title: Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression.
abstract: Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols.
authors.affiliation: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Miyauchi
authors.affiliation: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Youichiro
authors.initials: Y
authors.lastname: Abe
authors.affiliation: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Dušan
authors.initials: D
authors.lastname: Kojić
authors.affiliation: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Manami
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Department of Systems Medicine, Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Nana
authors.initials: N
authors.lastname: Chikazawa
authors.affiliation: Department of Systems Medicine, Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Yuhki
authors.initials: Y
authors.lastname: Nakatake
authors.affiliation: Department of Systems Medicine, Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Shigeru B H
authors.initials: SB
authors.lastname: Ko
authors.affiliation: Department of Physiology, Fukushima Medical University School of Medicine, Fukushima, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kobayashi
authors.affiliation: Department of Physiology, Fukushima Medical University School of Medicine, Fukushima, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Hazama
authors.affiliation: Department of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Fujiwara
authors.affiliation: Department of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Uchida
authors.affiliation: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Yasui
mesh_terms: D000818:Animals; D051398:Aquaporin 1 / Q000378:metabolism; D051401:Aquaporin 4 / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000235:genetics; D002470:Cell Survival; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D015925:Cryopreservation / Q000379:methods*; D004285:Dogs; D053595:Embryonic Stem Cells / Q000166:cytology; D005615:Freezing; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008822:Mice, Transgenic; D054258:Neural Plate / Q000378:metabolism; D009995:Osmosis; D010957:Plasmids / Q000378:metabolism; D044022:Receptor, Endothelin A / Q000378:metabolism
chemical_list: C496020:AQP1 protein, human; C496031:Aqp4 protein, mouse; D051401:Aquaporin 4; D044022:Receptor, Endothelin A; D051398:Aquaporin 1