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

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.

Tuesday/Thursday Yellow

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.

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.

Wednesday/Friday Red

We, along with the pink group, are testing the effects of mechanical stress on maintenance of chondrocyte phenotype. We are comparing our 2D and 3D control samples to a set of cells in 3D culture that are under constant mechanical stress in the form of two washers (49.2 g total) placed on top of our petri dish apparatus (Figure 1).

Sample	Cell density per ml	Alginate type	Medium
3D Control	5 million	1 % Sigma	normal
3D Weighted	5 million	1 % Sigma	normal

For 2D culture, we used 600,000 cells per flask (total 1.2 million), split them every 4 days at 1:10 and used normal medium.

Morphology:

As expected, the 2D cells exhibited fibroblast phenotype (strand-like) under the light microscope. For our 3D cultures, cells under mechanical stress maintained chondrocyte-like phenotype better than cells not under stress. (note: due to our experimental setup, the cells under stress were probably also exposed to less oxygen.

Viability:

In terms of cell count, we had very few cells in both of our 3D cultures. Yet in the live/dead fluorescence assay, the 2D and 3D cells looked similar.

Transcript:

After visualizing our RT-PCR fragments on agarose gel, our normalized data indicated that the weighted 3D sample expressed more CN II (chondrocyte-like) and unweighted 3D control expressed more CN I (fibroblast-like). With regard to the CN II : CN I ratio, the 3D weighted sample had a much higher ratio than all other samples including the collagen standards. As predicted, the 3D weighted cells exhibit chondrocyte phenotype, while the 2D and 3D control do not. For CN I, the control and 3D control had similar intensities and similar amounts of DNA (area of band on gel). For CN II, control and 3D weighted have similar intensities and amounts of DNA. Yet for the 2D sample, it was about half as intense and the band area was about 1/2 to 1/3 of the other two samples. These results confirmed our hypothesis that applying mechanical stress promotes chondrocyte phenotype.

Wednesday/Friday Green

We chose to vary the physical culture conditions. We originally planned to have four culture conditions: the 2D culture in a T25 flask, a 3D culture in 2% Sigma-brand low-viscosity alginate beads, a 3D culture in PEG-APGL-PEG-RGD and a 3D culture in a modified hollow-fiber bioreactor with 100 kDa cutoff cellulosic fibers. The PEG-RGD polymer contains the RGD peptide, which is an integrin binding sequence. We hoped that the cells would adhere to the polymer better with that peptide present. The bioreactor continuously circulates media and gas at a rate of 120 mL per minute and 27.5 mL per minute, respectively. It also puts shear stress on the cells at an adjustable quantity, which we hoped would stimulate the cells to produce collagen. Unfortunately, the PEG-APGL-PEG-RGD polymerization by UV light failed and made it impossible to work with our cells (e.g. exchange media). (Possible reasons for this were incorrect UV wavelength and old, degraded PEG and/or activator.) Also unfortunately, the bioreactor chambers melted during autoclave sterilization and were rendered unusable. This left us with just the first two described cultures (the 2D and 3D alginate). The 2D culture was seeded with approximately 500,000 cells, and the 3D culture was seeded with 1.1M cells (per replicate).

After seven days, we harvested some of our cells for a Live/Dead© (Invitrogen) cell count assay. Images of our assay can be found on the Day 4(?) talk page. Our results were these:

Culture	Live	Dead
2D	36	2
3D Alginate	15	2

Based on this data, both 2D and 3D cultures were quite viable, though as expected the 2D culture was somewhat more viable. We also looked at our cells under 40X magnification with visible light to observe morphology. The 2D culture cells were mostly fibroblasts, where as the 3D culture cells were mostly chondrocytes.

We then did RT-PCR on the RNA from the samples to determine relative production of type I and II collagen. The gel showed a higher percentage of chondrocytes in the 3D culture than 2D culture, based on the relative amounts of mRNA present in the cells. (This is based on the fact that chondrocytes express collagen II in higher amounts than fibroblasts do.)

(ELISA RESULTS)

Wednesday/Friday Blue

Background and Set-up:

From our research we found that the rate of attachment of cells increases with cross-link density. So we planned on increasing the cross-link density of one our 3D cultures(from now on 3D experimental) to test if this increased the dedifferentiation of chondrocytes into fibroblasts. We accomplished this by increasing the calcium concentration ten-fold (1002 mM instead of 102 mM). Our 3D control sample used a standard calcium concentration (102 mM). We used a 2% alginate for both samples with G/M ratio of 70/30, because the G segments are involved in the cross-linking. We actually noticed a difference in the size of our alginate beads: The higher calcium concentration beads were much smaller in size. Our 2D sample was set-up at normal conditions. We plated the 3D cultures with about 1.3 million cells per well and the 2D culture with 100,000 cells.

Morphology and Cell counts:

We saw differences between our samples, as expected. The 2D culture cells looked more like fibroblasts than chondrocytes. They were very flat and spread out. The 3D control cells looked more like chondrocytes since they were large and spherical. The 3D experimental cells still looked like chondrocytes but they were significantly smaller in size than the 3D control cells. Our 2D cells had about 720,000 cells in total. Our 3D control and experimental samples had 49,500 cells total.

Live/Dead Cell Assay:

Unfortunately, since we had a low cell count and then we also had low cell recovery, we were not able to get good data from this assay. We saw about 4-5 live cells and 1-2 dead cells in the 2D sample. For our 3D control, we could more live than dead cells, but this was for a small sample size. The live cells were in a clump, so we were not able to count them.

Analysis:

When we prepared our cells for analysis, we had a low cell count for our 3D control sample and no cells in the 3D experimental.

Transcript-Level

The gel results are mostly consistent with our predictions. The 3D control and 2D samples show a high CN II:I ratio, indicating that a significant portion of the cells remained as chondrocytes. However, our 3D experimental sample shows a low CN II:I ratio. There are many reasons that account for the low ratio. It is possible that the chondrocytes de-differentiated into fibroblasts; but the low cell count in the 3D experimental sample does not allow us to make that conclusion.

Protein-Level

(to be continued...)

Wednesday/Friday Purple

For our experiment, we decided to add a calcium chelating agent (mix of Sodium Citrate, EDTA, NaCl) to one of our 3D samples. We hypothesized that the addition of a calcium chelating agent would reduce the mechanical strength of our scaffold by inhibiting the G block interactions between our alginate beads. We predicted that this reduced mechanical support would be detrimental to chondrocyte phenotype preservation and would cause more chondrocyte de-differentiation to fibroblasts. The other 3D sample was used as a control (no calcium chelator), in order to have a basis of comparison for the level of de-differentiation. Our 2D sample was used to compare the effects of chondrocyte development in an environment lacking a scaffold. Due to low cell recovery, we initially seeded only about 100,000 cells in the 2D sample and about 1.5 million cells in each of our 3D samples. We added chelating agent to 3D-sample 2 at a concentration of 1%. We determined this concentration after some discussion of the fact that too high a concentration might completely obliterate our alginate matrix, while too low would yield no interesting results.

Upon trypan exclusion on day 3, we found cell counts to be extremely low. We determined that our 2D sample had around 500,000 cells while each of our 3D samples had only around 11,000 cells. Preliminary microscopy showed no difference in the morphology of our 3D samples. However, we noticed that most of the cells in our 2D sample had an oblong shape consistent with fibroblast morphology. Because of our low cell count, we took the maximum amount of each sample (1.5mL) for our Live/Dead fluorescence assay. Despite this, cell counts were too low for us to observe many live or dead cells.

We measured the collagen II to collagen I ratio of our samples by examining the RNA content as well as collagen expression of our samples. With the first half of our samples, we extracted all RNA for RT-PCR. Using collagen specific primers, we reverse transcribed the collagen RNA to cDNA and then amplified this cDNA through PCR. Upon running our RT-PCR products on an agarose gel we found that our Col II:Col I ratio was high in all of our samples. Using ImageJ, we analyzed the intensity of the agarose gel bands and found the following ratios:

Sample	CN-II : CN-I
2D	25.6
3D - Sample 1	18.0
3D - Sample 2

• • 2D Sample Results are inconsistent with our microscopy observations - we will continue to address this after the results of our ELISA.

Wednesday/Friday Pink

Our Experiment

For our experiments, we chose to test the effects of mechanical compressive stress on chondrocyte phenotype. Our 2D sample was cultured normally. Both of our 3D samples were cultured in Petri dishes: the control had no weight added and was cultured in 1% alginate, our test sample was compressed using the bottom of the inverted Petri dish, covered with filter paper to allow diffusion of nutrients and oxygen into the beads. We hypothesized that in compressive situations, similar to those seen by the cartilage in the knee, would result in maintained chondrocye phenotype.

Cell Observations

Observation of the cells after one week of growth confirmed our prediction, at least initially. Our 2D sample was mostly de-differentiated, but not completely. It was hard to tell what happened with our 3D samples, but the cells within the beads looked circular. We recovered 356000 cells/mL of our 2D sample, and less of both of our 3D samples; 33300 cells/mL for the weighted sample and 116600 cells/mL for the control sample.

Live/Dead assay

Our Live/Dead assay revealed very few dead cells, but also very few lives cells. This is most likely the result of relatively low recovery and incomplete staining. We think, after looking at the cells under normal lighting that it was a staining problem, and that most of our cells remained alive regardless of conditions.

Cell Concentrations

Concentration analysis of our samples showed high protein amount in two out of three cell conditions. Our 2D sample had a concentration of 120.4 ug/mL, 3D control was 32.4 ug/mL, and 3D weighted was very low, at 6.8 ug/mL. We decided to run the full 200 ng of RNA in each RT-PCR reaction, but ran out of our 3D weighted sample. We ended up using only 75% as much for the 3D weighted as for the other two.

RT-PCR Results

We performed RT-PCR on all three of our samples, but got very little yield. Our 2D samples yielded a strong band of collagen II (around 400bp), and a very faint but visible on the gel (not on the picture) around 100bp that was collagen I. None of our 3D samples showed visible bands, despite the high protein concentration evidenced in spectrophotometer. Regardless, based off the striking results of our 2D sample, we can conclude that even the cells displaying the more fibroblast phenotype were still producing markedly more collagen II than collagen I. Using the 2D results on the agarose gel, our collagen II to collagen I ratio was 23.25 (100/4.3).