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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.