Human Skeletal Muscle Myoblast Manual - Zen
Contents
1. It is not intended for human veterinary or in vitro diagnostic use Proper precautions and biological containment should be taken when handling cells of human origin due to their potential biohazardous nature Always wear gloves and work behind a protective screen when handling primary human cells All media supplements and tissue cultureware used in this protocol should be sterile Human myoblast viability depends greatly on the use of suitable media reagents and sterile plastic wear If these parameters are not carefully observed limited differentiation may occur and cell growth may be slow MATERIALS PROVIDED FOR EACH CATALOG ITEM Note Zen Bio recommends that the Human skeletal muscle myoblasts be processed immediately upon receipt Cryopreserved Human Skeletal Muscle Myoblasts catalog SKB F Frozen vial containing at least 0 5 x10 myoblasts store in liquid nitrogen upon receipt 50 ml Skeletal Muscle Cell Growth medium cat SKM M Rev OCT 2012 Page 3 of 10 MEDIA COMPOSTIONS Skeletal Muscle Cell Growth Skeletal Muscle Cell Skeletal Myoblast Medium Differentiation Medium Cryopreservation cat SKM M cat SKM D Medium Cat SKM 100 DMEM DMEM DMSO Fetal bovine serum Horse serum SKM M medium Bovine Serum Albumin Bovine Serum Albumin Fetuin Fetuin Human Epidermal Growth Factor Penicillin Dexamethasone Streptomycin Human Insulin Amphotericin B Penicillin Streptomycin Amphotericin B All media contain 1 02. see Table 1 Feeding Volumes Incubate the plate at 37 C and 5 COs Fresh Differentiation Medium will need to be added every 2 3 days Remove all of the medium and replace with fresh medium 3 After 6 days the cells should have fused to form myotubes These are elongated multinucleated cells They will appear to be lined up when viewed under a microscope 4 The myocytes may be used for assays 6 8 days after the initiation of differentiation and are suitable for most assays Table 1 Feeding Volumes Change SKM M to Change SKM D to Change SKM D to SKM D SKM D SKM D ee ee ee ie a eo ee 150 ul well 150 pl well 150ul well 150ul well 150u1 well 150ul well 150u well ee 500 ul well 500ul well 500ul well 500I well 500uI well 500ul well 500u1 well ps 1 0 mlwell 1 0 miwell 1 0 mi well 1 0 mi well 1 0ml well 1 0mi well 4 0mI well ne 2 0 mi well 2 0 mi well 2 0 ml well 2 0 ml well 2 0 ml well 2 0 ml well 2 0 mi well 6 well plate 3 0 ml well 3 0 ml well 3 0 ml well 3 0 ml well 3 0 ml well 3 0 ml well 3 0 ml well T 75 flask 20 mi flask 20 mi flask 20 mi flask 20 mi flask 20 ml flask 20 ml flask 20 mi flask T 25 flask 7 ml flask 7 ml flask 7 ml flask 7 mli flask 7 ml flask 7 mi flask 7 mi flask Rev OCT 2012 Page 6 of 10 A 80 Confluent B 3 day old myocytes C 1 week old myocytes myoblasts 3 days post differentiation 1 wk post differentiation MYOBLAST gt MATURE
3. 2 3 days with SKM M Replace all medium with fresh SKM M 6 Aspirate medium and wash myoblasts 4 5 times using sterile Phosphate Buffered Saline PBS to remove all traces of serum until there is no foaming of the medium Remove the PBS and release the cells from the flask bottom by adding 2 mL T 75 flask of 0 25 trypsin 2 21mM EDTA solution Allow cells to trypsinize for 5 minutes at 37 C Tap the flask gently to loosen the cells Rev OCT 2012 Page 7 of 10 7 Neutralize the trypsin using 7 ml Skeletal Muscle Cell Growth Medium cat SKM M per T 75 flask Check the flask under a microscope to ensure all cells are free of the flask bottom 8 Count the cells and plate in desired format see page 5 for plating protocol Ensure cells are evenly suspended when plating large numbers of plates or flasks Do not agitate plates and flasks after plating Place in a humidified incubator at 37 C and 5 CO making sure the surface is level for even cell distribution 9 Follow the differentiation protocol as outlined on page 6 or split the cells 1 4 for further expansion 10 We DO NOT recommend differentiating myoblasts that are older than Passage 6 Cells will arrive at Passage 3 or 4 TROUBLESHOOTING GUIDE Observation Possible causes Suggestions Myoblasts do not differentiate Cells have been passaged too many times ke Use cells of a lower passage number optimal media Make sure that wells are 8
4. 0 confluent BEFORE initiating differentiation Cells were plated at a low density 3 Use the cell density recommended in our manual Sona used not 4 Verify the surface area for the ppimaognuman pimay cultureware brand you are using myoblasts Myoblasts do not grow Cells have been passaged 1 Use cells of a lower passage too many times number Cells expanded too high 2 Do not exceed 1 4 expansion ratio Edge effects Medium in outside wells evaporated 1 Ensure a saturated humidity in the incubator and feed the cells no less than every 3 days Make sure multiple plates are stacked no more than 3 plates high Rev OCT 2012 Page 8 of 10 FREQUENTLY ASKED QUESTIONS e When do the cells differentiate Cells should begin to fuse and line up within 3 days after differentiation is induced See Figure 1 e Can pass the cells Myocytes cannot be passed because they are terminally differentiated Myoblasts can be trypsinized and replated several times Myoblasts grow slower with each passage and differentiate poorly after passage 6 Cells are shipped at Passage 3 4 e How fast do the cells replicate The average doubling time is 24 36 hours However keep in mind that the replication rate for human myoblasts varies slightly from patient to patient e How long do the cells last in culture Myocytes retain similar morphology and express myocyte specific genes for at least 1 week after induction of differenti
5. Plate approximately 5 000 15 000 cells cm using the media volumes from the table below Refer to the manufacturer s specifications for the specific cultureware brand you are using VOLUME TOTAL VOLUME PER FORMAT PER WELL FORMAT 96 well plate 150 ul 14 4 ml 48 well plate 500 ul 24 0 ml 24 well plate 1 ml 24 0 ml 12 well plate 2 ml 24 0 ml 6 well plate 3 ml 18 0 ml 10 cm dish 15 ml 15 0 ml T 75 flask 20 ml 20 0 ml T25 flask 7 ml 7 0 ml We recommend preparing slightly larger volumes to allow for loss due to foam and pipet error 5 Plate cells in desired format and place in a humidified 37 C incubator with 5 CO Do not agitate the plate as cells will not plate evenly To differentiate the cells please see the protocol on page 6 starting at step 1 Rev OCT 2012 Page 5 of 10 DIFFERENTIATION OF MYOBLASTS INTO MYOCYTES 1 Plated myoblasts in Skeletal Muscle Growth Medium cat SKM M can undergo differentiation using Skeletal Muscle Cell Differentiation Medium cat SKM D Differentiation should be initiated when the plated myoblasts reach 80 90 confluence The exact number of days necessary to reach 80 90 confluence will depend on your initial seeding density typically 1 3 days 2 To start the process aspirate the entire volume of Skeletal Muscle Growth Medium from all wells Add the appropriate volume of Skeletal Muscle Cell Differentiation Medium catalog SKM D to the wells
6. SKELETAL MYOCYTE SS Figure 1 Photographs of 80 confluent Myoblasts A 3 day old post differentiation cultured myocytes B and mature 1 week post differentiation cultured Myocytes C These are unstained photographs of human myocyte morphology 20X The cells should appear comparable in appearance to these pictures The myocytes should be 80 confluent after plating for differentiation If they are not 80 confluent the cells will not differentiate well Please see the Troubleshooting guide for any problems EXPANSION PROCEDURE Cryopreserved Human Skeletal Muscle Myoblasts Catalog SKB F 1 Remove cells from liquid nitrogen and place immediately into a 37 C water bath with agitation Be careful not to submerge the cap of the vial into water Do not leave the vials in water bath after most of the content has thawed Rinse the vials with 70 ethanol before taking them to the culture hood 2 Upon the thawing add the cells to a sterile conical bottom centrifuge tube containing 10 ml of Skeletal Muscle Growth Medium SKM M 3 Centrifuge at 280 x g 20 C 5 minutes Aspirate the medium and resuspend cells in a volume of SKM M appropriate for counting the cells Count using a hemacytometer 4 Place approximately 0 5 X 10 cells in T 75 culture flasks using Skeletal Muscle Growth Medium Incubate cells until they are 70 confluent in about 3 5 days Do not let the cells become 100 confluent Cells will need to be fed every
7. arch Triangle Park NC 27709 U S A Telephone 919 547 0692 Facsimile FAX 919 547 0693 Toll free continental US only 1 866 ADIPOSE _ 1 866 234 7673 Electronic mail e mail information zenbio com World Wide Web http www zenbio com Rev OCT 2012 Page 1 of 10 CONTENTS PAGE Introduction 3 Materials Provided for Each Catalog Item 3 Media Compositions 4 Plating Procedure for Cryopreserved Human skeletal muscle Myoblasts Differentiation of Myoblasts into Myocytes Expansion Procedure for Cryopreserved Human skeletal muscle Myoblasts Troubleshooting Frequently Asked Questions Pathogen Testing 10 Rev OCT 2012 Page 2 of 10 INTRODUCTION Cultured human skeletal muscle myoblasts Skeletal muscle is an important site of insulin stimulated glucose disposal and often the site of insulin resistance in obesity Human primary cultured skeletal muscle cells can directly reflect a patient s metabolic phenotype because many of the signaling pathways are maintained intact Zen Bio offers human primary skeletal muscle cells from a variety of donors including obese donors with Type 2 diabetes Skeletal muscle satellite cells are isolated from the rectus abdominus and propagated in culture as myoblasts Each lot is analyzed for myotube formation and the expression of myocyte specific markers The myoblasts are cryopreserved and guaranteed for use with Zen Bio support media PRECAUTIONS This product is for research use only
8. ation e Should antibiotics be included in the medium Yes Antibiotics and anti fungal agents are always recommended since the cells are primary cells All Zen Bio media contain antibiotics and anti fungal agents e Where are the cells from The myoblasts are isolated from human rectus abdominus muscle e How are the cells shipped Frozen cells will be packaged on dry ice and shipped to customers via Federal Express overnight delivery e How long do I have to wait before receiving the cells e We do not ship cells to domestic locations on Fridays In general myoblasts can be shipped the second day after the purchase order is confirmed e Do you test for pathogens Which ones Yes Samples from each donor are tested via PCR to confirm non reactivity for HIV 1 HIV 2 HTLV HTLV Il hepatitis B and hepatitis C However since we cannot test all pathogens please treat the culture as a potentially infectious agent e What donor information do I receive The donor s gender age and BMI will be provided e Are the cells from one donor Yes We can also provide lot numbers containing cells mixed donors to get average responses Please inquire about availability of single donor and mixed donor called a superlot lots at time order is placed PATHOGEN TESTING Rev OCT 2012 Page 9 of 10 Samples from each donor are tested via PCR to confirm non reactivity for HIV 1 HIV 2 HTLV I HTLV Il hepatitis B and hepatitis C However no known t
9. est can offer complete assurance that the cells are pathogen free Our products are tested and are free from mycoplasma contamination Proper precautions and biological containment should be taken when handling cells of human origin due to their potential biohazardous nature All human based products should be handled at a BSL 2 Biosafety Level 2 or higher Always wear gloves and work behind a protective screen when handling primary human cells REFERENCES Lists of articles using ZenBio Inc cultured human cultured products may be found at our website http www zenbio com under the COMPANY button zenbio inc e mail information zenbio com e http www zenbio com p 0 box 13888 e 3200 east nc highway 54 suite 100 e research triangle park e north carolina 27709 phone 919 547 0692 e fax 919 547 0693 Toll free 1 866 ADIPOSE 234 7673 Rev OCT 2012 Page 10 of 10
10. g L D glucose MEDIA EXPIRATION DATES e If placed at 4 C upon arrival the media is stable until the expiration date on the bottle label e If stored at 20 C upon arrival it is stable for 6 months Add fresh antibiotics when you are ready to use Rev OCT 2012 Page 4 of 10 Plating Procedure for Cryopreserved Human Skeletal Muscle Myoblasts Cryopreserved Human Skeletal Muscle Myoblasts Catalog SKB F 1 Remove cells from liquid nitrogen and place immediately into a 37 C water bath and agitate while in bath Be careful not to submerge the cap of the vial into water Do not leave the vials in water bath after most of the content has thawed Rinse the vials with 70 ethanol before taking them to the culture hood 2 Upon thawing transfer the cells to a sterile conical bottom centrifuge tube containing 10 ml of Skeletal Muscle Growth Medium cat SKM M Centrifuge 1 200 rom 282 X g 20 C 5 minutes Aspirate the supernatant TAKE CARE TO NOT ASPIRATE ANY OF THE CELL PELLET 3 The cell vial contains a minimum of 0 5 x 10 viable cells however we recommend performing a cell count to determine a more exact number of cells Resuspend the cell pellet in 0 5 ml Skeletal Muscle Growth Medium dilute an aliquot in 0 4 trypan blue solution We suggest withdrawing an aliquot of 50 ul of cells and mixing with 100 ul of the trypan blue solution resulting in a dilution factor of 3 Count live unstained cells on a hemacytometer 4
11. zenbio p gt Human Skeletal Muscle Myoblast Care Manual Maintenance and Differentiation from Myoblasts to Myocytes INSTRUCTION MANUAL ZBM0044 04 SHIPPING CONDITIONS Human Skeletal Muscle Myoblast Cells Orders are delivered via Federal Express courier All US and Canada orders are shipped via Federal Express Priority service and are usually received the next day International orders are usually received in 3 4 days Must be processed upon shipment receipt STORAGE CONDITIONS Media Short Term 4 C 6 months 20 C All Zen Bio Inc products are for research use only Not approved for human or veterinary use or for use in diagnostic or clinical procedures LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product No other warranties of any kind expressed or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Zen Bio Inc Zen Bio Inc shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product Zen Bio Inc warrants its cells only if Zen Bio media are used and the recommended protocols are followed Cryopreserved myoblasts are assured to be viable when thawed and maintained according to Zen Bio protocols ORDERING INFORMATION AND TECHNICAL SERVICES ZenBio Inc 3200 East NC Highway 54 Suite 100 PO Box 13888 Rese
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