Lecture 8 Flashcards
define the terms “clone” and “clonality” in the contest of cell culture
clone/clonality: group of identical cells with common ancestry, i.e. derived from the same cell
clonality: state of a cell/animal being derived from a single cell
clone: group of cells/organisms descended from and genetically identical to a single progenitor. the process of producing a group of cells that are genetically identical
list reasons for isolating cell clones
to isolate cells with special properties (eg cancer cells)
reduce heterogeneity of a culture, prevent wrong lineage from dominating over time
some assays need a homogenous population (survival assays, optimization of growth conditions, chemrsensitivity, radiosensitivity)
to select for a desire trait, eg genetic manipulation like immortalization
briefly describe what Hybridoma cells are
Hybridoma is the product of a mouse injected with antigen that then produces B-cells which are harvested and fused with immortal B-cell cancer cells. The mouse B-cell and immortal B-cell fusion is called a Hybridoma. they are tested for production of specific and single antibody
describe why it is not possible to isolate cell clones from primary cells
primary cells are not immortal. By the time the single cell you started out with has doubled enough times to grow a culture (10^6 cells) it will probably start to die as it reaches the hayflick limit (20-80 PDs)
describe the procedure for cloning adherent cells and for cloning suspension cells
adherent: use cell culture dishes, multi well plates, or flasks. it is easy to discern individual colonies. use dilution cloning
suspension: must seed cells into gel (agar/agarose) or viscous solution (methocel). methocel has fewer impurities than agar. perform an assay to detect transformed status of cells. use dilution cloning
list experimental considerations and conditions used to improve clonal growth
- rich medium with high serum
- Serums: FBS > CS > horse. pick the best stuff for your cells
- insulin hormone increases plating efficiency. hydrocortisone analogs also can enhance plating efficiency, but not all. use the best stuff for your cells
- supplement medium with keto-acids (eg pyruvate) and nucleosides. intermediary metabolites
- CO2 is essential for cloning efficiency. 5% is good or 20 mM HEPES (advantage of HEPES is cells can be out of incubator for long periods of time)
- treat vessel substrate with Poly-L-Lys to improve cell adherence
- purified trypsin is better than crude. cold is better than warm. recombinant trypsin (TrypZean and TrypLE) can be more gentle, pure, and room temp stable
describe how feeder layers work. what are benefits and considerations?
feeder layers provide nutrients, growth factors, and matrix constituents and help overcome cell inability to survive at low cell densities.
arrest the feeder layer by irradiation or mytomycin C. then seed the dilute cloning cell suspension with the feeder cells and incubate to allow colonies to form of clones. feeder cells are viable up to 3 weeks
describe how selective media are used to isolate cell clones
use a media that kills cells that are not the ones you want, useful for transfected cells that obtain drug resistance via a plasmid
positive and negative selection (select for the transfected cell or select against it)
selective substrates is also an option, they confer selective adhesion, detachment, feeder layers, and semisolid media (agar)
describe Microtitration plates
a specific strategy in dilution cloning used for suspension cells. the plates have many small wells and you must seed the plate for an average of 1 cell per 10 wells to ensure the cells are isolated. they will grow in their individual wells and can be checked for growth, trypsinized, and used as a clonal colony.
describe Microtitration plates
a specific strategy in dilution cloning used for suspension cells. the plates have many small wells and you must seed the plate for an average of 1 cell per 10 wells to ensure the cells are isolated. they will grow in their individual wells and can be checked for growth, trypsinized, and used as a clonal colony.
what is the conditioned medium considerations when cloning? what type of cells would you use for embryonic vs keratinocytes vs leukemic
using conditioned media (that has been used to grow other cells) can improve plating efficiency when diluted into regular growth medium. Must be clean!
Consider the conditioning cell type:
MEFs, BRL, STO are good for embryonic
3T3 is for keratinocytes
5637 is for leukemic cells
start collecting at late log/early plateau phase and condition for 4 days. collect multiple times and pool, avoid carry over of cells by filter or freeze/thaw sterilizing
what is the conditioned medium considerations when cloning? what type of cells would you use for embryonic vs keratinocytes vs leukemic
using conditioned media (that has been used to grow other cells) can improve plating efficiency when diluted into regular growth medium. Must be clean!
Consider the conditioning cell type:
MEFs, BRL, STO are good for embryonic
3T3 is for keratinocytes
5637 is for leukemic cells
start collecting at late log/early plateau phase and condition for 4 days. collect multiple times and pool, avoid carry over of cells by filter or freeze/thaw sterilizing
what additional things can be done to improve suspension cloning?
all the same as for monolayer and also sulfhydryl compounds can be helpful (mercaptoethanol, glutathione)
In general, suspension cloning is less efficient than monolayer. But isolation of colonies is much easier in suspension
options for isolation of monolayer clones
trypsin for Microtitration plates
cloning rings for dishes (literally just small rings that act as a barrier so you can trypsinize and remove)
flasks with top film
opticell chamber
seed on coverslips or fragments
capillary technique: grow colonies inside capillary tube then break on either side of colony and transfer to fresh plate
