Evidence for diminished functional expression of intestinal transporters in Caco-2 cell monolayers at high passages.
OBJECTIVE
To investigate the effects of passaging on the intrinsic membrane transport parameters of compounds absorbed by means of passive and carrier-mediated processes in the Caco-2 cell line.


METHODS
Caco-2 cells at low (28-36) and high (93-108) passage numbers were used to evaluate the transport characteristics of model compounds for paracellular diffusion (mannitol), transcellular diffusion (progesterone) and carrier-mediated transport (cephalexin, cephradine, phenylalanine, proline, and taurocholic acid) using side-by-side diffusion chambers. Intrinsic intestinal transport parameters were determined by correcting the effective permeability for potential biases introduced by the microporous filter and aqueous boundary layer. Intrinsic maximal flux (Jmax), Michaelis constant (K(m)) and carrier permeability (Pc) were determined as a function of passage number.


RESULTS
Compared to the low passaged cells, the high passaged Caco-2 cells were characterized by less morphological heterogeneity, higher transepithelial electrical resistance, higher transcellular diffusion, lower paracellular diffusion, lower carrier-mediated transport and lower alkaline phosphatase activity. The use of effective transport parameters overestimated the K(m) and underestimated Pc but had no effect on Jmax.


CONCLUSIONS
The current results provide experimental evidence that the passaging process significantly affects the biological characteristics and transport properties of Caco-2 cell monolayers. The effects are consistent with a reduction in the functional expression of a brush border enzyme and several transport proteins as passage number is increased. The underlying basis for this appears to be a selection of fast-growing subpopulations from the original heterogeneous Caco-2 cell line during passaging.
Yu
H
H
College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855, USA.
Cook
T J
TJ
Sinko
P J
PJ
D000469:Alkaline Phosphatase / Q000378:metabolism; D001692:Biological Transport; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002452:Cell Count; D002463:Cell Membrane Permeability; D004058:Diffusion; D017097:Electric Impedance; D004848:Epithelium / Q000502:physiology; D006801:Humans
D002352:Carrier Proteins; D000469:Alkaline Phosphatase