Organoids in Precision Medicine

Overview of Various Experimental Models & Organoid Technology

  • Traditional experimental tools for studying human diseases include 2D cell lines and animal models.
  • 2D cell lines, like those used in prostate cancer research, have limitations:
    • Too few available.
    • Collected over 40 years ago.
    • Minority derived from the prostate.
    • Do not share common alterations found in patients.
    • Questionable clinical relevance (Ahmed et al., Biorxiv 2024).
  • 3D cellular models increase complexity compared to 2D monolayers and more closely mimic in vivo conditions.
  • Key study showed cells in 3D form functional epithelial acini with secretory function (casein production) which is lost in 2D monolayers.
  • Examples of 3D models include:
    • Cells in hydrogel.
    • Spheroids.
    • Bioprinting.
    • Organoids.
  • The organization of cells in space is linked to their functionality through:
    • Cell-cell interactions.
    • Cell-extracellular matrix interactions.
    • Structural support and topology of specialized cells.
    • Growth factors and secreted factors.
  • The extracellular matrix (ECM) or "niche" is important for specialized cell function.
  • Example is the mammalian gut crypt, a tube of cells on a basement membrane where stem cells are located and regulated by mesenchymal cells from the ECM (Meran et al., SCI 2017).
  • Organoids can model multicellularity and topology (mini-guts).
  • Advantages of different experimental models:
    • 2D cell culture:
      • Advantages: Human origin, genetically stable, unlimited cell growth, easy to handle.
      • Disadvantages: Absence of microenvironment, lack of heterogeneity, loss of cell polarity.
    • GEMMs (Genetically Engineered Mouse Models):
      • Advantages: Defined genotype, recapitulate physiological complexity, presence of stroma and immune system, heterogenous cell populations
      • Disadvantages: Murine genetic background, Laborious and time-consuming, high cost, Absence of immune system, Tumour-host species differences
    • PDXs (Patient-Derived Xenografts):
      • Advantages: Human origin, Geneticly stable, Presence of stroma, Mantain original tumour heterogeneity
      • Disadvantages: Laborious and time-consuming, Slow expansion and high cost, *Patient-Derived Xenografts
    • Patient Derived Organoids:
      • Advantages: Human primary cells, Geneticly stable, Partial physiological complexity, Mantain original tumour heterogeneity
      • Disadvantages: Absence of microenvironment, Difficult to standarize, Moderately costly
  • Organoid terminology:
    • Organoids: Multicellular, self-assembling 3D structures mimicking specific organ/tissue functions, expandable in many cases.
    • Spheroids: Usually cell lines in aggregation, one or two cell types, in suspension/matrix, for specific experiments, not expandable.
    • 2D cell lines: One cell type, adherent conditions, immortalized, prolonged passaging leads to selection/adaptation.
  • Overview of organoid technology:
    • Cell source: Tissue-derived organoids (TDCs) or iPSC-derived organoids.
    • Soluble factors: Growth factors and small molecules (Wnt, EGF, HGF, IGF, FGF, BMP, TGF, ROCK, MAPK for TDCs; Activin-A, BMP4, Wnt, FGF, VEGF, BMP, TGF for iPSCs).
    • Matrix: Matrigel, collagen, or synthetic polymeric hydrogel. Gels composed of ECM components. Synthetic gels can decouple stiffness and increase variability.
    • Physical cues: Provide ECM support and signalling cues; mimic nutrient and waste diffusion of the basement membrane; dynamically tunable microenvironment as stem cell niche.
    • Integrating cues: Integrating key physiological and structural features of the organ. Bioprinting enables direct deposition of organoids to produce tissues.
  • Different organoid types include intestinal, hepatic, endometrial, renal, and cortical brain organoids.
  • The extracellular matrix (ECM) is a non-cellular component in connective tissues, with tissue-specific composition.
    • Composed of fibrous proteins (collagens) and glycosaminoglycan (GAG)-based components.
    • Provides shape/stability, regulates cell adhesion, and supports cell migration.
    • GAG-based components ensure hydration/lubrication and act as a reservoir/modulator of cytokine signaling (Theocharis et al., 2016; Yong et al., 2020).
  • ECM influences gene expression (Bissell, Hall, & Parry, 1982; Roskelley & Bissell, 1995).
  • Fibroblasts in 2D lose their contractility properties.
  • ECM deposition by cells leads to structural arrangement of ECM polymers, resulting in contractility (functionality) as in vivo.
  • Rapid evolution of organoid technology involves biology, biophysics, bioengineering, and computational sciences (Garetta et al., Nature Materials 2020).

Practical Aspects (Derivation, Sources)

  • Organoids require specific spatiotemporal cues, growth factors, nutrients, support matrices (e.g., Matrigel, collagen), or low adherence conditions.
  • Methods of Derivation:
    • ECM-based support matrices: Single cells embedded in a polymerizing matrix (temperature and/or pH) with growth factors in the cell culture media. Alternatively, cells are placed on top of preformed gel first.
    • Examples: Dome Culturing (cells suspended within ECM to generate a self-contained dome for self-organization) and Bioreactor Culturing (organoids encased in Matrigel droplets in a spinner flask or bioreactor for improved nutrient absorption).
    • Matrigel, derived from mouse sarcoma cells, provides physical scaffolding and a rich microenvironment.
    • ECM composition is tissue-specific, differing in proteins, proteoglycans, isoforms, and posttranslational modifications.
    • Permeable support (e.g., nitrocellulose membrane) with vessels such as Transwell® or Falcon® permeable supports for optimal structure and conditions for cell differentiation.
    • Low adherence conditions: Ultra-Low Attachment (ULA) surfaces in microplates prevent cell binding, creating 3D structures that can be embedded in an ECM.
      • Cell-cell contact is the determinant of organoid formation.
  • Derivation of organoids from tissues:
    • Tissue collection followed by digestion with buffer (Accutase) and incubation at 37C.
    • Passaging, cryopreservation, media change.
    • Organoid seeding for screening, drug addition, and readout.
  • Cellular sources of Organoids:
    • Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).
    • Somatic cells can be reprogrammed into iPSCs with transcription factors.
    • ESCs differentiate into three germ layers: endoderm, mesoderm, and ectoderm.
    • Adult stem cells (AdSCs) from organs can form organoids in vitro when treated with appropriate culture conditions.
  • Organoid biobanks offer the opportunity to generate expandable, living biobanks for translational research (Verstegen et al., Nature Medicine 2025).
  • Genetic and environmental factors can be studied in organoids at the individual and societal level.

Applications in Precision Medicine

  • Organoids can be initiated from many tissue types using similar work-up and culture conditions (Sato et al., 2009). *Organoids in Precision Medicine:
    • Suitable for disease modeling, drug response, dosage optimization, and regenerative medicine.
    • Allow recapitulating a patient’s own cells and 3D microenvironment in a dish to better mimic the in vivo environment for personalized treatments.
  • Applications in Homeostasis & Disease
    • Ideal models mimicking human genetic diseases caused by induced mutations.
    • CRISPR/Cas9 enables investigation of diseases associated with genetic defects.
    • Potential tool for high-throughput drug discovery, toxicity testing, and preclinical studies.
    • Potential for organoid transplantation therapy in the future.
    • Homeostatic processes in intestinal regeneration (Yang and Xue et al., Nature Cell Biology (2021).
    • Cell fate and mechano-osmotic forces in intestinal crypt formation (Nikolaev et al., Nature (2020), Park et al., Nature Methods (2022)).
    • Influence of microbiota in normal processes Cost-parasite interactions.
  • Conventional epithelial organoids can be propagated nearly indefinitely, but long-term culture requires continuous passaging.
  • Novel technologies such as OCTOPUS or Scaffold-based organoid tubular systems maintain tubular intestinal epithelia (mini guts) for several weeks without passaging.
  • Organoids in Disease Modeling Applications
    • Celiac disease modeling using intestinal organoids (Santos et al., 2024).
    • Drug Discovery: Resemble in vivo models more closely in a high throughput manner.
      • Screen millions of compounds against humanlike disease models.
      • Epithelial organoids from patient-derived tissues provide an in vitro screening tool for promising compounds.
      • Forskolin-induced swelling of intestinal organoids correlates with disease severity in cystic fibrosis and homozygous F508delF508del mutations.
  • Gene Editing:
    • Combination of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing solutions with organoids improves genetic and drug screening models (Geurts et al., Nature Communications, 2023).
      • Correct the mutated allele in monogenetic diseases like cystic fibrosis.
      • Dissect molecular alterations that lead to oncogenic transformation in cancer studies.
    • Understanding cellular functionality and drug toxicity:
      • Bioprinting creates 3D constructs using cells, spheroids, or organoids to study cellular relationships.
  • Renal organoids to model nephrotoxicity or screen for new compounds (Dilz et al., 2023 SciReports).
  • Organoids on Chip Models: Multiorganoid body-on-a-chip system for drug-screening.
  • Applications in oncology:
    • Tumor organoids can be derived from patient cancerous tissues/biopsies or from normal tissues which have undergone specific mutagenesis (Tuveson and Clevers, 2019, Sachs et al., 2018).
    • Disease subtyping & drug response (precision oncology).
    • Biobanking.
    • High conservation of molecular (genetic/transcriptomic) profile.
  • PDO-based co-clinical trials mimic primary and acquired resistance to regorafenib in mice and recapitulate intra- and interpatient heterogeneity in response to treatment in vivo.
  • There are ongoing clinical trials using organoids for personalized medicine.
  • Near-patient derived models consist of patient-derived xenografts (PDX bank) and Patient-derived Organoids.
  • Organoids Pipeline for modelling patient derived PCa Drug repurposing pipeline directly on PDOs.

Limitations

  • Limited standardization of protocols; different protocols are needed for the same tissue or different disease stages (Xu et al., J Hematology & Oncology, 2018).
  • Depending on the tissue type, organoids cannot always recapitulate the physiologically found tissue architecture.
  • Lack of neural, vascular, or immune cues.
  • Lack on interstitial pressure as in vivo
    • To address this, incorporation of endothelial cells with vascular networks generated around and within otherwise avascular tissue constructs (i.e., organoids) serves as a promising means of overcoming limitations (growth rate, necrotic parts).