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Permeability Assays using Caco-2 Cells - Coggle Diagram
Permeability Assays using Caco-2 Cells
Introduction
Caco-2 cells
immortalized cell-line of human colorectal adenocarcinoma cells
Characteristics that make into an enterocyte-like cell
grows as a polarize cell layer
has apical brush border villi
has intercellular tight junction complexes
expresses many of the enzyme markers and transport systems of primary epithelial cells
bidirectional transport studies
absorptive
secretory
properties
growth
adherent monolayer of epithelial cells
differentiation
takes 14-21 days after confluence under standard culture conditions
cell morphology
polarized cells with tight junctions under standard culture conditions
electrical parameters
high electrical resistance
Cultivation and Culturing
thawing
cell lines are shipped frozen on dry ice in cryopreservation vials
storage at temperatures below -130C
thaw the vial by gentle agitation in 37C water bath
culturing the cells
must be done in a laminar flow hood
should be carried out under strict aseptic conditions
prior to the operations, disinfect materials that will brought into the good
always wear gloves
transfer the vial contents to a centrifuge tube containing complete culture medium
spin for 5-7mins
discard supernatant
resuspend the cell pellet with the recommended complete medium and dispense into a 75cm Tflask
DMEM (dubelco's modified eagle medium)
nutrient supply
FBS (fetal bovine serum
growth factors
antibiotic (penicilin and strep)
incabate the cells at 37C in a 5% CO2 incubator
passaging the cells
the growth behavior of the cells are best shown through a semi-log plot: lag, log: stationary
log phase for subculture
process
tissue minced for culturing
disaggregation by use of enzyme
cells inoculated in fresh culture medium
confluent culture; cell separated using enzymatic disaggregation
cryopresevation of cells for further use
subculturing or passaging
confluence
refers to the percentage of the surface that is already covered
when the cells reach 80% confluency, perform subcultirng or passaging
remove the media from T75 flask by suction
add 10mL PBS slowly -> remove PBS
maintain isotonic envi
add 2mL typsin-EDTA
degrade enzymes in the wall
add 10mL complete mediu. transfer to centrifuge tube
add fresh media to anew t flask
get 1.5mL of the suspension and put it in the t- flask. make replicates
incubate
cell counting
reperform trypsinization, and resuspend into a new media
transfer a small amount to an eppendorf tube
mix 10uL of the cell suspension with 10uL of trrypan blue -> transfer to a hemocytometer
each well should contain 80k cells (optimized cell density)
seeding the cells in transwell plates
change media every 2-3days (every 48h)
assessment of the cell monolayer integrity
after 21 days, replace the old media inn both chambers with new opnes
assess the monolayer integrity: TEER > 300ohms/cm2
Permeability Assay Procedures
Buffer solutions
Hank's balanced salt solution (HBSS)
isotonic soln used to maintain osmalality and pH in biological applications; this include glucose and sodium bicarbonate for shortterm maintenance of cells outside of the growth medium
Donor solutions
Test drug
dissolve the compound of interest in a buffered HBSS at the desired concentration
high-permeability IS
C14 mannitol
replace the media in the transwell plate chambers with the following
AB (A->B)
1.5mL of HBSS
300uL donor soln
BA (B->A)
1.5mL donor soln
300ul HBSS buffer
sampling
incabate the plates at 37 C
take 100uL samples in the receiving chamber during the following recommended time points: 15, 30, 45, 60, 90 and 120min
take 100uL samples in the donor chamber after 120min
analysis
drug transport studies method validation
the suitability of caco2 cell should be demonstrated by establishing a rank-order relationship between experimental permeability values and the extent of drug absorption in human subjects using zero, low (<50%), medium (50-84%), and high (>= 85) permeability model drugs