Human Sebocytes Manual - Zen
Contents
1. 25 trypsin 2 21mM EDTA solution by adding an equal volume of 0 5 mg ml soybean trypsin inhibitor remove the solution along with any detached cells to a 15ml conical tube and then add 2ml of complete SEB 1 To harvest the remaining attached sebocytes wash the plate with PBS add another 1ml of trypsin to the flask and repeat step 4 Centrifuge to obtain cell pellets as described below and combine resuspended cells prior to counting Centrifuge at 300xg for 5 minutes at 20 C 8 Aspirate the medium and gently resuspend the cell pellet in a desired volume of SEB 1 and proceed to cell counting 9 Seed cells at 10 000 20 000 cells cm 0 25 0 5x10 cells per T25 flask in 6 ml of SEB 1 If using 48 or 96 well plates seed the cells at 20 000 40 000 cells cm Ensure cells are evenly suspended when plating large numbers of plates or flasks Do not agitate plates and flasks Rev 06 2014 Page 4 of 6 ZenBio Inc after plating Place in a humidified incubator at 37 C and 5 COs making sure the surface is level for even cell distribution 10 Replace the medium 24 hours after plating and then every 2 3 days until they are 70 80 HUMAN SEBOCYTES MORPHOLOGY Figure 1 Non confluent Figure 2 Sub confluent FREQUENTLY ASKED QUESTIONS 1 Can passage the cells a All cells are shipped at passage 3 after establishing a primary culture We guarantee performance up to 1 passage passage 4 2 How fast do the cells replicate a Th2. information do receive a The donor s age gender body mass index BMI and a current medications list are provided in the certificate of analysis that accompanies each lot of cells PATHOGEN TESTING 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 test 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 Rev 06 2014 Page 6 of 6
3. SEBOCYTE MEDIUM PREPARATION INSTRUCTIONS 1 Aseptically add the entire contents of 1 set Sebocyte Medium Supplements BPE hrEGF to 1 bottle Sebocyte Basal Medium 500ml to make complete Sebocyte Medium cat SEB 1 2 Gently invert to mix contents Store complete medium at 4 C for 21 days 3 The expiration date of the complete Sebocyte Medium will be exactly 21 days after the supplements are added to the Sebocyte Basal Medium 4 DO NOT FREEZE the Sebocyte Basal Medium Store at 4 C 5 DO NOT FREEZE the complete Sebocyte Growth Medium Store at 4 C THAWING AND CULTURING Human Adult Sebocytes Thawing 1 Pre warm the complete SEB 1 medium at 37 C Prepare all your pipets and vessels 2 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 For best results the thawing step should not take more than 2 minutes Stop thawing when there is still some ice in the vials Rinse the vials with 70 ethanol before opening 3 Transfer the cells to a sterile conical bottom centrifuge tube containing 9 mL of Sebocyte Growth Medium SEB 1 4 Centrifuge at 300 x g 20 C for 5 minutes Rev 06 2014 Page 3 of 6 5 Aspirate the medium and resuspend the cell pellet in a volume of SEB 1 appropriate for counting the cells Count cells using a hemacytometer 6 Seed the cells at 0 25 0 5 X 10 cells per T 25 culture flask 10 000 20 000 cells
4. cm in 6 mL of SEB 1 Note If using 48 or 96 well plates seed the cells at 20 000 40 000 cells cm 7 Incubate at 37 C in a humidified incubator with 5 COs Change the medium after 24 h in culture 8 Medium needs to be changed every 2 3 days until the cells reach 70 80 confluence see Figure 2 SUBCULTURE Human Adult Sebocytes Adult sebocytes should be passaged for subculture when they are no more than 70 80 confluent Note that all cells are shipped at passage 3 after establishing a primary culture For primary sebocytes we guarantee up to one passage 1 Pre warm complete SEB 1 medium 0 25 trypsin 2 21mM EDTA solution and sterile Phosphate Buffered Saline PBS Ca Mg free in a water bath at 37 C 2 Aspirate medium and gently wash the cells 2 3 times with sterile PBS to remove all traces of medium Remove the PBS and add 1ml T 25 flask of pre warmed 0 25 trypsin 2 21mM EDTA solution 4 Incubate the cells at 37 C Monitor cell detachment under the microscope after 2 minutes Tap the flask gently to loosen the cells If the cells are still attached place them at 37 C for another 1 3 minutes Note A longer incubation in trypsin can damage the sebocytes 5 Neutralize the trypsin using an equal volume of 0 5 mg ml soybean trypsin inhibitor Collect all the cells in a conical tube containing 2ml of SEB 1 6 Note If some of the cells remain attached after the recommended time we suggest that you neutralize the 0
5. duct is for research use only t 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 bionazardous 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 sebocyte viability depends greatly on the use of the recommended protocols suitable media reagents and sterile plastic wear If these parameters are not carefully observed this may results in poor growth viability and differentiation capacity of the cells MATERIALS PROVIDED FOR EACH CATALOG ITEM e Cryopreserved Human Primary Sebocytes Cat SEB F Frozen vial containing 500 000 viable human adult sebocytes Store in vapor phase nitrogen 150 C to 190 C immediately upon receipt Rev 06 2014 Page 2 of 6 MEDIUM COMPOSITION AND PREPARATION Sebocyte Growth Medium Cat SEB 1 2 component media kit A B Note This medium has been developed to optimize sebocyte proliferation and maintain sebocytes in an undifferentiated state A Sebocyte Basal Medium Storage 4 C Composition e Serum Free Base Medium MCDB153 e Penicillin e Streptomycin e Amphotericin B B Sebocyte Medium Supplement Set Storage 20 C Composition e Bovine Pituitary Extract BPE e Human Recombinant Epidermal Growth Factor hrEGF
6. e average doubling time is 48 72 hours However keep in mind that the replication rate for human sebocytes varies from donor to donor 3 Should antibiotics be included in the medium a Yes b Antibiotics and anti fungal agents are always recommended since the cells are primary cells 4 Where are the cells obtained a The adult sebocytes are isolated from micro dissected sebaceous gland from the facial skin of healthy consented adult donors undergoing elective surgery 5 Do you test for pathogens Which ones a Yes b Samples from each donor are tested via PCR to confirm non reactivity for HIV 1 HIV 2 HTLV I HTLV II hepatitis B and hepatitis C However since we cannot test all pathogens please treat the culture as a potentially infectious agent 6 What quality control tests are performed on the Sebocytes a They highly express the Sebocyte marker Cytokeratin 7 and MUC 1 when differentiated as assessed by immunostaining b Sebocyte cells exhibit an epithelial morphology with small cytoplasmic lipid droplets Rev 06 2014 Page 5 of 6 7 Do you sell a Sebocyte Differentiation Medium a At this time we do not have a Sebocyte Differentiation Medium available for retail 8 Can just buy and use the Sebocyte Basal Medium for my experiments a No Sebocyte Growth Medium cat SEB 1 is a 2 part medium kit comprised of both the base medium and the supplements Any other use negates the ZenBio product liability 9 What donor
7. ucts for all claims regarding shipment damage incorrect ordering or other delivery issues Delivery claims received after 7 days of receipt of products are not subject to replacement or refund ORDERING INFORMATION AND TECHNICAL SERVICES ZenBio Inc 3200 East NC 54 Suite 100 PO Box 13888 Research 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 06 2014 Page 1 of 6 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 without amendment or substitution Contact ZenBio Inc within no more than 24 hours after receipt of products for all claims regarding shipment damage incorrect ordering or other delivery issues Delivery claims received after 7 days of receipt of products are not subject to replacement or refund PRECAUTIONS This pro
8. zenbio gt Human Sebocytes Manual INSTRUCTION MANUAL 2ZBM0081 02 SHIPPING CONDITIONS Human Sebocyte 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 2 4 days Must be processed upon shipment receipt STORAGE CONDITIONS Media Store as indicated IMMEDIATELY UPON ARRIVAL e Sebocyte Basal Medium Store at 4 C DO NOT FREEZE e Sebocyte Supplements Store at 20 C Cells Sebocyte cells are to be stored in vapor phase nitrogen 150 C to 190 C IMMEDIATELY UPON RECEIPT 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 without amendment or substitution Contact ZenBio Inc within no more than 24 hours after receipt of prod
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