Lecture 2 Biology of Cultured Cells

Lecture 2 Biology of Cultured Cells

• Culture environment
  • physiochemical constitution of medium
  • incubation temp
  • constitution of gas phase
  • degree of contact with other cells
  • nature of substrate in which the cells grow
• Cell adhesion molecules
  • mediated by specific cell surface receptors
  • cadherin
    • Ca2+ dependent
    • c-c
    • adherens junctions or desmosomes→actin cytoskeleton
    • signaling, structural roles
  • c-c adhesion molecules
    • Ca2+ independent
    • NCAM homophilic in neural synapses
    • ICAM heterophilic in immunological synapses
  • integrin
    • c-m
    • interact with fibronectin, entactin, laminin & collagen via RGD seq
  • transmembrane proteoglycan
    • c-m
    • interact with other proteoglycan or collagen not via RGD motif
  • claudins & occludins
    • tight junctions
    • homophilic
    • help establish cell polarity
• Cytoskeleton
  • integrins + actin via linker protein↔reciprocal signaling between cell surface & nucleus
  • cadherins + actin in adherens→cell shape & morphogenesis
  • cadherins + intermediate in desmosome→structural & signaling
  • microtubules
• ECM (collagen, laminin, fibronectin, hyaluronan, proteoglycan) allows
  • attachment & spreading for cell proliferation
  • specific interactions of cells with basement membrane/adjacent tissue cells
  • enhance cell survival, proliferation & differentiation
• Effects of new microenvironment
  • lack heterogeneity & 3D architecture→absence of hormonal & nutritional stimuli; low c-c & c-m interaction
  • favors spreading, migration, proliferation of unspecialized progenitor cells w/o differentiated function
  • might not express correct in vivo phenotype
• Cell motility
  • ruffling (edges of moving fibroblasts in culture repeatedly folded upward and inward)
  • fibroblast exhibit high motility at low cell density
  • dense epithelial monolayer show reduced motility
  • contact inhibition→cessation of movement at confluence没位子, low PM ruffling, withdrawal of cells from division cycle
• Cell proliferation→G1, S, G2, M
• 
• Cell signaling
  • contact-mediated (cytoskeleton)
  • diffusible factors
    • autocrine→same cell
    • homocrine→adjacent similar cell
    • endocrine→insulin, glucocorticoid
    • paracrine→heterotypic interaction of differ cells
    • proliferation, migration, differentiation, apoptosis
• Energy metabolism
  • glucose as main carbon source for glycolysis under anaerobic→generate lactic acid
  • TCA remains active
    • amino acid as carbon source
    • deamination of glutamine produces ammonia
    • use of dipeptides minimizes ammonia production
• Senescence
  • normal cells can only divide a limited no of times
  • die out after a fixed no of population doublings
  • progressive shortening of terminal DNA seq in telomeres
  • mutate gene p53 deletion (transformation)→continuous cell lines
  • aneuploidy→transformed cells lose anchorage-dependance & often show some chromosome fragmentation