By Dr. Megha Agrawal, Dr. Shyamasri Biswas, Dr.

Kim Van Vliet, Contributing Editors

Vacuum Enabled Microfluidic Cell

Culture Technology:
Controlling Single Living Cells to
the Development of Organ on Chip

acuum assisted microfluidic cell culture technology is

an emerging powerful tool in advanced medical biotechnology research and development that allows temporal
control of the cellular environment. It enables the study of single
living cells in a precisely controlled microenvironment and offers a host of new applications that include generating concentration gradients, patterning substrates for interaction with cells
and stimulating cells with biochemical inducers in a dynamic
fashion. The study of a single living cell is of great significance
as it allows closely mimicking the ever-changing natural environment in which cells normally reside in the human body. The
controlled handling of single cells allows cell line development
or single-cell analysis, e.g., for cancer research or for emerging
diagnostic methods. Hence, the ability to control and manage cell
behavior in microenvironment is of great importance that allows
superior diagnosis and treatment of complex health conditions
that are caused by cell damages [1]. Also, ability to isolate and
control single cells paves the way for future developments of organs-on-chip for many applications in medical biotechnologies.
The vacuum assisted microfluidic technique has been employed for microfluidic perfusion cultures for mammalian cells
that provides a novel means for probing single-cell behavior.
Researchers presented a novel trapping design to easily and reliably load a high density of cells into culture chambers that were
extremely isolated from potentially damaging flow effects. A
transient on-chip vacuum was employed to remove air from the
culture chambers rapidly replaced the volume with a liquid cell
suspension (Figure 1). The researchers demonstrated the ability
and aptness of this method to load and culture three commonly
used cell lines. The authors showed that the incorporation of an

Figure 1. Microfluidic device design and vacuum loading of cells.

(A) Each of the 33 cuboid culture chambers is connected via a narrow
opening to a main perfusion channel that runs between ports 1 and 2.
Ports 3 and 4 with separate air channel allows the application of a temporary vacuum at the polydimethylsiloxane PDMS interface to draw
fluid from the main perfusion channel into the culture chambers. Ports
5-7 comprise the Dial-a-Wave function (DAW) dynamic stimulation
generator. (B) At time zero, upon application of a vacuum in the air
channel, fluid containing cells is rapidly drawn into the culture chambers and fills the traps within 2 minutes, at which point the vacuum is
turned off. HeLa cells then attach and begin to spread out within 1-2
hours after loading during continuous perfusion culture [Source: Lab
Chip. 2012, 12(22): 47324737].

Vacuum Technology & Coating September 2016 www.vtcmag.com 

Figure 2. HeLa and CHO-K1 cells rapidly colonize the glass growth
area of the device during continuous media perfusion after initial vacuum loading into the culture chambers [Source: Lab Chip. 2012, 12(22):
47324737].

on-chip function generator can be used for dynamic stimulation

of cells during long-term continuous perfusion culture [1]. Furthermore, researchers employed Hela and CHO-K1 cells types
that exhibited healthy morphology in all experiments and fully
colonized the uncoated glass surface of each culture chamber
within a few days, Figure 2 [1].

One of the most exciting developments of the control and

isolation of single cells is the fabrication of the lung-on-a-chip
microdevice. Such a device can recapitulate the key structural
and microenvironmental features of the living human lung that
induces the cultured endothelium and epithelium to express
complex integrated organ-level physiological functions that are
not observed in conventional in-vitro cultures [2]. Researchers
have demonstrated the ability to modify experimental design as
needed and to quantitatively analyze the functional behavior of
the cells in a physiologically healthy state. Investigations were
conducted to study responses to insults or to the introduction of
drugs into organ-on-chip microdevices [2]. The role of vacuum
technology is crucial in lung-on-a-chip microdevice functions
where vacuum is applied to the side hollow chambers to induce
outward bending of the elastic vertical walls of the central microchannels (Figure 3a). This results in mimicking physiological
breathing motions of the lung. This deformation laterally stretches the intervening PDMS membrane along with the attached epithelial and endothelial monolayers (Figure 3b) [2].
In another report on vacuum assisted microfluidic cell control, researchers developed hybrid microfluidic-vacuum device
for direct interfacing with conventional cell culture methods to
generate and maintain controlled soluble environments in a Petri

Figure 3. Mechanically active organ-on-chip microdevice with compartmentalized 3D microarchitecture. (a) The human lung-on-achip microsystem is constructed in a multilayered microfluidic device comprising the upper (blue) and lower (red) cell culture microchannels with a microfabricated porous elastic membrane sandwiched in-between. The microdevice is also equipped with two full-height, hollow microchambers alongside
of the cell culture channels. (b) Physiological breathing motions in the living human lung are reproduced by the application of vacuum to the side
chambers. This actuation causes the lateral elongation of the intervening elastic membrane, which induces mechanical stretching of the adherent
tissue layers in the central channels [Source: Nature Protocols, Vol.8 (2013), 2135].

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September 2016 Vacuum Technology & Coating

dish, Figure 4 [3]. The design of hybrid microfluidic-vacuum

device incorporates separate sets of fluidic channels and vacuum
networks on a single device enabling reversible application of
microfluidic gradients onto wet cell culture surfaces. The vacuum
channels are used for reversible bonding to a hard surface. The
researchers demonstrated stable, precise concentration gradients
of soluble factors that were generated using simple microfluidic channels that were attached to a perfusion system. Real-time
optical live/dead cell imaging of neural stem cells were shown
to be exposed to a hydrogen peroxide gradient and chemotaxis
of metastatic breast cancer cells in a growth factor gradient [3].

Concluding Remarks
Microfluidic based cell culture processes and methods have
entered into the advance phase of applications with the incorporation of vacuum technology. The combination of vacuum and
microfluidics paves the way to new developments in single cell
isolation for a range of applications in medical biotechnology.
We anticipate a range of research and developmental activities
at the interface of engineering, vacuum technology and medical science in the future to control and manage cell behavior in
microenvironment that would eventually lead to breakthroughs
in diagnosis and treatment of complex physiological conditions.
References for Further Reading
1 Martin Kolnik, Lev S Tsimring, and Je Hasty. Vacuum-assisted cell
loading enables shear-free mammalian microfluidic culture. Lab
Chip. 2012, 12(22): 4732-4737.
2. Dongeun Huh et. al. Microfabrication of human organs-on-chips.
Nature Protocols. 2013, 8: 2135-2157.
3. Bong Geun Chung et. al. A hybrid microfluidic-vacuum device for
direct interfacing with conventional cell culture methods. BMC Biotechnology. 2007, 7:60, DOI: 10.1186/1472-6750-7-60.

Figure 4, (A) A schematic illustration of a hybrid microfluidic-vacuum

platform for interfacing with Petri dish that show separate vacuum and
fluidic channels. Side reservoirs (purple) contain buffer while buffer
with FITC-Dextran (green) filled the middle reservoir. Parallel laminar
flows from the three reservoirs generate stable gradients by diffusion.
(B) A single device with generated gradients of varying slope and profile. Two different gradient profiles generated at 0.5 mm and 6 mm
downstream of the junction were visualized by FITC-Dextran simulation profile. Gradient profiles were generated using 1 l/min withdrawal rate. Experimentally measured and calculated gradient profiles
were normalized for comparison. Scale bars are 100 m [Source: BMC
Biotechnology, 2007, 7:60].

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