Biology Of Cultured Cells

Does Culturing Reflect Reality

• Culturing Deviates From In Vivo Environment

– 3-D matrix is disrupted (collagen, cell-cell contact)
– Heterogeneity is changed
– Local growth factors are removed
• New Environment Promotes New Properties
– Progenitors are encouraged to proliferate
– Differentiated cells might not have the same
function as starting differentiated cells
 Adhesion
• Majority Of Cells Adhere On Plastic (Treated) Provided
They Are Not Transformed
• It Was Observed That Cells Prefer –vely Charged Glass
Surface
• Plastic (polystyrene) Is Tissue Culture Treated
– With High Energy Ionizing Radiation
– Electric Ion Discharge
• Adhesion Is Mediated By Surface Receptors And Matrix
– Matrix Is Secreted By Cells, Adheres To Charged Plastic
– Receptors Bind to Matrix
Cell Surface Adhesion Molecules
– cell-cell Adhesion Molecules
• CAMs (Ca2+ Independent)
• Cadherins (Ca2+ Dependent)
• Primarily Between Homologous Cells
• Signaling occurs
– Cell-Substrate Molecules
• Integrins
• Bind to fibronectin, entactin, laminin, collagen
• Bind the specific motif (RGD, arginine, glycine,aspratic)
• Comprised of  and  unit
Cell Surface Adhesion Molecules

• 3rd Class Is Proteoglycans

– Also Binds Matrix or Other Proteoglycans
– Not Via RGD Motif
– Low affinity Growth Factor Receptors
– May Aid Binding To Higher Affinity Receptors
– No Signaling Capacity
 Extracellular Matrix (ECM)

• Spaces In Between Cells Filled With ECM

– Common constituents: fibronectin, laminin, collagen,
hyaluronan, proteoglycans, bound growth factors/cytokines
• ECM Is Dependent On Cell Types
– Fibrocytes secret collagen I and fibronectin
– Epithelial cells secret laminin
• In Most Cases Cell Lines Are Allowed To Make
Their Own ECM
• Sometimes We Provide ECM
Cell Proliferation
 Cell Cycle
• 4 Phases
– M Phase, mitosis occurs
• Chromatin condensation, sister chromatid separation
• Daughter cells
– G1 Phase
• Progression to DNA SYNTHESIS
• Alternatively Go OR differentiation
• Restriction Points
– S Phase
• DNA Synthesis
• Progression to G2
– G2 Phase
• Integrity of DNA Checkpoints
• Apoptosis is an option
– DNA fragmentation, cell shrinkage, formation of small vesicles
Sistem
pengontrolan
siklus sel
- Mengontrol siklus sel, melalui sistem “checkpoint “ , yaitu:

1. G1 “checkpoint”
Sistem pengontrolan siklus sel pada akhir fase G1,
untuk memasuki fase S
2. G2 “checkpoint”
Sistem pengontrolan siklus sel pada akhir fase G2,
untuk memasuki fase M
3. Metafase “checkpoint”
Sistem pengontrolan siklus sel pada akhir metafase
dari kariokinesis, untuk memasuki tahap anafase.
Sistem “checkpoint” dalam siklus sel
2 komponen utama dalam sistem pengontrolan
siklus sel:
1. Cyclin
Protein yang disintesis dan diuraikan dalam fase tertentu
dalam siklus sel
2. “Cyclin-dependent kinase” (CdK)
Enzim yang memicu kejadian-kejadian spesifik dalam siklus
sel.Konsentrasinya tetap dalam siklus sel

Tanpa protein Cyclin, enzim CdK

tidak aktif
Kombinasi
komponen
dalam sistem
pengontrolan
siklus sel
Deteksi kerusakan DNA oleh sistem kontrol siklus sel
(“DNA damage checkpont”)
• Pada fase G1 akhir
DNA yang rusak mengaktifasi protein kinase yang.
memfosforilasi protein 53 (p53), sehingga menjadi aktif dan
stabil. P53 yang aktif akan mengaktifasi transkripsi gen p21
(CdK inhibitor protein), selanjutnya p21 akan menginhibisi
aktifitas G1/S-CdK dan S-CdK.
* Pada fase G2 akhir
Kerusakan DNA akan stimulasi inaktifasi M-CdK sehingga
akan mencegah sel masuk ke fase M. Jadi pencegahan ini
karena replikasi DNA belum sempurna.
“DNA damage
checkpoint”
pada akhir fase G1
 Control Of Cell Proliferation
• Environment Regulates Entry Into Cell Cycle
• External Growth Factors Promote Cell Proliferation
– PDGF, EGF, FGF (+ve)
– TGF- (-ve)
– Interact with surface receptors
• High Density Inhibits Proliferation (Contact
Inhibition)
• Inside The Cell Both Positive and Negative Factors
– Positive, cyclins, Growth Factor Receptor Activation
– Negative, p53, Rb, Checkpoints
Cell signaling
 Proliferation vs Differentiation
• Proliferation Does NOT Promote Differentiation
• Differentiation Often Requires
– High density
– Cell-Cell Interaction
– Cell-Matrix Interaction
– Differentiation Factors
• The Above Conditions Can Be Antagonistic To
Proliferation
 Dedifferentiation
• Inability To Express In Vivo Phenotype Is Attributed
To Dedifferentiation
• Still Not Clear If Dedifferentiation Occurs
– Wrong lineage expansion is a possibility
– Undifferentiated cells dominate
– Absence of appropriate inducers, hormones, matrix
• Deadaptation vs Dedifferentiation
– Deadaptation-enviroment suppresses phenotype, reversible
– Dedifferentiation-conversion to primitive phenotype,
irreversible
 Evolution Of Cell Lines

• After 1st Passage Primary Culture Becomes Cell

Line (note  Between Finite and Continuous)
• By 3rd Passage Cell Line Stabilizes
• Survival Of Stronger Might Not Necessarily Be
The Objective
• Mesenchymal Cells Usually Dominate
– Ex. Fibroblasts
• It Is Hard To Avoid Overgrowth Of Specialized
Cells (Ex. Hepatic Parenchyma)
 Evolution Of Cell Lines

• Approximately 10 Passages
• Senescence Follows
– Thought To Be Due To Telomeres
– Every Division Telomeres Shorten
– Germ, Stem Cells Use Telomerase
• Transformation Is Needed If Division Will
Continue
 Continuous Cell Lines
• Finite Cell Lines Can Change To Continuous
• Often p53 Mutation or Deletion Occurs
• Overexpression Of Telomerase
• Transformation vs Immortalization
– Transformation-additional changes in growth
characteristics
– Immortalization-infinite lifespan
• Aneuploidy Is A Characteristic Of Cont. Cell Lines
– In between diploid and tetraploid
– Heteroploidy is also observed
• Most Cells Never Become Continuous Cell Lines