J. Biomedical Science and Engineering, 2010, 3, 20-26
doi:10.4236/jbise.2010.31003 Published Online January 2010 (http://www.SciRP.org/journal/jbise/
Published Online January 2010 in SciRes. http://www.scirp.org/journal/jbise
Applications of a new In vivo tumor spheroid based shell-less
chorioallantoic membrane 3-D model in bioengineering
Nzola De Magalhães1, Lih-Huei L. Liaw2, Michael Berns2, Vittorio Cristini3, Zhongping Chen2,
Dwayne Stupack4, John Lowengrub5
1Department of Biomedical Engineering, University of California, Irvine, USA;
2Beckman Laser Institute and Medical Clinic, University of California, Irvine, USA;
3Health Sciences Center, University of Texas, Houston, Houston, USA;
4Moores Cancer Center, University of California, San Diego, USA;
5Department of Mathematics, University of California, Irvine, USA.
Email: nmmagalh@uci.edu
Received 20 September 2009; revised 11 November 2009; accepted 3 December 2009.
The chicken chorioallantoic membrane (CAM) is a
classical in vivo biological model in studies of angio-
genesis. Combined with the right tumor system and
experimental configuration this classical model can
offer new approaches to investigating tumor proc-
esses. The increase in development of biotechnolo-
gical devices for cancer diagnosis and treatment, calls
for more sophisticated tumor models that can easily
adapt to the technology, and provide a more accurate,
stable and consistent platform for rapid quantitative
and qualitative analysis. As we discuss a variety of
applications of this novel in vivo tumor spheroid
based shell-less CAM model in biomedical engineer-
ing research, we will show that it is extremely versa-
tile and easily adaptable to an array of biomedical
applications. The model is particularly useful in
quantitative studies of the progression of avascular
tumors into vascularized tumors in the CAM. Its en-
vironment is more stable, flat and has a large work-
ing area and wider field of view excellent for imaging
and longitudinal studies. Finally, rapid data acquisi-
tion, screening and validation of biomedical devices
and therapeutics are possible with the short experi-
mental window.
Keywords: CAM; Cancer; Spheroid; Optical Coherence
Tomography; Photodynamic Therapy; Computational
Modeling; Angiogenesis
Effective investigations in cancer research, whether in
cancer dynamics, drug delivery, drug and diagnostic tool
development, involves the use biological models that
closely reflect realistic solid tumors.
While in vitro models may not provide a complete as-
sessment of the processes that occur only in the envi-
ronment of solid tumors [1], scientists have relied on
tumor in vivo models or the integration of tumor in vitro
models with in vivo models to circumvent the shortcom-
ings in in vitro models.
Current integration approaches typically introduce
tumor cells to in vivo models as single cell suspensions,
multi-layered systems such as biopsies, or embedded in
scaffolds such as gels or sponges prior to implantation [2,
3]. Additionally, some investigators add exogenous fac-
tors in the culture medium or scaffolds to induce certain
functions such as angiogenesis [4] and invasion, that
may manifest only in in vivo environments [5].
Single cell suspensions and biopsies may not be ideal
tumor systems for bioengineering applications requiring
quantitative analysis. This is because, biopsies cut di-
rectly from the animal host may not have a homogene-
ous cell population, and single cell suspensions lack a
uniform and constant shape.
In contrast, the symmetric 3D configuration of multi-
layered tumor spheroids, a sphere, and the radial de-
pendency of their proliferative and metabolic properties,
can facilitate the generation of boundary conditions and
fixed data points useful in quantitative investigations,
such as computational modeling of tumor growth [5].
Tumor spheroids have been used extensively in studies
of cancer dynamics both as an in vitro model [7,8,9,10]
and combination with an in vivo model [9] because they
manifest similar growth and morphological dynamics of
solid tumors. They begin with an avascular growth stage
where the tumor grows to a certain size in vitro generat-
ing a necrotic center. As we demonstrate later in the arti-
cle, tumor spheroids can induce angiogenesis and pro-
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SciRes Copyright © 2010 JBiSE
Figure 1. H&E histological section of a 1 mm diameter
ACBT glioblastoma spheroid embedded in CAM. Grey
area is the CAM; center region in purple denotes the tu-
mor spheroid. Dark purple regions along the top mem-
brane denote tumor cells invading regions of the CAM; (a)
Top view of tumor spheroid and CAM interface of the
formalin fixed spheroid inside CAM (mesoderm) after
removal from the chicken and embryo prior to histologi-
cal processing; (b) Tumor microvasculature as a result of
angiogenesis (1,2,3).
mote vascular growth when introduced to an in vivo en-
vironment with existing vascular network.
Biomedical imaging systems ideally require a wide
working area, and a steady flat environment for consis-
tent analysis. Imaging resolution and light penetration
deep into animal tissue can be compromised due to ex-
cessive light scattering and absorption from animal tis-
sues. Qualitative and quantitative data acquisition may
be facilitated with tissue of greater transparency such as
the chicken embryo chorioallantoic membrane (CAM).
The traditional in-shell CAM model [12], although
still useful as an alternative system to animal models for
studies of tumor angiogenesis[13] and tumor dynamics
[14,15], is not optimal for imaging and biomedical en-
gineering investigations due to its restricted and unstable
environment. Within the shell environment, the embryo
can move freely in the presence of perturbations. Drug
injections and imaging studies can be challenging with
constant movement of the medium. In addition, the ex-
perimental window of the shell model can be very small,
restricting access to the entire vessel network and limit-
ing the size of the experimental site. Surface tension may
cause the surface of the CAM in the shell model to
The shell-less CAM version was introduced to address
the drawbacks of the traditional shell CAM model
[16,17,18]. The shell-less CAM model not only offers a
stable and flat environment, it also offers a large ex-
perimental area and wider field of view useful for imag-
ing and biomedical engineering applications.
Unlike current shell-less models, our model is the first
model to use three-dimensional spheroids as the tumor
system of choice. The three-dimensional spheroids are
implanted directly into the CAM, and vascularized
spheroids are generated endogenously without the use of
growth factors to promote angiogenesis or scaffolds to
secure and confine the tumor inside the CAM.
This form of vascularization allows the study of the
tumorigenic behavior of three dimensional tumors with-
out the application of exogenous signals that could oth-
erwise interfere with the natural processes of tumors.
Tumor spheroids combined with the shell-less CAM
model can enhance the capabilities of bioengineering
modalities by providing a sophisticated tumor model
with excellent imaging properties that can facilitate the
studies of the angiogenic switch, and model the onset of
tumor progression quickly and more effectively.
The in vivo shell-less CAM tumor spheroid model was
prepared as follows:
Initially, monolayers of ACBT grade IV human glio
blastoma were cultured in T-75 culture flasks containing
enhanced Dulbecco modified Eagle medium (DMEM),
supplemented with 10% heat-inactivated fetal bovine
serum, 1% of streptomycin, penicillin, L-glutamine, and
non-essential amino acids. The cells were maintained at
37ºC and 8% CO2 in a tissue culture incubator. After
reaching 60% confluence, small tumor aggregates
started to form. Using the liquid overlay method [7],
ACBT suspension aggregates were collected from the
flasks and transferred to culture medium filled square
Petri-dishes with the bottom covered with 2% agar. Cul-
ture medium was changed three times a week. The tumor
spheroids derived from ACBT suspension aggregates
grew to 1 mm in diameter in approximately 30 days. For
consistency, 1 mm3 spheroids were selected from the
spheroid colony on the day of implantation.
Three days old white fertilized Leghorn chicken eggs
(AA Lab Eggs, Inc, Westminster, CA, USA) were disin-
fected with 70% alcohol wipes, and incubated in a ven-
tilated hatching incubator at 38°C for three hours. After
incubation, eggs were removed from the incubator, and
under light restricted conditions, egg contents were
carefully transferred from the egg shells to a condiment
cup. The cups are covered with a breathable polyethy-
lene sheet (fisher brand) and returned to the incubator.
On day seven of embryonic stage (EA), the cups con-
taining the chicken embryos were removed from the
incubators. After a small incision was made on the outer
membrane of the CAM, one tumor spheroid was im-
N. De Magalhães et al. / J. Biomedical Science and Engineering 3 (2010) 20-26
SciRes Copyright © 2010 JBiSE
planted onto the mesoderm of CAM. Chicken embryos
were returned to the incubator for 24 hrs. The implanta-
tion was assessed after 24 hours, and angiogenesis was
assessed 7 days post implantation (EA 14) using a ste-
reomicroscope (Olympus, model SZH) coupled to a
digital camera (Olympus DP 10). After visual assess-
ment, the CAM/spheroid interfaces were fixed with 10%
formalin, removed from the CAM, and stored in 10%
formalin solution overnight.
The samples were processed, embedded in paraffin
and cut in 6 µm serial sections. The sections were
stained with hematoxylin and eosin (H&E) to distinguish
the tumor, tumor microvasculature and CAM environ-
Microvasculature is observed around the spheroid region
in Figure 1(a). The middle H&E stained histological
section of the CAM/tumor spheroid interface (largest
cross-sectional area of tumor spheroid) shows three dis-
tinct microvessels at the center of the spheroid (Figure
1(b)). New tumor regions are observed, as well as inva-
sion in adjacent CAM areas.
Vascularized tumor spheroids were endogenously
generated with this spheroid based shell-less CAM
model using spheroids derived from human glioblastoma
(U-87 MG and ACBT), human breast cancer (MCF-7),
and human pancreatic cancer (BXPC-3) cell lines. En-
dogenous tumor induced angiogenesis is evident by the
penetration of the blood vessels to the center of the
ACBT tumor spheroid as shown in Figure 1(b). New
tumor regions on the CAM (grey regions) adjacent to the
tumor spheroid (middle purple mass) show the invasive
capability of the tumor.
Not only is this system adaptable to different types of
tumor cell lines to generate vascularized tumor spheroids,
it can be used to investigate multiple tumor related
processes including, tumor growth, angiogenesis, inva-
sion and metastasis. The experimental configuration of
this system allows its integration with biomedical engi-
neering platforms for more sophisticated analysis of
these biological processes.
The following section discusses applications of this
model in some areas of biomedical research, including
therapeutic, computational, and optical imaging studies.
4.1. Application in Therapeutic Studies:
Photodynamic Therapy
The CAM system has been used previously to study the
effects of therapeutic drugs, such as chemotherapy drugs
and photodynamic therapy (PDT) photosensitizers [19]
on tumor cells and microvasculature. The advantage of
Figure 2. ALA mediated Photodynamic Therapy (PDT) in
vascularized tumor spheroids implanted on the CAM of a
chicken embryo. (a) Normal vascularized ACBT glioma sphe-
roid pre-PDT; (b) Vascularized ACBT glioma spheroid post-
using this CAM-spheroid model to study therapeutic
efficacy is that, multiple spheroids can be implanted on
the same system for dosimetry analysis andmulti-param-
eter evaluations.
In a previous Photodynamic Therapy study, the
shell-less CAM-tumor spheroid model was used to ex-
amine the effects of combined Amino-levulinic Acid
(ALA) mediated PDT on tumor growth and microvascu-
lature [9]. Damage to the tumor cells, extracellular ma-
trix (ECM) and microvasculature (occluding) after acute
ALA-mediated PDT was observed in Figure 2(b). Indi-
vidual blood cells are no longer distinct, and microvas-
culature lining was no longer visible (Figure 2(b)), com-
pared to normal tumor vasculature observed in Figure
2(a). This shows that PDT was effective in causing vas-
cular damage inside the tumor and on the CAM. The
tumor cell shape and nucleus is no longer round. Previ-
ous studies have reported similar findings [19,20]. Addi-
tionally, the extracellular matrix (ECM) of the tumor
environment was observed to have a rough characteristic
after PDT treatment.
Standard assays have used tumor cell suspensions
embedded in the CAM to investigate the effect of PDT
on tumor growth and angiogenesis [5]. Because cells
suspensions tend to be more diffuse than spheroids, it is
possible that in cell suspension, the PDT effect could be
excessive since solid tumors do not appear in nature as
cell suspensions. Furthermore, the use of exogenous
angiogenic factors to induce angiogenesis may introduce
differences in PDT efficacy. This in vivo shell-less CAM
– tumor spheroid model would be a great model to con-
duct comparative studies since no exogenous factors are
used to induce angiogenesis, and the morphology of the
spheroid and its natural growth in the CAM represent a
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SciRes Copyright © 2010 JBiSE
Figure 3. (a) 3-D Optical Coherence Tomography (OCT)
and (c) Optical Doppler Tomography (ODT) imaging of
CAM-spheroid section of a live chicken embryo (Image
Acquisition Time ~2 min). (b) This figure shows real
time ODT imaging of blood flow in a major blood vessel
in the CAM next to the spheroid. The different colors
represent the velocity of blood flow. Black color repre-
sents zero velocity and red color represents maximum
velocity. Velocity increases from black, green, yellow to
more realistic model of in vivo tumors.
The use of this model is not limited to photodynamic
therapy. Nanotherapy has been a growing field of invest
tigation in recent years. Scientists are interested in de-
veloping effective delivery mechanisms for therapeutic
drugs of the same nanoscale as their biological targets.
Scientists have used the traditional CAM system to test
the delivery capabilities and target selectivity of thera-
peutic drugs and hydrophobic nanoparticles carrying
therapeutic drugs [19] for photodynamic therapy based
[21] and chemotherapy based [22] cancer treatments.
Nanoparticles for drug delivery and treatment pur-
poses could be introduced into the CAM vascular net-
work for analysis and validation of its effectiveness to
reach the tumor spheroid region penetrate the tumor mi-
crovasculature and release the therapeutic drug. When
used in conjunction with a color coded marker or visual
tag, the nanoparticles can be tracked by means of in vivo
imaging systems as they progress from the CAM vascu-
lar network to the tumor spheroid microvasculature.
As discussed in the following section, Doppler studies
[23,24], such as rate of nanoparticle delivery to the target
site could also be quickly assessed by coupling our tu-
mor system with a Doppler based imaging system.
4.2. Application in Optical Imaging Studies
Sophisticated imaging devices with cancer diagnostic
implications can be validated and optimized using this
fast and simple in vivo shell-less CAM – tumor spheroid
model. The large aperture and flat surface of the model
could facilitate imaging, system alignment and diagnos-
tic experiments. This shell-less CAM tumor spheroid
model was used to validate a combined Optical Coher-
ence Tomography (OCT) and Optical Doppler tomogra-
phy (ODT) system developed by Zhongping Chen et al
OCT has been used extensively in the clinical arena as
an alternative to conventional systems such as MRI and
CT to image the morphology of soft tissue [27]. In addi-
tion, ODT has been used to investigate and measure flow
of blood and other fluids in tissue [24,27,28,29,30]. Both
systems can be combined to provide both morphological
and functional information of the tissue of interest
The structure and morphology of the CAM-spheroid
interface (Figure 3(a) and 3(c)), as well as the blood
flow in the vessels (Figures 3(b) and 3(c)) were ana-
lyzed simultaneously using this combined system.
The morphology of CAM – tumor system was suc-
cessfully assessed using this combined OCT-ODT sys-
tem. The shape of the tumor spheroid as well as of the
blood vessels along the surface of the CAM is clearly
evident in Figures 3(a) and 3(c). However, any branch-
ing of vessels into the tumor spheroid cannot be detected
from the OCT and ODT images.
In addition, Figure 3(b) shows the real-time imaging
of a functional major blood vessel in the CAM with
large blood flow in the center, and less blood flow in the
periphery. The ODT channel of the system can detect
velocity changes based on color mapping, with black
color representing zero velocity and red color represent-
ing maximum velocity. Thus, velocity increases from
black, blue, and green, yellow to red. Figure 3(c) illus-
trates the variation of blood velocity along major vessels.
Yellow regions depict centers of high velocity, while
blue regions depict areas of low velocity. There is a blue
region on the major blood vessel adjacent to the tumor
spheroid. One can speculate that low blood flow in that
region may be an indication of branching of vessels near
the tumor causing a reduction of flow in main vessel.
Further work is necessary to investigate this phenomena
and any impact that tumor induced angiogenesis may have
on blood flow in major vessels adjacent to tumor masses.
Moreover, future advancements in the technology of the
ODT system may allow the direction of blood flow to be
detected, leading to the identification of different types of
vessels in tissue such as capillaries and veins.
4.3. Application and Integration in In Silico and
Computational Studies
Mathematical modeling and multi-scale computer simu-
lations of tumor dynamics have a promising future as
innovative diagnostic tools for treatment of cancer in
addition and complementary to experimental and clinical
N. De Magalhães et al. / J. Biomedical Science and Engineering 3 (2010) 20-26
SciRes Copyright © 2010 JBiSE
Figure 4. 3-D In-silico simulation of Tumor Spheroid in-
duced Angiogenesis: (a) Early time; (b) Later time. The thin
curves show vessel sprouts, the thick red curves describe
blood-carrying vessels. The inner surface bounds the perine-
crotic region. Figure courtesy of Dr. Fang Jin using methods
described in [36,39].
investigations [31].
Various mathematical models have been designed to
perform in silico (on the computer) experiments or
simulations to investigate and predict tumor behavior
and response to therapy both in vivo and in vitro [32,33].
These include modeling of tumor growth [34,35,36],
invasion [37], angiogenesis and vascular growth [36,38,
39,40], drug delivery [5,41,42] and therapeutic response
[43,44]. The in-silico results are compared with experi-
mental and histological in vitro and in vivo data to vali-
date and optimize the model’s predictions. In addition to
providing more insight into the cancer dynamics, the
predictive capability of the computer tumor simulator
may offer many future clinical applications.
To simulate tumor processes, these models require the
acquisition of parameters such as tumor and necrotic
core sizes before and after angiogenesis, density of
blood vessels, speed of vessel migration towards the
tumor, and gradients of growth factors involved in an-
giogenesis such as VEGF. The predictive capabilities of
these models can be improved and optimized with the
use of in vivo biological models that represent the dy-
namics of realistic tumors.
Current computational models often use tumor sphe-
roids to integrate in vitro experimental data with vascu-
lar tumor growth simulations and vascular extrapolations
[36,39]. An example of in silico tumor spheroid inducing
angiogenesis is shown in Figure 4. The in vivo shell-less
CAM 3-dimensional tumor spheroid model has the po-
tential to enhance the capabilities of computational
models by providing data for an in vivo and three-di-
mensional component representative of solid tumors.
Thus, by integrating this data, tumor simulations will
reflect more realistic representation of tumor vasculature
and behavior.
The shell-less CAM tumor spheroid system may be
used to acquire the parameters described above and
validate computational models because it can yield fast
results due to its short experimental period. Furthermore,
the transparency of the CAM may facilitate data proc-
essing using imaging systems. In addition to the CAM’s
flat surface and closed system, the spheroid’s initial
symmetrical geometry may facilitate the formulation of
equations and boundary conditions that represent the
processes such as growth and angiogenesis that govern
tumor proliferation. Thus, quantitative information can
be extracted from tissue samples of the shell-less CAM
tumor spheroid system. This model would be easily
adaptable to existing mathematical models already using
the tumor spheroid as an in vitro system, as the spheroid
is simply being transferred to an in vivo platform (the
CAM), enabling the incorporation of the in vivo compo-
nent to existing mathematical principles.
Once these models are optimized they have the poten-
tial to be used to predict the aggressiveness of a patient’s
tumor. In addition, they could also predict a patient’s
tumor response prior to treatment.
That is, the in silico model could be used as a diag-
nostic tool to recommend the most effective individual-
ized treatment based on the patient’s prognosis. This can
prevent the development of resistance to treatment by
eliminating trial and error treatment sampling. Other
advantages of the in silico model include providing fast
and non-invasive diagnosis thus minimizing the dis-
comfort to the patient, and improving overall quality of
life. Finally, while the application of in-silico models in
cancer therapy require improved understanding of cancer
behavior and mechanisms, their versatility allows opti-
mization and calibration of parameters to match with
new developments and knowledge of cancer dynamics.
The in vivo 3-dimensional tumor spheroid based shell-
less CAM model presented in this article is a new, at-
tractive and practical system to study multiple mecha-
nisms of tumor biology, including tumor growth, inva-
sion, and angiogenesis. In addition, this system has a
vast and dynamic application in many biomedical and
bioengineering studies including drug discovery, nano-
particle delivery, therapeutic efficacy, cancer diagnostic
imaging device development and validation, and
mathematical and computer simulations of cancer dy-
namics [45].
The development of this system successfully created
vascularized spheroids in the CAM in the absence of
exogenous factors four days after implantation. Due to
the system’s analogous representation of the microenvi-
ronment found in vivo solid tumors, this model can be
used as an alternative or complement to animal models,
and the research findings can provide preliminary im-
plications to clinical studies.
This article was made possible by the tremendous generosity, expertise
and mentorship of the honorable Ms. Li-Huei Liaw. We acknowledge
N. De Magalhães et al. / J. Biomedical Science and Engineering 3 (2010) 20-26
SciRes Copyright © 2010 JBiSE
Linda Li and Angela Giogys for the valuable contributions during the data
processing stage. We also extend our gratitude to Dr. Tromberg for the use
of the Beckman Laser Institute facilities. We further thank Dr. Fang Jin for
his assistance with the in silico vascular tumor shown in Figure 4.
Financial support for this project was provided by the NIH F31 Grants
CA12371-01 and CA12371-02, and the Merck-UNCF pre-doctoral fell-
owship, and NIH grant number EB-00293.
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