Animal Cell Culture Basics

Animal Cell Culture Introduction

• Animal cell culture is the growth of tissues or cells separated from an organism.
• In vitro culture maintains and/or proliferates cells, tissues, or organs.
• Liquid, semi-solid, or solid growth media (broth or agar) are used in this process.
• Growth occurs outside the natural environment of the animal cells, in controlled conditions.

Topics Outline

• What is animal cell culture?
• Historical background of animal cell culture methods.
• Types of tissue culture (e.g., adherent and suspension cultures).
• Advantages and limitations of animal cell culture techniques.
• Applications of animal cell culture.

What is Animal Cell Culture?

• The growth of tissues or cells from an organism, separated from the organism in vitro (outside the organism)
• In vitro culture of cells, tissues, or organs to maintain and/ or proliferate
• This is done through the use of a liquid, semi-solid, or solid growth medium (broth or agar)
• Animal cells are grown in vitro (outside the living organism), in controlled conditions.

Historical Background

• 1907: Ross Granville achieved the first successful tissue culture in vitro.
• 1911: Alexis Carrel and Montrose Burrows defined "tissue culture." They established the first cell line using diluted blood plasma.
• 1920s: Salt solutions were formulated for cell culture specifically.
• 1940s: Antibiotics began to be used in cell culture.
• 1960s: Widely available carbon dioxide incubators became readily available.
• Development of various culture media and equipment since.
• Recent advances include: DNA, methods for isolating cells to be used.

Three Types of Tissue Culture

• Adherent cultures: Cells grow in a monolayer attached to a solid substrate.
• Suspension cultures: Cells remain suspended in the liquid culture medium.
• Explant cultures: Tissue fragments are attached to a substrate, often glass or plastic, to create an interface between the tissue and the liquid medium.

Advantages of Animal Cell Culture

• Superior method in biotechnology to alter physiological and physiobiological conditions like pH and temperature
• Enable related studies on cell metabolism and the biochemistry of cells, like drug testing
• Allows observation of the effects of different compounds (e.g., drugs, proteins) on various cell types under controlled conditions
• Provides consistent results if a single cell type is used.

Disadvantages of Animal Cell Culture

• Requires trained personnel and aseptic conditions, so it's a costly technique using specialized equipment
• The subculture of cells might create differentiated properties compared to the original strain
• Results in proportionally very small amounts of recombinant proteins, increasing overall expenses
• Contamination with mycoplasma and viruses is frequent and challenging to detect.
• Instability of cells due to chromosomal aneuploidy.

Cell Culture Application

• Studying normal cell physiology and biochemistry
• Investigating the effects of drugs and toxic compounds on cells
• Analyzing mutagenesis and carcinogenesis
• Drug screening and development
• Large-scale manufacturing of biological compounds (vaccines, insulin)
• Cancer research, studying differences between cancer and normal cells
• Testing the effects of carcinogenic substances
• Examining drug and technique effectiveness for cancer treatment

Cell Culture Morphology

• Suspension cultures: Cells grow as single cells or small clumps. Derived from blood (leukemia, lymphoma).
• Monolayer cultures: Cells grow as a layer attached to a surface. Derived from solid tissues (lungs, kidney, breast, etc). These can include epithelial, neuronal, and fibroblast cells.

Biology of Cultured Cells

• The culture environment, characterized by factors like the nature of the substrate, degree of contact with other cells, culture medium (physicochemical and physiological constitution), gas phase, and incubation temperature, profoundly influence cells in vitro.
• These factors influence how cells proliferate, migrate, differentiate and undergo apoptosis.
• The environment can alter cell function in vitro, compared to in vivo.

Cell Adhesion

• Most cells from solid tissues grow as adherent monolayers.
• The adhesion is mediated by specific cell surface receptors for molecules in the extracellular matrix (ECM).
• Cells spread across the substrate with secretion of ECM protein and proteoglycans.
• Cells bind to the matrix via specialized cell receptors.
• Glass or plastic surfaces are often treated to provide specific binding interactions.

Cell Adhesion Molecules

• Cadherins (Ca2+ dependent): Interactions between homologous cells through adherens junctions or desmosomes.
• CAMs (Ca2+ independent): Cell-cell adhesion molecules that interact with integrins.
• Integrins: Mediate cell-matrix adhesion and interact with molecules like fibronectin, entactin, laminin, and collagen.
• Transmembrane proteoglycans interact with matrix components like proteoglycans and collagens.

Cell Motility

• Most cultured cells have the capacity to move.
• Fibroblasts are highly motile under standard cell growth conditions, and epithelial cells (monolayers) are less so.

Evolution of Cell Lines

• Primary cells have a finite lifespan.
• Primary cell culture goes through a lag phase, followed by exponential growth (log phase), a plateau phase, and finally, senescence (the cells stop dividing).
• Continuous cell lines can be created from primary cells via transformation or immortalization.
• These cells have an indefinite lifespan, meaning they continually divide and grow.

What is Senescence?

• Senescence is a state of permanent cell cycle arrest from which cells cells cannot subsequently re-enter the cell cycle.
• It is a cellular process that limits cell growth and division.
• Senescence is naturally regulated based on aspects like the shortening of telomeres through each replication cycle.

Initiation of Cell Lines

• Primary cultures: Derived directly from tissues, with limited capacity for division and time in culture.
• Cell lines: Derived from primary cultures through repeated propagation. These can be finite (have a limited time in culture) or continuous (capable of continued growth).

Continuous Cell Lines

• Continuous cell lines exhibit alterations like p53 mutation or deletion, overexpression of telomerase, and immortalization.
• Their chromosomal numbers are aneuploid (not a normal diploid or tetraploid).
• Most cells generally will not transform to become continuous cell lines.

Regulation of Cell Proliferation

• Proliferation is governed by factors like the culture environment, growth factors, confluency, and intracellular controls. These mechanisms also regulate normal cells entry into the 'cell cycle'.
• Factors promoting include: mitogens such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), Fibroblast growth factors (FGF).
• Factors inhibiting include the density of the cells and TGF. Internal regulatory mechanisms control this process.

Cell Proliferation vs Cell Differentiation

• Cell proliferation involves cells growing and dividing, while differentiation involves cells becoming specialized for particular functions.
• Normal culture conditions, with low density and presence of mitogens, typically favor proliferation. High density conditions promoting differentiation.

Dedifferentiation

• This is the loss of specialized characteristics or traits in a cell line or a cell.
• Factors leading to this include inability or problems with differentiation in vitro, and undifferentiated or wrong cell lines outgrowing cells in their normal differentiated states.

Dedifferentiation vs Deadaptation

• Dedifferentiation represents cells losing characteristic traits, often an irreversible process.
• Deadaptation reflects a reversible loss of phenotype by a change in the cell’s growth environment.

Tissue Retains Function Longer

• 3D tissue cultures have the advantage of better retaining properties, though often not easily propagated.
• Matrigels can be useful for supporting these 3D cultures.

Cell Signaling

• Cells communicate through signaling pathways that regulate cell activities such as cell proliferation, migration, apoptosis and more.
• These pathways include cell-cell communication and interactions, signaling via cell adhesion molecules, and hormonal stimuli. Autocrine, paracrine and endocrine pathways exist in vivo, but autocrine mechanisms are more likely to operate in vitro.

Checkpoint in Cell Cycle

• G1 checkpoint: Cell checks for DNA damage, nutrients, and growth factors to ensure the viability of the cell before it proceeds to the S phase.
• G2 checkpoint: Cell checks for DNA replication completeness and any damage or errors.

How Cell Cycle Inhibitors Regulate Cell Proliferation

• Cell division is regulated by genes that code for, and regulate cell cycle enzymes. These include cyclins and cyclin-dependent kinases.
• Inactivation of these genes and protein products like p53 and Rb proteins can lead to uncontrolled cell growth and division, commonly associated with the development of cancers.

This quiz explores the fundamentals of animal cell culture, including types of cultures, advantages, and disadvantages. Test your knowledge on the principles that govern this essential area of biotechnology and tissue engineering.