Effects of Cell Concentration on Chondrocyte Differentiation and
Viability in Dense 3% Alginate Beads
Emily Suter
Massachusetts Institute of Technology, Department of Biological Engineering
Introduction:
Through the use of a porous scaffold and soluble growth factors, tissue
engineering is engineering method that allows for the repair of tissues damaged by
trauma or disease. As a growing field, this means of manipulating biomaterials holds
great potential for in vivo regeneration of new tissue. For the development of new
cartilage tissue, chondrocytes or progenitor cells are applied to alginate scaffolds, yet
maintaining differentiated cells requires their growth in alginate beads as opposed to flat
plates. This presents issues of inconsistent movement and viability throughout the beads
due to inadequate transportation of nutrients and growth factors (Heywood). To test for
the effects of a high scaffold density bead on this cell viability, we designed an
experiment to evaluate cell concentration on the proliferation of chondrocytes in dense
alginate beads. Using 3% alginate, we plan on comparing 6.5 M/mL and 1.5 M/mL
concentrations of cells in cell viability and production of Collagen I and II to extrapolate
information on cell competition and differentiation. This information is valuable in the
design of future experiments as it provides a better understanding of cell viability and the
effects of scaffold preparation on the ultimate success of our assay.
Results:
RT-PCR analysis
We performed RT-PCR on both of our sample concentrations, and then isolated
the cDNA to run on a 1.5 % agarose gel. By comparing fluorescence to the housekeeping
gene GAPDH, we were able to determine relative expression of both Collagen I and
Collagen II in each. Across all four samples, our GAPDH expression was fairly
consistent, indicating that the samples began with the same overall amount of DNA. For
Collagen I, both samples had expression similar to that of GAPDH, and after we
normalized our data and subtracted background, we found that the Collagen I to GAPDH
ratio was 1.1 and 1.3 for our 1.5 M/mL and 6.5 M/mL samples, respectively. Collagen II
was expressed much more, with the Collagen II to GAPDH ratio being 3.1 and 2.7 for the
1.5 M/mL and 6.5 M/mL samples. The overall ratios of the corrected Collagen II to
Collagen I were 2.8 for the 1.5 M/mL sample and 1.9 for the 6.5 M/mL. These results
show that a much greater relative amount of Collagen II was produced in our less
concentrated sample. While this could have been due to inconsistencies in our GAPDH
expression across the samples or to an unequal amount of starting sample, these results
might indicate that a greater percentage of cells stayed as chondrocytes (the Collagen IIproducing form) in the 1.5 M/mL samples than in the 6.5 M/mL samples. The physical
number of chondrocyte cells was actually greater in the 6.5 M/mL concentration, but a
greater percentage of the lower concentration was able to remain differentiated.
Before RT-PCR, we measured RNA concentration through its absorbance at 260
nm and used these values to determine quantities of 100ng RNA for our RT-PCR
reactions. To confirm our RNA purity in these concentrations, we compared to
absorbance at 280 nm, and found our ratios to be 2.00 for the 1.5 M/mL sample and
1.625 for the 6.5 M/mL sample. While this confirms the purity of the less concentrated
samples, these ratios indicate some potential protein contamination of our more
concentrated sample. This gives some room for doubt regarding the overall ratios of
collagen expression previously described. It also might provide an explanation as to why
the 6.5 M/mL sample showed low relative amounts of collagen II as compared to the 1.5
M/mL sample. However, because relative collagen I remained fairly consistent, we can
still assume that lower collagen II expression is due to dedifferentiation.
Cell Viability
When imaged under a fluorescence microscope, no clear cell colonies were
visible. Background fluorescence levels were extremely high. While this could indicated
low cell count, it is more likely that the dye solution had trouble traveling quickly
through the thicker bead over a short time interval, creating a more uniform visualization
of the fluorescence. There was not a significant difference between the live (green) and
dead (red) fluorescence, again indicating that the dyes were just poorly distributed and
washed away due to thick alginate. However, we were able to see cells within the bead
under a light microscope. While this does not indicate whether the cells are live or dead,
it does indicate cell presence and movement as they have made their way within the
entire bead.
ELISA Protein Assay
After growing chondrocytes for 11 days, Collagen I and II proteins were isolated
and prepared for indirect ELISA analysis. Samples were treated with two antibodies and
secondary fluorescence was measured to determine relative protein concentration against
I and II standards, thereby measuring levels of cell dedifferentiation. Two different
samples were measured, a “less” and “more” sample referring to the amount of beads
originally contributing to the collagen level.
Due to low protein levels, however, ELISA results were fairly inconclusive for both, as
the highest concentration lay significantly below the lowest standard measurement. After
background subtractions and standards calibrations, most experimental values in fact
were below zero. These inconclusive results may be caused by multiple experimental
errors, including faulty antibody application or washing. However, this is not consistent
with the success of our standards measurements, meaning simply low concentrations of
protein and potentially plate reader error are the most likely causes of unproductive
results.
Discussion:
While our ELISA test did not yield significant results, we were able to show a
higher level of maintained differentiation in the lower concentration sample through our
RT-PCR analysis. With a 1.4 times increased Collagen II to Collagen I ratio in our 1.5
M/mL sample, a correlation between appropriate cell concentration and differentiation
can be established, leading us to speculations as to how a lower concentration might
promote this. We can consider whether the more crowded environment within the bead
negatively affects differentiation, possibly due to intercellular interactions that signal to
adjacent cells to become less chondrocytic. Our higher concentration sample also
contained a much higher level of dediffentiated fibroblasts on the bottom of the plate.
While we isolated our alginate beads for actual evaluation so these cells were not
included in the analysis, the beads were still grown in a medium that contained them. We
can speculate that these cells may have effected the dedifferentiation of chondrocytes
within our beads.
These experiments provide important considerations for further experimentation.
In the future, we may perform the same experiments but continually remove the
fibroblasts from our plates to determine the effect on cells within the beads. We might
also consider ways of improving ELISA tests. Low collagen levels may have been
caused by insufficient nutrients in the media or by an abbreviated cell growth period, so
adjusting these could impact the rates of protein production. Additionally, we could
further investigate dense bead effects by improving viability tests. Overall, despite some
inconclusive evaluations, our experiments help tissue engineering by increasing
knowledge and awareness of the effects of dense beads and cell concentration on
chondrocyte proliferation and differentiation. These effects can help future experiment
design and help improve the accuracy of the field.
References:
Heywood HK, Preetkamal KS, Lee DA, BADER DL. Cellular Utilization Determines
Viability and Matrix Distribution Profiles in Chondrocyte-Seeded Alginate Constructs.
Tissue Engineering 10 Volume 9/10: 2004
20.109 S’09 Lab Wiki: Module 3
http://www.openwetware.org/wiki/20.109(S09):Module_3_Accessed Monday May
4,2009
Figure 1. RT-PCR gel analysis of Collagen I and II DNA expression. After isolating
RNA and performing RT-PCR, 1.5 M/mL and 6.5 M/mL cell samples were run on a
1.2% agarose gel for 45 minutes at 125V. Across all four samples, GAPDH expression
was fairly consistent, indicating a consistent amount of initial DNA. For Collagen I,
bands were light and both samples had expression similar to that of GAPDH. For
Collagen II, the less concentrated sample had a much brighter band than our more
concentrated (6.5 M/mL) sample, though both were brighter than Collagen I samples.
This indicates overall greater expression of Collagen II, especially in the 1.5 M/mL
sample.
Table 1. RT-PCR Collagen I and II expression ratios as compared to housekeeping
gene GAPDH. Band images from RT-PCR gel were analyzed for size and pixel
brightness to determine relative Collagen I and II expression levels in our two samples.
Background was removed and sample values were normalized. As seen above, the
Collagen I to GAPDH ratios were much lower (1.1 and 1.3 for the 1.5 M/mL and 6.5
M/mL samples, respectively) than those of Collagen II to GAPDH (3.1 and 2.7 for 1.5
M/mL and 6.5 M/mL samples, respectively). Overall, the ratio of Collagen II to Collagen
I was 1.4 times higher in our less concentrated sample at a value of 2.8 as compared to
the higher concentrated sample, which had a value of 1.9.
Table 2: ELISA assay gives inconclusive, negative concentrations of Collagen types I
& II.
Less sample
More Sample
Cell-seeding
AvgCalc
AvgCalc.
density
bkgd
conc.
bkgd
conc
1.5
M/mL
0.0113
-247
0.0074
-250
CN I
1.5 M/mL
0.0264
-233
0.0314
-228
CN II
6.5 M/mL
0.0143
-244
0.0190
-240
CN I
6.5 M/mL
0.0630
-199
0.0514
-210
CN II
After isolating and plating Collagen I and II, antibodies were applied and an ELISA assay
was prepared. Fluorescnece levels were measured and background was subtracted. Two
different samples were measured, a “less” and “more” sample referring to the amount of
beads originally contributing to the collagen level in that sample. After background
subtraction, calculations placed concentrations below zero, indicating little to no collagen
present or experimental error.
A
B
Figure 2: Plot and linear fit to standards and plot of calculated protein
concentrations of collagen type I & II. After background removal, standard values of
Collagen I and II were calibrated to best fit and the linear values were isolated to an
equation of y=922x-257. Two different samples, a “less” and “more” sample referring to
the amount of beads originally contributing to the collagen level in that sample, for each
concentration was fit along the calibration. However, after background subtraction and
normalization calculation, all collagen levels lay below zero, rendering our results
inconclusive.