Tuesday, March 24, 2015

Phenotypic and MoA Analysis of Anti-Metastatic Molecules using 3D Tumoroid Invasion Assays, Kinetic Ligand Binding, and Cellular Microscopy Webinar Recap


Metastasis is the main cause of death in cancer patients and one of the most complex biological processes in human diseases. The development of therapies designed to forestall the metastatic activity of tumors has been met with multiple challenges, including the choice of an appropriate cell model. Tumors in vivo exist as a three-dimensional (3D) mass of multiple cell types, including cancer and stromal cells. Therefore, incorporating a 3D spheroid-type cellular structure that includes co-cultured cell types forming a tumoroid, provides a more predictive model than the use of individual cancer cells cultured on the bottom of a well in traditional two-dimensional (2D) format. A second hurdle has been accurate mechanism of action determination. Possessing knowledge of how invasion is interrupted provides a more complete picture before proceeding to clinical testing. A final hindrance which has been necessary to overcome is the proper capture and analysis of kinetic reader-based data and microscopic images during the tumor invasion process. Being able to monitor inhibitor binding, as well as tumor invasion through the matrix in a quantitative way is critical.

In the webinar presented last week, we demonstrated methods to perform live-cell, label-free, phenotypic analyses of 3D tumor invasion. Spheroid Microplates from Corning Life Sciences, coated with an ultra low attachment surface, were incorporated for tumoroid formation, and performance of the invasion process. Tumor invasion tracking and quantification was then performed via digital microscopy and cellular analysis using the Cytation 5 cell imaging multi-mode reader and Gen5 Data Analysis Software.

During the presentation, we also displayed the ability to determine inhibitor drug-target residence time using the Tag-lite® direct, homogeneous ligand binding assay from Cisbio. The compound set was then incorporated into the tumoroid invasion assay to determine their potential inhibitory effect on the invasion process. Finally, we illustrated how analysis of the involvement of matrix metalloproteinases during the invasion process could be assessed while simultaneously monitoring tumoroid invasion, using the EnSens Protease Activity Detection Technology from Enzium.

The combination of these techniques present accurate, yet easy-to-use methods to assess target-based and phenotypic effects of new, potential anti-metastatic drugs. We invite you to download and watch the recording of the webinar.

By: BioTek Instruments, Brad Larson, Principal Scientist

Tuesday, March 10, 2015

Spicy Surprise

Bugs usually don't bother me unless they are in my food. I found a couple critters crawling around my cupboard last night and traced it back to a whole colony in the paprika I brought back from a trip last year. Disposal of the colony was quick, except 1 bug. I decided to bring one to work and take a look at it in the Cytation 5 Cell Imaging Multi-Mode Reader.

The bug was placed in a clear 24 well costar plate and onto the carrier. Manual mode was launched and the 4x objective was selected. Texas Red seemed like a good wavelength to start with since it was living in the red paprika. The bug was mobile in the bottom of the well so I connected the joystick and began to play "catch the bug" with much ease. After a little practice with the joystick focus I managed to get an image of my former house visitor. A sharp focus was not obtained on the 1 mm thick sample using the 4x objective, but the image gave me a good idea of what my unwanted visitor looked like up close.

What’s in your spice?

By: BioTek Instruments, Kevin Myers, Service Product Manager

Tuesday, February 24, 2015

The Case for Vaccination

I remember travelling to Nigeria as a teenager and requiring a host of vaccines prior to travel:  yellow fever, hepatitis A, cholera, typhoid, diphtheria–pertussis–tetanus, polio booster - my right arm couldn’t take the full load, so my left was sore for days too.  Once there, I had to take weekly doses of chloroquine to combat malaria.  Then there were the infectious diseases that didn’t have a pill or vaccine: African trypanosomiasis or the sleeping sickness carried by the tsetse fly. All we could do was use DDT sprays to dissuade the flies from landing on us.  We have come a long way in our ability to combat infectious diseases, eradicating killers like cholera, smallpox and polio. So it mystifies me why there remain a significant number of parents who decline to have their children vaccinated with the measles-mumps-rubella (MMR) vaccine, as demonstrated in the measles outbreak last December in Disneyland.  Their decision to forego the vaccine is largely prompted by the belief that MMR may induce autism.

The 1998 publication by Andrew Wakefield in the Lancet that linked MMR to autism has been completely discredited.  The journal fully retracted the article in 2010 following the British General Medical Council’s finding that Wakefield had acted both against the interests of his patients and "dishonestly and irresponsibly" in his published research.  A few months later, Wakefield was struck off the Medical Register and is barred from practicing medicine in the UK. Yet there persists this fear of vaccination, in general. Wakefield himself still insists his MMR assertions are true, although he has no support whatever from the medical community.

Most scientists support vaccination programs as they can better understand the science behind infectious agents and medicine’s ability to combat them. BioTek Instruments supports vaccine research through the provision of many a microplate reader or liquid handling instrument into the various biosafety level laboratories at the Centers for Disease Control and Prevention (CDC). Only through this important work will medicine have a chance to eradicate emerging infectious diseases.

By: BioTek Instruments, Peter Banks Ph.D., Scientific Director

Wednesday, February 18, 2015

Detection of Residual Protein A in Biological Therapeutics

Over the past 10 years the pharmaceutical community has witnessed an explosion in growth of biotherapeutics. An increased focus on developing and marketing antibody based products resulted in a product shift to the point that in 2013 antibody based therapies represented one-third of all biotherapeutics and nearly half of all revenue in the product category. In fact, 2014 saw a new approval record set with nine new antibody products entering the market and many more in the pipeline or under review for approval in 2015.

Treatment of even a small population will require industrial scale production of therapeutic proteins, using a variety of bioprocessing methods including recombinant cell line expression systems, chromatographic purification methods and stringent purity assessment. Purity requirements include minimizing the concentration of host cell proteins and DNA ranging in the parts per million or lower relative to the product. Additionally, the formulation must be sterile insuring no viable microorganisms exist in the final product and void of any residual contaminants from the purification process itself.

A recombinant human monoclonal antibody is commonly produced in a mammalian cell line such as Chinese Hamster Ovary (CHO) cells during large scale manufacturing. Purification typically relies on the use of a three-column chromatography process to meet the stringent purification requirements: 1) Protein A affinity chromatography, 2) Cation exchange (CEX), and Anion Exchange (AEX); a viral filtration (VF) step is also generally used during the final stages of production. Resin with immobilized Staphylococcal Protein A (PA) has a high affinity for the crystallizable fragment (Fc) region present in rhuMAb IgGs allowing capture from the culture media or crude cell lysate of the host cell line. While these resins provide a high capacity and selectivity for the target protein, trace amounts of the PA ligand has been found to leach from the column contaminating the antibody product. Residual PA contamination of a biotherapeutic may result in immunogenic consequences as well as toxicological and/or mitogenic effects. Therefore, reliable, robust methods for the detection and quantification of trace amounts of PA are necessary and mandated in the US by the FDA.

Recently we demonstrated automation of a HTS compatible homogenous proximity assay for the detection of residual PA in biological therapeutics. The application includes screening results for detection of residual PA in a panel of ten (10) human IgG antibodies including samples of the  biologically active antibody components in the therapeutic products Herceptin®, Rituxan®, and Erbitux®.

D Ekers. (2015, Jan. 29) Antibodies: $75 million in sales and no slowing down in sight. [Web log comment] Retrieved from http://www.bioprocessblog.com/archives/699.
Mehta, A., et al. 2007. Purifying Therapeutic Monoclonal Antibodies. CEP. SBE Special Section: Bioprocessing. S14-S20.
Zhu-Shimoni, J.; Gunawan, F.; Thomas, A.; Vanderlaan, M.; Stults, J. 2009. Trace level analysis of leached Protein A in bioprocess samples without interference from the large excess of rhMAb IgG. J. Immunol. Methods, 341 (1-2), pp. 59–67.

By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA

Thursday, February 5, 2015

3D Cell Models Enhance Hepatotoxicity Screening

A few weeks ago I wrote a blog comparing the character "Flat Stanley" to the long-standing, traditional method of cell culture, and also explaining how three dimensional (3D) cell culture methods could help provide a more relevant model for drug discovery. Last week further evidence was seen to support this shift when BioTek's Flat Stanley hit the road with me to attend the High-Content Analysis and Phenotypic Screening Conference in San Diego, CA. This conference, apart from providing a wealth of information for those looking to learn more about cellular imaging, highlights another important shift in drug discovery, which is the re-emphasis of methods to analyze the final phenotypic effect of a test molecule during early screening; away from the target-based approach which has been in place since the 1990s. Here again, a significant amount of attention was placed on the incorporation of physiologically relevant 3D cell models into the screening process during plenary sessions of both conference tracks.

Of interest during the 3D focused talks was the high level of attention given to incorporating 3D cell models into toxicity screening…particularly hepatotoxicity. It has become generally accepted that primary human hepatocytes, when aggregated into a 3D configuration, maintain higher levels of cell viability and functionality compared to 2D cultured hepatocytes. Therefore, inclusion of these “microtissues” provides the ability to generate a clearer picture of the potential toxic effect of lead molecules.

BioTek has partnered with 3D cell culture providers to demonstrate how the combination of in vitro liver models, appropriate assay technologies, and image-based analysis can provide procedures for measuring hepatotoxic effects that are both robust, yet easy to perform.

Application Notes




By: BioTek Instruments, Brad Larson, Principal Scientist

Monday, January 26, 2015

Calling All Cooks!

Yes, you - the scientist reading this article. You see, I liken an experiment to a recipe. You have a list of materials needed and then you have your instructions, detailing how much and when to add a particular component. There might even be some cooking time (a.k.a incubation) at the end of which you get your final product. Of course, then comes the "taste test" or analysis. Did you add too much of this, too little of that... it might even take a few tries for optimization. Remember, you've got to satisfy the critics!

Just like in cooking, a scientist benefits from having the right tools. On television, a popular cooking competition show sabotages the cooks' efforts by substituting necessary kitchen tools like knives, bowls and pans with "unconventional" utensils. Hopefully, you don't have to deal with this kind of subversion, but it does make you realize how the right tools make tasks easier and cleaner.

Scene from BioTek’s Holiday 2014 video : The right tools make tasks easier and cleaner, but we can have fun once in a while, right?

Why do something manually when you can automate it and spend your time doing something else? For example, in cooking you can hand-whip cream or set your electronic mixer to do it while you slice some strawberries. In a lab, automating a task can have multiple benefits such as time, money and reproducibility. Yes, it may get done either way but is it efficient? For advice on automating your lab, check out this article from Lab Manager, with contributions from BioTek's own Jason Greene.

Cooks' and scientists' tools have to be dependable. At BioTek, we take pride in every step of the process -  from research and development to manufacturing, all of which is done in the United States. If you’re a cooking aficionado, you know which brands to buy. But whether you’re a new or seasoned scientist, you should know to contact BioTek Instruments first.

BioTek Instruments: Proudly made in Vermont, USA

By: BioTek Instruments, Ellaine Abueg Ph.D., Product Manager, Specialist

Friday, January 16, 2015

Flat Stanley…a Model for Cell Culture?

I’m sure most, if not all of you are familiar with Flat Stanley. Originally a series of books, he’s now more famous for the projects that are performed in elementary school with him. Students color in a picture, cut it out, and then take him on trips, or mail him to friends or family across the country and the world. The idea is to help kids learn about other people, places, and cultures that they might otherwise not even know existed. A great way to get children to learn in a fun, unique way; wouldn’t you agree?

But would you also agree that Flat Stanley makes a great model of the tissues and tumors that exist within the human body? Preposterous you would most likely say. Yet upon closer examination, drug discovery has been using the “Flat Stanley” method of tissue culture for decades. Cells are added to wells of a slide or microtiter plate, spread out, and attach to the bottom in a flat, two-dimensional manner. Unfortunately this cell culture model has led to numerous clinical failures of lead molecules, as in vitro results do not correlate to what is actually seen in vivo with animal or human subjects. Therefore a new cell culture model is needed. Three dimensional (3D) cell culture is poised to meet this demand. The 3D cell culture methods which now exist, including scaffold-, hanging drop-, or ULA plate-based models, provide a means in which cells aggregate together into structures that have length, width, and depth to them, as they would in the body.

The following links provide information regarding the projects that BioTek has performed, as well as the liquid handling, reading, and imaging instrumentation used to further enhance the results generated using 3D cultured cells.

Application Notes: http://www.biotek.com/resources/app_notes.html
Hot Applications: http://www.biotek.com/resources/hot_applications.html
www.cellimager.com: http://www.cellimager.com/#!applications/cjg9
Presentation: http://www.biotek.com/resources/articles/enabling-3d-cell-culture-methods-and-applications.html

As you will see, BioTek can help you out whether you are new to 3D cell culture, or would like to see what new opportunities exist using cells cultured in this manner.

Move over Flat Stanley…there’s a new cell culture model in town!

By: BioTek Instruments, Brad Larson, Principal Scientist