SIO2004 Animal Cell Culture Lecture 5

SIO2004 Animal Cell and Tissue Culture, Lecture 5

• Course: Biotechnology Program
• University: Universiti Malaya
• Instructor: Dr. Nuradilla Mohamad Fauzi

Maintaining Cell Cultures

• Maintaining cell cultures involves keeping cultured cells alive.
• Media changes are crucial for replenishing nutrients and removing harmful metabolic byproducts and dead cells.
• Adherent cultures have media removed and replaced through aspiration.
• Suspension cultures require separation of cells from the media via centrifugation, then resuspension in fresh media.

Checking Cell Morphology

• Regularly examine cell morphology (shape and appearance) under a microscope to assess successful cell culture experiments.
• Healthy cells exhibit no granularity around the nucleus, remain attached to the substrate, and have no cytoplasmic vacuolation.
• Signs of deterioration (granularity, detachment, vacuolation) should signal potential issues such as contamination, senescence, toxins in the medium, or the need for a medium change.

Cell Types

• Primary culture: Freshly isolated from a tissue source.
• Cell lines:
  • Finite cell lines have a limited lifespan and die after several subcultures.
  • Continuous cell lines are "immortal" and cancer-derived or transformed, able to be passaged and maintained.
  • General rule: Cells that can divide can be passaged and maintained (not all).

Contact Inhibition and Confluence

• Contact inhibition: Cell types avoiding each other, slowing proliferation.
• Confluence: Cell layer covers the dish surface; cells stop dividing.
• Confluence is used to estimate the number of adherent cells.

Subculturing / Passaging

• At high confluence, cells become crowded, nutrients become limited, and cells may undergo differentiation or death. Passaging is needed.
• Methods for detaching adherent cells include:
  • Enzymatic methods (Trypsin, Trypsin-EDTA, Dispase)
  • Mechanical methods (scraping, cell scrapers)
• Suspension cells are subcultured by taking a small sample and diluting to fresh medium.

Cryopreservation

• Freeze cells to maintain them when not needed.
• Cryoprotectants like DMSO prevent ice crystal formation during the process.
• Freeze at 1°C per minute.
• Store in liquid nitrogen.

Cell Cycle

• Cells regenerate from existing cells.
• Eukaryotic cell cycle is an orderly sequence of events where cells duplicate contents then divide.
• The cell cycle control system (regulatory proteins) governs progression and responds to internal and external signals.

Cell Cycle Phases

• Key phases (from figures): G1, S, G2, M.

• G0 phase: Cells that aren't dividing.

• Senescence: A non-dividing state entered by cells.

Telomeres

• Telomeres are repetitive sequences at chromosome ends.
• Protect chromosomal ends from degradation or fusion.
• Telomeres shorten with each cell division.
• Telomeres reach a critical length when cells stop dividing.
• Some cells activate telomerase enzyme to extend telomeres, therefore allowing continued cell division.

Telomerase

• Telomerase is a reverse transcriptase enzyme adding to telomeres.
• Prevents degradation of chromosomal ends.
• Its expression regulates development.
• Post-natal somatic cells often lack telomerase activity.

Cells that Express Telomerase

• Fetal tissues, adult germ cells, stem cells, cancer, transformed cells, express telomerase to extend telomere lifespans.

Explore the essential techniques in maintaining animal cell cultures in this lecture from the Biotechnology Program at Universiti Malaya. Understand the processes of media changes and the importance of monitoring cell morphology for successful cultures. The quiz will cover adherent and suspension cultures and their specific requirements.