Rapid, Repeated FISH in Human Amniocytes and Fibroblasts for ...

Rapid, Repeated FISH in Human Amniocytes and Fibroblasts for ...

Bone Matrix Production Using Canine Osteosarcoma Cells
Embedded in Calcium Alginate Beads
Cyndi H. T. Edwards, Aaron M. Fifer, Andrew K. Nickerson
School of Chemical, Biological, and Environmental Engineering
Oregon State University, Corvallis, OR

Results

Introduction
Demineralized bone matrix (DBM) is used in humans to induce bone formation
o Spinal fusion surgery, osteoporotic fracture healing, and dental procedures
o Harvested from human cadaver bones
o High variability in quality due to factors such as age, gender, and diet
o Major drawbacks can be overcome by producing osteoinductive proteins in vitro
The goal of the project is to determine if gel encapsulation of bone-forming osteoblasts is
a feasible method of cell stabilization for commercial DBM production.
o Osteoblasts prefer to grow attached to a surface the alginate matrix of a bead
provides sites for adhesion
o Bone cells respond to physical stress by producing more protein, so agitation may
increase yield
o Alginate weight percent is involved in shear stress transduction, and effects pore
size

Experiment 1

Value
3.75E-06

Definition
cell density [g cells/ml bead]

Vbead

0.05

volume of bead [ml]

Rp/c

0.1

cellular rate of protein production [g protein/g cell*h]

Rp

1.875E-08
150

bead rate of protein production [g protein/bead*h]
# beads per beaker

F

2.813E-06
0.5208333

total rate of protein production in a beaker [g protein/beaker*h]
perfusion rate [mL/h]

Vbioreactor

25

volume of bioreactor [mL]

Cp,bone

5.4

bone protein concentration in media coming out [ug/mL]

Cp,medium

2652

serum protein concentration in media coming out [ug/mL]

Cp,tot

2657.4

total protein concentration in the media [ug/mL]

RpTOT

Cells
The percentage of viable cells digested from 20
beads from each of the four treatments at Day 9.

A cross-sectional slice of a gel bead stained with
trypan blue shows the distribution of cells within a
bead taken at 4x magnification. Cells can be seen as
small dark specks.

Gel Beads

Incubator 37C, 5% CO2

Potential Improvements

Beakers
Orbital
Shaker

Variable
X

N

Experiment 2

Materials and Methods

Modeling

Tflasks

Water Pan

Day 2
After the medium is removed, the
gel beads remain pink because
they have taken up medium. Here
a bead is being taken for imaging.

Protein concentration as measured by Bradford
assay in samples being prepared for SDS-PAGE
(Sodium Dodecyl Sulfate PolyAcrylamide Gel
Electrophoresis).

Cell viabilities in all experiments were lower than anticipated. In the first experiment, it
was due to low inoculation density resulting in poor cell-to-cell signaling. In the second
experiment, the viability was higher at about 60% on Day 2, but still low.
In the future if work is continued on this project, the following improvements will be
made:
oPurchase Type-1 collagenase for bead digestion
oUse ELISA (Enzyme-Linked Immunosorbent Assay to determine trace protein
concentrations
oAdditional controls for the protein assay
oDo not use expired materials

Acknowledgements
We would like to thank Dr. Russell Turner and the Skeletal Research Lab for providing
lab space and equipment, Dr. Philip Harding for funding and support, Dr. Kevin Marley
for providing cells, supplies, and wisdom, Dr. Adam Higgins for advice on freezing cells,
and Dr. Christine Kelly for guidance, reference materials, and assistance with assays.

Day 6

Dead
Cell
Viable
Cell

A hemacytometer is used to determine cell viability.

Unhealthy
Cell

SDS-PAGE results: other than ladder, which is
composed of a mixture of proteins of known
molecular weight, most of the lanes resemble the
media control. Flask 2 and flask 6 are T-flask
controls. Exp. 2-A, 2-B, and 2-C are beads that
could not be digested that were soaked in protein
extraction solution.

Healthy
Cell

Healthy osteosarcoma cells appear stretched out,
while unhealthy cells appear spherical in a T-flask
(10x magnification).

Day 9

References
Bae et al. Intervariability and Intravariability of Bone Morphogenetic Proteins in Commercially
Available Demineralized Bone Matrix Products. Spine 2006; 31(12): 1229-1306.
Chen, Huang-Chi and Yu-Chen Hu. Bioreactors for tissue engineering. Biotechnology Letters 2006;
28: 1415-1423.
Freshney, R. Ian. Culture of Animal Cells. New York: Wiley-Liss, 2000
Phelan, Mary C. Basic Techniques for Mammalian Cell Tissue Culture. Current Protocols in Cell
Biology (1998) 1.1.1-1.1.10
Sandhu et al. Demineralized bone matrix, bone morphogenetic proteins, and animal models of spinal
fusion: an overview. European Spine Journal 2001; 10:S122-131.
Unpublished data, Oregon State University Skeletal Research Lab.

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