Talk:20.109(S11):Initiate cell culture (Day2)

Plan for Day 2

Group 1 should arrive by 1:05 pm at the latest and immediately go to the tissue culture room. When everyone has arrived, we will thaw your cells in the water bath. After you have finished your culture preparations (ideally by 3 pm), you can take a 10 minute break to refresh your minds, and then will take a short quiz.

Group 2 should arrive by 2:45 pm at the latest and will begin by taking the quiz. If all goes well, you will begin working in the tissue culture room at 3 pm. If your culture preparations that involve a special physical set-up, you can come earlier and work in the extra tissue culture hood.

Notebooks may be handed in by Friday the 15th at 5 pm for the T/R section, and by Wednesday the 20th at 5 pm for the W/F section. (Earlier is certainly welcome for W/F folks if you are not away for the holiday.)

T/R

Arrival time (at latest!)	1:05 pm	2:45 pm
Team colour 1	Pink	Green
Team colour 2	Purple
Team colour 3	Red
Team colour 4	Yellow

W/F

Arrival time (at latest!)	1:05 pm	2:45 pm
Team colour 1	Purple	Red
Team colour 2	Blue	Green
Team colour 3	Orange
Team colour 4	Pink

Designs (T/R)

Team	Design
Yellow	We decided to test the effects of stiffness, through the use of low and high calcium concentrations, on the rate of dedifferentiation of chondrocytes. We expect there to be less dedifferentiation on stiffer alginate (high calcium concentration). Thus, we expect that the cells on a stiffer alginate will maintain a chondrocyte-like phenotype better than those on a less stiff surface, because similar results have been recorded using a smaller range of calcium concentration (Genes et al. 2003). We also plan to compare our results using medium viscosity alginate with those of Red and Orange, who are using low viscosity alginate.
Red	We predict the alginate in 200mM calcium will maintain chondrocyte phenotype better than alginate in 20mM calcium because the 200mM calcium forms a stiffer porous scaffold, allowing for the cells to form a better matrix
Orange	We decided to compare the cross-linking produced by high and low Calcium concentrations (20mM, 200mM) with the Alginate density and viscosity to see how each of these affects stiffness and resulting phenotypes of tissues. We used the 1% low viscosity alginate, and we wish to compare this with the same Calcium parameters tested in 2% low viscosity by the Red Group. In particular We would like to observe effects of increased cross-linking in low density alginate, versus less cross-linking in a higher percent alginate. density was shown to increase retention of certain mechanotranducers in the literature, and stiffness influences dedifferentiation to fibroblast-like so we would like to see if cross linking produces a comparable stiffness to effect collagen I expression in chondrocytes as 2% alginate at constant viscosities. Then conpare this to the effects of varying viscosity in 2% alginate (from Yellow and orange data). By varying three parameters it may be possibly to quantify expression of the two collagen types as well as characterize morphological differences for each set of conditions.
Blue	We will be preparing alginate cell scaffolds supplemented with none, low (0.05 mg/ml final), or high (0.5 mg/ml final) levels of hyaluronic acid (HA) in the presence (1 mg/ml final) and absence of exogenous collagen II. We will be examining the differentiation of stem cells into chondrocytes under these conditions by assaying collagen II and collagen I mRNA transcript levels (markers of cell type). We expect the cells under conditions of high HA and no exogenous collagen II to exhibit more of a fibroblastic phenotype, because of the results of Yoon et al. (2009). On the other hand, we expect cells under conditions of exogenous collagen II and low HA to exhibit more of a chondrocytic phenotype, because of the results of Bosnakovski et al. (2005). The interesting case will be the scenario with high HA and exogenous collagen II. We expect this case to yield some intermediate level of differentiation.