Caco-2 cells, originally isolated in the year 1977, are the immortalized cell of heterogeneous human epithelial colorectal adenocarcinoma cells. Enterocytes or mucus cell features are “expressed differentially by the human Caco-2 cells that are characteristic of a mature intestinal cell.” Caco-2 cells are grown from a stock cells and such a well-defined and studied process of growing the cells is essential for productive use of Caco-2 cells in any study or research. Iron is an essential micronutrient that is involved in oxygen transport (Webb, 1992) and energy metabolism (Beard and Dawson, 1996) and since Caco-2 cells grown on a well define media is used as a rapid screening model they can be used to predict the absorption of potential iron availability. The serum used for growing the Caco-2 cells and the method of inactivation is a critical step in the growth process.
To determine if using heat inactivated foetal bovine serum (FBS) in comparison with non-heat inactivated FBS affected Caco-2 cell growth as measured by cell count and viability studies.
To determine if cell culture media ascorbate levels made with heat inactivated FBS were different compared to cell culture media made with non-heat inactivated FBS using High Pressure Liquid Chromatography and enzyme assay to measure ascorbate levels.
Hypothesis: – Caco-2 cell growth will be improved with the use of non-heat inactivated FBS compared with heat inactivated foetal bovine serum.
Cell culture media ascorbate levels will be higher in media made with non-heat inactivated FBS compared with heat inactivated FBS.
Methods and Material
- Caco-2 cells, obtained from (American Type Culture Collection) ATCC are grown as a confluent monolayer on a filter inserts (e.g. 96 well plates) and 6 well plates and their viability is assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay.
- HPLC analysis of Vitamin C in heat inactivated and non-heat inactivated FBS.
- EnzyChrom Ascorbic Acid Assay Kit method is employed in here to predict the presence of ascorbic acid (Vitamin C) in the Heat inactivated Foetal Bovine Serum (FBS) and non-heat inactivated Foetal Bovine Serum. The detailed methods of each of them have been given later below their respective titles.
Discussion: – Graphs show no clear evidence of growth of the cells in either of the heat inactivated FBS or the non-heat inactivated FBS. But there is an impact of the non-heat inactivated FBS media in the beginning indicating it suitable for the growth of the Caco-2 cells. No evidence of Vitamin C presence by the HPLC analysis of Vitamin C and by Ascorbic Acid Determination Using EnzyChrom Ascorbic Acid Assay Kit.
Conclusion: – The findings supports the recommendation made by ATCC that non-heat inactivated FBS is suitable for the growth of the Caco-2 cells.
Tables of Content (Jump to)
Caco-2 cells, originally isolated in the year 1977, are the continuous cell line of human epithelial colorectal adenocarcinoma cells. The parenteral cell line when grown under specific culture condition undergoes spontaneous differentiation which leads to formation of monolayer of cells, expressing several morphological and functional characteristics of a mature enterocyte present in the small intestine. Caco-2 cells express tight junctions, microvilli, and a number of enzymes and transporters that are characteristic of enterocytes: peptidase, esterase, P-glycoprotein, uptake transporters for amino acids, bile acids carboxylic acid. Hence Caco-2 cell is widely used as an in-vitro model in the pharmaceutical industries to predict the absorption of orally administered drugs through the human intestinal mucosa.
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Caco-2 cells are used as a confluent monolayer grown on a filter inserts (e.g. 96 well plates) and are not used as a single cell. Maintenance of the Caco-2 cells on these filter inserts allows to carry out the transport studies across cell monolayer and it has also to facilitate cell differentiation and polarity. A polarized epithelial monolayer of cell is formed when the Caco-2 cells are cultured in the above format providing a physical and biochemical barrier to the passage of ions and small molecules.
The secretion of iron is poorly regulated but its absorption is essential for maintenance of iron level in the body. Iron absorption is affected by many dietary factors including the low molecular weight substances like ascorbic acid, which not only acts as a weak chelator but also as a reducing agent, has shown to increase its transport across mucosal cell layer. The absorption of iron, entering the duodenum is greatly influenced by its physical state. In diet, iron exits in two forms, heme and non-heme (Hallberg, L., Hulthen, L., & Garby, L., 2000). Dr. Simpson (2010) “Acid digestion of the food in the stomach releases the non-heme iron and it must be reduce to ferrous (Fe2+) ion prior to uptake by duodenal enterocytes. Ascorbic acid can reduce the ferric (Fe3+) ion in the intestinal lumen.” Gastric acid also lowers the pH in the proximal duodenum thereby enhancing the solubility and uptake of ferric iron. So when gastric acid secretion is impaired, iron absorption is also reduced. The absorption of heme iron is not less understood and it is an area of active research. It is found that heme oxygenase degrades the heme form of iron in enterocytes releasing iron ions for efflux via ferroportin resulting in iron deficiency. (Kappas et al., 1993) In the year 2005, Shayeghi and his colleagues found that a new transporter SLC46A1, transferred dietary heme into the absorptive enterocytes from the intestinal lumen. Later based upon their study they named this molecule as the heme carrier protein 1 (HCP1). Qui et al. (2006) showed that these protein primarily transferred protein and they proposed that SLC46A1 be called as proton coupled folate transporter (PCFT). Later Simpson, 2010 reviewed that in the small intestine [the iron absorption] rate by enterocytes is controlled by transporters [Divalent Metal Transporter 1] DMT1 and ferroportin [activity] in the appropriate membrane.
Light and Olson (1990) demonstrated that heme, due to its hydrophobic nature, can diffuse across the model lipid membranes and also study of Noyer et al. (1998) in isolated hepatocytes and Worthington et al. (2001) intestinal cell line Caco-2 study have demonstrated that heme uptake takes place by a saturable, carrier-mediated process indicating that it is a heme transporter. Although many studies have utilized this model in assessing the factors influencing the bioavailability of iron bioavailability, no study have been made in comparing the effect of heat inactivated FBS and non-heat inactivated FBS on ascorbate levels.
The aim of the experiment is to investigate the effect of heat inactivated FBS on ascorbate level when compared to the cell media containing the non-heat inactivated. ATCC recommends using non-heat inactivated FBS for the growth of Caco-2 cells and our project study is based not only onto research on this recommendation but find out the ascorbate level in heat inactivated FBS and non-heat inactivated FBS.
The Caco-2 monolayer has been widely used in pharmaceutical industry as an in-vitro model to predict the absorption of orally administered drugs in human intestine mucosa.
As a theoretical predictions reference model of drug absorption.
To study intestinal transporters functionality in the Caco-2 cell model.
In the study of pre-systemic drug metabolism.
More recently, they have been used to determine the efflux mechanisms of phase II conjugates of drugs and natural products.
Widely used in pharmaceutical industry as an in-vitro model to study the absorption of many drugs like antibiotics and anti-cancer and also used to study the bioavailability of minerals.
Caco-2 cell model can be used as an aid in development of formulation strategies.
Have also been used to study the transport and toxicity of nutrients and xenobiotics
Drug candidates absorption potential, the transport and the drugs and dietary mechanism can be determine using the Caco-2 cell culture.
The genetic manipulation of Caco-2 cells will advance the utility of this model in the drug development process and also to establish this model as the “gold standard” for studying intestinal disposition of drugs.
In addition to variation in different culture protocol, the maintenance and existence of Caco-2 cells in different laboratories condition and their occurrence from different clonal origin makes it extremely difficult to compare the results in literature. The difference in cell culture condition plays an important role on morphological and functional expression of Caco-2 cells related to tight junction, biotransformation and barrier function on small intestine enterocytes. Impairment in the gastric acid secretion function and also presence of heme oxygenase leads to deficiency of iron in the body. Some of the cell and culture related factors affecting the performance of Caco-2 cells are given below: –
Passage number Medium Support
Brush Border enzyme activities pH Composition Material Pore size Attachment
TEER Motility Permiability Non-specific adsorption Dome formation Attachment
Proliferation rate Proliferation Differentiation Cell density Growth Spreading
Cell Density Differentiation Morphology Transport Density
Glucose transporter expression Permiability TEER Differentiation
Table 1: – Factors affecting the performance of Caco-2 cells. Sambuy, De Angelis, Ranaldi, Scarino, Stammati and Zucco (2005).
There is no simple straight forward method for determining the number of passage to be carried out for particular cell line until a reliable and reproducible data have been obtained. The different function and activities of cell lines have shown to be greatly influenced by the number of passage. Cell lines at high passage numbers experience alterations in cell morphology, response to stimuli, growth rates, protein expression, transfection and signalling, compared to lower passage cells (ATCC, Bulletin no. 7). For example, high-passage of Caco-2 cells shows an increase in the expression of Green Fluorescence Protein (GFP) reporter gene after transfection. (Hughes et al, 2007) There has been a marked increase in the expression of sucrose-isomaltase and glucose transporter GLUT-5, the differentiation intestinal enterocytes, from early to late passages. The late passages have also increase in proliferation rate and also increase in trans-epithelial electrical resistance. The appearance of multilayer area in cell population has also been contributed to the late passage.
Even with the importance of Caco-2 cells, conflicting evidence has also been reported regarding the expression of transport properties with high passage number. The two-fold down regulation for glucose transport GLUT-3 with higher passage number has been reported. There has also been reduction in functional expression of the transporter PepT1 as the passage number increased.
Passage number has shown to influence the metabolic activity. The variability in Cytochrome P450 3A4 (CYP3A4) mRNA expression, dependant on passage number, was reported in Caco-2 cell lines. When assay was conducted about 7 days after seeding, strong difference in CYP3A4 mRNA levels was observed among the cell lines indicating the effect of passage number and/or culture condition (Nakamura et al., 2003). And also a 20-fold increase was observed in CYP3A4 mRNA in one of the lines after 28 days of culture. (Nakamura et al., 2003)
There are several recommendations by ATCC for avoiding passage dependant effects and to ensure valid and reproducible experiments
Start with high-quality cells
Cells should always be used from well-known biological resource centres (BRCs) which are likely to be better characterized and should not be removed from their original sources material as far as possible.
Optimize the cells’ environment
Careful attention needs to be given regarding the selection of the media and sera, controlling pH with buffers and gases, temperature monitoring and also attention needs to be given to the growth surface.
Optimized cell culture technique
Focus on harvesting, feeding and storing cells.
Locally derived cell line or cell lines obtained from sources other than BRCs need to be tested.
Certain tumour derived cell lines may release certain growth promoting factors in the medium which may modulate the polarization and differentiation of cells in the culture. The Caco-2 cells expresses the epidermal growth factor receptor and its affinity, level of expression and related cellular response are strongly modulated by the degree of cell differentiation and the substrate on which the cells are grown. E.g. Vitamin D receptors are present, both in differentiated and undifferentiated cells, with less binding sites in the latter than the former. When the cells were exposed to 1 alpha, 25-dihydroxyvitamin D3, cell proliferation was drastically reduced and alkaline phosphatase was significantly increased.
The cell proliferation was found to increase in dose dependant manner when insulin-like growth factor IGF-I bind specifically to Caco-2 cells. The well differentiated cell expresses more binding cells than less differentiated cells.
When Caco-2 cells, secreting the growth factor with autocrine action, added to calf serum medium, the frequency of medium change may affect the growth and differentiation of Caco-2 cells. It has also been suggested that daily medium change may increase the antioxidant defence than less frequent medium change, not affecting alkaline phosphatase activity, a marker of differentiation (Bestwick and Milne, 2001)
Anastomotic dehiscence, ischemia, and malignancy are associated with abnormalities in mucosal ph. It has been postulated that intraluminal pH influences intestinal epithelial motility, proliferation, and differentiation and studied extracellular pH (7.0-8.5) effects on human (Caco-2) intestinal epithelial motility, proliferation, and differentiation (Perdikis et al, 1998)
In addition to the cell related factor mentioned above such as passage number and pH, the cell culture can also be influenced by culture related factor such as cell replication, senescence and differentiation by the time of culture and also by seeding cell density. In order to produce the reproducible and comparable data this factors should be optimized.
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Since the differentiation process only starts when the cells reaches confluence, so the seeding density plays an important role in the time-course of expression of differentiation related traits. Seeding at very different densities and difference in days of culture may represent different differentiation stages. A range of different cell density from 1x104cells/cm2 to 5x104cells/cm2 on 96 well plates has ideally been used. Higher seeding density has also been used with the idea that all the areas in cell monolayer reach confluence at approximate same time but it leads to the formation of multilayer. This multilayer formation is also dependant on the cell as few of the Caco-2 cell line have shown that. The recent study observed no significant difference on the TEER and paracellular permeability due to seeding density on cell differentiation, while monolayer structure and carrier mediated transport were greatly affected (Behrens and Kissels, 2003). Culturing the cells for about 3 weeks with the intermediate density of 6x104cells/cm2 showed good differentiation while higher or lower density showed a down regulation of active peptide transporter HPT1 and P-gp efflux pump (Behrens and Kissels, 2003).
Caco-2 cells differentiation appears to follow the strict time schedule in the expression of morphological and biochemical characteristics of absorptive enterocytes.
The trans-epithelial passage of nutritionally relevant molecules, from intestinal lumen to the blood circulation, has been expressed by several carrier proteins on the apical and basolateral membranes. Several intestinal carriers for amino acids, sugars and for other heavy metals have been identified and functionally characterized in Caco-2 cells. Functional assays or protein and mRNA expression studies have been used to study the variation in activity and expression of these transporters during the Caco-2 cell differentiation.
Some function of the Caco-2 cells like morphology, differentiation, proliferation and permeability can be modulated by the cell culture medium, either by its physiochemical properties or by its composition. Only the sucrase activity level was found to be lower and no other major change in morphological and functional characteristics was observed when Caco-2 cells were cultured in serum free defined medium containing insulin, transferrin and selenium than in serum containing medium (Jumarie and Malo, 1991).
In the research done by Talkvist, Bowlus and Lonnerdal, 2000, Caco-2 cells were grown in a modified eagles medium (MEM) in a specific condition and after 3 weeks they were treated for 24, 72 and 168 hours with one of the following: serum-free medium (MEM), iron-supplemented serum-free medium (MEM + Fe), serum-containing medium (MEM + FBS), or iron-supplemented serum-containing medium (MEM + FBS + Fe).
Cells treated with MEM + Fe were found to have significant greater intracellular iron concentration than those treated with MEM and even significantly greater in cells treated with MEM + FBS + Fe than those treated with MEM + FBS at 168 hrs. Cells treated with iron-supplemented media (MEM + Fe and MEM + FBS + Fe) were found to have approx. 50% of both the uptake and trans-epithelial movement of iron than those compared to their respective controls (MEM and MEM + FBS) at 72 and 168 hrs.(Tallkvist, Bowlus and Lonnerdal, 2000)
It has been reported by Alvarez-Hernandez et al, 1991 that “Caco-2 cells transport iron from the apical to the basal pole in a process responsive to the valence of iron and the iron status of the cell”. Two concurrent mechanisms have been proposed to explain iron absorption by Caco-2 cells (Halleux and Schneider 1994): nonspecific paracellular transport and a more selective mechanism that is valence dependent and saturable or reaches equilibrium between uptake at the apical pole and excretion at the basolateral pole.
The medium used for the growth of Caco-2 cells in this project are heat inactivated foetal bovine serum (FBS) and non-heat inactivated foetal bovine serum.
Heat inactivation is a process in which finished serum is placed in a heated water bath and maintained at a temperature of 56°C for 30 minutes. This is the most common method and it was used in the past to inactivate the complement system for immunoassays (Jungkind et al, 1986). Heat activation has also been reported to inactivate other undetermined inhibitors of cell growth in culture; however, the practice is labour-intensive and expensive. The protocol must be followed exactly as too high a temperature or too long a time may destroy some growth factors.
ATCC states that heat inactivation of foetal bovine serum or any other animal serum that is being used for cell culture can have detrimental effects on the serums ability to support cell growth as well as causing components to precipitate out giving the appearance of contamination.
In an article presented by (Danner et al, 1999) showed that a component in serum called complement was believed to be destroyed by heat inactivation process. The adventitious agents such as viruses and mycoplasma were also believed to be killed but when collection methods and filtration processes were improved along with the ability to gamma irradiate serum, these issues became irrelevant.
Leshem, Yogen and Florentini (1999: 249-254) recommends that heat inactivation is not mandatory and also heat inactivating serum should test samples side by side with normal serum. If the necessity arises in the particular application than heat inactivation of the serum should be carried out and it should be performed using a reliable, repeatable procedure.
In this procedure given by Davis, R. and Hsueh, R. in 2002, foetal bovine serum (FBS) is heated at 56o C in a water bath to destroy thermo labile complement proteins before using it in cell growth medium.
500 ml bottle of FBS is removed from -80O C freezer and is placed to thaw overnight in refrigerator. Complete thawing of the serum is done on the following day by placing it in a 37O C water bath and make sure that the water level are higher than that of the serum level and mix by inversion.
Once serum is completely thawed, incubate for an additional 15 min to allow serum to equilibrate with the 37 °C bath.
Temperature setting of the bath is raised to 56O C and timer is used to set the time for 30 minutes. During this incubation, invert the bottle to mix the serum every 10 min. If necessary, allow an additional 5 min for bath to reach 56 °C.
Incubate serum for 30 mins once the bath reaches 56 °C. Invert bottle every 10 minutes.
Remove serum from water bath and allow cooling at room temperature for 30 minutes. Reset water bath to the 37 °C mark.
Aliquot of 50 ml are prepared from that bottle into conical tubes and they are stored in a freezer at -20O C.
Caco-2 cell function may be affected by the nature of the substrate. The factors that need to be taken into account are material of the filter, pore size and the coating of the filter material. Different membrane materials having specific properties have been used to culture the Caco-2 cells. Nitrocellulose having the disadvantage of absorbing the molecules has led to the usage of inert materials like polycarbonates, aluminium oxide, polyester and poly (ethylene-terephthalate) membranes. Poly (ethylene-terephthalate) produces higher TEER value and lower Papp value compared to other supports (Behrens and Kissel, 2003). Permeability may also be influenced their density in filters of different materials apart from pore size (Delie and Rubas, 1997)
The in vivo epithelial cell’s growth and differentiation is affected by the extracellular matrix and the adhesion molecules such as integrins by interacting with them.
Laminar Air Flow (HEPASafe), Pipetting device, pipette 5ml or 10ml, stopwatch, incubator, conical tubes, centrifuge tubes, tube stand, Flask, 6-well plates, 96-well plates, microscope, neubauer slide, covering slip, tissue roll, water bath, Caco-2 cells obtained from ATCC, Heat inactivated and heat non-inactivated Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO), 0.25% Trypsin, 10% Phosphate buffer saline (PbS), Trypan blue.
Caco-2 cells were obtained from an American Type Culture Collection. All the experiments were conducted at passage 32-36. The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO) with 10% heat inactivated and with non-heat inactivated FBS (GIBCO). They were incubated at 37o C containing 95% atmospheric O2 and 5% CO2. At a decent confluency, the cells were trypsinized and seeded in a cell culture flask of 25-cm2 area (Corning Costar Corporation) at a density of 25 X 104 cells. Their viability was assessed using MTT assay.
Place heat inactivated and non-heat inactivated Dulbecco’s Modified Eagle’s Medium (DMEM) into water bath at 37 degree Celsius. Warm the medium for approximately 30 minutes. Also keep the trypsin for thawing.
In laminar flow hood, using a pipetting device pipette 2ml of the solution containing cell suspension and the heat inactivated medium into each of the 6 well plates. Same is done with the other 6-well plates containing non-heat inactivated medium.
Transfer 4ml of the solution to each of the two T25 flasks as well.
Add the solution in completely sterile condition as it may lead to infection.
Add 10 microliters of the solution to each of the well with two different sets of medium to two different 96-well plates (8 X 12) and keep it for incubation at 37O C with 95% atmospheric O2 and 5% CO2.
Change the medium on alternate days and the cell count is done on 3rd, 5th, 7th, 10th, 12th and 14th day. Subculture the cells on every 7th day.
When changing the media in T25 flask, empty all the media in waste media vessel and add 4ml of the fresh media.
For changing the media in 6-well plates, remove media, add PbS to one of the well, remove it and then add 900 microliter or 1 ml trypsin to it. Make sure that all the cells are detached form the surface. Pipette the solution in centrifuge tube and add 2ml of the fresh media in other 5 wells.
Cell count is done by taking the 10 micro-litre of the solution in the centrifuge tube and 10 micro-litre of the trypan blue is added to that.
Using the neubauer slide, count the cell present in the four chambers.
Report the number of cell present in each of the chamber and calculate the number of cells by taking the average.
For 96-well plate, carefully remove the media from the entire columns except for the one that we are going to count. Make sure that the cells are not withdrawn from that plate. Add the fresh media to the entire well.
Add 20 microliter of MTT to the column in which the cell are to be counted and then keep it for incubation for the period of 4 hours. After 4 hours, remove the solution from that column and add 100 microliter of Dimethly Sulfoxide (DMSO) to it.
Keep it for incubation for the period of 30 minutes and then take the readings in the plate reader at 580 nm.
Following the above procedure take the readings for all the columns on their respective days and then plot the graph.
Sub-culturing Protocol: –
Sub-culturing is an important process for the growth of the cells hence should be made sure that it is carried out in completely sterile condition as insterile condition leads to infection of the Caco-2 cells i.e. growth of mold takes place.
Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% (w/v) Trypsin – 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
Add 1.0 ml of Trypsin-EDTA solution to flask and observe cells under microscope until cell layer is dispersed (usually within 4 to 5 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Now based upon the calculation, pipette the amount of cell suspension and the growth medium required such that viable cell count is approximately 1 X 104 cells/ml.
Incubate cultures at 37C.
We used Agilent Technologies 1200 series HPLC instrument with UV detector and Chemstation for LC systems software for the detection of the ascorbic acid or Vitamin C. The chromatographic condition for the experiment is listed below
Ascorbic acid acts as a strong reducing substance. In vivo, vitamin C gets reduced to a radical intermediate to dehydroascorbic acid. It also plays an important role in synthesis in collagen and so it is also important for the de novo synthesis of cartilage, tooth and bone and also for healing of wounds and it is also important for the production of noradrenaline. Another important function of vitamin C is it acts as an antioxidant i.e it protects other substance from oxidizing effects of oxygen and it also promotes the resorption of iron in the intestine. The lack of vitamin C in the body is indicated by the following symptoms like tiredness, increased susceptibility to infection and physical and mental weakness. The advantage of HPLC analysis is that it can do the simultaneous handling of many analytes in a single test. Higher number of the samples can be handled by automating the sample.
Principle of the test
The determination of Vitamin C with HPLC is an easy, fast and precise method. Fast and easy sample preparation is the first step in the determination of Vitamin C. The heat inactivated
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