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.
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.
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 diverticulitis1,2. 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 unkown4,5.
Adipose tissue-derived stromal cells (ASCs) are associated with anti-inflammatory and trophic properties6,7, 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 oesophagus9,10,11,12,13. 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 technology16,17 offers an innovative method of ASC delivery18,19. 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.
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
2. ASC Sheet Preparation
3. Partial Colectomy and Colorectal Anastomosis
4. ASC Sheet Transplantation
5. Post-operative Evaluation and Follow-up
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 of the ASC sheet at the anastomotic line and prevention from leakage 3 days postoperatively.
The follow-up period allowed for colorectal anastomosis evaluation. The different assessments are shown in the figures and table. Compared to control animals, the transplanted animal groups showed less frequent anastomotic dehiscence and leakage on postoperative day 3 and less frequent abscess formation on postoperative day 7. Compared to control animals, transplanted animals did not develop significant adhesions or stricture formation. Table 1 shows the macroscopic findings at the end of the follow up periods in transplanted and non-transplanted animals.
Figure 1: Study flow chart. A) ASCs were isolated and expanded before seeding onto thermo-responsive culture dishes. ASCs were seeded at a density of 400,000 cells/cm2 and cultured for 48 h before the transplantation. On the day of transplantation, ASC sheet were detached by allowing the thermo-responsive culture dish to cool down to room temperature for 30 min. B) Scheme of surgical procedure; after ligation of supplying vessels a partial colectomy and colorectal anastomosis is performed. An ASC sheet was wrapped around the anastomosis in the ASC sheet group; a) caecum, b) terminal ileum, c) anus. The schematic overview of the surgical procedure was partly adapted with permission from Wu Z. et al.24 Please click here to view a larger version of this figure.
Figure 2: ASC sheet before and after detachment. A) ASC sheet in thermo-responsive culture dishes after 48 h of culture 40x magnification. ASC sheets were obtained by culturing ASCs on commercial thermo-responsive culture dishes. B-C) ASC sheet during (B) and after detachment (C). When the thermo-responsive culture dishes were allowed to cool down to room temperature, the ASC sheet spontaneously detached from the dish surface as one intact ASC sheet. No enzymatic treatment was necessary. The size of the ASC sheet reduced after the detachment. Please click here to view a larger version of this figure.
Figure 3: ASC sheet transplantation. A) Depiction of ASC sheet orientation in the thermo-responsive culture dish.26 B) Orientation of ASC sheet after the transplantation. The dish side of the ASC sheet was placed on top of the serosal surface of the anastomotic line (anastomotic line is indicated with crosses). C) Intra-operative views of the different steps of transplantation. Two atraumatic forceps were used to lift up the ASC sheet and wrap it around the anastomotic line. White arrows indicate ASC sheet location. The images of transplantation were partly adapted with permission from Sukho, P., et al.20 Please click here to view a larger version of this figure.
Figure 4: Colorectal anastomosis macroscopic and histological evaluation. A-B) Compared to the control group, macroscopic observation showed reduced leakage and abscess formation at post-operative day 3 (A) and 7 (B), respectively.White arrows point at anastomotic area and black arrows point at transplanted ASC sheets.C) Representative cross-sections of the colorectal anastomosis site in transplanted animals stained with H&E. The sheet structure could be identified at the anastomosis site up to 7 days postoperatively.The images were partly adapted with permission from Sukho, P., et al.20 Please click here to view a larger version of this figure.
Post-operative day 3 | Post-operative day 7 | |||||
Control (%) | ASC sheet (%) | p-value | Control (%) | ASC sheet (%) | p-value | |
Peritonitis | 1/14(7.1) | 0/14(0) | NS | 0/15(0) | 0/14(0) | NS |
Anastomotic disruption | 10/14(71.4) | 2/14(14.3) | 0.002 | 3/15(20) | 2/14(14.3) | NS |
Stricture | 2/14(14.3) | 2/14(14.3) | NS | 2/15(13) | 2/14(14.3) | NS |
Abscess at anastomosis | 14/14(100) | 12/14(85.7) | NS | 10/15(66.7) | 4/14(28.6) | 0.04 |
Adhesion at anastomosis | 14/14(100) | 14/14(100) | NS | 15/15(100) | 14/14(100) | NS |
Abscess elsewhere | 11/14(78.5) | 8/14(57.1) | NS | 6/15(40) | 4/14(28.6) | NS |
Adhesion elsewhere | 8/14(57.1) | 3/14(21.42) | NS | 9/15(60) | 7/14(50) | NS |
Table 1: Post-operative macroscopic findings.
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 results25,26,27. The aim of cell therapies is to facilitate tissue repair by tissue replacement or the stimulation of local healing through paracrine secretion.
In this rat model, ASC sheet transplantation was technically successful. Clinical and histological evaluations demonstrated the effectiveness of the ASC sheet at reducing leakage after colon resection with primary anastomosis.
The transplantation of an ASC sheet to colorectal anastomoses is a novel approach in colorectal surgery. In this study, for the first time, a high-density ASC sheet was transplanted. The choice for ASCs delivered as a cell sheet was based on the previously reported advantages of cell sheet technology28,29 over conventional cell transplantation techniques. In addition, the choice for ASCs was based on multiple studies that demonstrated their anti-inflammatory and trophic abilities, especially in cardiac muscle- and skin-healing11,12,19,32.
ASC sheet preparation and detachment can be challenging. In some ASC donors, ASC sheets detached prematurely from the culture dish and folded, precluding their use. In this study, the exact cause for this problem in these specific donors was not identified. Since a high consumption of culture medium was notified in these donors, it was assumed that individual variations in proliferation rate may play an important role. Serum coating assisted in cell adhesion to the surface. Together with careful distribution of the cells in the culture dish and minimizing opening of the incubator door after seeding ASCs (preventing temperature drops), less folding of the ASC sheets occurred. In this way, ASC sheets from all donors were successfully transplanted, confirming the feasibility of the technique.
On the day of the transplantation, most ASC sheets spontaneously detached after allowing the thermo-responsive culture dish to cool down to room temperature. However, a limited number of sheets did not fully detach. Gentle shaking of the culture dish or soft flushing at the rim using a pipette eventually assisted in complete detachments.
For successful ASC sheet transplantation to colorectal anastomoses, several critical steps need to be addressed. First, excessive blood at the anastomosis site should be removed to ensure the contact between the ASC sheet and the serosal surface. Second, the dish side of the ASC sheet should be placed on the serosal surface of the anastomosis. It is postulated that the function of cell sheets after harvest is maintained due to the preservation of cell surface proteins and cell-to-cell junction proteins. Additionally, the ability of ASC sheets to spontaneously adhere to the serosal surface in a short time might be facilitated by the presence of deposited ECM that is produced during in vitro culture19,20,21. Third, the size of the ASC sheet and the colon diameter should be comparable to allow complete coverage of the anastomosis site. When larger surfaces need to be covered, the use of several sheets can be considered.
There are some limitations of this protocol for ASC sheet preparation and transplantation. When using thermo-responsive culture dishes to prepare the ASC sheet, the temperature should strictly be maintained at 37 °C during the entire process to prevent premature detachment. After the transplantation, the ASC sheet survival rate is unknown. Although previous experiments showed that ASCs were viable at 3 days postoperatively, mitochondrial activity of these ASCs was diminished at 7 days postoperatively17. The cell survival rate and dose effect of the transplantation are important questions that need to be addressed in future studies. Observer bias cannot be completely ruled out since the ASC sheet can be identified macroscopically and microscopically at post-operative evaluations. Although transplanted animals did not show more postoperative adhesion formation compared to control animals17, the evaluation of the colorectal anastomosis can be hindered by the presence of these intra-abdominal adhesions.
An advantage of this application technique is that it is simple and easy to perform. With only a small amount of practice and using universal atraumatic forceps, intestinal surgeons should be able to wrap the ASC sheet around the anastomosis. In addition, surgery time is not greatly affected since ASC sheets immediately adhere to host tissues. Moreover, when using ASC sheets, no synthetic biomaterial remains in the body minimizing the risk of a foreign body reaction.
Transplanted ASCs organized in cell sheets demonstrated their ability to reduce colorectal anastomosis leakage. In view of these promising results, future studies should be conducted to evaluate long-term effects both for therapeutic and safety reasons. Besides that, several methods have been described to modify the therapeutic effects of ASC sheets such as inducing overexpression of vascular endothelial growth factor30. These methods may further enhance tissue repair using ASC sheets and should be considered in future studies.
The authors have nothing to disclose.
The authors are grateful to Prof. Dr. S.E.R. Hovius, Dr. M.A.M. Mureau and all surgeons of the department of Plastic Surgery for the collection of subcutaneous adipose tissue. P. Sukho is supported by a grant from The Netherlands Fellowship program (NFP-12/435), during the conduct of the study. Y.M. Bastiaansen-Jenniskens is supported by grants from Dutch Arthritis Foundation (LP11).
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 |