Talk:20.109(S12):Initiate cell culture (Day2)

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Contents

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.

Notebooks may be handed in by the following Tuesday and Wednesday (when we don't have lab), 1 pm outside my office.

T/R

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

W/F

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

Designs (T/R)

Team Orange (with Team Yellow): Papers have shown that TGF Beta 1 is an important growth factor in maintaining a chondrogenic phenotype. Our experiment will examine if there is a dose dependency of TGF Beta 1 on chondrocyte dedifferentiation. We propose examine how well 0 ng/mL, 2ng/mL and 10ng/mL of TGF B-1 maintains chondrogenic phenotype using 3% low viscosity agarose, and 20x10^6 cells/mL. I think that the chondrocytes with no TGF B-1 will dedifferentiate, because they will be growth factor deficient. The 2ng samples I would expect to maintain their phenotype. If the 10ng/mL is not cytotoxic to the cells, then I also expect them to maintain their phenotype.

Team Green: Our experiment will examine the effects on mesenchymal cell differentiation based off of initial cell concentration.To examine this we will grow two cells cultures on low viscosity,2 % agarose gel. One of the cultures will have initial concentration of 10^7 cells/mL, and the other of 10^8 cells/mL. We assume that the cells with initial concentration of 10^7 cells/mL will differentiate more effectively.

Team Blue: We will examine the effects of alginate gel viscosity on cell differentiation rate (and possibly cartilage structure formation). We will grow two cell cultures, 1 of low and 1 of high viscosity, in 2% alginate matrix, each at 10^7 cells/mL. We hypothesize that cells grown in the less viscous matrix will grow differentiate faster.

Team Red: We intend to investigate how changing the serum used in the cell media affects the phenotypic expression of chondrocyte cells. Our two cell cultures will be grown in low viscosity alginate mixture at approximately 10^6 cells/mL and either Fetal Bovine Serum or Newborn Bovine Calf Serum (NBCS). We anticipate that the proteoglycan level of the NBCS culture will be higher than that of the FBS grown culture.

Designs (W/F)

Green Group: We will investigate how transferrin levels in media may slow down levels of de-differentiation in chondrocytes.We hope that cells with more transferrin will exhibit slower de-differentiation than cells with less transferrin in the media. To the control group we added the basic ITS supplement whereas to the experimental group we added 6 ug/mL of transferrin along with ITS supplement (Note that the ITS supplement contains about 6 ug/mL). We are growing cells at a density of 7 million cells per mL and a scaffold of 2.0% alginate of low viscosity.

Blue Group (in association with Yellow Group): We are going to compare the effect of adding insulin to the scaffold and media versus adding it only to the media on chondrocyte dedifferentiation. We are going to grow cells at a density of 10^7 cells/mL with a 1.5% alginate medium viscosity. We will add 20 ug/mL of insulin to the scaffold and 15 ug/mL to the media in one scenario while adding only 15 ug/mL of insulin to the media in another. Yellow Group will perform a similar experiment but will use 5 ug/mL of insulin in the media. We expect that both adding insulin to the scaffold and increasing insulin concentration will improve chondrocyte growth and reduce dedifferentiation.

Pink Group: We are going to compare the effect of synthetic vs. natural serum on the growth and behavior of chondrocytes. We are going to grow cells at a density of 10^5 cells/mL with 1.5% alginate with medium viscosity. We will prepare the media according to the growth requirements for chondrocytes listed on the M3D1 page. However, in place of FBS, the experimental group will have SeraSub. **We have just changed our protocols and now will be investigating the differences between using 2% FBS and 10% FBS in our chondrocyte cultures since we learned that SeraSub is not meant to be used in cell culture, but only for assays.**

Purple Group: We are going to compare the effect of scaffold stiffness on the differentiation of mesenchymal stem cells into chondrocytes. We will vary the wt. percentage of low-viscosity alginate (1% and 2.5%) with MSCs at a density of 3.4 x 10^6 cells/mL. We hypothesize that MSCs grown in the 2.5% alginate scaffold will have more of a chondrocyte phenotype since it is closer to the stiffness at the bone-cartilage interface.

Red and Orange Group: We are going to compare the effects of TGFB and BMP II within the scaffolds of 1.5% alginate vs. 0.3% collagen and 1.5% alginate mixture. There is a known positive effect of bone morphogenetic proteins on growth and differentiation in agarose. We expect a similar effect in alginate and a minimal effect in collagen II. For the TGFB cases, we expect a greater effect in collagen than in alginate. See Bosnakovski et. al. 2006.

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