The overall goal of this essay is to isolate cancer stem cells from a finite cell line obtained from human osteosarcomas. The main advantage of this work is to clearly show others in the oncology field how to isolate, in vitro, cancer stem cells. These cells, can then be used to research therapies against osteosarcoma and other solid tumors.

Generally, individuals new to this matter will struggle if they don't know how to isolate the sarcoma colonies. Visual demonstration of this is critical, as the isolation step is difficult to realize, and to understand. We first had the idea for this technique, when we had read about the sphere formation essay.

Describe by Gypsum and collaborators. Demonstrating the procedure will be Dr.Gaia Palmini a PhD student, and Dr.Roberto Zonefrati, a technician from my laboratory. To begin, prepare all of the necessary culture medium.

Digestion buffers, and other solutions that will be needed for the procedure. Next, establish an osteosarcoma finite cell line, by isolating osteosarcoma from a confirmed needle or surgical biopsy. And sub-culturing the cells obtained as described in the accompanying text article.

Once the osteosarcoma cell culture has been established, and the plate is near confluence. Remove the medium by aspiration, and trypsinize the cells to release them from the plate. Transfer the cells to a fresh tube and gently pipette the cell suspension up and down in order to disperse any cell clumps.

Next, collect ten micro-liters of the cell suspension, and transfer it to a hemocytometer chamber. Place the hemocytometer chamber under a phase contrast microscope and calculate the concentration of cells in the suspension. Then, add 280, 000 cells to 35 milliliters of sarcosphere growth medium, containing methocellulose, and gently mix the solution using a pipette, to disperse any clumps.

Transfer five milliliters of the cell mixture into each well of six well ultra low attachment plates. Once plated, use an inverted microscope to observe how the cells appear and if they are well isolated. Incubate the cells in a 37 degree celsius, five percent carbon dioxide incubator.

Every three days, add fresh alcoholates of beta FGF and EGF to each well to refresh the concentration of the growth factors. Examine the progress of the sarcosphere assay by looking at the cells under the microscope at seven, 14, 20, 21, and 28 days. When you observe the presence of large black sarcospheres, that are all together in the center of the well, stop culturing and begin desolation process.

When it is time to isolate the sarcosphere, use a sterile 1, 000 microliter pipette to transfer the medium containing the sarcospheres to a syringe with a sterile membrane filter holder. After completely removing the medium containing the sarcospheres, add five milliliters of growth medium to the well, and rinse it to be sure. And collect any remaining spheres.

Then, use a microscope to confirm whether all the sarcospheres were recovered. If not, wash the well again until the collection of the spheres is complete. Next, filter the suspension through the membrane filter holder by gravity in order to not damage the spheres and make sure no sarcospheres cross through the filter.

During this process, remove any air bubbles by gently using a sterile glass pasture pipette. Once all the sarcospheres have been filtered, wash the filtration unit by adding ten milliliters of growth medium directly to the syringe and letting it filter without pressure to be sure to eliminate single cells. Once the suspension of sarcospheres has been filtered, it is necessary to discard the filter of the liquid and pay serious attention to the recovery of sarcospheres which are tangled into the net filter.

At this point, take a part the filter unit, removing it from the syringe. And place it into a 100 millimeter petri dish. Remove the net filter from the membrane filter holder using a pair of sterile peri tweezers.

And transfer the filter to a new 60 millimeter petri dish. Then, wash the net filter by shaking it gently. While adding five milliliters of growth medium, in order to release the sarcospheres from the tangle of the membrane.

Next, place the membrane in a well and confirm by microscopic observation that there are no more spheres tangled in the membrane. Wash the membrane again, and then recheck the membrane under the microscope if there are still spheres tangled in the membrane. When no more spheres remain in the membrane, incubate them in growth medium to grow the sarcospheres into a mono-layer.

Monitor the growth of the osteosarcoma cancer stem cells, which are obtained from the sarcospheres. Until cells reach about 90%confluence, in a 60 millimeter petri dish. In addition to the numerous standing protocols found in this article, methods to evaluate the differentiation capacity, ALDH activity, flow citometry, immunoflorescence, and RTPCR analysis, are included in detail in the accompanying text protocol.

Osteosarcoma samples are obtained by needle aspiration or surgical over-section. And if treated properly, take approximately one month for the primary cultures to reach confluence in a 100 millimeter petri dish. Following expansion, the cells are plated in six well ultra low attachment plates, to begin the sarcosphere assay.

This plate maintains the cells in a suspended state, and prevents the substrata attachment. After seven days, several small spherical colonies surrounded by single cells are observed. And after 28 days, several large sarcospheres are observed.

Once the spherical colonies are isolated, stem like cancer cells can be expanded from the single sarcospheres by placing them on normal attachment plates. The immunoflorescent stainings, have showed the mesenchymal stem cell like phenotype as the expanded cells were positive for CD44 for CD105, and moderately positive for Stro-1. RTPCR, also positively showed the embryonic stem cell phenotype by the expression of three embryonic stem cell marker genes.

Nanog, Oct 3/4, and Sox2. And the CD133 gene, a marker for osteosarcoma cancer stem cells. The detailed isolation technique which we have shown in this video, will pave the way for other researchers to study cancer stem cells by showing them how to understand and reproduce the most critical steps of the sphere formation assay.

These modified assay is a good technique for isolating cancer stem cells, and studying their biology. This matter, with additional adaptations, may also be used to isolate cancer stem cells from other finite cancer cell lines. Obtained by biopsies of solid tumors.

In conclusion, the isolation of cancer stem cells from several types of tumors, will allow for the study of their biology. With the final goal of finding molecular targets and developing very specific anti-cancer therapies directed against particular cellular subsets.

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