title: Trehalose effectiveness as a cryoprotectant in 2D and 3D cell cultures of human embryonic kidney cells.
abstract: Post cryopreservation viability of human embryonic kidney (HEK) cells under two-dimensional (2D) and three-dimensional (3D) culture conditions was studied using trehalose as the sole cryoprotective agent. An L9(34) Taguchi design was used to optimize the cryoprotection cocktail seeding process prior to slow-freezing with the specific aim of maximizing cell viability measured 7 days post thaw, using the combinatorial cell viability and in-vitro cytotoxicity WST assay. At low (200 mM) and medium (800 mM) levels of trehalose concentration, encapsulation in alginate offered a greater protection to cryopreservation. However, at the highest trehalose concentration (1200 mM) and in the absence of the pre-incubation step, there was no statistical difference at the 95% CI (p = 0.0212) between the viability of the HEK cells under 2D and 3D culture conditions estimated to be 17.9 ± 4.6% and 14.0 ± 3.6%, respectively. A parallel comparison between cryoprotective agents conducted at the optimal levels of the L9study, using trehalose, dimethylsulfoxide and glycerol in alginate microcapsules yielded a viability of 36.0 ± 7.4% for trehalose, in average 75% higher than the results associated with the other two cell membrane-permeating compounds. In summary, the effectiveness of trehalose has been demonstrated by the fact that 3D cell cultures can readily be equilibrated with trehalose before cryopreservation, thus mitigating the cytotoxic effects of glycerol and dimethylsulfoxide.
authors.affiliation: a Department of Bioengineering , Santa Clara University , Santa Clara , CA , USA.
authors.forename: Jared
authors.initials: J
authors.lastname: Hara
authors.affiliation: a Department of Bioengineering , Santa Clara University , Santa Clara , CA , USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Tottori
authors.affiliation: a Department of Bioengineering , Santa Clara University , Santa Clara , CA , USA.
authors.forename: Megan
authors.initials: M
authors.lastname: Anders
authors.affiliation: a Department of Bioengineering , Santa Clara University , Santa Clara , CA , USA.
authors.forename: Smritee
authors.initials: S
authors.lastname: Dadhwal
authors.affiliation: a Department of Bioengineering , Santa Clara University , Santa Clara , CA , USA.
authors.forename: Prashanth
authors.initials: P
authors.lastname: Asuri
authors.affiliation: a Department of Bioengineering , Santa Clara University , Santa Clara , CA , USA.
authors.forename: Maryam
authors.initials: M
authors.lastname: Mobed-Miremadi
mesh_terms: D000464:Alginates / Q000737:chemistry; D001692:Biological Transport; D002214:Capsules / Q000737:chemistry; D018929:Cell Culture Techniques; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects; D018914:Cells, Immobilized / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000378:metabolism / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000378:metabolism / Q000494:pharmacology; D005163:Factor Analysis, Statistical; D005782:Gels; D020723:Glucuronic Acid / Q000737:chemistry; D005990:Glycerol / Q000378:metabolism / Q000494:pharmacology; D057809:HEK293 Cells; D006603:Hexuronic Acids / Q000737:chemistry; D006801:Humans; D044367:Phase Transition; D014199:Trehalose / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000464:Alginates; D002214:Capsules; D003451:Cryoprotective Agents; D005782:Gels; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D014199:Trehalose; D005990:Glycerol; D004121:Dimethyl Sulfoxide
keywords: Alginate encapsulation; HEK cells; cryopreservation; cytotoxicity