title: Vitrification preserves murine and human donor cells for generation of tissue-engineered intestine.
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.
authors.affiliation: Division of Pediatric Surgery, Children's Hospital Los Angeles, Keck School of Medicine of the University of Southern California, Los Angeles, CA.
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Spurrier
authors.affiliation: Division of Pediatric Surgery, Children's Hospital Los Angeles, Keck School of Medicine of the University of Southern California, Los Angeles, CA.
authors.forename: Allison L
authors.initials: AL
authors.lastname: Speer
authors.affiliation: Division of Pediatric Surgery, Children's Hospital Los Angeles, Keck School of Medicine of the University of Southern California, Los Angeles, CA.
authors.forename: Christa N
authors.initials: CN
authors.lastname: Grant
authors.affiliation: Division of Pediatric Surgery, Children's Hospital Los Angeles, Keck School of Medicine of the University of Southern California, Los Angeles, CA.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Levin
authors.affiliation: Division of Pediatric Surgery, Children's Hospital Los Angeles, Keck School of Medicine of the University of Southern California, Los Angeles, CA. Electronic address: tgrikscheit@chla.usc.edu.
authors.forename: Tracy C
authors.initials: TC
authors.lastname: Grikscheit
mesh_terms: D053687:Adult Stem Cells / Q000473:pathology; D000818:Animals; D006801:Humans; D007421:Intestine, Small / Q000473:pathology; D008648:Mesoderm / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D023822:Tissue Engineering; D058989:Vitrification
keywords: Cryopreservation; Intestine; Regenerative medicine; Short bowel syndrome; Stem cells; Tissue engineering; Tissue-engineered small intestine; Vitrification