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

Summary

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.

Abstract

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.

Introduction

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 a....

Protocol

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....

Results

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.......

Discussion

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.<.......

Materials

List of materials used in this article

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