Transient transfected cell lines

COMMERCIALIZATION OF TRANSIENTLY TRANSFECTED CELL LINES FOR HIGH THROUGHPUT DRUG SCREENING AND PROFILING APPLICATIONS by KALPITA DEEPAK MEHTA Submitted in partial fulfillments of the requirements for the degree of Master of Science Thesis Committee: Christopher Cullis, Ph.D. Stephen Smith, Ph.D. James Zull, Ph.D. Emmitt Jolly, Ph.D. Department of Biology CASE WESTERN RESERVE UNIVERSITY May 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Kalpita Deepak Mehta candidate for the Master of Science degree *. (signed) Christopher Cullis Ph.D. (chair of the committee) Stephen Smith, Ph.D._______________________ James Zull, Ph.D._____________________________ Emmitt Jolly Ph.D._____________________________ (date) March 22, 2010 *We also certify that written approval has been obtained for any proprietary material contained therein. Dedicated to my parents and family Table of Contents TOC quot;
1-3quot;
List of figures: PAGEREF _Toc257380630 vi Abstract PAGEREF _Toc257380631 viii 1Recommendations and Conclusions PAGEREF _Toc257380632 1 2Introduction PAGEREF _Toc257380633 4 2.1Advantages of transient transfected over stably expressed cell lines: PAGEREF _Toc257380634 5 3ChanTest Corp. Overview PAGEREF _Toc257380635 9 4Background and Objective of the validation summary report PAGEREF _Toc257380636 10 5Validation results PAGEREF _Toc257380637 15 5.1 Specificity of fluorescent signals in transiently transfected HEK cells PAGEREF _Toc257380638 15 5.2Performance of previously frozen to fresh cells PAGEREF _Toc257380639 15 5.3Control of membrane potential by changes in external potassium concentrations………………………………………………………… PAGEREF _Toc257380640 15 5.4Activation and inactivation of the calcium signal by external potassium PAGEREF _Toc257380641 15 5.5Assay stability PAGEREF _Toc257380642 15 5.6Pharmacological sensitivity and use-dependence PAGEREF _Toc257380643 15 6Protocol: Transient transfection cell line construction: PAGEREF _Toc257380644 16 6.1Solutions and chemicals used in FLIPR TETRA TM assay PAGEREF _Toc257380645 18 6.2Experimental methods –FLIPR TETRA TM assay. PAGEREF _Toc257380646 20 7Discussions-Technical challenges and relative propositions PAGEREF _Toc257380647 23 7.1Problems and relevant suggestions PAGEREF _Toc257380648 23 8Commercial conclusions PAGEREF _Toc257380649 28 8.1Business proposition PAGEREF _Toc257380650 35 9Appendices PAGEREF _Toc257380651 37 9.1Appendix A: Estimated production cost for 2250 vials PAGEREF _Toc257380652 37 9.2Appendix B: Globally projected estimation of units sold per year. PAGEREF _Toc257380653 38 9.3Appendix C: Comparison of expenses (stable vs. transient) PAGEREF _Toc257380654 39 10Bibliography PAGEREF _Toc257380655 40 List of figures: TOC quot;
Figurequot;
Figure 1: Mean fluorescent signal in cells expressing Cav2.2/3/21 + Kir2.1 in response to K+ stimulation. PAGEREF _Toc257380692 12 Figure 2: Variability in results PAGEREF _Toc257380693 24 Figure 3: Compromise with expression level PAGEREF _Toc257380694 25 Figure 4: Low transfection efficiency PAGEREF _Toc257380695 27 Figure 5: Survey methodology PAGEREF _Toc257380696 28 Figure 6: Market potential is identified for biotechnology and pharmaceutical PAGEREF _Toc257380697 30 Figure 7 Demographics area of research PAGEREF _Toc257380698 30 List of tables Table 1: Assay Stability ………………………………………………………………..15 Table 2: Hill fit and reference values for selected reference compounds………………15 COMMERCIALIZATION OF TRANSIENTLY TRANSFECTED CELL LINES FOR HIGH THROUGHPUT DRUG SCREENING AND PROFILING APPLICATIONS Abstract By KALPITA MEHTA Drug screening and profiling in the drug discovery process can be carried out either by using stable transfected or transiently transfected cell lines. Transient expression system is a more appealing alternative in contrast to stable expression system because the latter is a very labor intensive, time consuming and expensive. It takes between eight to twelve weeks to develop a stable cell line as opposed to seven to ten days to develop a transient cell line. Moreover, given the size and cost of a High Throughput Screening (HTS) program, researchers cannot afford to perform an assay that has a high batch to batch variation. To meet these performance criteria, ChanTest has developed transiently transfected cell lines called Ion Channel EZCellsTM TT. These cell lines have been validated using voltage gated calcium channels into HEK293 cells (Human Embryonic Kidney) using scalable electroporation method and run calcium influx assay in FLIPR (Fluorometric Imaging Plate Reader). This report includes a detailed description of the validation results of Ion Channel EZCellsTM TT and its commercial feasibility including market entry and pricing strategies. Recommendations and Conclusions The conclusions are made based on the data obtained by learning psychographics, market trends, and challenges faced by the researchers using Ion Channel EZCellsTM TT. The process was adapted to include an understanding of customer needs by conducting a survey. The survey responses proved very valuable in making recommendations to support the technical feasibility. The main conclusion from the survey was that: transiently transfected cell lines are a platform that researchers identify as meeting performance standards, which is often a bottleneck in the lead discovery process. The scalable electroporation method used has the ability to reduce assay development time and provide more clinically relevant assays. The power of scalable electroporation technology is related to the speed, consistency, capacity and throughput efficiency in sterile and non–toxic environments, which supports the validation of drug targets and conducting HTS (High Throughput Screening). Advantages of the technology include: Transient cell lines eliminate in-house cell line development or costs to purchase replicating lines. They are designed to eliminate the cell culture and cell line maintenance costs. The transfected cells with transient expression are packed in vials, each vial contains 6 million cells resulting in ~15,000 cells per well of 384-well plate derived from each vial. A pilot study was conducted by ChanTest to demonstrate the performance standard and feasibility of the transient transfection approach. This study validates these cell lines using voltage gated calcium channels into HEK293 cells (Human Embryonic Kidney) run in calcium influx assay in FLIPR (Fluorometric Imaging Plate Reader). The FLIPR allows rapid assays of cellular signaling processes made feasible by simultaneous kinetics measurement of cell-based fluorescence changes in a 96- or 384-well format. This case study involves transfection of an equimolar ratio of the following four ion channel cDNAs in HEK293 cells: ,[object Object]

Inward rectifier potassium channel (Kir2.1) to allow modulation of resting membrane potential by external K+.Specific results involving assay stability, performance and specificity of fluorescent signals in transiently transfected HEK cells are shown later in this report. ChanTest`s ion channel-expressing EZCellsTM TT are commercially feasible having benefits over ion channel stable cell lines and division- arrested cell lines. Pricing is based on the cost per well – (6 million cells per vial result in a 384-well plate, with ~15,000 cells per well). Pricing: $599 per vial for one vial ($1.56 per well) and discounted pricing is allotted on the purchase of more than one vial. An addressable market of $4.5 million was estimated (Appendix A) for the ion channel EZCellsTM TT. Today, ChanTest Corp. acquires about 12% of the total Pharma/Biotech ion channel stable cell line market and is expected to grow up to 17% by 2011. Market entry strategy and financials give a clear picture of the commercialization plan, shown in detail later in this report. Introduction Drug discovery is one of the most important sectors in pharmaceutical research and development. It involves High Content Screening (HCS) and analysis, and includes applications that require sufficient levels of sample throughput along with the study of complex cellular events and phenotypes Choice of Targets Reagent Procurement Assay Development and Validation Mass Screening HTS implementation Data Capture, Storage and Analysis Leads Reference: (Macarron and Hertzberg 2002) Reagent appropriation (Reagent type such as cell line) is often a major bottleneck in the HTS (High Throughput Screening) process. This can delay the early phase of assay development. Elements of drug performance such as toxicity and specificity can be established simultaneously using mixed cell types. The ion channel is the most commonly used target type. Other target types are GPCRs (G-protein coupled receptors), nuclear hormone, kinases and protease. Time require to develop the test system is much shorter in transient transfected than stable cell lines. To meet assay performance standards, reduce assay development time and to obtain fast results ChanTest has developed transiently transfected Ion Channel EZCellsTM. They have similar expression levels as replicating cell lines. Transient gene expression is achieved by introducing foreign genes into eukaryotic cells, in particular mammalian cells, by non-viral methods; this process is called transfection. The gene is introduced with the help of chemical, lipid or physical methods. However, transient gene expression is temporary and is predicated on the burst of gene expression between 12 and 72 hours after transfection. This burst is followed by a rapid deterioration in expression of the transgene because of cell death or loss of the expression plasmid. Reporter gene activity is used to evaluate the transient expression system. Advantages of transient transfected over stably expressed cell lines: Development time: Developing a stably transfected cell line is a costly, multi-step process that normally takes at least several months. Transiently transfected cells, on the other hand, can be created very quickly once the DNA is obtained and engineered into a mammalian expression vector. This makes transient transfection ideal when a new protein target becomes of interest and results are needed quickly. It is also a good first step to determine whether a stably expressing cell line is likely to produce good results in a particular assay. 2. Expression level: Stably transfected cell lines have only a few copies of the transfected gene (those that are successfully integrated into the host cell genome). Transiently transfected cells often have a higher number of copies of the transfected gene immediately after transfection, which usually results in a higher expression level of the encoded protein (and higher current amplitudes for the ion channel). For most ion channels, higher expression is better because higher current amplitude produces better resolution between signal and noise, which allows the electrophysiologist to calculate channel block IC50 (half maximal inhibitory concentration) more precisely and reliably. There are a few ion channels for which we selected lower-expressing stable clones because a higher-expressing clone produced such high current amplitudes that saturated the amplifiers (making it impossible to determine IC50 of channel blockers), but generally, higher expression is better. The caveat of this argument is that with each cell division cycle, the expression level in transiently transfected cells decreases: since the DNA is not incorporated into the host genome, the same number of copies of the gene gets diluted with each cell division. This means that in order for the transiently transfected cells to be useful, they must be either used very soon after transfection, or frozen very soon after transfection and then used very soon after thawing. 3. Cell health: Over-expression of some proteins can be toxic to cells. For ion channel proteins, constitutive high expression of a number of calcium channels triggers apoptosis (Cell death). ChanTest's early attempts to develop stably transfected cell lines that constitutively expressed members of the calcium channel family failed because no high-expressing clones grew well in culture. One way to get around this problem is by using transiently transfected cells since these cells can be used soon after transfection so that any negative impact of expression on the rate of cell growth and division is minimized.Another way to work around this problem is to develop stably transfected cell lines where expression of the problem gene is inducible. Expression of the transfected gene is turned off during clone selection, and cell culture stocks are maintained without ever inducing expression. Only daughter cultures are induced. Both these options (transient transfection and inducible expression) are used successfully at ChanTest, with each approach being better for certain situations, depending on the ion channel and testing platform involved. 4. Versatility of subunit compositions: Most, if not all, ion channels exist in the cell as heteromers; several proteins encoded by different genes come together to form an active ion channel. In most cases, a single gene encodes the pore-forming subunit (through which ions flow across the membrane), and auxiliary subunits modulate gating of the channel. These auxiliary subunits can vary depending on tissue type, stage of development, and other factors, which can result in an associated varying degree of sensitivity of the channel to stimuli (including drug compounds). In other cases, two or more genes encode proteins that come together to form the pore structure. For example, many potassium channel pore structures are formed by four protein subunits, each containing six transmembrane domains. For hERG channels, four copies of the same gene (hERG) form a homomultimer, (Zhou, Qiuming Gong and January 1998) which acts as the pore structure (thus, the hERG protein can be referred to as the quot;
pore-forming subunitquot;
of hERG channels). For KCNQ3/KCNQ5 channels, two copies of each protein subunit (KCNQ3 and KCNQ5) form a heteromultimer that acts as the pore structure.For many ion channels, over-expression of only a single pore-forming subunit is sufficient to obtain electrophysiological recordings that approximate the response in the body to a drug compound. For these ion channels, stably transfected clones are very useful. However, for some ion channels, co-expression of multiple subunits is necessary to obtain meaningful electrophysiological recordings (for example Cav1.2/beta2/alpha2delta or KCNQ3/KCNQ5). Some investigators are interested in different variations of subunit compositions (because they exist in the body such as in different tissue types, different stages of development, etc.). It would be very costly and time-consuming to develop a stable cell line for each set of subunit compositions of interest. If there is little demand for a particular subunit composition, it may not be worth the cost of developing a stable cell line with that composition. Transient transfection allows us to quickly swap in or out different subunits as required. ChanTest Corp. Overview ChanTest was founded in 1998 by Arthur Brown, to meet the demand for cardiac safety testing services. This demand stemmed from the identification of the human ether-a-go-go related gene (hERG), which encodes a potassium channel as the principle target of concern for drug-induced sudden cardiac death (Brown and Rampa 2000). ChanTest is a science - driven Ion Channel and GPCR products and services company serving the Biotech and Pharmaceutical industry. It is recognized as “most trusted ion channel Services Company” by independent survey conducted by HTStec consultancy. (Comley, John HTStec 2007) Services are functional screens for Ion Channel and GPCRs on manual and automated platforms, comprehensive pre-clinical cardiac risk assessment services, cell culture services on large-scale cell culture, transfection and cryopreservation along with custom cell line development. ChanTest’s ion channel portfolio is the most comprehensive, commercially available library of ion channel - expressing cell lines. Cell lines are validated for structure, function and pharmacology, and performance is characterized by manual patch clamp and on one or more automated electrophysiology platforms. Cell lines are also offered in EZCellsTM format (division arrested or transient transfected) for evaluation, assay development and screening. Background and Objective of the validation summary report The objective of this study was to: 1. Validate the scalable transfection system for its suitability in creating an HEK cell line that transiently expresses human N-type calcium channels (Cav2.2) 2. To develop and validate a FLIPR (Fluorometric Imaging Plate Reader) that allows rapid assays of cellular signaling processes by simultaneous kinetic measurements of cell-based fluorescence changes in a 96- or 384-well format. 3. To assess the commercial feasibility of transient transfected cell lines. Specifications of Cav2.2/beta3/alpha2delta1 Calcium Channel cells +Kir2.1 Synonyms: N-type calcium channel; alpha1BHost cell: HEK293, transiently transfectedGene name CACNA1B/CACNB3/CACNA2D1 (Ca2+ channel); KCNJ2 (Kir2.1)Mycoplasma status: Negative (MycoAlert Kit)Packaging: Three vials of cryopreserved, transiently transfected cells, 2x106 cells/vial (6x106 cells total) Growth media: DMEM/F12; 10% FBSGrowth characteristics: cells remain viable 1–2 days post-thawExpression: Adequate expression for at least 36 hrs post-thawStorage recommendation: frozen under liquid nitrogenRecommended assay: FLIPR(Fluorometric Imaging Plate Reader)Recommended plating density (96-well format): 35,000-38,000 cells/well. Background HEK293 cells- Human Embryonic Kidney Cells The most commonly used cell types for monitoring conduction using the Fluorometric Imaging Plate Reader (FLIPR) assay are HEK and CHO (Chinese Hamster Ovary) cell types. In the current experiment, we use HEK293 cells that are transfected using target gene(s) using scalable electroporation method followed by calcium influx assay in FLIPR. HEK293 cells were generated by transformation of Human Embryonic Kidney cell cultures with sheared adenovirus 5 DNA. HEK cells are significant because they natively have a relative depolarized membrane potential (ChanTest 2009), which should inactivate and subsequently reduce availability of calcium channels for stimulation, which is a desired effect for the experiment. The N-type Calcium channel (Cav2.2) Calcium channels are selective to calcium ions and allow entry of calcium ions into the cell. Voltage gated calcium channels function to regulate gene expression and mediate cell death. Biophysical and pharmacological criteria are used distinguish various types of voltage gated Ca channels, labeled L, T, N, P, Q and R. In the current study, the potential therapeutic targets are inflammatory and neuropathic pain, the N-type calcium channel is more pharmacological sensitive. The N-type calcium channel is predominantly expressed in the nervous system where it is a main contributor to synaptic transmission. Incoming action potentials invade the synaptic terminal and calcium influx through N-type channels activates neurotransmitter exocytosis (secretion from the cell). In the spinal cord, N-type calcium channels play a prominent role in the pain-sensing pathway. In Ca2+ channels, the principal 1 subunits can co-assemble with 2, , and possibly subunits with profound effects on pharmacology (Mould J 2004). There are four different major forms of Calcium channel  subunits (1-4). There is some evidence that points to 3 as being the predominant  subunit that co-assembles with the principal Cav2.2  subunit (Nampkung 1998). A number of industrial research groups also seem to favor 3 for co-assembly with Cav2.2 (Benjamin, et al. 2006). We, therefore, choose to express Cav2.2 together with 2 and 3 in response to an inward rectifier. -152400341630 3162300166370 BA Figure SEQ Figure ARABIC 1: Mean fluorescent signal in cells expressing Cav2.2/3/21 + Kir2.1 in response to K+ stimulation. Reference: (Benjamin, et al. 2006) In figure 1 panel A: mean fluorescent signal in cells expressing Cav2.2/3/21 + Kir2.1 in response to K+ stimulation. The blue trace represents the control signal (n = 8) and the red trace the mean response in wells that were treated with the specific antagonist -Conotoxin GVIA (n = 4). Figure 2 panel B: mean fluorescent signal in untransfected HEK293 cells in response to the same experimental conditions that were used in A. Together, the experiments in A and B show that the fluorescence signal in the transiently transfected cells is due to expression of Cav2.2/3/21. Cotransfection with an inward rectifying potassium channel (Kir2.1) In addition, Kir2.1, an inward rectifier potassium channel was also co-transfected along with the calcium channel genes. Inward rectifiers conduct inward potassium current up to relatively depolarized membrane potentials, which stabilizes the membrane potential at more negative values. HEK cells natively have a relative depolarized membrane potential (ChanTest, unpublished) which should inactivate and subsequently reduce availability of calcium channels for stimulation. By co-transfecting the inward rectifier, the membrane potential can be kept hyperpolarized and calcium channel availability should be greatly enhanced. (Xia M 2004 Apr 1). Further, co-expressing the inward rectifier permits graded control of the membrane potential that can be utilized for identifying use-dependent interactions of pharmacological compounds with Cav2.2 and therefore, targeting of specific pathological states of Cav2.2 activity (Winquist RJ 2005). During high frequency action potential firing of neurons, N-type calcium channels in the synaptic terminals are thought to accumulate in the inactivated state. A drug that preferentially targets the inactivated state, should theoretically limit high frequency synaptic activity associated with certain pathologies such as neuropathic pain while normal synaptic activity should be unaffected. In the FLIPR assay, preincubating cells transfected with Cav2.2 with elevated K+ concentrations induces calcium channel inactivation and therefore simulates these pathological conditions. Validation results (Removed due to proprietary information) 5.1 Specificity of fluorescent signals in transiently transfected HEK cells 5.2Performance of previously frozen to fresh cells 5.3Control of membrane potential by changes in external potassiumconcentrations 5.4Activation and inactivation of the calcium signal by external potassium 5.5Assay stability 5.6Pharmacological sensitivity and use-dependence Protocol: Transient transfection cell line construction: Grow un-transfected CHO and HEK cells in 37°C incubator with 5% CO2 and 95% humidity, keep cell density in about 70-80% (number of cells per unit volume ~2 billion cells/plate for large scale transfection), pass them though the buffer at least twice a week. (use Ham’s/F12 for CHO cells and DMEM/F12 for HEK cells, supplemented with 10% FBS and 1% pen-strep) A day before transfection, plate cells in dishes/flasks/chambers (with appropriate size and number in order to have enough cells (~2 billion cells/plate) according to transfection scale, and let the cells reach about 70-80% confluent at harvesting point. Wash culture vessels with HBSS, and trypsinize for 1-5 minutes at room temperature, then resuspend cells in complete medium, count cells using hemacytometer. Spin down cells at 1000 g for 5 minutes at room temperature, and wash cell pellet once with transfection buffer (washing volume is determined by final cell suspension volume, at least 10 times as much as final volume) Spin down cells again at 1000 g for 5 minutes, and resuspend cells in certain amount of transfection buffer (final cell suspension volume is calculated based on the total cell number harvested) to make 1*108 cells/mL cell suspension. (final cell suspension – ready for transfection) Mix target plasmid DNAs (if the target has more than one subunit, mix different subunits at equal molar ration) and GFP plasmid DNA at 10:1 ratio Choose a transfection unit (OC-100, OC-400, CL-2, OC-100 holds 100ul, OC-400 holds 400 ul and CL-2 holds 50 ml) according to transfection scale. Mix DNA and cells together (DNA final concentration, 100ug/ml for HEK and 200ug/ml for CHO) Select appropriate programs on transfection machine, after electroporation, transfer cells into dish/plates, and let cells recover for 20 minutes at 37°C with 5% CO2 and 95% humidity. Plate cells into dishes/flasks incubate overnight at 37°C in a CO2 incubator and let GFP (Green fluorescent protein) and target genes express. Monitor transfection efficiency under fluorescent microscope or using FACS (Flourescent Activated Cell Sorting) sorter. Collect GFP positive populations by sorting on FACS machine, if needed Culture GFP positive populations for EP assays, or freeze cells for later use and sale (freezing solution=90% FBS+10% DMSO). CHO cells were transiently transfected with cDNA for Cav2.2 1, 2δ, and 3 subunits in addition to Kir2.1 cDNA using the scalable electroporation system. Cells were immediately frozen in liquid nitrogen for later use. For use in this study, cells were removed from storage, quick-thawed in a 37°C water bath, and then transferred to a conical tube also containing 30 mL cell culture medium - (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 g/mL streptomycin sulfate and 500 g/mL G418). The cell suspension will be centrifuged at 1,500 rpm for 2 minutes and the supernatant will be discarded. Cell will be re-suspended in 30 mL of fresh medium and 20 L of this suspension will be mixed with Trypan blue to determine cell viability and density. Cells will be counted immediately using a Hemacytometer. Cells will be diluted to a density of 35,000 to 38,000 cells/well of a 384-well FLIPR assay plate (Type: BD Biocoat Poly-D-Lysine Multiwell Cell Culture Plate) depending on measured viability and density. Using a multichannel pipettor, cells will be plated into the wells of the multi well plate. The day before a scheduled FLIPR experiment, cells will be plated onto a Polylysin-coated 384-well assay plates (BD Biosciences) using a multichannel pipettor (Titertek Multidrop) for overnight growth. Solutions and chemicals used in FLIPR TETRA TM assay Formulations All chemicals used in solution preparation were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted and were of ACS reagent grade purity or higher. Stock solutions of test and control articles were either prepared in dimethyl sulfoxide (DMSO) or deionised water and stored frozen. Test article (drug sample from sponsor) and positive control concentrations were prepared fresh daily by diluting stock solutions into appropriate buffer solutions. Solutions used in this study contained 0.01% Pluronic F-127. The composition of HEPES-buffered physiological saline (HB-PS), 10; Glucose, 10; pH adjusted to 7.4 with NaOH (prepared weekly and refrigerated until use. These formulations were then loaded in glass- lined 96-well compound plate of FLIPRTETRA instrument (Molecular Devices Corporation, Union City CA). Dye Solution Calcium 4 Assay Kit (Molecular Devices) Assay buffers Name:Preincubation buffer (0.5K/0Ca HB-PS)Storage Conditions:RefrigeratedComposition (in mM):NaCl, 140.2; KCl, 0.5; CaCl2,0; MgCl2, 1; HEBES, 10; Glucose, 10; pH adjusted to 7.4 with NaOHCalcium 4 No Wash Kit (Molecular Devices Corp.)0.5 K+ was included to achieve sufficient current through the inward rectifier and therefore a more negative membrane potential and sufficient availability of calcium channels for stimulation. Stimulation buffer (135K/10.8Ca HB-PS)Source:ChanTest Corp.Storage Conditions:RefrigeratedComposition (in mM):NaCl, 0; KCl, 135; CaCl2, 10.8; MgCl2, 1; HEBES, 10; Glucose, 0; pH adjusted to 7.4 with NaOHRationale:135 mM K+ is close to the maximum possible potassium concentration that can be achieved in a buffer that also includes 10 mM HEPES and 10.8 mM CaCl2, and 1 mM MgCl2 and that does not violate a quasi-physiological osmolarity of around 300m osmoles. A maximum K+ concentration is desirable to produce maximum stimulation of the expressed calcium channels. Test article carrier Name:Dimethyl sulfoxide (DMSO)Source:Sigma-AldrichStorage Conditions:Room temperatureRationale for Selection:DMSO aids in the dissolution of test articles. 1% of DMSO was included in the wells. Consumables Type:Compound plateCell plates (poly-D-lysine coated)FLIPR pipette tipsAutomated liquid handler tips Experimental methods –FLIPR TETRA TM assay. Fluid Addition Periods On the experimental day, after removal of the culture medium on a plate washer (Titertek MAP-C), 10 L/well of dye solution was added to the cells using a Multidrop (Titertek multidrop) bulk dispensing system. Cells were then incubated for 30 minutes at 37°C and 95% O2/5% CO2. In antagonist assays, 5 µL/well of antagonist compound dissolved in dye incubation buffer were added to the existing dye solution in the plates using the pipettor onboard the FLIPR. Plates were then preincubated in dye solution plus test compound for an additional 30 minutes at ambient temperature, before 25 µL/well of stimulation buffer addition activated the IC and the fluorescent response was measured. The stimulation buffer contained test article or control article concentrations that were equal to the test or control article concentrations in the wells. The test compound preincubation period was omitted in buffer assays and buffers of varying compositions were added after 60 minutes in dye solution. Data acquisition: Fluorescence changes due to ion channel activation and modulation by test articles were recorded by the FLIPR instrument (FLIPRTETRA, MDS, and Sunnyvale, CA) displayed with the FLIPR Screenworks software. Data were only recorded during the stimulus addition period, but not during the 30 minute-long preincubation period that preceded the stimulus addition. At the beginning of the stimulus addition period, a 30 second baseline period was recorded which then was followed by stimulus buffer addition to activate the ion channels. During the first 60 seconds of activation, data were acquired at the maximum rate (1 sample/second) and for the remaining 5 minutes, data were sampled every 5 seconds. Data Analysis: Data were stored on the ChanTest computer network (and backed-up nightly) for off-line analysis. Data acquisition was performed via the Screenworks software that is supplied with the FLIPR System (Molecular Devices, Union City, Ca) and data were analyzed using Screen works build-in functions for corrections and data reductions, Microsoft Excel 2003 (Microsoft Corp., Redmond, WA), and Igor Pro 5.0 (Wavemetrics, Inc., Lake Oswego, OR). Using Igor’s built-in fitting function, concentration-response data were fitted to a Hill equation of the following form: Where Base is the response at low concentrations of test article, Max is the maximum response at high concentrations, x half is the EC50, or IC50 (half-maximal inhibitory concentration of the drug), the concentration of test article producing either half-maximal activation or inhibition, and rate is the Hill coefficient. Non-linear least squares fits were made assuming a simple one-to-one binding model. If appropriate fits were weighted by the standard deviation. No assumptions about the fit parameters were made and the fit parameters were determined by the algorithm. In addition, the z and z’ factors for the assay will be calculated (Zhang, Chung and Oldenburg 1999). Discussions-Technical challenges and relative propositions Once the validation report was summarized, based on the advantages of transiently transfected cell lines over stably transfected cell lines, few problems with transient transfected cell lines are demonstrated along with the possible suggestions. Problems and relevant suggestions The following are the identified problems and corresponding suggestions for overcoming those problems seen in transient gene expression system. Problem: Variability in results Suggestion: We could work on the scale of 1 to 5 billion cells at a time, and the transfected cells would be frozen and saved in liquid nitrogen for assays, the cell numbers from one single transfection are more than enough for finishing multi-assays. Therefore, the variations are very limited. Another method to eliminate batch to batch variation in results could be minimized for larger screening projects by placing orders from same batch. Cells are stored at −150 degrees Celsius to ensure consistent performance. Figure 7 shows calcium influx measured with FLIPRTETRA® in HEK293 cells transiently transfected with Cav1.2, beta2, alpha2delta, and Kir2.1. When calcium influx with FLIPR in HEK 293 measured cells, Panel A before 24 hours post transfection and after 48 hours post transfection have similar signal strength (RFLU at 24 hours 3500 and 6500 after 48 hours). In fact, there is enhanced signal seen in panel A, 48 hours post transfection which means that there is no degradation in the signal strength and hence, there is no variation in cell qualitatively. Figure SEQ Figure ARABIC 2: Variability in results Reference: (Oestreich 2009) Problem: Compromise with expression level Suggestion: By adjusting DNA concentrations to control expression level expression level could be maintained. As ion channel and other cell surface proteins are shown to contribute to cell toxicity, the well-expressed cells might die out during the course of stable cell line development, and cause stable cell line instability while culturing. In certain cases, transient transfection is the way to reach high-level expression for some gene targets. (Figure 8) shows that transiently and stably transfected CHO cells exhibit comparable levels of assay performance. The curves are super imposed. Hence no compromise is seen with expression level. Figure SEQ Figure ARABIC 3: Compromise with expression level Reference (Maxcyte; Brady, James 2009) Problem: Consistency Suggestion: Since the Maxcyte STXTM transfection system uses electroporation, the only two variables that have major impacts on transfection efficiency are the DNA concentration and cell density. So, as long as these two factors are well controlled, consistency should not be a concern. Problem: Cell toxicity Suggestion: The Maxcyte STXTM electroporation buffer is very close to physiological conditions, so it is not stressful to any of the types of cells so far we have used, and no toxic effects have been detected in control group (with buffer and electroporation, but without DNA). Problem: Low transfection efficiency Suggestion: This is not a problem transfection system used by ChanTest since the transfection efficiency with GFP DNA as control in HEK, CHO and 3T3 cells is usually above 90%. Figure 9 shows transiently transfected cells perform comparably to stable cells in 2XFYVE- eGFP assay. Figure SEQ Figure ARABIC 4: Low transfection efficiency Reference (Maxcyte; Brady, James 2009) Commercial conclusions Survey methodology Figure SEQ Figure ARABIC 5: Survey methodology Survey conducted under the supervision of (Brown, Keck and Smith 2009) A web- based survey was prepared using Qualtrics.com professional software. This survey is conducted to meet the needs of survey sponsors, ChanTest Corp; who is interested in understanding customer needs for Ion Channel and GPCR cell lines, especially transiently transfected cell lines and division arrested cell lines used in HTS cell based assays. A questionnaire was prepared to know what the customers’ want and how we could find the best fit for them. The main objectives of this study: a) Experimenter’s specific requirement for cell lines b) To know experimenter’s specific cell type and species preference in their protocol. c) Cell lines market trends and demographics. d) To learn customers’ profile. The questionnaire consists of 10 questions related to cell lines and 5 questions on demographics. A total of 15 multiple choice questions and two open ended two are open ended questions. The contacts with majority of respondents were by email, mainly by using a professional website called linkedin.com. Another reminder email was sent in two weeks as a final call. The data collection phase of the questionnaire was limited to 4-8 weeks. The number of respondents from contacts was challenging to achieve. The total number respondents are 80 out of 7000 contact. Some of them emailed writing their views instead of taking the survey. There are 74 surveys started and 70 completed. Data was analyzed on line using www.Qualtrics.com web analysis tools to sort and filter specific group responses and to download results. Data was subsequently processed and presented graphically using Microsoft Excel. The survey is completely anonymous and no personal identifying information was collected without permission. The link to the survey: https://academictrial.qualtrics.com/SE?SID=SV_3Co3XlDXO6yzFfm&SVID=Prod Survey results Responses received are from the recipients of different background globally. Huge market potential is identified for biotechnology and pharmaceutical companies as illustrated in figure 11 Figure SEQ Figure ARABIC 6: Market potential is identified for biotechnology and pharmaceutical According to the demographics area of research, 72.34% comes from drug discovery group, mainly used for HTS applications, secondary screening and profiling. (Figure 12) Figure SEQ Figure ARABIC 7 Demographics area of research Looking at the market trend, 30-40 % of respondents plan to use ion channel / GPCR cell lines in their HTS cell based assays for screening drug candidates libraries and significant number (60%) of responses from biotech companies have agreed that transient gene expression system (that ChanTest offers) could cost effective and time saving alternative to stable expression system. Top choice from respondents for Ion channel cell lines from responses is ChanTest Corp. among the other competitive businesses such as Scottish Biomedical, Millipore, Invitrogen. ChanTest Corp. has the largest offering ion channel validated cell lines of about 70 whereas Millipore has about 35 , Bsys about 10, Invitrogen and Scottish Biomedical has a small group focus on ion channel. Industry overview Biotechnology CRO products and services is 17.5% of R&D expense. (IBIS World 2009). Products sold are usually a broad range from cell lines to media and service industry carries a huge portion of this. This expense trends slightly downwards since the number of Ion channel screening labs reduced due to recession in 2008 through 2009. Major market segment in Research and Development is private sector industry, 63.7%. Of which 29.9% is Federal Government and 25.5% is non-manufacturing industries. (IBIS World Report 2009) Leading businesses in the CRO biotechnology industry belongs to drug discovery. ,[object Object],Operational/management trends within the industry: MDS predicts that there will be a drop in net revenues by 28% in CRO market for 2010, which will account for discontinued operations of about $50 million (–Pharma, Outsourcing 2010). This is due to economic pressures, enhancing merger activity and reduced access to capital. ChanTest identifies major customer group with pharma/biotech company for its Ion Channel transient gene system as drug discovery group (HTS applications), secondary Screening and profiling. Market entry strategy for ChanTest`s EZCellsTM TT 1. Creating Awareness: Ion Channel EZCells TM TT is commercially new to the market. Hence, educating researchers about its benefits is very important. Suggestions include conducting webinars, distributing flyers, posting advertisements on popular e-journals and conducting survey. Strategic partnership with instrument makers like Nanion and Sophion who develop scalable transfection system Convert replicating cell line market into EZCellsTM TT (for assumptions see Appendix A and B) Market Overview: Focus and demographics: United States and Europe are the biggest market in the world for the use of Ion Channel Cell lines in drug discovery. The total European cell-based assays market is expected to be valued at greater than $380 million by 2011. (Frost and sullivan 2004) Japan is also a key market with upcoming markets forecasted in India. The outsourcing sector is expanding at the rate of 43% per annum in India. It is expected that ten drugs discovered worldwide in the next few years will come from India. (NickTaylor 2009). $2.46 billion in 2010 is accounted only for India’s contract manufacturing. Report released from KPMG. (Taylor 2008) Psychographics: 51% researchers who use cell-based assays perform transfection. (Biocompare 2007) Trends: Customer behavior from survey suggests that researchers are always looking for less time consuming and fast transfection gene system (GEN 2008). Major players involved with producing ion channel / GPCR cell lines are mainly ChanTest, Millipore, Perkin Elmer and Invitrogen, Bsys and Scottish Biomedical. Companies involved in producing reagents, kits, tools are Applied Biosystems (Foster, California), Ambion (Austin, Texas), Bio Rad Laboratories Inc. (Hercules, California), Qiagen Inc. (Europe) and Stratagene Corp. (California). Consumable costs per data point have ranged from $1 to $4- fluxionbio.com. (Lexis Nexis 2009) FLIPR assay pricing per point is about $10, FASTPatch costs about $100-$150 and Quattro $25 - $100 depending on the channel of choice. ,[object Object],Based on number of large pharma-biotech companies, small pharma-biotech companies and academics involved in ion channel activities(testing) and the estimated budget expenses made on ion channel cell lines correspondingly, in house and outsourced; global estimate of market size for the ion channel -expressing stable cell lines is made. The total market estimate for the global Pharma & Biotech market for ion channel testing in 2009 was estimated to $106M for in house consumables, $45M for outsourced testing at fee-for-service providers and $76M for capital expense purchases on instruments. Where appropriate these markets were broken down, segmented and CAGR estimates for 2011 made. (HTStec, 2009) Break down of these 2009 consumable budget for ion channel testing is shown in the figure below (HTStec 2009). 19% of this is indicated as cell lines and cell media budget. This account to $16 M of global pharma/ biotech in house ion channel cell lines and cell culture media. Estimation for the global pharma/ biotech market size for ion channel expressing stable or replicating cell line. Cell formats arranged based on extent of usage by customers from most widely used to the least: stable transfected cell lines > transient transfected > cryopreserved > division arrested. Therefore, global pharma/ biotech in house consumable budget market for ion channel stable cell lines estimated is $ 10 M of the $16 M from global in house ion channel cell lines and cell culture media. Pricing proposition: Pricing is based on per well cost and number of cells in each vial-transiently transfected ion channel cells. 6 million cells per vial; plate at ~15,000 cells per well of 384-well plate All price per well based on 384-well plate $599 per vial for one vial (Removed due to proprietary information) Pricing is still in debate, with suggestions for increasing it to $1000-$1500/vial based on benefit per cost and competitive pricing over stable cell lines.(Appendix C) Business proposition ChanTest Corp. acquires 12% of total pharma/biotech market for ion channel stable cell line and this is expected to grow up to 17% in 2011. The analyses reveal that the addressable market for Ion Channel EZCellsTM TT is $4.5 M at proposed pricing of $1000/vial. This implies that the required unit sales should be about 2250 vials per year (Appendix A). The target drug discovery market for Ion Channel EZCellsTM TT are secondary screening and profiling applications with large market potential in biotech and pharmaceutical companies and academics. Appendices Appendix A: Estimated production cost for 2250 vials Appendix B: Globally projected estimation of units sold per year. Appendix C: Comparison of expenses (stable vs. transient) The table below compares the cost of using EZCellsTM TT against replicating cell lines, depending on the expenses incurred during users’ research Bibliography Benjamin, ER, et al. quot;
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