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.
