Biomedical Engineering Reference
In-Depth Information
to engineer tissues with complicated 3-D geometry con-
taining multiple cell types. More significantly, bioreactors
can impart appropriate biochemical and mechanical
stimuli in a controllable environment to promote cell
growth, maturation, and tissue differentiation. Artificial
tissue development in bioreactors generally involves less
handling than static culture methods to significantly
reduce contamination risk and is amenable to scale up for
the generation of tissue products. Bioreactors can serve as
tissue growth systems as well as packaging and shipping
units that can be delivered directly to surgeons.
Bioreactors have been widely used from drug pro-
duction to beer brewing to create products in an efficient
manner. The basic concept of bioreactors that take ad-
vantage of microorganisms is to create an environment
that is advantageous for microorganisms to the creation
of a desired product, whether it be penicillin, alcohol, or
ECM. Nutrient, waste, and oxygen levels must be care-
fully controlled to prevent the organisms from dying.
There are three types for such bioreactors: batch, fed-
batch, and continuous systems. In a batch system, or-
ganisms are combined with the required nutrients in
a single-step process. A fed-batch process is similar to the
batch reaction in that nothing is removed during the re-
action. However, in contrast to the batch system, addi-
tional nutrients are added over a portion of the process to
keep the reaction rate at its maximum. A continuous
bioreactor adds nutrients and removes waste and prod-
ucts over the entire course of the reaction. Continuous
reactors are the most efficient and cost-effective once the
equipment is running.
The bioreactors used in engineering tissues are pri-
marily batch processes and there is no need to run a large-
scale operation. As custom design of engineered tissues is
often necessary because of immunological concerns,
a continuous reactor is not necessary. The bioreactors for
engineering tissues, as opposed to bioprocessing, provide
an environment in which cells continuously reside
throughout the culturing period. Mass transfer becomes
the main concern because nutrients and oxygen must be
provided in sufficient amounts to tissues to grow to
a usable size. The stimulus from a mechanical force can
still be beneficial. Articular cartilage in the knee experi-
ences shearing stresses during normal movement. On the
surface of cartilage, a thin layer of synovial fluid provides
lubrication, which reduces friction, but shearing of the
tissue still occurs because of the solid-on-solid contact.
Shear forces alter the phenotype of the cells seeded on
a scaffold to one that is close to native cartilage. These
forces can be emulated using a flowing fluid either across
or through the cell-seeded scaffold. Microcarrier beads
may effectively provide surface area for attachment of
anchorage-dependent cells. In addition, agitation of
microcarrier cultures leads to homogeneous culture
conditions and improved gas-liquid oxygen transfer.
If constructs must be produced using patient-specific
cells that do not produce an immune response, then
continuous bioreactors are not required but large-scale
bioreactors that resemble a batch process will have to be
created. This makes the task much more difficult be-
cause the samples must be separated at all times and the
equipment must be sterilized after each batch. A me-
chanical force must be applied to each scaffold in a con-
trolled manner without any mixing of cell batches.
Tissue culture systems that provide dynamic medium
flow conditions around or within tissue-engineered con-
structs should be designed to enhance nutrient exchange
and cell growth
in vitro.
Such tissue culture systems are
required as bioreactors to engineer thicker, more uniform
tissues for transplantation. In addition to enhancing mass
transport, bioreactor systems may be useful to deliver
controlled mechanical stimuli such as flow-mediated
shear stress, matrix strains, or hydrostatic pressures to
tissue constructs. This general approach has been used to
culture a variety of 3-D constructs including bone, car-
tilage, muscle, and blood vessels. Tissue culture systems
that incorporate dynamic medium flow conditions for
developing 3-D tissue constructs include spinner flasks,
perfusion systems, and rotary cell bioreactors. In general,
improved cell viability, proliferation, and ECM pro-
duction have been demonstrated in dynamic systems
relative to static controls. Internal fluid flow in each
system is achieved in different ways. In spinner flasks,
stirred culture medium is moved past the scaffolds at
fixed positions within the vessel. In rotating bioreactors,
the scaffolds are not fixed, but in continuous free fall
within the culture medium during bioreactor rotation. In
perfusion cultures, culture medium is directly perfused
using a uniform fluid pressure head applied to the scaf-
folds and fluid. In spinner flasks, flow and mixing of
culture medium is associated with turbulent shear at
construct surfaces. Mass transport between the con-
structs and culture medium is enhanced by convection,
whereas mass transport within the constructs remains
controlled by diffusion. In perfused reactors, interstitial
flow of culture medium enhances mass transport
throughout the construct volume, and exposes all cells to
laminar shear.
7.2.6.3.1 Spinner flask
One of the simplest bioreactors is the mechanically
stirred flask and the most common mechanically stirred
bioreactor is the spinner flask. Scaffolds seeded with cells
are attached to needles hanging from the cover of the
flask and suspended within a stirred suspension of cells
with addition of sufficient medium to cover the scaf-
folds, as shown in
Fig. 7.2-21 [17]
. This method has
produced favorable results, but potential weaknesses of
the technique include the amount of time required for
