The overall goal of the following experiment is to use in vivo bioluminescence imaging to study immune responses after implantation of engineered heart tissue or EHT in rats. This is achieved by first generating luciferase positive ehs. Next, EHS that stably express luciferase are transplanted into the greater omentum of recipient rats.
Then in vivo bioluminescence imaging can be performed on recipient animals to monitor noninvasive longitudinal EHT survival and rejection. Using this model, the intensity and kinetics of acute rejection, for example, can be correlated with the decline of the observed bioluminescence signal intensity over time. The main advantage of this technique over existing methods like histology is that daily measurements allow for non-invasive longitudinal assessment of graft survival.
This method can provide insight into the survival and rejection of engineered heart tissues, but it can also be applied to other questions. For example, metabolism of ehs. The bili technique will be demonstrated by Christiana Parman who's performing this technique on a daily basis.
In my lab, transgenic heart cells are first isolated from Luciferase Lu neonatal rats to generate fibrin based DHT for implantation purposes, prepare a master mix containing cells isolated from neonatal luciferase, lu transgenic, rat hearts, bovine fibrinogen, and matrigel to X-D-M-E-M and NKM. Prepare aros casting molds by adding nine milliliters of 2%aros in PBS to each well in six well culture dishes, and then placing Teflon spacers into the wells. After the aros solidifies, remove the spacers and place silicone post racks on the culture dishes with the post reaching into the molds.
To generate EHT, gently mix the master mix, then add a calculated amount of thrombin into the master mix and briefly mix. Then quickly pipette 1100 microliters of master mix into each mold. After the fibrinogen polymerizes, gently remove the racks with the posts that now have adherent EHT from the agros casting molds and transfer the racks to new six well cell culture dishes containing eight milliliters of custom made medium per well.
EHT are maintained for 10 days in a 37 degree Celsius, 7%carbon dioxide cell culture incubator. And the medium is changed three times a week. During this culture period, neonatal rat heart cells will begin to elongate, interconnect, and align along force lines in between silicone posts.
At the time of implantation, spontaneous coherent contractions will be observed. Sterilize the instruments for surgery by autoclave prior to use warm 30 milliliters of sterile saline solution. Rats for implantation should weigh 100 to 150 grams and be housed under conventional conditions provided with standard rat chow and water ad libido.
In this video, brown Norway rats are used after anesthetization, sedate the rats prior to surgery and confirm sedation by lack of response to toe pinch while the rat is under anesthesia, inject five milligrams per kilogram of body weight, carens subcutaneously into the plank, shave the abdominal area and apply eye ointment to prevent the animal's eyes from drying out. During anesthesia, disinfect the abdominal area using Betadine, and then 80%ethanol swabbing from clean to dirty. Repeat this disinfection.
Step three times, drape the animal and perform a midline abdominal incision, separating the skin and muscle in two steps to open the abdomen. Use hot bead sterilization on tools between cuts. Dampen a powder freak glove with the saline.
Place the intestines inside the glove. Fold the glove around the intestines to prevent moisture loss. Spread the incision and expose the spleen.
Identify and spread out the greater momentum with forceps. Carefully cut the EHT from the suspending silicone posts and transfer it onto the greater momentum. Wrap the greater momentum around the EHT and fix it closed using two single seven oh proline sutures.
Next, move the intestines back into the abdomen. Flush the abdomen with one milliliter of prewarm sterile saline. Close the muscle layer of the abdominal wall using six oh proline running sutures.
Use five oh proline running sutures to close the skin to provide sufficient analgesia for this type of procedure. At 50 milligrams per 100 milliliters of ole to the drinking water for three days post implantation. The IVUS Bioimaging platform is used for non-invasive bioluminescence Imaging in vivo and results are analyzed using the IVUS living image software.
Package imaging is performed daily, starting two hours postoperatively. Prior to visualizing the EHT bioluminescence, anesthetize the rat and confirm sedation by toe. Pinch disinfect the abdominal area.
Inject Lucifer at 375 milligrams per kilogram of body weight intitally and place the anesthetized rat into the ivu imaging chamber. Up to four rats can be analyzed at the same time. Use the IVUS living image software to observe and record the peak signal, which appears approximately 20 minutes after Lucifer injection.
Then establish the baseline signal intensity of luciferase positive cells inside the EHT in units of photons per second per centimeter squared per Ceridian in the region of interest, 120 minutes after implantation and at days 1, 2, 3, 5, 7, 10, and weeks two, three, and four. The data can be expressed in graph form, showing the signal intensity over time. The cellular in vivo immune responses in Immunocompetent Brown, Norway and Immunodeficient.
Nude rats were studied after EHT implantation recipient SP cytes were harvested five days after EHT implantation. Interferon, gamma and IL four expression were evaluated by lispa assays using mitomycin inhibited EHT as stimulators and five times 10 of the six recipient cytes as responder cells. TH one and TH two responses were significantly higher in the immunocompetent model compared to the Immunodeficient model as evidenced by interferon, Gemma and IL four production respectively.
LUCIFERASE positive EHS were transplanted into the greater omentum of either immunocompetent PN rats or immunodeficient nude rats. Cell survival was longitudinally followed by BLI. The rate of animals that rejected the transplanted EHS is presented.
All BN rats rapidly rejected the EHT transplants, whereas the cell survived in T-cell deficient Rats Once mastered surgeries can be done within minutes if the technique is performed properly after surgery. Other methods like immunological essays and histology can be performed in order to answer other questions regarding cell infiltration and humeral response.
