The overall goal of this rapid post-mortem cell culture protocol is to generate durable patient-derived cell cultures of diffuse intrinsic pontine glioma to facilitate the experiments necessary to understand and ultimately develop effective therapies for DIPG. This method can help to answer key questions about the fundamental biology of and therapeutic strategies for diffuse intrinsic pontine glioma, such as whether certain drugs work across different patient-derived cultures. The main advantage of this technique is that is allows direct study of the biology of patient-derived cells.
The implications of this technique extend toward therapy for DIPG, because it allows for the testing of different therapeutic strategies across different genetic subtypes of the disease. Though this method can provide insight into DIPG, it can also be applied to other diseases, such as other pediatric high-grade gliomas, adult glioblastomas, or other central nervous system tumors. Generally, individuals new to this technique will struggle, because it is difficult to procure and rapidly process autopsy samples in an efficient manner.
Prior to sample preparation, sterilize the tissue culture hood, along with razor blades, curved hemostats, and other non-sterile tools under the UV light for one hour, and perform all the following steps under sterile conditions. Next, transfer the tissue into a high-walled 100 millimeter by 20 millimeter cell culture dish. Remove the media left over from shipping, and replace it with 10 to 15 milliliters of cold culture media.
Then, using the curved hemostats to grasp a razor blade, mince the tissue finely while removing the blood vessels or meninges. The final tissue fragments should be smaller than one millimeter. Afterward, transfer the tissue into a clean 50-milliliter conical tube.
Wash the cell culture dish with an additional five milliliters of cold culture media and transfer the sample to the conical tube. Repeat this wash step as necessary to transfer the remaining tissue. Using a 10-milliliter serological pipette, triturate the sample gently for four to five times.
Allow larger tissue fragments to settle to the bottom of the tube. If necessary, briefly centrifuge the sample for one minute. To collect the dissociated fraction, remove the supernatant and filtrate through a 100-micron filter into a new 50-milliliter conical tube labeled Mechanical Dissociation.
Invert the filter over the original conical tube and wash it with culture media to recover the tissue fragments. Subsequently, centrifuge the mechanical dissociation fraction for five minutes. Afterward, remove the supernatant from the pelleted mechanical dissociation fraction and re-suspend the tissue in cold culture media.
If there is more than five milliliters of tissue in the mechanical dissociation conical tube, split the tissue into other 50-milliliter conical tubes such that no tube has more than five milliliters of tissue. Then, store the mechanically dissociated fraction on ice until the sucrose gradient centrifugation step. In this procedure, centrifuge the conical tube containing the remaining tissue fragments for five minutes.
After than, remove the supernatant and add the pre-warmed enzymatic digestion solution, such that there is five milliliters of digestion solution for every one milliliter of tissue. Then, seal the conical tube lid with laboratory film and incubate the reaction on a rotator at 37 degrees Celsius for 30 minutes. After incubation, triturate the samples gently.
Using a 10-milliliter serological pipette, pipette the sample up and down for six to eight times, and avoid generating excessive air bubbles. Next, add a 1, 000 microliter pipette tip to the end of the pipette and triturate the sample for an additional six to eight times. Allow any remaining chunks to settle to the bottom of the tube.
Then, remove and filter the supernatant with the cells still suspended through a 100-micron filter into a new 50-milliliter conical tube labeled Enzymatic Dissociation and store it on ice. Following that, centrifuge the enzymatic dissociation tube for five minutes, and continue to sucrose gradient centrifugation. If the sample is still suspended in solution, centrifuge it for five minutes.
Next, remove the supernatant and re-suspend the tissue in 20 milliliters of cold HBSS without calcium and magnesium. Then, bring the volume up to 25 milliliters with cold HBSS. Slowly add 25 milliliters of 1.8 molar sucrose solution, and invert the tube to mix.
This results in a 0.9 molar sucrose gradient. Subsequently, centrifuge the sample with no break for 10 minutes. Carefully aspirate the myelin debris in as much sucrose sucrose solution as possible.
Then, wash the sample by adding 30 milliliters of cold HBSS without calcium and magnesium, and mixing gently. After that, centrifuge it for five minutes. In this procedure, remove the wash supernatant.
Add five milliliters of ACK lysis buffer, and gently re-suspend the cell pellet, swirling the tube for one minute at room temperature. Then, quench the lysis by adding 30 milliliters of cold HBSS without calcium and magnesium. Subsequently, centrifuge it for five minutes.
Re-suspend the final cell pellet in 10 to 15 milliliters of warm complete TSM with growth factors, and quantify the viable cell density on a hemocytometer, using Trypan blue exclusion. Afterward, transfer the final cell suspension to a new T75 culture flask. Add additional growth factors every other day to maintain the overall growth factor levels, and monitor for the development of tumor cell neurospheres.
Here are various examples of how the samples can appear from the early stages of culturing to a durable culture. Initially, plated and early cultures often do not appear to contain many surviving cells. Furthermore, early cell clusters often do not exhibit the degree of contrast typically associated with neurospheres.
However, the initial passage of these cell clusters, combined with reverse filtering of cell clusters, can isolate healthy tumor cells that are able to form spheres. The filtrate typically contains leftover debris. These patient-derived samples can then be maintained in culture for multiple passages.
Once mastered, this technique can be done in three to four hours if performed properly. While attempting this procedure, it is important to ensure the sample is maintained at cold temperature through every step except the enzymatic dissociation, and minimize traumatic handling of the sample. Following this procedure, additional studies can be performed, such as in vitro drug screening, or orthotopic xenografting, to answer subsequent questions about DIPG pathobiology.
After its development, this culture technique has paved the way for researchers in the field of pediatric neuro oncology to explore new therapeutic avenues for children with DIPG. After watching this video, you should have a good idea of how to perform rapid post-mortem cell culture protocols on brain tumor samples. Don't forget that working with human tissue can be hazardous, and precautions, such as wearing personal protective equipment and sterile technique, should always be utilized.
