Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

The overall goal of this procedure is to demonstrate how to prepare and transplant normal and malignant muscle. From transgenic donor zebra fish into rag two immune deficient zebra fish recipients. Muscle transplantation involves first isolating fluorescently labeled muscle by dissection of the dorsal musculature.

After homogenization of the zebra fish muscle cells are filtered and made into a single cell suspension. Then cells are transplanted intramuscularly into rag two mutant zebra fish epi fluorescence microscopy is then used to follow tissue engraftment in the recipient animals as early as 10 days after transplantation. Similarly, a primary embryonal rhabdo myosis sarcoma can be harvested, made into a suspension and transplanted intraperitoneal into rag two mutant zebrafish.

Then microscopy is used to follow the tissue engraftment and the propagated tumor cells can be utilized in downstream applications. The main advantage of using adult immune compromised zebrafish cell cell transplantation is that it provides a universal platform for allograph transplantation of several tissues and tumor types. Tissues can be grafted from a variety of donor genetic backgrounds without a need for immune suppression in recipient fish.

This method can help answer key questions in stem cell and regenerative biology such as the self renewal capacity of progenitors. We can also use these methods to engrave tumors into rag two mutant fish, and then identify novel chemicals that, for instance, carb tumor growth Demonstrating normal and malignant muscle cell preparation and transplantation will be chin, tang and anes. 10 to two talented graduate students in the LAL lab Begin with sacrificing donor zebra fish with fluorescently labeled muscle.One.

Typical adult alpha acting RFP donor fish yield between 500, 000 to 2 million muscle cells. In our example experiment, we use 30 donor fish to transplant into 20 recipient fish with a million cells each. After euthanizing the donor fish dry the animal by placing it on a disposable paper towel.

Use a razor blade to harvest the dorsal musculature. The cut should be made at a 45 degree angle posterior to the anus region, including bones and skin tissue transfer 10 harvested dorsal muscular sections to a clean Petri dish. Then add 500 microliters of chilled 0.9 XPBS 5%FBS suspension Buffer.

Next, mince the tissue until a uniform suspension is obtained fully homogenizing the tissue using a five milliliter serological pipette, add two milliliters of suspension buffer to the homogenate, then tate the suspension through the pipette 20 times. Next, filter the suspension using a 40 micron strainer and collect cells in a 50 milliliter conical tube on ice. Keep the cells cold for the remainder of the procedure.

Wash the remaining tissue in the strainer with 2.5 milliliters of suspension, buffer and strain into the 50 milliliter conical tube. Now, count the total number of viable cells using trian blue and concentrate them by centrifugation at 1000 G for 10 minutes at four degrees Celsius. Then resuspend the cells in 0.9 XPBS with 5%FBS at 333, 000 cells per microliter.

If the donor cell number is limiting as few as 50, 000 injected cells can also lead to successful engraftment. Begin with anesthetizing two to four month old rag two deficient fish in trica. When a perm movements are slow and the fish is still move the fish to a damp towel with the left side of the fish facing up.

Now use a pre-cleaned 10 microliter 26 s gauge micro syringe to inject three microliters of the prepared muscle cell suspension injections should be completed at a 45 degree angle and injected directly into the dorsolateral musculature of recipient fish. It is important to note when performing intramuscular transplantation, a total volume of less than three microliters should be injected into the recipient fish. Then carefully transfer the recipient fish into a recovery tank containing clean fish system water.

Continue injecting the remaining fish and place them into one recovery tank. Later, assess the engraftment rates at 10, 20 and 30 days post transplantation using brightfield and epi fluorescence microscopy. Processing each tumor separately.

Sacrifice a fish with an embryonal rhabdomyosarcoma. Place the fish in a clean Petri dish and dissect the tumor away from the non-malignant tissues using a razor blade and fine forceps, Careful dissection around the tumor will lead to higher purity of the cell suspension obtained with minimal contamination of surrounding muscle tissue. Transfer the dissected tumor to a new dish and add 100 microliters of pre chilled 0.9 XPBS 5%FBS suspension, buffer.

Then mince the tissue with a razor blade until a uniform suspension is obtained. Then add an additional 900 microliters of buffer using a one milliliter pipette followed by trier. Rating the suspension through the pipette several times to dissociate the cells.

Now filter the cells through a 40 micron cell strainer. Rinse the plate by adding an additional two to four milliliters of suspension, buffer and filter through the same cell strainer. Concentrate the cells as before.

Keep cells on ice. Then resuspend the cells in 100 microliters of buffer and count the viable cells using trian blue. Adjust the concentration of the cell suspension to 5, 000 cells per microliter.

Next, anesthetize the rag two immune compromised recipient zebrafish and place it on a clean paper towel with the ventral side facing up. Using a cleaned 26 s gauge syringe needle. Inject the desired volume into the peritoneal cavity of the recipient zebrafish here.

25, 000 cells are injected in five microliters. However, up to 10 microliters can be successfully injected. Let the injected fish recover in a recovery tank before returning them to the main system.

Later, follow recipient fish for engraftment using epi fluorescence microscopy and harvest the cells for downstream studies. Using the described procedure skeletal muscle cells from alpha actin RFP transgenic donors were transplanted into immune compromised homozygous rag two mutant zebra fish. The skeletal muscle tissue produced a single cell suspension containing 84.3%viable cells as assessed by DPI exclusion.

Following flow cytometry analysis, RFP positive cells comprised 35.3%of this donor cell suspension. Transplantation of cells into the dorsal skeletal muscle of RAG two homozygous mutant recipient fish led to consistent and strong and graftman. This was confirmed by the formation of multinucleated fluorescent muscle fibers in the recipient fish wild type recipient fish failed to engraft muscle fibers.

However, by 10 days post transplantation, nine out of 14 mutant zebra fish contained RFP positive muscle fibers near the injection site. Importantly, the engrafted RFP positive muscle persisted to at least 30 days post transplantation. Similarly, robust engraftment was obtained following transplantation of RAG two mutant fish with embryonal rhabdomyosarcoma or ERMS.

The engrafted ERMS shared histological features with the primary ERMS tumor cell heterogeneity was maintained as assessed by fluorescence analysis. After watching this video, you should have a good understanding of how to performing muscular transplantation and intraperitoneal transplantation of normal and malignant tissues using recipient adult immune compromised be fish. Following this other methods like fluorescence activated cell sorting, limiting dilution transplantation analysis and real-time PCR can be performed to assess functional and molecular differences between different tumor subpopulations.

Overall, our approach permits the robust and long-term engraftment of fluorescent muscle and malignant tissue into immune compromised RAG two mutant fish. The benefit of this approach is that engraftment occurs in the absence of prior immune suppression of the recipient fish and does not require immune matching of donor and recipient animals.