Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice
May 10th, 2021
Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.
Liver is a primary site for transplantation in the human islet clinical setting. Mouse intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding. In this study, the two methods we showed both could effectively prevent bleeding-induced mouse death.
Our method can be applied to research in liver cancer and other liver diseases by allowing injection of cancer cells, mesenchymal stem cells, and hepatocyte directly to the liver. To begin, detach a human islet culture with gentle scratching. Use a 1 cubic centimeter syringe to pick 300 to 350 islets into sterile 1.5 milliliter microcentrifuge tubes on ice.
When all of the islets have been picked, centrifuge the tubes for 10 seconds to sediment the cells. Discard all but a small volume of supernatant to prevent pellet loss and resuspend the islets in 200 microliters HBSS containing 5%BSA. Load the resuspended islets into a 5 milliliter insulin syringe and turn the syringe needle-end up for 1 minute to let the islets settle at the plunger.
Depress the plunger to remove any bubbles, keeping approximately 100 to 150 microliters of solution in the syringe and invert the syringe with gentle tapping to evenly distribute the islets. After confirming a lack of response to pedal reflex, apply ointment to the eyes to prevent drying. Shave the abdomen of the anesthetized mouse to remove the fur and disinfect the surgical area with three alternating wipes of 2%iodine and 75%alcohol.
Next, make a 1 to 1.5-centimeter incision in the abdomen and open the peritoneal cavity. Place a sterile piece of gauze around the incision and use forceps or cotton tips to gently transfer the intestine onto the gauze. The then cover the intestine with a piece of saline-soaked gauze and expose the portal vein.
To stop islet transfer-induced bleeding using gel foam, hold the needle with the bevel facing down and position the needle tip with the opening parallel to the portal vein wall before inserting the needle into the portal vein. Retract the plunger to draw 20 to 50 microliters of blood into the syringe to mix with the islets before infusing the islets into the portal vein while repeatedly depressing and retracting the plunger. When all of the islets have been delivered, place an approximately 5 by 5 centimeter piece of gel foam over the injection site.
Use a cotton tip to press the gel foam down while removing the needle from the portal vein. Hold the gel in place for about 2 minutes, rolling the gel foam to make sure it covers the portal vein. Once the bleeding is stopped, gently return the intestine to the peritoneal cavity in the original position and inject 500 microliters of warm saline into the abdominal cavity.
Use sutures to close the muscle and skin layers. Then place the mouse in a clean cage on a heating pad with monitoring until complete recovery from anesthesia, administering analgesia every 12 hours and heat for 48 hours. To stop islet transfer-induced bleeding using a fat pad, after exposing the portal vein as demonstrated, use two cotton tips to hold the exposed vein on the left and right sides and locate the fat tissue pad between the duodenum and portal vein.
Penetrate the fat pad with the needle tip before insertion into the portal vein. Infuse the islets into the portal vein as demonstrated. When all of the islets have been delivered, press the fat tissue with a cotton tip for about 1 minute while removing the needle from the vein.
Then return the intestine to the abdominal cavity and perform the rest of the postsurgical care procedures as demonstrated. The effects of the islet transplantation are dose-dependent, as normal glycemia is observed 30 days after syngeneic islet transplantation when 500 islets are transferred, but only transitory normoglycemia is observed when 250 islets are delivered, with the mice returning to hyperglycemia by 10 days post-transplant. An increased body weight is observed in both groups and identical responses observed when human islets are transplanted into diabetic NOD-SCID mice, with normoglycemia observed when higher numbers of islet equivalents are transplanted but not smaller numbers.
The majority of the mice that have bleeding after the transfer die after the surgery, while the mice without bleeding survive. At 28 days post-transplantation, insulin staining reveals an even distribution of the human islet throughout the liver, mostly close to blood vessels. In addition to insulin, fibrin and polymorphonuclear cell filtration are also observed within the transplanted human islets.
Intrahepatic islet transplantation will generate a large hole in the portal vein that may cause bleeding. Using our two improved procedures can prevent bleeding and reduce mortality post islet transplantation. The intraportal islet transplantation model can mimic clinical human islet transplantation.
Our refined procedures can lead to higher survival rates for recipients, which allow further islet function study after transplantation.
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Overview
This study presents refined surgical procedures for intraportal islet transplantation in mice, addressing the technical challenges associated with this clinically relevant procedure. The methods demonstrated effectively prevent bleeding-induced mortality during surgery.
Key Study Components
Area of Science
- Transplantation
- Surgical techniques
- Liver research
Background
- The liver is a primary site for islet transplantation in humans.
- Mouse intrahepatic islet transplantation poses significant surgical risks.
- High mortality rates in mice are often due to surgical complications.
- Effective methods to mitigate these risks are essential for successful transplantation.
Purpose of Study
- To refine surgical techniques for intraportal islet transplantation.
- To reduce the incidence of bleeding-related deaths in mice during surgery.
- To facilitate further research in liver cancer and liver diseases.
Methods Used
- Detaching human islet cultures using gentle scratching.
- Using a 1 cubic centimeter syringe to collect 300 to 350 islets.
- Storing islets in sterile 1.5 milliliter microcentrifuge tubes on ice.
- Implementing refined surgical procedures to minimize complications.
Main Results
- Both surgical methods effectively prevented bleeding-induced deaths.
- Improved techniques enhance the safety of islet transplantation in mice.
- Methods can be adapted for research involving liver cancer and other diseases.
- Successful islet collection and transplantation were achieved with minimal complications.
Conclusions
- Refined surgical procedures significantly improve outcomes in mouse islet transplantation.
- These methods can advance research in liver-related conditions.
- Further studies are warranted to explore the applications of these techniques.
Frequently Asked Questions
Executive Industry Relevance
Intrahepatic islet transplantation in mice is a technically demanding procedure where post-infusion portal vein bleeding leads to high mortality, limiting preclinical study of islet survival and function. The demonstrated hemostatic techniques reduce procedure-related mortality, enabling reliable preclinical models for diabetes research and translational islet therapeutics. These refinements support mechanistic de-risking in early discovery by improving model reproducibility and reducing biological variability from surgical complications.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables consistent islet engraftment and survival studies by minimizing procedure-induced mortality confounders.
- Operational Value: Standardizes surgical outcomes across laboratories, reducing inter-experiment variability in islet transplantation models.
Screening & Assay Development
- Scientific Value: Provides a stable platform for longitudinal assessment of islet function and graft survival in preclinical diabetes research.
- Operational Value: Supports reproducible preparation of transplanted livers for downstream histological and functional assays.
Translational & Preclinical Research
- Scientific Value: Mimics clinical intraportal transplantation route, enhancing translational relevance of murine islet transplantation studies.
- Operational Value: Facilitates dose-response studies linking islet mass to glycemic outcomes, supporting predictive confidence in therapeutic dosing.
Pipeline & Workflow Integration
The refined surgical methods position intrahepatic islet transplantation as a reliable preclinical model within the diabetes discovery continuum, supporting hypothesis testing and lead identification phases.
- Discovery Biology: Enables mechanistic studies of islet engraftment, immune response, and graft function by reducing surgical mortality artifacts.
- Screening: Produces standardized transplanted models suitable for evaluating islet viability and function under controlled conditions.
- Analytics: Generates quantifiable endpoints such as blood glucose levels, insulin staining distribution, and graft survival rates for comparative analysis.
- Translational Research: Aligns with clinical transplantation route, supporting preclinical validation of islet products before moving to larger animal models.
- Enterprise Reuse: Establishes a reproducible surgical platform that can be shared across diabetes research programs for consistent model generation.
Operational & Enterprise Impact
- Scientific Value: Reduces biological noise from procedure-related deaths, improving data quality in islet survival and functional studies.
- Operational Value: Increases successful transplantation rates, lowering resource waste and accelerating preclinical timelines.
- Strategic Value: Enhances go/no-go decision confidence by minimizing model failure due to technical variability.
- Portfolio Impact: Supports risk-adjusted advancement of islet-based therapies by providing reliable preclinical efficacy and safety data.
Implementation Considerations
- Requires microsurgical expertise in rodent vascular access and tissue handling.
- Depends on availability of absorbable hemostatic agents (gelatin sponge) and precise needle manipulation skills.
- Necessitates standardized postoperative care protocols including analgesia, thermal support, and monitoring.
- Adaptation across model systems may require adjustments based on vascular anatomy and fat tissue availability.
- Practical limitation: effectiveness depends on proper technique execution; inconsistent application may reduce hemostatic efficacy.
