1. Host cell transfection
<ol>
	<li>Seed 2 x 106 CHO-DXB11 cells in T75 Corning cell culture flask in 15 ml growth medium (MEMa with nucleotides and nucleosides (Gibco, catalog number 12571) supplemented with 10% &gamma;-irradiated characterized fetal bovine serum (cFBS) (HyClone, catalog number SH30071.03) and 10 mL/L of 45% glucose solution (Sigma, catalog number G8769)) one day before transfection.</li>
	<li>At the time of transfection, prepare transfection complexes as follows:
		<ol class="lalpha">
			<li>Dilute 8 &mu;g DNA in 800 &mu;l of growth medium without serum in a sterile microcentrifuge tube. Mix gently.</li>
			<li>Dilute 40 &mu;l Lipofectamine (Invitrogen 18324012) in 800 &mu;l of growth medium without serum in a polystyrene tube. </li>
			<li>Add the diluted DNA to the diluted Lipofectamine. Mix gently and incubate for 30 minutes at room temperature. </li>
		</ol>
	</li>
	<li>Remove the growth medium from cells in T75 and replace with 6.4 ml of growth medium without serum. Add the 1.6 ml of transfection complexes to the flask. Mix gently by rocking the plate.</li>
	<li>Incubate cells at 37&deg;C in a CO2 incubator for 6 hours.</li>
	<li>Add 8 ml of growth medium to the flask and incubate at 37&deg;C in a CO2 incubator overnight.</li>
	<li>Remove medium by aspiration and add fresh growth medium to the flask. Incubate the cells at 37&deg;C in a CO2 incubator overnight.</li>
	<li>Replace the growth medium with selection medium (MEMa medium without nucleotides or nucleosides (Gibco, catalog number 12561) supplemented with 10% &gamma;-irradiated dialyzed fetal bovine serum (dFBS) (HyClone, catalog number SH30079.03) and 10 mL/L 45% glucose solution (Sigma, catalog number G8769)).</li>
	<li>Incubate the cells at 37&deg;C in a CO2 incubator for 2-3 weeks.</li>
</ol>
2. Cloning by Flow Cytometry Activated Cell sorting
<ol>
	<li>Dislodge cells from the flask and centrifuge at 800 rpm for 5 minutes at 4&deg;C.</li>
	<li>Resuspend the cells in selection medium supplemented with 2% bovine serum albumin and fluorescently labeled anti-human IgG antibody at 1:20 dilution.</li>
	<li>Incubate on ice for 15-30 minutes and then wash twice with cold PBS. Resuspend cells in 1x PBS in a polypropylene tube.</li>
	<li>Prepare for aseptic sort on a BD FACSAria. Prepare 96-well cell culture plate that contains 200 &mu;l selection medium in each well.</li>
	<li>Aseptically load the tube with cells and analyze on BD FACSAria, record the results for stained and unstained cells, respectively. </li>
	<li>Set the gates and settings to sort single cells of the desired fluorescence staining profile into 96-well plate(s). 
		<ol class="lalpha">
			<li>Transfected cells are first analyzed based on forward scatter versus side scatter. A gate for live cells is made based on forward scatter, which measures a cell's size, and side scatter, which measures a cell's complexity or granularity.</li>
			<li>The cells that fall within this gate are then examined for PE staining. PE negative gate is set for unstained transfected cells or stained host cells. As stained transfected cells are examined by FACS, the "High PE" gate is then delineated to encompass the top 5% of all cells that are positive for PE staining.</li>
		</ol>
	</li>
	<li>Incubate the plates in 37&deg;C incubator for two weeks.</li>
</ol>
3. Documentation of colony formation by CloneSelect Imager
Document colony formation in 96-well plates on CloneSelect Imager at day 0, day 3, day 7 and day 13 after FACS sorting. The image documentation will ensure selection of clones derived from a single cell. A critical step in this documentation is getting the focal plane adjusted correctly for the day 0 measurement, since there is only one cell in each well at that point. It is very important that the images of those single cells are in focus and attempting to adjust the focus on them can be misleading at best. The focal plane can vary significantly between brands of plates and possibly even from lot to lot of the same brand. Thus a pre-determined focal plane needs to be used. This can be accomplished by focusing on test micro-beads or high density cells deposited on plates from the same lot being used. Alternatively, fully grown plates from the same lot may be used for this purpose. After FACS sorting, it is also important to let the cells settle for a minimum of 2 hours prior to imaging, so that they will be 'fixed' in position on the plate.
4. Screening and selection of highly productive clones 
<ol>
	<li>Document sample position in the receiving 96-well assay plates.</li>
	<li>Transfer aliquots of 20 &mu;l supernatant (dilute if necessary) into 96-well assay plates by the TECAN Freedom EVO liquid handling system. This flexible and capable robotic platform has been adapted for this and other automated cell culture work. Special programs were written to best utilize and enhance the Freedom EVO robotic capabilities for cell line development. An example of this is setting the TECAN to interpret CloneSelect Imager data for sampling wells with single colonies. Other programs were required to perform additional cell culture tasks such as to make the appropriate dilutions of the samples taken, to interpret, catalog and sample manually marked plates if required, to scale up high producing clones, etc. Many of these programs can easily be applied to 384 well plates as well as the original 96 well format.</li>
	<li>Antibody production in the samples is analyzed by a sandwich human IgG detecting ELISA (Pierce, Rockford, IL) on the TECAN Freedom EVO liquid handling system.
		<ol>
			<li>Samples and standard (Human myeloma IgG1, kappa) (Sigma, St. Louis, MO) were loaded on pre-coated Bio Coat Anti-Human IgG Assay Plates (BD Biosciences, San Jose, CA).</li>
			<li>The plates were incubated at room temperature for two hours followed by three PBS washes.</li>
			<li>ImmunoPure Goat Anti-Human IgG, (Fc-gamma)-HRP (Pierce, Rockford, IL) was added to the plate at a 1:2000 dilution and incubated for one hour at room temperature. </li>
			<li>The plates were washed three times with PBS before adding ABTS substrate (Pierce, Rockford, IL) and were read on a plate reader using absorbance at 405 nm for detection.</li>
		</ol>
	</li>
	<li>Select highly productive clones and evaluate productivity in suspension culture. For fed-batch culture, cells were inoculated at 0.3X106 cells/mL in protein free medium JRH ImMEDIAte ADVANTAGE 65578 (SAFC Biosciences, Lenexa, KS) supplemented with 5 g/L soy hydrolysate (DMV International, catalog number SE50MAF-UF, lot number ) and incubated at 37&deg;C with 7.5% CO2. The culture continued and were supplemented with 2 g/L glucose (Sigma, catalog number G8769, lot number 098K2329) when glucose plus lactate concentration in the culture reached below 2 g/L, and fed 2 mM glutamine (Gibco, catalog number 25030, lot number 568177) when glutamine levels reached below 200 mg/L (metabolites measured by YSI Bioanalyzer). Fed-batch cultures were terminated on day 14 and antibody production was measured by reverse phase HPLC.</li>
	<li>Genetic characterization of candidate clones by fluorescence in situ hybridization (FISH)5.</li>
</ol>
5. Representative Results:
An example of developing antibody producing clones with the high throughput method is shown in Figure 1 and Figure 2. Transfected cell pool was stained with fluorescently labeled anti-human IgG antibody (Figure 1A), analyzed and sorted on BD FACSAria (Figure 1B). Colony formation in 96-well plate was imaged on CloneSelect Imager (Figure 1C). Cell culture supernatant was sampled from wells that contain single colony and assayed by ELISA on the TECAN Freedom EVO liquid handling system (Figure 1D). Highly productive clones were found from cells that exhibited higher surface antibody level reflected by staining with fluorescent labeled anti-human IgG. When comparing clones generated from two populations of the initial transfected cell pool, a vast difference in productivity in fed-batch culture was observed (Figure 2). For the top two clones generated from the cell population with high surface antibody expression (PE high), the specific productivity ranged from 50-70 pg/cell/day. Whereas the antibody productivity was ~20 pg/cell/day for the top two clones generated from the cell population with low surface antibody expression (PE low). Furthermore, cell lines developed by FACS sorting are more stable with respect to antibody production than those that were cloned through manual liming dilution (Figure 3A). The specific productivity of the top clone, "Clone 1", generated by manual liming dilution dropped from ~30 pcd to ~10 pcd after culturing for 80 cell doublings. On the other hand, the top subclone generated by FACS sorting, "Clone 2", was able to maintain the specific productivity around 20 pcd for at least 100 cell doublings. By fluorescent in situ hybridization (FISH), we demonstrated Clone 1 showed as a mixed population of phenotype A (85%) and phenotype B (15%). In contrast, FACS sorted clone (clone 2) showed to be a more homogeneous population, with 99.5% of the cells proved to be phenotype A (Figure 3B).
<img src="/files/ftp_upload/3010/3010fig1.jpg" alt="Figure 1" /> 
Figure 1. Development of manufacturing cell lines for the production of monoclonal antibodies from CHO cells. A) Surface staining of initial transfected cell pool with fluorescently labeled anti-human IgG antibody. B) Sorting of cell population with high fluorescence intensity (PE high) and low fluorescence intensity (PE low) into 96-well plate. C) Documenting colony formation from day 0 to day 13 post seeding on CloneSelect Imager. D) Screening clones by ELISA. 
<img src="/files/ftp_upload/3010/3010fig2.jpg" alt="Figure 2" /> 
Figure 2. Surface associated antibody level correlates with antibody productivity of the cell. Clones with high and low fluorescent staining were both developed into cell lines and evaluated in fed-batch culture. Volumetric titer and specific productivity were compared between top 2 clones of each population.
<img src="/files/ftp_upload/3010/3010fig3.jpg" alt="Figure 3" /> 
Figure 3. Clones generated from FACS sorting exhibit higher genetic homogeneity and clone stability. A) Antibody production stability in batch culture for ~80 cell doublings of culture elapse time. &Delta;, clone was generated through limiting dilution, where cells were seeded at 2000 cells/well, 1000 cells/well and 500 cells/well. <img src="/files/ftp_upload/3010/3010triangle.jpg" alt="Black Triangle" />, clone was generated through FACS at 1 cell/well. B) Transgene integration site in CHO cell chromosome is identified by Fluorescent in situ hybridization.

<ol>
	<li>Hung, F., Deng, L., Ravnikar, P., Condon, R., Li, B., Do, L., Saha, D., Tsao, Y. S., Merchant, A., Liu, Z., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+stability+and+antibody+production+in+CHO+cells:+improvement+through+gene+optimization.">mRNA stability and antibody production in CHO cells: improvement through gene optimization.</a> Biotechnol. J. 5, 393-401 (2010).</li><li>Brezinsky, S. C., Chiang, G. G., Szilvasi, A., Mohan, S., Shapiro, R. I., MacLean, A., Sisk, W., Thill, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+enriching+populations+of+transfected+CHO+cells+for+cells+of+higher+specific+productivity.">A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity.</a> J. Immunol. Methods. 277, 141-155 (2003).</li><li>Kromenaker, S. J., Srienc, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+producer+hybridoma+cell+lines+after+cell+sorting:+a+case+study.">Stability of producer hybridoma cell lines after cell sorting: a case study.</a> Biotechnol. Prog. 10, 299-307 (1994).</li><li>Marder, P., Maciak, R. S., Fouts, R. L., Baker, R. S., Starling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cloning+of+hybridoma+cells+for+enhanced+immunoglobulin+production+using+flow+cytometric+cell+sorting+and+automated+laser+nephelometry.">Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry.</a> Cytometry. 11, 498-505 (1990).</li><li>Henegariu, O., Heerema, N., Wright, L. L., Bray-Ward, P., Ward, D. C., Vance, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+cytogenetic+slide+preparation:+controlled+chromosome+spreading+chemical+aging+and+gradual+denaturing.">Improvements in cytogenetic slide preparation: controlled chromosome spreading chemical aging and gradual denaturing.</a> Cytomery. 43, 101-109 (2001).</li><li>Meng, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grenn+fluorescent+protein+(GFP)+as+a+second+selectable+marker+for+selection+of+high+producing+clones+from+transfected+CHO+cells.">Grenn fluorescent protein (GFP) as a second selectable marker for selection of high producing clones from transfected CHO cells.</a> Gene. 242, 201-207 (2000).</li><li>DeMaria, C. T., Cairns, V., Schwarz, C., Zhang, J., Guerin, M., Zuena, E., Estes, S., Karey, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+clone+selection+for+recombinant+CHO+cells+using+a+FACS-based+high-throughput+screen.">Accelerated clone selection for recombinant CHO cells using a FACS-based high-throughput screen.</a> Biotechnol. Prog. 23, 465-472 (2007).</li><li>Weaver, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+microdrop+technology+for+rapid+isolation+of+rare+an+high+producer+cells.">Gel microdrop technology for rapid isolation of rare an high producer cells.</a> Nat. Med. 3, 583-585 (1997).</li></ol>

The authors of this article are employees of Merck &amp; Co., Inc., who use this method for large scale production of protein therapeutics from mammalian cell lines.

We presented an automated manufacturing cell line development platform that enables isolation of highly productive clones, and in the mean while ensures clonality and clone stability. The engagement of surface antibody staining followed by FACSAria cell sorting is the key to obtaining single clones with high antibody productivity. Our results and other reports2-4 suggest that the antibody level transiently displayed on cell surface correlated well with productivity of the cell. Thus, a fluorescein-conjugated antibody can be used to recognize the membrane associated product and aid the isolation of high producers. Alternatively, coexpression of reporter genes and gel microdrop technology have been used to facilitate the selection of high-producing clones by FACS6-8. In addition to isolating high producing clones via FACS, other technologies have evolved to achieve the same goal. These include Trellis Bioscience CellSpot technology that uses microspere antibody detection systems and laser screening technology to identify highly productive clones and eliminate unwanted cells, Genetix ClonePix FL and StemCell Technologies ClonaCell EasyPick platform that perform high throughput screening of production cell lines using a fluorescent antibody detection system. We chose to implement the FACS cloning method based on surface associated antibody level because it combines the ease of operation, selectivity on productivity and clonality.
The employment of TECAN Freedom EVO liquid handling system significantly increased screening throughput, which provide the opportunity to select for high yield clones from a much larger cell pool. Other instrumentation for assay such as HTRF, BioForte Octet, HPLC, etc, are all possible means of selecting high producers and screening out low producers. By tracking clone formation with CloneSelect Imager, we generated necessary documentation to prove clonality with digitized cell line history. Clonality is further confirmed by genetically characterize single transgene integration site on the chromosome. A subcloning step, which is usually taken to ensure clonality, may thus be eliminated. This will save 4-8 weeks of time for cell line development. In summary, the quality cell lines generated by the high-throughput automated platform showed high productivity, production stability and documented cell line history that should meet demand for large-scale production and current regulatory requirements.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
		</tr>
		<tr>
			<td>BD FACS Aria&trade; Cell Sorter</td>
			<td>BD Bioscience</td>
		</tr>
		<tr>
			<td>CloneSelect&trade; Imager</td>
			<td>Genetix </td>
		</tr>
		<tr>
			<td>TECAN Freedom EVO&reg; liquid handling system</td>
			<td>Tecan Group </td>
		</tr>
	</tbody>
</table>

a high-throughput automated platform for the development of manufacturing cell lines for protein therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

Watch this Scientific Journal Video about A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics at JoVE.com

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

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22.1K Views.  Merck & Co., Inc. A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

Video: A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...