Frequently Asked Questions Stem Cells

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1 Q: Do you add antibiotics to your media? A: Coriell does not use antibiotics when culturing stem cells. Customers should be aware that inclusion of antibiotics in media may change growth characteristics of the cells. Also, continued culturing of cells in antibiotics can result in growth of antibiotic-resistant strains of bacteria Q: I am replenishing the culture medium daily; however, the medium is changing to a light orange/yellow color. Am I doing something wrong? A: The basal culture medium contains the ph indicator phenol red. When the media color changes from pink to yellow, it is indicative of the ph becoming more acidic, which commonly occurs when cells are dividing rapidly and producing acidic waste products. The medium should never be a bright yellow as this is an indication that the cells have overgrown. Q: Why do you recommend that I cryopreserve a small number of vials of stem cells for my laboratory? A: Establishing a master stock is critical so that a vial can be recovered and expanded if a problem arises with the growing stem cells such as: contamination, spontaneous differentiation and changes in chromosomal integrity. If any of these occur, you can recover a vial of your master stock and expand it to make a working stock for the laboratory. Q: Why do you recommend that I perform karyotype analysis on my stem cells? A: Due to the possible genomic instability that can be introduced with the reprogramming process, ipsc lines need to have routine genomic analysis performed. Additionally, chromosomal abnormalities may arise if ipsc lines are not cultured appropriately. We recommend that you analyze the stem cells using G-band karyotype analysis when you create a master stock, a working stock and every 10 to 15 passages. We also recommend that karyotyping be performed if there is a noted change in morphology or growth kinetics. Human induced pluripotent stem cells (ipsc) Q: I plan to obtain human induced pluripotent stem cells (ipsc) from Coriell. What reagents should I have on hand to successfully culture ipscs? A: The table below provides a list of reagents necessary for culturing human ipscs. There are no preferred vendors. Reagents can be obtained from a variety of vendors; the list below is given for convenience only. Refer to the appropriate Stem Cell Protocol for additional information.

2 Reagent Potential Sources 6-well plates Nunc, BD biosciences, Corning 0.1% gelatin Millipore, Stem Cell Technologies Mouse embryonic fibroblasts (MEFs) Global Stem, Applied Stemcell, ATCC DMEM/F12, Stem Cell Technologies Knock-out Serum Replacement L-glutamine, Basic fibroblast growth factor (bfgf), Global Stem, Stemgent Non-essential amino acids, 2-mercaptoethanol Rho Kinase (ROCK) Inhibitor Stemgent, Millipore Collagenase Sigma, Fisher TrypLE Express Dispase Versene Q: Why do you suggest using a Rho Kinase (ROCK) Inhibitor in the recovery medium? A: ipscs have a very low viability following cryopreservation. Inclusion of the Rho Kinase (ROCK) Inhibitor in the recovery medium increases the viability of the cells increasing the number of colonies observed within the newly thawed colony and decreasing the number of days it takes for the colonies to develop. Q: I have thawed my ipscs according to the stem cell protocol. It is 24 hours following recovery and I do not see colonies. What should I do? A: Continue replenishing the culture medium daily. The viability of ipscs following recovery from cryopreservation is very low (between 0.1% and 1%). Colonies may take up to one week to begin growing. If after one week, colonies have not developed contact customerservice@coriell.org. Q: How do I know when it is time to passage my ipscs? A: ipscs should be passaged for any of the following reasons:

3 1. MEFs are over 2 weeks old 2. Individual colonies are large (over 700 microns) 3. Colonies are close together and reaching confluence (~70-80%) 4. Significant differentiation is observed There are many factors that contribute to the successful culturing of ipscs. Cells should be observed microscopically daily and MEFs should be plated the day before an anticipated split. In general, ipsc colonies should be passaged every 4-7 days at a 1:3 to 1:6 split ratio. However, the number of days between passaging and the split ratio are dependent on the individual cell line and its growth. We recommend that you record the passage information for each cell line to determine the exact growth kinetics of a particular line. As soon as the ipscs have been passaged, we highly recommended that you cryopreserve, at miniumum, a small number of vials as a master stock. Refer to Cryopreservation of Human ipsc in the stem cell protocol. Q: Why do you suggest I centrifuge my cells at such a low speed? A: ipscs should be passaged as clusters and not as single cells. By using centrifuge speeds that are lower than typically used to pellet other cell types, the clusters will pellet to the bottom of the centrifuge tube while the single cells remain floating in the media. Q: Why is the concentration of basic fibroblast growth factor (bfgf) variable? A: Each ipsc line is grown in bfgf at the concentration specified by the submitter. The concentration of bfgf for a specific line is indicated on the Certificate of Analysis. Murine Embryonic Stem Cells (mes) Q: I plan to obtain murine embryonic stem cells from Coriell. What reagents should I have on hand to successfully culture mes cells? A. Refer to the Murine Embryonic Stem Cell Culturing Protocol for detailed information. The table below provides a list of reagents necessary for culturing mes cells. There are no preferred vendors. Reagents can be obtained from a variety of vendors; the list below is given for convenience only. Reagent Potential Sources 6-well plates Nunc, BD biosciences, Corning 0.1% gelatin Millipore, Stem Cell Technologies Global Stem, Applied Stemcell, Open Biosystems DR4 Mouse embryonic fibroblasts (MEFs) DMEM (high glucose), Sigma, Hyclone

4 ES cell Fetal Bovine Serum Applied Stem Cell, ATCC, Millipore, Invitrogen Glutamax B-mercaptoethanol, Sigma Non-essential amino acids, Sodium pyruvate, Leukemia Inhibitory Factor Millipore, Stem Cell Technologies, Stemgent Doxycycline Clontech Puromycin Q: I have thawed my mes cells according to the Murine Embryonic Stem Cell Protocol. What should my cells look like 24 hours after plating? A: The picture above is of a cell line 24 hours after thaw. Note the round colonies and even distribution of the cells throughout the dish. If colonies have not developed after two days, contact customerservice@coriell.org. Q: How do I know when it is time to passage my mes cells? A: Typically, mes cells passage every 2-3 days and plate at a density between 1.0 X 10 6 and 1.8 x 10 6 cells per well of a 6 well plate. The cells should be passaged when they are between 70-80% confluence using split ratios ranging from 1:4 to 1:10. However, the number of days between passaging and the split ratio are dependent on the

5 individual cell line and its growth. We recommend that you record the passage information for each cell line to determine the exact growth kinetics of a particular line. As soon as the mes cells have been passaged, we highly recommended that you cryopreserve, at miniumum, a small number of vials as a master stock. Refer to Cryopreserving mes cells. Q: Initially my cells were growing and passaging as expected. Now my cells are not growing and seem to be lifting off the plate. What is happening? A: The sudden change in growth kinetics of a cell line can be indicative of problems with media components. Specifically, puromycin concentration as well as FBS and MEF quality can impact the growth of your cells. Testing new lots of reagents prior to using them in growth medium for your cells can decrease the probability of observing negative effects on the growth of your cells. For information, we recommend testing new lots of MEFs to determine the optimal plating density, see the protocol Determining Optimal Plating Density for Mouse Embryonic Fibroblasts (MEFs). We further suggest performing a puromycin kill curve as the concentration can vary slightly with each lot of antibiotic.