Here, we present a protocol for in vivo validation of hydrogel-based cell therapy, illustrated by the example of islet transplantation. h-Omental Matrix Islet filliNG (hOMING) implantation allows implantation of a cell-hydrogel mixture between the omental layers, near to blood vessels, to maximize engraftment in a proper metabolic environment.
Regenerative medicine based on cell therapy represents a new hope for curing disease. Current obstacles include proper in vivo validation of the efficiency of the therapy. For transfer to the recipient body, cells often need to be combined with biomaterials, especially hydrogels. However, validation of the efficacy of such a graft requires the right environment, the right hydrogel, and the right recipient site. The omentum might be such a site. Based on the example of islet transplantation, we developed the hOMING (h-Omental Matrix Islet filliNG) technique, which consists of the injection of the graft inside the tissue, in between the omental layers, to improve islet implantation and survival. To achieve this, islets have to be embedded in a hydrogel with a viscosity that enables its injection using an atraumatic needle. Syringes are loaded with a combination of hydrogel and islets. Several injections are performed inside the omental tissue at different entry points, and the deposition of the islet/hydrogel mixture is made along a line. We tested the feasibility of this innovative approach using dextran beads. The beads were well spread throughout the omental tissue, in close proximity to blood vessels. To test the efficacy of the graft, we transplanted islets into diabetic rats and perform a metabolic follow-up over two months. The transplanted islets exhibited a high rate of re-vascularization around and inside islets, and reversed diabetes. The hOMING technique could be applicable for other types of hydrogel or cell therapy, for cells with high metabolic activity.
Cell therapy is a hot topic, as it aims to cure diseases based on the regenerative medicine. Biological material-assisted cell therapies have been increasingly studied in recent years, especially because cell implantation often requires a carrier for the transfer of the cells from the culture dish to the recipient. Biomaterial scaffolds are potentially valuable cell carriers that fulfill several roles1. A competent carrier should protect cells from mechanical stresses and provide favorable growth conditions, such as essential growth factors, metabolic waste excretion, exchange of nutrient substances, and oxygen2.
Among the different types of biomaterials used in cell therapy, hydrogels have many advantages. They are biocompatible, biodegradable, easy to handle, and facilitate oxygen diffusion3. Furthermore, current technology allows the use of hydrogels to help cells survive and engraft with, for example, supplementation with growth factors or extra-cellular matrix proteins4.
Hydrogel carriers containing stem cells can be injected as treatments, e.g., bone regeneration5 and nervous system disease6. The implantation of metabolically active cells is needed. While in vitro validation of the approach is possible, tools and techniques for in vivo validation remain to be refined.
Cell and hydrogel transplantation can easily be carried out by subcutaneous injection when biocompatibility tests are conducted. However, when grafted cells are meant to regulate systemic factors by their metabolic action, this subcutaneous localization is not optimal, essentially in terms of venous drainage7. Therefore, there are no current tools to quickly, safely, and efficiently evaluate the beneficial effects of a hydrogel. Based on the example of islet transplantation, which requires that hormones be released into the bloodstream from the graft in response to blood glucose levels, we developed a new method for cell/hydrogel implantation in vivo.
The first step was to identify an acceptor transplantation site, which can accept a hydrogel with cells. The omentum offers a large space for implantation, is highly plastic, and its dense vascularization combined with the intraperitoneal setting is interesting for studying cells having high metabolic activity8. We next needed to establish a surgical technique allowing the transfer of the cells and the hydrogel into the omentum. Inspired by the lipofilling used in plastic surgery9, we developed the h-Omental Matrix Islet filliNG (hOMING) approach. Islets embedded in hydrogel are injected inside the omental tissue. The technique also aimed to provide maximum engraftment by utilizing multiple depositions of the cell and hydrogel mixture into the omental tissue, where the large number of blood vessels also improves graft oxygenation.
In the present study, we describe a simple and innovative technique for islet implantation between omental sheets, inside the fat tissue closest to the blood vessels. This consists of micro-invasive surgery, which could be completed under laparoscopy, with the injection of islets contained in a hydrogel into the fat tissue. This technique is easily applicable to all hydrogel and cell combinations that need to be tested in a in a metabolically functional environment.
All animal experiments were performed according to the National Institutes of Health guidelines, with the authorization number: AL/60/67/02/13.
1. Recipient Preparation
2. hOMING: Intra-omental Matrix Islet Filling
3. Omental Graft Explantation
NOTE: This procedure will permit the confirmation of good graft function. After retrieval of a functional graft, rats should return to a diabetic state. This step is performed after 1 or 2 months of metabolic follow-up.
Figure 1: Omental artery distribution. For omentum graft explantation, the critical area composed of gastroepiploic arteries is represented in blue. During resection of this part of the omental tissue, attention must be to be paid to the right gastroepiploic artery section. Compression, ligature, or cauterization can be used to limit bleeding. Please click here to view a larger version of this figure.
4. Histological Analysis: Hematoxylin and Eosin Staining
5. Statistical Analysis
The hOMING method permits the avoidance of intravascular implantation and the confinement of islets in an organ. A maximum time of 8-10 min is required for the entire islet implantation procedure, including anesthesia, which a timeline comparable to classical liver transplantation.
To study the way islets are distributed inside the omental tissue, dextran beads were transplanted using the hOMING method (Figure 2). One day after implantation, rats were sacrificed, and omental tissues were retrieved for histological analysis. Hematoxylin and eosin staining revealed a uniform distribution of the beads throughout the tissue (Figure 2, bottom right). Very often, beads were close to blood vessels and were well-implanted in the fat tissue. Immediately after implantation, an inflammatory reaction occurs around the beads, resulting in tissue rearrangement to nest the islets in the tissue.
Figure 2: Description of hOMING technique and bead distribution through the omental tissue one day after implantation. (A) Illustration of the hOMING technique. After organ exposure (A, left), the islet-hydrogel mix (replaced here by blue-colored dextran beads for better visualization) was carefully injected in the tissue using an atraumatic needle (A, middle). Beads implanted in the tissue are visible (A, right). (B) Hematoxylin and eosin staining of omentum explanted 1 day after bead injection. Beads are found in the tissue with a uniform distribution. Scale bar = 100 mm. Please click here to view a larger version of this figure.
To validate this technique, we performed isogenic studies using Lewis rats (n=8). Diabetic rats receiving islet transplants (7660 islet equivalent, IEQ) per kg rat body weight using hOMING were monitored for glycemia and C-peptidemia for two months. Glycemia was controlled by implantation of insulin pellet (as attesting to the first drop in glycemia observed in the Figure 3A). Graft function was reflected by glycemia of approximately 2 g/L and C-peptidemia >500 pM. Before transplantation, rats were diabetic (glycaemia >5 g/L and C-peptidemia <200 pM). After transplantation and insulin pellet retrieval, glycemia maintenance and normalization was observed just 3 days post-hOMING transplantation and was maintained until graft retrieval (p < 0.05 compared to pre-transplant levels). After omental explantation, glycemia rose again to the pre-transplant level, attesting to the functionality of islets that were transplanted by hOMING (Figure 3A). The C-peptidemia pattern was exactly the opposite, with low to undetectable levels before the graft, followed by an increase and maintenance at this increased level throughout the course of the study (p <0.05), and, after omental explantation, a decrease to pre-transplant levels (Figure 3B). Analysis of the explanted omentum by histology revealed highly re-vascularized islets, most likely as a result of their proximity to blood vessels (Figure 3C).
Figure 3: Two-month metabolic follow-up of rats receiving hOMING and graft assessment. (A) Glycemia measurement and (B) C-peptide assessment after hOMING using alginate as an islet carrier (Tx: Transplantation and insulin pellet retrieval; Explantation: Explantation of the omentum). Grafts are functional, as is shown by the maintenance of normoglycemia after insulin pellet retrieval and increase in C-peptidemia after islet implantation. Gray shaded areas represent the minimal and maximal recorded values at each time point. (C) Hematoxylin and eosin staining of an omental section after islet transplantation using the hOMING method. Islets are well-integrated into the tissue two months after implantation without any surrounding fibrotic tissue. Vessels have grown around and inside islets, as shown by the arrows, and thus completely restore islet function. Morphology of the islets also seems well-preserved. Scale bars = 50 µm. (n = 8) (*p < 0.05; ** p < 0.01; *** p < 0.001 determined using repeated measures analysis of variance (ANOVA) with Tukey's honest significance difference test as a post hoc test). Please click here to view a larger version of this figure.
Some critical steps can be highlighted in this protocol. First, the person performing the surgery and manipulating the tissues must be delicate with the fat tissue, as it is fragile. Crushing or damaging the omentum needs to be avoided. Omentum, as a defensive tissue, is enriched in macrophages and other leukocytes. These immune cells can be activated by excessive manipulation and could negatively affect the graft. Secondly, graft (islet-hydrogel mixture) loading is also critical. The person performing the procedure must avoid dead volumes and must load all of the hydrogel-islet mixture into the injection device. Immediately after this step, another critical point is the injection itself. Injection must be performed slowly, with care, and delicately. The syringe must be flushed by the empty hydrogel loaded initially to retrieve any remaining islets. Thirdly, suturing must be done carefully and in two steps. The muscular plan should be sutured first, taking care not to suture the omentum with the muscle, and the skin should be sutured separately. Regarding the explantation procedure for the omental graft, special attention should be taken at the moment when the arteries close to the stomach (gastroepiploic arteries) are incised. It is important to pay attention to any hemorrhages that occur and stop them before suturing the animal, as any persistent bleeding leads to animal death within several days.
Troubleshooting may be necessary regarding the medium for carrying islets; the choice of hydrogel is up to the experimenter, as long as the hydrogel is injectable. The use of different materials can result in variable graft function (with or without supplementation, for example). The method described here has proven its efficiency for inert co-transplanting material with an islet ratio of 7660 IEQ/kg.
The present method may be limited by the omental size and hydrogel properties. To use a diabetic rodent model, insulin therapy is mandatory prior to islet implantation to provide sufficient grafting area. Diabetic rodents lose a lot of weight and fat mass due to STZ-induced diabetes. Considering the small weight of the animals enrolled in this study, grafting in an even smaller area is not possible.
Conservation of proper glycemia management (before transplant using pellets and thereafter using long-acting insulin) is mandatory. Here, we chose to conserve the insulin pellet until the day of transplantation for several reasons. First, the maintenance of a proper glycemic control considerably reduces oxidative stress induced by diabetes in the recipient organs, which can be deleterious for islet graft11 and allows continuation of intensive insulin therapy and recipient preparation observed in the clinic12. Second, we wanted to limit the maximum number of anesthesia procedures for the rats. Concerning the implementation of insulin therapy during the metabolic follow-up using long-acting insulin, the scheme used avoided massive complications due to glucotoxicity (which can destroy grafts) and to conserve a "real" glycemia value.
To monitor graft efficiency, glycemia is not a relevant parameter in the first few days if rats are receiving insulin therapy using pellets. This is why checking the C-peptide level several times prior to transplant is mandatory. These times include at the beginning of the study, to select animals after STZ injection, and then just preceding transplantation, to ensure that rats remain diabetic and that regeneration is minimal.
Hydrogel properties are also important. Liquid hydrogel will leak out of omental tissue and highly viscous hydrogel will not be injectable. Hydrogel viscosity and injectability has to be tested before use, but it appears essential to also test the material directly by evaluation of islet or another cells survival in vitro. Here, as islets are very sensitive to hypoxia and the use of a highly viscous carrier can affect oxygen diffusion.
In terms of the significance of the method described here with respect to existing methods, this intra-tissue transplantation technique has benefits in term of reproducibility. It is also atraumatic for the tissue and, because of its extra-vascular location, avoids bleeding and thrombosis risks. Also, when considering islet transplantation, instant blood-mediated inflammatory reaction is avoided14. Furthermore, hOMING allows transplanting islets into a non-vital organ, unlike transplantation into the liver, which poses risks in terms of hepatic function15.
To produce optimal performance, a hydrogel has to be adapted to the cells it will carry. Use of growth factors or specific proteins can have a positive effect on transplanted cells16,17.
To conclude, this technique can be used for in vivo validation of a hydrogel on different cell types, especially when an active metabolic environment is needed, as for islets. In addition, this surgical technique is applicable to multiple applications and could be, in the future, rapidly transferable to humans, as it can be easily performed using laparoscopy.
The authors have nothing to disclose.
This work was funded by Région Alsace, BioArtMAtrix-Pôle Alsace Biovalley-CQDM; 53/14/C1. The authors are grateful to the team of Pr. Bruant-Rodier from the Hôpitaux Universitaires de Strasbourg for helping develop this innovative technique.
Alginate (PRONOVA UP LMV) | Novamatrix | 4200206 | Hydrogel carrier |
Atraumatic needle (Blunt) | B.Braun | 9180109 | |
CMRL without FBS | Gibco | 11500576 | |
C-peptide ELISA kit | Mercodia | 10-1172-01 | |
Eosin | Leica Microsystems | 3801592E | |
Ethilon 4/0isoflo | Ethicon | F2414 | Surgical suture |
Hematoxylin | Leica Microsystems | 3801562E | |
Insulin pellets | Linshin | INS-B14 | |
Isofluorane | Centravet | ISO007 | |
Lantus (Insulin-Glargin) | Sanofi Adventis | Lantus SoloStar | Long acting insulin |
Metacam | Boehringer Ingelheim | MET019 | Anti-inflammatory drug |
NaCl (for saline 0.9%) | Sigma | 10112640 | |
Needle 26G | TERUMO | 050101B | |
Oxygen | Linde | 2010152 | For isoflurane use |
Pentobarbital sodique | Vetoquinol | Dolethal | For euthanasia |
Steranios 2% | Anios | 11764046 | |
Streptozotocin | Santa-Cruz | SC-200719A | |
Syringe – Injekt-F | B.Braun | 9166017V | |
Trocar & stylet (linshin) | Linshin | G12-SS | For pellet insertion |