Biomedical Engineering Reference
In-Depth Information
“novel” substrates. We stress that this preference is not merely for convenience; rather, given the
high sensitivity of cells to minute changes in surface composition, changing the protocols and
substrate may yield artifactual results in micropatterning experiments or afect future experi-
ments probing diferent cellular functions.
his need for the random-culture experiment to be an appropriate control for the
micropatterned-culture experiment places stringent constraints on the choices of micropat-
terning techniques that are appealing to the cell biologist. he substrates universally used for
cell culture in molecular and cell biology research are either glass or polystyrene—either coated
with proteins or bare. On the other hand, cell micropatterning approaches largely use one of
two strategies to deposit cells on designated areas of the cell culture substrates: (1) selective cell
attachment is guided by diferential adhesiveness of the substrate (a very simple and widely used
method to deter cell attachment consists of adsorbing albumin, a protein that lacks cell adhesion
motifs, on the cell culture substrate); or (2) cell attachment to a homogenously adhesive sub-
strate is blocked in selected areas with a removable physical barrier. For an extensive coverage of
these methods, see Sections 2.5 and 2.6.
6.1.3 From Cells in Large Static Volumes to Cells in Small Flowing Volumes
As pointed out previously, in high-density microluidic cell cultures, the microenvironment's
nutrients, pH, and gas concentrations cannot be kept constant for long periods of time, and
replenishing the volume of the microluidic chamber becomes necessary. An extreme deviation
of a “proper” cell culture environment results in obvious cell death. However, the efects can
be much subtler because (a) cells are exposed to shear stress from the low, and (b) at the cell
membrane surface, the actual concentration of growth factors (that either bind to cells or are
secreted by them) “seen” by the cell depends on the speed of the low, which efectively “washes
away” the growth factor molecules that happen to difuse into the stream. Some researchers
have argued that cells that normally do not grow well in culture, such as neurons and stem cells,
should grow better in small microluidic chambers where endogenous growth factors rapidly
accumulate.
Given that low/shear stress may produce unanticipated deleterious efects on the cells, sev-
eral groups have been implementing “
open microluidics
” systems whereby the cells are seeded
on open pools or reservoirs, and the luids are supplied to the cells via side channels (e.g., “micro-
jets,” see Section 3.9.3, or difusion ports). hese systems are inherently more benign to the cells
because at least in their no-operation mode they are guaranteed to produce the same results as
the control surface. (On the other hand, a closed-microchannel system in its no-operation mode
is guaranteed … to kill the cells!).
6.1.4 From a Homogeneous Bath to Microluidic Delivery
In traditional cell culture, cells are bathed in a homogeneous medium; thus, any cellular
response that relies on graded or focal exposure of the cell to a given factor will not be observ-
able, including many cell growth and motility phenomena. Traditionally, focal stimulation of
cells with luids has been possible only using micropipettes—which eject luid on application
of a pressure pulse (“puing”) or voltage pulse (“iontophoresis”)—or using chemically-caged
compounds that are “uncaged” (i.e., released) by a laser pulse. Clearly, these techniques result in
very low throughputs and poorly characterized volumes or gradients, they require bulky equip-
ment and substantial manual skill, and are unscalable (stimulation at more than three or four
sites is not practical, at least with pipettes). Microluidic systems, on the other hand, constitute a
technology for directing many diferent luids to a large number of (small) cell populations that
is scalable, amenable to luid dynamics modeling, and where the delivery system is prealigned
with the cells. In 1999, a collaborative team led by Don Ingber at Children's Hospital (Boston)
and George Whitesides at Harvard University irst demonstrated the use of multiple laminar
