Distribution of Growth Induced By Straight Versus Cuvilinear Distractive Enterogenesis

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 20.6. Formation of an Intestinal Epithelial Surface Using Collagen Gel. Z. Jabaji,1 V. Joshi,1 N. Lei,1 J. Wang,2 M. Lewis,3 M. Stelzner,1 M. Martin,2 J. C. Dunn1; 1Department of Surgery, UCLA, Los Angeles, CA; 2Department of Pediatrics, UCLA, Los Angeles, CA; 3Department of Pathology, West Los Angeles VA, Los Angeles, CA Introduction: Intestinal stem cells can form spheroids when they are cultured within Matrigel supplemented with the Wnt agonist R-spondin that interacts with the Lgr5 receptor. A critical barrier to translate this methodology to cell-based therapy is the ability to generate a sheet of intestinal epithelial cells using well-defined extracellular matrix. We hypothesize that collagen gel will induce the formation of such epithelial cell sheets and eliminate the need for Matrigel. Methods: Intestinal crypts were isolated from transgenic GFP mice between 4-6 weeks of age using EDTA chelation. Intestinal subepithelial myofibroblasts were obtained from murine C57BL/6 pups. Crypts were mixed in collagen gel and were seeded on top of myofibroblasts. Crypts were also seeded on top of collagen gel without myofibroblasts in some experiments. Spheroids were counted by light microscopy, and quantitative PCR and immunohistochemical staining were performed on cells cultured for 7 days in media with R-spondin. Results: Crypts initial formed spheroids when they were cultured within collagen gel supported by myofibroblasts that expressed smooth muscle actine and vimentin. Cells within the spheroids expressed E-cadherin and Cdx2, confirming their intestinal epithelial origin. the cell mass increased in culture as confirmed by microscopy and GFP-DNA PCR. Epithelial cells were observed to migrate from these spheroids and formed a sheet of columnar cells at the liquidgel interface. These cells possessed brush borders and basally located nuclei, and they also expressed E-cadherin and Cdx2. Goblet cells were observed in this newly formed epithelium. When directly seeded on top of collagen gel in the absence of myofibrolasts, crypts formed a sheet of cobblestone-patterned cells at the liquid-gel interface. Conclusions: Collagen gel can be used to support the in vitro growth of primary intestinal epithelial cells. the formation of an epithelial cell sheet instead of spheroids may allow scaling up of this methodology for clinical use. This technique eliminates the dependence of intestine epithelial cells on Matrigel and may facilitate the translation to cell-based therapy in the future.

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lengthening. Table 1 shows: percentage of BrdU+ epithelial cells; TER; and area of microvessels per 20x high powered field. Finally, because the degree and application of distractive forces varied between the two devices (curvilinear-shaped hydraulic device more even distribution vs. straight-shaped SMA device, force confined to both ends), the differences in growth between devices was examined. Growth, (BrdU+ epithelial cells) was higher in the mid-portion of the curvilinear case vs. concentrated near the ends for the straight device, correlating with higher microvessel growth distribution for each device. Conclusions: Longitudinal distractive forces appear to be an effective method for small bowel growth. Growth does not appear to occur evenly throughout the stretched segment, but is concentrated on the end for the SMA device and the middle for the hydraulic. These growth characteristics will prove useful in guiding future device development.

TABLE

BrdU SMA (%)U BrdU Hydraulic (%)U TER SMA (U*cm2) TER Hydraulic (U*cm2) Microvessel SMA (mm2)+ Microvessel Hydraulic (mm2)+

Control

End of Device

Middle of Device

31 27 30.2 25.1 27160 5223

39 22 42.9 27.4 20204 2393

20 32 37.9 25.7 16076 6920

U Mean of percentage of BrdU positive epithelial cells in the crypt villous complex over 20 complexes. þ Mean vessel area per 20x high powered field averaged over 30 fields.

20.7. Distribution of Growth Induced By Straight Versus Cuvilinear Distractive Enterogenesis. R. S. Herman,1 R. Sueyoshi,1 E. A. Miyasaka,1 M. Okawada,1 B. Utter,2 I. Czarnocki,2 N. Si,1 J. Luntz,2 D. Brei,2 D. H. Teitelbaum1; 1 University of Michigan Medical School Department of Pediatric Surgery, Ann Arbor, MI; 2University of Michigan, Mechanical Engineering Department, Ann Arbor, MI

20.8. In Vitro Crypt Culture from Small Bowel Maintains Cephalo-caudal Gradients of Gene Expression. M. K. Fuller,1 D. M. Faulk,2 N. Sundaram,2 S. J. Henning,3 M. A. Helmrath2; 1Department of Surgery, Chapel Hill, NC; 2 Department of Surgery, Cincinnati, OH; 3Department of Cellular and Molecular Biology, Chapel Hill, NC

Introduction: Length of remaining small bowel is an important prognostic factor in long term survival and ability to wean off parenteral nutrition in patients with short bowel syndrome(SBS). Distraction enterogenesis, using mechanical forces to lengthen intestinal tissue, is a novel, potential treatment option for SBS. Two newly developed intraluminal devices to achieve functional longitudinal small bowel growth in a pig model are described. the experimental aims were to test the characteristics of growth induced by both devices. Methods: Two engineered devices were implanted into live pigs. in one pig a straight-radio frequency controlled device driven using a shape memory alloy(SMA)-powered expansion mechanism(Figure 1A) was implanted and extended over an 8 day period. in a second pig, a curvilinear-shaped hydraulic-powered device was implanted and expanded over a 14 day period(Figure 1B). Devices were placed into roux-en-y segments to isolate them from enteric flow. Outcome measures were initial and final bowel length, epithelial cell proliferation using bromodeoxyuridine(BrdU), functionality using transepithelial resistance(TER) measured by an Ussing chamber, and calculating mesenteric microvessel growth using a-smooth muscle actin staining. Location of growth in the bowel relative to the devices was also examined. Results: Initial and final bowel lengths with the SMA device were 13 cm and 18.5 cm; 5.5 cm of lengthening. Initial and final lengths with the hydraulic device were: 15 cm and 24.9 cm; 9.9 cm of

Introduction: Recent methodology to expand intestinal crypts to polycryptoid structures (enteroids) has been previously described by our group. We sought to determine whether enteroids in vitro maintained their in vivo characteristics by comparing regional variation in vitro. Methods: After IACUC approval, crypts were harvested from 5 cm of proximal and distal small bowel of C57BL/6J mice aged 6-8wk (n¼3 animals). Crypts were cultured using a modification of the Sato technique. Proximal and distal small intestinal tissue, crypts, and enteroids (at 7, 14, and 28 days in culture) were collected. in addition, proximal and distal enteroids were co-cultured with both proximal and distal myofibroblasts and collected at day 14. mRNA was extracted via RNeasy Mini kit (Qiagen Biosciences), and reverse transcribed via High Capacity Reverse Transcription kit (Applied Biosystems). qRT- PCR with Taqman universal primer master mix (Applied Biosystems) for regionally expressed genes GIP, FABP1, GATA4, PYY, Asbt, and Cubulin was performed in triplicate. the data were analyzed using a relative standard curve method normalized for the expression of b-actin. *p < 0.05 was determined significant by two way ANOVA followed by Bonferroni post-test corrections. Results: Morphologically, proximal enteroids demonstrated significantly greater budding than distal at 7 days in culture (6.7 vs 5.1 buds per enteroid*). Gene expression of the proximal markers GIP*, FABP1*, and GATA4* was pronounced in proximal