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

Kato Y, Miyauchi T, Abe Y, Kojić D, Tanaka M, Chikazawa N, Nakatake Y, Ko SB, Kobayashi D, Hazama A, Fujiwara S, Uchida T, Yasui M - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.

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.

Show MeSH

Related in: MedlinePlus

Freezing tolerance of cells expressing AQP after ultra-quick freezing.(A) Cell viability assessed using trypan blue-exclusion immediately at cooling rates of −1, −30, −50, and −120°C/min. The cell viability of control CHO cells is indicated by the dotted line, while that of AQP4-CHO cells is indicated by the solid line. Data are shown as the mean ± standard deviation (n ≧ 3, **P<0.01). (B) Colony-formation as assessed by counting the number of growing cells within the visual filed with DAPI staining at 3 days after freezing and thawing at cooling rates of −1, −30, −50, and −120°C/min. The cell viability of control CHO cells is indicated by the dotted line, while that of AQP4-CHO cells is indicated by the solid line. Data are shown as the mean ± standard deviation (n ≧ 3, *P<0.05). (C) Flow cytometry analyses of membrane damage in CHO cells (upper panels) and AQP4-CHO cells (lower panel) at the following freezing rates: −1, −30, −50, and −120°C/min. Cells were stained with phycoerythrin (PE)-conjugated Annexin V and 7-amino-actinomycin D (7-AAD) and were analyzed using flow-cytometry. Living cells were identified as cells with negative PE-Annexin V and 7-AAD staining (lower left region). Cytoplasmic membrane-damaged cells were Annexin V-positive (lower right region), whereas membrane damaged and dead cells were both Annexin V and 7-AAD-positive (upper right region). 1×104 cells were analyzed by flow cytometry. (D) Scanning electron microscopy (SEM) study showing the surface characteristics of the membranes. Non-freezing (negative control, left panels) and ultra-quick freezing (−120°C/min, right panels) images show the morphological characteristics of CHO cells (upper panels) and AQP4-CHO cells (lower panels). Scale bar: 5 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3928110&req=5

pone-0087644-g001: Freezing tolerance of cells expressing AQP after ultra-quick freezing.(A) Cell viability assessed using trypan blue-exclusion immediately at cooling rates of −1, −30, −50, and −120°C/min. The cell viability of control CHO cells is indicated by the dotted line, while that of AQP4-CHO cells is indicated by the solid line. Data are shown as the mean ± standard deviation (n ≧ 3, **P<0.01). (B) Colony-formation as assessed by counting the number of growing cells within the visual filed with DAPI staining at 3 days after freezing and thawing at cooling rates of −1, −30, −50, and −120°C/min. The cell viability of control CHO cells is indicated by the dotted line, while that of AQP4-CHO cells is indicated by the solid line. Data are shown as the mean ± standard deviation (n ≧ 3, *P<0.05). (C) Flow cytometry analyses of membrane damage in CHO cells (upper panels) and AQP4-CHO cells (lower panel) at the following freezing rates: −1, −30, −50, and −120°C/min. Cells were stained with phycoerythrin (PE)-conjugated Annexin V and 7-amino-actinomycin D (7-AAD) and were analyzed using flow-cytometry. Living cells were identified as cells with negative PE-Annexin V and 7-AAD staining (lower left region). Cytoplasmic membrane-damaged cells were Annexin V-positive (lower right region), whereas membrane damaged and dead cells were both Annexin V and 7-AAD-positive (upper right region). 1×104 cells were analyzed by flow cytometry. (D) Scanning electron microscopy (SEM) study showing the surface characteristics of the membranes. Non-freezing (negative control, left panels) and ultra-quick freezing (−120°C/min, right panels) images show the morphological characteristics of CHO cells (upper panels) and AQP4-CHO cells (lower panels). Scale bar: 5 µm.

Mentions: In order to understand the mechanisms behind the effects of AQP on cell survival after ultra-quick freezing, we next examined cell survival rate at different cooling rate. No significant difference in the cell viability was seen at freezing rates of −1, −30, or −50°C/min between the control CHO cells (dotted line) and those expressing AQP4 (AQP4-CHO cells) (solid line). However, at −120°C/min, a significant difference in survival rate was observed: 2.4±1.4% for CHO cells, and 60.5±16.7% for AQP4-CHO cells, indicating that the expression of AQP resulted in freezing tolerance at high cooling rate (Figure 1A).


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

Kato Y, Miyauchi T, Abe Y, Kojić D, Tanaka M, Chikazawa N, Nakatake Y, Ko SB, Kobayashi D, Hazama A, Fujiwara S, Uchida T, Yasui M - PLoS ONE (2014)

Freezing tolerance of cells expressing AQP after ultra-quick freezing.(A) Cell viability assessed using trypan blue-exclusion immediately at cooling rates of −1, −30, −50, and −120°C/min. The cell viability of control CHO cells is indicated by the dotted line, while that of AQP4-CHO cells is indicated by the solid line. Data are shown as the mean ± standard deviation (n ≧ 3, **P<0.01). (B) Colony-formation as assessed by counting the number of growing cells within the visual filed with DAPI staining at 3 days after freezing and thawing at cooling rates of −1, −30, −50, and −120°C/min. The cell viability of control CHO cells is indicated by the dotted line, while that of AQP4-CHO cells is indicated by the solid line. Data are shown as the mean ± standard deviation (n ≧ 3, *P<0.05). (C) Flow cytometry analyses of membrane damage in CHO cells (upper panels) and AQP4-CHO cells (lower panel) at the following freezing rates: −1, −30, −50, and −120°C/min. Cells were stained with phycoerythrin (PE)-conjugated Annexin V and 7-amino-actinomycin D (7-AAD) and were analyzed using flow-cytometry. Living cells were identified as cells with negative PE-Annexin V and 7-AAD staining (lower left region). Cytoplasmic membrane-damaged cells were Annexin V-positive (lower right region), whereas membrane damaged and dead cells were both Annexin V and 7-AAD-positive (upper right region). 1×104 cells were analyzed by flow cytometry. (D) Scanning electron microscopy (SEM) study showing the surface characteristics of the membranes. Non-freezing (negative control, left panels) and ultra-quick freezing (−120°C/min, right panels) images show the morphological characteristics of CHO cells (upper panels) and AQP4-CHO cells (lower panels). Scale bar: 5 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928110&req=5

pone-0087644-g001: Freezing tolerance of cells expressing AQP after ultra-quick freezing.(A) Cell viability assessed using trypan blue-exclusion immediately at cooling rates of −1, −30, −50, and −120°C/min. The cell viability of control CHO cells is indicated by the dotted line, while that of AQP4-CHO cells is indicated by the solid line. Data are shown as the mean ± standard deviation (n ≧ 3, **P<0.01). (B) Colony-formation as assessed by counting the number of growing cells within the visual filed with DAPI staining at 3 days after freezing and thawing at cooling rates of −1, −30, −50, and −120°C/min. The cell viability of control CHO cells is indicated by the dotted line, while that of AQP4-CHO cells is indicated by the solid line. Data are shown as the mean ± standard deviation (n ≧ 3, *P<0.05). (C) Flow cytometry analyses of membrane damage in CHO cells (upper panels) and AQP4-CHO cells (lower panel) at the following freezing rates: −1, −30, −50, and −120°C/min. Cells were stained with phycoerythrin (PE)-conjugated Annexin V and 7-amino-actinomycin D (7-AAD) and were analyzed using flow-cytometry. Living cells were identified as cells with negative PE-Annexin V and 7-AAD staining (lower left region). Cytoplasmic membrane-damaged cells were Annexin V-positive (lower right region), whereas membrane damaged and dead cells were both Annexin V and 7-AAD-positive (upper right region). 1×104 cells were analyzed by flow cytometry. (D) Scanning electron microscopy (SEM) study showing the surface characteristics of the membranes. Non-freezing (negative control, left panels) and ultra-quick freezing (−120°C/min, right panels) images show the morphological characteristics of CHO cells (upper panels) and AQP4-CHO cells (lower panels). Scale bar: 5 µm.
Mentions: In order to understand the mechanisms behind the effects of AQP on cell survival after ultra-quick freezing, we next examined cell survival rate at different cooling rate. No significant difference in the cell viability was seen at freezing rates of −1, −30, or −50°C/min between the control CHO cells (dotted line) and those expressing AQP4 (AQP4-CHO cells) (solid line). However, at −120°C/min, a significant difference in survival rate was observed: 2.4±1.4% for CHO cells, and 60.5±16.7% for AQP4-CHO cells, indicating that the expression of AQP resulted in freezing tolerance at high cooling rate (Figure 1A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, Keio University, Tokyo, Japan.

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.

Show MeSH
Related in: MedlinePlus