MEL-1 and MEL-2 Human Embryonic Stem Cells (Catalog Nos. SCC020 and SCC021)

Transcription

1 MEL-1 and MEL-2 Human Embryonic Stem Cells (Catalog Nos. SCC020 and SCC021) PLEASE REVIEW LIMITED USE LICENSE ON PAGE 31 PRIOR TO OPENING PRODUCT FOR RESEARCH USE ONLY Not for use in diagnostic procedures. USA & Canada Phone: 1(800) Fax: 1 (951) Europe 44 (0) Australia Germany ISO Registered worldwide custserv@chemicon.com techserv@chemicon.com

2 Contents 1) Application ) Kit Components ) Storage a) Product Specifications: MEL-1 Cells (SCC020) b) Product Specifications: MEL-2 Cells (SCC021) ) Shipment Details ) Suggested Production Schedule for Routine Culture of mef and hes cells ) Thawing Passage 4 mefs ) Propagating mefs Passage 4 to Passage ) mef Crypreservation ) Preparation of Mitomycin C-Treated mef Feeder Layers ) Thawing hes Cells ) Manual Passaging of hes cells ) Enzymatic Passaging of hes cells ) Growth of MEL-1 and MEL-2 in HEScGRO Serum-Free Media ) Freezing hes Cells via Vitrification ) Formulation of Media and Solutions ) Warranty ) Limited Use License i

3 1) Application MEL-1 and MEL-2 cells are human Embryonic Stem (hes) cells designed for use in human embryonic stem cell research. Please see Limited Use License (page 31) for specific application details. For Research Use Only; Not for use in diagnostic procedures 2) Kit Components 1. hes Cells (MEL-1 or MEL-2), 2 straws containing 9-10 colony sections per straw 2. Primary Mouse Embryonic Fibroblasts (mef cells or mefs) (1 vial containing 5 x 10 6 cells/vial) 3) Storage ALL COMPONENTS MUST BE STORED IN LIQUID NITROGEN 4a) Product Specifications: MEL-1 Cells (SCC020) Source: Donated frozen IVF embryos no longer required for infertility treatment and approved for stem cell derivation by the Australian National Health and Medical Research Council Licensing Committee [Licence #309709]. Derivation conditions: Derived on mouse fetal fibroblast feeders using 20% Fetal Calf Serum in standard hesc culture medium [DMEM/Non-essential AA (1x)/β-mercapto (0.1mM)/glutamine (2mM)]. Shipment conditions: Supplied in 'open pulled straws' (x2) containing 9-10 colony sections in vapor phase Liquid Nitrogen. A single vial of mef cells is included. 1

4 Microbiology: Negative for mycoplasma, HTLV-1&2, Hepatitis B&C, HIV, and Treponema palladium. Passage # P Feeder cells Mouse Embryonic fibroblasts Karyotype analysis 46, XY (p5 and p21) Morphology: Cell Surface markers: Oct-4 GCTM2 TG30 (CD9) Podocalyxin CD24 TRA 1-60 TRA 1-81 SSEA-1 SSEA-3 SSEA-4 Well defined colonies with compact cells displaying high nuclear to cytoplasmic ratio and prominent nucleoli. - In vivo pluripotency: - Representative germ layers in SCID/teratomas Endoderm, ectoderm and mesoderm observed. 4b) Product Specifications: MEL-2 Cells (SCC021) Source: Donated frozen IVF embryos no longer required for infertility treatment and approved for stem cell derivation by the Australian National Health and Medical Research Council Licensing Committee [Licence #309709]. Derivation conditions: Derived on mouse fetal fibroblast feeders using 20% Fetal Calf Serum in standard hesc culture medium [DMEM/Non-essential AA (1x)/β-mercapto (0.1mM)/glutamine (2mM)]. Shipment conditions: Supplied in 'open pulled straws' (x2) containing 9-10 colony sections in vapor phase Liquid Nitrogen. A single vial of mef cells is included. 2

5 Microbiology: Negative for mycoplasma, HTLV-1&2, Hepatitis B&C, HIV, and Treponema palladium. Passage # P 12 Feeder cells Mouse Embryonic fibroblasts Karyotype analysis 46, XX (p5 and p36) Morphology: Cell Surface markers: Oct-4 GCTM2 TG30 (CD9) Podocalyxin CD24 TRA 1-60 TRA 1-81 SSEA-1 SSEA-3 SSEA-4 Well defined colonies with compact cells displaying high nuclear to cytoplasmic ratio and prominent nucleoli. - In vivo pluripotency: - Representative germ layers in SCID/teratomas Endoderm, ectoderm and mesoderm observed. 5) Shipment Details The cells are shipped in a liquid nitrogen dry shipper. The nitrogen is in vapor phase, so do not be concerned if there is no liquid in the container upon arrival. In this shipment you will receive the following: One 5 ml cryovial containing 2 open straws of requested human embryonic stem (hes) cells. One 2 ml cryovial containing Passage 4 mouse embryonic fibroblast (mef) feeder cells. 3

6 Protocol Manual on how to thaw, culture, freeze and mitotically inactivate mefs and a detailed manual on how to thaw, culture and freeze hes cells. Upon arrival of this shipment please transfer contents of shipper immediately to Liquid Nitrogen (LN 2 ) storage (preferably vapor phase, however liquid phase is also suitable). NOTES 1. hes cells are frozen in a volume of approx 3 µl and will thaw quickly at room temperature. The cells must be transferred into LN 2 storage quickly. 2. Due to poor viability obtained from freezing and thawing hes cells in vials, the hes cells that you have received have been frozen in open pulled straws using the vitrification method. Each straw contains 9-10 colony sections that have been manually cut. Before you thaw your first straw of hes cells: The following culture manual provides detailed instructions for thawing and culturing both mefs and hes cells. It is strongly recommended that the protocols provided be followed until such a time as MEL-1 and / or MEL-2 cells are well established in your laboratory. As with any fetal calf serum (FCS) dependent cell line, MEL 1 and MEL 2 hes cell lines are susceptible to FCS batch-to-batch variation. Therefore, it is recommended that a good quality serum be purchased. MEL-1 and MEL-2 can also be successfully cultured in HEScGRO Serum-Free Media (Cat No. SCM020) from Millipore. Please see page 20 for further information. Only a limited supply of mefs has been provided so provision of future stock needs to be considered. (Note: not all mef lines produced will support the growth of MEL 1 and/or MEL 2 hes cells, so we recommend that testing of new mef lines takes place in conjunction with culturing on supplied mef line.) mefs The mefs provided in this shipment are at Passage 4 and can be split to Passage 6 then inactivated for use to culture hes cells. Unfortunately the mefs lose their ability to support undifferentiated hes cell growth beyond Passage 6 so it is important that they are not expanded any further. To ensure sufficient stocks of mefs for future expansion of hes cells, it is recommended that stocks of mefs are frozen at Passage 5 and Passage 6. Take care when determining cell number to freeze per vial at Passage 6 as 4

7 the vial can only be thawed then inactivated when confluent, particularly if your method of inactivation is Mitomycin C treatment. The mefs in this shipment have tested negative for mycoplasma. hes Cells This shipment contains two straws of hes cells. It is recommended that one straw be thawed in the first instance, leaving the remaining straw as a back up. It is strongly recommended that further stocks of frozen hes cells be produced as soon as possible. hes cells are more sensitive to changes in culture conditions than other mammalian cells and will differentiate quickly if care is not taken to ensure that optimal conditions are maintained at all times. The hes cell lines contained in this shipment are fully characterized and have tested negative for viruses and mycoplasma. 6) Suggested Production Schedule for Routine Culture of mef and hes cells DAY Monday Tuesday Wednesay Thursday Friday Weekend ACTIVITY Mitomycin C treat mef cells and set up feeder dishes and plates Change media on hes plates/flasks Change media on mef dishes and flasks to hes media Passage hes colonies if ready for culturing Change media on hes plates/flasks Passage hes colonies if ready for culturing Change media on hes plates/flasks Passage hes colonies if ready for culturing Change media on hes plates/flasks Passage hes colonies if ready for culturing Thaw P6 mef cells Change media on hes plates/flasks Change media on mef flasks 5

8 7) Thawing Passage 4 mefs This shipment contains one vial of Passage 4 mefs that have been tested to support MEL-1 and MEL-2 hes cell lines. These cells can be passaged twice more before inactivation either chemically or with irradiation. The following procedures give instructions on how to thaw, propagate and freeze mefs. MATERIALS F-DMEM media (prewarmed; see section 16 for media formulation) 15 ml tube Haemacytometer Trypan blue 75 cm 2 tissue culture flask PROCEDURE 1. Transfer 5 ml F-DMEM media (prewarmed to 37 C) into a 15 ml tube. 2. Remove vial from LN 2 and quickly transport to 37 o C H 2 O bath. 3. Agitate vial to thaw quickly but not completely, leaving a sliver of ice in vial (Note 1). 4. Thoroughly spray vial with 70% ethanol and transport to biosafety cabinet. 5. Remove lid and add dropwise 1 ml prewarmed F-DMEM media. 6. Mix cells by gently pipetting up and down. 7. Transfer cell mixture to 15 ml tube containing 5 ml F-DMEM media. 8. Centrifuge cells at 2000 rpm (500 x g) for 2 minutes at room temperature. 9. Resuspend pellet in 5 ml prewarmed F-DMEM media. 10. Perform cell count and viability to determine recovery of cells after thawing. 11. Transfer cell suspension to tissue culture flask and place in 5% CO 2, 37 o C incubator. 12. Refresh media daily until confluent. NOTE It is important that the cells do not thaw completely as DMSO is toxic to cells at higher temperatures. 6

9 8) Propagating mef s Passage 4 to Passage 6 These feeder cells are cultured, passaged and frozen at intervals for manageability of resources and inventory. MATERIALS F-DMEM media (prewarmed; see section 16 for media formulation) 0.05% Trypsin EDTA (prewarmed) PBS- (PBS without Ca 2 and Mg 2 ) (prewarmed) 50 ml tubes Centrifuge 175 cm 2 Tissue Culture Flasks Haemacytometer Trypan Blue PROCEDURE 1. Check Passage 4 cells are 80-90% confluent under microscope. 2. Wash mefs twice with warm PBS-. 3. Suction off PBS- and add 4-5 ml 0.05% Trypsin EDTA to flask. 4. Gently tilt flask to ensure that entire cell surface is coated. 5. Lay flask flat for 2-3 minutes at RT (flask can be placed in 37 o C incubator to facilitate detachment of cells if required). 6. Tap side of flask with enough force to dislodge cells (you can be quite forceful here). 7. Add 20 ml warm F-DMEM media to flask and swirl flask using a tilting motion to mix. 8. Collect cell suspension and transfer to 50 ml tube. 9. Rinse the flask with a further 10 ml fresh F-DMEM media as above and add to tube. 10. Centrifuge at 2000 rpm (500 x g) for 3 minutes at room temperature. 11. Resuspend cells and perform a cell count and viability, mefs are now at Passage Determine number of flasks to be set up and transfer calculated volume of cells to each flask (approx 3 x 10 6 cells/175 cm 2 flask or 1.7 x 10 4 cells/cm 2 ) and place flasks in a 37 o C, 5% CO 2 incubator. 13. Refresh media everyday until confluent (approx. 2-3 days). 7

10 14. It is recommended to freeze stocks of Passage 5 mefs using the mef cryopreservation procedure below. 15. Repeat steps 1 11, mefs are now at Passage 6. Excess cells should be frozen here. Freeze in aliquots of 3 x 10 6 cells/vial. Thaw each vial into a 175 cm 2 tissue culture flask three days prior to treating with Mitomycin C. 9) mef Crypreservation It is recommended that stocks of mefs are frozen and then thawed when needed. MATERIALS mef freezing solution (see section 16 for formulation) 2 ml cryovials Mr Frosty freezing container Centrifuge PROCEDURE 1. Prepare mef freezing solution and place at 4 o C (Note 1). 2. Determine number of vials needed to be frozen at 3 x 10 6 cells/vial. 3. Label cryovials with cell line name, date, cell number, and passage number. 4. Centrifuge cells at 2000 rpm (300 x g) for 2 minutes at room temperature. 5. Suction off supernatant and resuspend pellet in required volume of mef freezing media (approx 0.8 ml per vial). 6. Transfer 800 µl of cell suspension to each of the cryovials. 7. Place cryovials in Mr Frosty and place at -80 o C as per instructions on freezing container. 8. Once cells have been at -80 o C for the required time, transfer cells to liquid nitrogen (LN 2 ) storage tank. NOTE 1. When adding DMSO to culture media, an exothermic reaction occurs which can damage cells, therefore make the freezing solution in advance and allow to cool at 4 o C. 8

11 10) Preparation of Mitomycin C-Treated mef Feeder Layers hes cells are grown on a layer of inactivated primary mefs to promote growth and prevent differentiation. The mefs can be inactivated either by chemical treatment or irradiation, however as access to an irradiator is not always available, the method of choice is Mitomycin C treatment. MATERIALS Mitomycin C (MITC) solution (see section 16 for formulation) Organ culture dishes and/or 25 cm 2 tissue culture flask 0.1% Gelatin Solution (see section 16 for formulation) F-DMEM media (prewarmed; see section 16 for formulation) 0.05% Trypsin EDTA ( prewarmed; see section 16 for formulation) PBS- (PBS without Ca 2 and Mg 2 ) (prewarmed) 50 ml tubes Centrifuge Haemacytometer Trypan Blue PROCEDURE 1. Passage 6 mefs need to be set up 3 days prior to Mitomycin C treatment. 2. Ensure the tissue culture flask is 90% confluent (Note 1), it is important that the flask is not over confluent as the MITC treatment will not be effective. 3. It is not necessary to suction off media at this stage as the MITC solution (2 mg/ml) can be added directly to the flask. Add MITC to a final concentration of 10 µg/ml (e.g. 150 µl MITC solution in 30 ml media). 4. Swirl flask to mix MITC solution and return flask to 37 o C, 5% CO 2 incubator for hours (Note 2). 5. Prepare required number of organ culture dishes/tissue culture flasks by coating with 0.1% gelatin and labeling with mef cell name and date (Pipette 4-5 ml sterile H 2 O into outer well of organ culture dishes). Dishes to be coated with gelatin for 1 hour (Note 3). 6. When MITC incubation period is complete, suction off media containing MITC solution and wash twice with PBS-. 7. Suction off PBS- and add 2-3 ml 0.05% Trypsin/EDTA. Tilt flask until entire cell surface is coated and lay flat for 2-3 minutes. 8. Tap flask gently to dislodge cells. 9. Add 20 ml prewarmed F-DMEM media to flask and gently pipette up and down rinsing the cell surface. 9

12 10. Transfer cell suspension to a sterile 50 ml tube. 11. Repeat step 10 and transfer to same tube. 12. Centrifuge cell suspension at 2000 rpm (500 x g) for 2 minutes at room temperature. 13. Suction off supernatant and resuspend cell pellet in 10 ml F-DMEM media. Perform cell count and viability. Determine volume of cell suspension required to seed organ culture dishes at 1.7 x 10 5 cells/dish (6 x 10 4 cells/cm 2 ) and 25 cm 2 tissue culture flasks at 1.5 x 10 6 cells per flask. 14. Suction off gelatin from dishes and flasks. 15. Organ culture dishes are plated at 1 ml/dish, therefore calculate volume of media required to add to the calculated cell suspension volume. For example, 3 ml cell suspension 7 ml F-DMEM media to seed 10 organ culture dishes. 16. T25 tissue culture flasks are plated at 5 ml/flask, therefore calculate volume of media required to add to calculated cell suspension volume. For example, 2.4 ml cell suspension 2.6 ml F-DMEM media = 5 ml total volume. 17. Carefully transfer dishes and flasks to 37 o C, 5% CO 2 incubator and leave O/N. Next day replace media with hes media. Feeder plates are now ready for use. NOTES 1. It is quite important that feeders are not over-confluent as the MITC solution will be ineffective resulting in further cell division of feeders after being plated. This will result in restricted growth of the hes colonies and the feeders being patchy and may pull away from edge of dish and curl up. 2. The time frame for incubation of MITC solution of the feeders is critical. If the solution is not on long enough then the feeders will continue to divide after they have been plated and if the solution is on the feeders for too long it may result in further cell damage and effect the viability of the feeders. 3. It is not important how long the 0.1% gelatin solution is on the dishes or flasks, rather that the entire surface is coated and the solution aspirated off just prior to seeding of feeders. Do not allow the dishes or flasks to dry out. 4. When coating the feeders with 0.05%Trypsin only the cell surface needs to be coated, not the entire inside of the flask. 5. Once mefs have been MITC treated and plated their lifespan is limited to 1-2 weeks, therefore it is preferable to only plate out what you need and freeze the remaining cells as a back-up. 10

13 11) Thawing hes Cells MATERIALS ES HEPES media (see section 16 for media formulation) 0.2M Sucrose Solution 0.1M Sucrose Solution Dissecting microscope with heated stage Inactivated pre-prepared feeder dishes Fine pointed forceps PROCEDURE 1. Prepare 4-well plate with 800 µl 0.2M Sucrose in well 1, 0.1M Sucrose in well 2, ES HEPES Media in wells 3 & 4 and place well plate in 37 o C 5% CO 2 incubator. 2. Locate hes cells to be thawed and remove from storage tank and place on cryocane in LN 2 holding vessel. 3. Remove 4 well plate containing warmed solutions from incubator and place on dissecting microscope with Well 1 in field of view. 4. Safety glasses should be worn, as 0.2M 0.1M Sucrose Sucrose 1 min 3 min well 1 well 2 well 3 well 4 ES HEPES ES HEPES Media Media 1 min 1 min LN 2 will spit out of end of straw. Using forceps remove straw from cryovial and quickly take hold with thumb and forefinger. 5. Transfer to 4 well plate and holding at a 45 o angle insert into well 1 observing under dissecting microscope. 11

14 6. When colony pieces begin to thaw, place forefinger over end of straw not in liquid and colony pieces will exit the straw via capillary action. 7. Leave colony pieces in well 1 for 1 min. 8. Using 20 µl pipette, transfer colony pieces to well 2 for 3 minutes. 9. Transfer colony pieces to well 3 for 1 minute. 10. Transfer colony pieces to well 4 for 1 minute. 11. Remove fresh feeder plate from incubator and label clearly with hes cell line and date of thaw. 12. Transfer colony pieces to feeder plate and carefully transfer plate to incubator taking care that the colony pieces do not merge together. 13. Change media daily for 7-8 days or until colonies are a reasonable size. NOTE Check the thawed cells the following day but do not be alarmed if there is a lot of cellular debris, this is normal. Colony pieces should be attached however with some slight outward growth. Colonies should be ready for passaging in 7-8 days. 12) Manual Passaging of hes cells This method allows the operator to be very selective of hes cell colony area to be transferred onto fresh feeder plates. The advantage of this method is it allows the scientist to be very selective when transferring hes colony pieces from week to week ensuring a greater likelihood of maintaining the hes cultures in an undifferentiated state. The disadvantage of this method is it is very laborious restricting large scale hes cell growth. The following protocol uses standard hes media (DMEM/FCS formulation, see page 25). MEL-1 and MEL-2 can also be successfully cultured in HEScGRO Serum-Free Media (Cat No. SCM020) from Millipore. Please see page 20 for further information MATERIALS hes media (prewarmed) (See section 16 for media formulation) Dissecting microscope and heated stage PBS 4 well plate Pulled glass capillary attached to an instrument sleeve P20 pipette and 20 µl sterile tips Inactivated pre-prepared feeder plates 12

15 PROCEDURE 1. On day of transfer, check each plate under a microscope (dissecting or inverted whichever you prefer). 2. Choose those plates that contain the healthiest colonies to transfer from and put them to one side in the incubator. 3. Change media on culture dishes and/or flasks with hes media. 4. Prepare a 4-well plate with 1 ml PBS in each of the wells and place in 37 C incubator. 5. Using a pulled glass capillary pipette attached to an instrument sleeve (Note 1) carefully cut the colonies into 8-10 pieces (depending on size of colony) of roughly equal sizes. Avoid transferring the dense center (central button) section of the colony. Also cut around perimeter of colony detaching colony from feeder layer. Day 7 hes colony ready for transfer Cut out central button area 6. Using a sterile 20 µl micropipette tip gently nudge the colony pieces until they completely lift off the feeder layer. 7. Collect colony pieces using 20 µl micropipette and transfer to PBS wash plate. 8. Remove required number of fresh feeder plates from incubator and label clearly with hes line name, passage number and date. 9. Using a 20 µl micropipette, pick up 8-10 colony pieces and transfer them to labeled feeder plate, spacing apart the colony pieces (Note 2). 10. Carefully transfer plate to 37 o C 5% CO 2 incubator making sure that colony pieces do not merge together. 11. Refresh media daily until day 7 (Note 3) when transfers take place again. 13

16 NOTES 1. Pulled glass capillary pipettes are used to manually cut the hes colonies, however a needle attached to a small syringe could also be used, however you may find that the needle is too pointy and tears the colonies rather than giving a nice straight cut. 2. It is important to space the colony pieces far enough apart so that by day 7 the colonies have reached their optimal growth size. If the colony pieces merge together then their growth is restricted and less undifferentiated cell area is available to transfer on. 3. The hes cell colonies will differentiate if media is not replenished on a daily basis, however usually one change over a weekend is acceptable. Cut colony pieces on fresh feeder plate Day 1 colonies Day 2 colonies Day 3 colonies 14

17 Day 4 colonies Day 5 colonies Day 6 colonies 13) Enzymatic Passaging of hes cells Day 7 colonies This method is chosen if a large volume of hes cells is required. Unlike the cut and paste method where the operator can choose only undifferentiated cell pieces to culture on, this method does not allow the operator such choice. Differentiated and undifferentiated hes cells will be transferred into fresh culture vessels often resulting in a mixture of cell types. MATERIALS Culture dishes or flasks seeded with Mitomycin C treated mefs Note: mefs need to be seeded at 1/3 the density as seeded for manual passaging. PBS- (prewarmed; see section 16 for formulation) Serum Replacement media (prewarmed; see section 16 for formulation) 4 mg/ml Collagenase IV solution (prewarmed; see section 16 for formulation) Cell Scraper 15 ml sterile tube Basic Fibroblast Growth Factor (bfgf) 10 ng/µl stock (see section 16 for formulation) 15

18 PROCEDURE 1. Suction off media from hes culture flask. 2. Wash flask twice with PBS-. 3. Add Collagenase IV solution to flask (1 ml for 25 cm 2 flask, 3 ml for 75 cm 2 flask). 4. Incubate flask 37 o C for 8 minutes. 5. Tap flask with enough force to dislodge any differentiated cells and suction off Collagenase solution. 6. Use a cell scraper to gently scrape the cells from the surface of the flask. 7. Add 5 ml Serum Replacement media to flask and gently pipette up and down to break up the cells then transfer cells to a 15 ml tube. 8. Rinse flask with 5 ml Serum Replacement media and add to tube. 9. Centrifuge cells at 1000 rpm (130 x g) for 2 minutes. Remove the supernatant and resuspend the hes cells in Serum Replacement media that contains 10 ng/ml bfgf. 10. Add cell suspension to inactivated pre-prepared mef flask. 11. Observe flask daily and passage when confluent. NOTES 1. To start enzymatic passaging from an organ culture dish, cut colonies using cut and paste method and transfer cut colony pieces to a tube containing 1 ml Collagenase IV solution and incubate at 37 o C for 8 minutes. Using a 1000 µl pipette, gently pipette up and down until colony pieces begin to break up. Then add 10 ml warmed Serum Replacement media to tube and follow steps You will need approximately five hes plates for each 25cm 2 tissue culture flask to be seeded. 2. If your starting material is of the highest quality (contains no differentiated cells) then there is a greater likelihood of maintaining the bulk cultures in an undifferentiated state. 3. Due to a higher volume of cells in the culture flasks, the hes cells may need to be passaged more regularly than the cut and paste cultures. 16

19 Colonies that have been passaged using Collagenase IV 14) Growth of MEL-1 and MEL-2 in HEScGRO Serum-Free Media Mel-1 and Mel-2 cells can be grown in serum-free/animal component-free media on human fibroblast feeders (see Note 4, page 21). In these conditions the colonies have a flatter morphology, however they have excellent growth and marker expression. The advantage of this method is that it avoids the need for serum batch testing as HEScGRO media is ready to use. This method should be only attempted with Mel-1 and Mel-2 after successful establishment of serum conditions and freezing of additional straws. MATERIALS HEScGRO media (prewarmed) (Millipore Cat No: SCM020) Dissecting microscope and heated stage Pulled glass capillary attached to an instrument sleeve P20 pipette and 20 µl sterile tips Inactivated pre-prepared Detroit 551 (ATCC) feeder plates PROCEDURE 1. On day of transfer, check each plate under a microscope (dissecting or inverted whichever you prefer). 2. Choose those plates that contain the healthiest colonies to transfer from and put them to one side in the incubator. 17

20 3. Change media on culture dishes and/or flasks with HEScGRO media at least 1 hour prior to transfer to allow equilibration. 4. Using a pulled glass capillary pipette attached to an instrument sleeve (Note 1) carefully cut the colonies into 8-10 pieces (depending on size of colony) of roughly equal sizes. Avoid transferring the dense center (central button) section of the colony. Also cut around perimeter of colony detaching colony from feeder layer in a manner similar to that shown in section 12, Manual Passage of hes cells. 5. Use edge of the needle to tease the pieces away from the edge of the bottom of the dish. 6. Remove required number of fresh feeder plates (Note 4) from incubator and label clearly with hes line name, passage number, and date. 7. Using a 20 µl micropipette, pick up 8-10 colony pieces and transfer them to labeled feeder plate, spacing apart the colony pieces (Note 2). 8. Carefully transfer plate to 37 o C 5% CO 2 incubator making sure that colony pieces do not merge together. 9. Refresh media daily for 5 to 7 days (See Note 3) when transfers take place again. NOTES 1. Pulled glass capillary pipettes were used to manually cut the hes colonies, however a needle attached to a small syringe could also be used, however you may find that the needle is too pointy and tears the colonies rather than giving a nice straight cut. 2. It is important to space the colony pieces far enough apart so that by day 7 the colonies have reached their optimal growth size. In HESGRO Media colonies can grow faster than in serum. If colonies have grown large by day 5 it is better to passage at this time. If colony pieces merge together then their growth is restricted and less undifferentiated cell area is available to transfer on. If the colonies grow too large in HESGRO conditions then colonies can start to show differentiation particularly neural rossetting or neuroectoderm formation. 3. The hes cell colonies will differentiate if media is not replenished on a daily basis, however usually one change over a weekend is acceptable. 4. Detroit 551 human feeders are recommended with HEScGRO media for Mel-1 and Mel-2. This media may also work with other feeders but they should be tested for survival in serum free conditions and also compared to Detroit 551 cells in side-by-side comparisons prior to their routine use. Detroit feeders are prepared by grown according to instructions from 18

21 ATCC. They are then inactivated in an identical manner to mef cultures using either Mitomycin C (as described in section 10 - Preparation of Mitomycin C-Treated mef feeder layers) and plated at 6x10 4 cells/cm 2 (1.7x10 5 cells per dish). 15) Freezing hes Cells via Vitrification Vitrification is a process of converting a material into a glass-like amorphous solid which is free of any crystalline structure, either by quick removal or addition of heat and/or by mixing with an additive. Slow rate freezing and rapid thawing is most commonly used for cyropreservation of cell lines. While these standard methods are efficient for many nucleated cells, it has been observed that the survival of undifferentiated human embryonic stem cells following slow-rate freezing is very poor and most cells either differentiate or die. MATERIALS ES HEPES Media (see section 16 for formulation) 10% Vitrification Solution (see section 16 for formulation) 20% Vitrification Solution (see section 16 for formulation) 4-well plate Dissecting microscope and heated stage Liquid Nitrogen Cryocanes 5 ml Cryovials with holes punched Open Pulled Straws PROCEDURE 1. Prepare 4 well plate with 800 µl ES-HEPES media in well 1, 10% Vitrification Solution in well 3 and 20% Vitrification in well 4 and place in 37 o C 5% CO 2 incubator (see figure on next page). 2. Collect Liquid Nitrogen (LN 2 ) into a holding vessel and attach a 5 ml cryovial that has been hole punched (Note 1) and labeled to a cryocane. Place cane in LN 2 and attach cryovial to cane ready for insertion of vitrification straws (Note 2). 3. Cut colonies from one culture plate as per section 12 Manual Passaging of hes Cells steps Transfer all the colony pieces to well 1 of the vitrification plate. 19

22 5. Pick up 8-9 colony pieces with a 20 µl micropipette and transfer to well 3 of the vitrification plate for 1 minute. 6. Using new 20 µl micropipette tip, transfer 20 µl 20% vitrification solution to lid of well plate creating a high drop. ES Hepes Media Lid well 1 well 2 well 3 well 4 10% Vit Soln. 20% Vit Soln. 1 min. 25 secs. 20 µl 3 µl Drop Droplet 7. In minimal volume as possible (Note 3) pick up the colony pieces from well 3 and transfer to well 4 for 25 seconds the pieces will swirl around so take care that they do not attach to air bubbles that may be present. 8. In minimal volume, transfer colony pieces from well 4 to droplet on lid. 9. Pick up the colony pieces with 3 µl micropipette and make a small, high droplet on the lid of the well plate. 10. Pick up sterile vitrification straw (Note 4) and holding it at a 30 o angle, touch the edge of the 3 µl droplet and the colony pieces will enter the straw via capillary action. 20

23 11. Transfer straw to LN 2 and holding at a 45 o angle, plunge straw into LN Quickly transfer straw to cryovial. 13. Repeat procedure until all colony pieces have been frozen. NOTES 1. Using an open flame, heat an 18 gauge needle to red hot then insert 1 cm from the bottom of the cryovial. Punching two holes in the cryovial allows a small volume of LN 2 to enter the cryovial. This volume ensures that the bottom of the straws that contain the frozen colony pieces stays frozen whilst transferring from LN 2 freezing vessel to LN 2 storage tank. 2. Attaching the cryovial to a cane makes it easier to place the frozen straw into it. 3. By keeping the volume to a minimum when transferring colony pieces from well 3 to well 4, it reduces the dilution of the final freezing solution. 21

24 4. The open pulled vitrification straws may be too tall for the 5 ml cryovial, so have a pair of scissors nearby to trim the end of the straws that will not contain the colony pieces to be frozen. 16) Formulation of Media and Solutions a) hes Media Component 200 ml 500 ml DMEM (high Glucose, with L-Glutamine) 155 ml 387 ml FCS 40 ml 100 ml 100X Non-Essential Amino Acids (NEAA) 2 ml 5 ml 100X Insulin-Transferrin-Selenium-G Supplements (ITS) Penicillin/Streptomycin 2 ml 5 ml 1 ml 2.5 ml 5000 U/mL Penicillin-G 5000 µg/ml Streptomycin Sulfate 55 mm β-mercaptoethanol 367 µl 918 µl 1. Combine all ingredients and sterile filter (0.2 µm). 2. Store at 4 o C wrapped in foil. 3. Take a 3 ml sample and place in 37 o C 5% CO 2 incubator for 5 days to check sterility. 22

25 b) F-DMEM Media Component 200 ml 500 ml DMEM (high Glucose, with L-Glutamine) 179 ml 448 ml FCS 20 ml 50 ml Penicillin Streptomycin 1 ml 2.5 ml 5000 U/mL Penicillin-G 5000 µg/ml Streptomycin Sulfate 1. Combine all ingredients and sterile filter (0.2 µm). 2. Store at 4 o C. 3. Take a 3 ml sample and place in 37 o C 5% CO 2 incubator for 5 days to check sterility. c) Serum Replacement Media Component 200 ml 500 ml DMEM/F12 (with L-Glutamine) 157 ml 392 ml KnockOut Serum Replacement 40 ml 100 ml 100X Non-Essential Amino Acids (NEAA) 2 ml 5 ml Penicillin Streptomycin 1 ml 2.5 ml 5000 U/mL Penicillin-G 5000 µg/ml Streptomycin Sulfate 55 mm β-mercaptoethanol 367 µl 918 µl 23

26 c) Serum Replacement Media, cont d 1. Combine all ingredients and sterile filter (0.2 µm). 2. Store at 4 o C wrapped in foil. 3. Take a 3 ml sample and place in 37 o C 5% CO 2 incubator for 5 days to check sterility. 4. Just prior to use, add 10 ng/ml Basic Fibroblast Growth Factor (bfgf). d) 1% Gelatin 200 ml 1. Weigh 2 gm Gelatin powder and add to 200 ml sterile H 2 O. 2. Autoclave (this will dissolve the gelatin). 3. When cool aliquot into 40 ml lots and store at -20 o C. e) 0.1% Gelatin 400 ml 1. Thaw one aliquot of 1% Gelatin. 2. Add to 360 ml sterile H 2 O and swirl to mix. 3. Sterile filter (0.2 µm) and store at 4 o C. f) 0.05% Trypsin EDTA 10 ml 1. Thaw 2.0 ml aliquot of 0.25% Trypsin EDTA. 2. Add 8 ml PBS (without Ca 2, Mg 2 ). g) 2 mg/ml MITOMYCIN C (MITC) Solution 1 ml 1. Remove plastic covering from lid of one vial of Mitomicin C (Sigma Cat. No. M0503) 2. Wipe top with 70% Ethanol. 3. Using a 22 gauge needle attached to a 3 ml syringe, draw up 1 ml PBS-. 4. Transfer PBS- to vial of MITC powder and swirl vial to dissolve. 5. When dissolved, draw up solution and carefully detach syringe leaving needle in the vial. 6. Transfer to a 15 ml tube. 7. Wrap tube with foil and store at 4 o C. h) Reconstituting Basic Fibroblast Growth Factor (bfgf) 10 ng/µl 1. Dissolve 50 µg bfgf (Millipore Cat. No. GF003) in 5 ml sterile water 2. Freeze aliquots of 0.5 ml 24

27 i) mef Freezing Solution 1. 10% DMSO / 90% F-DMEM media. 2. Prepare 30 minutes prior to use and store at 4 o C to cool down. j) 20 mg/ml Collagenase Type IV (from Clostridium histolyticum) mg Collagenase Type IV (Invitrogen Cat. No ). 2. Add 20 ml PBS and swirl to dissolve then aliquot into 1 ml aliquots and store at -20 o C. k) 4 mg/ml Collagenase Type IV (working concentration) 1. Thaw 1 ml aliquot of 20 mg/ml Collagenase Type IV and add to 4 ml PBS. Solutions and Medias for Freezing and Thawing hes Cells l) ES HEPES Media 20 ml ml DMEM 2. 4 ml FCS ml 1M HEPES Buffer 4. Combine ingredients and filter through a 0.2 µm syringe filter. m) 1M Sucrose Solution 10 ml 1. Weigh out 3.42 gm Sucrose into a 15 ml tube and add 6 ml ES-HEPES media. 2. Invert tube until sucrose has dissolved. 3. Add 2 ml FCS and mix. 4. Sterile filter through 0.22 µm syringe filter. 5. Store at 4 o C until use NB: Aliquots of this solution can be stored at -20 o C. n) 10% Vitrification Solution 5 ml ml DMSO ml Ethylene Glycol 3. 4 ml ES HEPES Media 4. Mix to combine ingredients. 25

28 o) 20% Vitrification Solution 5 ml 1. 1 ml DMSO 2. 1 ml Ethylene Glycol ml 1M Sucrose solution ml ES HEPES Media 5. Mix to combine ingredients. p) 0.2M Sucrose 5 ml 1. 1 ml 1M Sucrose 2. 4 ml ES HEPES Media 3. Mix to combine ingredients. q) 0.1M Sucrose 5 ml M 1M Sucrose ml ES HEPES Media 3. Mix to combine ingredients 26

29 17) Warranty These products are warranted to perform as described in their labeling and in CHEMICON literature when used in accordance with their instructions. THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS EXPRESSED WARRANTY AND CHEMICON DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CHEMICON s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be, at the option of CHEMICON, to repair or replace the products. In no event shall CHEMICON be liable for any proximate, incidental or consequential damages in connection with the products. 2006: CHEMICON International, Inc. - By CHEMICON International, Inc. All rights reserved. No part of these works may be reproduced in any form without permissions in writing. Millipore, Chemicon and HEScGRO are trademarks of Millipore Corporation. KnockOut is a trademark of Invitrogen Corporation. 27

30 18) Limited Use License READ THE FOLLOWING LIMITED USE LICENSE CAREFULLY BEFORE OPENING THE SEALED PACKAGE CONTAINING THE PRODUCT. OPENING THE PACKAGE/VIAL INDICATES YOUR ACCEPTANCE OF THESE LICENSE TERMS AND CONDITIONS. IF YOU DO NOT AGREE WITH THESE LICENSE TERMS AND CONDITIONS, PROMPTLY RETURN THIS PACKAGE UNOPENED TO CHEMICON AND YOUR MONEY WILL BE REFUNDED. This product is sold for research users only. The purchase of this product only conveys to you a non-transferable right to use the purchased amount of the product and components of the product for research use only, conducted solely by the purchaser. The purchaser has no right, and shall not, use this product for (a) use in or administration to humans, (b) any use for any clinical or therapeutic purposes, (c) any other purposes besides research use. Purchaser agrees to various research limitations while using this Product. Specifically, the purchaser agrees he/she will not perform any of the following experiments: (1) intermixing of the Product (or any of its components) with an intact embryo, either human or nonhuman; (2) implanting the Product (or any of its components) in a uterus; or (3) attempting to make whole embryos by any method using the Product or any of its components. The purchaser acknowledges and agrees that the Product, or one or more of its components meets at least one of the following conditions: (1) shall have been derived from excess embryos created for the purpose of in vitro fertilization with appropriate consent of the donor couple and not for the purpose of creating embryonic stem cells; or (2) shall have been derived from embryos created specifically for research purposes either by in vitro fertilization or by somatic cell nuclear transfer, for which the following additional conditions apply: (a) the embryo may not have been maintained in vitro for more than 14 days; (b) the gamete donor(s) and somatic cell donor (if any) made the donation without payment beyond reimbursement for reasonable expenses associated with donation; (c) in the case of egg donation, the donor was fully informed of the risks to herself; (d) the gamete donor(s) and somatic cell donor (if any) were fully informed of the purposes to which their donated materials would be put; (e) the research could not be done equally well using surplus IVF embryos originally created for reproductive purposes; (f) the research protocol, including gamete collection, somatic cell collection, embryo management and stem cell derivation is approved by an appropriate Institutional Review Board; and (g) protections are in place to prevent misappropriation of embryos created specifically for research. 28

31 The purchaser cannot sell, distribute or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for any commercial purpose. Such prohibited commercial purposes include (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data to others in exchange for consideration; (3) use of the product or its components (or any derivatives thereof) for therapeutic, diagnostic or prophylactic purposes (including as part of a device, chip, assay or other product); or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. The purchase cannot modify or create derivatives of this product. The purchaser cannot use this product or its components in humans or as food for humans or animals. The purchaser shall comply with all applicable laws, and shall, as appropriate, seek and comply with the decisions and recommendations of any applicable Institutional Review Board or similar body in the purchaser s use and handling of: (a) this product (b) its components or (c) materials made using this product or its components, and shall keep all of the foregoing under reasonable safe and secure conditions so as to prevent unauthorized use or access. If the purchaser is not willing to accept the limitations and obligations set forth in this product label, the purchaser may not use the product and shall promptly return it to Chemicon for a full refund. For information on other uses of this product beyond those permitted above, contact: Intellectual Property Department ( , ext. 4274), provided that purchaser is hereby put on notice that it can be given no assurances or representations that any such other uses can be permitted or authorized or rights to such other uses will be available. 29

32 SCC020/SCC021 October 2006 Revision A: