Please enable JavaScript.
Coggle requires JavaScript to display documents.
ANIMAL CELL AND TISSUE CULTURE - Coggle Diagram
ANIMAL CELL AND TISSUE CULTURE
I. Introduction and Objective
Objective: To introduce the basic understanding of the principles and essential techniques of animal culture.
Definition: The process of keeping tissue alive and growing outside the suitable body in a culture medium containing nutrients.
Significance: A cornerstone of modern biology, medicine, and biotechnology.
II. History and Milestones
Conceptual Beginnings (Late 19th - Early 20th Century):
1880: Arnold showed leucocytes can divide outside the body.
1907: Harrison observed development and outgrowth of nerve fibers in plasma clot culture.
1910: Burrows and Carrel refined the plasma clot culture method.
Breakthroughs (1940s–1960s):
1948: Wilton Earle established the first mouse cell line (L cells), which was aneuploid and immortal.
1952: George Otto Gey established the HeLa cell line, enabling large-scale cell culture and vaccine development (e.g., Polio vaccine).
1955: Harry Eagle developed Eagle's Minimal Essential Medium (MEM), the first successful chemically defined medium.
1959: Leonard Hayflick discovered the Hayflick Limit, observing that normal human fibroblasts divide a finite number of times.
Modern Era (1970s–Present):
1970s: Development of Hybridoma technology (monoclonal antibodies/mAbs).
1990s: Serum-Free Media (SFM) became standard for commercial applications to reduce cost and variability.
2000s–Present: Development of methods to culture Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs).
III. Advantages and Disadvantages of ATC
Advantages:
Control & Standardization: Researchers can precisely control the physico-chemical environment (temperature, pH).
Experimental Utility: Allows Direct Visualization of living cells and Isolation of Effects.
Biotechnology: Essential for industrial production of biologics (mAbs, vaccines).
Ethics & Cost: Reduces the use of live animals for research and toxicity testing.
Disadvantages:
Artificiality: Cells in 2D monolayer lose their native 3D architecture and cell-to-cell signaling.
Relevance: Many specialized cells may de-differentiate (lose specific functions) over time.
Genetic Drift: Continuous passaging can lead to genetic instability and aneuploidy.
Technical: High susceptibility to contamination (bacteria, fungi) and requires specialized, expensive equipment.
IV. Types of Cell Culture
A. Based on Passage and Lifespan:
Primary Culture: Isolated directly from tissue; unpassaged; has a finite lifespan (Hayflick limit); closest to in vivo properties.
Secondary Culture (Cell Line): Established after the first subculture (passage).
Finite Cell Line (Diploid Cell Strains): Limited divisions before senescence; retains normal karyotype; used for vaccine production.
Continuous Cell Line (Immortalized): Undergone transformation; unlimited capacity for subculture; characterized by aneuploid karyotype.
B. Based on Attachment:
Adherent Cells (Anchorage Dependence): Must attach to a substrate (e.g., plastic) to survive and divide. Requires Passaging using an enzyme (Trypsin-EDTA).
Examples: Fibroblasts (spindle-shaped), Epithelial Cells (polygonal sheets), Endothelial Cells.
Technique: Monolayer Culture (2D). Microcarrier Culture (for large-scale adherent production).
Suspension Culture (Anchorage Independence): Cells float freely; maintained by continuous agitation.
Ideal for: Hematopoietic Cells (e.g., hybridoma cells), and biopharmaceutical production.
Disadvantage: Shear Sensitivity complicates scale-up.
C. Tissue-Based and 3D Cultures:
Primary Explant Culture: Small tissue fragments anchored to a substrate; cells migrate outward, avoiding enzymatic damage.
Organ Culture: Cultures intact fragments to preserve 3D structure and functional differentiation. Cell division is minimal.
Techniques: Plasma Clot Method, Floating Raft Method (Trowell), Metal Grid Support.
Limitation: Diffusion Limit causes core necrosis if the explant is too large (< 2mm).
Organoid Culture: Self-organizing 3D structures derived from stem cells (ESCs or iPSCs) that resemble mini-organs.
V. Cell Culture Environment
Physical Parameters:
Temperature:37.0±0.5∘C (Ensures optimal enzyme kinetics).
pH: 7.2 - 7.4 (Critical for membrane integrity and enzyme function).
Osmolarity: 290±10 mOsm/L (prevents osmotic stress).
Gas Phase:
Carbon Dioxide ($CO_2$): Typically 5%, required to maintain the bicarbonate buffer system and pH equilibrium.
Oxygen ($O_2$): Ambient air (18-21%) standard; Hypoxic (2-5%) for specialized cells (e.g., stem cells).
Humidity: 95-100% (Prevents rapid evaporation of the medium).
VI. Cell Potency and Differentiation
Cell Potency Hierarchy:
Totipotent: Forms a complete viable organism (including extra-embryonic tissue; zygote). Cannot be stably cultured.
Pluripotent: Forms ALL cell types of the body (e.g., ESCs, iPSCs). Foundation for organoid culture.
Multipotent: Forms a limited family of cell types within a single lineage (e.g., Adult Stem Cells, Hematopoietic Stem Cells).
Differentiation:
Definition: The acquisition of a specific, restricted phenotype (structure and function).
Stages: Includes Commitment (Specification and Determination) leading to Terminal Differentiation (often post-mitotic).
Goal: Direct pluripotent stem cells into specific lineages for therapies or disease modeling.
VII. Applications of ATC
Model Systems.
Toxicity testing and Drug Screening.
Cancer Research.
Virology (e.g., vaccine production).
Cell-Based Manufacturing (recombinant proteins).
Genetic Engineering and Gene Therapy.