OBJECTIVE: To develop a methodology to separate intestinal stem cell (ISC)-enriched crypts from differentiated epithelial cell (DEC)-containing villi to improve the morphology of tissue-engineered intestine (TEI). METHODS: Small intestinal tissues from 5- to 7-day-old transgenic Lgr5-EGFP mice (with fluorescently labeled ISCs) were used to measure the height of villi and the depth of crypts. Based on the significant size difference between crypts and villi, a novel cell filtration system was developed. Filtration of mixed organoid units from full-thickness intestine of transgenic Lgr5-EGFP mice allowed determination of the percentage of ISCs in the different size-based filtration fractions obtained. In vivo, 5-7-day-old Lewis rat pups were used as cell donors to obtain purified crypts and villi, and the dams of the pups served as recipients. Flat and tubular polyglycolic acid (PGA) scaffolds were seeded with either ISC-enriched crypts or DEC-containing villi and implanted intra-abdominally on the anterior abdominal wall. After 1, 3, 7, 14, 21, and 28 days of in vivo incubation, explants were processed for histologic evaluation. RESULTS: Small intestine from transgenic Lgr5-EGFP mice contained villi with an average height of 134.89±41.91 μm and crypts with an average depth of 49.59±8.95 μm. After filtration, we found that the 100-200 μm fractions contained relatively pure villi in which DECs were located, whereas the 25-70 μm range fractions contained concentrated crypts in which ISCs were located. In vivo, flat PGA scaffolds implanted with purified crypts formed well-developed mucosa by day 14 postimplantation, whereas flat scaffolds seeded with villi were replaced with fibrous tissue. Tubular scaffolds seeded with the crypt fraction developed a well-formed mucosal layer on the interior surface, with 80.9% circumferential mucosal engraftment and an average villous height of 478±65 μm, which was very close to native intestine (512±98 μm), whereas tubular scaffolds seeded with the villous fraction only had 21.7% circumferential mucosal engraftment and an average villous height of 243±78 μm. CONCLUSION: The novel filtration system described can effectively and efficiently isolate ISC-containing crypts. TEI produced from ISC-containing crypts has an improved morphology that is similar to native intestine.
OBJECTIVE: To develop a methodology to separate intestinal stem cell (ISC)-enriched crypts from differentiated epithelial cell (DEC)-containing villi to improve the morphology of tissue-engineered intestine (TEI). METHODS: Small intestinal tissues from 5- to 7-day-old transgenic Lgr5-EGFP mice (with fluorescently labeled ISCs) were used to measure the height of villi and the depth of crypts. Based on the significant size difference between crypts and villi, a novel cell filtration system was developed. Filtration of mixed organoid units from full-thickness intestine of transgenic Lgr5-EGFP mice allowed determination of the percentage of ISCs in the different size-based filtration fractions obtained. In vivo, 5-7-day-old Lewis rat pups were used as cell donors to obtain purified crypts and villi, and the dams of the pups served as recipients. Flat and tubular polyglycolic acid (PGA) scaffolds were seeded with either ISC-enriched crypts or DEC-containing villi and implanted intra-abdominally on the anterior abdominal wall. After 1, 3, 7, 14, 21, and 28 days of in vivo incubation, explants were processed for histologic evaluation. RESULTS: Small intestine from transgenic Lgr5-EGFP mice contained villi with an average height of 134.89±41.91 μm and crypts with an average depth of 49.59±8.95 μm. After filtration, we found that the 100-200 μm fractions contained relatively pure villi in which DECs were located, whereas the 25-70 μm range fractions contained concentrated crypts in which ISCs were located. In vivo, flat PGA scaffolds implanted with purified crypts formed well-developed mucosa by day 14 postimplantation, whereas flat scaffolds seeded with villi were replaced with fibrous tissue. Tubular scaffolds seeded with the crypt fraction developed a well-formed mucosal layer on the interior surface, with 80.9% circumferential mucosal engraftment and an average villous height of 478±65 μm, which was very close to native intestine (512±98 μm), whereas tubular scaffolds seeded with the villous fraction only had 21.7% circumferential mucosal engraftment and an average villous height of 243±78 μm. CONCLUSION: The novel filtration system described can effectively and efficiently isolate ISC-containing crypts. TEI produced from ISC-containing crypts has an improved morphology that is similar to native intestine.
Authors: Mitchell R Ladd; Diego F Niño; John C March; Chhinder P Sodhi; David J Hackam Journal: Curr Opin Organ Transplant Date: 2016-04 Impact factor: 2.640
Authors: Yanchun Liu; Tyler Nelson; Jason Chakroff; Barrett Cromeens; Jed Johnson; John Lannutti; Gail E Besner Journal: J Biomed Mater Res B Appl Biomater Date: 2018-09-30 Impact factor: 3.368
Authors: Yanchun Liu; Yijie Wang; Jason Chakroff; Jed Johnson; Aidan Farrell; Gail E Besner Journal: Tissue Eng Part A Date: 2018-11-07 Impact factor: 3.845
Authors: Yanchun Liu; Barrett P Cromeens; Yijie Wang; Kelli Fisher; Jed Johnson; Jason Chakroff; Gail E Besner Journal: Tissue Eng Part A Date: 2018-03-01 Impact factor: 3.845
Authors: Laura Y Martin; Mitchell R Ladd; Adam Werts; Chhinder P Sodhi; John C March; David J Hackam Journal: Pediatr Res Date: 2017-11-01 Impact factor: 3.756