p3 Flashcards
organoid def and source
self-organized 3D tissue that is typically derived from stem cells (pluripotent, fetal, or adult) and mimics the key functional, structural, and biological complexity of an organ
Cells comprising organoids can be derived from induced pluripotent stem cells (iPSCs) or tissue-derived cells (TDCs), including normal stem/progenitor cells, differentiated cells, and cancer cells
Organoid cultures exhibit significant heterogeneity and variable complexity in cellular composition
lack stromal, vascular and immunological components
Organogenesis
The series of organized integrated processes that transform an amorphous mass of cells into a complete organ in the developing embryo.
How do starting cells influence organoids
Starting cell population
* Can affect the variability and heterogeneity
* Can affect the function (quality of isolated starting cells)
How is tissue dissociation done
enzymatic (to dissolve the ECM) and mechanical
iPSC maintenance
- iPSCs can be maintained and expanded as undifferentiated clonal populations over many generations.
- Undifferentiated human iPSCs are maintained on feeder cells or defined ECM substrates.
TDCs sources
- TDCs from solid organs are derived from either biopsy samples or surgical resections
- TDCs cells can also be isolated from liquid samples such as peripheral blood, ascites, and pleural effusions
What is matrigel made of
Matrigel is mainly composed of laminin, collagen IV, entactin, perlecan and growth factors
chemically heterogeneous and has a poorly defined
composition
Matrix for organoids
- Natural matrices can be recombinantly produced from proteins or polysaccharides
- Synthetic hydrogels allow independent manipulation of biochemical and biophysical matrix properties
- The ideal organoid matrix is stress-relaxing and dynamic
- Dynamic hydrogels based on polyethylene glycol (PEG) demonstrate how tunable hydrogel properties control stemness and differentiation in cultured organoids
What are soluble factors
Largely, proteins such as growth factors, or small-molecule drugs, which can activate or inhibit signalling pathways
Physical cues for organoids
- Nutrient supply and waste removal, which are diffusion-dependent, become less efficient during organoid growth into larger tissue structures
- Can be partially resolved using shaking cultures, spinning bioreactors or suspension under continuous agitation, or in continuously stirred bioreactors
- These bioreactors can monitor pH, temperature, oxygen and glucose levels to maximize mass transfer while minimizing shear stress
Microheterogeneity in Stem Cells
Definition: Variation in characteristics such as protein and RNA expression among neighboring cells within homogeneous stem cell colonies. Indicates diversity in gene expression and cell identity even in seemingly uniform cell populations.
Characterization of Stem Cells via RNA Analysis
Key Methods: RT-PCR, microarray, RNA-sequencing (RNA-seq).
Purpose: To quantify gene expression levels and identify the cell’s state or identity by analyzing specific RNA molecules.
Epigenetic Modifications in Stem Cells
Definition: Stable alterations in gene expression without changes to the DNA sequence, including DNA methylation, histone modification, and chromatin remodeling. Crucial for cell type differentiation and identity.
Embryonic Stem Cells (ES Cells)
Location: Derived from the inner cell mass of the blastocyst.
Properties: Pluripotent, capable of differentiating into all cell types.
Challenges: Ethical concerns, potential for immune rejection, and tumor formation.
Tissue Stem Cells (Adult Stem Cells)
Definition: Multipotent cells found in specific tissues, capable of differentiating into a limited range of cell types relevant to their origin, e.g., hematopoietic stem cells in bone marrow.
