This work was supported by NIH grants R01CA174388 and U54CA143868. The authors would like to acknowledge the PURA award from the Johns Hopkins University for support of Wei-tong Chen. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1232825.

The protocol provided follows the guidelines of The Homewood Institutional Review Board (HIRB).
1. Stable Expression of H2B-mCherry as a Marker for Cell Mitosis
<ol>
	<li>Generation of lentiviral particles from human embryonic kidney 293T (HEK 293T) cells
	<ol>
		<li>Plate the HEK 293T cells on a 10 cm cell culture dish at the density of 5 x 106 cells/dish in cell culture medium (Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) containing high glucose (4.5 g/L), ...

<ol>
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The previous study of cell division in 3D was not efficient due to experimental limitations and technical challenges18,19. The critical steps for efficient study of mammalian cell division in 3D collagen matrices are: (1) the incorporation of fluorescence-labeled mitotic markers to the cells; (2) the synchronization of cell division; and (3) the monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection micro...

Cell mitosis is a critical event in cellular life, the regulation of which plays crucial roles in tissue and organ development. Abnormal mitosis is implicated in natural genetic variations, human aging processes, and the progression of cancer1,2,3,4,5. The increased rate of proliferation of tumor cells compared with normal cells is one of the hallmarks of cancer, despite the fact that cell behaviors are quite heterogeneous among different types of tumors and even among patients. In spite of promising preclinical results, some newly-developed antimitotic drugs have not shown to be effective in clinical trials6,7,8,9,10,11.The relevance of experimental and preclinical models has to be considered. Many types of normal mammalian and cancer cells divide in three-dimensional (3D) matrices, such as fibroblasts and fibrosarcoma cells in collagen I-rich 3D connective tissues, and metastatic cancer cells in the 3D stromal extracellular matrix (ECM). However, the vast majority of mammalian cell division experiments and assays have been performed on cells cultured on two-dimensional (2D) substrates. An engineered 3D matrix could better recapitulate the microstructure, mechanical properties, and biochemical signals of the 3D ECM of both normal and pathologic tissues12,13,14,15,16,17.
The study of how mammalian cell division is regulated in 3D environments remains largely unexplored despite both the physiological relevance and the therapeutic significance18,19. Possible reasons include the technical difficulties and experimental challenges associated with studying cell division in 3D matrices. Cell mitosis constitutes a small temporal fraction in the whole cell cycle20. Previous work has shown that the proliferation rate of many mammalian cells, such as human breast adenocarcinoma MCF-7, human osteosarcoma U2OS, and human liver HepG2, is much lower in 3D matrices compared with their counterparts on 2D substrates21,22. Furthermore, cells embedded in 3D matrices move in and out of focus during live-cell imaging. All of these factors contribute to the extremely low efficiency of capturing cell-division events in 3D culture using imaging techniques.
Interactions between the ECM and cells play critical roles in regulating cell divisions. Here, we describe an approach to efficiently study mammalian cell division in 3D collagen matrices. The method includes the incorporation of mitotic markers to the cells, synchronization of cell division, as well as the monitoring of division events in 3D matrices using the live-cell imaging technique, time-resolved confocal reflection microscopy, and quantitative imaging analysis. Fluorescence-labeled histone protein H2B is first introduced into the cells as a marker to differentiate mitotic and interphase cells. Then the cells are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then directly encapsulated into 3D collagen matrices. Cell division events of multiple cells are monitored efficiently using low-magnification time-lapse live-cell imaging. The deformation of collagen fibers, which is an indicator of cell-matrix interaction, is monitored using confocal reflection microscopy at high-magnification.
We have previously used this technique to monitor and quantify cell-matrix interaction before, during and after the mitosis of two metastatic cancer cell lines, human invasive ductal carcinoma MDA-MB-231 and human fibrosarcoma HT1080 cells, in 3D collagen matrices19. The methods presented here provide an efficient and general approach to study both mammalian cell division in a 3D environment and cell-matrix interactions. The MDA-MB-231 cell line is used as an example throughout the paper. This protocol provides novel insights into the molecular basis of the development of normal tissue and diseases, and could also allow for the design of novel diagnostic and therapeutic approaches.

<table>
	<tbody>
		<tr>
			<td>Human embryonic kidney 293T</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>Physical Sciences Oncology Center, NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder</td>
			<td>ThermoFisher Scientific</td>
			<td>12100-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100X</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene HD</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life technologies</td>
			<td>11668-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid encoding H2B-mCherry in a lentiviral vector</td>
			<td>Addgene</td>
			<td>plasmid 21217</td>
			<td></td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T1895</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>GibcoBRL</td>
			<td>11810-025</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>113375-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>J.T. Bake</td>
			<td>3722-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV syringe filter unit, 0.45-&#956;m, PVDF, 33 mm</td>
			<td>Millipore</td>
			<td>SLHVM33RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon TE2000E epifluorescence microscope</td>
			<td>Nikon</td>
			<td>TE2000E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade 1K CCD camera</td>
			<td>Roper Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements AR imaging software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1 confocal microscope</td>
			<td>Nikon</td>
			<td>A1</td>
			<td></td>
		</tr>
	</tbody>
</table>

mammalian cell division in 3d matrices via quantitative confocal reflection microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

The goal of this article is to present an imaging-based method to study mammalian cell division processes in 3D matrices, and to quantify the interactions between the cell and the 3D extracellular matrix during and after cell division. To facilitate the imaging of cell mitosis, we incorporated H2B-mCherry into MDA-MB-231 cells using lentiviral transduction. H2B conjugated with fluorescent proteins is used as a mitotic marker to distinguish mitotic cells from interphase cells, and to defin...

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Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

The overall goal of this procedure is to study mammalian cell division and cell matrix interactions in a physiologically relevant 3D environment. The main advantage of this technique is that it provides an efficient and general approach to study mammalian cell division and cell matrix interactions. This method can help answer key questions in cell biology such as the molecular basis of the development of normal and disease tissue.A day prior to transfection, plate five million HEK-293 T-cells in a 100 milliliter cell culture dish in 10 milliliters of normal cell culture medium. Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day just before starting transfections, remove a vial of the transfection reagent from four degrees Celsius and leave it out to reach room temperature.Then to start, add one milliliter of reduced serum medium to a sterile microfuge tube. To this, add specified amounts of lentiviral vector plasmids used for transfection and mix by pipetting. Incubate the tube at room temperature for five minutes.After five minutes, add 48 microliters of the transfection reagent into this tube and mix gently by pipetting. Subsequently, incubate this mixture at room temperature for at least 15 minutes. Once the incubation is complete, remove the plate with HEK-293 T-cells from the incubator.Add the prepared DNA transfection reagent mixture drop wise onto the cells and gently swirl the plate to ensure even distribution in the plate. Place the plate back into the incubator. Approximately six hours after transfection, remove the plate and aspirate the medium.Add 10 milliliters of fresh culture medium and transfer the plate back into the incubator. At multiple time points, 24, 48, and 72 hours post-transfection, harvest the culture medium containing the lentiviral particles in separate tubes. Next, filter the harvested culture medium through a sterile 0.45 micron filter to remove cellular debris.Replace with fresh culture medium following each harvest. The filtrate containing the lentiviral particles can be used for transduction right away or can be stored at minus 80 degrees Celsius. Plate human breast carcinoma cells MDA-MB-231 in a 35 millimeter culture dish in normal culture medium.Incubate these cells at 37 degrees Celsius for 24 hours in a humidified incubator with 5%carbon dioxide. The next day, aspirate the culture medium from the plate and add one milliliter of the lentiviral particles together with one milliliter of fresh culture medium. Incubate the cells in the incubator for about 16 hours.After the incubation with viral particles is complete, remove the plate to aspirate the medium. Then add two milliliters of fresh medium and transfer back to the incubator for 24 to 72 hours. Check the expression of H2B-mCherry in these transduced cells using an epifluorescence microscope.To start the synchronization experiments, first plate MDA-MB-231 cells expressing H2B-mCherry at 50 to 60%confluency in a 24-well plate. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, replace the culture medium with 0.5 milliliters of medium containing two millimolar Thymidine.Leave the plate in the incubator for 24 hours. Once the incubation is complete, wash the cells three times with PBS to release them from the Thymidine treatment. Then leave the cells in 0.5 milliliters of normal culture medium for five hours in the incubator.After this, to block these cells in mitosis, add Nocodazole to the culture medium. Leave the cells in the incubator for 12 hours. After the Nocodazole treatment is complete, take out the plate and using an orbital shaker, shake the cells for 45 seconds to one minute to release mitotic cells.Subsequently, transfer the medium containing the extracted mitotic cells into a new centrifuge tube. Then add 0.5 milliliters of fresh medium to each well. After, repeat the shaking and extraction of cells two more times.Pool the total collected medium and then centrifuge it to pellet the mitotic cells. After the spin, resuspend the cell pellet in 0.25 milliliters of fresh cell culture medium. Using a hemocytometer, count the number of cells.At the start of this step, prepare 50 milliliters of 10x DMEM solution and filter sterilize it. Next, determine the volumes of all the components that will be used to make the 3D collagen matrix as specified in the text protocol. Pre-chill all these components on ice to slow down the gelation of collagen.Now, to a pre-chilled 1.5 milliliter centrifuge tube, add the required numbers of cells in medium then 10x DMEM, FBS, deionized water, collagen, and finally sodium hydroxide. Mix carefully using a one milliliter pipette. Once the solution is well mixed, add 500 microliters of the mixture into each well of the 24-well plate.Place the 24-well plate in a 37 degree Celsius incubator. After 30 minutes, take out the plate and add 500 microliters of pre-warmed culture medium on top of the gel. Prior to imaging, turn on the live cell units of the phase contrast and confocal microscopes.To capture dividing cells, place the 24-well plate containing the cells embedded in collagen matrices into the live cell unit of the fluorescence microscope. Using the 10X objective, find the bottom of the plate and then move it up until the microscope focuses at about 500 micrometers from the bottom of the plate. Move the stage around to find cells in different stages of mitosis and select multiple positions for imaging.Set up the timelapse experiment with two-minute intervals. Next, to image the collagen network, configure the confocal microscope to capture only reflected light from the 488 nanometer laser. Then place the plate containing cells in collagen matrices into the live cell unit of this microscope.Find the bottom of the plate and then move the objective lens up until it focuses at about 100 micrometers from the bottom of the plate. Move the stage around to find cells and select multiple positions for imaging. Set up the timelapse experiment with five-minute intervals.Fluorescent images of a dividing H2B-mCherry expressing MDA-MB-231 cell embedded in collagen matrix are shown here. Different phases of mitosis are readily observed as expected. The distribution of collagen fibers in white visualized simultaneously using confocal reflection microscopy changes very little during the course of mitosis.Cell synchronized G2-M phase and embedded in a collagen matrix complete cell division in the first two hours while control asynchronous cells divide randomly. Quantification of the distribution of collagen fibers during interphase and mitotic phases suggest that matrix deformation is minimal during cell division and higher in interphase cells. Once mastered, this technique can be completed in seven days starting from generating the stable cell line to analyzing the video cell division in a 3D matrix.After watching this video, you hopefully have a good understanding of how to synchronize and monitor mammalian cell division in 3D matrices using live cell imaging and how to determine matrix deformation using confocal reflection microscopy and quantitative imaging analysis techniques.

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9.0K visualizações.  Johns Hopkins University. The overall goal of this procedure is to study mammalian cell division and cell matrix interactions in a physiologically relevant 3D environment. The main advantage of this technique is that it provides an efficient and general approach to study mammalian cell division and cell matrix interactions. This method can help answer key questions in cell biology such as the molecular basis of the development of normal and disease tissue.A day prior to transfection, plate five million HEK-293 ...