From 2D Cells to 3D Organoids: Advanced Insights into Cancer Modeling and Personalized Medicine
Introduction and Speaker Background
Presenter: Jose, a Research Scientist at the Knight Cancer Center, part of the OHSU community.
Academic Background: Jose completed a PhD in oncology and subsequent post-graduate studies, followed by research in cancer metabolism.
Current Research Focus: His current work at the Knight Cancer Center involves: * Pancreatic cancer research. * Development of cancer vaccines. * Study of a specific crude sugar known as LFicus.
Lecture Theme: The transition "from 2D cells to 3D organoids" and the fundamental principles of modeling human diseases.
Modeling Human Disease: The Evolution of 2D Cell Lines
Defining the Goal: To study diseases ranging from simple forms to complex multifactorial conditions like cancer (the "emperor of maladies"), researchers must create models that capture both genetic and environmental factors in tumorigenesis.
Historical Origins of Cell Lines: * One of the first human cell lines was generated from cervical cancer at Johns Hopkins University in the laboratory of Doctor Yeh. * Methodology: A small piece of tumor was isolated from a patient and placed in a dish (in vitro). * Growth Conditions: Scientists provided growth factors, inhibitors, and maintained a temperature of to mimic the human body.
Significance of Cell Lines: * The discovery of these cells is considered one of the most significant revolutions of the 20th century because it demonstrated that tumors could be grown outside the body. * This led to the isolation and growth of various tissue types beyond tumors.
Logistical Advantages: * 2D cell lines can be frozen and stored in liquid nitrogen at for decades. * They are easily recovered (thawed) and passaged (subcultured). * They are relatively inexpensive for genomic studies and grow very quickly.
Limitations of Two-Dimensional (2D) Models
Surface Area Exposure: In a 2D petri dish, roughly of a cell's surface area is exposed to the culture medium, while the other is attached to the plastic plate. This does not represent the internal environment of a 3D organism.
Loss of Fidelity: Over excessive passages, cell lines undergo genetic and phenotypic drift, losing the properties of the original primary tumor. They may no longer provide data representative of the patient's actual disease.
Misidentification Issues: Historical publications have been retracted because cell lines supposedly representing human tissue were found to be mice or different tissue types altogether after years of study.
Missing Compartments: 2D cell cultures typically consist only of cancer cells. They lack the immune compartment and other supporting cell types found in a complex tumor microenvironment.
Animal Models in Cancer Research
Standard Usage: Mice are a primary animal model, though other facilities (such as those at OHSU) utilize primates, like monkeys, for specific studies such as HIV research.
Advantages: They provide a whole-organism perspective that cell lines cannot.
Disadvantages: * They are not fully representative of human biology. * They are expensive to maintain. * Generating specific genetic modifications, such as a localized knockdown of a gene, can take a very long time.
The Rise of Organoids: Advanced 3D Models
Definition: Organoids are advanced 3D cell culture systems that comprise multiple cell types found within a specific tissue. They are essentially "organs in a dish."
History and Proliferation: * Formally established around 2008. * The first small intestine organoid was introduced in 2009. * As of 2025, there have been more than published studies on organoids.
Personalized Medicine Applications: * Clinicians currently use generic treatments (chemotherapy or surgery) that may result in tumor recurrence because of specific genomic or transcriptomic profiles in individual patients. * Organoids allow for "screening on a trial": scientists isolate a patient's tumor, grow organoids, and test different drugs to see which is most effective before starting actual patient treatment. * Biobanking: Large-scale storage of organoids from different patients allows for extensive comparative research.
Ethical Impact: One of the major goals of organoid technology is to reduce the use of animal models in biomedical science.
Stem Cells and the Biological Foundation of Organoids
The Stem Cell Niche: Recreating an organoid requires a deep understanding of the stem cell niche. Previously, niches for the brain or skin were poorly understood, but current knowledge allows for the recreation of most tissues.
Adult Stem Cells: These reside in mature tissues (e.g., skin cells or intestinal crypts). They are responsible for wound healing but are multipotent rather than totipotent; they can only differentiate into the specific types of cells within their tissue of origin.
Pluripotent Stem Cells: These include embryonic stem cells that can differentiate into any cell type derived from the three germ layers: endoderm, mesoderm, and ectoderm.
Induction of Differentiation: Researchers use different growth factors and inhibitors to guide pluripotent cells through developmental stages. This is often used for regeneration and developmental studies, though it carries a risk of unwanted background mutations.
The Anatomy and Modeling of the Small Intestine
Structure: The small intestine contains finger-like structures called villi (for nutrient and mineral absorption) and the crypt of the epithelium.
Cellular Organization of the Crypt: * Cells: Found at the very bottom of the crypt, these are the primary adult stem cells used to generate intestinal organoids. * Paneth Cells: Located at the bottom of the crypt; they produce antibacterial molecules to protect the stem cells. * Transient Amplifying Cells: These cells move upward from the crypt toward the top of the villi, receiving cues to differentiate. * Goblet Cells: Differentiated cells that produce mucus. * Enteroendocrine cells: Other specialized differentiated cells.
Molecular Markers: Scientists use specific markers to identify cell types via molecular techniques like flow cytometry or histochemistry: * Stem cells are identified by high expression of the gene. * Stem cells can also be identified as in histochemistry.
Technical Procedures: Establishing Organoid Cultures
Isolating the Crypt: The intestine (mouse or human) is chopped, washed, filtered, and centrifuged to isolate pure crypts of the epithelium. This process must be precise to avoid bacterial contamination or the inclusion of non-stem cells (like villi).
Scaffold and Environment: Organoids are grown in a 3D scaffold called Matrigel, which is an extracellular matrix (ECM) rich in Collagen and Laminin. They require specific stiffness, cytokines, hormones, and growth factors.
Culture Methods: 1. Air-Liquid Interface (ALI): Uses a two-chamber system with a porous membrane. The organoid is exposed to air on the top while receiving medium from the bottom. This is ideal for skin or lung tissues and long-term co-culture studies. 2. Submerged Culture: The organoids are placed directly into Matrigel "nodes" in a plate and completely submerged in medium. This is the more common method for many lab studies.
Well Plates: Organoids are typically grown in 24, 48, or 96-well plates.
Comparing Organoid Growth: Normal vs. Tumor Tissue
The Importance of Normal Tissue: Isolating normal tissue from the same patient acts as a crucial control for comparing genomic and transcriptomic profiles against the tumor.
Growth Patterns: * Normal Organoids: Grow in a structured, branching manner that recapitulates the miniature architecture of the intestine within 4 to 7 days. * Tumor Organoids: Exhibit cystic, irregular growth patterns. This is often driven by mutations such as mutations or activation (suggestively Wnt pathway signaling), which cause the cells to proliferate without normal checks.
Patient Heterogeneity: Growth rates and phenotypes vary significantly between patients, often depending on the specific treatments (chemotherapy/radiation) the patient received before the biopsy.
The Tumor Microenvironment (TME) and Co-culture Systems
TME Challenges: Cancer is not just the cancer cell; the "neighborhood" around the cells significantly influences the outcome. Tumor cells can hijack immune cells to promote growth by producing factors like VEGF (vascular endothelial growth factor) for vascularization.
Hot vs. Cold Tumors: * Cold Tumors (e.g., PDAC/Pancreatic Cancer): Highly fibrotic and suppressive environments that act as a wall against drugs. * Hot Tumors (e.g., Melanoma): More permissive and easier to target with immunotherapies.
Immune Co-culture: Modern organoid models involve adding immune cell populations (Natural Killer cells, T cells, or Dendritic cells) to the organoid culture. This allows researchers to study if a vaccine or drug can boost immune activity against the tumor in a controlled, cost-effective way compared to animal models.
Advanced Modeling: Microfluidic Systems and Vascularization
Microfluidics: Instead of static dishes, researchers use chambers with channels to create a miniature circulatory system. This better replicates the constant fluid flow and circulatory exposure found in the human body.
Vascularization Experiment: A study using pluripotent stem cells generated neurons, tumor cells, and endothelial cells (vessels). When endothelial cells were added to the organoid in a microfluidic chamber, the vascularization significantly promoted organoid growth compared to non-vascularized controls.
Analysis: These systems allow for high-resolution imaging using fluorescent microscopes to track the movement of immune cells and their interactions with the tumor.
Applications in Drug Discovery, CRISPR, and Regeneration
Cytotoxicity Testing: Scientists can test small molecules on both normal and tumor organoids simultaneously to ensure a drug is toxic to the tumor but safe (non-cytotoxic) for normal tissue.
Gene Editing (CRISPR-Cas9): Jose used CRISPR-Cas9 to study genes in colon organoids. By knocking down the expression of specific genes, he identified two important F-box protein genes that significantly altered organoid phenotype.
Regenerative Medicine: A Nature 2024 paper described a study where pluripotent stem cells were used to generate photoreceptors in a dish. These were injected into mice with retinal degeneration, resulting in significantly improved vision.
Visualization Techniques: Organoids are characterized using stains like H&E (Hematoxylin and Eosin) to view the nucleus (dark) and cytoplasm (pink), or F-actin staining to view the cytoskeleton.
Questions & Discussion
Audience Question: I was curious, can you use organoids for blood cancers?
Jose's Response: (Transcript ends shortly after the start of the response). Jose begins by noting it is a very nice question.