#cell culture

Plant tissue culture 1

Increase in secondary metabolites in cell culture is commonly achieved by:

Increasing secondary metabolite accumulation in plant cell cultures is commonly achieved by supplementation with elicitors after achieving adequate biomass. Secondary metabolism is resource costly competing with primary growth for carbon nitrogen and energy, regulated by growth defense trade off controlled by TOR kinase promoting growth versus SnRK1 promoting defense. Under normal culture high auxin low stress conditions flux stays in primary metabolism. Elicitor addition artificially activates jasmonate and salicylate mediated defense transcriptome inducing genes encoding rate limiting enzyme

Ref: Mishra & Ranjan 2008 secondary metabolite; PubMed elicitor methyl jasmonate.

View Details

Animal Tissue Culture 3

Generation time is defined as:

Generation time, synonymous with doubling time, quantifies interval needed for population to double during exponential phase, reflecting duration of one complete mitotic cycle comprising G1 phase where cells assess nutrient sufficiency and growth factor signaling via Ras-MAPK and PI3K-Akt inducing cyclin D, S phase where DNA polymerase alpha-primase and delta synthesize new genome with fidelity checks, G2 phase where mitotic proteins such as cyclin B and Cdk1 accumulate, and M phase involving spindle formation, chromosome segregation, and cytokinesis. Mathematically derived from growth curves

Ref: Freshney Ch.13 Generation time one division; Lodish MBoC Ch.13 Cell cycle timing G1 S G2 M doubling time calculation.

View Details

Animal Tissue Culture 3

The senescence phase in cell culture corresponds to:

Senescence phase in cell culture corresponds to death or decline phase of growth curve where proliferative capacity permanently lost and net cell number decreases. Replicative senescence triggered by telomere attrition beyond critical threshold leads to uncapped chromosome ends recognized as DNA double-strand breaks activating ATM and ATR kinases that stabilize p53 and induce cyclin-dependent kinase inhibitors p21CIP1 and p16INK4a causing irreversible G1 arrest. Morphology changes to enlarged flattened cells with increased granularity, vacuolation, positive senescence-associated beta-galactosi

Ref: Hayflick L Exp Cell Res 1961 senescence death phase; Campisi J Cell 2005 Senescence SASP and p16 p53 pathways.

View Details

Animal Tissue Culture 3

Cells should ideally be passaged during:

Growth curve of cultured cells comprises lag adaptation, exponential log proliferation, plateau where division balances loss, and death where loss predominates. Ideal timing for passaging aligns with transition from late log to early plateau when cultures achieve sufficient density providing high yield of cells for splitting but before deleterious effects of overconfluence arise. At this point confluence typically 70-80% for adherent fibroblasts and epithelial lines, or suspension density half of maximal sustainable concentration, providing many cells yet maintaining health with active DNA syn

Ref: Freshney Ch.14 Passaging at plateau late log; Alberts MBoC Ch.17 Growth regulation subculture timing before overconfluence stress.

View Details

Animal Tissue Culture 3

The phase of cell growth most suitable for drug testing is:

Pharmacological testing and chemosensitivity evaluation are most predictive during logarithmic exponential growth phase when cell population is actively cycling, metabolically robust, and homogeneous. During lag, cells are still recovering and fraction in S phase low, causing underestimation of antimetabolite efficacy. In log phase, DNA synthesis enzymes including topoisomerase II, thymidylate synthase, and kinases targeted by many drugs are highly expressed, mitochondrial activity high, ATP levels stable. Dose-response curves generated in this window show reproducible IC50 values. Plateau or

Ref: Freshney Ch.21 Drug testing log phase; NIH Assay Guidance Manual 2021 Chemosensitivity assays log phase optimal viability.

View Details

Animal Tissue Culture 3

BHK21 cells are commonly employed in:

Plateau phase, also termed stationary phase, occurs when culture reaches carrying capacity constrained by surface area for adherent cells or nutrient limits for suspension cells. Cell number stabilizes as division rate equals death rate, curve flattening. Molecular mechanisms include contact inhibition mediated by Hippo pathway: high cell density activates LATS1/2 kinases phosphorylating YAP leading to cytoplasmic retention and degradation preventing transcription of cyclin D1 and survivin. Cadherin engagement also induces p27Kip1. Simultaneously growth factor exhaustion, accumulation of inhib

Ref: Alberts MBoC Ch.17 Plateau contact inhibition Hippo YAP; Freshney Ch.13 Growth curve plateau stationary quiescence balance.

View Details

Animal Tissue Culture 3

CHO cells are extensively used for:

Logarithmic or exponential phase follows lag and is characterized by maximal and constant specific growth rate where population doubles at regular intervals, producing straight line on semi-logarithmic plot of cell number versus time. Birth rate greatly exceeds death rate. Cellular activities peak including DNA replication by DNA polymerases alpha and delta, rRNA transcription, protein synthesis, high glucose consumption via aerobic glycolysis producing lactate, and oxygen uptake by mitochondria. For most continuous lines generation time 18-24 hours though HeLa approximately 20 hours. Contact

Ref: Freshney Ch.13 Log phase exponential growth; Lodish MBoC Ch.12 Cell cycle exponential proliferation and growth curve.

View Details

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.

View Details

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.

View Details

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.

View Details

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.

View Details

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.

View Details