progress and achievements between 2002-2007

ASSAY and Drug Development TechnologiesVolume 5, Number 4, 2007© Mary Ann Liebert, Inc.DOI: 10.1089/adt.2007.9990

An Interview with Lars J. Brandén, Ph.D.

Associate Director for Chemical Genetics, Judith P. Sulzberger Genome Center, Columbia University, New York

Lars J. Brandén, Ph.D., is Associate Director for Chemical Genetics atthe Judith P. Sulzberger Genome Center, Columbia University, in NewYork City, as well as Director of Automation for the Columbia MolecularLibrary Screening Center Network, in the Department of Biophysics andCell Biology. Dr. Brandén received his undergraduate degree inmolecular genetics from the University of Lund, Sweden, and completedhis doctoral studies in cell and molecular biology at the KarolinskaInstitutet, Stockholm, Sweden, under the guidance of Professor C.I.Edvard Smith. He pursued a postdoctoral research fellowship in theLaboratory of Cellular Biochemistry & Biophysics at the Sloan-KetteringInstitute, Memorial Sloan-Kettering Cancer Center, New York City, underthe direction of James E. Rothman, Ph.D.

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Dr. Brandén, how does the work underway in your labo-ratory at the Judith P. Sulzberger Genome Center fit in withthe National Institutes of Health’s (NIH’s) Molecular Li-brary Screening Center Network (MLSCN)?

The whole setup we have here was initiated becausewe received the MLSCN grant. The 6,000 square feet ofspace we received was intended to be refurbished and usedto support the NIH molecular library screening programand to establish a network lab. The work I do is almostexclusively for the MLSCN. We have another grant, aphilanthropic donation that supports our embryonic stemcell research; it is not related to the MLSCN activity.

The lab includes an automation suite where the screen-ing takes place, divided between three robotic platformsthat are clones of each other. All the automation equipmentis enclosed in Biosafety Level 2 enclosures, and the roomitself has been designed to be at Biosafety Level 2�. Thespace has welded floor seams, special paint on the wallsthat can withstand harsh detergents, ceiling tiles that aredust resistant, and magnetic locks on the automation roomso we can control access. The lab also has two cell culturerooms; one is dedicated to R&D cell culture, and the other

for high-throughput production of cells. The room for high-throughput cell production also houses embryonic stem cellculture. My intention was to have as few people in thatroom as possible, so I have separated it from the space usedfor everyday types of R&D cell culture applications, towhich a lot of people need in-and-out type of access daily.The high-throughput cell production room features an au-tomatic cell culture system we developed here. It was de-signed with an airlock, in which people switch gowns. Ithas in situ autoclavable incubators that can be cycled up toabout 120°C to avoid contamination risk. Their interiorsare copper, which minimizes growth inside the incubators.

The freezer room houses the cell bank. When we ex-pand the cells we can freeze them down at high densityin a controlled-rate freezer to maximize cell viability. Webank our cells in liquid nitrogen. We also have a roomwith a colony picker for library replication, but that isnot intended for use by the MLSCN project. In that roomwe also have the gas supply for the automation suite—carbon dioxide and nitrogen tanks—so we do not needto go into the automation room to switch gases.

The wet lab is a standard wet lab that can house up to18 people. In the equipment niche we have a Tecan

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MiniPrep, shakers, and incubators. The floor space neededfor supplies to support the R&D work and for high-throughput production is removed from the general lab tomaximize use of the space. The office suite was designedto be like a hub, where everyone can gather, to promoteinteraction between the HTS and the biology core, for ex-ample. My intent was for the core people to have easy ac-cess to each other, so the office suite is designed as an openstructure with a few dedicated offices. The chemistry groupworks across the street from us. We are integrating themquite heavily by bringing them over on a regular basis—once or twice a day—and providing open office space inwhich they can easily interact with the rest of the group.

Our informatics core is in Florida. We have a dedicatedT1 line, so we have no problem with dedicated data trans-fer. We can transfer as much as 50 terabytes of data peryear. The lab team has traveled to Florida on retreats, andwe have had very good feedback from those experiences;they engender a sense of belonging. We have dedicatedinformatics hardware and personnel in Florida—they workexclusively with us—and we benefit from the experienceand network located there. They are the best of the best!

I am trying to integrate the findings and reagents weproduce from my other projects into the MLSCN proj-ect. For example, our embryonic stem cell project in-volves cloning all the human genes and tagging them withgreen fluorescent protein (GFP). We have about 17,400genes now, and I am waiting for another 1,400 to beadded to our collection. Suitable GFP fusions can thenbe transferred into an assay, and we can capitalize on thatproject to introduce new assays into the network. We tryto synergize as much as possible.

What part does chemical genetics play in this overall ef-fort, and how are you applying HTS and robotics to studygene function and establish links between gene expres-sion and human disease?

We are gearing up to use HTS and robotics to studygene function. We need a gene expression library avail-able before we can start doing gene screens. The embry-onic stem cell project I am running is establishing the ge-nomic reagents needed to do these gene screens. The NIHallows us to use the equipment and personnel from theMLSCN project—when there is time left over—to pur-sue our other work, and we will take the genomic reagentswe produce and screen them against cell-based assays inwhich we have identified compound hits. We can look atwhich of the genes we have overexpressed or knockeddown that are able to shift the 50% inhibitory concen-tration of the drug, indicating that the gene plays an im-portant role in the pathway in which this compound in-teracts. There will be a lot of generic hits—hits affectingmultiple pathways, for example, drugs that disrupt vesi-cle transport, especially in an assay such as nuclear fac-

tor �B (NF�B), where you have recirculation of the re-ceptor. If you disturb that by disrupting vesicle transportit can mimic a hit, because you will be preventing translo-cation of the receptor back to the cell surface. You canno longer stimulate the cells and it will look as thoughyou are blocking the pathway.

You can generate the same kinds of generic hits whenit comes to genes—genes that affect a lot of differentpathways without necessarily targeting a particular path-way. An example would be some of the more generic ki-nases. When a particular gene is overexpressed you mightsee some functions appear that would not be present at alower expression level. Similarly, with some compoundsyou might see more specific effects when they are pres-ent at low levels, whereas at higher concentrations a com-pound may exhibit a greater breadth of functions butlower affinity for a particular target.

Gene screens will require high content analysis; bothtime course and concentration studies might be necessaryso you can see when certain effects appear and disappear.Our goal is to produce three sets of genomic reagents toenable us to convolute linked pathways: a cDNA over-expression library with corresponding short hairpin RNA(shRNA) constructs and the corresponding cDNA fusedto GFP.

With regard to establishing links between gene ex-pression and human disease, if you have an assay thatdescribes a disease state, you can overexpress gene bygene and look for a shift in the disease model. You cando the same thing with shRNA to knock down gene bygene. You can then establish which genes are involvedin a disease platform.

What screening platform are you using, and how was itcustomized for your lab? How are compound librariesinterfaced to the system?

We are using the Tecan screening platforms (Fig. 1).They are not really customized, but we have adapted themextensively. For example, we have added an extra lidchute that takes care of the lid problem. Once you seal aplate with sealing tape you cannot re-lid it, so the lid hasto go somewhere. When the robot stacks the plates in theplatform a chute going through an opening directs thelids into a trashcan.

We also have a custom-made hand-off station. Two ro-botic arms are located on either side of the platform. Theleft arm cannot reach all the way to the right side, and viceversa, because there is a liquid handling arm situated be-tween them. So the left arm moves a plate as far as it canto a hand-off position and stops there. The hand-off stationthen transfers the plate to the robotic arm on the right side.

We have not completely automated the compound li-brary handling. However, when we load the compoundlibrary into the automation platform the plates are bar-

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coded, so we can trace the location of each compoundand cherry-pick from a particular well. But the actual handling of the compounds, such as loading into thecarousels, is manual. Automating a complete freezer sys-tem, or even a room temperature storage unit, and au-tomating cherry-picking and loading into the automatedplatform would be very costly. With that same amountof money we can hire about three more full-time equiv-alents who can serve other functions as well. The trans-fer process only involves accessing the compounds be-fore a run and returning them to storage after a run. Whenyou start screening large compound libraries, then it ishelpful to have the automation in place; we are investi-gating how we will meet that need when it arises.

What is the follow-up procedure you use to classify ac-tives from your screens?

Once we have identified an active compound in a pri-mary screen we redo the screen on the identified com-pound, and once we have verified that it is an active, wethen run a titration curve. We are in the process of de-ciding whether we should run a titration curve right awayon a hit, or whether we should wait for the verificationthat it is a real hit. About 80% of hits are false-positives.

Do you have a different procedure for handling librariesof shRNAs or other biomolecules?

Compound libraries can remain at room temperaturefor about 3 or 4 months according to the Lipinski guide-lines. I would not let a short interfering RNA (siRNA)

or shRNA plasmid library sit out at room temperature forthat period of time. We have to aliquot those more strin-gently. We are more likely to go through three or fourcycles with siRNA, shRNA, plasmids, or cDNA overex-pression plasmids, for example. If we opt to use adeno-virus, we can keep them at �4°C for a couple of weekswithout losing the titer. So for adenovirus-based librariesyou can aliquot out what you need for 2–3 weeks.Lentiviruses are more problematic. You can freeze andthaw them, but you do lose titers, depending on the pro-cedures you use. With standard freeze/thaw proceduresyou can lose up to 70% of your titer. If you use a mod-ified protocol you may be able to maintain up to 80% ofyour titer through three or four freeze/thaw cycles, butthen it will drop off. Lentiviruses are usually very un-stable at 37°C—they have about an 8-h half-life. Youneed to have a good understanding of lentiviruses to workwith them efficiently.

What technology do you have in place for cellular imag-ing, and what advantages and limitations does it have?

We use the IN Cell Analyzer 3000 from GE Health-care. We have about 15 different algorithms for studyingvarious events within cells, including object intensity andnuclear translocation algorithms.

The main limitation is true confocality with high res-olution in the z orientation. We are working with rela-tively flat cells—they are at most a couple of micronsthick—but it would be beneficial to be able to work onother types of cells from a confocal standpoint. Severalsystems on the market can achieve that with high through-

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FIG. 1. Columbia screening center automation platform. The Columbia screening center automation consists of three identicalTECAN platforms housed in BLS-2 enclosures. Each system has been designed for maximum flexibility and can process a wideassortment of assay types as well as performing various types of cell culture and virus production (adenovirus primarily but alsolentivirus). Each platform has an integrated plate reader to enable seamless processing of low-content cell-based assays or enzy-matic assays.

put, such as the Opera™ system from Evotec Technolo-gies, the BD Pathway™ 435 system from Becton Dickinson, and the UltraVIEW™ LCI system fromPerkinElmer. I wouldn’t single out any one system—theyare all based on a spinning disk system and can give verygood four-dimensional resolution.

We also have plate readers, which are quite useful forlower content analysis. They can do virtually any type ofassay except Flash reading; they can do fluorescence res-onance energy transfer, fluorescence, luminescence, andabsorbance.

How are you using imaging to do assay characterizationof your candidate compounds from HTS?

We have a large set of assays developed in-house,about 130 of them. We select a cross-section of those de-termined to be most relevant for a particular study andrun the candidate compounds on them. These assays canhelp determine, for example, how specific the compoundsare; if a compound activates only a limited number of as-says, you can then determine whether those assays arerelated to each other. For instance, if you were to get ahit in the NF�B pathway, and you could see that it waspropagated through the vascular cell adhesion molecule(VCAM) and E-selectin pathways, and you knew thatthese pathways were on the same “line”—first NF�B,then E-selectin, then VCAM—then you would have avery interesting finding. If you had a “hit” in the VCAMpathway that did not activate the NF�B or E-selectinpathways, then you might have a very selective hit. Youcan do a lot of deconvolution by looking at the results ofspecific assays and determining where your hits pop upin them. You can also learn something about when thehits manifest in the different pathways. For example, ifyou have a block in an upstream pathway that is feedinga secondary assay downstream, you can say somethingabout when it should it be turned on.

Our aim, with regard to the use of cell-based assays, isto allocate them extensively and to annotate them carefully.We are trying to characterize the assays we have in-houseon one particular cell type, human umbilical vein embry-onic cell (HUVEC) primary endothelial cells. If we wereto switch from cell type to cell type, we would get lost. Byfocusing extensively on one cell type for the characteriza-tion studies, you can then switch to a different cell type andsee what differs. Then you can start understanding why dif-ferent drugs may have different potencies, for example, orwhy their side effects are different.

What are important considerations when setting up alarge-scale tissue culture lab for HTS?

The most important factor is to minimize the risk ofcontamination, to the nth degree. Our large-scale tissue

culture setup is an integrated system. It is based on spin-ner bottles and microcarriers (Fig. 2). When we build asystem, we fuse together all the associated tubes with asterile welder, and we use a 0.2-�m filter for gas ex-change, which is the only opening in the system. The en-tire system is autoclavable. We then connect the systemto the media supply using a sterile welder to seal the bot-tles to the tubing. There is never an opening to the ex-ternal world, not even inside laminar hosing. The onlytime that happens is when you put the sterile cap ontothe media bottles, which is done inside of a hood. We in-oculate via a septum.

To expand the cells, before we inoculate them into thehigh-throughput production unit, we use Corning® Cell-STACK® culture chambers and put them in incubatorsthat cycle up to high temperatures, up to 120°C, as de-scribed previously. The entrance to the high-throughputcell production lab has a small airlock; the door neveropens straight into the cell culture room.

How does one keep cell response stable over the dura-tion of a multiday screen?

First, you have to have done your homework on thebench regarding your assay. If the assay demands that thecellular response should be stable over the duration of amultiday screen, and that is not feasible with the proto-col as written, then that cell line cannot be used in a mul-tiday assay. If you have an assay that is supposed to bereactive on the first day and you have a plate that comesinto the process a day and a half later, for example, thenyou must determine the viability and utility of the cells.We have studied how long we can keep the cells in platesand maintain the same output and response. That timelimitation sets the limit for the batch size. The batch sizedepends completely on the biology.

One solution is to use a �80°C carrier, or “hotel,” onthe platform, which can house cryogenically preservedcells together with, for example, cytokines. We have atrough in the incubator filled with regular media (with-out cytokines). We can then thaw the cells and the cyto-kines and spike the media on demand. Even if the in-tegrity of the cells is such that we cannot use them formore than 24 h, we can feed out additional cells on de-mand using this in-house system. We can maintain com-plete homogeneity from the first plate to the last in a largebatch using this approach.

What are the best viability criteria and detection meth-ods to decide cell health for good assay response?

We always run a validation screen on the cells we planto use. We determine if they respond as expected to con-trol compounds. If, on the validation run, we see a lossof cell number or reduced activity of the cells, then we

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know that something is wrong, and we need to evaluatethe problem. Something as simple as cell count will tellus if there is a problem with cell viability, and there area variety of cell viability and toxicity assays available.We feel it is best to run the assay with a few control com-pounds and see if we get the expected results.

For engineered lines, are there specific cell types orparental lines that reliably give a good response?

HUVECs are primary cells, not a cell line, but that isthe specific cell type that gives us the best response. Theycontain many of the components of the signaling path-ways in which we are interested. These endothelial cu-bic cells are very useful—they line all the blood vesselsand are the first line of response in inflammation. They

have many receptors and are readily stimulated. They alsohave a good representation of the majority of linked path-ways.

Do you use transiently transfected cells in HTS? If so,how do you accomplish this?

No, we do not. If we get an assay that demands tran-siently transfected cells, then we take a close look at theprotocol to see if we could change to a cell line that ismore easily transfected. Personally, when it comes totransfection of cells, I prefer to use inexpensive trans-fection reagents rather than one of the more costly alter-natives. If we would use a transiently transfected cell linefor HTS, we would use something that could be donevery cheaply in-house.

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FIG. 2. Columbia screening center high-throughput cell culture system. The Columbia screening center high-throughput cellculture system is designed around three major components: (1) the spinner flask culturing system from Corning (schematics shownon upper left, spinner flasks shown on lower right); (2) the Delta V controller from Broadley-James; and (3) the BioNet soft-ware running the system (both upper middle). (Lower left) Top view of growth and harvest flasks. By integrating these systemswe have an in-house–developed high-throughput cell culture system that can produce in the range of 4 � 109 cells per batch. Thiscombined with cryopreservation of the cells at high density by controlled-rate freezing enables extensive quality control of thecells before they are used for screening (upper right and middle right). It will also minimize variation in the screens that oth-erwise would be present due to variations in cell quality/status. PBS, phosphate-buffered saline.

What are the critical factors for healthy cell suspensionswhile plating during screens—proper pH, temperature,time in suspension, oxygenation, etc.?

You can plate them in a water-jacketed spinner bottleand hook that up to recirculating troughs on the automa-tion platform. If it were a matter of seeding out cells topreserve homogeneity between the first plate and the lastplate, then we would use the �80°C carrier on our plat-form and cryogenically preserve the suspension cells athigh density. We can set up smaller batches of plates onthe platform and when a batch is done seed out morecells. One vial of frozen cells can cover about 30 plates,and we can have up to 12 vials on a platform. The incu-bator can only accommodate 220 plates at a time, so wehave overcapacity when it comes to cryogenically pre-served cells on the platform.

Proper pH, temperature, and oxygenation are all dic-tated by the incubators. Cell seeding is a fast process, sowe do not worry too much about the physical conditionswhile we seed out the cells. If we work with suspensioncells, the time in suspension is irrelevant. But if we useadherent cells that are trypsinized and kept in a spinnerbottle as a source of cells for seeding, then we are lim-ited to about 2 h before we start to induce apoptosis. Non-suspension cells cannot be kept indefinitely in a spinnerbottle.

What are the critical factors in maintaining assay sensi-tivity over cell passages (e.g., avoiding confluency, min-imizing the number and extent of trypinizations, allow-ing sufficient recovery of cells following plating beforestimulation, etc.)?

We do all of those types of studies as part of the as-say development process. We know, for example, that ifwe go above passage 8 or 9 with the HUVECs, they startto lose response to many of the ligands we use for stim-ulation. We prefer to use passage 6 or 7. We are now inthe process of implementing a diabetes-related assay thatincludes three different stably integrated factors. Weknow that if we let them go for about 10 passages, thegene expression response goes down quite dramatically.We have to use passage 3 or 4 to get a good response.Part of assay development is determining what passageis optimal to get the response you need.

What efforts do you take to detect and prevent Mycoplasma contamination?

First, we purchase certified HUVECs from Cambrex.We expand them very rapidly and freeze them down. Ifwe would see that the effect of an assay goes down, wewould have a direct indication that something is wrongwith the cells. We also have checkpoints to test for con-

tamination, and we recently implemented an in-housepolymerase chain reaction-based Mycoplasma test. If wedetect contamination, then of course we have to discardthe whole batch of cells.

For those who wish to adopt the tissue culture paradigmyou have developed, can you comment in general on howcells grown on microcarrier beads subsequently behavewhen plated onto plastic microtiter plates? Is this pro-cess best used with a limited set of cell lines or can a va-riety of cells be processed on microcarrier beads andtransferred to plastic surfaces?

Cells cultured on microcarriers have the same charac-teristics when it comes to subsequent culturing on stan-dard cell culture plastic as they would have if you wereto expand them in regular cell culture vessels (plastic).A large variety of cells can be cultured in this fashion.Human embryonic kidney 293 cells and other cells thatadhere poorly might be the problematic ones.

Parallel processing of multiple cell lines is restrictedto the number of spinner bottles you can connect to yoursystem, and that is dependent on the number of con-trollers that you have access to. The controllers can eas-ily be daisy-chained together. At present we would beable to parallel process four separate cell lines if we hadthree more controllers. The software is already set up forthat.

Has your group achieved the goal set of being able totest 100,000 or more different genes in a single day? (Ifnot, what challenges remain?)

We are trying to establish the genomic reagents atpresent. The limiting factor is the automation. Testing100,000 genes in a single day would mean that if youcould transfect the genes into cells, you would then beable to run them on multiple assays. For example, if youwere looking at 20 different genes, then you would haveto run them on five different assays to achieve testingof 100,000 genes. It would not be a problem for us todo that in one day. The challenge that remains is to es-tablish all the necessary genomic reagents in the properformat.

How did your dissertation work on developing syntheticgene delivery systems, under Professor Edvard Smith atthe Karolinska Institutet in Sweden, lead to your positionat Columbia and the research you are now pursuing?

I knew that I wanted to study gene therapy when I was12 years old. I read about retroviruses, which are nature’sway of inserting a gene of interest into a cell. I wonderedwhether retroviruses could be used to insert new genesto correct diseases. This is still a viable concept. People

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tried to take this concept into the clinic too early on, with-out knowing exactly what was going on at the geneticlevel, and unfortunately this led to at least one patientdeath.

Retrovirus elements are recognized as foreign by thehuman immune system. So, if you use a retrovirus ele-ment to drive gene expression you will get methylationof those elements, which will shut down gene expression.If you use non-retroviral vectors, such as adenovirus vec-tors, you will have a problem with immune response. Be-cause adenoviral vectors would yield transient expres-sion, you would have to re-transduce the patient, whichwould trigger an immune response.

I read a lot about gene delivery techniques and genetherapy when I was younger—I was not your typicalteenager. I traveled in the United States and visited NewYork for the first time with my cousin when I was 15years old. After finishing my high school degree in Swe-den I spent almost two years in the Swedish military andthen started my university studies in southern Sweden. Ifocused on biology and genetics, with a lot of chemistryalso. During my last half-year of studies I approached aPrincipal Investigator at the Karolinska Institute, EdvardSmith, who had started experimenting with gene therapy.He suggested that I also study immunology, and that iswhat I focused on my last half-year. I then pursued myPh.D. with Edvard Smith, and I essentially wrote my ownPh.D. program in gene therapy. He was very supportive.He sent me to work with Donald Kohn at Children’s Hos-pital in Los Angeles, who taught me how to make theretrovirus vectors.

I worked on the concept of treating X-linked agam-maglobulinemia (XLA) using gene therapy. It turned outthat to correct XLA, which is caused by a deficiency inthe Bruton’s tyrosine kinase, you need to introduce a va-riety of control elements, and you cannot fit all the nec-essary regulatory elements into a retrovirus. I was doingthis work in mice and nothing was working. After twoyears, I began to look for a new gene delivery systemand invented Bioplex, or biological complexes, for genedelivery.

Did this lead you to co-found Avaris, the company youstarted while completing your Ph.D. work?

In the process of doing all these transductions with retro-viruses I noticed that sometimes I would get really badtiters, and you want as efficient gene delivery as possible.I invented a device, basically a bioreactor that enhanced thetransduction efficiency by about 10-fold. That was aboutthe same time I invented these biological complexes. Youcan read about this in an article by Brandén and Smith inMethods in Immunology.1 Those patents became the basisof the company. In Sweden you have the advantage of hav-ing the company activities go on side-by-side with the workof your academic laboratory In that way you can capital-ize on the space and the equipment. You just pay a nomi-nal fee for the use of the equipment and the space.

After some time I wanted to expand my experience,and I had always wanted to live in New York City. Iwanted to work outside the field of gene delivery andgene therapy as I was starting to get bored. Boredom ismy main enemy. I sent out two letters, one to James Roth-man, who responded rather quickly and invited me to Co-lumbia to give a seminar. I went in June 2002, gave aseminar, had an interview with Jim, and met the post-docs in the lab. I moved to New York City later that sum-mer to begin work on a proteomics project Jim was run-ning at Sloan Kettering. My job was to find problems inthe project and fix them. After about a year and half westarted talking about moving over to Columbia, and Jimoffered me the position as Associate Director of the Ge-nome Center. He subsequently offered me the opportu-nity to become Director of the MLSCN project.

—Interview by Vicki Glaser

Reference

1. Brandén LJ, Smith CIE: Bioplex technology. A novel, syn-thetic gene delivery system based on peptides anchored tonucleic acids. Methods Enzymol 2002;346:106–124.

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Yale Center for High Throughput Cell Biology

Yale Center for High Throughput Cell Biology P.O. Box 27381

West Haven, CT 06516 www.yale.edu/htcb

Center Infrastructure

The Yale Center for High Throughput Cell Biology utilizes Tecan Freedom EVO 200 platforms -- highly advanced, fully automated liquid and plate handling platforms capable of processing 96- to 1536-well plates. Our platforms can deliver liquid volumes from 10 nanoliters to 125 microliters and can process over 200,000 wells per day. Barcoded screening plates allow digital plate tracking and real-time data streaming to our enterprise-scale sample, plate, and results database. Three platforms are housed in custom biosafety enclosures allowing for processing of BSL-2 samples including human cells and certain pathogens. A fourth platform is used exclusively for immunostaining and aids in maintaining high throughput.

The Center utilizes a Perkin Elmer Opera confocal imaging system containing four laser excitation lines

(405, 488, 561, and 635 nm) for high-content image acquisition. Capable of acquiring over 100,000

image sets per day, the Opera provides the Center with high throughput image acquisition capabilities

with integral image analysis scripting. With its Microlens-Enhanced Nipkow Disk, the Opera is

capable of imaging optical sections as thin as 1.3 µm for 3-dimemsional imaging. 4-D imaging is

accomplished using the Perkin Elmer UltraVIEW system. Designed for low throughput high-content

data acquisition the UltraVIEW system uses up to six laser lines to provide detailed images of expression

pattern changes through time. Fluorescence-based assays are read using Infinite Plate Readers integral

to the automation platforms.

The Center has deployed a highly scalable bioinformatics hardware infrastructure housed within a

modern industrial-scale data center. The data center is supported by layered security (physical,

hardware, software), redundant power supplies, and advanced environmental controls. The Center has

deployed databases and data processing applications that, for the purposes of high content imaging and

analysis, primarily utilize a range of high-performance IBM servers. Auxiliary computational resources

are available for high-throughput image analysis via a Center-administered Linux cluster as well as the

Yale High Performance Computing Center, the latter a massive cluster of over one thousand CPUs.

Computer disk space, always a concern with high content imaging, is available in ample quantities to

satisfy present and future needs; the currently deployed architecture is quickly scalable to 80 TB on

existing disk controllers and equipment, and can easily grow with minor enhancements.




Biological Profiling Assays

Yale Center for High Throughput Cell Biology (HTCB) has established core services group providing low- and high- content screening for biopharmaceutical and academic partners. Utilizing expertise in automation technology, high content imaging, and bioinformatics, the Center specializes in developing and executing novel cell-based gene-splicing assays.

Assays Table

Type Assay Target Stimulation

Structural

p230 Golgi structure None

FAK FAK localization None

Phalloidin Actin structure None

Paxillin Paxillin localization None

Tubulin Tubulin structure None

Inflammation

E-selectin E-selectin expression IL-1B, TNFa, CD40L, LPS, Etoposide, None

IkB IkBa (pSer32) phosphorylation TNFa, IL-1B, None

NFkB p65 translocation IL-1B, TNFa, CD40L, LPS, Etoposide, None

NFkB (2) NFkB (pSer 529) phosphorylation

IL-1B, TNFa, CD40L, LPS, Etoposide, None

P-selectin P-selectin expression IL-4, None

VCAM VCAM expression IL-1B, TNFa, CD40L, LPS, None

STAT Signaling

IRF9 IRF9 expression IFNg, None

STAT1 STAT1 pTyr 701 phosphorylation IFNa, IFNg, None

STAT1 (2) STAT1 pSer 727 phosphorylation TNFa, IL-1B, None

STAT1 (3) STAT1 translocation IFNa, IFNg, None

STAT2 STAT2 translocation IFNa, None

STAT6 STAT6 translocation IL-4, None

Oxidative Stress HO1 HO1 expression None

PKC Signaling PKCdelta PKCdelta (pThr505)

phosphorylation TPA, Etoposide

PKCmu PKCmu (pSer916) phosphorylation TPA, None




Assays Table (continued)

Type Assay Target Stimulation

DNA Damage Apoptosis Cell Cycle

p53 Pathway

Alamar Blue Cytotoxicity None

ATM ATM (pSer1981) phosphorylation

Hydroxyurea, Etoposide, None

Caspase3 Caspase 3 (Asp175) cleavage Staurosporine, None

CHK1 CHK1 (pSer345) phosphorylation


CHK2 CHK2 (pThr68) phosphorylation


Histone H2A.X Histone H2A.X (pSer139) phosphorylation


Histone H3 Histone H3 (pSer10) phosphorylation


HSP27 HSP27 (pSer82) phosphorylation

Anisomycin, Etoposide, None

p53 p53 (pSer15) phosphorylation


PKC Signaling PKCdelta PKCdelta (pThr505)

phosphorylation TPA, Etoposide

PKCmu PKCmu (pSer916) phosphorylation TPA, None

MAP Kinases

ERK1/2 ERK1/2 (pThr202/Tyr204) phosphorylation TPA, None

JNK JNK (pThr183/Tyr185) phosphorylation

Anisomycin, TNFa, IL-1B, None

JNK (2) JNK localization Anisomycin, TNFa, IL-1B, None

MAPKAPK2 MAPKAPK2 (pThr334) phosphorylation Anisomycin, None

MAPKAPK2 (2) MAPKAPK2 (pThr222) phosphorylation Anisomycin, None

MKP1 MKP1 (pSer359) phopsphorylation TNFa, IL-1B, TPA, None

MNK1 MNK1 (pThr197/202) phosphorylation TNFa, IL-1B, TPA, None

MSK1 MSK1 (pThr581) phosphorylation TPA, None

p38 p38 (pThr180/Tyr182) phosphorylation

Anisomycin, TNFa, IL-1B, Etoposide, None

Protein Synthesis

p70-S6K p70 (pThr389) phosphorylation

Anisomycin, Hydroxyurea, None

p90-RSK p90 (pThr360/Ser364) phosphorylation TPA, None

S6 ribosomal protein S6 (pSer235/236) phosphorylation Anisomycin

If you are interested in an assay not listed, please feel free to contact us to discuss assay development appropriate to your research.




Biological Profiling & Counter Screening

Yale Center for High Throughput Cell Biology (HTCB) has developed an extensive set of secondary assays to counter-screen hits from primary screens to provide a deeper understanding of targets and pathways, mechanisms of drug efficacy and toxicity, and ultimately human variation in therapeutic response. In general, for phenotypic assay development, we have used a single cell type, HUVEC (human umbilical vein endothelial cells). These cells have the advantages of being non-transformed and of human origin, thus ensuring the biological validity of the assays. In addition, endothelial cells, which in vivo are situated at the junction between the circulatory system and the solid tissues of the body, play an active role in assimilating and transmitting signals. In this respect, it is noteworthy that endothelial cells have been shown to possess a broad assortment of receptors capable of triggering many of the key intracellular signaling pathways.

As shown in the table below, our repertoire of secondary assays monitors a variety of physiological states and signaling pathways. Technically, these assays are divided between those that employ antibody detection of a target protein and those that monitor transcriptional activation using luciferase reporter gene readout. Many of the assays monitor specificity of kinase or transcription factor activation. Other assays flag compounds that are toxic in some way or that cause a stress response in the cells.

Selected Counter-Screening Assays developed by Yale University Center for High Throughput Cell Biology

Growth Factors Cytokines Apoptosis

Cell Cycle

Stress Structural Integrity

AP1 RE cJun NFAT RE Caspase 3 Cell Division ATF6

RE Actin

ATF2 MapkapK2 JNK Caspase 6 CHK2 ARE Fak

CRE Mnk1 STAT1 Caspase 7 cMyc DRE P230

(Golgi) CREB p38 STAT2 Caspase 8 Histone H3 Y-BOX

E2F RE PKCµ STAT6 HistoneH2a ThRE ERK1/2 PMA RE NFkB ATM HO1

cFos cSrc E-Selectin p53 Hsp27 SRE VCAM-1 JNK(1) C-MARE

Abbreviations used: RE, response element; CRE, cAMP response element; CREB, cAMP response element binding protein; SRE, serum response element. ARE, antioxidant response element; NFAT, nuclear factor of activated T cells; DRE, dioxin response element; C-MARE, CRE-like maf response element; NFkB, nuclear factor kB; HO1, heme oxygenase-1; ThRE, Thrombin response element.




We are also incorporating over 50 additional assays based on adenoviral vectors into our array of profiling assays. Of these, 25 assays use a fluorescent protein reporter gene to monitor protein translocations and require an image-based readout suitable for multiplexing. Another 20 assays feature and enzymatic reporter to measure transcriptual activation from a variety of response elements.

SID 855810 SID 855758 SID 856923 SID 857745 SID 858409 SID 861918

An example of compound activity in 42 secondary assays

Red indicates inhibition; blue activation. Each compound was tested at 10 serial 2-fold dilutions with the highest concentration (10 µM) on the left. The overall inhibition level of all the assays, in red above, indicates the level of promiscuity of each of the assay compounds.

O

O H

H

H

O

N NH OH O

O

N N

O

O H

O O O

O

O

O N H SO

O

N N O S H O

N

N H N

N

N

O

O N N

N N S

NFkB TNFa NFkB TNFa NFkB IL-1B NFkB IL-1B

E-selectin IL-1BE- VCAM TNFa

VCAM IL-1B p38 Anis

p38 IL-

ERK1/2 TPA STAT1 IFNg STAT6 IL-4

JNK Anis JNK TNFa JNK IL-1B

p53 No ne p230(Agrains) No ne

Histone H3 No ne

Tubulin (4hr) No ne Cell Num ber (4hr) No ne

Cell Number (24hr) No ne AP1 PMA

PMA PMA

NFAT PMA E2F PMA

Rb PMA

CRE PMA ATF6 No ne

ARE No ne ThRE No ne

Y-BOX No ne




High Content Screening

The Yale Center for High Throughput Cell Biology has established a core services group specializing in high content screening and other technologies. Utilizing expertise in automation technology, high content imaging, and bioinformatics, the Center develops and executes novel cell-based assays. The Center provides flexible cost-effective solutions to biopharmaceutical and academic partners and provides novel insights about biological phenomena.

Whenever feasible, we multiplex readouts for each assay used as a primary screen. This increases the depth of the data collected as well as the biological significance of the data. Our imaging platform, the Perkin Elmer Opera, can utilize up to three different laser lines, in addition to UV excitation, for either sequential imaging or simultaneous imaging of multiple channels.

For most assays, one channel is dedicated to visualizing the cells’ nuclei. Thus all image-based assays are already multiplexed for cell number and nuclear size and shape – useful controls as decreased numbers or condensed nuclei can be indications of toxicity or apoptosis. The majority of image-based cellular assays use either a fluorescent protein, such as GFP, or antibodies coupled to fluorescent markers to visualize cellular components. In designing a multiplexed assay, the technical requirements include having fluorescent readouts that are on different channels (e.g. red and green) and that have well separated spectra of emitted light.

As an example, the first assay that we multiplexed was VCAM-1, which was screened against a 100k compound library. During inflammation, cytokine activation of the NFkB pathway in vascular endothelial cells results in cell surface expression of VCAM-1. Adhesion and subsequent transmigration of circulating monocytes is carried out by VCAM-1 anchored to the cytoskeleton by its cytoplasmic domain. Following migration out of the lumen, monocytes are transformed into macrophages that can accumulate lipid, a critical early step in the development of atherosclerosis1, 2. Thus, it is possible to block monocyte adhesion by two independent routes, decreased VCAM-1 expression or disruption of F-actin fibers3, 4. A recognized anti-inflammatory agent, simvastatin, inhibits monocyte adhesion despite increased VCAM expression because it simultaneously disorganizes the cytoskeleton5

1 O’Brien, et al., 1996, Circulation (93), 672 2 Ross, 1999 N Engl J Med (349), 115 3 Wojciak-Stothard, et al., 1999 J Cell Physiol (176), 150 4 Vandenberg, et al., 2004 J Cell Biochem (91), 926 5 Pozo, M., et al., 2006, Eur J Pharm (548), 53

. Clearly, a screen for small molecules that block monocyte adhesion to Human Umbilical Vein Endothelial Cells (HUVEC) would have both biological and therapeutic significance. However, such a screen would be difficult and costly to perform in a high




throughput mode. Fortunately, similar results can be obtained by multiplexing the assay for VCAM-1 expression with detection of F-actin. The actin cyctoskeleton is easily visualized using phalloidin, which is specific for polymerized actin, (i.e. F-actin). Phalloidin, which is read in the green channel, is comparable with visualization of VCAM-1 using an antibody visualized in the red channel. For a multiplexed VCAM-1 assay, Human Umbilical Vein Endothelial Cells (HUVEC) are dosed with compounds, stimulated with IL-1b, and 24 h later fixed and stained for VCAM-1 and F-actin fibers.

Multiplexed VCAM-1 assay monitors VCAM-1 expression and actin cyctoskeleton

These images provide information such as:

• Nuclear shape (the size, blebbing) which can be an indication of apoptosis and nuclear fragmentation;

• The general morphology of cells which can indicate stress and show blebbing, which may indicate apoptosis

• Shape and appearance of the cytoskeleton (visualization by phalloidin staining) can indicate whether there is a problem with the actin polymerization or a more general problem with the cytoskeleton; and

HUVEC, 24 h after treatment with TNFa 10ng/ml, and stained with anti-VCAM-1 (A and B) and phalloidin (A and C)




• The level and location of the VCAM-1 which can indicate protein distribution problems in the intracellular transport machinery (such as vesicular transport).




Informatics

Informatics at The Yale Center for High Throughput Cell Biology (HTCB) is redefining modern

genomics analysis. As a vital element of modern life sciences research, informatics is fundamental to our

Center’s mission of providing world-class, publication-level results.

Genome-wide RNAi analysis at HTCB is enriched by our inherently collaborative processes. Our

informaticians and biologists focus on understanding the research objectives of our collaborators. In

order to translate experimental results into broad biological context, our informatics scientists study

relevant cellular pathways of interest in silico, apply novel methods to discover relevant marginal hits

from our in vitro screens, and fit confirmed gene hits into broader protein interaction networks. As a

result, we can help prioritize high impact follow-up experiments.

HTCB Informatics also provides expert level cheminformatics support, exploiting the synergies between

compound profiling results (chemical activators and inhibitors) and gene hits from RNAi campaigns.

Combining data sets, we can overlay novel screening data with, for example, known protein/tissue

interaction data and can speed the discovery of novel biomarkers.

We have developed unique approaches to exploring hit-space via our novel software development

efforts; via machine learning and other techniques we uncover the biological significance of subtle

patterns of gene activity across seemingly disparate systems.

Advanced analyses are available that focus on subjects of highest interest to our collaborators. Advanced

techniques can include access to custom databases and tools specifically scripted per project.

Hit-space analysis:

• Clustering and annotation

• Inferred hit analysis

• Pathways analysis

• Protein network analysis

• Disease interactions

• Chemical interactions

• Tissue interactions

Profiling analysis:

• Chemical epistasis analysis

• Gene profiling analysis

High content analysis:

• Image analysis script authoring and

optimization

• Multiparametric analysis




Selected Assay Publications

IL-1B Induced NFkB Translocation Keywords: high content, cell-based assay, primary screens http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=796

Chemical Inhibitors of TNF Alpha Stimulated VCAM1 Expression Keywords: high content, multiplexed, cell-based assay, biological signal transduction http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=802

Chemical Modifiers of Cytoskeleton Assembly

Keywords: high content, multiplexing, microscopy http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=836

Clearance of Mutant Huntintin Protein Keywords: high content, cell-based assay, neurodegenerative disease mode/poly Q http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=853

Novel sHE Inhibitors for the Therapeutic Treatment of Hypertension and Inflammation

Keywords: enzymatic assay, biochemical assay, low-content http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=707

LYP Inhibitors-An Autoimmunity Target

Keywords: enzymatic assay, biochemical assay, low-content http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=640

http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=796�









siRNA Genome-wide Screening

The Yale Center for High Throughput Cell Biology has established a core services group specializing in genome-wide RNAi screening. Utilizing expertise in automation technology, high content imaging and bioinformatics, the Center develops and executes novel cell-based gene-silencing assays. The flexible, automated infrastructure allows for high throughput RNAi screening of various assay formats. The Center provides cost-effective solutions to biopharmaceutical and academic partners and provides novel insights about biological phenomena.

We collaborate closely with principal investigators from the initial assay design phase through confirmation and characterization of hits. We create assays capable of interrogating biological pathways of interest and screen them against the Dharmacon siGenome library. Using our automation platforms we conduct genome wide screens at high efficiency and with high precision. Project objectives can be met by single parameter plate reader assays for endpoint detection such as luminescence, fluorescence, absorbance, and FRET. Using the Opera confocal imager, we capture and analyze multi-parametric data sets to measure biological events such as:

• Phosphorylation • Translocation • Change in expression • Redistribution • Co-localization

Our imaging capabilities allow us to multiplex up to three different primary readouts as well as secondary readouts such as cell number and morphology. We have four laser lines at present, 405, 488, 561, 640 measuring from UV to far red. Customized image algorithms offer multiple analysis options that allow for alternative hit classification and definitions.

We offer a suite of profiling assays designed to further elucidate the mechanisms of action of the hits generated in the primary screen. Our assays cover a range of areas and can be used to rule out non-specific hits to help focus follow-up work. We can additionally design assays tailored to areas of interest in the given pathway. Using existing knowledge in the field we can choose key areas of regulation for assay development and provide an insight into which part of the pathway is being affected and at what level.

Our bioinformatics tools have been developed to utilize screening campaign results to characterize genes. Our goal is to systematically discover functionality shared by genes by identifying similar activity profiles between genes screened across our wide range of cellular assays.

We encourage interested parties to contact us to discuss opportunities or to schedule a tour of our facilities. Please contact Dr. Lars Branden at the Yale Center for High Throughput Cell Biology at [email protected] for further information.

mailto:[email protected]�




Pricing and Expertise Guide

The Yale Center for High Throughput Cell Biology is pleased to offer low and high-content high

throughput screening to corporate and academic partners. Pricing for screening campaigns is highly

variable, depending on conditions such as preexisting high-throughput assay readiness, number and

type of cellular markers used, and informatics needs. Our pricing structure is based on a flat fee for

library access and literature reviews with variable costs for laboratory consumables, assay development

and informatics labor. We encourage interested parties to contact the Center’s Director, Dr. Lars

Branden, [email protected] for more information on services and fees.

Every service that Yale HTCB provides has an intrinsic and vital component of bioinformatics

incorporated. HTCB informatics experts provide support including sample tracking, data capture, data

storage, data analysis, backup, and retrieval. The close collaboration between Center biologists and

informaticians allows for iterative analysis of screening data sets, thus providing Center collaborators

deeper insights into the systemic biological meaning of screen results.

Our expertise includes:

• Researching, preparing, and executing high-content screens

• Developing assay descriptions/protocols

• Optimizing assays for high-throughput screening

• Process flow

• Consulting on assay development

• Biological profiling & analysis of genes

• Defining biological activity of chemical analogs

• Preparing literature reviews on relevant areas

• Library reformatting

• Bulk production of cells/viruses

• Aliquoting

• Screening consultancy

progress and achievements between 2002-2007

Documents