Visceral Adipocyte Care Manual - Zen
Contents
1. from the plate Open the lid and remove the white liner using sterile forceps or a hemostat and discard Carefully remove the clear adhesive seal by grabbing the edge with sterile forceps or hemostat and lifting the film slowly towards the other end Discard adhesive film in appropriate biohazard waste container Replace lid on plate The excess medium added to each well for shipping should be removed for incubation in a CO2 incubator When changing medium do not remove all the liquid as the cells will detach and float Depending upon the plate configuration please use the chart below as a guide to determine medium volumes to remove from each well Please note some settling may occur during shipping Please leave sufficient volume of medium to cover the cell monolayer 1 3 miwel Keep the plates at 37 C with 5 COs in a humidified incubator until ready for use The adipocytes should be fed with Omental Adipocyte Medium OM AM if not used within 5 7 days of arrival When feeding we recommend you remove and replace approximately half of the volume of each well The adipocytes should remain healthy and responsive for at least three weeks after induction of differentiation Unless otherwise stated on the plate cultured adipocytes will be 2 3 weeks old upon receipt Human differentiated adipocytes do not replicate and cannot be trypsinized without cell lysis Different lots will vary due to patient variation When large numbers of a particu2. 24 well plate 1 ml 24 0 ml 12 well plate 2ml 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 6 Plate cells in desired format and place in a humidified 37 C incubator with 5 COs Do not agitate the plate as cells will not plate evenly 7 Twenty four hours after plating check the plates for confluence If they are not completely confluent leave for and additional 24 hours maximum before inducing differentiation If the cells are not confluent after 48 hours DO NOT INDUCE DIFFERENTIATION differentiation will be poor Contact Zen Bio immediately 8 To differentiate the cells please see the protocol on page 7 starting at step 2 Rev Oct 2010 Page 9 of 12 US PATENT 6 153 432 TROUBLESHOOTING GUIDE ZenBio Inc Observation Possible causes Suggestions Preadipocytes do not differentiate 5 Cells have been passaged too many times Visceral cells will arrive at passage 2 3 or 4 We do not recommend expanding the visceral cells Use our defined Differentiation Medium OM DM Make sure that 2 Differentiation conditions not optimal wells are confluent BEFORE initiating differentiation Use the cell density recommended 3 Cells were plated at a low density in our manual 4 Cultureware used not optimal for Zen Bio does not recommend the h
3. second day after the purchase order is confirmed Please inquire as to the availability of the adipocytes when ordering All visceral cultured adipocyte orders require 2 weeks to prepare e Can differentiate the cells myself Yes You can order preadipocytes and pre made culture media for adipocyte differentiation Simple instructions for differentiating the cells are found in this manual 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 I HTLV Il hepatitis B and hepatitis C However since we cannot test all pathogens please treat the culture as a potentially infectious agent Rev Oct 2010 Page 11 of 12 US PATENT 6 153 432 ZenBio Inc e What donor information do I receive The donor s gender age and BMI will be provided Additional clinical information such as fasting blood glucose or medications taken may be available Please contact Zen Bio for more specific details e Are the cells from one donor Yes We can also provide lot numbers containing cells mixed from 5 to 8 donors to get average responses Please inquire about availability of single donor and mixed donor called a superlot lots at time order is placed e What if I want to test my own compounds in differentiation Please call or fax special media requests e What is the formulation of Zen Bio s serum free media Zen Bio s serum free media are not enhanced to supplement t
4. 7 4 Fetal bovine serum Penicillin Streptomycin Amphotericin B Omental_ Basal Medium cat OM BM DMEM Ham s F 12 medium 1 1 v v HEPES pH 7 4 Biotin Pantothenate All media contain 3 15g L 17 5 mmol L D glucose All media are also available as phenol red free and or without serum added Please inquire for custom media requests 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 the media is stable for 6 months Add fresh antibiotics when you are ready to use The media will expire 30 days after the thaw date Rev Oct 2010 US PATENT 6 153 432 ZenBio Inc MAINTENANCE OF VISCERAL PREADIPOCYTES Your human visceral preadipocytes have arrived in our patented CellPorter packaging system Upon receiving the plates please follow the instructions carefully to ensure your safety and the optimal performance of these cells 1 Check the seal for each plate Discard any plate where the vacuum seal has been compromised during shipment Please be aware that these cells are of human origin Please treat them as potentially infectious since we cannot test for all pathogens ALWAYS WEAR GLOVES AND USE PROTECTIVE MEASURES WHEN HANDLING HUMAN PRIMARY CELLS Place the package into a sterile environment THIS IS VERY IMPORTANT SINCE BREAKING THE VACUUM SEAL MAY POTENTIALLY INTRODUCE CONTAMINATION INTO THE PLA
5. NICAL SERVICES ZenBio Inc 3200 Chapel Hill Nelson Blvd Suite 104 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 Oct 2010 Page of 12 US PATENT 6 153 432 CONTENTS Introduction Materials Provided for Each Catalog Item Visceral Media Compositions Maintenance of Visceral Preadipocytes Maintenance of Visceral Adipocytes Differentiation of Visceral Preadipocytes into Adipocytes Plating Procedure for Cryopreserved Visceral Cells Troubleshooting guide Frequently Asked Questions Pathogen Testing ZenBio Inc PAGE Rev Oct 2010 Page 2 of 12 US PATENT 6 153 432 ZenBio Inc INTRODUCTION Cultured human visceral adipocytes Visceral preadipocytes can be cultured as growing precursor cells or differentiated into adipocytes using medium supplemented with adipogenic and lipogenic hormones This instruction manual describes procedures required to induce human preadipocytes to differentiate into mature adipocytes as well as culturing methods for human preadipocytes and adipocytes The process of differentiating preadipocytes to adipocytes has been patent protected by Zen Bio under US patent number 6153482 A complication to commercial development of cultured visceral adipocytes is the varying definition o
6. TE Use scissors to snip open the bag at any end The vacuum seal should be released at this time You may notice some bubbling of the medium in the plate at this time This is normal and will not affect cell performance In a sterile environment remove the plate from the bag taking care to not disturb the cover top from the plate Open the lid and remove the white liner using sterile forceps or a hemostat and discard Carefully remove the clear adhesive seal by grabbing the edge with sterile forceps or hemostat and lifting the film slowly towards the other end Discard adhesive film in appropriate biohazard waste container Replace lid on plate The excess medium added to each well for shipping should be removed before incubation in a humidified atmosphere CO incubator Depending upon the plate configuration please use the chart below as a guide to determine medium volumes to remove from each well Please note some settling may occur during shipping Please leave sufficient volume of medium to cover the cell monolayer Keep the plates at 37 C with 5 CO in a humidified incubator until ready for use Differentiation into adipocytes should be initiated immediately see page 7 If cells are to be maintained as preadipocytes they should be fed with Omental Preadipocyte Medium OM PM every other day Preadipocytes are flat phase dark spindle shaped cells The cells have a similar appearance in culture to fibroblasts or smooth muscle cells T
7. ZenBio Inc zenbio gt Visceral Adipocyte Care Manual Maintenance and Differentiation from Preadipocytes to Adipocytes INSTRUCTION MANUAL ZBM0022 03 SHIPPING CONDITIONS Human Visceral Adipocyte Preadipocyte 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 Cells Frozen liquid nitrogen Plated 37 C incubator 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 visceral preadipocytes are assured to be viable when thawed and maintained according to Zen Bio protocols ORDERING INFORMATION AND TECH
8. at which you have normally observed in the subcutaneous human adipocytes e Can l pass the cells Adipocytes cannot be passed since they float after trypsinization We do not recommend expanding the visceral preadipocytes Cells are shipped at Passage 2 3 or 4 please see vial or plate label to determine passage number of the lot of cells you have received Please contact Zen Bio for your studies in which large numbers of visceral cells are required e How long do the cells last in culture Adipocytes retain similar morphology and express adipocyte specific genes for at least 3 4 weeks NOTE Cultured adipocytes are usually shipped at 2 weeks old 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 except Omental Basal Medium OM BM e From where are the cells sourced The preadipocytes are isolated from human visceral adipose tissue obtained under informed donor consent mesenteric or peri renal or omental adipose tissue e How are the cells shipped Cells cultured in multiple well plates are sealed using our patented CellPorter package method and shipped to customers via Federal Express overnight delivery e How long do I have to wait before receiving the cells We do not ship to domestic locations on Fridays In general preadipocytes in culture or cryopreserved can be shipped the
9. e culture treated plates and flasks Please contact us if you have any questions 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 Upon thawing transfer the cells to a sterile conical bottom centrifuge tube containing 10 ml of Omental Preadipocyte Medium cat OM PM Centrifuge 1 200 rpm 282 X g 20 C 5 minutes Aspirate the supernatant TAKE CARE TO NOT ASPIRATE ANY OF THE CELL PELLET The cell vial contains a minimum of 1 0 or 2 0 x 10 viable cells see vial label however we recommend performing a cell count to determine a more exact number of cells Resuspend the cell pellet in 2 ml Omental Preadipocyte Medium and 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 Plate approximately 40 625 cells cm using the media volumes from the table below Refer to the manufacturer s specifications for the specific cultureware brand you are using see Table 2 FORMAT VOLUME PER WELL TOTAL VOLUME PER FORMAT 96 well plate 150 ul 14 4 ml 48 well plate 500 ul 24 0 ml
10. f visceral within the scientific community Some define omental and mesenteric fat as the only true visceral fat whereas others include all intra abdominal adipose tissue Some researchers do not define it further than simply not the subcutaneous layer The omentum OM is an immunologic organ composed of adipose blood vessels and lymph nodes which overlays the abdominal organs within the peritoneal cavity Mesenteric adipose tissue contained within the peritoneal cavity is associated with the vasculature of the intestines and colon Peri renal adipose is attached to the kidneys Navel SQ Adipose Abdominal Muscle Peritoneal Wall Liver Skin Kidney Perirenal Adipose Spinal Column Omental jfdipose Mesenteric Adipose Intestine Colon Rev Oct 2010 Page 3 of 12 US PATENT 6 153 432 ZenBio Inc 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 preadipocyte viability depends greatly on the use of suitable media reagents and sterile plastic wear If these parameters are not carefully ob
11. he absence of serum These media are available for assay procedures where cells are rested from serum Do not differentiate preadipocytes in serum free medium PATHOGEN TESTING 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 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 REFERENCES Lists of articles using Zen Bio Inc cultured human cultured preadipocytes and adipocytes may be found at our website http Awww zenbio com under the COMPANY button zenbio inc e mail information zenbio com e http www zenbio com p 0 box 13888 e 3200 chapel hill nelson blvd suite 104 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 2010 Page 12 of 12 US PATENT 6 153 432
12. he majority of the preadipocytes will differentiate into adipocytes using Omental Differentiation Medium cat OM DM and Omental Adipocyte Medium cat OM AM as described in this manual The differentiation efficiency varies depending on the donor Please see the Certificate of Analysis that came with your order for information specific to the cells you have received Rev Oct 2010 Page 6 of 12 US PATENT 6 153 432 ZenBio Inc MAINTENANCE OF VISCERAL ADIPOCYTES Your adipocytes have arrived in our patented CellPorter packaging system Upon receiving the plates please follow the instructions carefully to ensure your safety and the optimal performance of these cells 1 Check the seal for each plate Call Zen Bio if there is any problem with the shipment Please be aware that these cells are of human origin Please treat them as potentially infectious since we cannot test for all pathogens ALWAYS WEAR GLOVES AND USE PROTECTIVE MEASURES WHEN HANDLING HUMAN PRIMARY CELLS Place the package into a sterile environment THIS IS VERY IMPORTANT SINCE BREAKING THE VACUUM SEAL MAY POTENTIALLY INTRODUCE CONTAMINATION INTO THE PLATE Use scissors to snip open the bag at any end The vacuum seal should be released at this time You may notice some bubbling of the medium in the plate at this time This is normal and will not affect cell performance In a sterile environment remove the plate from the bag taking care to not disturb the cover top
13. l 150 ul well 150 ul well 90 ul well 120 ul well 90 ul well 90 ul well 48 well plate 500 ul well 500 ul well 500 ul well 300 pl well 400 pl well 300 ul well 300 pl well 24 well plate 1 0 mlMwell 1 0 mlAwell 1 0 ml well 0 6 mlMwell 0 8 mi well 0 6 mlwell 0 6 miMwell 12 well plate 2 0 mlWwell 2 0 mlWwell 2 0 ml well 1 2 ml well 1 6 ml well 1 2 mlwell 1 2 ml well 6 well plate 3 0 miMwell 3 0 mlWwell 3 0 mlMwell 1 8 ml well 2 4 ml well 1 8 ml well 1 8 ml well T 75 flask 20 mi flask 20 ml lask 20 mlflask 12mlAlask 16 mli flask 12 mlflask 12 ml flask T 25 flask 7 mi flask 7 mi flask 7 mi flask 4 2 mlAlask 5 6 mlflask 4 2 mi flask 4 2ml flask Cat 657160 665 Multi well Plate Format Greiner Bio One Cellstar cm2 well 3 Table 2 Summary Culture area Zen Bio Recommended Cultureware 7 1 94 1 02 0 35 180 662160 677180 655180 Nunc cm well Cat 3516 3513 3526 3548 3595 152795 150628 143982 150687 167008 Rev Oct 2010 Page 8 of 12 US PATENT 6 153 432 ZenBio Inc PLATING PROCEDURE Cryopreserved Visceral Preadipocytes Catalog MSN F PR F OP F Please note Primary cells can be very sensitive to brands of cultureware Zen Bio does not currently recommend the use of Falcon or Sarstedt brand plates or flasks Our scientists are using Nunc Costar Corning or Greiner bio one CellStar tissu
14. lar lot are needed please contact Zen Bio to reserve a lot for any specific orders Rev Oct 2010 Page 7 of 12 US PATENT 6 153 432 ZenBio Inc DIFFERENTIATION OF PREADIPOCYTES INTO ADIPOCYTES 1 Visceral preadipocytes are plated to be confluent in Omental Preadipocyte Medium cat OM PM and shipped the same day via overnight delivery Differentiation should be initiated within 24 hours after receiving the cells Please contact Zen Bio Inc to coordinate the shipping date with your schedule To start the process aspirate the entire volume of Omental Preadipocyte Medium from all wells 3 Add the appropriate volume of Omental Differentiation Medium catalog OM DM to the wells see Table 1 Feeding Volumes Incubate plate for 7 days at 37 C and 5 COs After 7 days cells should be fed by removing some of the medium and replacing with fresh Omental Adipocyte Medium catalog OM AM See Table 1 Feeding Volumes Caution Do not dry the wells Add new medium gently If using an automatic feeder set the slowest flow rate possible Two 2 weeks after the initiation of differentiation cells should appear rounded with large lipid droplets apparent in the cytoplasm Cells are now considered mature adipocytes and are suitable for most assays Table 1 Feeding Volumes Format Plating Change OM PM to Change OM DM to Change OM AM to OM DM OM AM OM AM IN OUT IN OUT IN OUT IN 96 well plate 150 ul wel
15. served limited differentiation may occur and cell growth may be slow MATERIALS PROVIDED FOR EACH CATALOG ITEM Note Zen Bio recommends that the Human Preadipocytes and Adipocytes be processed immediately upon receipt e Human Visceral Preadipocytes Includes Mesenteric peri renal omental Cat MP 2096 2048 2024 2012 2006 75 25 PR 2096 2048 2024 2012 2006 75 25 OP 2096 2048 2024 2012 2006 75 25 Approximately 100 confluent e Human Visceral Adipocytes Includes Mesenteric peri renal omental Cat MA 1096 1048 1024 1012 1006 75 25 PR 1096 1048 1024 1012 1006 75 25 OA 1096 1048 1024 1012 1006 75 25 e Cryopreserved visceral preadipocytes Includes Mesenteric peri renal omental Catalog MSN F PR F OP F Frozen vial containing 1 x10 viable visceral preadipocytes store in liquid nitrogen upon receipt 50 ml Omental Preadipocyte Medium cat OM PM Rev Oct 2010 Page 4 of 12 US PATENT 6 153 432 ZenBio Inc VISCERAL MEDIA COMPOSTIONS Omental Adipocyte Medium cat OM AM DMEM Ham s F 12 medium 1 1 v v HEPES pH 7 4 Fetal bovine serum Biotin Pantothenate Human insulin Dexamethasone Penicillin Streptomycin Amphotericin B Omental Differentiation Medium cat OM DM Omental Adipocyte medium OM AM Isobutylmethylxanthine IBMX PPARy agonist Omental Preadipocyte Medium cat OM PM DMEM Ham s F 12 medium 1 1 v v HEPES pH
16. uman primary adipocytes use of Falcon or Sarstedt cultureware for all cell culture applications 5 Differences in cultureware brand Verify the surface area for the surface area may affect plating cultureware brand you are using density if unknown Edge effects 1 Medium in outside wells evaporated Ensure a saturated humidity in the incubator Make sure multiple plates are stacked no more than 3 plates high Adipocytes appear 1 Medium was completely removed Make sure to follow instructions listed uneven in each well during feeding in Table 1 Feeding Volumes 2 Fresh medium was added too quickly Add media slowly to each well Position the pipet tips halfway down pressing on the side of the wells and slowly release the medium 3 Cells placed on uneven surface in the Place cultureware are on a level incubator surface in the incubator to ensure cells attach evenly Rev Oct 2010 Page 10 of 12 US PATENT 6 153 432 ZenBio Inc FREQUENTLY ASKED QUESTIONS e When do the cells differentiate Oil droplets should appear within 7 8 days after differentiation is induced They look extremely small initially Lipid accumulation continues throughout the first two weeks The oil droplets gradually fuse to several big locules Please note that omental preadipocytes and adipocytes are distinct from subcutaneous preadipocytes and adipocytes The level of lipid accumulation and morphology in culture may appear different from th
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