【Zenogen Pharma】STEM-CELLBANKER® GMP grade

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Zenogen STEM-CELLBANKER® GMP grade

Description

(Chemically defined cryopreservation solution)

STEM-CELLBANKER^® is a chemically defined freezing solution with optimized formulation for stem cells and iPS cells storage as well as other valuable cells. Furthermore, recent data supports it's ability of cryopreservation for tissues. It is completely free of serum and animal derived component, and contains only USP, EP, JP graded ingredients or JP excipients. It is ready-to-use with simple protocol while provides a safer and promising cryopreservation of stem cells and iPS cells.

STEM-CELLBANKER^® GMP grade is manufactured in a facility compliant with JP, EU, US and PIC/S GMP guidelines and the production and the quality are controlled in compliance with JP GMP guidelines.

Package:100ml/bottle

Advantages

●Significantly increased cell viability while maintaining cell pluripotency, normal karyotype and proliferation ability after freeze-thaw procedure.

●Useful for the cryopreservation of tissues without damages at DNA and RNA levels.

●Safer cryopreservation with high quality ingredients, no risk of contamination animal-derived components, batch-to-batch stability.

●Save cost/time/steps with complete formulation, no further preparation, direct freezing at -80℃, no programmable freezer required, etc.

●High quality product carefully monitored in compliance with JP GMP.

※This product has been registered in MF within PMDA in 2013 and BBMF within CBER (Center for Biological Evaluation and Research)/FDA (the U.S. Food and Drug Administration) in 2013.

Protocol (CELLBANKER^® series)

Freezing

For optimum results, cells for cryopreservation should be in log phase of growth. Similar or standard freezing protocols may be substituted.

1.Examine and make sure the cell culture free of contamination, in healthy situation and proper confluency, etc.

2.Perform a cell count to determine the viability of cells

3.Gently pellet the cells centrifugation (3 - 5 minutes at 1,000~2,000rpm, 4℃). Remove the supernatant using a aspirator.

4.Gently suspend the cells with CELLBANKER cryopreservation medium (1 ml for 5x10⁵ - 5x10⁶ cells)

5.Dispense the cell suspension in 1ml aliquots to cryopreservation vials that have been labeled with the cell line name, cell concentration, passage date and other essential information.

6.Place the vials directly in a -80℃ for storage. If necessary, transfer the frozen vials to a liquid nitrogen storage tank after the vials have been frozen for at least 24 hours.

IMPORTANT: Optimum protocol may change with the cell types.

Thawing

1.Remove the frozen cell from storage and quickly thaw in a 37°C shaking waterbath.

2.Immediately dilute and gently mix each 1ml of cells with 10ml of complete cell culture medium.

3.Gently pellet the cells centrifugation (3-5 minutes at 1,000 - 2,000rpm, 4°C). Remove the supernatant aspirator.

4.Gently suspend the cells with appropriate volume of complete cell culture medium. And plate in a culture flask.

5.Continue the further culture procedures according to standard protocols.

Storage and Stability

Store at 2 to 8 or below -20 degrees Celsius. In unopened condition and under storage temperatures (2-8℃ ), the product will be stable for 3 years after the date of manufacture.

Application

① Cryopreservation analysis on hESC and iPS cells with STEM-CELLBANKER®

Tested cell lines: HS293 & HS306 (hESC); ChiPSA (human iPS)
Freezing medium: STEM-CELLBANKER® vs Conventional freezing medium (10% DMSO in serum replacement containing culture medium)
Cell viability assessment: trypan Blue & calcein-esterase based live-dead assay

Result: Significantly higher cell viability was observed while cell pluripotency, normal karyotype and proliferation ability were maintained in HS293, HS306 and ChiPSA cell lines.

＊We are deeply grateful to Prof. Outi Hovatta and Ms. Frida Holm for above analysis.

② Cryopreservation analysis on Mesenchymal Stem Cells (MSCs) and Endothelial Progenitor Cells (EPCs) with STEM-CELLBANKER®.

MSCs EPCs
STEM-CELLBANKER® >95% >90%

Tested cell lines: Human UCB-derived MSCs and EPCs
Freezing medium: STEM-CELLBANKER®
Freezing protocol: Cell froze directly at -80℃ for 3days, transfer to -192℃ for 72 days.
Cell viability assessment: trypan Blue staining method.

Result: Cell viability as high as or more than 95% for MSCs and 90% for EPCs was observed while cell proliferation ability and differentiation ability were maintained in tested cells.

③ Cryopreservation analysis on Mouse ES cell (129SV)

Tested cell line: 129SV (Mouse ES cell)
Freezing medium: CELLBANKER 1, 1plus, 2 and STEM-CELLBANKER®

④ Cryopresevation of mouse kidney tissue using STEM-CELLBANKER®

Mouse kidney was minced into about 5 mm square. The tissue samples were either immediately fixed with 10% formalin or cryopreserved with 1 ml of STEM-CELLBANKER at -80°C for one week. The cryopreserved samples were fixed with 10% formalin immediately after thawing. Paraffin sections of the both samples were stained with either H&E or anti-active caspase 3 antibody. Sign of necrosis or apoptosis was negligible in both of the fresh and cryopreserved tissue samples. Similar results were obtained in the cases of the tissue samples from a variety of mouse organs.

⑤ Preservation of mRNAs after cryopreservation of cells with STEM-CELLBANKER®

Total RNA was extracted from the cells of a mouse lung fibroblast cell line either immediately after harvesting or after cryopreservation with STEM-CELLBANKER at -80°C for one week and thawing. mRNA levels in the two samples were compared DNA microarray analysis, and the results showed that 93% of mRNAs was within the range of + 1.6-fold difference.

Reference (Most popular publications)

●Shimazu et al. (2015) Serum- and xeno-free cryopreservation of human umbilical cord tissue as mesenchymal stromal cell source. Cytotherapy, 2015; 17: 593 - 600.

●Nakagawa M et al. (2014) A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Scientific Reports 4 Article, number:359.

●Miyamoto Y et al. (2012) Cryopreservation of Induced Pluripotent Stem Cells. Cell Medicine., Vol. 3, pp. 89-95, 2012.

●Saliem M et al. (2012) Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution. World J Hepatol. May 27;4(5):176-83.

●Holm et al. (2010) An effective serum- and xeno-free chemically defined freezing procedure for human embryonic and induced pluripotent stem cells. Hum Reprod. 25(5):1271-9. Epub 2010 Mar 5.

●Nakamura A et al. (2010) Human primary cultured hepatic stellate cells can be cryopreserved. Med Mol Morphol. 43:107-115.

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