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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 andÂ 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: -
1. 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.
2. HPLC analysis of Vitamin C in heat inactivated and non-heat inactivated FBS.
3. 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.
Table of Content Page No.
Caco-2 cell Backgroundâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.... 1
Importance of Caco-2 cellsâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.... 3
Factors Affecting the performance of Caco-2 cellsâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦. 3
Cell related factors affecting the performance of Caco-2 cellsâ€¦â€¦â€¦â€¦.. 4
Passage Numberâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦ 4
Essential first steps to minimize cell line passage effectsâ€¦â€¦â€¦â€¦.. 5
Paracrine and Autocrine Factorsâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦. 5
Effect of ph on performance of Caco-2 cellsâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦ 6
Culture related factors affecting the performance of Caco-2 cellsâ€¦â€¦â€¦.â€¦ 6
Seeding Densityâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦. 6
Time of Cultureâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. 7
Cell differentiationâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. 7
Expression of nutrient transportersâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦. 7
Influence of medium compositionâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. 7
Heat inactivation most acceptable procedureâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. 8
Characteristics and Nature of Substrateâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦ 9
Extracellular matrix and adhesion moleculesâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦ 9
Cell Count Culture Methodâ€¦..â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦... 10
HPLC analysis of Vitamin C................................................................................. 12
Ascorbic Acid Determination Using EnzyChrom Ascorbic Acid Assay Kitâ€¦. 14
Cell Count Culture Methodâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦ 15
HPLC analysis of Vitamin Câ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.. 17
Ascorbic Acid Determination Using EnzyChrom Ascorbic Acid Assay Kitâ€¦. 19
Caco-2 cells Background:-
Caco-2 cells, originally isolated in the year 1977, are the continuous cell line ofÂ humanÂ epithelialÂ colorectalÂ adenocarcinomaÂ 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,Â microvilli, and a number ofÂ enzymesÂ and transporters that are characteristic of enterocytes:Â peptidase,Â esterase, P-glycoprotein, uptake transporters forÂ amino acids, bile acidsÂ 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.
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.
Importance of Caco-2 cells: -
Importance Of Caco-2 Cells
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.
Factors Affecting the performance of Caco-2 cells:-
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).
Cell Related Factors Affecting the Performance of Caco-2 cells:-
Passage Number: - 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)
Essential first steps to minimizing cell line passage effects.
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.
Paracrine and autocrine factors:-
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)
Effect of pH on performance of Caco-2 cells: - 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)
Culture Related Factors Affecting the Performance of Caco-2 Cells: -
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.
Seeding density: - 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).
Time of Culture:-
Cell differentiation: - Caco-2 cells differentiation appears to follow the strict time schedule in the expression of morphological and biochemical characteristics of absorptive enterocytes.
Expression of nutrient transporters: - 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.
Influence of medium composition: - 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 56Â°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.
General Heat Inactivation: Most Accepted 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.
Heat Inactivation Procedure
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 Â°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 Â°C.
Incubate serum for 30 mins once the bath reaches 56 Â°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 Â°C mark.
Aliquot of 50 ml are prepared from that bottle into conical tubes and they are stored in a freezer at -20O C.
Characteristics and Nature of substrate: - 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)
Extracellular matrix and adhesion molecules: - 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.
Materials and Methods: -
Cell Culture Method: -
Apparatus and Materials Used: -
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.
HPLC Analysis of Vitamin C: -
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:-
Figure 1: - HPLC Instrument
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 and non-heat inactivated medium samples are kept for centrifugation and the supernatant is infected into the HPLC system. The separation of Vitamin C via HPLC follows an isocratic method using Sagal column and one run lasts for 3 to 4 minutes. Vitamin C is detected by using a UV- detector. In this experiment, spiking of the sample is done in order to calculate the concentration of Vitamin C present in the sample. The quantification is performed with the delivered calibrator and external standard methods are used for calculating the concentration of Vitamin C via integration of the peak areas.
Apparatus and Chemical Required: -
HPLC pump with UV detector, C-18 Sagal column, high precision pipette, Centrifugation tubes, centrifuge, ice and printer, solvent (methanol: water: acetic acid in the conc. of 30: 70: 1), heat inactivated and non-heat inactivated medium samples.
Method and Chromatographic Condition: -
Chromatographic condition: -
Column: - C-18 Column Sagal.
Mobile phase: - methanol: water: acetic acid (30: 70: 1)
Flow rate: - 1.0 ml/min
Injection volume: - 1 ml
Detector parameters: - UV detector at 245nm wavelength.
Run time: - 4 to 5 minutes.
Response time: - 1.5 to 2 minutes.
Accurately prepare the solvent containing methanol: water: acetic acid in the concentration of 30: 70: 1.
Calibration of the HPLC pump is done for about 5 to 10 minutes using the above mentioned solvent.
Once the calibration is done, obtained the peak value of Vitamin C using varied concentration: 0.002 mg/ml, 0.004 mg/ml, 0.006 mg/ml and 0.008 mg/ml.
Keep the heat inactivated and non-heat inactivated samples for centrifugation at 13,000 rpm for 15 minutes.
During the entire HPLC analysis process, place the sample, Vitamin C and blank media in the ice, to stop the interference of the heat as it reduce the vitamin C to dehydroascorbic acid.
Take the chromatogram of plain heat inactivated and non-heat inactivated samples.
For 0.004mg/ml conc.: - Mix 1ml of blank media and 1 ml of vitamin C (0.004 mg/ml) in a bottle and take the chromatogram.
Mix 1ml of no heat inactivated medium and 1ml of Vitamin C (0.004 mg/ml) in a bottle and take the chromatogram.
Follow the same procedure by taking 0.006 mg/ml and 0.008 mg/ml conc. of Vitamin C and also for non-heat inactivated medium.
Calculate the amount of amount of Vitamin C present in the sample by looking at the chromatogram obtained.
Ascorbic Acid Determination Using EnzyChrom Ascorbic Acid Assay Kit:-
Qualitative Colorimetric / Fluorometric Ascorbic Acid Determination.
As Clayden et al., 2001 states ascorbic Acid (the L- enantiomer commonly known as Vitamin C) is an important antioxidant found in living organism and applied as additives in food and other industrial processes. By reacting with reactive oxygen species, it protects the cell from oxidative damages. Bioassay system method provides a simple, direct and high-throughput assay for measuring ascorbic acid. In the assay, ascorbic acid is oxidized by ascorbate oxidase resulting in the production of H2O2 (hydrogen peroxide) which reacts with a specific dye to form a pink coloured product. The colour intensity at 570 nm or fluorescence intensity (530/585 nm) is directly proportional to the ascorbic acid concentration in the sample.
Use 20 microliter samples. Linear detection range, colorimetric assay 6 to 1000 micro-molar, fluorometric assay 1 to 100 micro-molar ascorbic acid.
Apparatus Used: -
Pipetting devices, centrifuge tubes, clear flat bottom uncoated 96 well plates, optical density plate reader.
Assays: - Detection of ascorbic acid in biological samples such as serum, plasma, urine, saliva, milk, tissue and cell cultures.
Drug discovery and pharmacology: - Effects of drugs on ascorbic acid metabolism.
Kit Content: -
Assay Buffer: - 10 microliter. Enzyme Mix: - 120 microliter.
Dye Reagent: - 120 microliter. Standard: - 400 microliter 10 micro-molar ascorbic acid.
Storage Condition: - The kit is shipped on ice. Store all components at -20o C. Shelf life of 3 months.
Precaution: - Normal precautions for laboratory reagents should be exercised while using the reagents.
Colorimetric Assay: -
Sample treatment: - Samples of serum and plasma can be assayed directly while that of tissue and cell (105 - 107) lysate can be prepared by homogenization in cold 1x PBS and centrifugation (5 min at 14,000 rpm). Use clear supernatants for assay. Milk samples should be cleared by mixing 600 microliter with 100 microliter 6N HCL. Centrifuge 5 min at 14,000 rpm. Transfer 300 microliter supernatants into a clear tube and neutralize with 50 microliter 6N NaOH. The neutralized supernatant is ready for assay (dilution factor n= 1.36).
Equilibrate all components to room temperature. Briefly centrifuge the tubes before opening. Keep thawed tubes on ice during assay.
Standards: mix 22 microliter 10mM standards with 198 microliter dH20 (final 1000 micro-molar). Dilute standard in dH2O as follows: -
1000 micro-molar STD + H2O
Ascorbic acid (micro- molar)
100mL + 0mL
60mL + 40mL
30mL + 70mL
0mL + 100mL
Transfer 20 microliter diluted standards into separate wells of a clear flat-bottom 96-well plate.
Samples: - transfer 20 microliter of each samples into separate wells of the plate.
Colour reaction: - prepare enough working reagent by mixing for each reaction well. 85 microliter Assay Buffer, 1 microliter Enzyme Mix and 1 microliter Dye Reagent. Add 80 microliter Working Reagent to each well. Tap plate to mix. Incubate 10 mins at room temperature.
Read optical density at 570nm (550-585nm)
From the cell growth graph and also from the MTT graph it is seen that there is no substantial growth of Caco-2 cells in either of non-heat inactivated FBS or heat inactivated FBS. However there are small amount of evidence that there is an impact of the non-heat inactivated in the beginning and also there appears to be a trend in the beginning for non-heat inactivated FBS which indicates that the non-heat inactivated FBS is suitable for the growth of the Caco-2 cells. However this "no clear evidence" is highly contributed to the large number of the experimental errors which increases through time. The experimental errors includes in-sterile condition during the procedure, no precise pipetting of the volume of media fed to cell, remaining of the trypsin in the well that may kill the cell due to break sown of proteins.
Figure 2: - Caco-2 cell response to the non-heat inactivated foetal bovine serum from passage 32 to 36. Each of the cell passage was grown for the period of 14 days in non-heat inactivated FBS and sub-cultured every 7th day.
Figure 3: - Caco-2 cell response to the heat inactivated foetal bovine serum from passage 32 to 36.
Figure 4: - Caco-2 cell growth comparison between the heat inactivated FBS and of non-heat inactivated FBS. In the graph it shows no significant difference between the cells grown with heat inactivated FBS or non-heat inactivated FBS.
Figure 5: - Caco-2 cells heat inactivated FBS vs non-heat inactivated FBS MTT assay. The Caco-2 cells treated with non-heat inactivated FBS shows a high growth rate in the beginning after which there is a sudden fall indicating either the heat affecting the growth only in the early stages or heat may be affecting in the later stages but it's not conclusive due to experimental errors.
It is also seen that heat inactivated serum hurts in the early stages i.e. before errors. So we can conclude that
The heat may matter but only in the early stage or
The heat may also matter in the later stages but due to experimental error it is not evident clearly.
The 2nd conclusion due to experimental errors at the later stages can be fixed experimentally by giving proper attention to the media used during the experiment i.e. it is not over heated in the water bath during the 1st step of bringing the media to the room temperature as it may degrade the proteins present in the media and may reduce the ascorbic acid present in the media to dehydroascorbic acid. Also during in-process procedure by making sure that the volumes of the media used during feeding the cells are precise, making sure that the media used for feeding the cells has been supplied to them in gentle way as pipetting the media hard on to the cells may kill the cells and also by maintaining the sterile condition while growing the cells.
The statistical data can be fix and the conclusion can be drawn from the graph by only looking at the left portion of the graph and by ignoring discontinuity. If we look only at left portion of the graph and try to make it a linear graph, it indicates that the cells fed with non-heat inactivated media reaches a higher growth than those treated with heat inactivated media.
HPLC analysis of Vitamin C: - Vitamin C is potential essential nutrient involved in large number of cellular processes. Human are not able to synthesized Vitamin C so it has to be supplied through diet. The average concentration of Vitamin C present in the serum is about 60-120 micromol/L. (Na, N. et al, 2006).
Ascorbic acid Chemical Structure
IUPAC name: - 2-oxo-L-threo-hexono-1, 4- lactone-2, 3-enediol or
(R)-3, 4-dihydroxy-5-((S) - 1, 2-dihydroxyethyl) furan-2(5H)-one.
Molecular Weight name: - 176 grams per mole.
Concentration of Vitamin C in serum = 60-120 micromol/L.
Now, 60 micromol/L gives 0.06 micromol/ml
Therefore, converting to mg/ml = 176 x 0.06 divide by 1 mol
= 10.56 mg/ml.
Similarly, 120 micromol/L gives 21.12 mg/ml.
500 ml of bottle of media contains: -
1ml à 10.56mg
60ml of serum à 60 x 10.56 divide by 1ml
= 633.6 mg / 500 ml of media
Similarly for 120mg/ml gives 1267.2 mg / 500ml of media.
So, the concentration of Vitamin C present in 500ml of bottle of lies between 633.6 mg to 1267.2 mg in 500 ml of media bottle.
Graphs of HPLC analysis of Vitamin C.
Concentration of Vitamin C = 1.03mg in 100 ml in the mobile phase.
Mobile phase use is Methanol: Water: Acetic Acid in the concentration of 30: 70: 1
Graphs to be kept here.
In the graph there is no evidence of Vitamin C for both of the concentration of 0.06 mg/ml and 0.08 mg/ml for heat inactivated FBS and also for non-heat inactivated FBS.
These results are highly attributed to less efficiency of the instrument to measure such minute concentration. The point that needs to be taken into consideration is that there was no HPLC analysis of FBS done of both the heat inactivated and also the non-heat inactivated serum during the term time of growth of the cells. So perhaps heat may have played an important factor for the insignificant results which may have reduced the Vitamin C present in the sample to L-dehydroascorbate. So it is recommended that HPLC analysis of Vitamin C is being done at regular interval to find out the presence of Vitamin C in the media. This can help check the maintenance of healthy media composition during the term time of growth of the cells.
Ascorbic Acid Determination Using EnzyChrom Ascorbic Acid Assay Kit: -
This method is useful in detection of the ascorbic acid in the biological samples such as serum. In this project, colorimetric method of detection of ascorbic acid has been carried out. The readings are as follows: -
From the reading produced there was no evidence of Ascorbic Acid or Vitamin C. The few of the higher reading may be attributed to the interference of the air bubbles present during preparation as a result may improper detection.
From the results obtained it can be concluded that non-heat inactivated FBS shows faster growth in the beginning of the experiment than heat inactivated FBS. So it supports the recommendation provided by ATCC of using the non-heat inactivated FBS over heat inactivated FBS. However from the experiment it is not clear that the faster growth of the Caco-2 cells in the non-heat inactivated FBS in the beginning is either due the presence of media or it is due to the natural rate.
There is no detection of Ascorbic Acid or Vitamin C in the media by HPLC analysis. This is due to instruments incapability to measure the concentration of ascorbic acid at such a minute level and also there was no analysis done for the period during the entire time of cell culture. So it is recommended that the analysis of ascorbic acid is done on alternate days to find out the effect of heat on the media as it may have reduce the vitamin C present in the media to dehydoascorbic acid. There was no detection of Vitamin C by Using EnzyChrom Ascorbic Acid Assay Kit.