Volume 17, Issue 12 (December 2019)                   IJRM 2019, 17(12): 891-906 | Back to browse issues page

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Akyash F, Tahajjodi S S, Farashahi Yazd E, Hajizadeh-Tafti F, Sadeghian-Nodoushan F, Golzadeh J, et al . Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report. IJRM 2019; 17 (12) :891-906
URL: http://ijrm.ir/article-1-1511-en.html
Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report
Fatemeh Akyash1 , Somayyeh Sadat Tahajjodi1 , Ehsan Farashahi Yazd2 , Fatemeh Hajizadeh-Tafti3 , Fatemeh Sadeghian-Nodoushan3 , Jalal Golzadeh3 , Hassan Heidarian Meimandi4 , Harry Moore5 , Behrouz Aflatoonian * 6
1- Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Department of Reproductive Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2- Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Department of Reproductive Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3- Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
4- Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
5- Department of Biomedical Sciences, Centre for Stem Cell Biology, University of Sheffield, Western Bank, Alfred Denny Building, Sheffield S10 2TN, UK.
6- Department of Advanced Medical Sciences and Technologies, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Department of Reproductive Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. , b.aflatoonian@gmail.com
Abstract:   (3090 Views)
Background: Cell banking of initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws.
Objective: To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification.
Materials and Methods: Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method.
Results: The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping.
Conclusion: Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient method of preserving hESCs.
 
 
Keywords: Derivation, Human embryonic stem cells, Human foreskin fibroblast, Microdrop, Vitrification
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Type of Study: Original Article | Subject: Stem Cell & Cloning
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