Name: PuraMatrix Encapsulation of Cancer Cells (Video) | JoVE Journal
Uploaded: 2009-12-17T17:00:00.000+00:00
Duration: 7 min 8 s
Description: Learn PuraMatrix encapsulation of CAR5 ovarian cancer cells to create 3D culture models that mimic in vivo micrometastatic nodules. This protocol enables researchers to study treatment responses in realistic tumor environments.

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5 minutes

Technical Notes:
<ul>
 <li>Gelation is initiated by the addition of salts. Therefore, it is CRITICAL that no salts come in contact with PuraMatrix until gelation is desired; exposure to salts of very low ionic strength (e.g., &lt; 0.05 M) will cause irreversible gelation.</li>
 <li>Leftover stock solutions that are prepared can be used at a later date provided they have not come in contact with salts. You do not need to worry about repeat sonication.</li>
 <li>Air bubbles can be removed aliquoting PuraMatrix into sterile tubes followed by centrifugation.</li>
 <li>Be very careful during media changes as aspiration can easily disrupt the PuraMatrix bed.</li>
</ul>
Materials:
BD PuraMatrix RAD16 Peptide Hydrogel-1% (Cat # 354250): The total peptide concentration of the undiluted mixture is 10 mg/ml.
<ul>
<li>Water Bath Sonicator or Vortex</li>
<li>96-well cell culture dish</li>
<li>Cell Culture Media</li>
<li>Sterile Filtered H2O</li>
<li>Sterile Filtered 20% Sucrose</li>
<li>Cells (all at 5,000 cells/well): OVCAR-5</li>
</ul>
All steps must be performed under aseptic conditions.
Procedure:
<ol>
	<li>Sonicate the 1% PuraMatrix (RAD16) in a water bath sonicator for 30 minutes to reduce the viscosity prior to use.</li>
	<li>The 1% PuraMatrix solution can now be aliquoted into sterile tubes to simplify future experiments.
	<ol class="lalpha">
		<li>Avoid the formation of air bubbles; if they form simply spin down the sterile tubes containing PuraMatrix at 1000 rpm (300 x g) for 5 minutes.</li>
		<li>The total peptide concentration of the undiluted mixture is 10 mg/ml.</li>
	</ol>
	</li>
	<li>Two sets of 4 wells will be plated at 2.5 mg/ml and 1.25 mg/ml total peptide concentration, respectively.</li>
	<li>Dilute the PuraMatrix in each well set with 0.2 μm sterile filtered 20% sucrose and 13.3% sucrose to total peptide concentrations of 5 mg/ml and 2.5 mg/ml, respectively to achieve a 2x concentration of BD PuraMatrix in 10% sucrose.
	<ol class="lalpha">
		<li>To Make 20% sucrose, dilute 10 g of sucrose, then volume to 50 ml water.</li>
		<li>13% and 10% sucrose can be prepared by diluting the 20% stock solution.
		<ol class="lroman">
			<li>13% Sucrose (for every 10 ml):  6.5 ml 20% sucrose + 3.5 ml sterile H2O.</li>
			<li>10% Sucrose (for every 10 ml): 5.0 ml 20% sucrose + 5.0 ml sterile H2O.</li>
		</ol>
		</li>
	</ol>
 </li>
	<li>Prepare a master mix of PuraMatrix peptide based on the number of wells you intend to plate. (See example below.) Then aliquot into individual tubes containing 25 ml of the master mix.
	<ol class="lalpha">
		<li>The master mix should be prepared at twice the desired final concentration of total peptide as it will be diluted with an equal volume of cells during plating. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="Peptide concentration diagram with PuraMatrix dilution process for research experiments." data-altupdatedat="1788235205000" data-alt="figure 1"></li>
	</ol>
	</li>
	<li>Prepare cell suspensions by trypsinizing cell monolayer cell cultures.
	<ol class="lalpha">
		<li>2 ml of 0.05% Trypsin EDTA can be used for each T-75 Flask.</li>
	</ol>
	</li>
	<li>Neutralize the trypsin by addition of cell culture media containing 10% heat inactivated fetal bovine serum (use at least 2 ml of media per ml of trypsin used in the previous step.)</li>
	<li>Centrifuge the cells at 1000 rpm (~ 300 x g) for 5 minutes to create a cell pellet.
 <ol>
		<li>Resuspend the cells in 10% sterile filtered sucrose.</li>
		<li>Count the cells and spin down again at 1000 rpm (~ 300 x g) for 5 minutes to remove trace amounts of salt found in the media.</li>
	</ol>
	</li>
	<li>Resuspend the OVCAR-5 cells in 10% sucrose at 2.0 x 105 cells/ml so that the final cell count per well will be 5,000 cells in each well. 
The following steps should be performed rapidly. </li>
 <li>Add 25 μl of the 2.0 x 105 cells/ml suspension to the first individual tube containing 25 μl of the master mix of PuraMatrix as shown in the above schematic.
 <ol class="lalpha">
 <li>Quickly mix by pipetting (~5 times up and down) in the tube without introducing air bubbles.</li>
 <li>Then pipette the 50 μl cell suspension/PuraMatrix mixture into the appropriate well of a 96 well dish.</li>
 <li>Initiate gelation of the BD PuraMatrix by gently pipetting 150 μl cell culture media (RPMI + 10% FBS)  to the side of the well <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="Cell suspension process, dilution into Puramatrix, diagram of experimental setup, tube distribution." data-altupdatedat="1788235205000" data-alt="figure 2"></li>
 </ol>
 </li>
 <li>Repeat the steps outlined in 8 until all wells have been plated.</li>
 <li>After 30 minutes Very Gently remove ~2/3 of the media using a 200 μl pipette to equilibrate the pH of the hydrogel.
 <ol class="lalpha">
 <li>DO NOT USE A VACUUM ASPIRATOR AS THIS WILL DISRUPT THE MATRIX </li>
 <li>Place the pipette tip in the well so that it does not touch the PuraMatrix bed and gently remove media from the well. </li>
 <li>Care should be taken to not disrupt the matrix during the culture of cells in PuraMatrix. </li>
 </ol>
 </li>
 <li>Very Gently add 150 μl of fresh cell culture media to the side of the well containing cells encapsulated in PuraMatrix.</li>
 <li>Change the cell culture media in each well every 48 hours with fresh culture media by carefully repeating steps 10 and 11.</li>
 </ol>
Representative Results:
<img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="Cell growth progression, microscope image, days 1-7, showing size and structure changes, 10 µm scale." border="0" data-altupdatedat="1788235205000" data-alt="figure 1"> Figure 1. OVCAR-5 cells were encapsulated as described in the above protocol. Images were acquired using an inverted Olympus FV1000 microscope equipped with Spectra-Physics DeepSee Ti:Sapphire laser tuned to 750 nm. Imaging sessions were conducted on days 1, 3, 5 and 7 post-plating. A long working distance, water dipping 40x objective (Olympus LUMPLFLN 0.8 NA) was used to image through the PuraMatrix. Three-dimensional volumes were collected by acquiring image stacks in 1.47 μm axial steps. Two non-descanned detectors collected the autofluorescence emission using violet (440-490 nm) and green (510-550 nm) bandpass filters. The final images and depth stack movies were processed using ImageJ by combining both detection channels. Please <a href="https://www.jove.com/files/ftp_upload/1692/1692_fig1.jpg">click here</a> to see a larger version of figure 1.

<ol><li>Gelain, F., Bottai, D., Vescovi, A., Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Designer+self-assembling+Peptide+nanofiber+scaffolds+for+adult+mouse+neural+stem+cell+3-dimensional+cultures%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Designer self-assembling Peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures.</a> PLoS One. 1, e119-e119 (2006).</li>
</ol>

puramatrix encapsulation of cancer cells

PuraMatrix Encapsulation of Cancer Cells

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these models utilize reconstituted basement membrane derived from animals which contain a variable amount of growth factors and cytokines that can influence the growth of these cell culture models. Here, we describe in detail the preparation and use of PuraMatrix, a commercially available self assembling peptide gel that is devoid of animal-derived material and pathogens to encapsulate and propagate the ovarian cancer cell line, OVCAR-5. We begin by describing how to prepare the PuraMatrix prior to use. Next, we demonstrate how to properly mix the PuraMatrix and cell suspension to encapsulate the cells in the hydrogel. Upon the addition of cell culture media or injection into a physiological environment, the peptide component of PuraMatrix rapidly self assembles into a 3D hydrogel that exhibits a nanometer scale fibrous structure with an average pore size of 5-200 nm1. In addition, we demonstrate how to propagate cultures grown in encapsulated PuraMatrix. When encapsulated in PuraMatrix, OVCAR-5 cells assemble into three dimensional acinar structures that more closely resemble the morphology of micrometastatic nodules observed in the clinic than monolayer in vitro models. Using confocal microscopy we illustrate the appearance of representative OVCAR-5 cells encapsulated in PuraMatrix on day 1, 3, 5, and 7 post plating. The use of PuraMatrix to culture cancer cells should improve our understanding of the disease and allow us to assess treatment response in more clinically predictive model systems.

This video demonstrates how to encapsulate and culture cancer cells in PuraMatrix, a commercially available self assembling peptide gel.

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these ...

Monolayer cell culture models have been very useful tools for studying various diseases including cancer. However, these models fail to recapitulate the three dimensional environment observed in vivo. This video will demonstrate the use of pure Matrixx, a commercially available self assembling peptide gel to encapsulate and propagate the ovarian cancer cell line of CAR five.The cell suspension is mixed with the pure Matrixx and cell culture. Medium is added to initiate ation of the pure Matrixx when encapsulated and cultured in pure Matrixx of Carv five cells assemble into 3D ASIN R structures that resemble the morphology of micrometastatic nodules. Hi, I'm Adnan Abbi.I'm a research fellow in the lab of Dr.TBA Hassan at the Woman Center for Photo Medicine at Massachusetts General Hospital. Today we're gonna show you a protocol for how to encapsulate and culture an ovarian cancer cell line in a synthetic matrix called pure matrix. We use this model in order to evaluate treatment response for ovarian cancer in our lab.So let's get started. The BD 1%pure matrix RAD 16 peptide hydrogel has a total peptide concentration of 10 milligrams per milliliter prior to use. Reduce the viscosity of the pur matrix by fornicating it in a water bath sonicate for 30 minutes following sonication while maintaining sterile conditions.Aliquot the 1%pure matrix solution into sterile tubes To simplify future experiments, avoid the formation of air bubbles if they form, simply spin down the sterile tubes containing pure matrix at 1000 RPM for five minutes. In this demonstration, we will plate two sets of four wells at 2.5 milligrams per milliliter and 1.25 milligrams per milliliter. Total peptide concentration respectively prepare two master mixes of pure matrix peptide based on the number of wells you intend to plate, which is eight in this case.Each master mix should be prepared at twice the desired final concentration of total peptide as it will be diluted with an equal volume of cells during plating to make the master mixes dilute the pure matrix with sterile 10%sucrose and 13.3%sucrose to total peptide concentrations of five milligrams per milliliter and 2.5 milligrams per milliliter respectively to achieve a two x concentration of pure Matrixx in 10%Sucrose then aliquot 25 microliters of the master mixes into eight individual tubes. The pure matrix master mixes are now ready for encapsulating cells. The ovarian cancer cell line of CAR five will be encapsulated and propagated in this experiment prior to trypsin incubate cells with 10 milliliters of sterile PBS without calcium and magnesium for 15 to 20 minutes.Aspirate PBS and trypsin a cell monolayer cell culture, neutralize the trypsin by adding cell culture media containing 10%heat inactivated fetal bovine serum. Use at least two milliliters of media per milliliter of trypsin used in the previous step. Next, transfer the cells into centrifuge tubes and centrifuge the cells at 1000 RPM for five minutes to create a cell pellet.After centrifugation reus, suspend the cell pellet in 10%sterile filtered sucrose. Count the cells and spin down again at 1000 RPM for five minutes. To remove trace amounts of salt found in the media reus suspend the O car five cells in 10%sucrose at two times 10 to the fifth cells per milliliter so that the final cell count per well will be 5, 000 cells in each.Well.The cells are now ready for encapsulation in the pur Matrixx. The following steps for encapsulating cells in the pure matrix must be performed rapidly at 25 microliters of the previously prepared cell suspension to the first individual tube containing 25 microliters of the master mix of pure Matrixx. Quickly mixed by pipetting about five times up and down in the tube without introducing air bubbles.Then pipette the 50 microliters cell suspension pure Matrixx mixture into the appropriate well of a 96 well cell culture dish. Initiate ation of the BD pure matrix by gently pipetting 150 microliters of cell culture media to the side of the well. Repeat the steps for encapsulation of the cells for the remaining wells.Remember that these steps must be performed quickly and the entire encapsulation procedure should be completed for one well before moving on to the next. After all eight wells have been plated, lead the cells for 30 minutes After 30 minutes using a 200 microliter pipette, place the pipette tip in the well so that it does not touch the pure matrix bed and very gently remove media from the well. About two thirds of the media must be removed to equilibrate the pH of the hydrogel.Do not use a vacuum aspirator as this will disrupt the matrix. Next very gently, add 150 microliters of fresh cell culture media to the side of the well containing cells encapsulated in pure Matrixx. In the same way as shown previously, remove two thirds of the media and add fresh media to the remaining wells.Replace the cell culture media in each well every 48 hours with fresh culture media. Ocar five cells were encapsulated as demonstrated in the proceeding. Protocol representative 3D renderings of images of OR five cells encapsulated and cultured in pure matrix on days 1, 3, 5, and seven are shown.So we've just shown you how to encapsulate in culture the ovarian cancer cell line of CAR five and a synthetic matrix called pure Matrixx. So when using pure Matrixx, it's important to realize that this protocol has been optimized for the O Car five cell line. Each researcher will have to optimize and fine tune the protocol to their particular cell line.And remember when doing media changes, it's very important to not disrupt the matrix bed as it's very fragile, never aspirate and always use a pipette. So that's it. Thanks a lot for watching and good luck with your experiments.

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Learn PuraMatrix encapsulation of CAR5 ovarian cancer cells to create 3D culture models that mimic in vivo micrometastatic nodules. This protocol enables researchers to study treatment responses in realistic tumor environments.

PuraMatrix Encapsulation of Cancer Cells

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