Talk:20.109(S14):Initiate cell culture (Day2)

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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 color 1 Red
Team color 2 Orange Blue/Green!
Team color 3 Yellow/Pink

W/F

Arrival time (at latest!) 1:05 pm 2:45 pm
Team color 1 Yellow White
Team color 2 Orange Pink
Team color 3 Red Silver
Team color 4 Blue Purple
Team color 5 Green

Designs (T/R)

Green/Blue (CB/CR, KT/MG)

Research has revealed that estrogen-related receptor γ (ERRγ) negatively regulates chondrocyte proliferation in vivo in transgenic mice, but no literature was found on the effects of ERRγ on in vitro mesenchymal stem cell (MSC) differentiation. Therefore, we will be testing the effects of different concentrations (0μM, 0.5μM, 1.0μM, and 5.0μM) of 4-hydroxytamoxifen, an inhibitor of ERRγ, on MSC differentiation. We predict that increasing the dose of tamoxifen will increase the amount of differentiation in our cells because it will inhibit the ERRγ and induce chondrocyte proliferation . Because the tamoxifen needs to be solubilized in ethanol, we need to make sure that an equal volume is added for each concentration dose, as different volumes of ethanol may cause a variation in cell viability among samples.

Red/Orange (AN/CT, AK/LT)

To mimic osteoarthritis joints, we will add 400 ug/mL fibronectin. (http://www.sciencedirect.com/science/article/pii/S1063458404001347). We will add ADAMTS-5 aggrecanase inhibitor (http://www.emdmillipore.com/life-science-research/adamts-5-inhibitor/EMD_BIO-114810/p_tCSb.s1L7sgAAAEW0mEfVhTm) at concentrations of 0, 2, 7, and 10 uM (IC-50 = 1.1) after 2 days to 1 mL total volume for each culture. The time delay will allow us to evaluate cell growth before the inhibitor is added. Aggrecans are responsible for chondrocyte-chondrocyte and chondrocyte-matrix interactions, and are critical for cartilage structure. Therefore, the misbehavior of aggrecanase, the enzyme that breaks down aggrecan, would result in weakened cartilage structure. We expect the inhibitor of aggrecanase, ADAMTS-5, to cause more cells to adhere to the scaffold, thus when applied to an OA model, decreases symptoms. We expect that the most chondrocyte-like cells after 9 days will be cells cultured with 10 uM inhibitor, followed by 7 nM, 2 nM, and finally 0 nM.

Yellow/Pink (EL/SW, CB/HH)

The Yellow and Pink groups have paired up to test the effect of collagen 1 concentration in the alginate gel on growth and viability of chondrocytes. Previous research has indicated that adding collagen 1 to the alginate scaffold causes greater cell growth and appears to assist in maintaining differentiation, but there have been no investigations into which concentrations of collagen 1 are ideal for these purposes. We are testing four conditions of collagen 1 concentration - 0mg/mL, 100ug/mL, 0.95mg/mL, and 1.9mg/mL - and examining the ability of each to promote cell growth and maintain chondrocyte differentiation. We expect that our samples with higher collagen 1 concentration, at 0.95mg/mL and 1.9mg/mL most of all, will be best for maintaining a differentiated state and allowing the chondrocytes to proliferate.

Designs (W/F)

White/Silver

Research has shown that presence of Collagen I or Collagen II makes mesenchymal stem cell (MSC) differentiation more effective. While it was shown that Collagen II is more effective than Collagen I, it was not shown what concentrations were most effective. Therefore, we will be testing the effects of Collagen II concentration on MSC differentiation, by mixing alginate beads with volumes of 0, 50, 250, and 500 μL of collagen II (final concentrations currently unknown, as they are dependent on what stock is available).

Yellow/Green Research has shown that the presence of Collagen I or Collagen II helps the differentiation process of Mesenchymal Stem Cell (MSC). It was also shown that Collagen II is more effective than Collagen I. We are curious to see whether this observation can be translated into the phenotypes maintenance of Chondrocyte. Therefore, we will be testing the effect of Collagen II at different concentrations in the maintenance of Chondrocyte phenotypes by mixing various volume (0, 50, 250, and 500 µL) of Collagen II with alginate. We expect that the highest concentration of Collagen II will maintain Chondrocyte phenotypes best because Collagen II will provide support for the Chondrocyte to grow.

Blue/Pink/Purple Some experimental evidence has shown that stem cells respond differently to low vs. high concentrations of retinoic acid. This response could be either differentiation or de-differentiation. Our goal is to investigate the effects of low (10^-7 M) and high (10^-5 M) concentration of retinoic acid on the differentiation/de-differentiation of both mesenchymal stem cells and chondrocytes. We expect that the difference in retinoic acid concentration will have some effect on the fate of both the mesenchymal stem cells and chondrocytes, namely that the high concentration will cause the chondrocytes to dedifferentiate and the low concentration will cause the stem cells to differentiate and proliferate.

Red/Orange It has been concluded that hydroxyapetite (HA) increases the strength of the scaffold used in tissue engineering, when compared to a negative control. We will be doing a dose response experiment with varying conditions: (a) 0.0075 g, (b) 0.015 g, (c) 0.03 g, and (d) 0 grams of HA. We predict that the highest concentration of hydroxyapetite added to the alginate solution will show the highest amount of collagen II, thereby corroborating the previous results. We also curious to see if our results biologically relevant and could pose as a viable clinical solution to replacing cartilage.

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