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Silicon Microchip Capable of High-Frequency Fluidic Valving at Heart of Technology
Jim Linton, Ph.D. Shane W. Oram, Ph.D.
The purification of cellular populations and individual cells is pivotal for the reliable characterization of gene expression, many aspects of life science research, and the development of cellular therapies. Cell sorting routinely involves fluorescence-based separation through flow cytometry, which has proven superior to crude techniques such as differential sedimentation. Through this process, large numbers of cells are rapidly analyzed for specific fluorescence signatures. Traditionally, cell samples are parsed in charged aerosol droplets, which are electrostatically sorted, enabling purification at thousands of cells per second at purities often greater than 90%.
This technology enables exceptional specificity using multiple fluorescent signatures (e.g., cell surface labels, cell size, and granularity).
However, the contributions of current flow-sorting platforms are balanced against significant limitations, including: high processing pressures that can result in loss of function and/or cell death; sample processing speeds/volumes that make processing clinical-scale samples (>500 million cells) unfeasible; a high degree of technical expertise needed to manage device complexity; increased risk of sample contamination through the use of open systems; and user safety concerns when processing aerosolized patient samples.
These limitations, plus the high unit and sample processing costs, must be overcome to enable further clinical application and commercialization.
To address these issues, Owl Biotech developed a fully closed cell-sorting system. This microchip-based technology employs closed fluid path cartridges with aseptic ports that permit the straightforward administration and collection of cell samples. At the heart of the cartridge is a patented microchip capable of very high-frequency fluidic valving (Figure 1).
Propelled by modest positive pressure, typically less than 0.2 atmospheres, cells pass through microchannels where laser-directed fluorescent signals are detected with photo-multiplier tubes. Upon identification of a positive target cell, the microchip valve opens, redirecting the cell to a collection chamber. Both positive and negative selected cells can then be retrieved from the cartridge and used for any number of downstream applications.
Improving Valve Speed Click Image To Enlarge +
Figure 2. Mechanism of microchip-based sorting: Labeled cell samples enter the chip from the input sample, as the cells approach the sort area each cell is analyzed. When a selected cell is identified a magnetic pulse opens and closes the valve and the cell is redirected to a collection chamber. An integrated single-crystal silicon spring returns the valve to its original position, and undesired cells are allowed to flow through.
One key design goal of all cell sorters is to maximize the speed at which the device can segment a stream of cells. In the case of Owl’s microchip-based technology, a fluidic valve determines the rate at which cells are isolated. Valve speed in a fluid microenvironment is in turn controlled by several factors, including the acceleration and magnitude of the opening and closing forces, and the inertia of the valve and the fluid surrounding it.
In the case of Owl technology, valve speed is controlled by its engineered magnetic properties and a powerful return-spring force, which serves to close the valve (Figure 2). Careful modeling and empirical testing has led to a design that allows μsec opening times, a user-selected sort collection delay, and μsec closing times.
A typical total cycle of around 50 μsec allows separation rates similar to a traditional droplet sorter, although with microchip-based sorting no aerosols or droplets are used.
Sorted cells are typically used for molecular analysis or sample preparation, for example, cellular expansion for research or therapeutic purposes. In such cases, those cells need to be in a healthy metabolic state ideally retaining their complete array of functional capabilities. Current flow cytometric sorting has distinct challenges in that respect due to technical requirements such as high pressure, extended shear rate, severe decompression upon aerosolization, and impact trauma during cell collection. Often, these factors result in cell isolates with compromised function and/or viability.
Using Owl’s microchip-based technology, the pressure applied to cells is minimal and the trauma associated with droplet sorting is removed, resulting in high cell viability. In addition, a wide variety of cell types have been sorted using the Owl technology, all with a high retention of cell functionality. For example, antigen-primed T cells have been shown to retain their cancer-specific cytotoxic capabilities in chromium-release assays.
Figure 3. Sorting target cells from dilute whole blood or PBMCs: Pre-sort fraction of diluted whole blood with CD4+ cells (A) Post-sort fraction showing enrichment of CD-4 positive cells (B) Pre-sort fraction of the spiked MART-1-specific T-cell clone into PBMCs (C) Post-sort fraction showing enrichment of MART-1 positive antigen specific T cells (D).
While the utility of microchip-based cell separation is being tested in many different applications, current studies show its utility in processing clinical samples for diagnostic and therapeutic applications. For these purposes it is best to process the samples with as little manipulation as possible.
Here, CD4+ cells were sorted from a diluted whole blood preparation by adding a fluorescent CD4 antibody marker to 8 mL PBS and 2 mL of whole blood. Results show that while the presorted fraction contained less than 0.01% CD4 positive cells, the sorted fraction contained more than 91% CD4 positive cells (Figures 3A and B). The effective purification was close to 10,000-fold in a single-step process and with a simple no-lyse, no-wash sample preparation.
Investigations have also been done to study the ability of antigen-specific T cells from a patient’s own blood to recognize tumor cells and trigger an immune system response. To explore the feasibility of this clinically important strategy, cells from a MART-1-specific T cell clone were spiked into a patient’s peripheral blood mononuclear cells (PBMCs), stained with a PE-conjugated tetramer loaded with a MART-1 peptide, and then sorted using the Owl’s microchip-based sorting technology.
Results show tetramer-positive cells were enriched from less than 1% to greater than 95% (Figures 3C and D). Importantly, sorted cells maintained their ability to kill MART-1+ tumor cells and to proliferate in vitro.
Acceleration of research for effective cancer and cellular therapies has increased the need for cell-sorting technologies that are safe, efficient, and easy to use. Owl’s cartridge-based closed system has effectively demonstrated its capability to sort a wide variety of cells at high speeds without impacting cell viability. This platform offers the additional advantage of protecting sample integrity while permitting quick sample-to-sample changeover—a feature that avoids the inter-sample cleaning and validation required for traditional cell sorting.
That this technology does not utilize sheath fluids opens the future potential for sequential sorting of large numbers of samples within a short timeframe, making practical clinical applications a possibility. In addition, combining the underlying microchip technology with other cell isolation techniques such as magnetic beads has the potential to empower processing of samples greater than 1 billion cells.
Jim Linton, Ph.D. (jim@owlbiomedical.com), is chief business officer at Owl biomedical, and Shane W. Oram, Ph.D. (shaneo@miltenyibiotec.com), is global marketing manager—cell analysis at Miltenyi Biotech.
Source: https://technical.sanguinebio.com/current-options-for-isolating-pure-cell-populations/
Author: Colt Egelston
Antibody based isolation kits for isolating immune cell populations have become a standard protocol in the toolbox of every immunologist over the last two decades. In fact, many new scientists are shocked to learn that lymphocytes used to be isolated from PBMCs and other tissue sources by filtering through nylon wool. How archaic! Here I will describe the various options cell isolation technologies available to biologists today.
FACS: Fluorescence Activated Cell Sorting
FACS is the most sophisticated way of isolating various cells of interest from your tissue source. You have the ability to incorporate up to 10 or so different fluorescent antibodies into your stain, which allows you to sort on cells of interest with exquisite precision and specificity. Another powerful tool is the ability of many FACS machines to do four-way sorts or even single-cell sorts.
However, sorting can be relatively time consuming, depending on your sample size and the percentage of cells of interest. Use of FACS machines are also fairly expensive, whether it be your laboratory’s investment in acquiring its own machine and committing to its maintenance or the hourly rates your institution’s core will charge you (averaging around $100 per hour in my experience).
Magnetic Antibody Based Cell Isolation
Cell separation reagents are available from the three main players in the cell isolation kit world: Stem Cell Technologies, Miltenyi Biotec MACS Technology, and Life Technologies Dynabeads. Though the technology varies slightly from company to company, they basically boil down to the same principles. Usually an antibody cocktail will bind either your cell of interest (positive selection) or your cells of non-interest (negative selection). After a short incubation the addition of magnetic nanoparticle beads to your cell mixture then binds the antibodies from the previous incubation. After another short incubation, cells can then be placed into the magnet purchased from the company. After a few minutes, the antibody bound cells will be drawn towards the magnet and the unbound cells can be collected. Bound cells can then be washed out and collected separately. This technology allows rapid and easy isolation of cell populations from bulk populations.
However, magnetic antibody based cell isolation involves some upfront investment in the purchasing of magnets (approaching $1000) and antibody kits (ranging from $300-$700). Because of this it is important to fully research which companies’ technology is right for you. I also highly recommend sampling the technology on some extra PBMCs you have if at all possible and finding an experienced colleague that can advise when you have questions.
RosetteSep Whole Blood Based Cell Isolation
RosetteSep kits from Stem Cell Technologies allow researchers to quickly isolate cells of interest directly from whole blood and without the investment in magnets. Furthermore it combines the Ficoll gradient isolation step with the isolation of specific target cells, making for an efficient and economical protocol. Instead of using magnetic nanoparticles, RosetteSep uses antibodies that conjugate directly to the RBCs in whole blood. When the blood is Ficolled the RBCs go to the bottom layer along with all the cells that you have targeted with antibody. Your top layer is left with untouched cells of your interest! Of course this protocol only works from whole blood, so it will not work on PBMCs or cells from other tissue sources.
Keep in mind that both FACS and antibody based cell isolation require starting with a single cell suspension of cells. It is important to think about whether you want touched or untouched cells (positive or negative selection) for your downstream assays. I also highly recommend doing purity checks (see figure below) by flow cytometry as often as you can, especially when first adapting any isolation technology to your lab.
Further Reading:
Stem Cell Technologies: https://www.stemcell.com/en/Products/Product-Type/Cell-isolation-products.aspx
Life Technologies Dynabeads: https://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Dynal/Dynabeads-Types-and-Uses.html
Miltenyi Biotec MACS Technology: https://www.miltenyibiotec.com/en/Products-and-Services/MACS-Cell-Separation.aspx
RosetteSep: https://www.stemcell.com/en/Products/Popular-Product-Lines/RosetteSep.aspx
About the Author:
Colt Egelston is currently a post-doctoral fellow at the Beckman Research Institute of the City of Hope, in Duarte, CA. He received his Ph.D. from Rush University in Chicago and is interested in all things immunology.
Source: https://www.bioopticsworld.com
08/27/2013
Scientists at McGill University (Montreal, QC, Canada) have crystallized a short RNA sequence, poly (rA)11, and used data collected at the Canadian Light Source (CLS; Saskatoon, SK, Canada) and the medical diagnostics (Ithaca, NY) to confirm the hypothesis of a poly (rA) double helix. Their discovery, building on 50 years' worth of work by various scientists, will have interesting applications for research in biological nanomaterials as well as in fabricating bionanomachines (devices derived from living organisms that can perform Related: AFM collaboration produces first in-situ view of DNA's double helix).
https://onlinelibrary.wiley.com/doi/10.1002/anie.201303461/abstract
“Bionanomachines are advantageous because of their extremely small
size, low production cost, and the ease of modification,” explains Kalle
Gehring, a biochemistry professor at McGill University who led the
work. “Many bionanomachines already affect our everyday lives as
enzymes, sensors, biomaterials, and medical therapeutics.”
Gehring adds that proof of the RNA double helix may have diverse
downstream benefits for the medical treatments and cures for diseases
like AIDS, or even to help regenerate biological tissues. His team
initially was looking for information about how cells turn mRNA into
protein when they made their discovery.
For the experiments, Gehring and a team of researchers used data
obtained at the CLS Canadian Macromolecular Crystallography Facility
(CMCF) to successfully solve the structure of poly (rA)11 RNA. CMCF
Beamline Scientist Michel Fodje said the experiments were successful in
identifying the structure of the RNA and may have consequences for how
genetic information is stored in cells.
Structure of poly (rA) duplex showing the two strands in
orange/yellow and green/blue. Ammonium ions that stabilize the structure
are shown as black balls. (Image courtesy of Kathryn Janzen, Canadian Light Source)
“Although DNA and RNA both carry genetic information, there are quite
a few differences between them,” says Fodje. “mRNA molecules have poly
(rA) tails, which are chemically identical to the molecules in the
crystal. The poly (rA) structure may be physiologically important,
especially under conditions where there is a high local concentration of
mRNA. This can happen where cells are stressed and mRNA becomes
concentrated in granules within cells.”With this information, researchers will continue to map the diverse structures of RNA and their roles in the design of novel bionanomachines and in cells during times of stress.
Research on the poly (rA) structure was funded by grants from the Natural Sciences and Engineering Research Council of Canada with support from the Canada Foundation for Innovation, the Government of Quebec, Concordia University, and McGill University.
The research team's paper appears in the journal Angewandte Chemie International Edition; for more information, please visit https://onlinelibrary.wiley.com/doi/10.1002/anie.201303461/abstract.
There are several ways to "trap" a beam of light -- usually with mirrors, other reflective surfaces, or high-tech materials such as photonic crystals. But now researchers at MIT have discovered a new method to trap light that could find a wide variety of applications.
The new system, devised through computer modeling and then demonstrated experimentally, pits light waves against light waves: It sets up two waves that have the same wavelength, but exactly opposite phases -- where one wave has a peak, the other has a trough -- so that the waves cancel each other out. Meanwhile, light of other wavelengths (or colors) can pass through freely.
The researchers say that this phenomenon could apply to any type of wave: sound waves, radio waves, electrons (whose behavior can be described by wave equations), and even waves in water.
The discovery is reported this week in the journal Nature by professors of physics Marin Soljačić and John Joannopoulos, associate professor of applied mathematics Steven Johnson, and graduate students Chia Wei Hsu, Bo Zhen, Jeongwon Lee and Song-Liang Chua.
"For many optical devices you want to build," Soljačić says -- including lasers, solar cells and fiber optics -- "you need a way to confine light." This has most often been accomplished using mirrors of various kinds, including both traditional mirrors and more advanced dielectric mirrors, as well as exotic photonic crystals and devices that rely on a phenomenon called Anderson localization. In all of these cases, light's passage is blocked: In physics terminology, there are no "permitted" states for the light to continue on its path, so it is forced into a reflection.
In the new system, however, that is not the case. Instead, light of a particular wavelength is blocked by destructive interference from other waves that are precisely out of phase. "It's a very different way of confining light," Soljačić says.
While there may ultimately be practical applications, at this point the team is focused on its discovery of a new, unexpected phenomenon. "New physical phenomena often enable new applications," Hsu says. Possible applications, he suggests, could include large-area lasers and chemical or biological sensors.
The researchers first saw the possibility of this phenomenon through numerical simulations; the prediction was then verified experimentally.
In mathematical terms, the new phenomenon -- where one frequency of light is trapped while other nearby frequencies are not -- is an example of an "embedded eigenvalue." This had been described as a theoretical possibility by the mathematician and computational pioneer John von Neumann in 1929. While physicists have since been interested in the possibility of such an effect, nobody had previously seen this phenomenon in practice, except for special cases involving symmetry.
This work is "very significant, because it represents a new kind of mirror which, in principle, has perfect reflectivity," says A. Douglas Stone, a professor of physics at Yale University who was not involved in this research. The finding, he says, "is surprising because it was believed that photonic crystal surfaces still obeyed the usual laws of refraction and reflection," but in this case they do not.
Stone adds, "This is in fact a realization of the famous 'bound state in the continuum' proposed by von Neumann and [theoretical physicist and mathematician Eugene] Wigner at the dawn of quantum theory, but in a practical, realizable form. The potential applications the authors mention, to high-power single-mode lasers and to large-area chemical [and] biological sensing, are very intriguing and exciting if they pan out."
Cellular measurements by
flow cytometric analysis constitute an important step toward
understanding individual attributes within a population of cells.
Assessing individual cells within a population by protein
expression using fluorescently labeled antibodies and other
fluorescent probes can identify cellular patterns. The technology
for accurately identifying subtle changes in protein expression
within a population of cells using a vast array of technology has
resulted in controversy and questions regarding reproducibility,
which can be explained at least in part by the absence of standard
methods to facilitate comparison of flow cytometric data. The
complexity of technological advancements and the need for
improvements in biological resolution results in the generation of
complex data that demands the use of minimum standards for their
publication. Herein we present a summarized view for the inclusion
of consistent flow cytometric experimental information as
supplemental data. Four major points, experimental and sample
information, data acquisition, analysis, and presentation are
emphasized. Together, these guidelines will facilitate the review
and publication of flow cytometry data that provide an accurate
foundation for ongoing studies with this evolving technology.
D.F. Alvarez et.al. AJP - Lung Physiol February 1,
2010 vol. 298 no. 2 L127-L130
Download PDF here
William G. Telford
Curr. Protoc.
Cytom. 59:9.38.1-9.38.12. © 2012 by John
Wiley & Sons, Inc.
Abstract:
This unit describes the use of a novel violet-excited
membrane-binding probe F2N12S [4′-N,N-diethylamino-6-(N,N,N-dodecyl-methylamino-sulfopropyl)-methyl-3-hydroxyflavone]
for the flow cytometric detection of the changes in membrane
asymmetry, fluidity, and charge that accompanies apoptosis. This
reagent inserts into the plasma membrane and undergoes a shift in
emission from orange to green during the membrane alterations
that occur during apoptosis. Since its mechanism of action
differs from annexin V, it can be used in situations where
annexin V binding is problematic, as in adherent cells removed
from their growth substrate. Like annexin V, it can also be
readily combined with other flow cytometric assays for apoptosis,
allowing detailed multiparametric analysis of the apoptotic
process.
Download PDF here (Free)
