Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The overall goal of the following experiment is to isolate purified muscle cells from human patient muscle biopsies and immortalize them for downstream studies. This is achieved by first weighing mincing and digesting the collected tissue to dissociate the mononuclear cells. As a second step, the myogenic cells are purified from non myogenic cells by facts.

Next, myogenic cells are infected with both CDK four and H turt to generate immortalized cells. The results show that patient-derived living muscle cells can be expanded for several passages and differentiated based on cell fusion assays, the presence of twitching myo tubes, and the expression of myogenic markers. The implications of this technique extend beyond diagnosis of muscle disease because it can potentially provide unlimited number of myogenic cells for drug screening or other downstream applications where high number of cells are required.

I will be demonstrating the procedure along with Jerome Robbin, a postdoctoral fellow from the laboratory of Dr.Woody Wright in Dallas. Begin with dissociating the muscle biopsy to purify the myogenic cells. First in a tissue culture biosafety hood, record the mass of the muscle biopsy.

Next in a 10 centimeter dish, use sterile scalpels to mince the tissue into finely chopped pieces. Add a few drops of one X-H-B-S-S so the tissue does not dry out per gram of tissue. Add 3.5 milliliters of disc paste two and 3.5 milliliters of collagenase D solutions.

Then mix the cells with a 25 milliliter pipette and transfer the plate into an incubator every 15 minutes. Mix the cells with a five milliliter pipette within 90 minutes. There should be a complete tissue dissociation.

Once the tissue is dissociated, add two volumes of growth media and filter it through a 100 micron strainer. Collect the filtrate in a 50 milliliter tube. Pellet the cells from the filtrate at 329 G for 10 minutes at room temperature.

Then resuspend the cells in HBSS with 0.5%BS, A, and count them. Adjust the volume to get 1 million cells per 100 microliters. Set aside a 250, 000 cell aliquot as an unstained control and set aside two similar aliquots for single stain controls.

These are all required for the fax analysis. Now, stain the cells with five microliters of anti CD 56 antibody per million cells, and incubate all the samples on ice for half an hour. Next, quickly wash the samples with 10 milliliters of HBSS.

Then pellet the cells as before, but at four degrees Celsius after the cells have been pelleted, discard the supernatant and resuspend the cell pellet in one X-H-B-S-S at 5 million cells per milliliter. Then add propidium iodide to a final concentration of one microgram per milliliter. Now purify the myogenic CD 56 positive cells from the non myogenic cells using facts following the purification plate.

The myogenic cells at 50, 000 per well in six well plates coated with 0.1%gelatin culture, the cells until they are attached, then infect them with virus for cell line immortal. In the text protocol, there is an additional review of how to purify and culture dermal fibroblasts as described in the text protocol. Prepare phoenix tropic cells after incubating the phoenix tropic cells overnight with the plasmid PolyJet mixture.

Feed the cells fresh, medium, composed of four parts of DME M1, part of medium, 1 99 and 10%calf serum. Also prepare inotropic packaging. PA three 17 cells in a six well plate with about 400, 000 PA three 17 cells per well.

12 hours later, collect the virus containing supernatant over the Phoenix tropic cells and pass it through a 0.45 micron filter. Then use one milliliter of the supernatant to infect the PA three 17 cells and culture them overnight. The next day, select for a virus producing cell line from the PA three 17 cells by applying antibiotics at 0.5 milligrams per milliliter to the medium when the cells are near co fluency, harvest the supernatant off the cells three times on 12 hour intervals.

Filter the collections after each harvest. The filtrate is the working viral supernatant. Divide it into one milliliter.Aliquots.

Set aside a few aliquots for immediate use. The extra aliquots can be stored at minus 80 degrees Celsius, but will lose 50%efficiency with each freeze thaw cycle. Take note that throughout the process equipment that has touched the viral particles must be bleach washed before it is discarded into the waste bin.

To permanently store the stable PA three 17 virus producing cell line, freeze the cells in 10%DMSO 90%serum and store them at minus 150 degrees Celsius to each well of fax purified cells. In a six well plate, add 400 microliters of freshly produced viral supernatant. Leave two wells without virus as controls.

Incubate the plate overnight and the next day change the medium in each well to 2.5 milliliters of fresh muscle media. Be sure to discard the media and pipettes that contain viral particles into the bleach container. Let the cells incubate in the fresh muscle media for three days.

Then select the cells for antibiotic resistance using 400 micrograms of neomycin per milliliter for CDK four infection, or 300 micrograms of hygromycin per milliliter for human telomerase. Reverse transcriptase infection for a week or two. Maintain the cells under the selective conditions until the cells and the control wells are all dead.

Always passage the cells before they become confluent, even during selection when they reach 60 to 80%Co fluency use a 0.05%tripsin EDTA solution to collect and pass the cells. Pass the cells to 10 centimeter dishes with fresh myoblast, medium with antibiotics. First seed the cells at a very low density of three to 500 per 10 centimeter dish.

Maintain the cells in these dishes for about two weeks until small colonies of 10 to 20 cells form when the small colonies are evident. Remove most of the media leaving just a thin film of media to keep the cells from drying out. Now, dip one side of a cloning ring in silicone vacuum grease and the ring over a desired clone.

Put a different ring around each desired clone in each ring. Add a few drops of trypsin EDTA solution. When the cells get rounded, use a microscope to carefully aspirate each ball of cells into a 200 microliter pipette tip.

Transfer the cell balls to wells of the smallest available multi-well plate. Then expand each clone as needed to prevent co fluency. Using the described protocol, myogenic cells were isolated along with fibroblasts, adipogenic cells, and possibly immune cells, and then purified by facts.

The cells were then immortalized as described using viruses expressing cyclin dependent kinase four, CDK four, and human telomerase reverse transcriptase h turt. It was crucial to keep the dishes below co fluency at each step as they could enter the differentiation program and form myo tubes comparing myo tube formation and contraction in non immortalized control cells and the immortalized cells. No differences were seen.

Immortal of the cell line was confirmed by measuring telomere length and telomeres activity. Immortal was also evident from cell growth curves. After watching this video, you should have good understanding of how to dissociate primary muscle cells from patient biopsy.

Immortalize the primary myoblast and isolate and select immortalize myogenic clones. Don't forget that working with virus requires a use of specific tissue culturehood and the secure ventilation system. In addition, everything involved in the virus manipulation such as pipettes, tips, dishes, syringes, and filters must be bleached to avoid any unwanted contamination.

Viral particles can be hazardous and per precautions should always be taken while performing this procedure.