Evaluation of Distinct Freezing Methods and Cryoprotectants for Human Amniotic Fluid Stem Cells Cryopreservation

Janz, Felipe de Lara; Debes, Adriana de Aguiar; Cavaglieri, Rita de Cássia; Duarte, Sérgio; Romão, Carolina Martinez; Moron, Antônio Fernandes; Zugaib, Marcelo; Bydlowski, Sérgio Paulo

doi:10.1155/2012/649353

Cited by 35 publications

(27 citation statements)

References 25 publications

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“…Due to the ability of penetrating cell membrane, DMSO protects the cell from rupture by removing the water within the cell to prevent intracellular ice crystals formation 35 . Janz et al 40 also showed that DMSO is better than trehalose in maintaining amniotic fluid-derived stem cells viability. Therefore, CPA containing trehalose alone cannot be used to replace DMSO in cryopreservation of ASCs.…”

Section: Resultsmentioning

confidence: 99%

Phenotypic and Functional Characterization of Long-Term Cryopreserved Human Adipose-derived Stem Cells

Yong

Pingguan‐Murphy

et al. 2015

Sci Rep

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Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.

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Section: Resultsmentioning

confidence: 99%

Phenotypic and Functional Characterization of Long-Term Cryopreserved Human Adipose-derived Stem Cells

Yong

Pingguan‐Murphy

et al. 2015

Sci Rep

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“…If the device is left undisturbed and the isopropanol is changed frequently to avoid loss of alcohol through evaporation, as per manufacturer guidelines, passive freezing may be employed in the absence of a control-rate freezer. Janz Fde et al., reported that both approaches display similar potential to maintain phenotype, viability and function of certain tissue [8] . Finally, our methodology may be unsuitable for whole tissue freezing of cervical tissue should a larger sized sample be required.…”

Section: Discussionmentioning

confidence: 99%

Methodology for reliable and reproducible cryopreservation of human cervical tissue

et al. 2017

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BackgroundIn order to conduct laboratory studies on donated cervical tissue at suitable times an effective and reliable cryopreservation protocol for cervical tissue is required.MethodsAn active freezing approach was devised utilising 10% dimethyl sulfoxide in foetal bovine serum as a cryoprotective agent with a cooling rate of 1 °C/min to −50 °C then 10 °C/min to −120 °C; a related thawing protocol was also optimised which would allow for the bio-banking of cervical tissue. Viability of freshly harvested cervical tissue was compared to frozen-thawed samples utilising colorimetric MTT assay. In parallel, fresh and freeze-thawed samples were cultured and tested on days 1, 7 and 14 to determine whether bio-banking had detrimental effects on tissue viability over time.ResultsRepeat testing revealed that tissue viability between fresh and freeze-thawed samples was comparable at all four time points (days 0, 1, 7 and 14) with no apparent reductions of viability, thus demonstrating this method of cryopreserving cervical tissue is reliable and reproducible, without detrimental effects on live tissue culture. We believe this methodology creates the opportunity for bio-banking donated cervical tissues, which aids improved experimental design and reduces time pressures and wastage.

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“…MSCs for application in functional tissue engineering needs to be abundantly available, harvested with minimal morbidity, cryopreserved with viability maintenance and be able to be transplanted safely and efficaciously (JANZ, DEBES, CAVAGLIERI et al, 2012). These multipotent stem cells can be isolated from various mesenchymal tissue sources in adults, most commonly bone marrow (BARRY and MURPHY, 2004).…”

Section: Discussionmentioning

confidence: 99%

Isolation and cryopreservation of adipose tissue derived stem cells from different adipose tissues in obese patients

et al. 2017

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Introduction: Adult stem cells (ASCs) are a population of tissue-resident cells that have the capacity for self-renewal and differentiation into different cell types with potential for cell therapies. New approaches for ASCs isolation, including many tissue sources and new protocols that are more effective and less expensive are under investigation. Thus this work aim is to isolate, maintain in cell culture and evaluate cryopreservation protocols for adipose derived stem cells (ADSCs) from different tissues such as subcutaneous adipose tissue, visceral mesenteric and omental visceral taken from the same individual. Material and Methods: The techniques of mechanical and enzymatic dissociation were used, in order to investigate the most appropriated method to ADSCs isolation. Dimethylsulfoxide (DMSO) and dimethylformamide (DMF) in different concentrations were tested as cryoprotectors in 24, 48 and 72 hours thawing protocols. The samples were collected from obese patients with associated diseases undergoing bariatric surgery, between 30 to 45 years old. Results: Among 10 collected samples it was possible to measure cell viability from 4 patients. The higher cell rate was obtained from the visceral tissue of omentum. Conclusion: DMSO was the more efficient cryopreservant for this condition. This adipose tissue source could be explored for ADSCs isolation and future clinical investigations.

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Evaluation of Distinct Freezing Methods and Cryoprotectants for Human Amniotic Fluid Stem Cells Cryopreservation

Cited by 35 publications

References 25 publications

Phenotypic and Functional Characterization of Long-Term Cryopreserved Human Adipose-derived Stem Cells

Phenotypic and Functional Characterization of Long-Term Cryopreserved Human Adipose-derived Stem Cells

Methodology for reliable and reproducible cryopreservation of human cervical tissue

Isolation and cryopreservation of adipose tissue derived stem cells from different adipose tissues in obese patients

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