J. Biomedical Science and Engineering, 2011, 4, 435-442 JBiSE
doi:10.4236/jbise.2011.46055 Published Online June 2011 (http://www.SciRP.org/journal/jbise/).
Published Online June 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Bioreactor for the reconstitution of a decellularized vascular
matrix of biological origin
Matthew G. Geeslin, Gabriel J. Caron, Stefan M. Kren, Ephraim M. Sparrow,
David A. Hultman, Doris A. Taylor
Department of Mechanical Engineering, University of Minnesota, Minneapolis, USA;
Center for Cardiovascular Repair, University of Minnesota, Minneapolis, USA.
Received 11 April 2011; revised 1 May 2011; accepted 11 May 2011.
ABSTRACT
Acellular matrices derived from animal and human
cadaveric donor vessels or other tubular matrices are
appropriate candidates for the creation of tissue- en-
gineered, small-diameter, muscular arteries. Engi-
neering principles have been used to design a biore-
actor and the necessary auxiliary systems for the re-
constitution of a previously decellularized vascular
matrix. The bioreactor enables the attachment of
cells to the luminal and/or exterior surfaces of the
matrix. For the recellularization procedure, the ma-
trix is situated within a sealed compartment in order
to maintain a sterile environment. The matrix is ro-
tated continuously to assure a spatially uniform re-
constitution. The auxiliary systems that serve the
bioreactor are: (a) an oxygenator, (b) peristaltic
pumps, one for conveying the internal cell medium
and the other for conveying the external cell medium,
(c) motor and gearing to create steady and controlled
rotation, (d) reservoirs for the containment of the two
media, and (e) tubing to convey the respective fluids
and to interconnect the bioreactor culture chamber to
the various auxiliary components. A recellularized
matrix produced by the bioreactor demonstrated its
capabilities to reconstitute a previously decellularized
scaffold.
Keywords: Bioreactor; Decellularize; Recellularize;
Blood Vessel; Tissue Engineering
1. INTRODUCTION
There is curren tly signific ant interest in crea ting human
vasculature by making use of scaffolds taken from de-
cellularized vessels or other tubular matrices [1]. One
attractive feature of this tissue engineering approach is
that those matrices can be harvested from animal and
human donors and recellularized with host-specific
(autologous) cells. Harvesting appropriate cells from a
patient, expanding the cells in culture, and applying
them to the decellularized matrix, accomplish recellu-
larization.
This interest has motivated innovative procedures
both for decellularization and for reconstitution of the
matrix with cells. In this paper, the design and imple-
mentation of a device for reconstitution is described.
The capabilities of the device have been verified by
laboratory experiments. This device will be termed the
Internal and External Flow Bioreactor. The bioreactor
facilitates the reconstitution process on both the inner
and outer surfaces of the vascular matrix. In particular,
the utilization of the device provides a coating of cells
on the inn er surf ace of the dec ellu lar ized tu bul ar matr ix
constructs-no matter the origin. To complete the pro-
duction of replacement vessels requires an additional
step in which smooth muscle cells (SMCs) are added to
obtain a co-cultured arterialized construct. This task is
also accomplished by the bioreactor described here.
The engineering of small-diameter artificial arteries
in-vitro has recently been reviewed [1]. That review
reveals a variety of methodologies for tissue-engineered
arterial replacements in various stages of development.
In the present paper, attention is focused on the use of
decellularized vascular or tubular matrices as scaffolds
to which cells may be attach ed. The literature contains a
number of papers [2-12] that deal with various issues
related to recellularization of decellularized vascular
matrices, but it appears that a goal-oriented, first-
principles-engineered, operation-proven device to per-
form recellularization has not been created. As dis-
cussed in the preceding paragraph, the goal of the pre-
sent work is to design, fabricate, and validate the effi-
cacy of such a device.
2. DESIGN METHODOLGY
The initial vision of the bioreactor was a configuration

M. G. Geeslin et al. / J. Biomedical Science and Engineering 4 (2011) 435-442 436
which would enable a continuous flow of tissue-culture
medium both within the lumen of the decellularized ma-
trix and over its external surface. That vision also in-
cluded the idea of matrix rotation to ensure the spatial
uniformity of the final cellular deposition. The minimum
requirements that were specified at the initiation of the
design pr oc e ss included:
a) Continuous, automated, and low-RPM matrix rota-
tion;
b) Closed-loop, operator-controlled perfusion of both
the lumen and the external surface of the vascular matrix;
c) Leakage-free interfaces which enforce the mainte-
nance of a sterile environment within the processing
chamber;
d) Sterilizability of device components that interact
with biological products.
The need for Requirement A is to increase the likeli-
hood of confluent distribution of the endothelial cells
(ECs) in the lumen and smooth muscle cells on the outer
surface of the vascular matrix. Requirement B was mo-
tivated by the desire to separate and separately control
perfusion of the interior and exterior of the matrix to
fulfill the goal of depositing potentially different cell
types on the interior and exterior surfaces in recognition
of different biological needs. Requirements C and D,
taken together, provide for the sterility of the culture
environment.
3. DEVICE OVERVIEW
The description of the device will be subdivided into two
main sections. The first will convey a schematic of the
overall system; describe its functio nality, and its mode of
operation. Subsequently, a number of photographs will
be displayed to illu strate the practical implementation of
the device.
The device schematic is displayed in Figure 1. The
figure is a schematic flow diagram of the internal and
external flow bioreactor. The heart of the device is the
bioreactor culture chamber. It is a cylindrical vessel
having a diameter and leng th of about 25mm and 75 mm,
respectively, with its axis horizontal. It is made of trans-
parent polycarbonate. The function of the culture cham-
ber is to provide a sterile environment in which a decel-
lularized tubular matrix is supported at its ends. The ma-
trix is separately perfused bo th internally and externally.
Internal perfusion is accomplished by connecting the
respective ends of the matrix to the cell-culture-medium
supply and extraction ports that are respectively located
at either end of the bioreactor. External perfusion is ac-
complished via ports into and out of the bioreactor cul-
ture chamber.
It can be seen from the figure that the two flow cir-
cuits are independent and have their own auxiliaries.
One of the auxiliaries is an autoclavable cell-culture
flask that is sealed by a rubber stopper equipped with
inlet and outlet ports. A second is a peristaltic pump that
is situated downstream of the flask. The pump is able to
provide flow-rates between 0.06 mL/min and 43 mL/min.
The discharge of the pump is delivered to the pressur-
ized oxygenator, the third auxiliary, whose function is to
maintain the oxygen content to replace that lost in cellu-
lar respiration.
A key feature of the bioreactor is the capability to ro-
tate the vascular matrix simultaneously with its perfu-
sion. The RPM of the rotation is adjustable from one to
three revolutions per minute. The rotational components
Figure 1. Schematic flow diagram of the bioreactor and its auxiliary components.

M. G. Geeslin et al. / J. Biomedical Science and Engineering 4 (2011) 435-442 437
include not only the vascular matrix, but also the ma-
trix support members to which the matrix is attached.
Rotation is achieved by means of a low-RPM DC
motor. The external shell of the bioreactor does not
rotate.
Figure 2 illustrates the key components of the device.
As can be seen in the figure, the underlying structure of
the device is made of a plastic, and Delrin was chosen
because of machinability and autoclavability. The
bioreactor culture chamber is seen to be the central
feature of the device. Provision was allowed in the
structure for a drive shaft which extended from the
proximal and distal ends of the device. The shaft was
driven by a motor situated at the distal end. By means of
a pair of gears which mate with the driveshaft, rotation is
imparted to the matrix and its supporting structures. The
entire assembly is laid out on a 6.25-mm-thick aluminum
plate.
Figure 3 is a downward-directed view of the fabricated
device in its operational environment. The setup of the
device is specific to the perfusion of the lumen.
Therefore, the exhibited components relate to that mode
Figure 2. Overall view of the fabricated device displaying certain key components.
Figure 3. Downward directed overview of the fabricated device in its operational envi-
ronment illustrating lumenal perfusion.

M. G. Geeslin et al. / J. Biomedical Science and Engineering 4 (2011) 435-442 438
of operation. The fully assembled device perfuses both
the interior and exterior surfaces of the vascular matrix.
The identification labels show the oxygenator, the
culture medium flask, and the drive motor. Also seen in
the photograph are the front and rear drive gears for
rotation.
4. DEVICE DETAILS
The design of the bioreactor includes a number of fea-
tures that guarantee its proper operation. These design
details will now be displayed.
Figure 4 displays the bioreactor culture vessel in
more detail than could be observed in the preceding
overview figures. The figure shows the reactor to
consist of a transparent cylindrical vessel within which
certain critical fixtures are contained. One of these
fixtures is the pair of vascular matrix support members.
The origins of these structures are syringes of suitable
size. The inflow and outflow ports used to perfuse the
exterior surface of the vascular matrix are clearly shown
in this photograph. Other features that are somewhat
obscured in Figure 4 will be called out in later figures
where they are more easily identified.
A dissassembled view of the bioreactor and its
components is presented in Figure 5. Shown there are
the parts of the parent syringes th at were utilized for the
support of the matrix and for the delivery and extraction
for the perfusing fluid. The matrix support structure is
seen to consist of a barrel-like disc at either end and a
bridging structure that interconnects them. A critical
Figure 4. Detail of the bioreactor culture vessel.
Figure 5. Disassembled view of the bioreactor culture vessel.

M. G. Geeslin et al. / J. Biomedical Science and Engineering 4 (2011) 435-442 439
issue was the attainment of a leak-free seal between the
matrix support structure and the inner surface of the
bioreactor culture vessel. The seal was accomplished by
the use of O-rings set into suitible-dimensioned grooves.
The matrix support members are highlighted in Figur e 6.
Each member consists of the delivery end of a syringe
enhanced by a trio of O-rings to obtain a leak-free seal at
the interface of the support members and the culture
chamber. Provision was made, by means of a knurled
handle, for compressing the O-rings to the appropriate
sealing pressure. The funnel-like plastic pieces that convey
and extract the perfusi ng fluid are als o cleary shown.
The sleeve interconnect provides the rigidity needed
for stability during the attachment o f the vascular matrix
to the support structure and subsequent insertion of the
loaded matrix into the polycarbonate cylinder. The
vascular matrix support member is the needle component
of a luer-lock syringe.
The last of the detail figures is Figure 7. This figure is
focused on displaying the components that comprise the
inflow end of the system. Shown there is the drive
mechanism which includes the drive shaft, the vascular
matrix rotation gear, the idler shaft, and the fluid
delivery path. The fluid passes from right to left through
a fluid coupling clamp and the rotating fluid coupling.
5. APP ARATUS ASSEMBLY AND OPER-
ATING PROTOCOL
5.1. Assembly of the Bioreactor
The first step in the assembly of the bioreactor is de-
scribed by making reference to Figure 5. The gap be-
Figure 6. Vascular matrix support structure.
Figure 7. Detail of the drive mechanism and the fluid delivery path at the inflow end of
the system.

M. G. Geeslin et al. / J. Biomedical Science and Engineering 4 (2011) 435-442 440
tween the vascular matrix support members is bridged by
the matrix which is sutured in place. The positioning of the
matrix is defined so that the suture is situated just upstream
of the flaired polyethylene fitting displayed in Figure 6.
The next step also makes use of Figure 5. The O-rings
displayed in the foreground of the figure are put in place in
the vascular matrix support member reinforcement sleeve.
Then, the bioreactor culture chamber is forced over the
O-rings to create an air-tight seal. This sub-assembly is
mated to the gear-ensleeved rotating fluid coupling that is
called out in Figure 7. Prior to the mating, a bolus of cells
must b e inj ec ted to lin e th e int erior o f the mat rix by me ans
of a syringe. The injection is accomplished here through
the ports that are called out in Figure 5.
At this point, the Delrin blocks, visible in the fore-
ground of Figure 7, are adjusted so that the idler gear
mates perfectly with the driving gear. Next, the gear-
ensleeved rotating fluid coupling is inserted into the
adjustable clamp that is called out in Figure 7. Then,
the various connections to facilitate the management
of the flowing media are made. These connections can
be seen in Figure 1.
5.2. Operating Protocol
There are a number of operating protocols that may be
used to achieve different objectives. These include: (a)
uniform coating of the interior surface of the decellular-
ized vascular matrix with endothelial cells, (b) uniform
coating of the exterior of the vascular matrix with
smooth muscle cells or other cells, and (c) reconstitution
of both matrix surfaces with their respective native cell
lines. The description of the operating protocol for the
latter will be conveyed here, since it is the most encom-
passing. To begin the description, it is to be noted that
the interior of the vascular matrix has already received a
bolus of endothelial cells. Then, the rotation of the ma-
trix is initiated and, simultaneously, an endothelial cell
culture medium is circulated about the outside of the
vascular matrix to provide diffusion-mediated nourish-
ment for the adhering layer of the endothelial cells on
the interior surface of the matrix. The nourishment phase
is continued for about eigh t hours and then discon tinued.
At this point, a large suspension of smooth muscle cells
is injected into the culture vessel which is filled to a
height that envelops the vascular matrix. Simultaneously,
the nourishment stream that formerly passed over the
outer surface of the matrix is now ducted to flo w through
the lumen of the matrix. These conditions are continued
for a period of approximately 12 hours. Then, a slightly
different arrangement is initiated. While the nourish ment
stream passing through the lumen is continued, a second
stream consisting of smooth muscle cell culture medium
is passed through the culture vessel as a continuous flow.
To decide on a stopping point of these operations, it is
necessary to weigh the probability o f a more confluently
reconstituted interior and exterior matrix against the
probability of compromising sterility. From experience
with various methods of matrix reconstitution, a period
on the order of three to four days should be allowed
prior to the stoppage of matrix perfusion and rotation.
Prior to the stoppage, preparations have to be made for
histologic analysis of the reconstituted matrix.
6. HISTOLOGIC ANALYSIS OF THE
RECONSTITUTED MATRIX
The sought-for information from the histologic analysis
is the uniformity of the cell coverage and evidence that
recellularization is achieved on both interior and exterior
surfaces of the reconstituted matrix. To convey this in-
formation, a representative sample of fluorescently
stained histologic images is presented in Figures 8, 9, 10
and 11. The first of these figures shows the recellulariza-
tion of decllularized rat aorta. Two flourescent stains
were used to confirm the presence of endothelial cells
and smooth muscle cells, one for each cell type. A third
stain, which binds to DNA, was used to indicate the
presence of either cell type and to show spatial uniformity.
The type of stains used and the colors which they
fluoresce include: DAPI (blue), CMFDA (green), and DiI
(red). DAPI stains the DNA of either cell type, CMFDA
stains endothelial cel l s , and D i I, smooth m uscle cell s.
7. CONCLUDING REMARKS
The key functional features of the device designed and
implemented here are the following :
Figure 8. Recellularization of decellularized rat aorta
showing en face cells of the endothelial type at a vascular
branch point. Stain: DAPI (blue) and CMFDA (green).

M. G. Geeslin et al. / J. Biomedical Science and Engineering 4 (2011) 435-442 441
Figure 9. Lumen surface and exterior surface recellulari-
zation of decellularized rat aorta. The displayed colors in-
dicate: red = smooth muscle cell, green = endothelial cell.
Stains: CMFDA (green), DiI (red), and DAPI (blue).
Figure 10. Interior and exterior surface recellularization
of decellularized rat aorta. The figure shows from right
(interior surface) to left (exterior surface): ECs, elastic
lamina (not stained), tunica adventitia (not stained), and
SMCs. Stain: CMFDA (green), DiI (red), and DAPI
(blue).
 Continuous, automated, and low-RPM vascular ma-
trix rotation
Closed-loop, operator-controlled perfusion of the lu-
men of the vascular matrix
 Independent control of the intra-lumenal stream and
of the extra-lumenal stream
 Vascular matrix can remain in rotation during simul-
Figure 11. Interior surface of the matrix as seen by splaying
the vessel open. Uniformity of the recellularization is clear-
ly in evidence. Stain: CMFDA (red), DiI (green), and DAPI
(blue).
taneous intra-lumenal and extra-lumenal perfusion
Leakage-free interfaces which favor the maintenance
of a sterile environment within the bioreactor culture
chamber
 Autoclavability of all device components that interact
with the culture medium
 Autoclavability of every device component except
the motor
 Custom-desig n e d, hi g h -capacity oxygenato r
 Three degrees of freedom to adjust for vessels of
different lengths
 Easy operator access to vascular matrix installation
and removal
 Positive anchoring of vascular matrix
A laboratory application of the device confirmed the
capability of the Internal and External Flow Bioreactor
to recellularize previously decellularized matrices of
biological origin. In conclusion, this apparatus enables a
more standardized approach for the recellularization of
decellularized matrices and in doing so, expands inves-
tigative opportunities in the area of tissue-engineered
blood vessels.
8. ACKNOWLEDGEMENTS
This work was supported by funds from the Medtronic Bakken Chair,
the University of Minnesota’s Office of Technology Commercializa-
tion “DECELL|RECELL” grant numbers Z05118 and Z05240, the
American Heart Association’s Jon Holden Dehaan Cardiac Myogenesis
Center AHA 0970499N, and the National Institutes of Health Mid-
western Progenitor Cell Consortium NIH U01-RFA-HL-09-004.

M. G. Geeslin et al. / J. Biomedical Science and Engineering 4 (2011) 435-442 442
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