SLAS 2017 Conference Recap
SLAS – Society for Laboratory Automation and Screening https://www.slas.org
Annual Conference February 6-8, 2017 in Washington DC
Attended by Tom Gaborski for informative purposes. Collaborators/colleagues in attendance: Vinay Abhankar (UT-Arlington), Andrea Mazzocchi (alum @ Wake Forest), August Allen (alum @ Recursion Pharmaceuticals)
My goal in attending this conference was to learn more about high content high-throughput screening (HTS). Historically HTS has involved high-throughput robotic biochemical assays in 96, 384 and 1536 well plates. New assays on the same platform but involve image cells and even organoids to derive information at the same throughput. These assays require high quality and high-throughput imaging as well as advanced automated image processing analysis. This is akin to flow cytometry but conducting 384 or 1536 parallel experiments in a series of plates that might all be handled by a robot. Our laboratory’s focus on developing membranes for barrier models that enable high quality imaging might be amenable to HTS and automated handling used by biopharma discovery teams. I was impressed by the advancement of the technology and its use by biopharma (high-throughput confocal microscopes that image multi-well plates hooked up to robotic arms that feed the cells and store them in incubators called hotels).
Below are some of the talks and topics that I found of most interest that I want to return to at some in the future. I focused on talks related to cellular HTS, and Micro & Nano Technologies.
Jennifer Lippincott-Schwartz (HHMI – Janelia) gave the opening keynote on advanced fluorescence microscopy focusing on new super-resolution (SR) techniques that exceed the conventional diffraction limit. The links below gives a great overview to each of the techniques.
Live Cell Multicolor Structured Illumination Microscope (SIM)
Structured illumination microscopy is an imaging method capable of doubling the spatial resolution of conventional widefield fluorescence microscopy by using spatially structured illumination light. The same idea is behind the well-known moiré pattern — the coarse pattern that appears when two much finer patterns overlap and is therefore much easier to see than either of the original patterns. In the context of microscopy, think of one of the original patterns as some undetectable super-resolution sample structures and the other pattern as a designed illumination pattern. The resultant moiré pattern is much coarser than the sample structure and thus readily detectable by a microscope.
Interferometric photoactivation and localization microscopy (iPALM)
“Interferometric photoactivation and localization microscopy (iPALM) is a novel technique that combines single molecule localization (SML) with multi-phase interferometry. Because it offers unparalleled spatial, nearly isotropic resolution in the lateral and axial directions, to well below the diffraction limit (<25nm), it is suitable for studies that require near electron microscopy levels of resolution, but within intact, fluorescently labeled samples.”
Lattice Light Sheet Microscopy (LSM)
The original implementation of a non-diffracting Bessel beam in light sheet microscopy solved this problem by delivering a virtual light sheet of submicron thickness. [4] The lattice light sheet imaging system currently housed at the AIC further improves upon this first generation configuration. By introducing a massively parallel linear array of coherently interfering Bessel beams, the microscope can generate a true light sheet, which significantly reduces data acquisition time. [7] One additional feature that is of great practical importance for biologists is the remarkably low photodamage caused by the lattice light sheet microscope. Tens of thousands of images have been taken from many specimens using this instrument without the sort of adverse effects that can be observed orders of magnitude sooner with other live cell imaging systems. The AIC lattice light sheet microscope is a powerful tool for gaining unprecedented mechanistic insights into biological processes that occur even on very fast time scales.
Daniel Hoeppner (The Lieber Institute for Brain Development) – Parsing cell homogeneity from heterogeneous stem cell cultures
There is significant spatial heterogeneity in hESCs and hiPSC cultures. There are domains within colonies as they develop. Higher intercellular tension at the borders of the colonies may lead to this heterogeneity. He cited this 2014 Nature Methods paper by Warmflash et. al. that showed… “geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development.” http://www.nature.com/nmeth/journal/v11/n8/abs/nmeth.3016.html
Han Xu (Amgen) – Developing assay technologies to realize the potential of iPSC technology in drug discovery
iPSC derived podocytes (kidney). Ideally show foot processes and cytoplasmic bridges – compare to rat primary podocytes. Investigate fluorescent BSA uptake, compare to HEK293 as negative control. iPSC podocytes are heterogeneous when it comes to podocin and podocalyxin immunofluorescence. What is the best method to culture and maintain mature podocytes in culture? (TG – co-culture with endothelial cells across a membrane?)
Chueh-Yu Wu (DiCarlo Lab @ UCLA) – 3D Shaped Cell Microcarriers for Cell Culture, Manipulation, High-throughput Analysis, and Sorting of Adherent Cells
One method to scale-up production of adherent cells is to grow them on beads in a large suspension cell culture system. A downside of this approach is the high shear forces during the stirring in the suspension culture system. This group developed an interesting and odd-shaped particle that has a recessed flat region. The recessed region is “sticky” to support cell attachment and at the same time shielded from the high shear forces present in these stirred reactors.
Robert Damoiseaux (California Nanosystems Institute) – Reconciling throughput and physiological relevance in HTS
A lot of groups are using high speed automated spinning disk confocal microscopy to image multiwell plates. He is looking at “force phenotyping” and screening on patterned ultrasoft PDMS – these substrates have patterned crosses and other features similar to the CYTOO company. They use Hyaluronic Acid functionalized with thiol groups that are bound to 4-arm PEGs.
Shane Horman (Genomics Institute, Novartis) – HTS Tumor:Stroma Co-Culture Spheroid Platform
I found this Nature Methods paper from 2013 that seems to cover some aspect of his talk: http://www.nature.com/nmeth/journal/v10/n10/abs/nmeth.f.370.html#affil-auth
Cancer associated fibroblasts (CAFs) recruit and promote immune cells and also stimulate neoangiogenesis. CAFs are main cancer stromal component. Cancer spheroids often 350 microns in diameter in a 384-well hanging drop plate. They use HuBiogel, which is a human ECM cocktail from placenta – non-animal and lacks growth factors in Matrigel and Geltrex.
$425 for 5mL http://www.vivobiotech.com/product_services.php
Ilyas Singec (Director of Stem Cell Translation Lab at NIH NCATS) – Progress and Challenges in iPS Cell Research
- Making and using iPSCs in standard fashion (SOPs, etc.)
- Directed differentiation
- Safety
24/7 Imaging of iPSCs reveals cell selection rather than uniform differentiation. Some cells die off during colony formation, others take over and proliferate. Similar to other talk describing outer shell cells have different properties (tension, etc.) compared to inner cells.
Ke Cheng (UNC/NCSU) – Angiopellosis as Alternate Mechanism of Cell Extravasation
Endothelial cells removel and engulf stem cells, then push them out of the vessel. Very different from leukocytes. ECs can push out clusters of 3 stem cells. Polymer microspheres do not get pushed out unless coated with stem cell plasma membrane. http://www.nature.com/articles/ncomms13724
Leukocytes need CD11a to extravasate, MSCs do not – they still get pushed out when CD11a is blocked. Cancer cells may get pushed out with the same mechanism – this is a big deal when clusters of cancer cells extravasate – much high risk of new cancer site. It is not clear how this relates to pericytes and MSCs stabilizing new blood vessels.
Joseph Charest (Draper Lab) – Microfluidic Model of Kidney for 96-well Plate
Kidney
Epithelial and endothelial cells (microvascular) across patterned microporous membrane. The created a grooved pattern in a TE membrane to align the cells – it sounded like they probably hot embossed the membrane – still showed irregular pore patterns like most TE membranes. Flow on both sides of membrane ~1 dyne/cm2. Studied glucose reabsorption.
There is a ZO-1 border analysis software toolbox from the BROAD that he used to quantify junctions. Published in a 2012 Integrative Biology paper: http://pubs.rsc.org/en/Content/ArticlePDF/2012/IB/c1ib00096a
Interestingly – he is the inventor on several microfluidic lung assist device patents, including this one: Microfabricated artificial lung assist device, and methods of use and manufacture