PROTOCOL PluriQ Serum Replacement
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- Magnus Cameron
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1 (PluriQ SR) is a serum free supplement formulated as a direct replacement for FBS (fetal bovine serum) used in the growth and maintenance of undifferentiated human embryonic stem cells (hesc) and induced pluripotent stem cells (ipsc) in culture. PluriQ SR has been shown to maintain undifferentiated healthy pluripotent stem cell cultures with normal karyotype for at least 20 passages and to support embryoid body formation and stem cell differentiation. Product Components, 500 ml PluriQ Supplement A, 500 L, 100 ml PluriQ Supplement A, 100 L Storage Conditions: Shipped frozen on dry ice Storage & Handling: Store at 20 C on arrival; protect from light Shelf Life: 1 year at 20 C GSM 6101 GSR 6101 GSM 6102 GSR 6102 Recommended Reagents (not included) ES DMEM/F12 (GSM 1002, GlobalStem) ES DMEM (GSM 2001, GlobalStem) FGF 2 (GSR 2001, GlobalStem) Collagenase IV ( , Invitrogen) I. ACCLIMATION TO PLURIQ SERUM REPLACEMENT Pluripotent stem cells, notably ipsc cells, can have different culturing characteristics that often require an acclimation period when introduced to new reagents depending on the culturing procedure that is used. Similarly, an acclimation period may be required when using PluriQ SR for the first time with cells that have been grown in either FBS or a different serum replacement. During this period, which may last up to three passages, a slightly shorter or longer population doubling time may occur. Carefully follow the guidelines and recommendations below to minimize the acclimation period: 1. Add Supplement A: Thaw PluriQ SR and Supplement A in a 37 C water bath as instructed in Section II. Once thawed add the total vial contents of Supplement A to the PluriQ SR. 2. We recommend using not less than 10ng/mL of FGF during the acclimation period. 3. Carefully review all the Points to Consider in Serum Free Culture in Section III. 4. If the cells are growing slower when first plated into complete supplemented PluriQ SR, it may be required to follow the Weaning Protocol in Section V. 5. Passage the cells only when they are ready to be passaged. Passaging cells too soon or too late will introduce stress to the cells and increase the risk of differentiation (see Figure 1).
2 II. MEDIUM PREPARATION A. Thawing and Addition of Supplement A Thaw 5X PluriQ SR and Supplement A (Rock inhibitor Y 27632) in a 37 C water bath. Alternatively, thaw PluriQ SR and Supplement A at 4 C overnight. When thawing PluriQ SR at 37 C, be sure to occasionally swirl the bottle to mix. Once thawed, add the total vial contents of Supplement A to the thawed PluriQ SR which will give a 1 M working concentration of Y Thawed PluriQ SR plus Supplement A can be stored at 4 C in the dark for up to 4 weeks. NOTE: Supplement A (Rock inhibitor Y 27632) significantly enhances plating efficiency and colony growth of ES and ips cells in PluriQ SR culture medium. B. Preparation of Complete Serum Free Medium a. Use Table 1 below for preparation of complete serum free culture media for human ES and ips cells when using GlobalStem ES DMEM or ES DMEM/F12 media. b. Use Table 2 below when not using GlobalStem ES DMEM or ES DMEM/F12 semi complete media. Table 1. Stock Conc. GlobalStem Cat No. Final Conc. For 100 ml ES DMEM or ES DMEM/F12* GSM 2001/ GSM X 80 ml w/ Supplement A (recommended) GSM 6101/ GSM % 20 ml FGF 2 10 g/ml GSR ng/ml Add daily to needed amount of medium * GlobalStem ES DMEM and ES DMEM/F12 are semi complete media, pre formulated with 2 Mercaptoethanol (0.1 mm), Nonessential Amino Acids (NEAA, 0.1 mm), and Dipeptide Glutamine (2 mm). Table 2. Stock Conc. GlobalStem Cat No. Final Conc. For 100 ml DMEM or DMEM/F12 1X 80 ml w/ Supplement A (recommended) NEAA 10 mm GSM 6101/ GSM % 20 ml 0.1 mm 1 ml L Alanyl L Glutamine 200 mm 2 mm 1 ml 2 Mercaptoethanol 55 mm 0.1 mm 182 L FGF 2 10 g/ml GSR ng/ml Add daily to needed amount of medium Complete medium is stable for 7 days when stored at 4 C in the dark. For best results, warm to 37 C only the amount of medium needed for that day s work. Avoid repeated warming and chilling of complete medium. Page 2
3 III. POINTS TO CONSIDER IN SERUM FREE CULTURE Typically, pluripotent stem cells in serum free culture are more sensitive to extremes of ph, temperature, osmolality, mechanical forces, and enzyme treatment. When introducing a new reagent extra care should be taken to minimize the stress to the cells. The following are points to consider when culturing your cells in serumfree medium and recommendations that will help minimize the effect of stress due to the acclimation period. Reagents Growth Factors Only use pluripotent stem cell qualified reagents. While some protocols call for 4 ng/ml of bfgf to culture pluripotent stem cells, we recommend using a higher concentration (10 ng/ml) of bfgf. This higher concentration of bfgf can help to minimize stress during the first few passages in PluriQ SR. Passaging Cells During the acclimation period, cells in the PluriQ SR may grow faster or slower than what you typically observe. This is not uncommon and is only temporary during the first few passages. If you observe faster or slower growth, it is important that you passage the cells only when they are ready to be passaged, not too early or too late (see below, Figure 1). Note that if using PluriQ SR in a side by side comparison with your current serum or serum replacement media, initially you may need to passage the different conditions on different days. To this end, we recommend using separate culture plates for the different conditions. Cell Dissociation Method We highly recommend using collagenase IV to dissociate the cells. Using a low concentration of the collagenase (2 mg/ml) for 30 to 40 minutes at 37 C without manual scraping will also be less stressful to the cells. Thus, we recommend following the protocol for Passaging Human Pluripotent Stem Cells Using Collagenase IV on page 4 (Section IV). Morphology Slight changes in cellular or colony morphology may be observed during acclimation to serum free media. As long as doubling times and viability remain good, slight changes in morphology should not be a reason for concern. Figure 1. Passage times should be determined by colony size and density of the culture: A typical culture will have colonies that vary in size. At passage, the majority of the colonies should be at the optimal size (Image B). It may be necessary to wait for smaller colonies (Image A, black arrow) to grow, but do not wait so long that the larger colonies (Image C) are lost to differentiation. 10x Magnification. Page 3
4 IV. PASSAGING HUMAN PLURIPOTENT STEM CELLS USING COLLAGENASE IV Collagenase IV is commonly used to passage pluripotent stem cells. It is gentler than trypsin, but requires longer incubation times. We recommend using a 2 mg/ml solution to dissociate and passage the cells. Collagenase solutions should be stored no longer than 6 months at 20 C and no more than 10 days at 4 C. The method outlined below is proven to selectively remove the undifferentiated cells from the feeder layer and other strongly adherent cell types. As always, periodic tests to confirm karyotypic stability during long term culturing and expansion is recommended. 1. Plate the feeder cells the day prior to passaging your human stem cells. 2. Aspirate the medium from the culture vessel and wash with enough 1X PBS to cover the entire cell growth area. 3. Cover the entire growth area of each vessel with Collagenase IV solution (2 mg/ml) and return vessel to 37 o C incubator. 4. Observe stem cell colonies under the microscope after 20 minutes. Some of the colonies will start to curl up on the edges. 5. Continue incubation until a majority of the colonies are curled up or floating. This can take between minutes. Note that the incubation times may vary among different batches of collagenase and different cell lines; therefore, examination of the colonies is needed to determine the appropriate incubation time. 8. Remove most of the supernatant. Using a 5mL pipette, pipette the cell suspension in order to break the colonies into smaller pieces. Be careful not to pipette too much. Note: Avoid breaking colonies down to single cells. 9. Resuspend the cell suspension in complete growth medium. 10. Plate stem cells into a new cell culture vessel. We recommend 1:3 1:5 split ratio depending on the growth rate of the individual cell line. Note the colony density and colony size at each passage. If the colony density is too sparse, decrease the split ratio at the next passage. If the colonies are either too large or too small, increase or decrease accordingly the amount of pipetting performed after collagenase incubation. 6. When the cells are ready to passage, add complete growth medium such as ES DMEM/F12 (GSM 1002) supplemented with PluriQ SR and bfgf. Wash the cell growth area with a pipette (5mL or 10 ml), depending on the size of the vessel, to dislodge colonies from the surface. In order to avoid pipetting the colonies too much, transfer the detached colonies to a tube. More media can be added to help dislodge additional colonies. 7. Collect resulting suspension cells and centrifuge at 270 x g for 5 min. (Alternatively, allow the colonies to settle by gravity. This decreases the transfer of any remaining fibroblasts to the new culture). Page 4
5 V. WEANING PROTOCOL (Optional) To minimize the impact of the acclimation period on cells to PluriQ SR, the following standard weaning protocol may be used. Here, cells are acclimated to PluriQ SR by gradually reducing the concentration of your current serum or other serum replacement supplement using sequential ratios of the serum supplemented medium and PluriQ SR over three passages. Day 1: Initial Plating Initially plate cells in 100% of your current medium. Upon first medium change after Day 1, use 75/25 ratio as illustrated. 1 st Medium Change 75% Complete medium containing current serum replacer/serum + 25% Complete medium containing PluriQ SR Passage 1 50% Complete medium containing current serum replacer/serum + 50% Complete medium containing PluriQ SR Passage 2 25% Complete medium containing current serum replacer/serum + 75% Complete medium containing PluriQ SR Passage 3 100% Complete medium containing PluriQ SR Page 5
6 Limited Use Label License The purchase of this product conveys to the buyer the non transferable right (a) to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for profit entity) but not to use the product or components of the product for therapeutic, diagnostic or prophylactic purposes or (b) to manufacture products or provide services. The buyer cannot sell or otherwise transfer for commercial purposes this product or its components to a third party. The buyer may transfer this product or its components or materials made using this product to a scientific collaborator, provided that, in conjunction with such transfer, such collaborator agrees in writing (a) not to transfer such product, components or materials to any third party, (b) to use such transferred product, components or materials and/or information solely for research and not for any therapeutic, diagnostic or prophylactic purpose; (c) not to resell the product or its components, whether or not such product or its components are resold for use in research; (d) neither analyze the chemical composition of the product nor reverse engineer the product. For products that are subject to multiple limited use label licenses, the terms of the most restrictive limited use label license shall control. GlobalStem will not assert a claim against the buyer of infringement of patents owned or controlled by GlobalStem which cover this product based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product or any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement GlobalStem is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research contact GlobalStem at info@globalstem.com. CONTACT / ORDERING INFORMATION GlobalStem 9430 Key West Ave. Ste Rockville Maryland info@globalstem.com phone / fax GlobalStem. All Rights Reserved. Printed in USA Page 6
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