The study under consideration is Development of Simulated Intestinal Fluids Containing Nutrients as Transport Media in the Caco-2 Cell Culture Model: Assessment of Cell Viability, Monolayer Integrity and Transport of a Poorly Aqueous Soluble Drug and Substrate of Efflux Mechanisms. The research was conducted by a group of scientists: Marianne L. Lind, Jette Jacobsen, Rene Holm and Anette Mullertz. The description of the study has been published in the European Journal of Pharmaceutical Sciences, Volume 3, Issue 2 (2007).
As such, it is a peer-reviewed article that, by the very nature of its publication, can be certified original. According to the study by Patel et al. (2006), in most in vitro studies of oral drug permeability, little attempt is made to reproduce the gastrointestinal lumenal environment. The current research is aimed at identifying a transport medium that would have greater physiological relevance in the realm of caco-2 cell culture studies.
As the use of physiological conditions in Caco-2 cell transport studies affects the transport of compounds, the general purpose of the research “was to identify a simulated intestinal fluid containing physiological relevant surfactants (TC and PC/lyso-PC) and nutrients compatible with the Caco-2 cell culture model and to examine the impact of the identified SIF on the transport of a poorly aqueous soluble compound and a substrate of efflux mechanisms.” (Lind et al. 2007, p. 262)
The present study extends the knowledge on the problem that other authors have reached. For example, Ingels’s and Augustijins’s researches have shown that solutions such as dimethyl sulfoxide (DMSO) or ethanol or solubilised by cyclodextrins or surfactants reduce the physiological relevance of the transport studies, and there is a need to study simulated intestinal fluids by the addition of bile salts, phospholipids and/or fatty acids (Ingels and Augustijns 2003).
Many studies have focused on the components of intestinal fluids and intestinal processes (Mansbach et al., 1975; Persson et al., 2006). Experiments on rats and Caco-2 cells conducted by Patel et al. (2006) have shown that using physiological solutions can influence permeability measurements. Studies also illustrated that absorption of lipophilic compounds, cholesterol and carotenoids were dependent on the type of phospholipid present (Hamada et al., 2005). Lyso-PC enhanced the absorption of cholesterol and carotenoids from micelles compared to PC.
In the current study, Leibovitz’s L-15 medium (L-15) has been used as the transport solution. It has been identified as the simulated intestinal fluid containing physiological relevant surfactants that are independent of the supply of CO2. By identifying such a transport solution and by examining its impact on the transport of a poorly aqueous soluble compound, the study adds to the existing literature. It further examines its impact on a substrate of efflux mechanisms.
We should admit that the authors of the report under analysis always make appropriate references to the previous studies explaining whether the current work is in line with their findings or contradicts them. For example, the authors refer to Martin et al. studies and suggest that their researches have shown that lyso-PC is toxic to cells because lyso-PC solubilises constituents of the membrane, and as a result, the cells lyse (Lind et al. 2007, p. 266). The findings of the present study are often compared with the previous ones and are given appropriate consideration. For example,
Compared to previous studies (Ingels et al., 2002), the osmolality of the media containing 15mM TC is markedly decreased from 635 to 370mOsm/kg, and pH is raised from 5.0 to 6.0, but these improvements were not sufficient to prevent decreased viability. Other studies have shown that Caco-2 cells were compatible with similar media of 15mM TC based on HBSS with an osmolality of 327–343mOsm/kg (Kataoka et al., 2006; Patel et al., 2006) (Lind et al. 2007, p. 266).
Where the study gets the findings similar to the previous researches, the report also states this. LDH release was only increased in 3 out of the 12 comparisons and was therefore considered not to have a major effect on the viability. This observation is in accordance with previous findings, which reported that mixed micelles of TC and linoleic acid do not decrease the viability of Caco-2 cells compared to TC (Meaney and O’Driscoll, 2000). The work’s relevance and close interaction with other studies contribute to the overall understanding of the problem both by the scientists and the general reader.
Though a bit difficult because of the constant use of the relevant terms, the paper is still comprehensible to the general audience. What contributes to it are the logical structure and the clear organization that the paper has. The paper contains Introduction, Materials and Methods, Results and Discussion, Conclusion, Acknowledgements and References. The report begins with an introduction that explains the important role of transport media in the absorption of a drug.
After the introduction, the materials and methods of this study are explained in detail. This explanation contains details of materials, preparation of transport media, characterization of transport media, cell culture model, the viability of caco-2 cells, viability assays, the solubility of estradiol, transport of estradiol, transport of etoposide and data analysis. We believe that these sections have sufficient information needed in terms of the studied problem.
The explanation is followed by a discussion of results regarding osmolality and stability of pH and monolayer integrity and viability of caco-2 cells, including transepithelial electrical resistance, the apical release of lactate dehydrogenase, and cellular protein content. Results regarding the transport of estradiol and etoposide are finally discussed, and conclusions are postulated.
We consider that the experiment conducted by the authors is thoroughly reported, the reader can find an exhaustive presentation on the given topic. The experiment has been covered in great detail: the composition of the apical media is presented in a tabular form; the reader is also informed about the percentage concentration of the solutions used in the experiment presented to the reader. In the case of the cell culture model, the temperature and humidity are also given.
The authors explain that Caco-2 cells were obtained from ATCC and were cultured at 37 ◦C in an atmosphere of 5% CO2/95% air and 90% relative humidity in DMEM supplemented with 1% penicillin-streptomycin, 1% non-essential amino acids, and 10% foetal calf serum. Then, the Caco-2 cells were maintained in T-75cm2 flasks and split when reaching 80–90% confluence using trypsin-EDTA. Cells, passage 30–50 and plated at a density of 105 cells/filter and cultured for 21–25 days before the initiation of an experiment. These details make it possible for the experiment to be replicated.
The experiment can be said to be fairly conclusive as it is backed up by data analysis. The obtained data have been given both in tabular form and in graphic form, increasing its comprehensibility. The experiments have been repeated on at least one additional passage number of Caco-2 cells. The statistical difference between the two mean values has been calculated using an unpaired t-test (two-tailed).
When more than two means were compared, a single factor analysis of variance followed by a Tukey’s test was used. p < 0.05 is considered statistically significant. The report can be considered complete; it even contains the details regarding the software used for statistical calculations. One of the most sensitive assays in the study is the measurement of apical LDH, which is considered indicative of cell damage. It was only by the use of LDH assay that a difference was detected between incubation with SIFs based on L-15 or HBSS.
Apart from the findings and implications of the research, the strength of the report is that where necessary, the general theory behind the reaction has always been included. It is explained that the addition of OA and MO to the SIFs could change the composition of the micelles, and different structures may induce different rates of drug transport across unstirred water layers and different access and affinity to the surface of the Caco-2 cells.
After explaining this, the report elaborates on the results. It was found that SIF composed of 5mM TC and 1.25mM also-PC only contained small micelles, and the addition of 0.5 mM OA and 0.25 mM MO increased the size slightly. The findings are conclusive and based on data and deductive reasoning. The release of LDH and the cellular protein content illustrated that SIFs containing 10mM TC and 2.5mM PC/lyso-PC or 15mM TC and 3.75mM PC/lyso-PC, induced damage to the Caco-2 cells and are therefore not suitable as transport media in this screening model.
As all experiments were repeated on at least one additional passage number of Caco-2 cells, we suggest that there are no serious omissions of the results. The conclusion made justifies the results achieved and, what is more, important it stresses the practical implication of the results:
Using this new transport medium for transport studies in the Caco-2 cell culture model, it is possible to use a physiologically relevant concentration of the drug since the micelles are able to increase solubility (Lind et al. 2007, p. 269).
An adequate discussion of the findings is present in the third part of the report, which examines various aspects of the problem. The findings inspire future work, authors suggest that in future studies, “the use of higher concentrations of lipolysis products should be evaluated.” (Lind et al. 2007, p. 266) Further research in the field should be focused on the exploration of the uptake mechanisms, “and due to decreased accumulation of drug intracellular and enhanced recovery, it might be possible to reliably measure the transepithelial permeability.” (Lind et al. 2007, p. 269)
B) The opportunities of modern medicine are really enormous. Human knowledge does not stop discovering the mysteries that are subordinate to chemistry rules. At the current stage of medicine development, special consideration is given to cell studies. The present article is concerned with colon cancer cells, or, to be more exact, with the Caco-2 cell culture model and the search for a new transport medium that can be used in transport studies.
Caco-2 cells are colon cancer cells that are similar to intestinal cells and can be grown in the lab relatively quickly. The simplicity of the experiments aimed at growing these cells allows the scientists to use them for the study of the absorption of drugs in the intestine.
During their growth, these Caco-2 cells form many layers with minute projections on the surface, also known as monolayers with microvillous structures. These layers can act as carrying media for sugars, amino acids and several drugs.
Most Caco-2 cell transport studies are performed with salt solutions or using organic solvents. As these solutions differ from intestinal fluids scientists, try to use simulated intestinal fluids.
During the absorption process in the intestine, mixed micelles are formed. These cells can dissolve drugs that dissolve in fats. The first simulated intestinal fluids (SIFs) were found in 1998 and were used to predict the dissolution of drugs within the intestine. Two kinds of such fluids were used for experiments: fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF). Of these two SIFs, it has been found that FaSSIF was compatible with Caco-2 while FeSSIF was found to be poisonous. Also, fluids from the human duodenum and jejunum have been collected, diluted and used as transport media.
A compound phospholipid known as lyso-PC is commonly used in the simulated intestinal fluids we have described above. Studies in rats and Caco-2 cells have shown that absorption of compounds is dependent on the type of phospholipid present. When Lyso-PC is used in the simulated intestinal fluids, the absorption of drugs is much enhanced compared to the absorption level when PC is added to the fluid. This indicates that including lyso-PC in the simulated intestinal fluid in the Caco-2 cell culture model will make the fluid behave more like the natural intestinal fluids.
When a solution is prepared to mimic the small intestinal fluids, fats and results of its reaction with drugs must be added to it. To add bile salts and lyso-PC to the simulated intestinal fluid in the Caco-2 cell culture model, it is necessary to increase the cell strength by adding nutrients, for example, the Leibovitz’s L-15 medium (L-15), that was used in the study conducted by a group of scientists: M. Lind, J. Jacobsen, R. Holm and A. Mullertz (Lind et al.)
This study had a two-fold purpose: to identify simulated intestinal fluids containing nutrients compatible with the Caco-2 cell culture model and to examine the impact of the identified medium on the absorption of a compound called estradiol and its impact on absorption reactions during absorbing etoposide. The identified medium was Leibovitz L-15.
It has been found that the viability of Caco-2 cells was enhanced by the use of the CO2 independent nutritional medium, Leibovitz’s L-15 compared to Hanks’ balanced salt solution. Moreover, it was found that the addition of lipolysis products to this medium did not affect the strength of Caco-2 cells. The presence of SIFs was shown to decrease absorption of estradiol due to the incorporation of estradiol in micelles, and hence there is less amount of free estradiol for absorption purposes. Further, there was a change in the apparent permeability of etoposide, which indicated that components in the SIFs affect absorption mechanisms.
Thus, this new transport medium can be used in transport studies in the Caco-2 cell culture model in a physiologically relevant manner. Moreover, the micelles will increase their solubility. Studies are needed for further exploration of the uptake mechanisms that will help to reliably measure the transepithelial permeability.
Works Cited
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Ingels, F.M., Augustijns, P.F. (2003). Biological, pharmaceutical, and analytical considerations with respect to the transport media used in the absorption screening system, Caco-2. J. Pharm. Sci. 92, 1545–1558.
Lind, L. Marianne, Jacobsen, Jette, Holm, Rene and Mullertz, Anette (2007). Development of simulated intestinal fluids containing nutrients as transport media in the Caco-2 cell culture model: Assessment of cell viability, monolayer integrity and transport of a poorly aqueous soluble drug and a substrate of efflux mechanisms. European Journal of Pharmaceutical Sciences. Vol. 3. Issue 2. pages 261–270.
Mansbach, C.M.; Cohen, R.S.; Leff, P.B. (1975). Isolation and properties of the mixed lipid micelles present in intestinal content during fat digestion in man. J. Clin. Invest. 56, 781–791.
Meaney, C.M. and O’Driscoll, C.M. (2000). A comparison of the permeation enhancement potential of simple bile salt and mixed bile salt:fatty acid micellar systems using the CaCo-2 cell culture model. Int. J. Pharm. 207, 21–30.
Patel, N.; Forbes, B.; Eskola, S.; Murray, J. (2006). Use of simulated intestinal fluids with Caco-2 cells in rat ileum. Drug Dev. Ind. Pharm. 32, 151–161.
Persson, E.M.; Nilsson, R.G.; Hansson, G.I.; Lofgren, L.J.; Liback, F.; Knutson, L.; Abrahamsson, B.; Lennernas, H. (2006). A clinical single-pass perfusion investigation of the dynamic in vivo secretory response to a dietary meal in human proximal small intestine. Pharm. Res. 23, 742–751.