Lernzettel: Animal Cell Culture Biotechnology

📋 Course Outline

1. Animal Tissue Culture Foundations
2. Culture Types and Applications
3. Cell Culture Laboratory Infrastructure
4. Culture Media and Serum
5. Media Formulation and Buffering
6. Cell Culture Contamination
7. Decontamination and Biosafety
8. Cell Growth and Culture Metrics

📖 1. Animal Tissue Culture Foundations

🔑 Key Concepts & Definitions

• Tissue culture : The in vitro maintenance and/or proliferation of animal cells, tissues, or organs.

★ Must-know

• Cell and tissue culture provide greater control of the physical environment, more homogeneous and reproducible samples, lower compound requirements than animal models, and shorter characterization times.

Further detail

• The main disadvantages of cell and tissue culture are loss of differentiated characteristics, difficult maintenance, limited tissue yield at high cost, dedifferentiation, instability, aneuploidy, and loss of the original tissue organization.

• Animal tissue culture is used in developmental biology, pharmacology, toxicology, and regenerative medicine.

📖 2. Culture Types and Applications

🔑 Key Concepts & Definitions

• Cell culture : The growth of cells as an adherent monolayer on a solid substrate or, for some cell types, in suspension in culture medium.
• Primary explant culture : Grows a fragment of tissue on a solid substrate, where attachment and cell migration occur in the plane of the substrate.
• Organ culture : Maintains an excised organ or embryo in a spherical or three-dimensional form with specific histological interactions.

📝 Essential Points

• Organ and embryo cultures maintain normal physiological functions and fully differentiated cells, but they grow slowly, are difficult to scale up, and require fresh explantation for each experiment.

• Primary cultures resemble their parent tissue and are useful models of in vivo conditions, but they have limited divisions, are initially heterogeneous, become dominated by fibroblasts, and are susceptible to contamination.

💡 Memory Hook

Cell → explant → organ: increasing structural complexity.

📖 3. Cell Culture Laboratory Infrastructure

🔑 Key Concepts & Definitions

• Laminar-flow hood : Protects the working environment from dust and contamination by maintaining a constant flow of filtered air across the work surface.

★ Must-know

• A cell culture laboratory requires a sterile and quiet workspace separate from animal and microbial laboratories, culture incubators, preparation and wash-up areas, sterilization equipment, and storage at 4°C, −20°C, −80°C, and liquid nitrogen temperature.

• A cell culture incubator requires controlled temperature, high humidity, and controlled CO₂ tension; it should provide temperature control within +0.5°C, forced air circulation, and a safety thermostat.

Further detail

• Horizontal laminar airflow moves from the rear toward the user parallel to the work surface, whereas vertical airflow moves downward from the top and is drawn through or vented from the work surface.

• Important cell culture equipment includes an autoclave, refrigerators and freezers, a liquid nitrogen tank, an inverted microscope, aspiration equipment, culture vessels, and multiwell plates.

📖 4. Culture Media and Serum

🔑 Key Concepts & Definitions

• Embryo extract : A clarified crude homogenate of a 10-day-old chick embryo that may contain peptide growth factors.

★ Must-know

• A typical culture medium contains amino acids, vitamins, inorganic salts, glucose, and serum.

• The low-molecular-weight fraction of chick embryo extract promotes cell proliferation, whereas the high-molecular-weight fraction promotes pigment-cell and cartilage-cell differentiation.

• Serum supplies electrolytes, lipids, glucose, amino acids, proteins, hormones, and growth factors, and the commonly used sera include calf serum, fetal bovine serum, adult horse serum, and human serum.

Further detail

• Fibronectin promotes cell attachment, fetuin in fetal serum enhances attachment, transferrin binds iron and makes it less toxic and more bioavailable, and α₂-macroglobulin inhibits trypsin.

📖 5. Media Formulation and Buffering

🔑 Key Concepts & Definitions

• Chemically defined medium : Contains contamination-free, ultrapure inorganic and organic ingredients and may include purified protein additives such as growth factors.
• Conditioned medium : Contains undefined components released or supplied by cells and should be replaced when its active constituents have been determined.

★ Must-know

• Artificial media may support immediate survival, prolonged survival, indefinite growth, or specialized cellular functions.

📌 Natural buffering uses equilibrium between gaseous CO₂ and carbonate/bicarbonate in the medium and requires an atmosphere containing 5–10% CO₂, whereas HEPES chemical buffering does not require a controlled gaseous atmosphere.

Further detail

• Protein-free media contain only non-protein constituents, and protein supplementation is added when required; RPMI-1640 is an example of a protein-free formulation.

📖 6. Cell Culture Contamination

★ Must-know

📌 Chemical contaminants are usually difficult to detect and include endotoxins, plasticizers, metal ions, and traces of disinfectants, whereas biological contaminants produce visible effects and include mycoplasma, yeast, bacteria, fungi, and cross-contaminating cells.

• Biological contaminants compete with host cells for nutrients, secrete acidic or alkaline by-products, degrade arginine and purine intermediates, inhibit histone and nucleic-acid synthesis, and may produce cell-toxic hydrogen peroxide.

📌 Contamination introduces unwanted microorganisms or substances into a culture, whereas cross-contamination introduces cells from another cell line.

Further detail

• Endotoxin contamination can be detected using the Limulus amebocyte lysate assay.

📖 7. Decontamination and Biosafety

★ Must-know

• When contamination is detected, the culture should first be identified as bacterial, fungal, mycoplasmal, or yeast contamination and then isolated from other cell lines.

• Antibiotics and antimycotics should be tested by dose response because high concentrations can be toxic to some cell lines.

• The safest response to an unrecoverable contaminated culture is to discard it, decontaminate the workspace, and restart with a fresh culture.

Further detail

• Decontamination requires cleaning incubators and laminar-flow hoods with laboratory disinfectant, checking HEPA filters, and diluting or isolating the contaminated culture when appropriate.

• Before each use, a laminar-flow hood should have its exposed work surfaces sterilized with ultraviolet light between uses and wiped with detergent and 70% alcohol.

💡 Memory Hook

Identify → isolate → disinfect → test → discard or restart.

📖 8. Cell Growth and Culture Metrics

🔑 Key Concepts & Definitions

• Finite cell line : A culture of normal cells that divides only a limited number of times before genetically determined senescence prevents further proliferation.
• Senescence : The genetically determined loss of proliferative ability that occurs in normal cells after a limited number of divisions.

★ Must-know

• Finite and continuous cell lines are used to study normal cell physiology, drug and toxin effects, mutagenesis, and carcinogenesis and to screen drugs and manufacture vaccines, protein hormones, and therapeutic proteins.

Further detail

• Cell culture growth is commonly described by lag, log, and plateau phases, corresponding respectively to adaptation, active proliferation, and reduced or stopped net growth.

📌 Plating efficiency measures the proportion of seeded cells that attach and grow into colonies, whereas cloning efficiency measures the ability of individual cells to generate colonies.

💡 Memory Hook

Lag → log → plateau.

📊 Synthesis Tables

Culture Types Compared

Culture type	Structure	Main strength	Main limitation
Cell culture	Adherent monolayer or suspension	Control, reproducibility, and scale-up	Some differentiated functions may be lost
Primary explant culture	Tissue fragment on substrate	Retains many parent-tissue characteristics	Heterogeneous and short-lived
Organ or embryo culture	Three-dimensional organ or embryo	Maintains physiological functions and differentiation	Slow growth and poor scale-up