Name: sample preparation strategies for mass spectrometry imaging of 3d cell culture models
Uploaded: 2014-12-05T13:00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models at JoVE.com

This work was supported by the Walther Cancer Foundation under the Advancing Basic Cancer Research program to the Harper Cancer Research Institute of the University of Notre Dame (DARW) and the 2011 Starter Grant from the Society for Analytical Chemists of Pittsburgh (ABH). Thanks also to the staff of the Mass Spectrometry and Proteomics Facility for useful discussions.

1. 3D Cell Culture and Preparation for Imaging
<ol>
	<li>Culture an appropriate cell line, i.e., HCT116 colon carcinoma cell line, in two dimensional, monolayer culture.</li>
	<li>Grow HCT 116 cells in a T-25 flask with McCoy&#39;s 5A media + 10% fetal bovine serum in a 5% CO2 incubator until 50-70% confluency, which generally takes a few days.</li>
	<li>Release cells with 0.25% trypsin-EDTA solution and count a portion of the cells using a hemocytometer and trypan blue staining to check for cell density and viability.</li>
	<li>After counting, dilute cells with complete medium to achieve an appropriate seeding density of 6,000 cells/well, or 30,000 cells/ml.</li>
</ol>
2. Prepare Agarose Coated 96 Well Plates
<ol>
	<li>Add 0.19 g of agarose to 10 ml of standard media in a 50 ml conical tube (1.5% agarose solution in media) 10. Ensure incorporation by gentle mixing. Autoclave the agarose in a water bath for 20 min.</li>
	<li>Using a multichannel pipette, aspirate 50 &#181;l of the agarose + media mixture into the 60 central wells of the flat bottomed 96 well plate culture plate. As the wells at peripheral edges of the plate have slightly higher evaporation rates, add 200 &#956;l 1x phosphate buffered saline (PBS) instead of the agarose to these edges.</li>
	<li>Once the agarose mixture has been added to the inner wells, set the plate aside to cool to ~37 &#176;C before the addition of the cells.</li>
</ol>
3. Seed and Tend the Three-dimensional Culture
NOTE: More or fewer cells may be seeded depending on the requirements of the culture involved, but it has been determined that these conditions result in 3D cell culture structures of approximately 1 mm in diameter after 10-14 days of culture (data not shown).
<ol>
	<li>Add 200 &#956;l of the appropriate cell solution (diluted to contain ~6,000 cells) to the agarose-coated 96 well plate. Cover the plate and set in humidified incubator with 5% CO2 at 37&#160;&#176;C for cell growth.</li>
	<li>Change cell media a minimum of every 48 hr, or when the media appears to be changing color.
	<ol>
		<li>Change media by carefully aspirating the old media from around the small 3D cell culture (visible to the naked eye by day 2) at the bottom of the agarose. Add 200 &#181;l of fresh media gently over the 3D cell culture.</li>
		<li>(Optional step) During these media changes, add chemotherapeutics or other drugs for testing. Dilute the drug of choice to the final concentration with complete media and add the requisite 200 &#181;l to each well.</li>
		<li>Monitor 3D cell culture growth by optical microscope, until the 3D cell cultures are approximately 1 mm in diameter.</li>
	</ol>
	</li>
</ol>
4. Harvest the 3D Cell Cultures
<ol>
	<li>Use a 2 ml serological pipette to gently remove the 3D cell culture from the agarose bed.</li>
	<li>Wash the 3D cell cultures with PBS by gently pipetting them into approximately 1 ml PBS in a waiting 24 well plate.</li>
	<li>Transfer harvested 3D cell cultures via serological pipet to centrifuge tubes for protein or metabolite extraction, or embed them in a cutting medium to assist in slicing for imaging applications.</li>
</ol>
5. Embed the 3D Cell Cultures in Gelatin
<ol>
	<li>Prepare a solution of ~175 mg/ml gelatin in high purity water (18 M&#8486; preferred).22,23 Mix the gelatin vigorously. Observe the solution becoming viscous and difficult to manipulate. Place the gelatin-containing conical tube in a water bath at ~60 &#176;C and warm until the solution becomes clear and easy to aspirate. 
	NOTE: The warm gelatin solution hardens rapidly as it cools, so perform all of the following steps rapidly before freezing.</li>
	<li>After it is heated, take the gelatin immediately to the cell culture hood. Keep the tube with gelatin in a beaker with preheated water at about 60 &#176;C to avoid hardening of the gelatin solution.</li>
	<li>Using a 2 ml serological pipette, deposit 0.6 ml of the warm gelatin solution into the well of a 24 flat-bottomed plate. This volume is sufficient to cover the bottom in a thin layer. Gently deposit the 3D cell cultures immediately on top of the gelatin layer using a different 2 ml pipette to avoid rupturing the 3D structure. 
	NOTE: Care must be taken at this stage not to place PBS into the gelatin. If this happens, however, remove the PBS from the top of the gelatin using a 200 &#956;l pipette.</li>
	<li>After the 3D cell cultures are placed on the surface of the gelatin layer, carefully pipette another 0.6 ml of the warm gelatin mixture over the 3D cell cultures, so as to not disturb the position of the cultures. After the second layer is placed, freeze the gelatin-embedded 3D cell cultures at -80&#160;&#176;C.</li>
</ol>
6. Slice and Thaw Mount the Gelatin-embedded 3D Cell Cultures for Mass Spectrometric Imaging
<ol>
	<li>Remove the 24 well plate containing the 3D cell cultures from the freezer. Warm the bottom of the plate with the heat from your hand until a tweezer or scalpel will slide gently down the side of the well.</li>
	<li>Smoothly slide the tweezer or scalpel, freeing the gelatin without thawing the center. Take a drop of water to the cryostat support and use this to adhere the gelatin disk to the support.
	<ol>
		<li>3D cell cultures embedded in gelatin are most amenable to slicing at -30&#160;&#176;C. Clean the cryostat blade and glass with 70% ethanol before use. Use a different part of the blade or a new blade for each 3D cell culture-gelatin disc to prevent dulling of the blade.</li>
	</ol>
	</li>
	<li>Using the cryostat, gently face off the excess gelatin until the 3D cell cultures become visible.</li>
	<li>At this point slowly slice the 3D cell cultures with a smooth motion in 12-16 &#956;m sections. 
	NOTE: Critical factors to slicing include the distance of the glass to the blade, the blade angle, the temperature of the cryostat, and the angle of the disk on the support, so all of these must be checked to ensure consistent results.</li>
	<li>Carefully thaw the slices and mount them on indium titanium oxide (ITO)-coated glass slides labeled appropriately and store it in a desiccator. Use the slices as quickly as possible for maximum quality.</li>
</ol>
7. Matrix Application and Sample Preparation for MALDI Imaging
<ol>
	<li>Prior to MALDI imaging, prepare slices with the appropriate matrix.
	<ol>
		<li>For small molecule analysis, no washing steps are generally required. For intact protein detection, wash the slices in cold 100% acetone, Carnoy&#39;s fluid, or 70%/95% isopropanol to remove small molecules such as lipids that could mask the signal.24&#8211;26</li>
		<li>Gently wash for no more than 30 sec at a time. Do not to disturb any slices.</li>
	</ol>
	</li>
	<li>Prepare matrix in a solution of organic solvent and water, with 0.1% TFA. For small molecule such as &#945;-Cyano-4-hydroxycinnamic acid (CHCA), use 60% ACN:40% H2O:0.1% TFA (10 mg/ml). For protein detection such as sinapic acid (30 mg/ml), use the same solvent solution. For optimal solubility of the sinapic acid, dissolve first in acetonitrile before addition of the TFA:H2O solution. Other matrices may be used as appropriate. 
	NOTE: Matrix can be applied with several different methods, but it should be noted some matrixes have been found to co-crystalize with the gelatin, such as ferulic acid, rendering the sample unusable. Smaller crystals produce more consistent mass spectra, allowing for more molecular species to be detected and attributed to biological differences rather than matrix effects.</li>
	<li>Apply matrix by handspotting method.
	<ol>
		<li>Use a fine-tipped syringe to apply 0.5 &#181;l of the matrix solution on the 3D cell culture slice and allow the matrix to crystalize.</li>
	</ol>
	</li>
	<li>Alternatively, use the airbrush method. Fill a commercial grade airbrush with the matrix solution.
	<ol>
		<li>Hold the sprayer 8-12 inches away from the slide, aim a fine spray at the slice. In between passes, allow the matrix to dry for 1 min to allow for the best crystal formation. Up to 10-20 passes of the airbrush is required to produce an optimal layer of matrix22.</li>
	</ol>
	</li>
	<li>Alternatively, use the sublimation method detailed elsewhere.25,27</li>
	<li>Dry slides in a desiccator for at least 30 min prior to MALDI-MSI, regardless of the method of matrix application.25</li>
</ol>
8. MALDI-MSI Analysis Using a MALDI-TOF System (i.e.,&#160;AutoFlex III)
NOTE: This section contains instructions and advice for setting parameters for a MALDI-TOF imaging experiment. Depending on the instrument manufacturer and software used, the process may vary.
<ol>
	<li>Fit the slides in an adapter made to securely hold 75 mm x 25 mm slides to image the 3D cell culture slices attached to ITO coated slides. Load the slide adapter into the instrument.</li>
	<li>Prepare the instrument for MALDI-MSI by selecting an appropriate calibration standard for the mass range in question. For interrogation of small molecules, the signals detected that correspond to the matrix used, &#945;-CHCA, are one common group of calibrants. For analysis of proteins, known protein standards (i.e., ubiquitin, trypsinogen) in the mass range of interest are selected and spotted adjacent to the 3D cell cultures as calibrants. Calibrate the instrument before proceeding with method development.</li>
	<li>Prior to imaging, develop data acquisition methods for the mass range of choice using the software provided by the instrument manufacturer. See Figures 1 and 2 for small molecule and protein imaging. For small molecule imaging, use reflectron mode for the highest mass resolution.
	<ol>
		<li>Adjust the mass range for small molecule data acquisition between 100-1,000 m/z and for proteins between 8,000- 25,000 m/z. Adjust the mass range to encompass the region of choice while providing the highest mass resolution possible with the instrument.</li>
		<li>Adjust laser power, frequency, number of laser shots and spot size using the calibration solution and a nearby &#39;sacrificial&#39; 3D cell culture slice. Observe these settings in the main instrument control software. Use the following settings as a recommended starting point: 60% power, 400 laser shots, ultra small spot size. These settings will vary across instruments and methods, so optimize instrument parameters to give the highest quality spectra for each specific instrument. Other parameters that can be adjusted include the detector gain, sample rate, and electronic gain. Save this method for use in the imaging software (i.e., AutoExecute Method that FlexImaging will draw upon). 
		NOTE: Suppress ions below the mass range of interest (again found in the instrument control) so as to not interfere with detection.</li>
		<li>Develop protein data acquisition methods for the mass range of choice. Follow the same steps outlined above, but for the mid-to-high mass ranges, above about 3 kDa, use linear mode. 
		NOTE: Settings will differ from those used for small molecule methods.</li>
	</ol>
	</li>
	<li>Setup specific MS instrument parameters using the imaging software provided by the instrument manufacturer, such as FlexImaging for Bruker MALDI-TOF systems.
	<ol>
		<li>Create a new imaging file and folder, using the methods developed prior and saved. Select the instrument parameters such as raster spot size (change from the default about 200 &#181;m to 50 &#181;m), set up the instrument control method (setup in step 8.3.2 or 8.3.3), and position where to scan over the 3D cell culture.</li>
		<li>Select three appropriate teach points on the sample, moving the laser to each in turn. Obtain the optical photograph from the MALDI-TOF laser control software using &#39;print screen&#39;. 
		NOTE: Many imaging software programs require an optical photograph of the sample to &#39;teach&#39; the software where the laser is located, which can be obtained with a scanner or often the instrument camera.</li>
	</ol>
	</li>
	<li>Then start the imaging process from the imaging software (not the instrument control) and acquire mass spectra from each slice. Observe most masses across the 3D cell culture and others localized to particular areas.</li>
</ol>
9. Optional Data Analysis Methods
<ol>
	<li>For targeted analysis, perform manual analysis of the spectra by clicking a mass in the mean spectrum, or allow the imaging software to automatically pick masses above a particular threshold (e.g., peaks above the top 20% threshold). For quality control, examine the distribution of matrix peaks or the mean spectrum.</li>
	<li>Perform statistical analyses with extracted datasets in mathematical software such as R or Matlab.
	<ol>
		<li>Export single spectra in ASCII format, a readable text file, for loading into the software of choice.</li>
		<li>Convert the individual spectra to ASCII , mzXML, of mzML format for reading by several different software programs,28&#8211;31 or export the data as an Analyze (.img) format file, a common format used for other imaging applications such as MRI.32 Two open-source options for analysis are MSiReader and BioMap32,33.</li>
		<li>Once the files are exported into a format readable by the software, load the dataset via the instructions in the respective software. After loading, perform post-acquisition processing, such as baseline removal and normalization.</li>
		<li>With this dataset, perform statistical treatments such as principal component analysis of hierarchical clustering using either custom scripts or built in algorithms in software such as Matlab18.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Schober, Y., Guenther, S., Spengler, B., R&#246;mpp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+matrix-assisted+laser+desorption/ionization+mass+spectrometry+imaging.">Single cell matrix-assisted laser desorption/ionization mass spectrometry imaging.</a> Anal Chem. 84 (15), 6293-6297 (2012).</li><li>Cornett, D., Frappier, S., Caprioli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-FTICR+imaging+mass+spectrometry+of+drugs+and+metabolites+in+tissue.">MALDI-FTICR imaging mass spectrometry of drugs and metabolites in tissue.</a> Anal Chem. 80 (14), 5648-5653 (2008).</li><li>Reyzer, M. L., Hsieh, Y., Ng, K., Korfmacher, W. a., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+drug+candidates+in+tissue+by+matrix-assisted+laser+desorption/ionization+mass+spectrometry.">Direct analysis of drug candidates in tissue by matrix-assisted laser desorption/ionization mass spectrometry.</a> J Mass Spectrom. 38 (10), 1081-1092 (2003).</li><li>Reyzer, M. L., Chaurand, P., Angel, P. M., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+molecular+analysis+of+whole-body+animal+tissue+sections+by+MALDI+imaging+mass+spectrometry.">Direct molecular analysis of whole-body animal tissue sections by MALDI imaging mass spectrometry.</a> Methods Mol Biol. 656 (18), 285-301 (2010).</li><li>Cornett, D., Reyzer, M., Chaurand, P., Caprioli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI+imaging+mass+spectrometry:+molecular+snapshots+of+biochemical+systems.">MALDI imaging mass spectrometry: molecular snapshots of biochemical systems.</a> Nat Methods. 4 (10), 828-833 (2007).</li><li>Guenther, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology+by+mass+spectrometry:+label-free+tissue+characterization+obtained+from+high-accuracy+bioanalytical+imaging.">Histology by mass spectrometry: label-free tissue characterization obtained from high-accuracy bioanalytical imaging.</a> Angew Chem Int Ed Engl. 49 (22), 3834-3838 (2010).</li><li>Schwamborn, K., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+by+mass+spectrometry--looking+beyond+classical+histology.">Molecular imaging by mass spectrometry--looking beyond classical histology.</a> Nat Rev Cancer. 10 (9), 639-646 (2010).</li><li>Kunz-Schughart, L. a., Kreutz, M., Knuechel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+spheroids:+a+three-dimensional+in+vitro+culture+system+to+study+tumour+biology.">Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology.</a> Int J Exp Pathol. 79 (1), 1-23 (1998).</li><li>Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., Kunz-Schughart, L. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+an+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: an underestimated tool is catching up again.</a> J Biotechnol. 148 (1), 3-15 (2010).</li><li>Friedrich, J., Seidel, C., Ebner, R., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid-based+drug+screen:+considerations+and+practical+approach.">Spheroid-based drug screen: considerations and practical approach.</a> Nat Protoc. 4 (3), 309-324 (2009).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat Methods. 4 (4), 359-365 (2007).</li><li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130 (4), 601-610 (2007).</li><li>Fernandes, T. G., Kwon, S. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+microarray+for+high-throughput+studies+of+stem+cell+fate.">Three-dimensional cell culture microarray for high-throughput studies of stem cell fate.</a> Biotechnol Bioeng. 106 (1), 106-118 (2010).</li><li>Meli, L., Jordan, E. T., Clark, D. S., Linhardt, R. J., Dordick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+a+three-dimensional,+microarray+environment+on+human+cell+culture+in+drug+screening+systems.">Influence of a three-dimensional, microarray environment on human cell culture in drug screening systems.</a> Biomaterials. 33 (35), 9087-9096 (2012).</li><li>Weaver, E. M., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mass+spectrometry:+From+tissue+sections+to+cell+cultures.">Imaging mass spectrometry: From tissue sections to cell cultures.</a> Adv Drug Deliv Rev. 65 (8), 1039-1055 (2013).</li><li>Li, H., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mass+spectrometry+of+three-dimensional+cell+culture+systems.">Imaging mass spectrometry of three-dimensional cell culture systems.</a> Anal Chem. 83 (22), 8794-8801 (2011).</li><li>Liu, X., Weaver, E. M., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Therapeutics+in+Three-Dimensional+Cell+Culture+Systems+by+MALDI+Imaging+Mass+Spectrometry.">Evaluation of Therapeutics in Three-Dimensional Cell Culture Systems by MALDI Imaging Mass Spectrometry.</a> Anal Chem. 85 (13), 6295-6302 (2013).</li><li>Ahlf, D. R., Masyuko, R. N., Hummon, A. B., Bohn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+Mass+Spectrometry+Imaging+and+Confocal+Raman+Microscopy+for+Studies+of+Three-Dimensional+Cell+Culture+Sections.">Correlated Mass Spectrometry Imaging and Confocal Raman Microscopy for Studies of Three-Dimensional Cell Culture Sections.</a> Analyst. 139 (18), 4578-4585 (2014).</li><li>Derda, R., Tang, S. K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multizone+paper+platform+for+3D+cell+cultures.">Multizone paper platform for 3D cell cultures.</a> PloS one. 6 (5), 18940 (2011).</li><li>Imbeault, S., Valenzuela, N., Fai, S., Bennett, S. a. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+protein+in+3D+neural+stem+cell+culture:+a+hybrid+visualization+methodology.">Localizing protein in 3D neural stem cell culture: a hybrid visualization methodology.</a> J Vis Exp. 2 (46), 2-7 (2010).</li><li>He, W., Kuang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Comparison+of+3D+and+2D+Glioma+Models+Reveals+Increased+HLA-E+Expression+in+3D+Models+is+Associated+with+Resistance+to+NK+Cell-Mediated+Cytotoxicity.">Proteomic Comparison of 3D and 2D Glioma Models Reveals Increased HLA-E Expression in 3D Models is Associated with Resistance to NK Cell-Mediated Cytotoxicity.</a> J Proteome Res. 13 (5), 2272-2281 (2014).</li><li>Chen, R., Cape, S. S., Sturm, R. M., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometric+Imaging+of+Neuropeptides+in+Decapod+Crustacean+Neuronal+Tissues.">Mass Spectrometric Imaging of Neuropeptides in Decapod Crustacean Neuronal Tissues.</a> Methods Mol Biol. 656, 451-463 (2010).</li><li>DeKeyser, S. S., Kutz-Naber, K. K., Schmidt, J. J., Barrett-Wilt, G. A., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry+of+Neuropeptides+in+Decapod+Crustacean+Neuronal+Tissues.">Imaging Mass Spectrometry of Neuropeptides in Decapod Crustacean Neuronal Tissues.</a> J Proteome Res. 6 (5), 1782-1791 (2007).</li><li>Van Hove, E. R. A., Smith, D. F., Fornai, L., Glunde, K., Heeren, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+paper+based+tissue+washing+method+for+mass+spectrometry+imaging:+localized+washing+and+fragile+tissue+analysis.">An alternative paper based tissue washing method for mass spectrometry imaging: localized washing and fragile tissue analysis.</a> J Am Soc Mass Spectrom. 22 (10), 1885-1890 (2011).</li><li>Yang, J., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Sublimation/Recrystalization+for+Imaging+Proteins+by+Mass+Spectrometry+at+High+Spatial+Resolution.">Matrix Sublimation/Recrystalization for Imaging Proteins by Mass Spectrometry at High Spatial Resolution.</a> Anal Chem. 83 (14), 5728-5734 (2012).</li><li>Seeley, E. H., Oppenheimer, S. R., Mi, D., Chaurand, P., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+protein+sensitivity+for+MALDI+imaging+mass+spectrometry+after+chemical+treatment+of+tissue+sections.">Enhancement of protein sensitivity for MALDI imaging mass spectrometry after chemical treatment of tissue sections.</a> J Am Soc Mass Spectrom. 19 (8), 1069-1077 (2008).</li><li>Gemperline, E., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-mass+spectrometric+imaging+for+the+investigation+of+metabolites+in+Medicago+truncatula+root+nodules.">MALDI-mass spectrometric imaging for the investigation of metabolites in Medicago truncatula root nodules.</a> J Vis Exp. 1 (85), 1-11 (2014).</li><li>Deutsch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mzML:+a+single,+unifying+data+format+for+mass+spectrometer+output).">mzML: a single, unifying data format for mass spectrometer output).</a> Proteomics. 8 (14), 2776-2777 (2008).</li><li>Pedrioli, P. G. a., Eng, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+open+representation+of+mass+spectrometry+data+and+its+application+to+proteomics+research.">A common open representation of mass spectrometry data and its application to proteomics research.</a> Nat Biotechnol. 22 (11), 1459-1466 (2004).</li><li>Lin, S., Zhu, L., Winter, A., Sasinowski, M., Kibbe, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+mzXML+good+for.">What is mzXML good for.</a> Expert Rev Proteomics. 2 (6), 839-845 (2005).</li><li>Orchard, S., Hoogland, C., Bairoch, A., Eisenacher, M., Kraus, H. -. J., Binz, P. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+the+data+explosion.+A+report+on+the+HUPO-PSI+Workshop.+August+2008,+Amsterdam,+The+Netherlands.">Managing the data explosion. A report on the HUPO-PSI Workshop. August 2008, Amsterdam, The Netherlands.</a> Proteomics. 9 (3), 499-501 (2009).</li><li>Robichaud, G., Garrard, K. P., Barry, J. a., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSiReader:+an+open-source+interface+to+view+and+analyze+high+resolving+power+MS+imaging+files+on+Matlab+platform.">MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform.</a> J Am Soc Mass Spectrom. 24 (5), 718-721 (2013).</li><li>Andersson, M., Groseclose, M. R., Deutch, A. Y., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mass+spectrometry+of+proteins+and+peptides+3D+volume+reconstruction.">Imaging mass spectrometry of proteins and peptides 3D volume reconstruction.</a> Nat Methods. 5 (1), 101-108 (2008).</li></ol>

This article is part of a special issue on Multimodal Pre-Clinical Imaging, sponsored by Bruker Biospin. The MALDI-TOF system used to obtain the representative results is a Bruker AutoFlex III.

Using the methods outlined above, 3D cell cultures may be grown and analyzed with MALDI-MSI. Many immortalized cell lines will form 3D structures when cultured appropriately.9,10 Care should be taken to cultivate cells that have not been passed too many times, that is, have low passage numbers. In general, cells with passage numbers of ten and lower are optimal for growing 3D cell cultures. Growing the 3D cell cultures in a traditional agarose support provides a cost effective way for research groups interested in 3D cell culture to try this system. This method utilizes few components not already in most mammalian cell culture laboratories. Careful attention must be paid to the preparation of the agarose plates for growth so that the 3D cell cultures are consistent in size and shape; some aspects that require careful attention include checking that no air bubbles are entrapped to the mixture and that a proper meniscus is formed at the bottom of each well. If the meniscus does not form properly, the cells may grow in non-spheroid shapes or in small clumps. Also, care must be taken not to place PBS into the gelatin with the spheroids. If this happens, remove the PBS from the top of the gelatin using a 200 &#956;l pipette. If cost is not a factor, ultra-low binding round bottom plates are commercially available and offer simplification of these steps. While other tissue embedding methods can also be costly and non-mass spectrometry compatible, gelatin-embedding has been shown to be both inexpensive and versatile. Preparation strategies outlined above have been optimized for 3D cell cultures to obtain the highest quality data. While gelatin-embedding has been shown to be compatible with mass spectrometry analysis, this process does narrow the available matrices due to co-crystallization. Once frozen, the 3D cell cultures are stable for months as long as they are not freeze-thawed. The gelatin becomes opaque when frozen, and the 3D cell cultures are of a similar color, so it is advisable before freezing to document the 3D cell culture placement to aid in slicing.
When preparing slides, matrix can be applied with several different methods, but it should be noted that some matrixes have been found to co-crystalize with the gelatin, such as ferulic acid, rendering the sample unusable. Smaller matrix crystals produce more consistent mass spectra. While handspotting is the simplest method of matrix application, the larger solvent volume can result in larger crystal sizes and significant analyte migration.
At the mass spectrometer, advances in MALDI-MSI technologies have reduced the expertise required for beginning analyses. Data acquisition and analysis is straightforward on most systems, allowing even a novice to obtain high quality information from ITO-coated slides. Careful selection of the instrument parameters, optimized on nearby non-image worthy 3D cell cultures, is critical to obtaining high quality data. For instance, increasing laser power may increase peak intensity but decreasing laser power may improve resolution and give more symmetrical peak shapes. 3D cell cultures should be used quickly after slicing to ensure optimal sample quality. Degradation of protein signal may be seen in less than a week without proper storage (dessicator/-80&#160;&#176;C freezer), particularly in the high mass region (above 20 kDa). Due to growing demand, many different visualization software programs have been developed and are free to the research public. Most imaging mass spectrometers have installed the software necessary to visualize single masses with the proprietary formats used on each instrument. These software programs may be used to obtain single-mass images and export the data. Most software will also allow the overlay of two or more masses of interest. Additionally, many different viewers for MSI data are free to download, such as MSiReader and BioMap.32,33 The mass spectrometry data obtained may also be processed post-acquisition with ease, bringing more useful information to the forefront.
From pharmaceuticals to lipids to proteins, the system outlined above allows for rapid acquisition of data to evaluate the distribution of molecules of interest across a 3D cell culture structure. Serial sections can be taken to image the entire 3D structure by building from each 2D image of a slice of the 3D cell culture. The techniques and notes in the protocol will be of use to researchers wishing to begin three-dimensional cell culture as a bridge between monolayer culture and animal models. Once mastered, these techniques can be applied to multiple types of 3D cell cultures, tissues, and cell cultures, allowing researchers to image whichever samples they choose.

Mass spectrometry imaging (MSI) is a powerful technique to examine the distribution of molecular species across samples of interest. Researchers have imaged biological samples from whole animals down to single cells with this technique.1&#8211;4 Using MSI hundreds of analytes may be imaged at a single time, rather than single-substrate techniques requiring the development of additional fluorophores for traditional confocal microscopy, or staining for histology.5&#8211;7 Additionally, MSI can be used in either a targeted or a discovery-based fashion, making it an attractive approach to look at the overlap between the distribution of chemical species. Presented within are methods for the growth, preparation, and matrix assisted laser desorption ionization/desorption (MALDI) imaging of small samples (~1 mm in diameter) of three-dimensional multicellular cell cultures.8&#8211;11
3D cell cultures are of particular interest to the biological community as an intermediary between traditional monolayer culture and in vivo tumors. As the name implies, 3D cell cultures are heterogeneous cellular aggregates that contain many of the chemical gradients in the microenvironment observed in small avascular tumor nodules, making them a more realistic model system to the native tumor environment than monolayer culture.10 Many human cell lines can be grown as 3D cultures, including cell lines derived from colon, breast, ovary, prostate, kidney and lung tissues.10 There are inherent challenges in imaging three dimensional cell cultures as they are smaller than many samples commonly used for mass spectrometry imaging and therefore require careful sample preparation.7 However, the advantages of these tractable model systems include lower cost and faster growth and analysis time compared to animal models. As a result, studies involving three dimensional cell cultures can be higher throughput than traditional animal studies.12&#8211;14
All of these advantages have resulted in increasing popularity for three dimensional cell cultures in biological research; there is a corresponding need to develop new analytical strategies to explore both the composition and spatial distribution of molecules in these systems.12,15&#8211;21 The goal of this manuscript is to describe the adaptation of mass spectrometry imaging methods and sample preparation to three dimensional cell cultures.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Name of Material/Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:175px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCT116 Cell line</td>
			<td>ATCC</td>
			<td>ATCC CCL-247</td>
			<td>Potential hazard, handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">McCoy&#39;s 5A media</td>
			<td>Gibco</td>
			<td>16600-108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine serum</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA solution</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin, unflavored</td>
			<td>Knox</td>
			<td>--</td>
			<td>Available at most grocery stores in single-packets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ITO-coated glass slides</td>
			<td>Delta Technologies, Limited</td>
			<td>CG-90IN-S115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Cyano-4-hydroxycinnamic acid (CHCA)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C8982</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sinapic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>85429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.3mm Gravity Feed Dual-Action Airbrush Paint Spray Gun Kit Set</td>
			<td>RoyalMax Airbrush</td>
			<td>--</td>
			<td>Many options available on sites such as Amazon.com</td>
		</tr>
	</tbody>
</table>

sample preparation strategies for mass spectrometry imaging of 3d cell culture models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

MSI imaging has the potential to reveal many different molecular distribution in 3D cell cultures. Using the method outlined above, species from small molecules (Figure 1) to large proteins (Figure 2) may be tracked across the culture. These results show visualization of a single ion with the free tool MSiReader.32 Results may be analyzed for single ions of interest across the 3D cell culture with visualization software either in a region of interest or the entire scanned region. Additionally most software will automatically visualize ions above a certain threshold set by the user, which may aid discovery efforts. Once single ion maps are obtained, most software includes an ability to overlay the images to view the colocalization of ions. Either in discovery-based approaches or in a targeted fashion, mass spectrometry imaging is a powerful tool to analyze 3D cell cultures.
<img alt="Figure 1" src="/files/ftp_upload/52313/52313fig1highres.jpg" /> 
Figure 1. Representative results of 3D cell culture growth and sectioning. Left) Optical image of an intact colorectal HCT-116 3D cell culture structure as viewed on day 14 prior to harvesting. Right) Optical image of an approximately equatorial slice of a colorectal 3D cell culture thaw mounted onto an ITO-coated slide for imaging. <a href="https://www.jove.com/files/ftp_upload/52313/52313fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52313/52313fig2highres.jpg" /> 
Figure 2. Representative results of small molecule MALDI-MSI of an equatorial section of a 3D cell culture. Top) Average mass spectra achieved with &#945;-CHCA in the low mass (small molecule) range. Bottom) Representative images of a single mass: 327.8 m/z primarily localized to the interior of the 3D cell culture and 429.7 m/z primarily localized to the edge of the 3D cell culture. Dashed line indicates the approximate edge of the spheroid. <a href="https://www.jove.com/files/ftp_upload/52313/52313fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52313/52313fig3highres.jpg" /> 
Figure 3. Representative results of protein imaging by MALDI-MSI of an equatorial section of a 3D cell culture. Top) Average mass spectra achieved with sinapic acid in the higher (protein) mass range. Bottom) Representative images of a single mass: 10,871 m/z primarily localized to the edge of the spheroid, and 12,184 m/z localized more toward the interior of the spheroid. Dashed line indicates the approximate edge of the spheroid. <a href="https://www.jove.com/files/ftp_upload/52313/52313fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models at JoVE.com

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

sample-preparation-strategies-for-mass-spectrometry-imaging-3d-cell

Research

JoVE Journal

Bioengineering

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Establishing the 3D Stromal-Endothelial Cell Co-Culture

To begin, take the pellet of the GFP-HUVECs grown to a confluence level of 80 to 100%and resuspend the cells in an appropriate volume of basal medium to achieve a concentration of one times 10 to the seventh cells per milliliter of stock solution. Prepare 50-milliliter conical centrifuge tubes containing the required volume of basal medium supplemented with the respective growth factors. Add the GFP-HUVECs from the stock solution at a dilution of one to 66.67 to achieve a concentration of 1.5 times 10 to the fifth cells per microliter.Then aspirate the medium from the plate containing the stromal monoculture and add 200 microliters per well of the prepared GFP-HUVECs suspension. Incubate at 37 degrees Celsius in 5%carbon dioxide in a humidified atmosphere, changing the medium every three to four days. Monitor the development of the culture by bright-field and fluorescence microscopy using a five times objective.Differences between the cultures could be observed from the bright-field and fluorescence images showing only the GFP-HUVECs. The cultures appeared less developed without any growth factor, However, faster development was observed in the presence of FGF-2. In contrast, the bright-field images showed the densest cultures in the presence of BMP-2.However, vascular like networks formed in both growth factor-containing conditions and the most extensive and interconnected networks were formed with FGF-2.