title: A study of the osmotic characteristics, water permeability, and cryoprotectant permeability of human vaginal immune cells.
abstract: Cryopreservation of specimens taken from the genital tract of women is important for studying mucosal immunity during HIV prevention trials. However, it is unclear whether the current, empirically developed cryopreservation procedures for peripheral blood cells are also ideal for genital specimens. The optimal cryopreservation protocol depends on the cryobiological features of the cells. Thus, we obtained tissue specimens from vaginal repair surgeries, isolated and flow cytometry-purified immune cells, and determined fundamental cryobiological characteristics of vaginal CD3(+) T cells and CD14(+) macrophages using a microfluidic device. The osmotically inactive volumes of the two cell types (Vb) were determined relative to the initial cell volume (V0) by exposing the cells to hypotonic and hypertonic saline solutions, evaluating the equilibrium volume, and applying the Boyle van't Hoff relationship. The cell membrane permeability to water (Lp) and to four different cryoprotective agent (CPA) solutions (Ps) at room temperature were also measured. Results indicated Vb values of 0.516 V0 and 0.457 V0 for mucosal T cells and macrophages, respectively. Lp values at room temperature were 0.196 and 0.295 μm/min/atm for T cells and macrophages, respectively. Both cell types had high Ps values for the three CPAs, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) (minimum of 0.418 × 10(-3) cm/min), but transport of the fourth CPA, glycerol, occurred 50-150 times more slowly. Thus, DMSO, PG, and EG are better options than glycerol in avoiding severe cell volume excursion and osmotic injury during CPA addition and removal for cryopreservation of human vaginal immune cells.
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, WA 98195, USA; School of Mechanical and Materials Engineering, Washington State University, Everett, WA 98201, USA.
authors.forename: Zhiquan
authors.initials: Z
authors.lastname: Shu
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Hughes
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, WA 98195, USA.
authors.forename: Cifeng
authors.initials: C
authors.lastname: Fang
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, WA 98195, USA; College of Information Technology, Beijing Union University, Beijing 100101, China.
authors.forename: Jinghua
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, WA 98195, USA; College of Information Technology, Beijing Union University, Beijing 100101, China.
authors.forename: Baiwen
authors.initials: B
authors.lastname: Fu
authors.affiliation: Department of Electronic Science & Technology, University of Science and Technology of China, Hefei 230027, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Zhao
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Fialkow
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Gretchen
authors.initials: G
authors.lastname: Lentz
authors.affiliation: Department of Obstetrics and Gynecology, University of Washington, Seattle, WA 98195, USA; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. Electronic address: fhladik@fredhutch.org.
authors.forename: Florian
authors.initials: F
authors.lastname: Hladik
authors.affiliation: Department of Mechanical Engineering, University of Washington, Seattle, WA 98195, USA. Electronic address: dayong@u.washington.edu.
authors.forename: Dayong
authors.initials: D
authors.lastname: Gao
mesh_terms: D001692:Biological Transport; D002463:Cell Membrane Permeability / Q000502:physiology*; D048429:Cell Size; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D019855:Ethylene Glycol / Q000378:metabolism; D005260:Female; D005990:Glycerol / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000276:immunology*; D009995:Osmosis / Q000502:physiology; D009997:Osmotic Pressure / Q000502:physiology*; D019946:Propylene Glycol / Q000378:metabolism; D012996:Solutions; D013601:T-Lymphocytes / Q000276:immunology*; D014621:Vagina / Q000166:cytology / Q000276:immunology; D014867:Water / Q000378:metabolism
chemical_list: D003451:Cryoprotective Agents; D012996:Solutions; D014867:Water; D019946:Propylene Glycol; D019855:Ethylene Glycol; D005990:Glycerol; D004121:Dimethyl Sulfoxide
keywords: Cryobiological characteristics; Cryopreservation; Human vaginal mucosa; Macrophage; Microfluidic perfusion channel; T cell