Human Keratinocyte Manual - Zen
Contents
1. Transfer the cells to a sterile conical bottom centrifuge tube containing 9 ml of Keratinocyte Growth Medium KM 2 Centrifuge at 300 x g 20 C for 5 minutes Aspirate the medium and resuspend the cell pellet in a volume of KM 2 appropriate for counting the cells Count cells using a hemacytometer Seed the cells at 0 37 0 75 X 10 cells per T 75 culture flask 5 000 10 000 cells cm in 20 ml KM 2 Incubate at 37 C in a humidified incubator with 5 CO2 Change the medium after 24 h in culture Medium needs to be changed every 2 3 days until the cells reach 70 80 confluent see Figure 1 Do not allow the cells to reach 100 confluency SUBCULTURE Human Adult Keratinocytes Adult keratinocytes should be passaged for subculture or cryopreservation when they are no more than 70 80 confluent in about 5 6 days in culture 1 Pre warm KM 2 medium 0 25 trypsin 2 21mM EDTA solution and sterile Phosphate Buffered Saline PBS Ca Mg free in a water bath at 37 C Aspirate medium and wash the cells 2 3 times with sterile PBS Ca Mg free to remove all traces of medium Remove the PBS and add 2mL T 75 flask or 6 ml T 225 flask of pre warmed 0 25 trypsin 2 21mM EDTA solution 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 A longer incubation2. Keratinocyte Growth Medium KM 3 4 Centrifuge at 300 x g 20 C for 5 minutes 5 Aspirate the medium and resuspend the cell pellet in a volume of KM 3 appropriate for counting the cells Count cells using a hemacytometer 6 Seed the cells at 0 37 0 75 X 10 cells per T 75 culture flask 5 000 10 000 cells cm in 20 ml KM 3 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 confluency see Figure 1 Do not allow the cells to reach 100 confluency Rev 01 11 2012 Page 6 of 9 SUBCULTURE Human Neonatal Keratinocytes 1 Human neonatal keratinocytes should be passaged for subculture or cryopreservation when they are no more than 70 80 confluent in about 5 6 days in culture Pre warm KM 3 medium 0 25 trypsin 2 21mM EDTA solution and sterile Phosphate Buffered Saline PBS Ca Mg free in a water bath at 37 C Aspirate medium and wash the cells 2 3 times with sterile PBS Ca Mg free to remove all traces of medium Remove the PBS and add 2mL T 75 flask or 6 ml T 225 flask of pre warmed 0 25 trypsin 2 21mM EDTA solution 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 A longer incubation in trypsin can damage the keratinoc
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4. in trypsin can damage the keratinocytes Rev 01 11 2012 Page 5 of 9 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 4ml of KM 2 Centrifuge at 300xg for 5 minutes at 20 C 7 Aspirate the medium and resuspend the cell pellet in a desired volume of KM 2 and proceed to cell counting 8 Seed cells at 5 000 10 000 cells cm2 0 37 0 75x10 cells per T75 flask in 20 ml of KM 2 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 COs making sure the surface is level for even cell distribution 9 Replace the medium 24 hours after plating and then every 2 3 days until they are 70 80 confluent see Figure 1 PLATING AND EXPANSION PROCEDURES Cryopreserved Neonatal Keratinocytes THAWING AND CULTURING Human Neonatal Keratinocytes 1 Pre warm the KM 3 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 Neonatal
5. inocyte markers keratin 5 and keratin 14 as assessed by immunostaining PRECAUTIONS This product 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 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 keratinocyte 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 e Cryopreserved Human Adult Keratinocytes Cat KR F Frozen vial containing 0 5 x10 viable human keratinocytes store in liquid nitrogen upon receipt 50 ml Human Keratinocyte Growth Medium cat KM 2 NOTE expiration date is 3 weeks from media ship date e Cryopreserved Human Neonatal Keratinocytes Cat KRN F Frozen vial containing 0 5 x10 viable human neonatal keratinocytes store in liquid nitrogen upon receipt 50 ml Human Neonatal Keratinocyte Growth Medium cat KM 3 NOTE expiration date is 3 weeks from media ship date Rev 01 11 2012 Page 3 of 9 MEDIA COMPOSTIONS Adult Keratinocyte Growth Medium Cat KM 2 MCDB153 Human Epidermal growth facto
6. lephone 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 zen bio com World Wide Web http www zenbio com Rev 01 11 2012 Page of 9 CONTENTS Introduction Materials provided for each catalog item Media compositions Plating and expansion of cryopreserved adult keratinocyte cells Plating and expansion of cryopreserved neonatal keratinocyte cells Cryopreservation procedure for human keratinocytes Troubleshooting guide Frequently asked questions Pathogen testing Rev 01 11 2012 Page 2 of 9 PAGE O O O OH INTRODUCTION ZenBio s adult human keratinocytes are isolated from the epidermis of healthy non diabetic donors between 18 and 60 years old who have undergone elective surgery ZenBio neonatal keratinocytes are isolated from the foreskin of healthy males aged newborn to infant from elective circumcisions The cells are isolated by trypsin digestion of the epidermal sheet and collected by centrifugal force This instruction manual describes procedures to passage and culture the adult and neonatal human keratinocytes For the adult keratinocytes donor matched dermal fibroblasts and preadipocytes are also available for many samples All samples are negative for common pathogens HIV 1 HIV 2 HTLV I HTLV Il hepatitis B and hepatitis C All keratinocytes exhibit the cobblestone morphology and highly express the basal kerat
7. man male foreskin from elective circumcisions 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 I HTLV Il hepatitis B and hepatitis C However since we cannot test all pathogens please treat the culture as a potentially infectious agent What quality control tests are performed on the keratinocytes All samples are negative for common pathogens HIV 1 HIV 2 HTLV I HTLV Il hepatitis B and hepatitis C All keratinocytes exhibit the cobblestone morphology and highly express the basal keratinocyte markers keratin 5 and keratin 14 as assessed by immunostaining What donor information do receive The donor s age gender and body mass index BMI 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 01 11 2012 P
8. r rEGF Human Insulin Human apo transferrin Bovine serum albumin fatty acid free low endotoxin Phosphoethanolamine Ethanolamine Hydrocortisone Calcium Chloride Epinephrine Bovine Pituitary Extract BPE Penicillin Neonatal Keratinocyte Growth Medium Cat KM 3 MCDB153 Human Epidermal growth factor rEGF Human Insulin Human apo transferrin Phosphoethanolamine Ethanolamine Hydrocortisone Calcium Chloride Epinephrine Bovine Pituitary Extract BPE Penicillin Streptomycin Amphotericin B e Streptomycin e Amphotericin B Keratinocyte Basal Medium suitable for both neonatal and adult keratinocytes Cat KB 1 e MCDB153 e Penicillin e Streptomycin e Amphotericin B Keratinocyte Cryopreservation Medium suitable for both neonatal and adult keratinocytes Cat KF 100 e Keratinocyte Growth Medium KM 2 e DMSO e FBS Please inquire for custom media requests Rev 01 11 2012 Page 4 of 9 PLATING AND EXPANSION PROCEDURES Cryopreserved Adult Keratinocytes THAWING AND CULTURING Human Adult Keratinocytes Pre warm the KM 2 medium at 37 C Prepare all your pipets and vessels 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
9. y 1 4 days after the cells have reached 80 C Rev 01 11 2012 Page 7 of 9 HUMAN KERATINOCYTES Morphology Figure 1 70 confluent Figure 2 100 confluent 7 ASS Mtr OA Pr POR DRS Pas Se GARG y y KERATINOCYTE TROUBLESHOOTING GUIDE Observation Possible causes Suggestions keratinocyte cells 1 Cells have been passaged too 1 Use cells of a lower passage number do not grow many times Edge effects 1 Medium in outside wells 1 Ensure a saturated humidity in the evaporated incubator and feed the cells no less than every 2 3 days Make sure multiple plates are stacked no more than 3 plates high If the wells are not all used fill the empty wells with medium Rev 01 11 2012 Page 8 of 9 FREQUENTLY ASKED QUESTIONS Can pass the cells Keratinocytes can be trypsinized and replated up to passage 4 or 5 All cells are shipped at passage 2 or 3 after establishing a primary culture How fast do the cells replicate The average doubling time is 48 84 hours However keep in mind that the replication rate for human keratinocytes varies from donor to donor Should antibiotics be included in the medium Yes Antibiotics and anti fungal agents are always recommended since the cells are primary cells Where are the cells obtained The adult keratinocytes are isolated from human epidermal tissue obtained from consented adult donors undergoing elective surgery The neonatal keratinocytes are isolated from hu
10. ytes Neutralize the trypsin using an equal volume of 0 5 mg ml soybean trypsin inhibitor Collect all the cells in a conical tube containing 4ml of KM 3 Centrifuge at 300xg for 5 minutes at 20 C Aspirate the medium and resuspend the cell pellet in a desired volume of KM 3 and proceed to cell counting Seed cells at 5 000 10 000 cells cm2 0 37 0 75x1 0 cells per T75 flask in 20 ml of KM 3 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 COs making sure the surface is level for even cell distribution Replace the medium 24 hours after plating and then every 2 3 days until they are 70 80 confluent see Figure 1 CRYOPRESERVATION Procedure for Human Keratinocytes 1 Cryopreserve adult or neonatal human keratinocytes after counting 2 Centrifuge at 300 x g 20 C 5 minutes 3 Suspend in cold Keratinocyte Cryopreservation medium Cat KF 100 at a concentration of 0 5X10 cells ml Do not exceed a 6 1 ratio of cells per million volume freeze medium per ml If using a controlled rate freezer Freeze by reducing the temperature 1 C per minute until the temperature reaches 80 C If using a cell cryopreservation container prepare according to the manufacturer s instructions 4 For best results we recommend transferring the vials to the vapor phase of a liquid nitrogen storage facilit
11. zenbio gt Human Keratinocyte Manual INSTRUCTION MANUAL ZBM0032 07 SHIPPING CONDITIONS Human Keratinocyte 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 3 weeks from ship date 4 C Cells Frozen liquid nitrogen 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 human adult keratinocytes are assured to be viable when thawed and maintained according to Zen Bio protocols ORDERING INFORMATION AND TECHNICAL SERVICES Zen Bio Inc 3200 Chapel Hill Nelson Blvd Suite 104 PO Box 13888 Research Triangle Park NC 27709 U S A Te
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