Stability, Consistency and Reproducibility
Many tumor-derived cells have been reported to show lack of culture stability and reproducibility based on the existence of irregular growth and non-specific genetic alterations (Lipps et al. 2013). To minimize these handicaps, the establishment of large cell banks is recommended to allow disposing cells at early and similar passages. On the contrary, the consistency of the assumed cell properties should be verified for highly different passages. For instance, Chen et al. (1987) proved that karyotypes were comparable for HT29 cells along more than 100 passages, which represents valuable data in the assessment of the cell line evolution. Likewise, derived cells adapted by specific treatments must be evaluated under different culture conditions as time and presence/absence of responsible agent(s). Lesuffleur et al. (1990) tested the irreversibility of the differentiation of MTX adapted cells by the comparison of the growth curve for several passages. No significant differences were observed for both 10−7 and 10−6 M MTX-derived cell lines between cells cultured with drugenriched media and drug-free media. The stability of other differentiation characteristics was also confirmed for MTX-free cells compared with equivalent MTX-treated cells as well as themselves along several passages before.
Relevance to Human In Vivo Situation
As in other cancer-derived cell line models, significant differences in the gene expression of transporters and metabolic enzymes from the normal human intestinal cells can affect the suitability of the model in reflecting the in vivo permeability (Langerholc et al. 2011). Similar protein expression was found in HT29 cells and the corresponding intestinal scrapings, of which some appear to be characteristic to human intestinal epithelium in vivo (Lenaerts et al. 2007). Nevertheless, several proteins, including transporters, were overor underexpressed. Bourgine et al. (2012) compared the expression of 377 genes in HT29 and other intestinal cell lines that are used as in vitro models of the epithelium with the corresponding tissue biopsy. The results showed that differentiated HT29 cells and human colonic tissues do not appear to be significantly different.
The use of HT29 as in vitro model of intestinal cells has some advantages and limitations that have been summarized by Zweibaum et al. (2011). This cell line in its differentiated phenotype is similar to small intestine enterocytes with respect their structure, and the presence of brush border-associated hydrolases and the time course of the differentiation process which is also comparable to that found in the small intestine. In addition, the amount of villin expressed in differentiated HT29 cells is close to the value observed for normal freshly prepared colonocytes. However, these cells also have some limitations, because (1) they are malignant cells with a high rate of glucose consumption and an impairment of the metabolism of glucose; (2) although they mimic some characteristics of small intestine enterocytes, they are colonic cells; (3) they cannot be compared with enterocytes from normal colon since they express brush border-associated hydrolases; (4) but they cannot be compared with absorptive enterocytes because not all hydrolases are present (e.g. lactase and maltase-glucoamylase are absent) and the ion transport properties are different. It has been postulated that these cells are close to human fetal colonic cells because of the type of hydrolases present and the intracellular concentration of glycogen accumulated (Hekmati et al. 1990).
Regarding the expression of cell surface receptors, it has been reported that differentiated HT29 cells express several receptors for peptides, such as vasoactive intestinal peptide, or insulin but also for non-peptide substances. In general, the receptors found in this cell line have their equivalent in normal intestinal cells, except for the receptor to neurotensin, which has been characterised in HT29 cells (Kitabgi et al. 1980), but it is not detectable in normal human colonic epithelium. On the contrary, receptors for peptide YY or neuropeptide Y which are well characterised in normal small intestine epithelial cells have not been reported in HT29. It has to be taken into account that these receptors are located at the small intestine and that HT29 are of colonic origin. It is also remarkable that when using cell cultures, the expression of a particular receptor may depend on the degree of cell differentiation and the growing conditions. Recently, the presence of opioid, serotonin, muscarinic, PPARβ/δ receptors has been reported in this cell line (Zoghbi et al. 2006; Ataee et al. 2010; Belo et al. 2011; Foreman et al. 2011). Information on the studies on receptors carried out in this cell line can be found at Zweibaum et al. (2011).