Viability of Ultrafoam as a cell seeding

scaffold and a comparison between dynamic

and static seeding methods
Nicolas Etcheverry, Erin Kizer, Michael Zhao, Yichen Zhao
Department of Bioengineering, University of California, San Diego
Correspondence Email: Ekizer@ucsd.edu

Abstract
Murine 3T3 fibroblast cells were seeded onto an Ultrafoam scaffold to compare seeding efficiencies
between static and dynamic methods, and cell density was measured using a PicoGreen DNA assay. The total
average number of cells seeded in the static and the dynamic methods were 210300 41570 and 498400 29610 cells,
respectively. The t-test for p<0.001 yielded a value of 11.29 and a critical t-value of 5.96, confirming statistical
difference between the seeding efficiency in each method. The seeding efficiencies for the static and dynamic
methods were 29.96 5.924 % and 71.06 4.220%, respectively. A t-test for p < 0.001 yielded a value of 11.29
with a critical t-value of 5.96, verifying statistical difference between the seeding efficiencies. With convection, cell
seeding is much more effective than traditional cell culture methods when plated on an Ultrafoam scaffold.
Introduction
Current medical research highlights a growing need to develop tissue engineered medical products for
purposes ranging from medicinal alternatives to organ replacements. In most cases, tissue engineering begins with
the important step of cell seeding and growth on a scaffold made from a variety of both natural and synthetic
materials. Thus it is of interest to explore and examine cell seeding methods that could potentially lead to improved
seeding efficiency, homogeneity, larger cell densities, and increased cell viability within the supporting scaffold.
Scaffold design and construction, a crucial aspect of tissue engineering, can often be very complicated. An
affordable, effective, and scalable cell seeding scaffold is highly desirable; however, there are currently few options
to choose from. Testing various natural and synthetic polymers to find the optimal material to develop scaffolds can
greatly assist the seeding process for the eventual proliferation of cells to create tissue. Ultrafoam, a commercially
available bovine collagen sponge, is marketed as a topical hemostatic, but its adhesive properties, large surface area
and availability make it a potential material for future scaffold designs (Micou 2012). Studying Ultrafoams ability
to support seeded cells may identify a new use for commercially available product.
In addition to selecting a viable scaffold, the technique used to seed cells will lead to different results.
Seeding can either be done statically in the absence of mechanical forces or dynamically in containers that
introduces mechanical shear forces on the seeding suspension (Micou 2012). It is important to compare the efficacy
of both static and dynamic seeding techniques in order to develop a full optimized cell seeding paradigm. Greater
homogeneity and increased cell density on a scaffold may be achieved by introducing shearing forces to a seeding
suspension compared to static techniques that rely on gravity to bring the cells in contact with the scaffold.

Double stranded DNA from seeded cells will be targeted with using the PicoGreen fluorescent assay in
order to quantify the ability of Ultrafoam, paired with static or dynamic seeding techniques, to support cell seeding.
Methods
First, eight scaffolds are cut out of the sheet of Ultrafoam using a sterile biopsy punch. Four of the scaffolds
are placed into four wells of a 24-well tissue culture plate for the static seeding condition, while the other four are
assembled in the spinner flasks for the dynamic seeding condition. A spacer is placed between each pair of scaffolds
while threaded onto the cap of the spinner flask. The cap assembly is attached tightly with scaffolds inside the
spinner flask after placing a stir bar at the bottom said flask.
Next, the 3T3 media is made by adding 25ml of bovine calf serum and 2.5ml of 100X
penicillin/streptomycin to 222.5ml of DMEM (high glucose), and then warming the solution in a 37C water bath.
Scaffolds in the flask are hydrated by gently adding 100ml of 3T3 media whereas 1ml of it is added to each of the
wells containing a scaffold.
Four plates of 3T3 cells are removed from the incubator, examined under a microscope, and then
trypsinized. The media is aspirated from the plate leaving the cells on the bottom, the cells are washed with 2 ml of
PBS per plate, and 2 ml of warm trypsin is applied to each cell monolayer to release the cells. Trypsinization occurs
in the incubator for 2-3 minutes, and excess media is added to deactivate the trypsin. The four plates are combined
into a flask, and the flask is then centrifuged at 300rcf for 5 minutes. The media in the flask is aspirated., and the
cells re-suspended in 5ml of 3T3 media. After mixing the cells thoroughly, 50l of cells are mixed with 450l of
DMEM and counted using a Scepter. Calculations dilutions are performed to create a cell solution of 1x10 6 cells/ml.
Then, 1ml of the suspension is transferred to each well in the static plate, and 4 ml are added to the flask through a
side port, which is left loosened to allow gas exchange. All the samples are placed in the incubator. Also, 250l of
cells are collected and stored in a -20C freezer for future use.
After 18-48 hours of incubation, the cell-seeded scaffolds and cell suspensions are digested. A 0.5mg/ml
proteinase-K solution is made by combining 100l of the 50X(25mg/ml) stock solution with 4.9ml PBE. 250l of
proteinase-K is added into each cryovial containing a scaffold so that the tissue is completely immersed in the digest
solution. The cells are digested by placing the tube in the 50C water bath and waiting at least 8 hours for complete
digestion, then stored in the freezer.

Lastly, a DNA assay is performed to quantify cell attachment by using two 5l PicoGreen aliquots, the
DNA standards, and the samples. PicoGreen dye solution is made by adding 5l of PicoGreen aliquot into 995l of
TE buffer (1:200) while DNA standards are made by diluting two 5l aliquot of 100g/ml DNA to 1g/ml with
495l of TE buffer (1:100). All dilutions are vortexed. DNA standard solution each has a volume of 500l with the
following concentrations: 200, 100, 50, 25, and 0 ng/ml by adding corresponding amount of TE buffers for dilution
and duplicated.
To measure fluorescence using the fluorometer, 50l of each standard and 50l of the PicoGreen dye are
added to a separate minicuvette and left to sit for 2 minutes. The fluorometer is calibrated by first measuring the
fluorescence of the minicuvette with no DNA component and then the one with a high DNA concentration. Then,
the fluorescence of each standard is measured to show an approximately linear curve on Excel. After vortexing the
samples, a portion of each sample in TE buffer is diluted to an approximate concentration of 25-200ng/ml with a
final volume of 250l and duplicated. 50l of each sample and 50l of the PicoGreen dye are added to separate
minicuvettes and fluorescence is measured. The following equations are used to calculate the seeding efficiency and
other relatable values. The percentage difference in duplicates of the samples are calculated as follows:
|

(1)

whereas a and b are the fluorescence measurements of the duplicates. Cells in sample is
calculated by:
(2)
while DNA in the samples are calculated by:
(3)
Therefore, we will be able to calculate the seeding efficiency using the following equation:
(4)
The data is entered into Excel and graphed along with the standards generated earlier. If any sample is out of range
of the standards or the fluorometer detection limit, the samples are diluted more until the reading falls within the
working range of the calibration curve.

Results
Figure 1 shows the data collected from the fluorometer using known concentrations of DNA to obtain a
standard curve used to analyze the samples. Table 1summarizes the numerical data obtained from the samples.
Percent error was calculated with the
250
.
Fluorescence

following formula:

DNA concentrations in the cuvettes were

obtained by solving the equation of the fitted
line in Figure 1 using recorded fluorescence

y = 0.8435x + 5.0506
R = 0.9806

200
150
100
50
0

values. The concentration of DNA in the

100

150

200

250

DNA Concentration (ng/mL)

samples were obtained by multiplying the

concentration in the cuvette by the dilution

50

Fig. 1 Standard curve of DNA concentration and PicoGreen

fluorescence

factor, giving the original concentration of

the cell samples before the samples were mixed with the PicoGreen. The amount of DNA per cell in the original
cell suspension was found to be 9.61 pg /cell, in contrast with the known value of 13.7 pg/cell that was used for all
subsequent calculations in order to obtain cell concentration values. The percent difference between the empirical
and known values of DNA content of the cell was calculated to be 35.05%. The concentration of cells

sample by 13.7 pg/cell. The number of

total cells in scaffold was obtained by
multiplying the concentration by 476L,
the sum of the volume of the 250 L of

Seeded Cell Count (cells)

dividing the DNA concentration in

600000

80
70

500000

60
400000

50

300000

40
30

200000

20
100000

10

Proteinase K used to disintegrate the

0

scaffold and the 226 L volume of the

scaffold itself. The scaffold was 12mm in

Seeding Efficiency (%)

in solution was then calculated by

Count

Static

Efficiency Efficiency Count

Dynamic

Fig. 2 Comparison of average number of cells and average seeding

efficiency for each seeding method

diameter and 2mm in height. Cell seeding

efficiencies were calculated by dividing total cells found on the scaffold by total cells in the cell suspension added to
the scaffolds and the values are shown in Table 1.

Table 1 Summary of cell concentrations of the samples from PicoGreen assay

Sample Description
Cell S.

Dynamic 1

Dynamic 2

Dynamic 3

Dynamic 4

Static 1

Static 2

Static 3

Static 4

Fluorescence
Fluorescence of
duplicate
Mean
fluorescence
Duplicate
Difference (%)
[DNA] in
cuvette (ng/mL)

28.20

68.83

69.26

63.54

66.64

32.50

34.85

29.15

23.22

22.45

61.60

70.98

59.14

64.19

32.65

37.47

29.30

25.39

25.33

65.22

70.12

61.34

65.42

32.58

36.16

29.23

24.31

22.70

11.09

2.453

7.173

3.745

0.4604

7.246

0.5133

8.928

24.04

71.33

77.14

66.73

71.56

32.63

36.88

28.66

22.83

Dilution factor
[DNA] in
sample (ng/mL)
[Cells] in
sample
(cells/mL)
Total cells in
sample
Seeding
Efficiency (%)

400

200

200

200

200

200

200

200

200

9,614

14,270

15,430

13,350

14,310

6,526

7,376

5,732

4,565

701,800

1,041,000

1,126,000

974,200

1,045,000

476,400

538,400

418,300

333,200

175,400

495,800

536,200

463,900

497,500

226,800

256,400

199,200

158,700

N/A

70.65

76.42

66.10

70.89

32.32

36.53

28.39

22.61

Figure 2 shows that the dynamic seeding method resulted

in more cells seeded in the scaffold, and had a higher seeding
efficiency compared with the static method. A two-tailed unpaired
T-test was done for the comparison between number of cells
seeded and seeding efficiency in dynamic and static methods.
Table 2 summarizes the results of the unpaired t-tests.
Discussion

Table 3 A comparison between of average

cell counts and seeding efficiency between
the dynamic and static methods
Average Dynamic
Cell Count (cells)
Average Static
Cell Count (cells)
Average Dynamic
Seeding
Efficiency (%)
Average Static
Seeding
Efficiency (%)

Average

Standard
Deviation

498400

29610

210300

41570

71.06

4.220

29.96

5.924

After a 48 hour seeding period followed by proteinase k digestion of the cell seeded scaffolds, the
Picogreen DNA assay revealed that the dynamic spinner flask method of cell seeding was much more effective than
the static method due to the higher DNA count and therefore cell count in the dynamic method. The fluorometer
readings pointed towards a two-fold increase in cell attachment to the scaffolds for the dynamic method compared to
the static method, and this is exemplified by the different seeding efficiency values. The dynamic method resulted
in a seeding efficiency of 71.06 4.220% , while the static method resulted in a seeding efficiency of 29.96
5.924%, indicating that the convective forces employed in the dynamic method led to a higher cell seeding by a
factor of 2.37. These results prove that cell movement in solution is an important factor when attempting to
improve cell seeding onto scaffolds.

In research conducted at the University of Twente in the Netherlands, dynamic cell seeding methods
displayed a similar two-fold increase in cell seeding efficiency compared to static methods (Van Blitterswijk
1999). Dermal fibroblasts were seeded onto porous PEO/PBT (Polyactive) scaffolds and seeding efficiency was
measured after 24 hours. While the scaffold, cell type, and seeding period differed when compared to the procedure
in this lab, the seeding efficiencies were almost identical to the results obtained in this lab. The seeding efficiency
was 76 3.6% for the dynamic seeding method, and the static method produced a seeding efficiency of 30 5%
(Van Blitterswijk 1999). The results of the study from the Netherlands as well as this report both confirm that
dynamic cell seeding is much more efficient than the static counterpart.
The traditional static cell seeding mechanism is mainly limited by its lack of convectional flow necessary
for the movement of cells in suspension, and the implementation of this force in the dynamic cell seeding method
exposed the weakness of static cell seeding. While the static method is much less effective due to its lack of cell
movement by convection, the dynamic seeding method fixes this, but this does not mean that the dynamic method is
not without its flaws. The stir bar used in the spinner flask was much smaller than the stir bar used by the other lab
group, and this resulted in a lower cell seeding efficiency compared to those where the stir bar covered a larger area
of the bottom of the flask. The revolutions per minute (rpm) of the spinning stir bar could greatly affect the seeding
efficiency as well, and further testing should be done to make sure that 60 rpm is the ideal setting. Also, the thin bar
holding the scaffolds and directed downwards into the media was much shorter compared to the other group; this
points toward the possibility that the elevated placement of the scaffolds may have affected cell seeding efficiency.
While this hypothesis should be considered, it is weakened by the fact that there was no statistically meaningful
difference between highest and lowest scaffolds in the spinner flask. Other possible sources of error include not
resuspending the cells properly and thus miscalculating and incorrectly diluting the original cell seeding solution.
However, we are still confident in the results. Other mistakes could be attributed to incorrectly diluting the DNA
standards in the cuvettes leading to a skewed standard graph, and therefore resulting in incorrect DNA and cell
concentrations in the samples.
In order to improve the cell seeding efficiency of the static method, a slow pipetting tactic dropping cells
directly onto the top of the scaffold may help in improving how many cells attach. One would want to avoid
pipetting the cell solution directly onto the bottom of the well in order to give more opportunities for cells to interact
with the scaffold with the aid of gravity. More experiments should be run to examine the effects of these different

techniques. With respect to the dynamic method, future experiments should employ the use of a much larger stir bar
in order to increase the cell seeding efficiency, and more tests should be performed with variations in the rpm of the
spinning stir bar. Future testing should also examine effect of the heights of the scaffolds on cell seeding efficiency
in greater detail because larger height differences between the scaffolds may point to a certain height position that is
most effective for attaching cells. These tactics would fine tune the dynamic method for better seeding, and the
analysis of more novel bioreactors may provide some useful design ideas to increase cell seeding efficiency.
The findings presented by this experiment clearly signify the importance of convection when applied to cell
seeding mechanisms. In traditional static cell seeding protocols, the cells are dropped onto the scaffold, and while
some cells attach to the scaffold, many fall to the bottom of the plate well without proper attachment. Without
movement of the solution, those unattached cells do not have the opportunity to come in contact with the scaffold
again for possible attachment. The dynamic cell seeding method fixes this issue by utilizing convection and
allowing for unattached cells to continue flowing in solution until they successfully attach to the scaffold. Many
variations of dynamic cell bioreactors have been designed with this premise in mind, and further understanding of
solution movement with respect to cell seeding will lead to improved cell densities on certain scaffolds in the future.
References
[1] Micou, Melissa Kurtis., and Dawn M. Kilkenny. "Dynamic versus Static Seeding of Cells onto Biomaterial
Scaffolds." A Laboratory Course in Tissue Engineering. Boca Raton: CRC, 2012. 101-06.
[2] Micou, Melissa Kurtis., and Dawn M. Kilkenny. "PicoGreen DNA Assay." A Laboratory Course in Tissue
Engineering. Boca Raton: CRC, 2012. 245-48.
[3] Xiao, Y. -L., J. Riesle, and C. A. Van Blitterswijk. "Static and Dynamic Fibroblast Seeding and Cultivation in
Porous PEO/PBT Scaffolds." Journal of Materials Science: Materials in Medicine (1999): 773-77. Web. 28 Oct.
2012.
Acknowledgements
We would like to acknowledge Dr. Melissa Micou and Andrew Richards for their guidance and contributions.