1. Biology of Cultured Cells
Chapter 3

2. Influence of environment on culture -four routes
The nature of the substrate on or in which cells grow
The degree of contact with other cells
The physicochemical and physiological constitution of the medium
The constitution of the gas phase
The incubation temperature

3. What factors allow for cell adhesion?
Attach and spread out on substrate before they proliferate
Grow in T-flasks or Petri dishes or Roux Bottles
Combination of electrostatic attraction and Van der Waal’s forces
Ca 2+ and basic proteins

4. Different Cell Adhesion Molecules
CAM’s
Cadherins
Integrins
Transmembrane proteoglycans

6. Cell-Cell Adhesion molecules
CAMs (Ca2+ independent) and Cadherins (Ca 2+ dependent)
Homologous cells interact
Cell-cell recognition generates signaling role in cell behavior

7. Cell-Substrate adhesion molecule
Integrins allow for cell-substrate interactions
Cell surface receptors for ECM (fibronectin, extractin, laminin and collagen)
Bind via RGD
Two polypeptide chains – α and β

8. Cell adhesion molecules
Transmembrane proteoglycans interact with matrix constituents such as proteoglycans or collagen
No RGD motif

10. What are intercellular junctions?
Cell adhesion molecules diffusely arranged in plasma membrane
Organized into intercellular junctions
Desmosomes, adherens, gap and tight junctions

11. What is extracellular matrix?
Intercellular spaces in tissues filled with ECM
Regulates phenotypic expression
Produced by cell cultures
Exogenous provision - experiments

12. How does protease act?
Digests some ECM and some extra cellular domains of transmembrane proteins
Epithelial and endothelial cells – resistant
Mesenchymal cells – less resistant
Confluency of epithelial cells

13. What elements of cytoskeleton are attached to cell adhesion molecules?
Integrins and Cadherins – actin cytoskeleton – in adherens junction
Cadherins – intermediate cytoskeleton – in intermediate junction – desmosomes
Microtubules

14. Do cells show motility? Fibroblasts show directional migration
Polar movement
Contact inhibition – directional migration ceases + reduction in plasma membrane ruffling
Leads to withdrawal of cell from division cycle

15. Eukaryotic Cell Cycle interphase
The eukaryotic cell cycle has 5 main phases:
1. G1 (gap phase 1)
2. S (synthesis)
3. G2 (gap phase 2)
4. M (mitosis)
5. C (cytokinesis)
The length of a complete cell cycle varies greatly among cell types.
interphase

16. Interphase Interphase is composed of:
G1 (gap phase 1) – time of cell growth
S phase – synthesis of DNA (DNA replication)
- 2 sister chromatids are produced
G2 (gap phase 2) – chromosomes condense
G1 phase – major portion of the cell cycle period, is the gap between cytokinesis and DNA synthesis.
S phase- replica of genome is synthesized
G2 phase – Prepares cell for mitosis, fills gap between DNA synthesis and beginning of mitosis, mitochondria and other organelles replicate, chromosomes begin to condense and microtubules begin to assemble at spindle
G0 phase-Cells often pause in G1 before DNA replication and enter a resting stage G0. ex: muscle and nerve cells remain there permanently others such as liver cells can resume G1 phase in repsonse to factors release during injury.

17. Control of the cell cycle
The cell cycle is controlled at three checkpoints:
1. G1/S checkpoint
-the cell “decides” to divide
2. S/G2 checkpoint
- the cell “decides” DNA repair or apoptosis
G1/S checkpoint –acts by deciding whether the cell should divide or not based on the availability of growth factors, the nutritional condition of the cell (whether starving or not). Once a cell has crossed this checkpoint then it has made a commitment to divide and any damage to DNA can result in halting cell cycle at this point.
G2/M checkpoint- The Cdks identified at this point are known as M phase-promoting factor. Passing this phase means the cell has made commitment to mitosis based on proper DNA replication. Without successful DNA replication the cycle can stop at this point.
Activation and inactivation at each checkpoint happens because of phosphorylation (but for activation cdk should bind to appropriate cyclins)
Spindle checkpoint – Arrangement of chromosomes on the metaphase.

18. Control of the cell cycle
3. G2/M checkpoint
-the cell makes a commitment to mitosis
4. late metaphase (spindle) checkpoint
-the cell ensures that all chromosomes are attached to the spindle

19. How growth factors control the cell proliferation?
- Epidermal growth factor (EGF), Fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF)
- Allows low cell density population to enter into cell cycle
- Does not allow proliferation of high cell density population
Inhibition of proliferation is initiated by cell contact, accentuated by crowding and resultant change in shape of cell and reduced spreading

20. How cell cycle inhibitors control cell proliferation?
Rb gene product, p53, and p16
Block cell cycle progression/arrest
Damage leads to cancerous cells
Inactivation by phosphorylation-cell cycle progression
Cell cycle inhibitors block cell cycle progression at restriction points or check points.

21. Factors that lead to Dedifferentiation
Inability of cell lines to differentiate
Wrong lineage of cells is selected in vitro
Undifferentiated cells of same lineage overgrow terminally differentiated cells of reduced proliferative capacity
Absence of inducers (hormones, cell or matrix interaction) can lead to reversible loss of differentiated properties
Inability

22. Different ways of cell-signaling
Signals reaching cells from another tissue via the systemic vasculature – Endocrine
Signals reaching cells from adjacent cells without entering bloodstream – Paracrine
Signals arising and interacting with same cell – Autocrine signaling

23. Different ways of cell-signaling
Signals arising and interacting with adjacent cells - Homotypic paracrine or homocrine signaling
Signals arising and interacting with different cells –Heterotypic paracrine

24. Types of cell-signaling in in vitro
Autocrine and homocrine signaling will occur
Slow growth due to dilution of autocrine or homocrine factors
Paracrine and endocrine factors are added

25. Energy metabolism 4-20mM glucose – carbon source for glycolysis
Glutamine – carbon source for citric acid cycle
- Deamination of glutamine produces NH3 - toxic and can limit cell growth
- Use of dipeptides such as glutamyl-alanine or glutamyl-glycine minimize production of NH3

26. Initiation and Evolution of Cell lines
Culture derived from main tissue – Primary Culture
Culture derived from primary culture – Cell Line
Continuous cultures or passage of cell cultures derived from cell line – Subculture
Only cells that survive disaggregation technique and adhere to substrate or survive in suspension will form PC. Cells that undergo proliferation will survive and others will not survive in cell line (because of repeated trauma of trypsinization and transfer). Some selection and phenotypic drift will continue by third passage the culture becomes more stable and becomes a proliferating cell. Some famous cell lines like Baby hamster kidney fibroblasts, CHO cells and L-cell is a mouse subcutaneous fibroblast

28. What is Senescence? Why and how does it happen?
Normal cell lines will die after fixed number of population doublings – Senescence
Inability of terminal sequences of DNA in telomeres to replicate at each cell division
Progressive shortening of telomeres – cell cannot divide

29. Why Senescence does not take place in all cells?
Germ cells, stem cells, transformed cells, tumor lines etc
Express enzyme telomerase which is capable of replicating the terminal sequences of DNA in telomeres
Extends life span of cells
They are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types
Extends life span of tumor cells infinitely

30. What are Continuous Cell Lines?
Ability of cell lines to grow indefinitely in vitro
Alteration of a culture – TRANSFORMATION and giving rise to continuous cell line – IMMORTALIZATION
Shows capacity for genetic variation/instability
p53 is mutated or deleted (if tumor cell line)
A group of morphologically uniform cells that can be propagated in vitro for an indefinite time . Chromosome number varies in continuous cell lines.
Immortalization means indefinite life span. Transformation means additional alteration in growth characteristics (anchorage independence, loss of contact inhibition and density limitation of growth) – morphological and kinetic alterations.

31. How Continuous Cell Lines develop (Heteroploidy)?
Aneuploidy – chromosome number lies between diploid and tetraploid
Mouse fibroblasts and cell cultures from variety of human and animal tumors
Genetic instability is present
Human continuous lines develop tetraploidy

32. Origin of Culture
Cell lines derived from embryo are good for culturing than adult cells
Identity of cultured cell depends on two factors
Lineage of cell in vivo (hematopoietic, hepatocyte, glial etc)
Position of cell in that lineage (stem cell, precursor cell or mature differentiated cell)
Embryo has more stem cells, precursor cells and mature differentiated cells.

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