Lecture 2 Flashcards
describe the difference between cell culture in vitro and in vivo conditions. what factors determine culture environment?
cell cell contacts, cell matrix contacts (3D structure), and hormonal and nutritional control differ in vitro vs in vivo.
5 factors that determine the culture environment:
1. Substrate/matrix
2. density
3. medium
4. gas phase composition (CO2)
5. incubation temp
list the steps of cell adhesion
- adsorption of attachment factors to the culture surface
- contact between the cell and the surface of the substrates
- attachment of cells to the coated surface
- spreading of the attached cells
define the relevance of cell proliferation (and its control) for cell cultures
Remember the cell cycle? go through that on slide 21. G1 is a key check point! G2 and M have checkpoints as well for DNA integrity
cell cycle is entered when there’s low cell density and mitogenic factors (growth factors), receptor activation, restriction point inactivation, and cyclins active
cell cycle arrests when no mitogenic growth factors and check points block. (cells get around this and are tumors)
describe the steps that are required for initiating a new cell culture and explain the selective pressure involved on each step
Disaggregate or Outgrowth: surviving and growing pressure
Attach and spread: adhering pressure
Proliferate/Differentiate: contact inhibition and GF pressure
1st Culture: trypsin sensitivity when removing, proliferating pressure
Ongoing culture: fastest growing strain, senescence, and transformation pressure
explain the “evolutions” of a cell line. explain hay flick limit and transformation
- Fibroblasts dominate cultures, slower cell types need special media. The cells a rapidly growing and lots of subculturing is needed
- Senescence: the hay flick limit is reached as population doublings are limited (around 30 pd’s for human). mass extinction occurs due to telomere shortening.
- Some cells survive by transformation: cells become continuous immortal cell lines with an infinite lifespan and increased tumorigenicity
describe the accurate nomenclature of cultured cells (primary culture, cell line, finite, continuous, strains)
Primary culture: 1st culture derived form explant or tissue
cell line: cells following the 1st culture
finite cell line: has a limited lifespan (hay flick limit)
continuous cell line: immortal lifespan
cell strains: cell line with defined special properties
types of junctions
mechanical junctions: cell-cell (desmosomes)
tight junctions: seal space between cells
gap junctions: allow exchange of small molecules between cells, small channel
how is cell adhesion done in lab?
many membrane glycoproteins and glycolipids contribute to negative membrane charge. if culture vessel surface is positively charged, attraction occurs. vessel surface must be hydrophilic and densely positive. cells attach to it and secrete ECM proteins which bind vessel and cells bind matrix via receptors. can also coat dish with ECM proteins to help attachment if you want.
what is Preconditioning of culture dishes?
grow cells in the dish that secrete the appropriate ECM proteins. remove the cells and leave the proteins to be used by the next cell culture. helps with attachment
list some cell adhesion substrates in nature and artificial
natural: ECM proteins like collagen, laminin, fibronectin, etc.
artificial: glass with alkali treatment, BD matrigel has ECM proteins isolated from mouse tumor to coat, plastics (polystyrene) treated to increase neg charge, positively charged polymers to coat
describe cell motility
some cells move on substrates, macrophages, fibroblasts, myoblasts, and endothelial cells using lamellipodium (polymerizing of actin). When cells touch, contact inhibition occurs and they stop moving/growing
define the relevance of differentiation for cell cultures
organs are made of different tissues which are made of different cell types. commitment (differentiation) must happen along the way to get to organs!
differentiation blocked by promoting proliferation (these two processes are opposite each other and must be balanced)
differentiation induced by cell type specific conditions
factors that affect if differentiation or proliferation occurs I vitro?
age of donor, embryonic will proliferate
cell source, cancer proliferates while normal tissues differentiate
tissue type, like brain cells are terminally differentiated while others might change like a skin cell
define the relevance (and control) of differentiation for cell cultures
organs are made of different tissues which are made of different cell types. commitment (differentiation) must happen along the way to get to organs!
differentiation blocked by promoting proliferation (these two processes are opposite each other and must be balanced)
differentiation induced by cell type specific conditions
define the relevance of cell signaling for cell cultures
signaling systems like the autocrine and homocrine still work in vitro with soluble factors. signals depend on cell-cell interactions (gap junctions, cytoskeletal tensions)
