Talk:20.109(S10):Initiate cell culture (Day2)

Experimental Plans and Predictions

T/R Lab

Red:

Experimental Plan: Assessing the effect of decreasing the calcium amount in the beads on the growth and phenotype expression. Expectations: We know that the beads affect the mechanical properties around the cells, and we expect that we are making the environment less appropriate for the cells. We are unsure whether this will affect the growth of the cells, phenotype expression, or collagen production ratio, and we will leave it for the results to determine it.

Orange:
Experimental Plan: Compare the effect of increased Proline concentration in media. One sample will be grown in media with the standard 400uM of Proline; the other sample will be grown in media with 1200uM of Proline. Expectations: The cells under the condition with 1200uM Proline is expected to have increased viability and remain differentiated as chondrocytes.

Yellow:
Experimental plan: Add .66 mmol sodium bicarbonate per 1 mL media (total 1.8 mL bicarbonate in 6 mL media) to raise the pH of the media to 8.0.

Cell type: chondrocytes

Cell density: 10^6 cells/6.4 mL

Total # cells needed: 6 million

Standard media other than bicarbonate addition

scaffold: Sigma Aldrich low viscosity 2% high M

Expectations: We expect the viability of the chondrocytes to drop at the higher pH; however, we do not expect the pH to be so far out of the biological range as to completely kill the chondrocytes. We expect the cells at biological pH (7.2-7.4) to maintain chondrocyte phenotype better than the cells at high pH.

Green:
Experiment: Compare acidic vs. neutral conditions. We added extra HEPES (acid) at 1:10 volume of media to lower pH to ~7 for the acidic conditions.
Expectations: We expect that the control neutral-pH chondrocytes to maintain chondrocyte phenotype while the acidic media causes its chondrocytes to lose their phenotypes and decrease their viability.

Blue:
Plan: Assess the effects of increasing ascorbate levels in the media that bovine chondrocytes are grown in. One set of samples will contain 40 uL/mL ascorbate, and another set of samples will contain 80 uL/mL ascorbate. All other conditions standard. We seeded the cells in 1% Protanal LF 10/60 alginate beads.
Expectations: According to existing research, increasing ascorbate levels will likely increase expression of collagen (types I and II). If we had more samples, we could better determine the long-range behavior of ascorbate modulation of collagen expression (i.e. is there a threshold level of ascorbate that decreases collagen expression?), but with our current setup, we'll only be able to quantify the effects of doubling ascorbate levels.

Pink:
Plan: Since mechanical loading and pressure in the joints is one of the factors that contributes to cartilage degradation in secondary osteoarthritis, through our experimental design, we seek to evaluate the effect of compression and pressure on the ability to grow a 3D chondrocyte culture. We will grow two cultures in a six well plate: both cultures will be covered with a square glass slide (m = 0.189 g, A=22 mm x 22 mm), and one of them will also be subjected to additional pressure by placement of a metal mass (m= 24.608 g) on top of the glass slide. (Cell culture conditions: Sigma Aldrich "low viscosity" alginate at 1.5%; Differentiated chondrocytes, 10^7 cells/mL, media conditions are standard).
Expectations: We think that the control (weightless) sample will preserve a chondrocyte-like phenotype, whereas the compressed sample will lose chondrocyte phenotype and will possible not be as viable, since we think compression and confinement in the scaffold will contribute to chondrocyte degradation.

Purple:
Experiment: Add ITS to growth medium at 2% as opposed to just FCS and determine any growth differences stemming from this change in environment. We also plan to possibly coordinate with another group working with 0.2% ITS for additional data.
Cell type: chondrocytes
Cell density: 10^7 cells/0.2 mL
Total # cells needed: 10 million
Alginate: Sigma Aldrich "low viscosity 2%" high M
Expectations: We believe that the cells treated with ITS will show just as little if not less de-differentiation from chondrocytes as compared to the untreated (-)ITS culture.

W/F Lab

Red:
Experimental plan: Make beads in different concentrations of CaCl2: 51 mM and 204 mM, so that the alginate will have different degree of cross linking and thus different stiffness.

Cell type: chondrocytes

Cell density: 5 million cells/ mL

Total # cells needed: 10 million

Alginate: Protanal LF120M

Expectations: A paper by Gene et al reported that the mechanical properties of the scaffold affect the cell phenotype. The stiffer the alginate is, the more they found fibroblast phenotype. Ca2+ will cause cross linking of the alginate so we expect that the higher [CaCl2] (204 mM), the stiffer the alginate will be, and the more fibroblast phenotype will be found compared to the 51 mM. We expect that more fibroblast phenotype will correspond to a higher Collagen II expression (compared to Collagen I).

Orange:
Experimental plan: We added 15ng/mL bFGF to media and poured 6 mL bFGF-laced media over alginate beads after last wash.

Cell Type: chondrocytes

cell density: 7 million cells/mL

Total # of cells: 13 million

Aliginate: Protanal LF 70/30 (2%)

Expectations: Based on past data, 5 ng/mL of bFGF fails to significantly alter dedifferentiation of chondrocytes into fibroblasts, so by increasing concentration three-fold to 15 ng/mL we expect to limit that process. Previous work has shown successful recreation of cartilage through use of bFGF laced microspheres, so we expect to mimic these results.

Yellow:
Experimental plan: Add 0.1mg/mL of chondroitin sulfate to one plate and 1mg/mL of chondroitin sulfate into the other plate (Ideally, we would like to add the chondroitin sulfate into the scaffold itself.

Cell type: chondrocytes

Cell density: 7million cells/mL

Total # cells needed: 14 million

Alginate: Protanal LF 120M (2%)

Expectations: As chondroitin sulfate is one of the components of cartilage and may contribute to maintaining phenotypes of chondrocytesWe expect the chondrocytes experiencing a higher concentration of chondroitin sulfate to maintain its phenotype better. Hence, the collagen type II to collagen type I ratio should be higher in this plate than the other one.

Green:

Experimental plan: We plan to compare stem cell only growth vs. stem cell + chondrocyte growth vs. chondrocyte only growth. One of our samples will contain stem cells only, and the other will have 40% stem cells and 60% chondrocytes.

Cell type: chondrocytes and mesenchymal stem cells

Cell density: 5 x 10^6 cells/mL (both)

Total # cells needed: 1 x 10^7 (stem cells); 5 x 10^6 chondrocytes (safe estimate)

Alginate: Sigma Aldrich 250 cps @ 2 % Alginate

Expectations: We expect that stem cells will respond to chondrocytes in the surrounding environment by differentiating into chondrocytes. So the sample containing stem cells and chondrocytes will hopefully produce more collagen II. We do not expect chondrocytes to affect stem cell viability.

Blue:

Experimental plan: Add different amounts of cell density to each sample; 10^6 cells/mL to one and 10^7 cells/mL to the other one

Cell type: chondrocytes

Cell density: 10^6 cells/mL and 10^7 cells/mL

Total # cells needed: 1.5 million

Alginate: FMC Biopolymen, protonal LF 120M, 10-50cps at 1%

Expectations: We expect that the chondrocytes will not have as much space to grow at density of 10^7 cells/mL; therefore, the cells should be more pecked together than the other sample. They should also grow less well, so the collagen type 2 to collagen type 1 ratio in the other sample should be higher.

Pink:

Experimental plan: Compare the difference in adding ITS to the FBS versus pure FBS.

Cell type: chondrocytes

Cell density: 2 x 10^6 cells/mL

Total cells needed: 4 million.

Alginate: 2% Sigma Aldrich "low viscosity" 250 cps at 2% "high M"

Expectations: We expect the +ITS sample to show greater retention of the chondrocyte phenotype and less de-differentiation into fibroblasts. We therefore expect to see more Collagen II in the +ITS sample and less Collagen I in comparison to the -ITS sample.

Purple:

Experimental plan: Grow two cultures of cells where one is in constant motion (a vortex machine at low speed setting) and the other is stable.

Cell type: chondrocytes

Cell density: 10^6 cells/mL

Total cells needed: 3 million.

Alginate: 2% Sigma Aldrich "low viscosity" 250 cps at 2% "high M"

Expectations: we expect the cells grown in constant motion will grow better because the constant fluid motion will allow a constant exposure to different media with fresh nutrients. The stagnant sample would not have any force mixing the media and cells would take all the nutrients away from the media immediately around them and grow less efficiently.