Animal Tissue Culture 2

Animal Tissue Culture 2

Excessive confluency in culture may lead to:

Exceeding optimal confluency and leaving cells at 100% coverage without timely subculture triggers multiple stress responses. Contact inhibition mediated by Hippo signaling phosphorylates YAP causing cytoplasmic retention halting proliferation and upregulating cyclin-dependent kinase inhibitors p27. Simultaneously culture medium undergoes rapid acidification due to aerobic glycolysis generating lactate, accumulation of ammonium from glutamine metabolism raising intracellular pH stress, and depletion of essential nutrients like glucose and growth factors. Cells may undergo spontaneous different

Ref: Alberts MBoC 6th Ed Contact inhibition Hippo pathway; Freshney Ch.14 Overconfluence stress differentiation and cell loss.

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Animal Tissue Culture 2

Cell confluency refers to:

Confluency describes percentage of available growth surface covered by adherent cells, assessed visually or quantitatively via image analysis. At low confluence 20-30%, abundant space available, cells spread widely, proliferation rapid. At mid 50%, actively growing but spaces remain. At 70-80%, monolayer approaching but gaps still exist providing window for transfection, infection, and passaging. At 100%, surface completely covered with no further expansion possible without overlapping or piling for most contact-inhibited lines. Estimation performed daily using inverted phase-contrast microsco

Ref: Freshney Ch.14 Confluence definition % surface covered; ATCC Cell Culture Basics: assessing confluency by phase-contrast.

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Animal Tissue Culture 2

Macroculture is preferred when:

Macroculture denotes large-scale culture formats including T-75 and T-175 flasks, roller bottles, multi-tray CellSTACKs and Cell Factories, and stirred-tank bioreactors producing volumes from hundreds of milliliters to thousands of liters. Preferred when substantial cell biomass required for downstream processing such as purification of therapeutic recombinant proteins like erythropoietin, monoclonal antibodies from CHO cells, viral vectors for gene therapy, vaccine virus harvest from Vero, or preparation of whole cell lysates for proteomics and metabolomics. Macro scale enables control over d

Ref: Freshney Ch.27 Scale-up macroculture; Wurm Nat Biotechnol 2004 Production of recombinant proteins in CHO large scale.

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Animal Tissue Culture 2

Microculture is characterized by:

Microculture systems utilize miniaturized formats including 96-well, 384-well, and 1536-well plates with working volumes of 20-200 microliters. Characteristic feature is very low consumption of expensive reagents such as recombinant growth factors, fetal bovine serum, Matrigel, and small molecule libraries, making them economical for screening and optimization experiments. Environmental control is achieved via highly controlled incubators with plate hotels and automated liquid handling robots. Cells grow as small monolayers suitable for colorimetric assays like MTT for viability, fluorescence

Ref: Freshney Ch.8 Microculture low reagent use; Zhang et al Nat Rev Drug Discov 2005 High throughput screening in microplates.

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Animal Tissue Culture 2

Cells that grow freely in suspension include:

Cells that proliferate freely without attachment are predominantly of hematopoietic origin including T lymphocytes, B lymphocytes, monocytes, neutrophils, erythroid progenitors, and leukemic lines derived from them such as Jurkat, Raji, THP-1. These cells physiologically exist in circulation or bone marrow where immobilization is unnecessary and survival signals arise from cytokines activating JAK-STAT pathways rather than integrin-ECM engagement. In culture they grow as spherical single cells or small clusters suspended in medium, requiring agitation only for oxygenation in large scale stirre

Ref: Freshney Ch.14 Hematopoietic suspension growth; Lodish MBoC Ch.24 Blood cells suspension culture cytokine dependence.

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Animal Tissue Culture 2

Anchorage-dependent cells require:

Anchorage-dependent cells require physical attachment to solid substratum coated with extracellular matrix proteins such as collagen type I, fibronectin, or laminin to progress through cell cycle. Attachment occurs via transmembrane integrin heterodimers α and β binding to RGD sequences in matrix, clustering into focal adhesions recruiting focal adhesion kinase autophosphorylation at Y397, Src recruitment, and downstream activation of PI3K-Akt survival pathway and Ras-MAPK mitogenic cascade inducing cyclin D1 transcription. Without this signaling, normal cells arrest in G1 and undergo anoikis

Ref: Alberts MBoC 6th Ed Ch.19 Integrin signaling anchorage dependence; Lodish MBoC Ch.24 FAK anoikis and cell survival.

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Animal Tissue Culture 2

Which cells do NOT require trypsin for passaging?

Adherent cell types such as fibroblasts, epithelial, endothelial, and myoblasts develop integrin-mediated focal adhesions and cadherin-based cell-cell contacts requiring proteolytic cleavage for detachment. Trypsin-EDTA treatment disrupts these connections enabling replating. Hematopoietic lineage cells including lymphocytes, monocytes, erythroblasts, mast cells, and numerous leukemia derivatives such as Jurkat, K562, HL-60 naturally grow in suspension, circulating in bloodstream and bone marrow without organized extracellular matrix attachment. They rely on cytokines like IL-2, IL-3, GM-CSF f

Ref: Freshney Ch.14 Suspension vs adherent cultures; Abbas Cellular and Molecular Immunology 10th Ed hematopoietic cells suspension growth.

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Animal Tissue Culture 2

Continuous cell lines are formed due to:

Continuous cell lines emerge when primary cultures escape senescence crisis and acquire capacity for indefinite proliferation, process termed immortalization through transformation. Transformation may occur spontaneously during prolonged passaging due to mutations inactivating tumor suppressors p53 and retinoblastoma protein, amplification of oncogenes like c-Myc, activation of telomerase reverse transcriptase hTERT, and aneuploidy. It can also be induced deliberately by chemical carcinogens, gamma irradiation, or introduction of viral oncogenes such as Simian Virus 40 large T antigen which bi

Ref: Freshney Ch.15 Transformation continuous lines; Cooper Cell A Molecular Approach 8th Ed oncogene mediated immortalization p53 Rb inactivation.

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Animal Tissue Culture 2

The Hayflick limit refers to:

Hayflick limit defines finite number of times normal somatic cells can divide before entering senescence, discovered by Leonard Hayflick and Paul Moorhead in 1961 using human fetal lung fibroblasts WI-38. They observed progressive decline in doubling capability leading to arrest after approximately 40-60 population doublings, refuting earlier claims by Alexis Carrel that cultured cells were immortal. Molecular basis is incomplete replication of linear chromosome ends due to end-replication problem: DNA polymerase cannot fully replicate 3 prime overhang, causing loss of 50-200 base pairs of tel

Ref: Hayflick L Exp Cell Res 1961 finite division; Watson et al Molecular Biology of the Gene 7th Ed Telomere shortening and replicative senescence.

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Animal Tissue Culture 2

Primary cell cultures are characterized by:

Primary cell cultures are established directly from normal tissue explants via enzymatic disaggregation and initially retain diploid karyotype, tissue-specific morphology, hormonal responsiveness, and differentiated functions such as albumin secretion by hepatocytes. However they possess finite proliferative lifespan limited to few dozen population doublings typically 20-50 depending on species, age of donor, and culture conditions. After this, cultures undergo replicative senescence with enlarged flattened morphology, increased cytoplasmic granulation, positive staining for senescence-associa

Ref: Alberts MBoC 6th Ed Primary cultures finite lifespan; Freshney Ch.11 Characteristics of primary vs continuous cell lines.

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Animal Tissue Culture 2

The temperature of liquid nitrogen used for cryopreservation is:

Liquid nitrogen provides ultralow storage temperature of -196°C at atmospheric pressure, close to its boiling point. At this temperature, kinetic energy of molecules becomes extremely low, effectively halting all biochemical reactions, enzymatic activity, solute diffusion, and ice recrystallization processes. Cells suspended in DMSO-containing freezing medium enter glassy state preventing further ice growth. Long-term viability preserved for decades to theoretically indefinitely. Mechanical ultra-low freezers at -80°C slow metabolism but do not fully arrest chemical reactions; free radical acc

Ref: ATCC Animal Cell Culture Guide Liquid Nitrogen -196°C arrests metabolism; Freshney Ch.22 Vapor vs liquid phase storage principles.

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Animal Tissue Culture 2

Cryopreservation of animal cells commonly uses:

Cryopreservation of mammalian cells routinely employs dimethyl sulfoxide as penetrating cryoprotectant at 5-10% concentration. DMSO is small amphipathic molecule freely crossing lipid bilayer, replacing intracellular water and disrupting hydrogen bonding network required for ice lattice formation. During controlled slow cooling at -1°C per minute achieved using isopropanol containers like Mr. Frosty or programmable freezers, extracellular ice forms first creating osmotic gradient that draws water out of cells dehydrating them. DMSO lowers freezing point, promotes vitrification where residual i

Ref: Freshney Ch.22 Cryopreservation DMSO prevents intracellular ice; Mazur Science 1984 kinetics of water loss and vitrification by DMSO.

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