LAB 1: CYTOTOXICITY SCREENING Materials testing is an important phase of modern biomanufacturing in everything from cell culture to joint replacement or cosmetics development [1-3]. A basic cytotoxicity study is an essential preliminary step for any new material produced with potential to be applied to a biological field. With respect to bioengineering, materials testing is used heavily in prosthetics development, and is often applied when looking at such effects as inflammation response and immune rejection. For the purposes of this class, the skeletons of biobots are built using a 3D printer (4). The materials used to print these skeletons must be biocompatible to ensure that 1) they do not induce toxicity in the cell line that we will use and that 2) the cells can proliferate and differentiate properly in contact with the skeleton. The choice of suitable biomaterials is critical for the success of the overall procedure. In this lab, you will be culturing C2C12 cells in the presence of different concentrations of Irgacure 2959 or Irgacure 651, two photoinitiators commonly used for UV curing of systems comprising of unsaturated monomers and prepolymers such as those used in 3D printing. In subsequent labs we will be using the photoinitiator the exhibits lower toxicity in these initial experiments. There are many products available that can help quantify results from such studies: the most simplistic being a live/dead stain, which uses a dye or marker to selectively stain either live or dead cells. More complex assays, such as the MTS assay, quantifiably measures proliferation to achieve a similar end. In the first week of this lab we will perform a live/dead assay. In the second week of the lab we will perform a quantitative MTS assay.
LIVE/DEAD ASSAY PROTOCOL Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually non-fluorescent cell-permeant calcein-am to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells (ex/em ~495 nm/~515 nm). EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em ~495 nm/~635 nm). EthD-1 is excluded by the intact plasma membrane of live cells. The determination of cell viability depends on these physical and biochemical properties of cells. Cytotoxic events that do not affect these cell properties may not be accurately assessed using this method. Background fluorescence levels are inherently low with this assay technique because the dyes are virtually non-fluorescent before interacting with cells. Day 1 (08/31/2015) 1) Preparation of reagents: Resuspend Irgacure 2959 at.5 M in 1 ml of 70% methanol and Irgacure 651 at.125 M in 1 ml of 70% methanol Irgacure 2959: FW 224.3 Irgacure 651: FW 256.3 2) Harvest a plate of C2C12 and resuspend them at 100,000 cells/ml 3) In a 24-well plate add the following treatments to 50,000 cells:
a) 3 Untreated controls b) 1 Methanol treated negative control c) 1 mm Irgacure 2959 d).1 mm Irgacure 2959 e) 1mM Irgacure 651 f).1mm Irgacure 651 * It is recommended that in a 1.5 ml tube you add the volume of photoinitiator needed for the treatment, then add.5 ml of cell suspension, mix well and plate. 4) Plate the remaining C2C12 cells in a 10 cm dish at an appropriate concentration. Day 3 (09/02/2015) 1) Preparation of control samples: Make sure that the live cells (positive control) are healthy and prepare some samples of dead cells by killing cells in a different well using 70% methanol for 10 minutes at 37 C. 2) Preparation of reagents: Add 5 µl of the supplied 2 mm EthD-1 stock solution to 5 ml of sterile PBS and vortex to ensure thorough mixing. Add 2.5 µl of the 4 mm calcein AM stock solution to the 5 ml EthD-1 solution and vortex the resulting solution to ensure thorough mixing. 3) Perform the Viability Assay: a) Remove culture medium and wash gently with PBS once. b) Add 200 µl (enough to cover the cells) of LIVE/DEAD solution to the wells and incubate the cells for 15 30 minutes at 37 C. c) Image the labeled cells under the fluorescence microscope and take pictures of representative fields. d) Use ImageJ to quantify the impact of these two photoinitiators in cell viability.
MTS ASSAY PROTOCOL The CellTiter 96 AQueous One Solution Cell Proliferation Assay(a) is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The CellTiter 96 AQueous One Solution Reagent contains a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium, inner salt; MTS(a)] and an electron coupling reagent (phenazine ethosulfate; PES). PES has enhanced chemical stability, which allows it to be combined with MTS to form a stable solution. The MTS tetrazolium compound (Owen s reagent) is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium. This conversion is presumably accomplished by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells (5). Assays are performed by adding a small amount of the CellTiter 96 AQueous One Solution Reagent directly to culture wells, incubating for 1 4 hours and then recording the absorbance at 490nm with a 96-well plate reader (6). During the first week we observed that Irgacure 2959 is biocompatible while Irgacure 651 is cytotoxic. In this lab we will determine Irgacure 651 lc50. Day 1 (09/14/2015) 1) Reagents: You will receive Irgacure 651 at.125 M in 1 ml of 70% methanol 2) Harvest a plate of C2C12 and in a 96-well plate (100 µl/well) seed 40,000 cells, 20,000 cells, 10,000 cells, 5,000 cells and 2,500 cells. These cells will be used to build the standard curve. You can easily do serial dilutions by resuspending some cells at 40,000 cells / 100 µl and using the following technique:
3) Treat 10,000 cells with the following concentrations of Irgacure 651: 1 mm 0.8 mm 0.6 mm 0.4 mm 0.2 mm 0.1 mm 4) Prepare control wells as needed 5) Plate the remaining C2C12 cells in a 10 cm dish at an appropriate concentration. Day 3 (09/16/2015) 1) Preparation of samples: Add 10 µl of MTS reagent to each well and mix by pipetting gently 2-3 times. Incubate for 30-40 minutes at 37 C. 2) Read Absorbance: Open Gen5 Data Analysis Software and create a protocol to read absorbance at 490 nm. Place the plate in the carrier and read absorbance. 3) Save and analyze your data. 4) Prepare a 3-5 minutes presentation describing the results from these 2 assays and your interpretation/conclusions.
REFERENCES 1. Rehfeldt, F., A. Engler, A. Eckhardt, F. Ahmed, and D. Discher. "Cell Responses to the Mechanochemical Microenvironment Implications for Regenerative Medicine and Drug Delivery." Advanced Drug Delivery Reviews 59.13 (2007): 1329-339. Print. 2. Na, S., O. Collin, F. Chowdhury, B. Tay, M. Ouyang, Y. Wang, and N. Wang. "Rapid Signal Transduction in Living Cells Is a Unique Feature of Mechanotransduction. Proc Natl Acad Sci U S A. 105.18 (2008): 6626-631. Print. 3. B. EKWALL, V. SILANO, A. PAGANUZZI-STAMMATI AND F. ZUCCO. "Toxicity Tests with Mammalian Cell Cultures." (1990): n. pag. Stanford. Web. 25 Sept. 2012. 4. Cvetkovic C, Raman R, Chan V, Williams BJ, Tolish M, Bajaj P, Sakar MS, Asada HH, Saif MT, Bashir R. Three-dimensionally printed biological machines powered by skeletal muscle. Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):10125-30. 5. Barltrop, J.A. et al. (1991) 5-(3-carboxymethoxyphenyl)-2-(4,5-dimenthylthiazoly)-3- (4-sulfophenyl)tetrazolium, inner salt (MTS) and related analogs of 3-(4,5- dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reducing to purple watersoluble formazans as cell-viability indicators. Bioorg. Med. Chem. Lett. 1, 611 4. 6. Bernabei, P.A. et al. (1989) In vitro chemosensitivity testing of leukemic cells: Development of a semiautomated colorimetric assay. Hematol. Oncol. 7, 243 53.