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Feeder-Free Culture Techniques for Pluripotent Stem Cells using PeproGrow-ESC

A. IntroductionPeproGrow-ESC is a serum- and phenol red-free medium of a complete, chemically-definedformulation designed for feeder-free maintenance and expansion of both human embryonicstem cells (hESCs) and human induced pluripotent stem cells (iPSCs) using BD Matrigel™

as a surface-coating matrix. This medium is intended for the culturing of hESCs and iPSCsin the undifferentiated, pluripotent state (SSEA4+/Oct4+), and demonstrates less than 15%spontaneous differentiation as indicated by flow cytometry (Figure 1). The proprietary formulation of the medium includes relevant growth factors, such as FGF2 (FGF-basic),but does not contain the insulin found in the majority of other hESC/iPSC media currently available on the market. PeproGrow-ESC, which was designed and developed byPeproTech in collaboration with the Stem Cell Training Course at Rutgers University, issupplied as a 100mL, or 500mL, bottle of basal medium and a separate, lyophilized growthfactor component.

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Figure 1: iPSCs were cultured in PeproGrow-ESCmedium, and prepared forflow cytometry analysis with a BD Stemflow™ kit as per the manufacturer’s instructions (see Appendix I).Cells stained with Oct4,SSEA1, and SSEA4 were analyzed and gated to include 64% of events for the elimination of debris and clumps (top left). Triple staining revealed that93% of cells were Oct4+ and SSEA4+, or SSEA4+ and SSEA1- (bottom left and right). Stringent gating with Oct4 and SSEA1 revealed roughly 86% were Oct4+/SSEA1-.

B. Materials and Reagents1. PeproGrow-ESC:

Medium Catalog Number Volume PricePeproGrow-ESC BM-hESC* 500mL $200PeproGrow-ESC BM-hESC* 100mL $80*Each medium bottle is supplied with a lyophilized growth factor component.

2. Refer to Appendix I for additional materials and reagents.

C. Preparation of Medium and Growth Factor Component 1. Reconstitute the lyophilized PeproGrow-ESC growth factor component with 500µL

of sterile cell-culture grade water.2. Aseptically add the entire reconstituted PeproGrow-ESC growth factor component

to the basal medium, and mix well by swirling. Filtration is not necessary if preparedaseptically.

3. Label the bottle with both the date of mixture and the newly calculated expirationdate (2 weeks from date of mixture). Store at 2°C to 8°C.

4. Remove only the necessary amount of medium for immediate use and warm to 37°Cin a water bath prior to feeding cells.

Storage/Stability: Keep medium in the dark. Product Form Temperature Storage TimeLiquid Basal Medium 2°C to 8°C; Keep in the dark. 6 monthsLiquid Basal Medium after preparation 2°C to 8°C; Keep in the dark. 2 weeksPeproGrow-ESC Growth Factor Component -20°C to -80°C 5 years(lyophilized) 2°C to 8°C 6 monthsAvoid repeated freeze-thaw cycles.

General recommendations: • Recommended Coating for Cell Culture Ware. It is recommended to first use BD

Matrigel™ when adapting to PeproGrow-ESC media, however, other matrices havealso been tested and shown to function well with PeproGrow-ESC media. Preliminaryresults are available using human recombinant Vitronectin (10 µg/mL) and Synthemax,which appear to perform optimally when using CellBind® dishes (Corning).

• Exceptions: During the enzymatic passaging of cells, it is recommended to use dispase at 0.5 mg/mL, which represents half the widely accepted concentration of 1 mg/mL and to adjust the time needed, from a standard of 5 minutes, to as short as3 minutes. If this level of enzymatic activity does not appear to be sufficient, then theconcentration can be adjusted to 1 mg/mL, and the time lengthened up to 7 minutes.

• WARNING: If cells are over digested with the enzyme, then these may fail to adhere/survive even in the presence of recommended enhancers such as Y-27632 orthiazovivin. The Accutase™ enzyme may be used at the standard concentration ordiluted 1:1 with PBS/EDTA.

• Seeding efficiency and/or viability is greatly improved when the thawing, passaging,or freezing procedures include an enzymatic treatment step (Accutase™, dispase oranother enzyme), as well as the addition of 2-5 µM Y27632 or 1-2 µM thiazovivin.

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When passaging or freezing cells with PBS/EDTA, there appears to be no substantialimprovement when either Y-27632 or thiazovivin is used.

• Refer to Appendix II for additional figures and descriptions.

D. Maintenance of Pluripotent Stem Cells NOTE: Cultures may appear sparse within a day of thawing or splitting, howeverhESC/iPSCs grow very rapidly after 3-4 days in culture. Cells need to be maintained by changing the media on a daily basis, with the exception of accepting one “doublevolume” feed over the weekend. When supplementing with a “double volume”, it is recommended to passage the cells on a Thursday, so that the double feed occurs whenthe cells are at a lower density (e.g. cells can receive 4mL/well on a Friday, to avoid theneed for a feed on a Saturday).

EXAMPLE: 6 well dish1. Check every well for growth, and monitor cultures for bacterial or fungal

microorganisms.2. Aspirate each well to remove old media, and add 2 mL of new, warmed media

into each well.

E. Thawing Cryopreserved Pluripotent Stem Cells NOTE: Cells of an ultra-frozen sample should be thawed to 37°C slowly and consistently in order to avoid temperature shock and shear stress. The use of cold andcool solutions allows for the gradual introduction of a newly thawed, and still cold, sample to warmer environments in a controlled manner. Once the cryopreserved samplehas been thawed, it is important to dilute out the DMSO as soon as possible, and to transfer the sample to a suitable culture vessel. This procedure has been streamlinedbelow so that it can be performed within 15 minutes.

1. Place a 15 mL conical tube in a rack (pre-cooled Biocision CoolRack™ 15 mL)in a Biosafety Level 2 (BL2) Laminar Flow hood, and fill with 9 mL of coldmedia. To improve thawing efficiency, add 2 µM Y-27632 or thiazovivin to allsolutions during the thawing procedure.

2. Remove a vial of cells (hESC or iPSC) from liquid nitrogen (LN2) vapor phasestorage tank, place in floater rack and transfer to 37°C water bath.WARNING: Instructions for the majority of cryo-vials caution against storageof samples in the liquid phase of LN2 due to the possibility for the accumulationof LN2 in the vial, which can cause an explosion during rapid thawing. To reducethe risk of an explosion, vials should be transferred to a -80°C freezer the nightbefore use to allow for the slow evaporation of LN2 that might have leaked intothe vial.

3. Do not shake the vial! To ensure that the average temperature in the vial remainscool, observe occasionally and remove the vial when a small amount of ice remains.

4. Spray the vial generously with 70% ethanol, wipe and transfer into a sterile environment in a BL2 laminar flow hood onto the appropriate rack. To preventfurther temperature fluctuations, it is highly recommended to use a pre-cooledBiocision CoolRack® XT CFT24. Most freeze racks (Biocision, Corning) havelocking grooves to unscrew the top of the ampoule, however, the majority ofthese racks do not secure the vial vertically into the rack. Be careful not to spillthe cells if working with multiple vials.

5. Using a disposable, sterile, plastic transfer pipette remove 1 mL of media fromthe 15 mL tube previously prepared in step 1, and slowly back-fill the cryo-vial

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with gentle stirring. Alternatively, use a 1-2 mL serological pipette, or a 1000µLsized micropipette.

6. Remove contents of the vial to a 15 mL conical tube.7. OPTIONAL: Rinse the vial with a second round of 2 mL diluted cells/media

from the 15 mL tube using the same transfer pipette.8. If recovering cells from dispase frozen stocks, then allow larger aggregates

of cells to settle at 4°C in a precooled Biocision CoolRack™ 15 mL tube on ice (or placed in the refrigerator) for 5-10 minutes, and skip the following centrifugation step and proceed to step 11.

9. Remove the BD Matrigel™ coated dish from the incubator and place in a laminarflow hood. This step allows the dish to cool to room temperature (RT).

10. Centrifuge the cells for 2-5 minutes at 120 x g at 4°C. Alternatively, if using aBiocision CoolRack™ (or ice) and the medium is still cool to the touch, then spinthe tube at RT.

11. Remove the supernatant and gently resuspend the cells in 2 mL of cool medium.Keep the tube in hand to warm for 30 seconds or alternatively, place the tube inthe rack for 1-2 minutes.

12. Place the cell suspension into an appropriate number of desired BD Matrigel™

(or other appropriate matrix) coated culture ware wells. Use RT medium to perform further dilutions for cells. For example, if the vial contains 1 confluentwell of a 6-well dish, then plate in 1-3 wells. Adjust the volume of cells to 3 mL,and dispense 1 mL per well in a dish already containing 1 mL media per well. TIP: When using 6-well dishes only, plate cells out by running the pipette tip aroundthe rim of the well while slowly dispensing the cell solution. It is recommend touse a 5 mL glass serological pipette for this step, as the bore is smooth comparedto the plastic version and may offer less shearing stress to the cell clumps. Not plating on a 6-well dish? While it is recommended to passage the cells at least once before using them for analytical purposes (immunostaining, flowcytometry, western blot analysis, Q-RT-PCR, etc.), cells can be plated directlyon other sized vessels at this point. Please be aware that the freeze/thaw processcan introduce a relatively noticeable amount of differentiation. For an indirectimmunofluorescence technique, it is recommended to use 24-well dishes. For freezing/passing cells with a 96-well size dish or greater than 96 wells/dish,it is recommended to avoid using dispase and to use Accutase™ or PBS/EDTA.

F. Passaging Pluripotent Stem Cells CulturesNOTE:When passaging cells into a 96-well dish (or any small vessel with high surfacetension such as a 4-well or 8-well chamber slide), it is recommended to remove the cellsfrom the culture dish using Accutase™ or PBS/EDTA. Both of these treatments dissociatethe colonies into single cells, or very small clumps and will ensure a more even spreadin the well. To ensure even spreading and to overcome surface tension, the cells shouldbe spin-seeded at 80 – 120 x g for 2-5 minutes, onto the culture dish using appropriateplate carriers. Without spin-seeding, cells will tend to clump towards the center of thewell. If plate holders for appropriate centrifuge are not available, then minimal volumesshould be used to ensure a faster attachment. Cells passaged in the morning should beattached by early afternoon, at which time the low volume of media can be gently toppedoff with incubator-warmed, pre-equilibrated medium. When using Accutase™, it is recommended to include 2-10µM Y-27632 to increase post-split viability. Dispase dissociation is not recommended for a 96-well-size dish, or smaller.

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a. Passaging Pluripotent Stem Cell Cultures Using DispaseNOTE: Volumes given in this section are for 6-well culture dishes; however cells canbe passaged onto any sized dishes. For a 96-well size dish or greater than 96 wells/dish,it is recommended to use Accutase™ or PBS/EDTA to ensure the even spread of cells onthe surface. Refer to Appendix I for selected dispase preparation protocols.

1. Remove medium from cultures (plated in a 6-well dish), add 1 mL/well dispase(0.5 mg/mL) and return dish to incubator for 3 minutes. After incubation the colonyedges may appear slightly folded back, i.e. like a thick pizza crust, but the coloniesshould still be well attached to the plate. If the edges have not yet begun to curl,then return the dish to the incubator, and check at the 5 minute time point.

2. Aspirate dispase, and gently rinse each well twice with 2-5 mL of warmedDMEM/F-12 (or any suitable media) per well. This step dilutes out the dispase.

3. Add 2-4 mL/well of media and remove cells with gentle motion of a cell scraper(Sarstedt, 83.1830). After 2-3 swipes with the scraper, large clumps of cellsshould be observable. Do not continue to break off the pieces with the scraperonce all the cells are removed from the dish’s surface. Protocol Tip: When scraping, tilt the blade down (clockwise when held in theright hand), scraping away from oneself, and when returning towards one’s body,tilt the blade downward (counter-clockwise). This angling helps to avoid “stuttering” or skipping of the blade, and prevents the loss of cells resulting fromsplashes. The use of a Sarstedt brand product is highly recommended for thisstep as these products have the softest blade and appear easiest to use.

4. Gently triturate cells using a 5 mL glass, serological pipette up to 3 times to dissociate colonies into smaller aggregates. WARNING: It is important to observe the granularity of the solution after eachtrituration step. The final desired texture should resemble “sandy water.” If thefragments are too small, then the cultures may not survive the disaggregation.This procedure can be practiced by using 3 mL volume in the well, trituratingonce, plating 0.5 mL of the cells into a well containing 1.5-2 mL of media, andrepeating the trituration step up to 6 times. Each well will contain cell clumpsof differing size. Record optimal user conditions and repeat with other wells.

5. Gently swirl the material in the well, and take up the needed split volume into a5 mL serological or 1000µL, micropipette, depending on the seeding volume.For example, if splitting the well 1:4 with a 4 mL scrape volume, then 1 mLshould be added per well. If splitting the well 1:8, then 0.5 mL should be addedto each well. A 1000µL pipette is recommended for the plating of any volumesbelow 500 µL, however cell volume should be aspirated and dispensed slowlyto avoid further shearing of aggregates.

6. Transfer the portion to a new well/dish by distributing around the rim of thewell. Cells will naturally gravitate towards the center as these plate out in theincubator; this technique leads to the even spreading of aggregates. This workswell for 6-well dishes, but not for dishes of other dimensions. If cells are denseand the split volume is 3 mL, then cells should be plated at 1:6-1:12 using 0.5to 0.25 mL of cell suspension.

7. If the colonies are at an optimal density, then the cells can be split every 5 - 7days using 1:6 to 1:12 splits (i.e. the aggregates from 1 well of a 6-well platecan be plated in 6 - 12 wells). If the colonies are too dense or too sparse, thenadjust the split ratio accordingly. Passaging cells either too early, or too late, mayresult in decreased attachment or increased differentiation. Please note that these

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guidelines are based on the growth characteristics of the H1 hESC and two Rutgers Stem Cell Training Course iPSC lines, and may vary between differentlines and laboratories. Ensure that newly seeded colonies are evenly distributedon the new BD Matrigel™ coated plate, as failure to do so may also result in increased differentiation in culture

b. Passaging Pluripotent Stem Cell Cultures Using Accutase™ or PBS/EDTA NOTE: Accutase™ is available as a ready-to-use solution, however it can be diluted as needed with PBS/EDTA (340 mOsm, 0.5 mM EDTA). When using Accutase™, it isrecommended to include 2-10µM Y-27632 to increase post-split viability. Please referto the above two notes.

1. Aspirate the media from cultures, and rinse once with 2 mL of warmed PBS (no Ca2+/Mg2+).

2. WARNING: Without rinsing, inhibitors in the media may prevent the Accutase™ or EDTA from working properly within the expected 4-5 minute incubation period. However, usually after 7-8 minutes, the enzyme or EDTA isable to disaggregate the colonies for passaging techniques.

3. Aspirate the PBS, and add 1 mL Accutase™ or PBS/EDTA. Larger volumes are not necessary, however, adding 2 mL is recommended when cultures are particularly dense.

4. Place the plate in the incubator and observe after 3 minutes; returning the plateto the incubator as needed. After 5 minutes, the colonies that were previously“smooth” should have many holes (resembling lacey Swiss cheese), and a muchbrighter phase contrast around many of the cells. Protocol Tip: If the cells have not yet begun to detach, then quickly and gentlyaspirate the Accutase™ or PBS/EDTA. Immediately and somewhat forcefullysquirt the desired volume of medium onto the cells using a circular motion fromthe center to the outside of the well. Triturate only twice, and proceed directlyto step 12 following centrifugation.

5. Once the cells have begun to detach from the plate, gently squirt 3-4 mL of culture medium or suitable base medium onto each well in a circular patternfrom the outside towards the middle. This will wash all cells from the plate.

6. Gently transfer the cell suspension to a 50 mL conical tube. A 50 mL size tubeis recommended, as it can be difficult to remove the media/enzyme solution froma 15 mL conical tube following centrifugation.

7. CELL ENUMARATION: Split the cells empirically (continue to step 8, orcount these as described in the below example). Remove a small aliquot of cellsfrom the split volume, and gently triturate an additional 2-3 times using a pipette.This helps break down aggregates and, therefore, gives a more accurate cell count.At this point, dilute the cells (remember to back multiply the dilution factor) and/oradd 1:1 volume trypan (2x = 0.08%) blue to help visualize viable cells.With a standard hemacytometer load 10 µL, count and multiply by the conversion factor (many use 1 x 104):

For example: (45 + 47 + 49 + 51)/4 = 4848 x 1 x 104 = 480,000 cells/mL

(If trypan blue was added, then x 2 to adjust the calculations.)Volume = 10 mL

480,000 cells/mL x 10 mL = 4,800,000 cells total.

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Recommended Seeding Densities for 1 well of a BD Matrigel™ coated 6 well dish:

Media Alone Media + Thiazovivin/Y-27632PBS/EDTA >3 x 105 >1 x 105

Accutase™ 0.5 - 1 x 106 >3 x 105

8. Centrifuge for 2-5 minutes at 120 x g at 4°C.9. Aspirate the supernatant and add the appropriate amount of medium for the given

split volume. For example, if splitting 1 well 1:6, then add 6 mL of medium, andwith a 5 mL glass serological pipette, gently triturate once to disaggregate cellsinto a “sandy-water” consistency. WARNING: Do not over triturate the cells atthis point as it can lead to excessive cell death.

10. Gently swirl the material in the well, and take up the needed split volume into 5 mLserological, or 1000µL, pipette depending on the seeding volume. For example,if splitting the well 1:4 with a 4 mL scrape volume, then 1 mL should be added perwell, while a 1:8 split would use 0.5 mL/well. A 1000µL pipette is recommendedfor the plating of any volumes below 500µL. However, the cell volume shouldbe aspirated and dispensed slowly to avoid further shearing of the aggregates.

11. Transfer the portion to a new well/dish by distributing volume around the rimof the well. Cells will naturally gravitate towards the center of the wells duringincubation; this technique leads to the even distribution of aggregates. Thisworks well for 6-well dishes, but not for dishes of other dimensions. If cells aredense and the split volume is 3 mL, then cells should be plated at 1:6-1:12 using0.5 to 0.25 mL of the cell suspension.

12. If the colonies are at an optimal density, then the cells can be split every 5 - 7days using 1:6 to 1:12 splits (i.e. the aggregates from 1 well of a 6-well platecan be plated in 6 - 10 wells). If the colonies are too dense or too sparse, thenthe split ratio should be adjusted accordingly. Passaging cells either too early,or too late, may result in decreased attachment or increased differentiation, respectively. Please note that these guidelines are based on the growth characteristics of the H1 hESC and two Rutgers Stem Cell Training Course iPSClines line, and may vary between different lines and laboratories. Ensure thatnewly-seeded colonies are evenly distributed on the new BD Matrigel™ coatedplate as failure to do so may also result in increased differentiation in culture.

G. Freezing Pluripotent Stem Cells a. Freezing Pluripotent Stem Cells with Dispase

1. Remove the media from the cultures (plated in a 6-well dish), add 1 mL/welldispase (0.5 mg/mL), and return the dish to the incubator for 3 minutes. Afterincubation the colony edges may appear slightly folded back, resembling a thickpizza crust, but colonies should still be well attached to the plate. If the edgeshave not yet begun to curl, then return to the incubator, and check at the 5-minutetime point.

2. Aspirate dispase, and gently rinse each well twice with 2-5 mL of warmedDMEM/F-12 (or any suitable media) per well. This step dilutes out the dispase.

3. Add 2 mL/well of media and remove cells from the dish with a gentle motion ofa cell scraper (Sarstedt, 83.1830). After 2-3 swipes with the scraper, large clumpsof cells should be observable. Do not continue to break off the pieces with thescraper once all the cells are removed from the dish surface.

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4. Collect cells in a 15 mL tube, and place into a Biocision CoolRack™ 15 mL, oron ice. At this point, allow the clumps to settle for 5 – 10 minutes, or centrifugeat 120 x g (800 RPM) for 2-5 minutes at RT.

5. Remove media to ½ the freezing volume. EXAMPLE: If freezing 1 well, then aspirate the media and adjust thecells+media volume to 0.5 mL. For 2 wells, adjust to 1 mL, and so forth.

6. Add equal volume of 2x freeze media to a final volume of 1 mL, triturate once,and transfer the entire volume into a cryo-vial using a 1000µL pipette. For largervolumes, use a 5 mL glass serological pipette to add 2x Freeze Medium, thentriturate gently, and transfer to cryo-vials.

7. Place the cryo-vials in a Biocision CoolCell®, or suitable alternative (e.g. Mr. Frosty), and store at -80°C. Transfer to LN2 the following day.

b. Freezing Pluripotent Stem Cells with Accutase™ or PBS/EDTA1. Aspirate the media from the cultures, and rinse once with 2 mL of warmed PBS

(no Ca2+/Mg2+).2. WARNING: Without rinsing, inhibitors in the media may prevent the

Accutase™ or EDTA from working properly within the expected 4-5 minute incubation period. However, usually after 7-8 minutes, the enzyme or EDTA isable to disaggregate the colonies for passaging techniques.

3. Aspirate the PBS, and add 1 mL Accutase™ or PBS/EDTA. Larger volumes are not necessary, however it is recommended to add 2 mL when cultures areparticularly dense.

4. Place the plate in the incubator and observe after 3 minutes; returning the plateto the incubator as needed. After 5 minutes, the colonies that were previously“smooth” should have many holes (resembling Swiss cheese), and a muchbrighter phase contrast around many of the cells. Protocol Tip: If the cells have not yet begun to detach, then quickly and gentlyaspirate the Accutase™ or PBS/EDTA. Immediately and somewhat forcefullysquirt the desired volume of media onto the cells using a circular motion fromthe center to the outside of the well. Proceed to step 6.

5. Once the cells have begun to detach from the plate, gently squirt 2 mL of culturemedia, or suitable base media, onto each well in a circular pattern from the outside towards the middle. This will wash all the cells from the plate.

6. Gently transfer the cell suspension to a 50 mL conical tube. A 50 mL size tubeis recommended, as it can be difficult to remove the media/enzyme solution froma 15 mL conical tube following centrifugation.

7. CELL ENUMARATION: At this point, freeze portions of a well into severalvials: it is strongly recommended that the cells be counted at this point. Removea small aliquot of cells and gently triturate an additional 2-3 times using a pipette.This helps break down aggregates, and therefore, gives a more accurate cellcount. At this point, dilute the cells (remember to back multiply the dilution factor) and/or add 1:2 volume trypan blue to help visualize viable cells.

With a standard hemacytometer load 10 µL, count and multiply by the conversion factor (many use 1 x 104):

For example: (45 + 47 + 49 + 51)/4 = 4848 x 1 x 104 = 480,000 cells/mL

(If trypan blue was added, then x 2 to adjust the calculations.)

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Volume = 10 mL480,000 cells/mL x 10 mL = 4,800,000 cells total.

NOTE: Freeze no less than 1,000,000 cells per cryo-vial. In the above example,there is 4.8 million cells, therefore, divide into 4 vials with 1.2 million cells pervial. In the steps below, follow centrifugation by adjusting the volume ofmedia/cells to 2 mL.

8. Centrifuge for 2-5 minutes at 120 x g at 4°C. 9. Aspirate the supernatant and add the appropriate amount of media for the given

volume (for the above example, add 2 mL). 10. Add equal volume of 2x freeze media, and mix gently.11. Place 1 mL of cells/1x freeze media per cryo-vial for a total of 4 vials.12. Place the cryo-vials in a Biocision CoolCell®, or suitable alternative

(e.g. Mr. Frosty), and store at -80°C. Transfer to LN2 the following day.

Appendix I –Reagents and MaterialsReagents and Materials for Cell Culture

1. PeproGrow-ESC (PeproTech, BM-hESC); Refer to “C. Preparation of Mediaand Growth Factor Component” for instructions.

2. DMEM/F12, 340 mOsm or equivalent3. Phosphate buffered saline/EDTA 0.5 mM, pH 7.4, 340 mOsm 4. Phosphate buffered saline, 340 mOsm5. Cell culture grade sterile water (ddH2O)6. Accutase™ (Innovative Cell Technologies, Inc., Stem Cell Technologies, or

BD Biosciences)7. Dispase (Life Technologies, BD Biosciences, or Stem Cell Technologies)8. DMSO (Sigma, D2650) 9. Y-27632 (Tocris, 1000x or 5 mM Solution in ddH2O = 10 mg/ 6.25mL) 10. Thiazovivin (Tocris, 3845; 1000x or 2 mM Solution in DMSO = 10 mg/16 mL)11. Human serum albumin (Sigma, A5843; powder)12. Trehalose (Sigma,T0167)13. HEPES (Mediatech, 25-060-CI) 14. BD Matrigel™ (BD Biosciences, 354227; hESC qualified or equivalent)15. Cell culture plastics (many vendors, for 6 well dishes Corning, BD Biosciences,

and Sarstedt can be used)16. Cell scraper (Sarstedt, 83.1830)

Reagents and Materials for Immunostaining1. Oct4 (1:1000-2000, monoclonal rabbit antibody, Life Technologies, A13998)2. Tra-1-60 (1:200-300, mouse IgM, Life Technologies, 411000)3. Nanog (1: 250; mouse IgG1, Sigma, N3038, clone NNG-811)4. DAPI (5 mg/mL stock, dilute 1:10000 to 500 ng/mL in PBS for use; Sigma,

D9542)5. Goat-anti-mouse-IgM-AlexaFluor 594 (1:500-1000, Life Technologies)

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6. Goat-anti-mouse-IgG1-AlexaFluor 488 (1:500, Life Technologies, A21121)7. Goat-anti-rabbit-AlexaFluor 647 (1:500-1000, Life Technologies)8. ProLong® Gold mounting medium (Life Technologies)9. Glass coverslips (12 mm)10. Fine forceps11. PBS12. HEPES (1 M, Mediatech) 13. ddH2O 14. Normal goat serum (EMD/Millipore, S26-100ML)15. Triton X-100 (diluted to 10% in PBS)16. Bovine serum albumin (low endotoxin, Fatty Acid, IgG Free)17. Blocking solution (10% goat serum, 1% BSA, 0.1% Triton X-100, 20 mM

HEPES in PBS)18. Antibody Solution (0.5 X Blocking Solution in PBS)

Reagents and Materials for Flow CytometryNOTE: When using iPSCs that have been reprogrammed using Oct4, it is highly recommended to substitute Oct4 with the Nanog antibody. Inclusion of a viability dyeis optional.

1. Human And Mouse Pluripotent Stem Cell Analysis Kit (BD Biosciences)2. Nanog- PerCP-Cy™5.5 (BD Biosciences, 562259)3. LIVE/DEAD® Fixable Green Dead Cell Stain Kit, for 488 nm excitation

(Life Technologies, L-23101)

Preparation of 2X Non-Serum Based Freeze Medium; 20% DMSO, 10% Trehalose (50 mL)

1. Measure 25 mL KOSR (Life Technologies).2. Dissolve 5 grams trehalose into 10 – 12 mL of DMEM/F12.3. Add 10 mL DMSO.4. Fill the volume to 50 mL using DMEM/F12.5. Filter using a 0.2 micron SFCA filter unit.6. Aliquot into 15 mL conical tubes and freeze at -20°C until further use.

Preparation of 2X Defined – Humanized Freeze Medium (10% HSA)1. Measure 20 mL 25% HSA in DMEM/F12.2. Dissolve 5 grams trehalose into 12-15 mL DMEM/F12. 3. Add 10 mL DMSO.4. Fill the volume to 50 mL using DMEM/F12.5. Filter using a 0.2 micron SFCA filter unit.6. Aliquot into 15 mL conical tubes and freeze at -20°C until further use.

Preparation of Dispasea. Preparation of Stem Cell Technologies Dispase, 5x stock

1. Dispase (Stem Cell Technologies,07913) is prepared as follows: Aliquot a 100mL bottle of stock dispase solution (5 mg/mL) by thawing and dispensing intosmaller volumes (10 or 20 mL).

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2. Store the dispase aliquots at -20°C and thaw as required. Aliquots should onlybe thawed once; repeated freeze/thaw cycles are not recommended.

3. Prepare a working solution of dispase at 1 mg/mL by diluting 1 in 5 withDMEM/F-12 (e.g. 10 mL of dispase plus 40 mL of DMEM/F-12). Diluted dispase can be stored at 2 - 8°C for up to 3 weeks. It is highly recommendedthat the diluted dispase solution be aliquoted into smaller working volumes toavoid repeated warming.

b. Preparation of Life Technologies Dispase1. Dissolve 500 mg of dispase into a final volume of 10 mL PBS + 20 mM HEPES.

PBS does not have to contain Ca2+/Mg2+ when making stock solutions; however,those ions are needed when making working stock solutions. Alternatively youcan use DMEM/F12+20 mM HEPES when making dispase stock solutions.

2. Gently rotate until most of the powder is dissolved.3. Sterile filter with 0.2 micron PES or SFCA filter (Corning) with syringe or tube

top unit, aliquot and store at -20°C until needed.4. Once thawed, keep at 4°C for up to a week, and make dilutions as needed.

Appendix II: Figures and Descriptions• Refer to Figure 1 of “A. Introduction” for flow cytometry analysis information.

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Figure 2: Immunostaining of iPSCs cultured in PeproGrow-ESC media. Cells plated on 24-welldishes were rinsed in PBS, fixed using 4% paraformaldehyde, rinsed 3x in PBS, incubated withblocking media for 1 hour, then stained with indicated antibodies diluted in antibody solution (see Appendix I) overnight at 4°C. The following day the samples were washed 3x in PBS, and incubated with secondary antibodies (see Appendix I) diluted in antibody solution for 1 hour at roomtemperature. Samples were rinsed once in PBS/DAPI for 5 minutes, twice more in PBS, and once in water before mounting with ProLong® Gold and 12 mm glass coverslips. Samples were imagedusing an Olympus IX71.

DAPI NANOG

Oct4Tra-1-60

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Figure 4: Cell Morphology of hESCs cultured in PeproGrow-ESC medium. This medium contains a unique blend of cytokines that at first may alter the cellular or colony appearance, making themmore flat, and elastic, however this morphological change appears less dramatic as the cell densityincreases, and over several passages. Stem cells will form standard tight circular colonies (left), andhave an expected morphology of a large nucleus and small cytoplasm (right).

Figure 3: The Growth of H1 hESCs Expressing turboGFP-NEO under the control of the Oct4 promoter. H1 hESCs modified by lentivirus to contain the Oct4 promoterdriving turboGFP, were cultured in PeproGrow-ESCmedia, were passaged using Dispase and plated in PeproGrow-ESC media containing 2 µM Y-27632 onto BD Matrigel™ coated 6-well dishes. Cultures were fed daily and photographed 24, 48, and 72 hours post split. Green fluorescence represents maintenance of pluripotency as indicated by Oct4 promoter activity.

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