Talk:20.109(S08):Protein-level analysis (Day6)

Tuesday/Thursday Blue

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.

Tuesday/Thursday Red

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.