hpsc Maintenance Media

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1 hpsc Maintenance Media Brigitte Arduini, version 2, 2013-Jun-12 Initially, it was found that pluripotency of human pluripotent stem cells (hpscs) can be maintained when plated in co-culture with mouse embryonic fibroblasts (MEFs) or in media conditioned by MEFs (MEF- CM). In the latter case, also known as feeder-free culture, additional treatment of tissue culture vessels with Matrigel or comparable substrate is necessary (see Matrigel Plate Coating). Subsequently, defined media formulations have been developed and marketed. However, MEF-CM is currently the only medium that allows the maintenance or spontaneous differentiation of hpscs with the change of a single parameter (ie: conditioned medium versus base medium). At this time, commercially available defined media do not have complementary formulations that lack pluripotency maintenance factors while supporting robust cell survival and differentiation. On the other hand, MEF- CM contains multiple components that are not completely defined, including Knockout Serum Replacement and factors released by the MEFs. The choice of pluripotency maintenance medium, therefore, must be determined by the experimental questions being pursued and the needs of the investigator. Defined media Many attempts have been made to develop defined media for the maintenance of hpscs, but a 2010 study by the International Stem Cell Initiative indicates that only a handful of these formulations support long-term self-renewal and pluripotency (Akopian et al., 2010). mtesr1 and STEMPRO (both available from ) were found to be successful. Another commercially available medium, Nutristem (Stemgent), also appears to maintain pluripotency. However, increasing the bfgf concentration from 4 ng/ml to 20 ng/ml is necessary for sustained growth rates. Finally, a new maintenance medium called Essential 8 was developed by the Thompson lab at the University of Wisconsin (Chen et al., 2011), and is available in prototype from. Early indications suggest that maintenance of hpscs is supported. However, I have found that it is not possible to grow hpscs to uniform confluence when cultured in Essential 8. This observation was confirmed by other stem cell facility directors, and applies to mtesr1, as well. This is a critical consideration for procedures such as neural induction, as differentiation of central nervous system neurons is optimal with a nearly 100% confluent population of hpscs to start. Additional considerations: Colony morphology in the various media hpscs cultured in CM form flat monolayers in adherent culture, whereas colonies in mtesr1 may be slightly domed. Essential 8 appears to support the flattened colony morphology. Cells are more tightly packed in mtesr1 and Essential 8 than in MEF-CM. Although pluripotency does not appear to be affected, these features may come into play during imaging.

2 Live cell imaging and FACS Unlike imaging of fixed cells, which may be performed in PBS, live cell imaging requires that cells be kept in the maintenance medium of choice. This may have consequences for detection of green and red fluorescent proteins, due to background fluorescence from media components. See Live Cell Imaging and FACS protocols for more information.

3 hpsc Growth Medium (HUESM) Brigitte Arduini, version 1, 2013-Jan-17 Medium formulation adapted from original H1 medium by Scott Noggle. Modifications: DMEM base rather than DMEM/F12 Addition of B27 supplement containing antioxidants (microarray data indicate that hescs express genes associated with oxidative stress (Sato & Brivanlou, 2006)) Increase of bfgf concentration from 4 ng/ml to 20 ng/ml (seems to compensate for variation between lots of MEFs when making conditioned medium) Materials: Filter system (bottle + filter), 0. 2µm PES membrane (VWR cat# ) Luer-lock syringe Syringe filter, 0.2µm PES (VWR cat# ) Microfuge tubes Conical tubes Tris-HCl Bovine Albumin Fraction V, 7.5% solution ( cat# ) Media components (see below) Component Catalog No. Final Conc. For 500ml Knockout Serum 20% 100ml Replacement (KSR) GlutaMAX 2mM MEM non-essential 0.1mM amino acids Penicillin-steptomycin 100U/ml-0.1mg/ml (Pen-strep) Mercaptoethanol 0.1mM 900µl DMEM % 37 Filter prior to adding supplement B27 Supplement 1X 10ml without Vitamin A bfgf PHG ng/ml - KSR, GlutaMax and Pen-Strep may be thawed at 4 o C, aliquoted, and re-frozen once at -20 o C. HUESM may be stored at 4 o C for up to two weeks. Pre-warm only what is needed for 20 to 40 minutes at 37 o C. To complete growth medium, bfgf should be added just prior to use (see below).

4 bfgf/aa (catalog no. PHG0021/PHG0023) is available in bulk quantities at substantial long-term savings. Stocks are made to 100µg/ml in sterile 10mM Tris-HCl ph % BSA. 20µl to 100µl aliquots should be made at room temperature, then snap-frozen with liquid nitrogen and stored at -80 o C. Once thawed, aliquots should not be re-frozen.

5 MEF-Conditioned Medium (CM) Brigitte Arduini, version 1, 2013-Jan-17 Modified from Scott Noggle, 2007-Sep-28 Mouse embryonic fibroblasts (MEFs) may be obtained from multiple commercial sources or isolated from E13 mouse embryos. The quality of this reagent is crucial for the production of reliable hpsc maintenance media, and we have found that GlobalStem ( is a consistently dependable source. MEFs must be Mitomycin-C treated or gamma irradiated to inhibit cell division. This will result in more consistent numbers of MEFs during the conditioning process and prevent contaminating MEFs from outgrowing hesc cultures. Materials: MEFs (GlobalStem, catalog no. GSC-6001M, 4-5x10 6 cells/vial) Filter system (bottle + filter), 0. 2µm PES membrane 10cm tissue culture dishes or T175 flasks 0.1% Gelatin Media components (see below) FM10 Medium Component Catalog No. Final Conc. For 500ml DMEM 88% 440ml Fetal Bovine Serum 10% 50ml L-glutamine 1X Penicillin-Streptomycin 1X Mercaptoethanol mM 900µl Add bfgf as needed bfgf PHG ng/ml - Plating MEFs: 1. Prepare dishes or flasks by coating in 0.1% gelatin at room temperature for minutes. 2. Thaw two vials of MEFs rapidly at 37 o C. 3. Resuspend in 2 FM Aspirate gelatin from dishes and immediately plate entire MEF volume. 5. Move dish side-to-side to distribute cells. 6. Incubate overnight to attach.

6 Conditioning HUESM: 1. Aspirate FM Rinse MEFs with HUESM and replace with 30ml HUESM plus bfgf. 3. Incubate overnight. 4. After 24 hours (+/- 1 hour), remove conditioned medium to a conical tube and replace with fresh HUESM. 5. Observe confluence of MEFs daily. After 4 5 days, reduce volume of HUESM being conditioned to 2 per dish. Dishes may be used to condition HUESM repeatedly for approximately 7 10 days. CM can be used immediately, stored at 4 o C for up to a week or stored at -80 o C up to several months. For most consistent results, media should be frozen between collection and use. When ready to feed hpsc cultures, add fresh bfgf at 20ng/ml.