoids and Organ-on-a-chip 11  Derived from an adjacent piece of non-tumorous tissue. 2. Purpose of Normal Organoid Culture Enables comparison between tumor and normal tissues. Important for identifying drugs that: Specifically target tumor tissue. Spare normal, healthy tissue. 3. Application in Drug Screening Dual cultures are used in drug screening assays to test the specificity and safety of potential treatments. Tumor Organoids and Their Reflection of In Vivo Tumors What Are Tumor Organoids? Epithelial Models: Organoid cultures grow the epithelial components of tissues. Growth Factors: Added to mimic signals from surrounding cells in vivo. Limitations: Lecture 17: Organoids and Organ-on-a-chip 12  Do not naturally include immune or stromal cells, but these can be added for specific studies. Do Organoids Retain Tumor Features? Histological Similarity: Tumor organoids closely match the pathological features of the primary tumor (e.g., differentiation state). Key tissue-specific markers are expressed in organoids, reflecting their tumor of origin. Morphological Diversity: Organoids display varying shapes (e.g., spherical or budded), correlating with tumor characteristics like signaling levels. Molecular Similarity Mutation Retention: Most organoids retain the same mutations as their parent tumors. Some differences arise due to: Tumor heterogeneity in the patient. Lecture 17: Organoids and Organ-on-a-chip 13  Continuous mutation in the tumor and during organoid culturing. Ongoing Research: Studies show organoids are genetically similar to their source tumors but allow investigation of evolving mutations. Tumor and Organoid Heterogeneity Within Organoids: Variability exists even within organoid cultures from the same tumor. Across Patients: Organoids differ significantly between patients, mirroring the unique nature of each individual tumor. Drug Screening Using Organoids Overview of Drug Screening Process Automated Systems: Robots set up organoids in multi-well plates. Drug Testing: Apply multiple drugs and combinations. Screen hundreds to thousands of drugs for their effects on organoids. Lecture 17: Organoids and Organ-on-a-chip 14  Applications: Identify drug vulnerabilities based on molecular characteristics of organoids. Discover useful drug combinations for clinical trials. Serve as a platform for new drug discovery. Drug Testing Methods Cell Viability Assays: Test the ability of drugs to kill organoids across a range of concentrations. Example Observations: Resistant Cultures: Some organoids show significant resistance to drugs, with surviving cells. Sensitive Cultures: Others are entirely eliminated by the drug treatment. Patient-Specific Variability: Organoids from different patients show varied responses to the same drug. Primary Tumors vs. Metastases Lecture 17: Organoids and Organ-on-a-chip 15  Background: Primary tumors may be surgically removed, but metastases often appear in other organs (e.g., liver in colorectal cancer). Drug Sensitivity Comparison: Example Study: A primary tumor and two metastatic samples were tested with a drug combination. Results indicated similar drug sensitivity between the primary tumor and metastatic samples. Research Aim: Determine if drug vulnerabilities and mutation spectra of metastases differ from primary tumors. Significance Enables testing and prediction of individualized drug responses. Provides insights into the treatment of primary and metastatic tumors, aiding precision medicine. Lecture 17: Organoids and Organ-on-a-chip 16  Can we use organoids as a direct preclinical test to determine how individual patients will respond to different treatments? 1. Concept of Organoids in Preclinical Testing Tumor Sampling: A piece of the patient’s tumor is taken during surgery. Drug Testing in the Lab: Organoids derived from the tumor are treated with a panel of drugs in vitro. Responses are evaluated using assays to determine drug effectiveness. Goal: Develop an "organoid score" based on drug responses. Provide this score to oncologists to guide personalized treatment. 2. Timeline While the Patient Recovers: Organoids are tested during the patient’s postoperative recovery period. Treatment Guidance: By the time the patient is ready for further treatment, results from the organoid screen would be available to assist in therapy selection. 3. Current Status Not Yet Routine: This method is still under research and development. Evidence of Potential: Compelling early results suggest organoids could become a useful tool for guiding treatments, particularly in specific cancers. 4. Future Vision To integrate organoid-based drug testing as a routine part of precision cancer medicine, improving treatment outcomes and minimizing trial-and-error approaches. Lecture 17: Organoids and Organ-on-a-chip 17  Outline the advantages of organoids to study infection Using Organoids to Study Gut Regeneration - NGR1 Regeneration Challenges: The gut lining has an innate ability to regenerate after damage from infection or trauma. However, in conditions like ulcerative colitis or Crohn's disease, regeneration may be incomplete, leading to epithelial denudation. Study Overview: Researchers aimed to identify factors promoting regeneration and repair using organoid models. Key Findings: Neuregulin 1 (NRG1) was identified as a promising candidate growth factor. Experimental conditions: 1. Control: Normal growth over 3–5 days shows baseline organoid development. 2. NRG1 Addition: Enhanced organoid growth, increased budding, and higher proliferation rates. 3. Combined EGF + NRG1: Synergistic effects observed. BRDU Staining: Demonstrated an increase in dividing cells with NRG1 treatment. Lecture 17: Organoids and Organ-on-a-chip 18  Conclusion: NRG1 significantly promotes gut lining repair and regeneration. Organoids were critical for identifying NRG1's activity and mechanism through biochemical inhibitor assays. Recapitulating human brain development using organoids Introduction to Brain Organoids Brain organoids represent a significant advancement in studying human brain development and related diseases. Challenges in studying the human brain include: Complex cellular and structural organization not replicated in animal models. Limited access to specific brain cell types affected by various conditions. Development of Brain Organoids Origins: Derived from induced pluripotent stem cells (iPSCs). Self-Patterning and Directed Development: Brain organoids can undergo self-patterning. They can also be directed to develop into specific regions of the human brain. Key Characteristics Size and Growth Time: Lecture 17: Organoids and Organ-on-a-chip 19  Brain organoids grow quite large compared to other organoids. They often require months of cultivation for desired cell differentiation. Nutrient Diffusion and Necrosis: As organoids grow, nutrient diffusion becomes a challenge, leading to necrotic zones in larger organoids. Applications and Research Highlights Disease Modeling: Used to model complex diseases such as Alzheimer’s, autism, and specific genetic syndromes. Future Potential Continued refinement is needed to create organoids that fully replicate the human brain. Increasing focus on: Enhancing structural and functional fidelity. Addressing issues like nutrient diffusion and long-term viability. Growing interest in using brain organoids for mechanistic studies of neural development and disease. Lecture 17: Organoids and Organ-on-a-chip 20  Normal human colonic organoid growth Applications of Normal Intestinal Organoids While part one focused on tumour-derived organoids, normal intestinal organoids are equally valuable for studying: Inflammatory diseases. Pathogenic and protective bacteria in the bowel. Traditional models, such as 2D cell lines and animal models, often fail to capture the complexity of the intestinal epithelium. Establishing Normal Intestinal Organoid Cultures Crypt Preparation: Tissue is incubated with EDTA, which disrupts junctional complexes and allows the epithelium to detach from the underlying cell layers. Crypt Structures: Funnel-shaped crypts are harvested, containing both stem cells and differentiated cells. Seeding in Matrigel: Crypts are seeded in Matrigel with added growth factors. Differentiated cells die off, leaving stem cells to proliferate. Development and Structure of Intestinal Organoids Early Growth (Day 3): Stem cells establish colonies visible as small clusters of cells. Structural Maturation: Organoids progress from simple cysts to budding structures, mimicking the architecture of intestinal crypts and villi. Cellular Composition: Buds feature crypt-like regions with paneth cells and other epithelial cell types organized in the same pattern as in vivo. Organoid Growth Lecture 17: Organoids and Organ-on-a-chip 21  Organoids grow with epithelial cells forming a circular structure surrounding an empty lumen. Over time, the organoids develop budding structures, replicating the crypt-villus organization. Future Directions Intestinal organoids derived from normal epithelium offer exciting opportunities to study: Host-microbe interactions, including both pathogenic and beneficial bacteria. The effects of environmental stimuli on gut health and disease. Using organoids to decipher how Clostridium difficile causes infectious diarrhoea Focus on Gut Infections A primary focus when investigating bacterial pathogens that cause gut infection is on Clostridium difficile, a serious pathogen responsible for infectious diarrhea. Toxin Effects on Gut Cells Lecture 17: Organoids and Organ-on-a-chip 22  Clostridium difficile releases toxins that damage gut cells, particularly: Toxin A: Causes some cell death. Toxin B: More potent, causing significant cell death quickly. Organoid Assay: Control organoid cultures were stained with PI dye, which marks dead cells red. Adding Toxin A resulted in a few red (dead) cells. Adding Toxin B caused widespread cell death, visualized as many red-stained cells. Applications of Organoids in Toxin Research Receptor Identification: Organoids help in identifying the receptors that toxins bind to. Drug Screening: Potential molecules that interfere with toxin binding can be tested in organoids. Treatment Development: Molecules identified could mitigate the downstream effects of toxin-induced damage, complementing treatments that eliminate the infection itself. Viral Infections and Organoids Intestinal organoids are increasingly used to study viral infections, with research expanding into a range of viruses that affect the gut. Lecture 17: Organoids and Organ-on-a-chip 23  Using Organoids to Study Coronavirus (COVID-19) Infection and Impact Organoids for Studying Coronavirus Infection Intestinal Organoids: On the left: A control intestinal organoid with no infection. On the right: An infected intestinal organoid, showing: White staining: Indicates virally infected cells. Green staining: Marks the luminal apical surface. Blue staining: Highlights the nuclei. Lecture 17: Organoids and Organ-on-a-chip 24  These organoids allow researchers to observe how the virus targets specific cells in the gut. Other Organoid Applications for COVID-19 Research Liver-Derived Organoids: Also used to study infection pathways and cellular responses. Cardiac Organoids: Reflect how COVID-19 impacts organs beyond the lungs, such as the heart and kidneys. Brain Organoids: Demonstrated the virus's ability to infect human brain cells directly, providing insights into neurological symptoms associated with COVID-19. Research and Drug Screening Global Efforts: Labs worldwide, including those in Australia, are leveraging organoids to: Study COVID-19's effects on different tissues. Conduct high-throughput drug screens for: Lecture 17: Organoids and Organ-on-a-chip 25  Inhibiting viral infection. Mitigating damage to affected tissues. Implications of Organoid Research Organoids are essential tools for understanding the direct impact of the coronavirus on various tissues and organs. This work is contributing to the development of targeted therapies and a deeper understanding of the virus’s systemic effects. Describe organ on a chip strategies for disease modelling and drug discovery Limitations of Current Organoid Models Predominantly Epithelial: Most adult organoid models consist only of epithelial cells, limiting their ability to mimic the complexity of an organ. Missing Surrounding Components: Lack of interaction with: Immune cells. Luminal contents and shear stress (as seen in the gut). Dynamic bacterial interactions (currently studied by injecting bacteria into organoids). Structural Limitations: Current models do not fully replicate the intricate architecture of organs. Organs on a Chip: Advancements Structural Engineering: Designed to better mimic the in vivo structure of organs. Example: Gut organoid chips with engineered crypt-like structures. Microfluidics Integration: Lecture 17: Organoids and Organ-on-a-chip 26  Allows for dynamic interaction with: Drugs. Bacteria. Other cells. Coupled with imaging systems for real-time observation. Enhanced Functionality: Supports differentiated cell types and stem cells. Replicates turnover processes (e.g., division, sloughing off into the lumen). Applications 1. Pharmaceutical Industry: Drug Screening: High-throughput screening for drug effects on organoids. Cardiac Organoids: Toxicity screening for cardiovascular impacts of drugs. 2. Gut Organoids on a Chip: Research Capabilities: Mimics crypt structures of the gut. Allows fluid flow and interaction with luminal contents. Experimental Setup: Drugs, bacteria, or other stimuli can be introduced into the lumen to study interactions with the epithelium. Comparison: Organoid vs. Organ on a Chip Suitability: The choice depends on the specific assay and research objective. Organoids are ideal for certain types of high-throughput screening. Lecture 17: Organoids and Organ-on-a-chip 27  Organs on a chip offer a more dynamic and physiologically accurate system for certain applications. Organoids are best for static, scalable screens where the goal is to study the response of 3D tissue structures to different treatments, especially in personalized medicine and cancer research. Organs on a chip excel in dynamic screening that requires real-time interaction, monitoring of multi-organ responses, or studying how substances are processed across different tissues. They are particularly useful for understanding complex physiological systems like drug metabolism, organ toxicity, and the effects of infection. Lecture 17: Organoids and Organ-on-a-chip 28