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Home > ÀüÁ¦Ç°º¸±â > Stem Cell > Áٱ⼼Æ÷ À¯ÀüÀÚÆíÁý > hiPSCÀÇ À¯ÀüÀÚ ÆíÁýÀ» À§ÇÑ ´ÜÀϼ¼Æ÷¹è¾ç

hiPSCÀÇ À¯ÀüÀÚ ÆíÁýÀ» À§ÇÑ ´ÜÀϼ¼Æ÷¹è¾ç

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À¯ÀüÀÚ ÆíÁýµÈ hiPSCÀÇ ´ÜÀϼ¼Æ÷ È®´ë¹è¾çÀ» À§ÇÑ
Cellartis¢ç DEF-CS¢â Culture System

ÃÖ±Ù À¯ÀüÀÚ ÆíÁý ±â¼úÀÇ ¹ßÀüÀ¸·Î ¼¼Æ÷³ª ´Ù¾çÇÑ À¯±âüÀÇ ¸ñÀû À¯ÀüÀÚ¸¦ º¯ÇüÇϰųª, µµÀÔÇÏ´Â °ÍÀÌ °¡´ÉÇØÁ³´Ù. human induced Pluripotent Stem Cells (hiPSC)¿¡µµ ÀÌ·¯ÇÑ À¯ÀüÀÚÆíÁý ±â¼úÀÌ Àû¿ëµÇ¾î Àΰ£ Áúº´ ¸ðµ¨ Á¦ÀÛ ¹× À¯ÀüÀû º¯È­·Î ÀÎÇÑ ±â´É º¯È­ ¿¬±¸, Àç»ýÀÇÇÐµî ´Ù¾çÇÑ ºÐ¾ß¿¡ Àû¿ëÇÏ°í ÀÖ´Ù.
ÀÌ·¯ÇÑ ¿¬±¸¸¦ À§Çؼ­´Â À¯ÀüÀÚ ÆíÁýµÈ hiPSC¸¦ È¿À²ÀûÀ¸·Î ´ÜÀϼ¼Æ÷·Î ¹è¾ç (Single Cell Cloning)ÇÏ¿©, ¿øÇÏ´Â À¯ÀüÀÚ°¡ Á¤È®ÇÏ°Ô ÆíÁýµÈ ¼¼Æ÷¸¦ ¼±º°ÇÏ´Â °úÁ¤ÀÌ ²À ÇÊ¿äÇÏ´Ù. ±×·¯³ª on-feeder ¶Ç´Â colonyÇüÅ·Π¹è¾çµÈ hiPSC´Â ´ÜÀϼ¼Æ÷·Î ¹è¾ç ½Ã ¼ºÀå°ú »ýÁ¸ °ü·Ã signalÀÌ °¨¼ÒµÇ¾î »ýÁ¸·ü (cell viability)ÀÌ ¸Å¿ì ³·À¸¸ç, »ýÁ¸ÇÑ ¼¼Æ÷´Â ¹ÌºÐÈ­ À¯Áö°¡ ¾î·Æ±â ¶§¹®¿¡ ¿¬±¸¿¡ ¾î·Á¿òÀ» °Þ°í ÀÖ´Ù.
Takara Bio¿¡¼­´Â DEF-CS¢â Culture SystemÀ» ÅëÇØ hiPSC¸¦ feeder-free, non-colony ÇüÅ (monolayer)·Î ¹è¾çÇÏ¿©, on-feeder ¹× colony ¹è¾ç¿¡¼­ ¹ß»ýÇÏ´Â ¹®Á¦Á¡À» ÇØ°áÇÏ¿´´Ù. À̸¦ ÅëÇØ, ´ÜÀϼ¼Æ÷¸¦ È¿°úÀûÀ¸·Î ¹è¾çÇÏ¿©, ¼¼Æ÷ÀÇ »ýÁ¸·üÀ» ȹ±âÀûÀ¸·Î Áõ°¡½ÃÅ°°í, °­·ÂÇÏ°Ô ¹ÌºÐÈ­¸¦ À¯ÁöÇÔÀ¸·Î½á hiPSCÀÇ À¯ÀüÀÚ ÆíÁý ½ÇÇèÀÇ È¿À²À» Áõ°¡½Ãų »Ó¸¸ ¾Æ´Ï¶ó, ½ÇÇè ½Ã°£µµ ´ëÆø ´ÜÃàÇÒ ¼ö ÀÖ´Ù.



hiPSCÀÇ À¯ÀüÀÚ ÆíÁý ½Ã, ´ÜÀϼ¼Æ÷ ¹è¾ç (Single cell Cloning)ÀÌ ÇÊ¿äÇÑ ÀÌÀ¯?
À¯ÀüÀÚ ÆíÁý È¿À²Àº 100%°¡ ¾Æ´Ï±â ¶§¹®¿¡, ½ÇÁ¦ À¯ÀüÀÚ°¡ ÆíÁýµÈ ¼¼Æ÷¸¦ ¼±º°ÇÏ´Â °úÁ¤ÀÌ ÇÊ¿äÇÏ´Ù.
    1. ¸ñÀû À¯ÀüÀÚÀÇ À¯ÀüÀÚ ÆíÁý ¿©ºÎ È®ÀÎ ÇÊ¿ä (monoallelic, biallelic)
    2. ¸ñÀû À¯ÀüÀÚ°¡ ÆíÁýµÈ ¼¼Æ÷¸¸ ¼±ÅÃÇÏ¿© ½ÇÇè
    3. ÆíÁýµÈ ¼¼Æ÷ Áß¿¡¼­µµ È¿°úÀûÀÎ Knock-out ¼¼Æ÷ ¼±º° ÇÊ¿ä





1. ´ÜÀϼ¼Æ÷ ºÐ¸®½Ã, limiting dilution (Clone Çü¼º È¿À² Áõ°¡)

ChiPSC cell line
Isolation method
Single clones
Double clones
Total clones (proportion)
Total clones (%)
ChiPSC 18
FACS
52
0
52/96
54%
ChiPSC 18
Limiting dilution
46
12
58/58
100%
ChiPSC 22
Limiting dilution
39
15
54/55
98%


2. ¼¼Æ÷´Â ¼ºÀå (proliferation)»óÅÂÀÇ hiPSC¸¦ »ç¿ë: ³·Àº ¹Ðµµ(0.8-1.5 x 105 cells/cm2)¿¡¼­ ¹è¾çµÈ hiPSC¸¦ »ç¿ë
    (¾Æ·¡ À̹ÌÁö ÂüÁ¶)

3. ÃÖÁ¾ Èñ¼® ¼¼Æ÷ ³óµµ¸¦ 1 cell/well ¶Ç´Â 0.5 cell/well·Î °è»ê
    - ¿¹) 50~100 cells/10 §¢·Î Èñ¼® ÈÄ, 100 §¡ ¾¿ 96 well plate¿¡ ÷°¡

4. Transfection ¶Ç´Â À¯ÀüÀÚ ÆíÁý µÈ ¼¼Æ÷ÀÇ ¾ÈÁ¤È­¸¦ À§ÇØ ÃÖ¼Ò 5ÀÏ ÀÌ»ó ¹è¾ç ÈÄ ´ÜÀϼ¼Æ÷·Î ¹è¾ç




Code
Á¦Ç°¸í
¿ë·®
Monolayer ÇüÅ (Non-colony)·Î ¹è¾çÇÏ¿©, °£ÆíÇÑ iPSC/ESC¹è¾ç°ú ¶Ù¾î³­ ´ÜÀϼ¼Æ÷ ¹è¾ç È¿À² (~30%)
Cellartis¢ç DEF-CS¢â 500 Culture System
1 Kit
´ÜÀϼ¼Æ÷¹è¾ç¿¡ ÃÖÀûÈ­µÈ ÷°¡Á¦ Á¶¼ºÀ¸·Î ÃÖ°í ¼öÁØÀÇ ´ÜÀϼ¼Æ÷¹è¾ç È¿À² Á¦°ø (¡Â50%)
Cellartis¢ç iPSC Single-Cell Cloning DEF-CS¢â Culture Media Kit
1 Kit
hIPSCÀÇ RNP (Ribonucleoprotein) ¹æ½ÄÀÇ À¯ÀüÀÚ ÆíÁý ÈÄ ´ÜÀϼ¼Æ÷¹è¾çÀ» À§ÇÑ System
Cellartis¢ç iPSC rCas9 Electroporation and Single-Cell Cloning System
1 Kit
hIPSCÀÇ GesicleÀ» ÀÌ¿ëÇÑ À¯ÀüÀÚ ÆíÁý ÈÄ ´ÜÀϼ¼Æ÷¹è¾çÀ» À§ÇÑ System
Cellartis¢ç iPSC CRISPR/Cas9 Gesicle and Single-Cell Cloning System
1 Kit