January 16, 2019 by Thamarasee Jeewandara
a) Cells are introduced into the cross-junction of the
microchannel. The stress applied on the cell is optimized to disrupt the
cell membrane and release subcellular components, while maintaining the
integrity of mitochondria. Mitochondria are dynamic, bioenergetic
intracellular organelles, responsible for energy production via ATP
production during respiration. They are involved in key cellular
metabolic tasks that regulate vital physiological responses of cells,
including cell signaling, cell differentiation and cell death. Defective mitochondria
are linked to several critical human genetic diseases, including
neurodegenerative disorders, cancer and cardiovascular disease. The detailed
characterization of functional mitochondria remains relatively
unexplored due to a lack of effective organelle extraction methods. For
instance, the extraction process must sustain sufficient functionality
of the organelle ex vivo to illuminate their cytosolic functions in the
presence of cytoskeleton and other subcellular organelles. Since
mitochondria grow in a complex reticular network within cells to undergo structural alternations, their intracellular characterization is further complicated. As a result, in vitro analysis of mitochondria remains the mainstream method,
to separately extract and understand the intrinsic properties of
mitochondria, without the interference of other subcellular organelles.
In a recent study, now published in Microsystems & Nanoengineering,
Habibur Rahman and colleagues at the Department of Biomedical
Engineering explored the possibility of controlling hydrodynamic stress
for efficient mitochondrial extraction. For this, they used
cross-junction microfluidic geometry at the microscale to selectively
disrupt the cellular membrane while securing the mitochondrial membrane's integrity.
3D geometry of the cross-slot microfluidics channel. (a) Overall
geometry and the boundary conditions of the model. (b) Meshing of the
elements as zoomed in the cross-slot region. Credit: Microsystems and
Nanoengineering, doi: https://doi.org/10.1038/s41378-018-0037-y
Advances in microfluidics have demonstrated the advantages of on-chip laboratory procedures
with significantly reduced sample size and increased experimental
reproducibility. Hydrodynamic stress produced in microfluidic chips can
be used to open cellular or nuclear membranes transiently during
intracellular gene delivery. The potential of such techniques has rarely been examined for extracting subcellular organelles since the constrained geometries of microchannels can cause subcellular component clogging in the micromachines.
The authors optimized the experimental conditions of operation based on previous studies
to effectively shred cell membranes while retaining intact mitochondria
in model mammalian cell lines. The model cell lines of interest were
human embryonic kidney cells (HEK293), mouse muscle cells (C2C12) and
neuroblastoma cells (SH-SY5Y).
In the working principle of the proposed microscale cell shredder, the scientists measured the difference in elastic modulus
between the mitochondrial membrane and the cellular membrane to disrupt
the cell while retaining the mitochondrial membrane. An increased
stress level in the system could disrupt cell membranes with higher
elastic moduli (as seen with the neuroblastoma cell line). The study
compared the protein yield and the concentration of extracted functional
mitochondria using the proposed method vs. commercially available kits
for a range of cell concentrations.
Cell disruption and protein extraction efficiency using the
microscale cell shredder, the Dounce Homogenizer and Qiagen Mitochondria
Isolation Kit. The
findings showed the proposed microscale cell shredder method was more
efficient than the commercial kits by yielding approximately 40 percent
more functional mitochondria. The scientists were able to preserve the
structural integrity of the extracted organelles even at low cell
concentrations. The method could rapidly process a limited quantity of
samples (200 µl).
The detailed outcomes were a first in study demonstration of intact
and functional mitochondria extraction using microscale hydrodynamic
stress. The possibility of processing a low concentration and small
sample size is favorable for clinical investigations of mitochondrial
disease. To test the stress exerted by the designed cross-junction, they
used a COMSOL Multiphysics simulation model first. Thereafter, Rahman
et al. experimentally determined the volumetric flow rate for three
model cell lines. During experimental cell membrane disruption, under
mean shear stress (16.4 Pa, for a 60 µL/min flow rate), subcellular
organelles were released and detected with increased mitochondrial
positive signals.
The scientists compared the capacity of the miniaturized cell
shredder with that of two commercial kits: the Dounce homogenizer
(mechanical method of cell disruption) and the Qproteome mitochondria
isolation kit (chemical method of cell disruption) to extract
mitochondria. To determine the number of functional mitochondria
extracted, the scientists used MitoTracker—a fluorescent dye that stains
mitochondria during flow cytometric
analysis. The results showed that the microscale cell shredder was able
to extract 40 percent more functional mitochondria compared to the
commercial kits for both HEK 293 and C2C12 cells.
Disruption of neuroblastoma cells (SH-SY5Y) and the subsequent
mitochondrial extraction. a Total protein yield and b concentrations of
functional mitochondria obtained from the three extraction methods. Rahman et al. conducted the citrate synthase assay to determine mitochondrial integrity through enzymatic activity of damaged mitochondria.
As before, compared to the commercial kits, mitochondrial integrity was
higher for those extracted using the microscale shredder in HEK293 and
C2C12 cells.
The study demonstrated the importance of membrane stiffness by
validating the proposed concept to disrupt neuroblastoma cell membranes
(SH-SY5Y). Since the SH-SY5Y cell membrane had a higher elastic modulus
than both HEK293 and C2C12 cell lines, the scientists had to optimize
the volumetric flow rate in the microscale shredder for effectively
disrupting SH-SY5Y cell membranes. Again, compared with the commercial
kit extractions, using the proposed method delivered a significantly
higher concentration of protein and functional mitochondria for the cell
line of interest.
A necking section is included in the channel design of the
microscale cell shredder to ensure the cells are focused laterally to
the center of the flow stream in the microfluidics bioreactor. Credit:
Microsystems and Nanoengineering, doi:
https://doi.org/10.1038/s41378-018-0037-y In this way, Rahman et al. investigated the possibility of disrupting the cell membrane to retain the integrity of mitochondrial membranes in diverse mammalian model cell lines. They determined the optimal extensional stress
and flow rate inside a microfluidic cross-section bioreactor, based on
the Young's modulus of the model cell line of interest. During channel
design, the scientists included a necking section in the microfluidic
bioreactor manufactured using soft lithography .
The proposed microfluidics microscale cell shredder demonstrated superior capability for extracting functional mitochondria
and proteins by controlling hydrodynamic stress for the first time,
compared with commercially available cell organelle extraction kits. The
experiments were feasible even with minute quantities of samples (200
µl volume, containing 104 cells/mL) for potential clinical
applications. Rahman et al. were able to faithfully replicate the
protocol across three cell lines. The experimental work can be
translated to a clinical setting to understand mitochondrial dysfunction
related disorders in depth.
More information:
Md Habibur Rahman et al.
Demarcating the membrane damage for the extraction of functional
mitochondria,
Microsystems and Nanoengineering (2018).
DOI: 10.1038/s41378-018-0037-y
Young Bok Bae et al. Microfluidic assessment of mechanical cell damage by extensional stress,
Lab on a Chip (2015).
DOI: 10.1039/c5lc01006c
