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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.

SUBMITTER: Kato Y

PROVIDER: S-EPMC3928110 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression.

Kato Yasuhiro Y Miyauchi Takayuki T Abe Youichiro Y Kojić Dušan D Tanaka Manami M Chikazawa Nana N Nakatake Yuhki Y Ko Shigeru B H SB Kobayashi Daisuke D Hazama Akihiro A Fujiwara Shoko S Uchida Tatsuya T Yasui Masato M

PloS one 20140218 2

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 ...[more]

PMID: 24558371

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Project description:BackgroundGrowing evidence has demonstrated immune reactivity as a confirmed important carcinogenesis and therapy efficacy for clear cell renal cell carcinoma (ccRCC). Aquaporin 9 (AQP9) is involved in many immune-related signals; however, its role in ccRCC remains to be elucidated. This study investigated AQP9 expression in tumor tissues and defined the prognostic value in ccRCC patients.MethodsA total of 913 ccRCC patients with available RNA-sequence data from the Cancer Genome Atlas (TCGA) database and Fudan University Shanghai Cancer Center (FUSCC) were consecutively recruited in analyses. Differential transcriptional and proteome expression profiles were obtained and validated using multiple datasets. A partial likelihood test from Cox regression analysis was developed to address the influence of independent factors on progression-free survival (PFS) and overall survival (OS). The Kaplan-Meier method and log-rank test were performed to assess survival. Receiver operating characteristic (ROC) curves were used to describe binary classifier value of AQP9 using area under the curve (AUC) score. Functional enrichment analyses and immune infiltration analysis were used to describe significantly involved hallmark pathways of hub genes.ResultsSignificantly elevated transcriptional and proteomic AQP9 expressions were found in ccRCC samples. Increased AQP9 mRNA expression was significantly associated with advanced clinicopathological parameters and correlated with shorter PFS and OS in TCGA and FUSCC cohorts (p < 0.001). ROC curves suggested the significant diagnostic and prognostic ability of AQP9 (PFS, AUC = 0.823; OS, AUC = 0.828). Functional annotations indicated that AQP9 is involved in the most significant hallmarks including complement, coagulation, IL6/JAK-STAT3, inflammatory response and TNF-alpha signaling pathways.ConclusionOur study revealed that elevated AQP9 expression was significantly correlated with aggressive progression, poor survival and immune infiltrations in ccRCC patients, and we validated its prognostic value in a real-world cohort. These data suggest that AQP9 may act as an oncogene and a promising prognostic marker in ccRCC.

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Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression.

Publications

Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression.

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