This video will demonstrate our technique for generating tissue engineered small intestine in the mouse using an organoid units on scaffolded approach. In this experiment, animals were treated in accordance with institutional animal care and use committee and the Children's Hospital, Los Angeles Savon Research Institute Animal Care Facility guidelines. The first step is the procurement of whole mouse intestine.
The humanely euthanized mouse and instruments are prepped with 70%ethanol in water, and a midline incision is made to gain entry to the peritoneal cavity. The small bowel is eviscerated and divided just distal to the ligament of trites. It is then sharply and bluntly dissected away from the mesentery and its vessels.
Finally, the ileum has transected five millimeters Proximal to the ileocecal valve. The harvested small intestine is shown to scale. Here we proceed to open The intestine lengthwise along the anti mesenteric border in a hank's buffered saline with one X antibiotic antifungal solution.
The intestine is then cleared a fecal Matter in the dish, followed by three repeat washings with HBSS and a test tube. The intestinal tissue sinks to the bottom of the tube and a floating material is Mesenchymal debris and can be discarded. The washing solution is removed carefully with a pipette.
The intestine is then minced to less than one millimeter square Pieces using scissors. The minced material is gathered up with an automatic pipette. The minced wash material is now ready for digestion.
The digestion solution contains DASE 0.125 milligrams per milliliter and collagenase 800 units per milliliter in HBSS with antibiotic antifungal. Note the brownish color of the digestion solution compared to the HBSS with antibiotic antifungal. It is freshly prepared and 10 milliliters are added to the mince material.
This is incubated at 37 degrees Celsius for 20 minutes on a rotator. The tissue is further disrupted by iteration with a pipette as shown here. This is repeated from 20 to 50 times until a uniform appearance is obtained.
The 10 milliliter pipette tip is optimally Sized. For this, the reaction is stopped With three milliliters of four degree DMEM with 10%fetal bovine serum. The material is centrifuged for 10 minutes.
At 500 RPM, the supernatant is discarded and the pellet is resuspended in a wash of 10 milliliters of four degree DMEM with 10%fetal bovine serum. This is then centrifuge for five minutes at 800 RPM to recover all tissue except for single cells which remain in the S supernatant and are discarded. The pellet contains the organoid units, the lighter material at the top, indicated by the forceps and waist material.
Below this layer, the DMEM with serum is removed with a pipette. Care is taken not to suspend the pellet in the pipette. The last few drops can be removed more safely with the disposable plastic pipette.
Shown here is the polyglycolic acid or PGA polymer scaffold used in our experiments. The distal two millimeters of a disposable 1000 microliter pipette tip are trimmed with sterile scissors. We load the polymer with organoid units in four wall culture plates to minimize spread of the material away from the polymer.
The organoid units are loaded onto the scaffold with the pipette first into the lumen, and then On the outer surface. A second forceps is used to ensure Adequate coating of the lumen care is taken not to disrupt the cylindrical shape of the polymer. The host mouse used in the case of a transgenic donor is an immunocompromised non-obese diabetic.
Severe combined immunodeficient gamma chain deficient or nod skid gamma animal. Otherwise, syngeneic hosts can be implanted for implantation. After general anesthesia is induced, the abdomen is shaved, prepped and draped.
A 0.5 centimeter Midline incision is made. The greater Omentum is identified and carefully eviscerated. The loaded polymer is wrapped in the omentum and secured with a five oh Monocryl suture.
It is critically important that the omentum is not torn, or the cells may leak into the Peritoneum. The Loaded polymer Secured the omentum is replaced in the abdomen, which is closed in layers. A four oh Vicryl suture is used in an interrupted fashion to close.
First the muscle layer and then the skin for Postoperative analgesia, two milligrams per kilogram. Ketoprofen in sterile water is injected as a subcutaneous wheel adjacent to the incision four weeks after implantation, the host animal is humanely euthanized and the original incision is reopened. The tissue engineered construct is identified as a globe and may be adherent to the stomach and intraabdominal viscera.
The adhesions are sharply taken down and a steroid of tissue engineered Intestine is retrieved. Shown here is the harvested Steroid in direct comparison to the starting polymer. In this image, the lumen of the engineered intestine has been opened sharply to reveal its interior.
The tissue can be fixed for histologic analysis or used freshly for various molecular biologic assays, as we have demonstrated in other publications. Shown here is a low power hematin and eosin stain micrograph of a typical successful tissue engineered small intestine construct. The left arrow indicates the lumen lined with intestinal mucosa, and the right arrow shows host pancreas.
That has adhered to The construct.
