Industrial Applications of Cell Cultures PDF

Industrial applications of animal cell and tissue cultures Cytotoxicity tests and determination of cytotoxic doses Cytotoxicity Viability levels and/or proliferation rates of cells are good indicators of cell health. Physical and chemical agents can affect cell health and metaboli...

Industrial applications of animal cell and tissue cultures Cytotoxicity tests and determination of cytotoxic doses Cytotoxicity Viability levels and/or proliferation rates of cells are good indicators of cell health. Physical and chemical agents can affect cell health and metabolism. These agents may cause toxicity on cells via different mechanisms such as destruction of cell membranes, prevention of protein synthesis, irreversible binding to receptors, inhibition of polydeoxynucleotide elongation, and enzymatic reactions. In order to determine the cell death caused by these mechanisms, there is a need for cheap, reliable and reproducible short-term cytotoxicity and cell viability assays.  In vitro cell viability and cytotoxicity assays with cultured cells are widely used for cytotoxicity tests of chemicals and for drug screening. Application of these assays has been of increasing interest over recent Trypan blue years. Currently, these assays are also used in oncological researches to evaluate compound toxicity and tumor cell growth inhibition during new drugs development. Because, they are rapid, inexpensive and do not require the use of animals. Furthermore, they are useful for testing large number of samples. Cell viability and cytotoxicity assays are based on various cell functions such as cell membrane permeability, enzyme activity, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. Cytotoxicity Cell and tissue model system for toxicity testing The preliminary stage: - preparation of the cell or tissue culture model, - determining the dose range of the tested substance, - determining the exposure time of the model to the tested compound, - Choice of toxicity assessment method (cell viability, metabolic activity, proliferation rate)  MTT assay The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells (Fig. 1). The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT to formazan. The insoluble formazan crystals are dissolved using a solubilization solution (like isopropanol or DMSO) and the resulting colored solution is quantified by measuring absorbance at 500-600 nanometers using a multi-well spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells. Determination of resveratrol cytotoxicity using the MTT test in HeLa cells. Resveratrol is a polyphenol, phytoalexin with the structure of stilbene. Sources of resveratrol: dark grapes, wine, especially red, and fruits, among others. cranberry, blueberry, mulberry, blackcurrant, and also some herbs. It has a specific spectrum of pharmacological properties. It has anti-inflammatory and antioxidant properties, as well as strong cardioprotective, neuroprotective and anticancer effects. Lee SR, Jin H, Kim WT, Kim WJ, Kim SZ, Leem SH, Kim SM. Tristetraprolin activation by resveratrol inhibits the proliferation and metastasis of colorectal cancer cells. Int J Oncol. 2018 Sep;53(3):1269-1278. doi: 10.3892/ijo.2018.4453. Epub 2018 Jun 25. PMID: 29956753. Materials: Procedure of MTT assay 96-well plate Cells of interest and media tested compound Incubator MTT reagent (typically 0.5mg/ml in PBS or DMEM without phenol red) Solubilization solution (DMSO or isopropanol) Microplate reader Steps: 1. Cells seeding: - seed the cells in a 96-well plate, adding an appropriate numer of cells per well (density of 0.4 x 105 cells/cm2). Add enough culture media to each well to make up a total volumeof 100-200 µl per well - Include control wells with no cells (just medium) for background correction 2. Incubation: - Incubate the cells at 37 C with 5% CO2 (usually 24 hours) - If testing drug cytotoxicity or treatment effect, add the compound or treatments to the wells at this step and incubate 24-72 hours - the 24-h HeLa cultures were treated with resveratrol in various concentrations: 0 (control), 10,50, 100, 200, 300, 400, 500 µM and incubated for 72 h under standard culture conditions 0 10 50 100 200 300 400 500 3. Adding MTT reagent: - Prepare a fresh MTT solution (0.5 mg/ml in PBS or culture media without phenol red) - Remove tested compound and add 100 ul MTT solution to each well 4. Incubation with MTT: - Incubate the plate 1-4 hours at 37 C at dark, this allows the living cells reduce MTT to formazan crystal 5. Removing MTT and dissolving fromazan crystals: - After the incubation, carefully remove MTT solution from each well without disturbing the formazan crystal - Add 100 ul of a solubilization solution ( eg. DMSO or isopropanol) to each well. This dissolves the formazan crystal , turning the solution purple - Gently mix the plate on a plate shaker 6. Measuring absorbance: - Measure the absorbance using microplate reader at 570 nm, you can use a reference wavelenght of 690 nm to correct the background absorbance  Determination of cytotoxicity of resveratrol by MTT test 3. Analysis of cell viability and metabolic activity by MTT test 1. Remove media from wells of a 96-well cell culture plate 2. Add MTT reagent 100 ul / well 3. Wrap the plate in aluminum foil and incubate 30 min – 1h at 37°C 4. After incubation, remove MTT reagent from the wells 5. Add 100 ul isopropanol / well 6. Wrap the plate with parafilm and aluminum foil 7. Incubate on a shaker 15 minutes, 8. Read absorbance at λ = 570 nm and 690 nm 9. Based onthe results of the analysis: 1- Determine the percentage inhibition of cell growth. 2- Plot the dose response curve, calculate the EC50 dose Determination of the percentage inhibition of cell growth depending on the concentration of the analyzed compund 1. Determine the percentage inhibition of cell growth according to the following relationship: - absorbance for control - 100% - absorbance for the sample - x% 2. Plot a graph of inhibition of cel proliferation against concentration of resveratrol Dose response curve Based on a linear fragment of the curve, three doses are determined, which are the basis for assessing the cytotoxicity of the test compound:  I mathematical model II mathematical model [C] Log10[C] MTT eksperymentalne MTT obliczone F. straty Ymin Ymax LogIC50 S dane eksperymentalne dane obliczone 0 0,7121 0,000346 0,7504053 1,490777 -1,15177 AKTYWNOŚC MITOCHONDRIALNA KOMÓREK CACO-2 W STOSUNKU 0,1 -1 0,7439 0,749392044 3,02E-05 120 0,5 -0,30103 1 0 0,7639 0,7198 0,74398417 0,736286145 0,000397 0,000272 Model matematyczny Y = Ymin + (Ymax-Ymin)/(1+10^((LogIC50-X)*Slope)) 5 0,69897 0,671 0,668569863 5,91E-06 100 20 1,30103 0,4674 0,467794346 1,56E-07 Wyznaczenie dowolnego IC[?] DO KONTROLI [%] 50 1,69897 0,27 0,274393524 1,93E-05 logIC[?] = logIC50 - (1/Slope)*log(F/(100-F)) 250 2,39794 0,06 0,062396625 5,74E-06 80 1000 3 0,01 0,013802795 1,45E-05 Symbol Wartość 0,000744 Ymin 0,000346 60 1 Ymax LogIC50 0,750405 1,490777 S -1,15177 40 0,8 IC50 30,95827 Absorbancja (570-630 nm) X log stężenia 20 0,6 IC10 IC[?] 0,6622774 0 4,594914 10 0 0,4 0,6622774 IC50 0,9 4,594914 10 0 25 50 75 100 125 150 175 200 0,2 1,4907767 1,4907767 0 30,95827 0,9 30,95827 STĘŻENIE [% V/V] 2,319276 IC90 0 208,5816 IC[?] 90 0 2,319276 0,9 208,5816 90 -1 -0,5 0 0,5 1 1,5 2 2,5 3 3,5 4 Y max. Log10 [C] Yobl.   Y min. MTT obliczone IC10 IC50 IC90 MTT eksperymentalne   [C ]  s  1      IC 50     max. Y min. Y IC50 S A B C D Ymax, Ymin - respectively the maximum and minimum value of the 100,8448 0,0000 9,4445 1,9192 tested characteristic, estimated by the designated model [C] - concentration of the tested inhibitor IC50 – seeked lethal or inhibitory dose s - is the slope of the curve - the greater is the greater influence of the inhibitor on the tested object - the curve is steeper https://www.youtube.com/watch?v=XnwzSFJvC3A