Biochem more

Tissue culture

general name for removing cells, tissues, or organs from an animal/plant and placing them in an artificial environment conducive to growth (a glass/plastic vessel with liquid or semi-solid medium supplying essential nutrients).

Cell culture

when cells are removed from organ fragments, disrupting their normal relationship with neighboring cells, before being cultured

Primary cells
Cell strains
Cell lines

Classes of Culture Cells (3 Types)

Primary cells

Directly isolated from an organism; finite lifespan (5–10 divisions)

Cell strains

Adapted, selected primary cells with constant growth rate; finite lifespan (40–60 divisions)

Cell lines

Transformed / immortalized cells; capable of dividing indefinitely

Primary Culture

Cells are surgically removed from an organism and placed in a suitable culture environment to divide and grow. • Finite lifespan: 5–10 divisions in vitro fi long-term experiments discouraged • Considered more physiologically similar to in vivo cells

Isolation
Primary Culture & Subculture
Cryopreservation

General Process of Primary Cell Culture

Isolation

Preparing tissue fi isolating and plating cells in dishes (skipped if using commercial primary cells

Primary Culture & Subculture

Change medium 24 hrs after seeding; monitor confluence/mitotic activity; subculture at proper tim

Cryopreservation

View culture under microscope; cryopreserve when ~90% confluent and actively growing

Tissue Acquisition
Dissection
Disaggregation
Incubation & Growth
Separation & Purification

Isolation of Primary Cells — 5-Step Workflow

Explant cultures
Enzymatic dissociation

Two Basic Methods for Obtaining Primary Culture

Explant cultures

Small tissue pieces attached (plasma clots/fibrinogen) to a glass/plastic vessel and immersed in culture medium

Enzymatic dissociation

More widely used. Proteolytic enzymes (trypsin, collagenase) dissolve the cementing matrix, creating a single-cell suspension for plating

Hayflick's Phenomenon

• Cells grow/divide normally for a limited number of passages
• At a certain point, cells stop dividing and eventually die, even with proper nutrients
• Correlation exists between maximal passage number and aging
• Cells from older individuals show a decreased number of possible passages

Cell Lines

• A cell strain that undergoes transformation (spontaneous/induced changes in karyotype, morphology, or growth properties) becomes “immortal” fi called a ________

Cell Strains

are cells that have been adapted to culture but, unlike cell lines, have a finite division potential
Formed via serial transfer/gradual selection of primary cells until one cell type predominates
• Finite lifespan: 40–60 divisions in vitro
• Useful in vaccine production

Anchorage-dependent (adherent)

must attach to a surface to survive; forms monolayers; growth limited by surface area; ceases at confluence

Anchorage-independent (suspension)

divides/survives without attaching to a substrate (e.g., haemopoietic lineage cells); requires agitation for gas exchange

Lag Phase
Log Phase
Plateau Phase

Eukaryotic cells in attachment culture follow a characteristic 3-phase growth cycle (similar to bacteria)

Lag Phase

Time after subculture/reseeding with little increase in cell number; cells replace elements lost during trypsinization, attach to the surface, and spread out

Log Phase

Period of exponential increase in cell number; length depends on seeding density & growth rate; optimal time for sampling (most uniform population, high viability)

Plateau Phase

Culture becomes confluent (surface fully occupied, cells touch neighbors); growth rate drops, sometimes proliferation nearly stops; growth fraction falls

Epithelial-like
Fibroblast-like

Morphology(shape & appearance) Type

Fibroblast-like

Thin, elongated shape

Epithelial-like

Flattened, polygonal shape

Well-Equipped Facility
Sterile Technique
Quality Reagents & Supplied
Technique Knowledge

Basic Requirements for Successful Cell Culture

• glucose,
• fats and fatty acids,
• lipids, phospholipids and sulpholipids,
• ATP and amino acids
• Vitamins
• Minerals

Nutrient & Supplement Requirements for cell culture medium

Serum

can provide various growth factors, hormones and other factors needed by the most mammalian cells for their long term growth and metabolism

7.2–7.4

Optimum pH for mammalian cells

L-Glutamine

is an essential amino acid required by virtually all mammalian cells grown in culture
• It is used for protein production, as an energy source, and in nucleic acid metabolism
• It is also more labile in liquid cell culture media than other amino acids

Penicillin
Streptomycin
Gentamycin
Nystatin

Antibiotic Supplements

37°C

Optimum temperature for mammalian cells

Culture Vessels

form a contamination barrier while maintaining proper internal conditions. For anchorage-dependent cells, they provide a consistent attachment surface, easy culture access, and optically clear viewing surfaces

Fetal Bovine Serum (FBS)

Common serum supplement providing growth factors and hormones