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	Platinum	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 to supplement the media with growth factor BMP2. BMP2 has been found to induce MSC cell chondrocyte proliferation, via certain signaling pathways, such as the Wnt/β-catenin pathway, at certain concentrations: specifically around 200 ng/mL. 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 112.5 ng/ mL BMP2 per 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&Green: We intend to culture chondrocytes under three different conditions: the first under standard conditions, the second with the addition of fibronectin, and the third with the addition of an RGDS amino acid sequence. Since chondrocytes normally recognize the RGDS aa sequence on fibronectin to promote dedifferentiation to fibroblasts, we hypothesize that by adding RGDS to compete with the fibronectin, no dedifferentiation will take place. This is because the cells will not be able to bind to the ECM via fibronectin adhesions.

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.

Green/Pink: We intend to study the effects of varying the alginate bead conditions (using the medium viscosity) upon mesenchymal stem cells. One will use 0.8% and the other will use 2%. We expect that the higher alginate concentration will yield better differentiation to chondrocytes due to the increased modulus of the substrate/scaffold.

Blue/Red/Purple: We plan to assess the effects of an aggrecanase inhibitor at two concentrations on the maintenance of the chondrocyte cells. We will introduce two different concentrations of ADAMTS-5 Inhibitor, with the goal of affecting only ADAMTS-5 for one sample (lower concentration) and both ADAMTS-5 and ADAMTS-4 for the other. We hypothesize that in both cases the chondrocyte state will be better maintained. We will also set a control with no inhibitor.

Yellow/Platinum: We propose to study the effects of increasing concentrations of fibronectin on the maintenance of chondrocyte. It is hypothesized that cells cultured in protein monolayers containing higher concentrations of fibronectin would better maintain chondrocyte phenotype as a result of increased active integrin binding. We also expect the fibronectin to affect matrix formation and increase proteoglycan production