Stem Cell

Frequently Asked Questions (FAQs)

Q:	*What method of reprogramming source cells is the most efficient?*
A:	The retroviral method of reprogramming source cells is the most efficient and also the most well-characterized. Typically the efficiency is about 1% for retroviral reprogramming.
Q:	*How should I culture my iPS cells?*
A:	Most iPS cells from SBI must be cultured on feeder cells. The conditions for feeder cells and media recipes are found within the product manuals for each product. Please follow the protocols specified by the product you are working with. See User Manuals.
Q:	*How long does it take to recover frozen iPS cells?*
A:	Typically recovery takes between 7-10 days.
Q:	*How should I handle frozen iPS cells?*
A:	SBI highly recommends that protective gloves, a lab coat, and a full face mask always be worn when handling frozen vials. While SBI stores cells in the gas phase of liquid nitrogen, it is possible that some liquid nitrogen can leak into the tubes inadvertently. Upon thawing, it is possible for the liquid nitrogen to return to the gas phase, resulting in excessive pressure within the tube that can cause the tube to explode or expel the cap with dangerous force. 1. All cell lines from SBI should be stored in gas phase liquid nitrogen and should not be immersed in liquid nitrogen. 2. When thawing frozen cells, SBI recommends transferring the tubes to dry ice for 10-15 minutes to allow any liquid nitrogen to evaporate. 3. SBI recommends wearing protective gloves, a lab coat, and a full face mask when transferring the tubes to a 37°C water bath.
Q:	*My iPS cells mysteriously died? What happened?*
A:	Mycoplasma is extremely toxic to iPS cells. SBI recommends testing your cell culture facility for Mycoplasma before attempting to grow new iPS cells.
Q:	*Why couldn’t I find iPS cell colonies after thawing?*
A:	Recovering iPS cells from a frozen vial takes about two weeks. It takes at least 5 days for colonies to appear. ROCK inhibitor is required for the first day of thawing as it prevents single iPS cells from undergoing apoptosis. Maintain ROCK inhibitor in the media at lower concentrations after the first day may also increase survival rates. Refer to the appropriate user manul for more details and media recipes.
Q:	*How can I avoid excess differentiation of iPS cells?*
A:	iPS cells should be cultured at optimal conditions using feeder cells, coated plates and stem cell medium. The culture medium needs to be changed every day. The morphology of iPS cells should be examined under the microscope daily. The culture should be split when it reaches 80% confluency or when some colonies become too large and are about to differentiate. The differentiated colonies should be removed before splitting by picking with a pipette tip so that they won’t be carried over into the next passage.