Talk:20.109(S08):Protein-level analysis (Day6)
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Revision as of 13:11, 7 May 2008
For our experiment, we chose to vary the cell density added to the 1% alginate beads. All other conditions were held standard. Additionally we prepared a 2D sample with 1 million cells, in the interest of comparing to other groups who prepared 2D samples with varying cell counts. Our two 3D samples were .5 million cells/mL (3D-1) and 10 million cells/mL (3D-2). The results of our light microscope cell count showed no cell recovery for 3D-1. However, 2D and 3D-2 showed comparable cell counts of 310,000 and 270,000 cells/mL, respectively. In all cases the cell count was lower than expected. This most likely due to poor recovery of cells from media and not cell death, since we did not observe dead cells. Our live-dead assay showed no cells for 2D and 3D-1, which could be due to poor staining, dye bleaching, or, again, poor cell recovery from the media. For 3D-2, we observed one live cell (fluoresced green but not red). We then ran RT-PCR on the cDNA from the samples to test for collagen 1 and collagen 2 production. The gel showed no collagen of either type for our 2D sample. This could be due to any number of problems, from low cell viability rate, to poor purification of mRNAs from the cells, or inefficient reverse transcription of the RNA to form cDNA (perhaps because of issues with the primer), or even problems with the PCR step itself. However, for 3D-1, the RT-PCR gel shows that a small amount of collagen 2 was produced, and that for 3D-2, a small amount of collagen 1 and a relatively large amount of collagen 2 were produced. The presence of collagen 2 in these two samples seems to indicate that the cells mostly retained chondrocytic phenotypes, as it is in this phenotype that higher amounts of collagen 2 are produced. These results could also indicate that growing cells in a 3D alginate setting causes increased production of both collagen types, since our 2D culture did have a significant cell count, and it seems unlikely that the RT-PCR failed to work properly only in both 2D samples.
We chose to vary the viscosity of our alginate beads. We used 500,000 cells for our 2-D cultures and a density of 5,000,000 cells/mL in our 3-D samples. For our low-viscosity 3-D sample, we used Sigma-Aldrich alginate, while for our high-viscosity sample, we used FMC 10/60; both alginates were used at 2%. In addition, we added ascorbate to our 3-D samples only to enhance the contrast between our 2-D and 3-D culture results. Our light microscope cell count showed very few cells in our 3-D samples; however, a microscope examination of our alginate beads showed extensive cell populations. This discrepancy was possibly due to errors made in the cell isolation process. As a result, our cell viability assay was not very informative, since so few cells were present for analysis. To analyze the differentiation state of our cells, we performed RT-PCR on our cells to isolate cDNA for collagens I and II. RNA extraction was very successful for all of our samples, especially the 3D-High sample. Analysis of the cDNA gel showed a sizeable presence of cDNA in each sample. The 2D sample yielded the highest Collagen II : Collagen I cDNA ratio, followed closely by our 3D-High sample. Our 3D-Low sample yielded the least cDNA, and also had almost a 1:1 ratio of Collage II : I. These transcript results seem to indicate that our cells were relatively healthy and maintained chondrocyte differentiation in 2D culture and high viscosity alginate, but were not so in low viscosity alginate.
In our experiments we added Collagen II to one of our 3D 1% alginate samples. Our other 3D and 2D samples were cultured normally. Our hypothesis was that the presence of collagen II would reduce the amount of dedifferentiation of the chondrocytes to fibroblasts. When we observed the cells after they were grown for a week, the morphology of the cells in our 3D cultures retained a round phenotype, suggestive of a chondrocyte phenotype. However, the cells in our 2D cultures appeared more spread out, suggesting dedifferentiation info fibroblasts. For each of our cultures, we recovered about 100,000 cells/mL but recovered slightly more (about 155,000 cells/mL) for the 3D culture with collagen. We noticed that the cells in the 3D cultures were very small, but rounded, and we found it difficult to distinguish between simply debris or a very small cell. However, few cells, live or dead, were observed under microscopy in the LIVE/DEAD assay. This may be due to the fact that it was difficult for us to observe cell pellets before aspirating our supernantants and we could have lost cells at this step in the LIVE/DEAD assay. Despite our concerns with having low cell yield and lack of results for the LIVE/DEAD assay, isolation of the RNA for our RT-PCR reactions showed mRNA concentrations of 2D: 6.8ug/mL, 3D+coll: 14 ug/mL, and 3D+none: 8.4 ug/mL. We used 100 ng of RNA for each PCR reaction. We ran the PCR fragments from our RT-PCR on an agarose gel with the intention of comparing the relative intensities of Collagen I and Collagen II cDNA. Unfortunately, faint bands in many of the lanes could not be photographed and therefore full image analysis of the gel could not be performed. Even so, our image analysis of our 2D sample surprisingly suggested that our 2D samples produced more collagen II than collagen I despite their previously observed fibrobast phenotype. No 3D collagen ratios could be calculated by detection by the computer since no 3D bands were visible on the photo. In general, our collagen II bands, though faint, were stronger than the collagen I bands (many of the collagen I bands were completely missing all together). Our calculated 2D collagen II to collagen II ratio was 1.30.
Our prove conclusively that protein is the hereditary material.