Talk:20.109(S13):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.

Note that you may be asked to switch your group number based on grouping together people working with the same type of cells, asking people who need special reagents/equipment to go in the second group, etc.

T/R

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

W/F

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

Designs (T/R)

Purple: Our plan is to culture chondrocytes under standard system conditions, but add an insulin supplement to the media at two concentrations higher than that already present in the ITS/FCS media. Insulin has been found to inhibit dedifferentiation of chondrocytes to the fibroblast state. We think our first 3D sample, subjected to 10 ug/day of insulin will inhibit dedifferentiation less than our second 3D sample, subjected to 20 ug/day, because a higher concentration of insulin will result in a larger inhibitory effect in the cell culture.

Platinum: Our plan is to culture stem cells under standard low viscosity alginate conditions but add growth factor BMP2 to the media. BMP2 has been found to induce chondrogenesis at certain concentrations. We think our first 3D sample, a negative control not subjected to BMP2 will show less of a chondrocyte phenotype than our second 3D sample, subjected to ___ ug/ mL BMP2 / day.

Yellow: We predict that the 3D sample of mesenchymal stem cells with coumarin and TGF-beta added to the media will be more differentiated than the 3D sample of MES with coumarin alone. TGF-beta and coumarin enhanced media has been shown to improve MES differentiation in 2D cell cultures, and we believe it would have the same synergistic effect in a 3D environment.

Blue: We intend to culture chondocytes under two different conditions: the first under standard conditions, and the second with the addition of fibronectin. We predict that the chondrocytes with the fibronectin will promote differentiation of chondrocytes to fibroblasts. This is because fibronectins contain integrin recognition sites that would enable chontrocytes to better attach to the alginate.

Designs (W/F)

Orange: We propose to study the effect of varying initial cell densities to determine an optimal starting density for the maintenance of chondrocyte differentiation. We hypothesize that lower cell density will result in a slower rate of central viability loss from due to the fact that in high density, cells can signal those around them and potentially disrupt central viability.