Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Podsumowanie

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Streszczenie

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

Wprowadzenie

Partial colectomy with a primary anastomosis is a commonly performed surgery that can be done for many diseases affecting the colon such as colorectal cancer, Crohn's disease and diverticulitis^1,2. The most dreaded complication after colorectal anastomosis is anastomotic leakage³. Although several risk factors associated with anastomotic leaks have been identified, solutions for preventing leakage remain unkown^4,5.

Adipose tissue-derived stromal cells (ASCs) are associated with anti-inflammatory and trophic properties^6,7, which makes these cells promising candidates for regenerative therapies⁸. The effectiveness of ASCs to promote tissue healing was shown in various tissues such as cardiac muscle, skin, and oesophagus^{9,10,11,12,13}. However, there are few reports on the use of ASCs to promote intestinal healing. Local transplantation of ASCs to experimental colorectal anastomoses via ASC-coated biosutures or serosal injections of ASCs showed either no improvement in healing¹⁴ or did not prevent anastomotic leakage despite a more favourable anastomotic healing¹⁵.

Local transplantation of ASCs in suspension or combined with biomaterials might be associated with insufficient cell retention or an inadequate distribution of transplanted cells¹¹. Cell sheet technology^16,17 offers an innovative method of ASC delivery^18,19. Therefore, in a previous study, a novel approach was proposed in which ASCs organized in a cell sheet could be applied on experimental colorectal anastomosis²⁰. This study demonstrated that ASC sheet transplantation is successful in reducing colorectal anastomosis leakage after partial colectomy in a rat model. This article reports ASC sheet preparation and surgical transplantation technique.

Protokół

Subcutaneous abdominal adipose tissue was obtained from human donors with approval of the Medical Ethical Committee (#MEC-2014-092), Erasmus MC University Medical Center, Rotterdam, The Netherlands. All animal experiments were approved by the Ethical Committee of Animal Experimentation, Erasmus MC University Medical Center, Rotterdam, The Netherlands (133-14-01).

1. Human ASCs Isolation and Culture

1. Prepare 3 mL of isolation medium for 1 g of adipose tissue. Mix low glucose Dulbecco's Modified Eagle's medium (LG-DMEM) with 10 g/L Bovine Serum Albumin (BSA) and 1 g/L collagenase type 1.
2. Dissect human subcutaneous abdominal adipose tissue (n = 8, all female, mean age 40 ± 9 years old) to small pieces (0.5 cm) using a sterile surgical blade size 10, Adson-Brown tissue forceps and Metzenbaum scissors in a sterile Biological SafetyCabinet.
3. Store the dissected tissue in a sterile glass media storage bottle. Weigh the total amount of dissected tissue and start the digestion with the prepared isolation medium (50 g of adipose tissue with 150 mL of isolation medium per bottle). The protocol can be paused here by storing the bottle with the dissected adipose tissue and isolation medium at 4 °C on a roller mixer overnight. Then prewarm the bottle the next day to room temperature 25 °C for 15 min before continuing with the next step.
   NOTE: Subcutaneous abdominal adipose tissue was obtained as leftover material from donors undergoing breast reconstruction surgery with approval of the Erasmus MC Medical Ethical Committee (# MEC-200) and according to the Code of Conduct: "Proper Secondary Use of Human Tissue" (<http://www.federa.org>). Leftover adipose tissue was only used from donors who did not opt-out to secondary use.
4. Digest the dissected adipose tissue in a sterile glass media storage bottle in a shaker-incubator at 150 rpm and 37 °C for 1 h.
5. Divide the digested solution into 50 mL tubes and centrifuge the tubes for 10 min at 390 x g.
6. Prepare culture medium: LG-DMEM supplemented with 10% fetal bovine serum (FBS), 50 µg/mL gentamicin and 1.5 µg/mL ampothericin B.
7. Following the centrifugation, remove the supernatant and resuspend the cell pellet in 20 mL of culture medium (LG-DMEM supplemented with 10% fetal bovine serum (FBS), 50 µg/mL gentamicin and 1.5 µg/mL ampothericin B). Centrifuge the cells again for 5 min at 390 x g and remove the supernatant.
8. Resuspend the cell pellet with 10 mL of culture medium (LG-DMEM supplemented with 10% FBS, 50 µg/mL gentamicin and 1.5 µg/mL ampothericin B) and filter the cell suspension through a 100 µm filter.
9. Add 50 µL of 3% acetic acid/methylene blue solution (for red blood cell lysis) to 50 µL of cell suspension, mix the solution and use 10 µL to count the cells. Perform cell counting with a hemocytometer. Then plate the cells at a density of 40,000 cells/cm² in culture medium (LG-DMEM supplemented with 30% FBS, 50 µg/mL gentamicin and 1.5 µg/mL ampothericin B).
10. Incubate the cells at 37 °C in a humid atmosphere with 5% CO₂ for 24 h.
11. Following the incubation, wash the cells with warm (37 °C) phosphate buffered saline (PBS) to remove cell debris and replace the media with 10% FBS culture medium with freshly added ascorbic acid-2-phosphate (25 µg/mL) and human recombinant fibroblast growth factor 2 (FGF2, 1 ng/mL).
12. Subculture ASCs at 90% confluence using standard 0.25% trypsin EDTA solution (3 mL for a T175 flask). After 3-5 min, neutralize the 0.25% trypsin EDTA solution with 10 mL of culture medium (LG-DMEM supplemented with 10% FBS, 50 µg/mL gentamicin and 1.5 µg/mL ampothericin B).
    NOTE: Flow cytometry analysis using common ASC surface markers and tri-lineage differentiation assays were performed previously and revealed that ASCs isolated using this protocol displayed ASC specific characteristics^21,22.
13. Wash the cells with 10 mL of culture medium (LG-DMEM supplemented with 10% FBS, 50 µg/mL gentamicin and 1.5 µg/mL ampothericin B) for a T175 flask and centrifuge the cells for 8 min at 250 x g. Remove the supernatant and resuspend the cells in 1 mL of culture medium. Count the cells with a hemocytometer.
14. Plate the cells in T175 culture flasks at a density of 8,000 cells/cm².
15. Freeze the remaining ASCs in liquid nitrogen with 10% dimethyl sulfoxide (DMSO) in culture medium before further use.

2. ASC Sheet Preparation

1. Pre-coat thermo-responsive culture dishes (3.5 cm diameter) with 1 mL of FBS, then place the dishes in an incubator at 37 °C for at least 30 min before cell seeding.
2. Trypsinize ASCs using standard 0.25% trypsin EDTA solution for 3-5 min (3 mL for a T175 culture flask). If there are any cell clumps after trypsinization, filter the cells through a 100 µm filter before counting.
3. Neutralize the trypsin solution with LG-DMEM supplemented with 10% FBS (10 mL for a T175 flask) and centrifuge the cell suspension for 8 min at 250 x g. Remove the supernatant and resuspend the cells in 1 mL of LG-DMEM supplemented with 10% FBS.
4. After coating the thermo-responsive culture dishes with FBS, move the dishes from the incubator to a warming plate at 37 °C and remove FBS from the dish.
5. Seed ASCs at 400,000 cells/cm² (in total, 3.52 × 10⁶ cells diluted in 2 mL of culture medium per dish).
6. Carefully distribute the cells as evenly as possible by gently swinging the dish.
7. Culture ASC sheets for 48 h at 37 °C in a humid atmosphere with 5% CO₂.
   NOTE: Try to minimize opening the door of the incubator after seeding ASCs to prevent temperature drops that may interfere with ASC sheet formation or cause premature detachment of formed ASC sheets.

3. Partial Colectomy and Colorectal Anastomosis

1. Induce and maintain Male Wistar rats (weighing between 250-350 g) with 1 L/min of isoflurane (1.5-5%)/oxygen inhalation and inject 0.05 mg/kg buprenorphine intramuscularly.
2. Once the animals are under general anesthesia, assess anesthetic depth using reflex testing. Apply vitamin A containing eye ointment to the eyes to prevent dryness. When anesthesia is considered sufficient for surgery, aseptically prepare the abdomen by shaving hairs and spraying the surgical area twice with 70% ethanol. Drape the abdomen with sterile paper drapes. Use an aseptic technique and maintain the sterile field during the whole surgical procedure.
3. Make a midline abdominal incision of 5 cm with a sterile surgical blade size 10 and extend the incision with Metzenbaum scissors. Identify and exteriorize the colon and pack the colon off from the remainder of the abdominal cavity with saline moistened gauzes. Identify the right, middle, and left colic arteries in the mesentery, bluntly dissect around each vessel using Halsted mosquito and ligate each individual vessel with non-absorbable braided silk 4/0.
4. Isolate the colonic segment between 1.0 cm aborally to the cecum and 0.5 cm above the caudal mesenteric artery. Transect through healthy colon using Metzenbaum scissors. After the resection of the colonic segment, bring the proximal and distal ends of the colon together by introducing two long cotton swabs trans-anally through the distal colon, and then into the proximal colon end.
5. Using a surgical microscope, create an insufficiently sutured end-to-end anastomosis by one-layer inverting suturing using 5 interrupted sutures (non-absorbable monofilament polyamide 8/0). Place the interrupted sutures through all layers of the colon wall and position the knots extraluminally.
6. Divide the rats randomly into control or ASC sheet group.

4. ASC Sheet Transplantation

1. Allow the culture dishes to cool down to room temperature 40 min before in vivo transplantation, to facilitate ASC sheet detachment.
   NOTE: After the detachment, ASC sheets shrink to approximately 2 cm in diameter. ASC sheet viability remains high for at least 3-6 h after the detachment²⁰.
2. Remove the culture medium and replace it with 1 mL of serum free LG-DMEM.
3. Gently grab the rims of the ASC sheet with atraumatic forceps and place the dish-side of the ASC sheet on top of the anastomotic line.
4. Carefully stretch out the ASC sheet approximately 0.25 cm above and below the anastomotic line using the atraumatic forceps and wrap the sheet around the colon. Lift the colon up to wrap the ASC sheet around the dorsal side.
   NOTE: ASC sheets adhere spontaneously to the anastomotic line, there is no need to use tissue glue or any other biomaterial. The control group does not receive any additional treatment.
5. Remove the cotton swabs and change the gloves and instruments. Replace the colon in the abdomen. Close the abdomen-linea alba with one layer of running sutures using absorbable braided polyglycolic acid 5/0. Suture the subcutis and the cutis together in one layer of running sutures using absorbable braided polyglycolic acid 5/0.

5. Post-operative Evaluation and Follow-up

1. Immediately rehydrate the animals with a subcutaneous injection of 5 mL warm saline postoperatively. Place the animals under a heat lamp to maintain body temperature and monitor the vital signs until animals regain sufficient consciousness to maintain sternal recumbency.
   NOTE: Animals that had undergone surgery were not returned to the company of other animals until fully recovered. After recovery, allow free access to water and regular rat chow. For post-surgical pain relief, administer 0.05 mg/kg buprenorphine subcutaneously every 6-8 h for 3 days. No antibiotics were administered at any time in this study.
2. Obtain daily clinical evaluations of wellness and weight. In case of a very low wellness score or severe weight loss, animals should be euthanized by exsanguination via cardiac puncture while still under anesthesia.
3. On postoperative day 3 or day 7, anesthetize the rats again with 1 L/min of isoflurane (1.5-5%)/oxygen inhalation without buprenorphine, and perform a re-laparotomy with a U-shaped incision. Check the abdomen for the signs of peritonitis, stricture, fibrous adhesions, and the presence of abscesses. Score the severity of adhesions and abscess both in the abdomen and at the anastomotic area.
4. Determine the anastomotic bursting pressure by the insufflation of air in a closed segment of the colon including the anastomosis. Record the air pressure and the place of rupture when the first air leak as bursting pressure.
5. Remove the colon from each sacrificed animal and fix the cut segment of the colon -containing the colorectal anastomosis- with 4% buffered formaldehyde, following by paraffin embedding and cutting the colon into 4 μm thick sections.
6. Euthanize the animals while under anesthesia directly after collection of the anastomosis segment by exsanguination via cardiac puncture. Confirm animal death with the absence of breathing movement and a heartbeat.
7. Perform histochemical stainings such as Hematoxylin & Eosin and Picro Sirius red, and immunohistochemical stainings with antibodies against human mitochondria, rat endothelial cells (anti-CD34) and cells of the immune system (CD3+, CD163+).
8. Digitalize the slides for computerized analysis and compare the animal groups.

Wyniki

A flow chart of this study depicting both ASC sheet culture and the procedure of colon resection and anastomosis is shown in Figure 1. Figure 2 shows ASC sheet microscopic morphology and the macroscopic appearance of the ASC sheet during and after the detachment. Figure 3 shows the different steps of ASC sheet detachment and transplantation. Figure 4 shows the presence o...

Dyskusje

Anastomotic leakage is the most serious adverse event following colon resection with a primary anastomosis. Optimal techniques to prevent anastomotic disruption and leakage are still lacking. Local application of an array of biomaterials has been conducted, with varying results^25,26,27. The aim of cell therapies is to facilitate tissue repair by tissue replacement or the stimulation of local healing through paracrine secretion.<...

Materiały

Name	Company	Catalog Number	Comments
LG DMEM	Gibco, Life technologies	22320022	ASC isolation and culture
Collagenase type I	Gibco, Life technologies	17100-01	ASC Isolation
Bovine Serum Albumin	Sigma-Aldrich	A9418	ASC Isolation
Fetal bovine serum	Gibco, Life technologies	10270-106	FBS, ASC isolation and culture
3% acetic acid with methylene blue	Stemcell Technologies	7060	ASC Isolation
Gentamicin	Gibco, Life technologies	15750-037	ASC isolation and culture
Ampothericin B	Gibco, Life technologies	15290-018	ASC isolation and culture
Shaker (Gallenkamp Environmental Shaker Model 10X 400)	Akribis Scientific	F240	ASC Isolation
Centrifuge	Hettichlab	Rotina380	ASC isolation and culture
Phosphate buffer saline	Gibco, Life technologies	14190-094	PBS, ASC isolation and culture
Ascorbic acid-2-phosphate	Sigma-Aldrich	A8960	ASC isolation and culture
Human recombinant fibroblast growth factor 2	AbD Serotec	AF-100-15	FGF2, ASC isolation and culture
Trypsin EDTA	Gibco, Life technologies	25200-056	ASC culture
Dimethyl sulfoxide	Sigma-Aldrich	D2650-5x	DMSO, ASC freezing
Thermo-responsive culture dishes	Nunc Thermo scientific	174904	ASC sheet preperation
Non-absorbable braided silk 4/0	B Braun	21151042	surgery
Non-absorbable monofilament polyamide 8/0	B Braun	G1118170	surgery
Absorbable braided polyglycolic acid 5/0	B Braun	C1048207	surgery