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There is nothing scary and disheartening than coming in Monday morning after a nice relaxing weekend, open your laboratory cell culture incubator and check your cells in your culture flasks to see them all contaminated.  There goes the week’s worth of experiments, time wasted in expanding the cells from the limited number, cost of wasted reagents.   I have done mammalian cell culture for more than 12 years and during this time I have picked up some wisdom regarding sterile techniques which may be useful.

Any reagent which may come in contact with the cells has to be sterile.  It is a good practice to write on the bottle if the reagent is sterile, especially if it is used by more than one person in the lab.  This will give the other colleague to use caution when using the reagent to use it sterilely.  If you are not sure if the reagent or culture medium is sterile, sterile filter the media. This is easier than using the media without filtering to contaminate the culture and waste time and cells.

Wear gloves when handling culture, this will protect both you and the cells from contamination. Since the gloves may not be sterile, rinse the gloves with 70% ethanol.

Use alcohol swab and wipe the mouth of the reagent bottles before using media to feed the cells.

When using Pasteur pipette always be aware of where the tips of the pipette is.  If you touch any surface by accident and put that pipette into the cell culture you can easily contaminate your cell culture. It is a good practice to keep the tip of the pipette far away, to avoid touching any surface.

When culturing multiple cell lines, handle one cell line at a time. There is a good chance of cross contamination if not careful by keeping different cell lines in the work space.

Check the cell culture everyday to make sure they are not contaminated.  Check frequently for mycoplasma contamination for purity and integrity of the culture. Mycoplasma contamination is difficult to see unlike bacterial or fungal contamination.

Make it a habit to get rid of the contaminated culture immediately by treating with chlorine. If you keep it around, the chances are the contamination will spread to other cultures.

Only use cell lines from reliable vendors or labs where they certify the cultures are tested negative for mycoplasma.  If cell lines are obtained from other local labs, confirm if that culture is tested negative for mycoplasma.

Keep the opening and closing of the incubators to a minimum. More often the incubator doors are open, the better the chance of the getting the cell culture contaminated.

If you are using a tray of water inside the incubator for humidity, make sure the water is always clean and changed frequently. Keeping dirty water is a great way to invite contamination.

Clean the warm water bath frequently and add fresh water for warming up medium, and other cell culture reagents.

If possible, reduce the foot traffic to the cell culture room. If the cell culture room is separated with a door, keep the door closed to avoid constant foot traffic to the cell culture lab. The more turbulent the air in the room, the more chances of bringing in contaminating organisms, especially during summer months fungal spores travel in the air.

If possible use filtered cell culture flasks instead of keeping the cap loose for aeration inside the incubator.

In recent years a confluence of factors in the life science industry research and development has contributed to the increase in productivity in the drug discovery we see today.  This includes development of exciting new technologies, a better understanding of human genome for target selection, development of ultra fast computational and IT infrastructure, vast array of sophisticated instrumentation for every conceivable application, and equally vast supply of reagents to support various R&D efforts.  In spite of these impressive advancements, one aspect of this equation which needs attention is the human factor.  Why is human factor important?  Because the cost in lost productivity from disengaged employees are billions of dollars per year in US alone.

The culture of an organization determines the culture of the individual’s engagement with the organization, its teams, technology, and the goals. The culture of the organization determines how the decisions are made, in the organization, and how the goals and objectives are expected to be met.  Lack of communication and clarity of purpose at various levels lead to misunderstandings, in fighting, missed targets, unnecessary delays of critical projects, and cost over runs.  The culture is a set of beliefs, objectives, or purpose understood by the members of the organization at every level and clearly communicated throughout the organization. In large organizations different units may have their own sub culture.

One of the major changes in culture is when an employee hired from an academic background. In academia the priority is in publications in the peer reviewed journals.  In academic context, usually investigators keep their findings to themselves and their group and guard it from competing lab for being able to publish first.  In industry, publication of the results from a study is much less important than sharing the data with the team and uses the data in a collaborative effort to advance the drug discovery development.  This change of culture is a very striking difference during the transition from academia to industry. The key differences between the two are in academia the goal is to advance the science and knowledge through scientific publications. In industry the goal is to advance the knowledge to develop a product, accumulate intellectual property, and get a competitive edge over your competition.  In industry the management has to answer to the board, work towards a healthy balance sheet.  Naturally, new projects, new product development effort would be under strict time line, with a clear development path.  These goals and deadlines have to be communicated to the groups with absolute clarity and make sure everybody buy into that.

One of the ways in large companies they organize their drug development efforts are by dividing the groups into separate groups based on therapeutic areas. These groups function independently and control their own budgets, setting targets for their own portfolio, and compete for resources with the other similar groups.  These independent groups function as if they were a separate biotech division fostering the small company feel and nimbleness within the large organization. In this system individual unit head to have to give clear leadership for the group, communicate the goals to the group, and make sure the goals are in line with the corporate strategy.

Some of the key areas where the winning culture can be fostered are by valuing each member’s opinion, whether it is regarding the time line or selection of targets, or new product development.  Especially in group meetings, listen and value the member’s opinion as well as concerns. Encourage to ask questions, or suggest ideas and solutions.  Encourage to think outside the box for unique solutions for the problems, or for a new product idea.  During these exchanges value the member’s comments.  Never put down the member’s ideas with comments such as “it is the dumbest thing I have ever heard”, or even before the ideas are completely explained, make the comments “it won’t work”.  Even if the comments are not very useful, or the product ideas are not very practical, respond such a way that the team and its leader appreciated the comment and encourage coming up with more ideas in future.  Putting down a member for suggesting an idea or asking a question creates ill feeling, and could lead to disengagement and could cost the team tremendously by way of lost productivity.

Make sure that the members of the team understand and believe that they have significant stake in the project and their contributions are critical for the success of the project.  When the project succeeds, compliment them and reward them. If the project fails, instead of singling out a person or group of people and blame them, share the responsibility.

Provide a clear path for careen development in the organization. Encourage the members to take on more responsibility and help them achieve their goals. If they are successful they will have the expectations of promotions as a supervisor, manager or director. Make sure the members are clear on what it takes to be in their chosen career path.

Open an effective communication channel between the staff members and human resource department.  Encourage  the members to discuss any issues within the team or between teams to the HR personnel and try to resolve the problem as quickly and effectively as possible. It may be useful from the human resource department to be proactive, and have periodic discussions with groups and individuals to share their concerns, or excitement and could be a very valuable bridge between the management and the staff of the organization.   In summary, I have described various ways to enhance the company culture to a winning culture which leads to enhanced productivity and happy work force.

Drug discovery has undergone transformational changes in recent years. In the past purified cell membrane preparations were commonly used in receptor binding assays to identify compounds which bind to specific target receptors. This provided a homogeneous assay with end point readout which was easy to adopt in high throughput screen (HTS).  As physiological importance of using the whole cell rather than the membrane fraction was realized, whole cell assays were adopted in drug discovery. This was done using immortalized, engineered cell lines representing specific disease models.  These immortalized cell lines were easy to culture and propagate in large numbers needed for HTS. Results from these screens gave good signal to noise ratio, reproducibility, and good CV for the data because of the homogeneous nature of the cells. For several years this was the system of choice for cell based assay in drug discovery research.

During the last few years,  pharmaceutical industry has seen an increasing number of failures of their lead compounds at late stage trials with various reasons including, lack of efficacy in target population, increased toxicity, and unpredictable side effects. This high rate of failures at late stage of development cost tremendously for the industry. This has led to re-evaluation of their HTS for target validation and later development efforts. They also questioned the physiological relevance of the immortalized cell lines for the cell based assay screen for the specific disease models and targets.  This change in attitude has led to the use of disease and target relevant primary cells derived from corresponding tissue types in high throughput screen. In primary cells the target is expressed in physiologically relevant environment and closely resembles that found in human disease and at levels physiologically relevant. Consequently the results of the screen are more relevant to the disease they are targeting. The availability of numerous varieties of primary cell types from leading suppliers ( Life Technologies, Lonza)  and optimized culture medium for the growth of these cells has made the transition at an accelerated pace.  Primary cell based assays are now commonly employed in identification of compounds targeting GPCRs, kinases, nuclear receptors, and ion channels. In GPCR assays, most of them measure second messenger accumulation, or more complex, protein translocation, or activation of kinase signaling pathways.

Another convergence of technology is the highly sophisticated instrumentation available for HTS. This ranges from precision liquid dispensers to high content imaging platforms to complex software programs for the cell based assay image data acquisition, storage, and analysis. Some of the leading providers of the liquid dispensers include, Beckman Coulter, Caliper, and Tecan.  Instruments offered by the leading manufactures can dispense into  96, 384, or 1536 well plates with precision and usually results in very good CV. This was instrumental in miniaturization of the assay saving costly reagents and the number of primary cells needed to screen a compound library.

Several leading instrument companies in life science space offer very complex high content image acquisition and analysis instrumentation. This includes Cellomics, now part of Thermo Fisher, Perkin Elmer, GE Health care, Evotec Technologies, Beckman Coulter and BD.  The BD Pathway from BD Biosciences is an automated CCD imager using spinning disc confocal device.  This system is designed to do end point assay or kinetic assay.  It can also acquire several images per second even during the addition of drug compounds to the cells made possible with available liquid handling system. Evotec’s Opera, another high content screening instrument also can screen kinetic live cell assays. Screening can be done in 96, 384 or 1536 well format.  Available environmental chamber controls CO2, humidity, and temperature within the cell chamber.  This system also offers liquid handling unit to do kinetic assays.

Most of the high content imaging systems comes with very sophisticated software to acquire and analyze the data. Some of the leading software for image acquisition and analysis packages are provided by Cellomics, MetaXpress (Molecular Devices), Acapella ( Evotec Technologies ), and Harmony software from PerkinElmer.

In summary the convergence of technology in the form of efficient instrumentation for liquid handling, sophisticated high content imaging systems for end point as well as kinetic assays, readymade reagent kits for fluorescently labeling cells, software packages to analyze the data, and the use of primary cells in the HTS screen has all came together and made significant progress in the drug discovery in recent years and will continue to impact the field for the  foreseeable future.

In the last article we discussed the increasing use of HCS in drug discovery and its important applications in drug discovery. Here we will discuss the leading providers of hardware, software and consumable kits used in the high content screen.  One of the leading companies in this area for instrumentation and ready to use assay kits is Cellomics, currently part of Thermofisher Scientific.  Cellomics has automated the HCS for cell based assay platform by offering complete system. They offer HCS instrumentation- ArrayScan^TM- informatics, cellular image analysis software, ready to use reagent kits, fluorescent reagents, engineered cell lines, and multiparametric assays. Cellomics constantly develops new software updates, new bioapplications and make sure that their customers can upgrade their system to the newest version, and application. They also provide excellent customer support and user forums. Their systems are used in most of the major pharma and biotech companies, which allows Cellomics to constantly develop new products in this niche market by adopting features requested by its users.

Another HCS instrument is GE Healthcare’s  IN Cell Analyzer^TM, which is an automated cellular imaging system which can be used for live or fixed cells. This platform can be integrated with laboratory automation modules for smooth and convenient operation.  This instrument can do either an end point assay or with available upgrade can do kinetic assays, transmitted light imaging, and slide handling. INCell Developer is the image analysis software which is a very flexible program for cellular analysis. This system is known for its sensitivity of imaging and flexibility for integration with other automation systems.

BD Pathway^TM from BD Biosciences is a unique confocal imaging system with endpoint and kinetic analysis capabilities.  This system uses a spinning disk confocal device and uses white light source for illumination.  This system can acquire several images per second even when adding stimulants to the cells with its on stage liquid handling system. The environmentally controlled image chamber, with onstage fluid dispensing capability, and rapid rate of image acquisition, is an ideal system to screen time lapse observation of living cells. This system is very useful in the study for neurite outgrowth assay, cellular translocation and trafficking, morphology changes, endothelial tube formation assay in a fully automated or semi automated mode.

Evotech Technologie’s Opera^TM is another confocal image analysis platform used in HCS.  It uses four lasers plus a Xenon lamp for fluorescent excitation. The image is acquired by multiple CCD cameras in parallel, enabling rapid multicolor image acquisition. Acapella ^TM is the software used for image analysis in this platform and can analyze images as it is acquired in a high throughput mode.  Some of the available programs are useful in analyzing cytotoxicity assays, translocation of signaling molecules, endocytosis, neurite outgrowth assays, etc.

Molecular Devices is another provider of HCS instrumentation as well as image analysis software. Their HCS system, Image Express is a high resolution CCD imager which can be expanded by adding different functional modules. Their powerful software suite Metamorph, controls all their HCS systems.

Reagents:

Novel reagents, fluorescent probes, assays kits for specialized assays optimized for HCS which provide improved quality of data are the driving the adoption of this HCS platforms in drug discovery.  One of the new developments in this area is the ability for single cell analysis, and being able to query multiple analytes per well. Some of the most commonly used fluorescent proteins for labeling probes are Green Fluorescent protein from GE Healthcare and Living Colors from Clontech, both of these are available to use with license.  Molecular probe, now a division of In Vitrogen sells a wide variety of fluorescent proteins for labeling various molecules.  Their popular Alexa Flour dyes are available as conjugation-ready dyes for labeling primary or secondary antibodies. Their Vibrant classes of dyes are optimized for HCS screen for apoptosis, cell proliferation, and cell viability assays.

These are some of the major companies operating in the HCS space for instrumentation, software and reagents.  Adoption of high content screen in drug discovery is growing rapidly, and additional players will emerge in the next few years with exciting new offerings in this niche market.

During the last few years pharmaceutical and biotech companies have adopted high content screening (HCS) extensively in their drug discovery research.  High content analysis uses automated microscopy, to capture cellular images, and analyze them using software to quantitate complex cellular events.  This includes cellular morphology, biochemistry, and differentiation, translocation of proteins, proliferation, and apoptosis. The specialized software recognizes patterns from the images and quantify the differences between wells as accurately as possible without bias.  Novel reagents, instruments and technology have significantly improved the quality and speed of the screen and have opened up the technology to a wider user base.   In the two part series we will discuss some of the most commonly used application for high content screen. We will also discuss some of the reagents, and hardware currently available on the market n this fast growing field.  There has been a steady increase in the adoption of HCS in pharmaceutical and biotech companies specially employing engineered cell lines or primary cells.

Most commonly used application for the HCS is in oncology, neurobiology, in vitro toxicology, immunology, cardiovascular biology, and endocrinology. HCS is widely used for target identification/validation, secondary screen, lead optimization and compound profiling.  Some of the most relevant application for the HCS include, signaling pathway analysis, morphological changes, multiplexed assays, translocation, cell proliferation, cell migration, cell differentiation, kinases, toxicological studies, and reporter assays. Some of the assays such as cell migration assays include trans-well migration, lateral migration or invasion of tumor cells through a matrix coated membrane towards a chemotactic  stimuli. High content analysis for neuroscience application include, quantifying  neurite outgrowth, transcription factor translocation, neuron and synapse number, cell proliferation, receptor internalization, migration and apoptosis.

Data management and informatics are critical aspects of the high content screen.  Typically multiple images of the micro plate wells are captured at different magnifications and sometimes stitched together and analyzed using specialized software programs.  Usually raw images are stored in high capacity storage devices.  Users need to implement both hardware and software systems in place to handle the large volumes of data generated after each screen in order to successfully execute a high content screen. From the images captured, software programs are able to recognize patterns and extract relevant quantifiable data in order to compare between compounds screened.  This will allow comparisons of multiple parameters of complex cellular characteristics in response to various potential drug candidates.

In summary we have discussed the increasing adoption of high content screen in drug discovery by small to medium biotech companies, and established pharmaceutical companies. Potential applications in various areas of drug discovery are also discussed. The critical nature of specialized hardware and soft ware necessary to store and analyze large volumes of data is also emphasized.  In the next article we will cover some of the major vendors offering reagents, hardware, and software systems to implement the high content screen.

During the past decade a large number of potential drug candidates successfully selected at the early stages of screening, have failed to make it to the market due to various issues including pharmacology.  This alarming rate of failure has led the biotech and pharmaceutical industry to re -evaluate their drug discovery models. While last few years have seen a general shift towards employing cell based assays, emergence of the use of stem cells as part of the screen has the potential for new opportunities for a new paradigm in drug discovery research.  Human stem cells with their potential to differentiate in vitro into a various cell types may become a very important tool for drug discovery.  Stem cell assays could replace traditional cell lines in the cell based assay screens with increasing relevance to pre-clinical development.

During the last several years pharma and biotech drug discovery employed cell based assays utilizing immortalized cell lines derived from various tumor or engineered immortalized cell lines to screen drug compounds.  The advantage of using these cell lines includes easy maintenance, scalability to accommodate large screens, homogeneous population of cells, with good reproducibility of data. In spite of these advantages, these screens may have contributed to the high rate of failures at the later stages of the drug development.  Primary human cells such as human umbilical endothelial cells (HUVEC), hepatocytes, and keratinocytes are being used for the compound screen; however, these primary cells are difficult to expand to get enough cells to use in a large screen.  After few passages these cells differentiate into cells with few characteristics of the original primary cells.

Another choice in selecting the cells for the primary drug screen is the human adult stem cells. Human adult stem cells can be isolated from a variety of issue types as listed below:

Adult Stem Cells –   Tissue

Embryonic  stem cells- Blastocytes

Induced pluripotent Cells- Reprogrammed from somatic cells

Adipose stem cell-Adipose

Hematopoietic stem cells (HSCs)- Bone marrow, Umbilical cord blood

Cardiac muscle stem cells- Heart

Liver stem cells- Liver

Neural stem cells- Brain

From the list above, only a few types of adult stem cells can be expanded to obtain enough cells for cell based assay screen. Stem cells isolated from liver, adipose, and brain has limited ability to expand, and when expanded, lose its ability to differentiate. Two of the well characterized, most commonly used stem cell preparations are hematopoietic stem cells (HSC), and mesenchymal stem cells which is derived from bone marrow (BM-MSC).  HSC when grown in the presence of certain cytokine and growth factor cocktail under specific culture conditions can differentiate into all types of blood cells. Bone marrow derived stem cells( BM-MSC)  can be easily expanded  in vitro and can differentiate into a variety of cell types such as adipocytes, osteocytes, chondrocytes, and to a lesser degree into neurons, and muscle cells.  After expansion these cells can be used in HTS screen for obesity, osteoporosis, diabetes, metabolic disorders, and joint diseases. MSCs isolated from human umbilical cords, like the bone marrow derived stem cells can be expanded and has the potential to differentiate, which could be used in large scale screening.  Both the BM-MSCs and MSCs from cord blood are available at early passage from various companies as frozen vials, and can be expand in culture to required numbers for the screen.  When using these cells from the vendors, lot to lot variability in its ability for proliferation and differentiation can vary widely. To avoid this problem, these vials should be expanded and prescreened to meet the optimum response and those closely match the required response set aside as frozen stock to be used for larger screen.

BM- MSCs can go 50 doublings before they lose their differentiation potential.  These doublings has to be evaluated with respect to the individual growth conditions which may vary widely. Even with this variability, cells started at passage 2 can be expanded considerably to get enough cells to screen close to 500,000 compounds in multi-well format  screen.  Like primary cells BM- MSCs shows variability from donor to donor, and some lots double more rapidly than the others.  To avoid this variability in the screen, these lots must be screened to identify the lots which behave closely in its response to differentiation.

BM-MSCs provide a great system to examine the molecular and cellular regulation of progenitor proliferation, differentiation, and eventual commitment.  During the last few years, scientists have developed specialized growth factor cocktails to modulate differentiation towards certain pathway.  These stem cell systems offer best tools to identify the druggable targets critical in cell proliferation, differentiation and function as part of drug discovery screen.

Stem cells besides providing a valuable tool for high throughput screen can also provide valuable basic information identifying certain pathways involved in the proliferation and differentiation process offering more druggable target selection. Some these studies are done not in the 96 well plates but in large cell culture dishes. After transfection of stem cells, or just growing cells as colonies, the new cell culture dish with numbered grids offered in the market will offer great way to locate and keep track of colonies for further selection, isolation and analysis. One of the commonly employed colony formation assay by hematopoetic stem cells, the new tool multi-well plates with grid bottom will offer great way to identify, and quantitative the colony size and number.  These are great new tools for the stem cell  and hematopoetic cell research.

In summary, cell lines derived from tumors which are commonly used in the cell based assay screen for target validation has been un- successful  in pharmacology, and may have contributed to the  drug failures for the pharma industry.  Stem cells offer a very valuable alternative tool to identify targets involved in proliferation and differentiation pathways. If we can manage donor to donor variability, and reproducibility, this will be great improvement over the current system of cell based compound screen.

Several years ago, while I was working at life science  laboratory I had to routinely prepare samples of RNA, DNA, Protein, etc. Many of these samples were temperature sensitive, and get ruined if kept at room temperature. I had very difficult time dealing with tiny 0.2 ml clear PCR tubes containing RNA and PCR mix kept on ice in an icebucket. It was very difficult to add or retrieve samples from the tiny tubes placed on ice. Most of the time it was difficult to keep the tubes straight. If the ice is partly melted, the tubes will float in the bucket. I had to deal with this problem for a long time and was very frustrated. I knew there has to be a better and efficient way to do this. After brainstorming I came up with the solution. I designed a Metal Chamber for 0.2 ml Tubes where  0.2 ml PCR tubes, strips or trays will fit nicely on the pre-made cavities. I placed the whole metal chamber on a cooling chamber or ice bucket. The tubes placed on the metal chamber was straight, organized, and even when some of the ice melted the tubes did not float in the ice bucket. The benefits of using the solid metal chamber are as follows:

• You can manage tiny 0.2 ml, 0.5 ml tubes, strips or trays easily on the metal block.
• The tubes can be arranged in an organized fashion, and easy to identify each sample.
• Because the metal surrounding the sample tubes are cold, the whole sample stays cold. In the ice bucket, if there is an air pocket, the sample may not be completely or evenly cold.
• It is easy to transfer metal chamber with samples from ice bucket to refrigerator, and back to bench top.
• The metal chamber can also be used to heat the sample by placing it on a hot plate.
• Since the chamber is solid aluminium, it conducts heat and cold instantly, and the whole sample tubes get chilled quickly.
• The Chiller blocks  functions as good platform surface to organize the samples.

Besides Chiller blocks designed for 96 well plates, we have designed blocks for 1.5 ml – 2ml eppendorph tubes, 5 ml tubes, and even chiller blocks for petridish.

The chill block for 0.2 ml PCR tubes or trays can also be placed on a cooling box ( Pioneer Scientific – CoolPod). The chiller block will be kept cold by a cool pack inside the CoolPod. This cool pack can keep the samples cold for 4-6 hours without any ice.  If you need to keep the samples longer, just replace the cool pack contained in the CoolPod with another cool pack and you can get another 4-6 hours. Coolpod, and Thermopod have small footprint and can be  easily carried.

Another very useful chill block is the Step Tube Holder (SSTH).  This block is designed with tiers to hold 1.5 ml to 2 ml centrifuge tubes. If you are working with large number of tubes in an assay, it is ideal to place the tubes on these chiller blocks on ice, because one can easily see different tubes at once. It is a great way to arrange the reagent tubes when adding or removing samples from large number of tubes during an assay.

Another very versatile chill block is PSMC90. This is a large chamber with the capacity of 90 X  1.5 ml – 2 ml tubes. This gives a large capacity for sample tubes. This chill block can be placed on a rectangular ice bucket to keep the samples cold. Pioneer Scientific also offers a variety of other metal chill blocks for 15 ml and 50 ml centrifuge tubes.

By using these metal chill blocks in combination with ice, one can work with samples of any size or type very efficiently and in a well organized fashion. These blocks can also be used to heat samples on a heat block.