Lecture 1a: Cell and Tissue Flashcards
Define cell culture
the maintenance and growth of the cells of multicellular organisms outside the body under precise conditions of temperature, humidity, nutrition, and freedom from contamination
Define tissue culture
fragments of tissue from an animal or plant are transferred to an artificial environment where they can continue to survive and function. The cultured tissue may consist of a single cell, a pop of cells, or a whole part of an organ
Primary cell culture
first line of cells taken directly from the target
Secondary cell culture. What do they tend to be?
Primary cell cultures that have been sub-cultured
For example, they have been split and transferred into fresh media. They tend homogenous
What is a cell line?
a cell culture capable of continued growth and passage
What are the two types of cells lines?
Finite cells lines
Continuous cell lines
Finite cell lines (4). Give two examples
- Limited replicative potential
- 20-100 generations before senescence (cell cycle arrest - stop dividing)
- Contact inhibition and anchorage dependence (need a solid surface)
- Grow slowly - double in 24-96hours
Ex? WI-38, HUVEC
Continuous cell lines 4
- Indef replication
- No contact inhibition nor anchorage dep
- Growth rate is rapid - 12-24 hrs
- Also know as “transformed”
Ex/ HeLa cells
Transformed cells have undergone a ______ change in _____ through a _______ ____
permanent, phenotype, genetic transformation
What is the most common transformation?
Viral
How does the virus SV40 transform cells?
SV40 T antigen distrupts the host cell tumor suppressor proteins RB and p53, by binding to them and deregulating cell prolif and cell cycle progression. Cells are not immortal
Papillomavirus produces ___ and ___ which bind the the host cells p53 and RB proteins
E6 and E7
E6 protein binding leads to the _______ of ______
ubiquination of p53
E7 binding to ___ prevents its action as a brake on cell prolid
Rb
What can be used instead of or in addition to viral oncogenes?
hTERT human telomerase reverse transcriptase
Why is hTERT useful?
It is useful because it can yield cells that behave like primary cultures but propagate like immortalized cell lines. It can establish non-cancer, normal cell line
HeLa cells
Human cervical cells transformed by HPV18
Jurkat cells
Derived from human T-cell leukemia and grown in suspension culture
HEK293 cells
from human embryonic kidney and transformed by adenovirus
What are advantages of HEK293 cells?
They are easy to grow, transfect
They are used lots in transfection-based experiments, protein expression, and vaccine production
HUVEC cells
primary cell line
Human umbilical vein endothelial cells
Used in CVD and solid tumor progression
Transfection
Overexpress a protein of interest
Knock-down expression of a protein of interest (siRNA)
Transient vs stable
What are 4 methods of transfection
Calcium phosphate
Lipid based (lipofectionO)
Electroporation
Viral systems
What can be used to increase the surface area and increase the number of cells?
Polystyrene beads
How do HEK293 grow?
In a monolayer on a coated surface (polystyrene beads)
Why do tissue culture plates/flasks not accurately mimic in vivo conditions?
Cells do not normally grow in a monolayer in an organism
Matrix environment mimics..
native cell-cell contact and signaling
What are some considerations for stem cell cultures? 5
Theoretical infinite lifespan
Specialized media
Prone to contamination
“feeder cells” need food
Induced differentiation
List some pros and cons of tissue cultures
Pros
Low cost
minimal time involved
easily manipulated
cell type specificity
Cons
contamination
physiological relevance??
Not every cells type is culturable
List some pros and cons of animal models
Pros
True in vivo model
physiologically relevant
knock in and knock out models
Tissue, organ, and whole organism anaylses
Cons
$$$
Very time consuming
ethics
Transformed cells have ____ gene expression
altered
Do transformed cells have contact inhibition?
no
What type of lifespan do transformed cells have?
infinite
What type of lifespan do primary cells have?
Finite
What type of gene expression occurs in primary cells?
normal
Quiescence
lack of nutriton and grown factors and the cell goes into a quiet state where it is not in the cell cycle but it can divide
What are the 3 main issues with cell lines?
- All cell lines exhibit altered expression of p16
which is a CDk inhibitor and is responsible for cell cycle control - Cell culture does not mimic native environment
- Altered function of p53 and Rb tumour suppressors
What affects p53?
E1A, SV40 T-antigen
What affects Rb?
E7
Normal cells have competent ____ whereas transformed cells do not
growth arrest, growth arrest
