Name: a strategy to identify compounds that affect cell growth and survival in cultured mammalian cells at low-to-moderate throughput
Uploaded: 2019-09-22T13:00:00
Description: Watch this Scientific Journal Video about A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput at JoVE.com

This work was supported by the NINDS Intramural Research Program.

1. NSC culture
NOTE: Manipulation of a human NSC line will be described below but any cell line can be used for this protocol. All cell culture work is performed in a biological safety cabinet.
<ol>
	<li>Coat a 96-well plate with basement membrane/extracellular matrix (ECM).
	<ol>
		<li>Thaw aliquot of ECM (Table of Materials), which will facilitate attachment of NSC, on ice. Dilute ECM to the appropriate concentration (generally 1:100) in 10 mL base medium (Table of...

<ol>
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The primary goal of this article was to describe a strategy that could efficiently and inexpensively identify compounds affecting cell growth in a low- to moderate-throughput screening. Two orthogonal techniques were utilized to assess cell number to increase confidence in the conclusions and offer additional insights that would not be available if only a single assay was used. One of the assays used a fluorescent cell imager to directly count tdTomato-positive cells and the second was dependent on the well-characterized...

One of the most important characteristics that needs to be determined for a chemical compound that has therapeutic potential is its toxicity to animal cells. This characteristic will determine whether a drug is a good candidate for more extensive study. In most instances, compounds with minimal toxicity are sought but there are situations in which a compound with the capacity to kill specific cell types is of interest, e.g., anti-tumorigenic drugs. Although whole animals are the best model systems to determine systemic toxicity, the cost and labor involved is prohibitive when more than a few compounds need to be tested. As such mammalian cell culture is generally used as the most efficient alternative1,2. Small to medium throughput drug screens are an important modality through which toxicity can be assessed in cell culture. These screens can be used to interrogate annotated libraries targeting individual signaling pathways. The general format of such a screen is to initially test all the compounds in the library at a single dose (generally 10 &#181;M) in an exploratory primary toxicity screen, and then perform an in-depth secondary dose response screen to fully characterize the toxicity profile of hits from the primary screen. The methods to implement this strategy will be described here and provide a quick, efficient, and inexpensive way to identify and characterize toxic compounds.
Multiple methods have been developed to assess cytotoxicity of small compounds and nanomaterial in mammalian cells3,4. It should be noted that certain materials can interact with the assay providing misleading results, and such interactions should be tested when characterizing hits from toxicity screens4. Cytotoxicity assays include trypan blue exclusion5, lactate dehydrogenase (LDH) release assay6, Alamar blue assay7, calcien acetoxymethyl ester (AM)8, and the ATP assay9. All these assays measure various aspects of cell metabolism which can serve as a proxy for cell number. While all offer benefits, tetrazolium salt-based assays such as 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis(2-methoxy-4-nitro-5-sulfopheny)-2H-tetrazolium-5-carboxyanilide inner salt (XTT)-1, and 4-(3-[4-Iodophenyl]-2-[4-nitrophenyl]-2H-5-tetrazolio)-1,3-benzene disulfonate (WST-1)10,11 provide good accuracy and ease of use at low cost. MTT, which will be used in this demonstration, is reduced to an insoluble formazan by a mitochondrial reductase and the rate of this conversion correlates strongly with cell number. This assay has been routinely utilized at both a small scale and for screening libraries with up to 2,000 compounds12. Direct counting of cells by a labeled marker offers another method to assess the cellular number, and unlike the MTT assay it can provide additional information about the dynamics of cellular growth. Several publicly available algorithms are available to perform automated cell count analyses and there are also proprietary algorithms that are part of software packages for imaging readers13,14. In this method description, a human neural stem cell (NSC) line that has been genetically edited to constitutively express tdTomato15 will serve as a test line to compare cellular viability results between an MTT assay and an automated cell counting assay in a screen assessing toxicity of 57 test compounds. Although the primary goal of this strategy was to identify and characterize toxic compounds, it has the additional benefit of potentially identifying growth inhibitory and growth enhancing compounds and thus provides an effective method for identifying drugs that can modulate cellular growth.

<table border="0" cellpadding="0" cellspacing="0" style="width:1292px;" width="1292">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B-27 (50X)</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">17504001</td>
			<td style="width:720px;">Neural stem cell medium component.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BenchTop pipettor</td>
			<td style="width:237px;">Sorenson Bioscience</td>
			<td align="right" style="width:119px;">73990</td>
			<td>Provides ability to pipette compound library into a 96-well plate in one shot.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BioLite 96 well multidish</td>
			<td style="width:237px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">130188</td>
			<td>Any 96 well cell culture plate will work.&#160; We use these in our work.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture microscope</td>
			<td style="width:237px;">Nikon</td>
			<td style="width:119px;">Eclipse TS100</td>
			<td>Visual inspection of cells to ensure proper density.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytation 5/ Imaging reader</td>
			<td style="width:237px;">BioTek</td>
			<td style="width:119px;">CYT3MFV</td>
			<td>Used for cell imaging and absorbance readings.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:237px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">610420010</td>
			<td>Solvent for compounds used in screen. Dissolves MTT precipitates to facilitate absorbance measurements.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FGF-basic</td>
			<td style="width:237px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>Neural stem cell medium component.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GelTrex</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td style="width:119px;">A1413202</td>
			<td>Neural stem cell basement membrane matrix.&#160; Allows cells to attach to cell culture plates.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gen5 3.04</td>
			<td style="width:237px;">BioTek</td>
			<td style="width:119px;"></td>
			<td>Analysis software to determine cell counts for tdTomato expressing cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamine</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">25030081</td>
			<td>Neural stem cell medium component.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microtest U-Bottom</td>
			<td style="width:237px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">3077</td>
			<td>Storage of compound libraries.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td style="width:119px;">M6494</td>
			<td>Active assay reagent to determine cellular viability.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multichannel pippette</td>
			<td style="width:237px;">Rainin</td>
			<td style="width:119px;">E8-1200</td>
			<td>Column-by-column addition of cell culture medium, MTT, or DMSO.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal medium</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">21103049</td>
			<td>Neural stem cell base medium.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RFP filter cube</td>
			<td style="width:237px;">BioTek</td>
			<td align="right" style="width:119px;">1225103</td>
			<td>Filter in Cytation 5 used to image tdTomato expressing cells.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TrypLE</td>
			<td style="width:237px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">12605036</td>
			<td>Cell dissociation reagent.</td>
		</tr>
	</tbody>
</table>

a strategy to identify compounds that affect cell growth and survival in cultured mammalian cells at low-to-moderate throughput

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug screening assays utilize cytotoxicity as one of the critical characteristics to be profiled for individual compounds. Cells in culture are a useful model to assess cytotoxicity before proceeding to follow up on promising lead compounds in more costly and labor-intensive animal models. We describe a strategy to identify compounds that affect cell growth in a tdTomato expressing human neural stem cells (NSC) line. The strategy uses two complementary assays to assess cell number. One assay works via the reduction of 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a proxy for cell number and the other directly counts the tdTomato expressing NSCs. The two assays can be performed simultaneously in a single experiment and are not labor intensive, rapid, and inexpensive. The strategy described in this demonstration tested 57 compounds in an exploratory primary screen for toxicity in a 96-well plate format. Three of the hits were characterized further in a six-point dose response using the same assay set-up as the primary screen. In addition to providing excellent corroboration for toxicity, comparison of results from the two assays may be effective in identifying compounds affecting other aspects of cell growth.

The automated cell count data identified eleven compounds with less than 25% viability when normalized to the DMSO control while the MTT data identified these same compounds plus two additional ones (Table 1 and Table 2, shaded red). The two compounds found to be toxic only in the MTT assay (wells F3 and G10) had 31% and 39%, respectively, the number of tdTomato-positive cells as the control and by rank order were the next two most toxic compounds in this library after those deemed to be...

Watch this Scientific Journal Video about A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput at JoVE.com

A Strategy to Identify Compounds that Affect Cell Growth and Survival in Cultured Mammalian Cells at Low-to-Moderate Throughput

It is often necessary to assess the potential cytotoxicity of a set of compounds on cultured cells. Here, we describe a strategy to reliably screen for toxic compounds in a 96-well format.

Cytotoxicity is a critical parameter that needs to be quantified when studying drugs that may have therapeutic benefits. Because of this, many drug ...

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