Tissue Engineering of the Intestine in a Murine Model.

Children's Hospital Los Angeles, Division of Pediatric Surgery, Saban Research Institute, Keck School of Medicine of the University of Southern California.
Journal of Visualized Experiments (Impact Factor: 1.33). 12/2012; DOI: 10.3791/4279
Source: PubMed


Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.(1) In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.(2) In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,(3) the relationship between which preserves the intestinal stem cell niche.(4) In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.(5)The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

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    ABSTRACT: Short bowel syndrome (SBS) is the most common cause of intestinal failure in children. It is defined as the inability to maintain adequate nutrition enterally as a result of a major loss of the small intestine. SBS is a life-threatening entity associated with potential significant morbidity and mortality. The etiology in the pediatric age group includes necrotizing enterocolitis (32 %), atresia (20 %), volvulus (18 %), gastroschisis (17 %), and aganglionosis (6 %). It is characterized by substrate malabsorption, electrolyte imbalance, intestinal bacterial overgrowth, steatorrhea, and weight loss. Current medical management includes parenteral nutrition, progressive feeds as tolerated, various medications, and surgical manipulations. However, frequently this management is not successful in achieving the goal of attaining normal growth and development without parenteral nutrition. It has been known for decades that there is a normal physiologic response of the residual intestine to massive bowel resection referred to as intestinal adaptation. The mechanisms that control this process are unknown. Unfortunately, intestinal adaptation and the current management are not always successful. As a result of new knowledge regarding the pathophysiology of SBS over the past two decades, several novel strategies have been developed in experimental animal models as well as limited clinical trials in infants and children. They can be divided into several categories that potentially influence intestinal (1) absorption, (2) secretion, (3) motility, and (4) adaptation. More recently, newer modalities have been studied including small intestine transplantation, and the use of specific intestinal growth factors. Ultimately, tissue and organ engineering will become the treatment for infants and children with SBS.
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    ABSTRACT: Background: Short bowel syndrome causes significant morbidity and mortality. Tissue-engineered intestine may serve as a viable replacement. Tissue-engineered small intestine (TESI) has previously been generated in the mouse model from donor cells that were harvested and immediately reimplanted; however, this technique may prove impossible in children who are critically ill, hemodynamically unstable, or septic. We hypothesized that organoid units (OU), multicellular clusters containing epithelium and mesenchyme, could be cryopreserved for delayed production of TESI. Methods: OU were isolated from <3 wk-old mouse or human ileum. OU were then cryopreserved by either standard snap freezing or vitrification. In the snap freezing protocol, OU were suspended in cryoprotectant and transferred directly to -80°C for storage. The vitrification protocol began with a stepwise increase in cryoprotectant concentration followed by liquid supercooling of the OU solution to -13°C and nucleation with a metal rod to induce vitrification. Samples were then cooled to -80°C at a controlled rate of -1°C/min and subsequently plunged into liquid nitrogen for long-term storage. OU from both groups were maintained in cryostorage for at least 72 h and thawed in a 37°C water bath. Cryoprotectant was removed with serial sucrose dilutions and OU were assessed by Trypan blue assay for post-cryopreservation viability. Via techniques previously described by our laboratory, the thawed murine or human OU were either cultured in vitro or implanted on a scaffold into the omentum of a syngeneic or irradiated Nonobese Diabetic/Severe Combined Immunodeficiency, gamma chain deficient adult mouse. The resultant TESI was analyzed by histology and immunofluorescence. Results: After cryopreservation, the viability of murine OU was significantly higher in the vitrification group (93 ± 2%, mean ± standard error of the mean) compared with standard freezing (56 ± 6%) (P < 0.001, unpaired t-test, n = 25). Human OU demonstrated similar viability after vitrification (89 ± 2%). In vitro culture of thawed OU produced expanding epithelial spheres supported by a layer of mesenchyme. TESI was successfully generated from the preserved OU. Hematoxylin and eosin staining demonstrated a mucosa composed of a simple columnar epithelium whereas immunofluorescence staining confirmed the presence of both progenitor and differentiated epithelial cells. Furthermore, beta-2-microglobulin confirmed that the human TESI epithelium originated from human cells. Conclusions: We demonstrated improved multicellular viability after vitrification over conventional cryopreservation techniques and the first successful vitrification of murine and human OU with subsequent TESI generation. Clinical application of this method may allow for delayed autologous implantation of TESI for children in extremis.
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    ABSTRACT: Transplantation of tissues and organs is currently the only available treatment for patients with end-stage diseases. However, its feasibility is limited by the chronic shortage of suitable donors, the need for life-long immunosuppression, and by socio-economical and religious concerns. Recently, tissue engineering has garnered interest as a means to generate cell-seeded three-dimensional scaffolds that could replace diseased organs without requiring immunosuppression. Using a regenerative approach, scaffolds made by synthetic, non-immunogenic, biocompatible materials have been developed and successfully clinically implanted. This strategy, based on a viable and ready-to-use bioengineered scaffold, able to promote novel tissue formation, favouring cell adhesion and proliferation, could become a reliable alternative to allotransplatation in the next future. In this paper, tissue engineered synthetic substitutes for tubular organs (such as trachea, esophagus, bile ducts and bowel) are reviewed, including a discussion on their morphological and functional properties.
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