Week 1 Flashcards
(14 cards)
Why cell cultures are used
Allow researchers to test substances on the human body. More ethical and related to humans. Fullfls the 3 Rs replacement, reduction, refinement.
The basics of growing cells
Need a very specific culture to survive and grow. Require a rich culture medium to maintain the right temperature 37C and a 5% CO2 to help regulate pH. Similarity must be controlled between 260-320. If not maintained cells could stop growing or die
Equipment
A laminar flow hood is used to protect cells from airborne microbes. Researchers use sterile pipettes, culture flasks, plates and centrifuges. Ethanol wipe surfaces and gloves are used. Incubators are used to maintain optimal temperature and CO2 levels
Types of growth surfaces
Attach to a solid surface from treated plastics like polystyrene. Some cells need and EMC like collagen to mimic human conditions.
Culture medium
Include essential nutrients like amino acids, sugars, fatty acids, salts for osmolaorty and trace elements. They may add fetal bovine serum which contains hormones, growth factors, proteins. May include antibiotics to prevent contamination, fungicides, pH buffers like HEPES and phenol red
PH and osmolarity
Maintaining pH at 7.4 is the best. If pH drifts away from 6.5-7.7 cells become stressed and may die. Cells that overthrow can acidify the medium by producing lactate. PH is regulated using bicarbonate buffer system. CO2 dissolves in water to form carbonic acid and bicarbonate keeps the pH steady.
Fetal Calf Serum
FBS is used to provide hormones, lipids, proteins, and growth factors that help cells grow. The composition between batches however varies too much and it’s also ethically controversial. There’s a risk of pathogen contamination
Contamination
Contamination include bacteria, yeast and mycoplasma. Mycoplasma is sneaky as it’s too small to see and can subtly alter cell behaviour by changing gene expression. It’s detected through PCR or fluorescent DNA staining and must be regularly screened for in long term cultures
Antibiotics
Usually added to prevent bacterial contamination. Aren’t necessary and can slow down cell growth, overuse can lead to antibiotic resistance. Primary cells are sensitive and Benefit from short term antibiotic use.
Types of cell culture
Primary cells - taken directly from tissues and closely mimic in vivo physiology, but have limited lifespans
Cell lines- modified to grow indefinitely - either spontaneously through viral oncogenes or introducing telomerase
Stem cells- are capable of differentiating into many cell types
Primary vs immortalised cells
Primary cells respond like real tissue. They are hard to maintain don’t divide much and lose functionality over time. Immortalised cells grow indefinitely and are easier to standardise through testing. They behave abnormally due to mutations and genetic instability
Cellular aging
Normals cells eventually get too short and the cell enters senscense as the telomeres shorten. Cancer cells and stem cells get around this limit by activating enzyme telomerase.
Immortalisation methods
Immortalised cell lines can come from spontaneous mutation, cancer or intentional genetic modification. Some methods include oncogenes from viruses like SV40 EBV or HPV. Another strategy is to overexpress telomerase
Comparing cancer vs non-cancer lines
Cancer lines grow rapidly and lack tumor suppressor genes like p53. They’re unstable and consume nutrient quickly. Non-cancerous are more stable retain physiological traits and are often preferable when studying toxicology
