title: Tolerance of human embryonic stem cell derived islet progenitor cells to vitrification-relevant solutions.
abstract: We have previously shown that human embryonic stem cell derived islet progenitors (hESC-IPs), encapsulated inside an immunoprotective device, mature in vivo and ameliorate diabetes in mice. The ability to cryopreserve hESC-IPs preloaded in these devices would enhance consistency and portability, but traditional 'slow freezing' methods did not work well for cells encapsulated in the device. Vitrification is an attractive alternative cryopreservation approach. To assess the tolerance of hESC-IPs to vitrification relevant conditions, we here are reporting cell survival following excursions in tonicity, exposure to fifteen 40% v/v combinations of 4 cryoprotectants, and varied methods for addition and elution. We find that 78% survival is achieved using a protocol in which cells are abruptly (in one step) exposed to a solution containing 10% v/v each dimethyl sulfoxide, propylene glycol, ethylene glycol, and glycerol on ice, and eluted step-wise with DPBS+0.5M sucrose at 37°C. Importantly, the hESC-IPs also maintain expression of the critical islet progenitor markers PDX-1, NKX6.1, NGN3 and NEURO-D1. Thus, hESC-IPs exhibit robust tolerance to exposure to vitrification solutions in relevant conditions.
authors: Development, Aging, and Regeneration Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.
authors: Reyhaneh
authors: R
authors: Lahmy
authors: CELLTRONIX, San Diego, CA 92126, USA; National Technical University "KhPI", Kharkov, Ukraine.
authors: Vladimir F
authors: VF
authors: Bolyukh
authors: Department of Reproductive Medicine, University of California, San Diego, La Jolla 92093, CA, USA.
authors: Sergio Mora
authors: SM
authors: Castilla
authors: Department of Reproductive Medicine, University of California, San Diego, La Jolla 92093, CA, USA.
authors: Louise C
authors: LC
authors: Laurent
authors: CELLTRONIX, San Diego, CA 92126, USA; Amorphous State Laboratory, Institute of Engineering Technology and Natural Science, Belgorod State University, Belgorod, Russian Federation. Electronic address: prodvincell@hotmail.com.
authors: Igor I
authors: II
authors: Katkov
authors: Development, Aging, and Regeneration Program, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA; Department of Pediatrics, University of California, San Diego, La Jolla 92093-0816, CA, USA. Electronic address: pitkin@sanfordburnham.org.
authors: Pamela
authors: P
authors: Itkin-Ansari
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005615:Freezing; D005990:Glycerol / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics; D000066449:Human Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology / Q000196:embryology*; D009419:Nerve Tissue Proteins / Q000235:genetics; D019946:Propylene Glycol / Q000494:pharmacology; D012996:Solutions; D013395:Sucrose / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D058989:Vitrification
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D019855:Ethylene Glycol; D005990:Glycerol; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D019946:Propylene Glycol; D012996:Solutions; D013395:Sucrose; D015534:Trans-Activators; D014157:Transcription Factors; C498274:DPF1 protein, human; C498695:NEUROG3 protein, human; C105201:NKX6-1 protein, human; C084010:pancreatic and duodenal homeobox 1 protein
keywords: Beta-cells; Cryopreservation; Diabetes; Insulin progenitors; Macroencapsulation; Osmotic tolerance; Toxicity; Vitrification