Ovarian Cancer Lecture 2.doc

4 Pages
Unlock Document

Western University
Anatomy and Cell Biology
Anatomy and Cell Biology 4461B
Geordie Shepherd

TS Lecture 2: Cell Culture Systems Generating Cell Culture Systems Using Cells from Patients • Isolate pure cultures of epithelial cells from normal tissue Isolate pure cultures of epithelial ovarian cancer cells from • • Malignant ascites Dissociated solid tumor tissue • • Primary culture: cells directly isolated from tissue • Advantages • Samples are more representative of a patient • Amenable to molecular characterization • Disadvantages • Primary cells are more difficult to manipulate • Limited lifespan • Possess high patient to patient variability • Cell Lines: immortalized cells derived from primary culture • Advantages • Well-characterized and very consistent • Long-lived • Can manipulate easily • Disadvantages • Cell lines are far removed from original patient • Need to consider: contamination, lifespan in culture, stability of genotype and phe- notype Isolation and Primary Culture of Human Ovarian Surface Epithelium (OSE) Cells • Receive ovarian biopsy from patients undergoing oophorectomy for benign conditions • Incubate in collagenase to detach surface epithelial cells from ovarian surface • Plate cells on growth factor rich culture media and allow primary cells to attach to tissue culture plastic. • Typically come off in sheets and will attach as “nests” of cells Slowly proliferate over several days to form monolayer • • Difficulties include: • Scraping can lead to contamination with underlying stromal cells • Eventually senesce Immortalization of OSE Cells into Cell Lines • Spontaneous Immortalization: cultured primary cells will undergo “crisis” where most cells die, some escape and become immortal. It is expensive and time-consuming. • Passage through immuno-compromised mice as tumor xenografts: rare cells will emerge like in spontaneous immortalization Ectopic Expression of oncogenic transforming proteins • SV40 T Antigen • Inactivates p53 and pRb • Temperature-sensitive mutants facilitate reversal of immortalization • HPV E6 and E7 • Inactivates p53 and pRb • hTERT • Human telomerase reverse transcriptase • Regenerates telomerase at the termini of chromosomes to bypass senescence and render primary human cells immortal Example of Immortalization • Karst et al isolated human fallopian tube epithelial cells • Immortalized using SV40 T antigen and hTERT • Transformed using oncogenic Ras or Myc • Xenograft the cell lines into nude mice: tumors that arise are histologically similar to high- grade serous ovarian cancer Ascites Fluid - ovarian cancer cells exist in ascites as single cells or multicellular spheroids • Cellular Fraction • ovarian cancer cells • red blood cells, white blood cells • mesothelial cells • Acellular Fraction • Proangiogenic factors • Growth factors • Cytokines • Matrix proteins • Isolation and primary culture of cells from patient ascites • Receive ascites from operating room or the clinic (paracentesis) • Mix with cell culture media and plate into tissue culture flasks • Better yields achieved if patients are not undergoing chemotherapy • Leave for three days then change
More Less

Related notes for Anatomy and Cell Biology 4461B

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.