ZBM0001.01SQAdipocyt... - Zen
Contents
1.2. 24 well plates 3 0 ml well 2 0 ml 12 well plates 5 8 ml well 3 8 ml 6 well plates 8 8 ml well 5 8 ml 75cm flask 260ml flask 240 ml 25cm flask 72 ml flask 65 ml 5 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 Preadipocyte Medium PM 1 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 see Figure 1 A The majority of the preadipocytes will differentiate into adipocytes see Figure 1 C using Adipocyte Differentiation Medium cat DM 2 and Adipocyte Maintenance Medium cat AM 1 as described in this manual The differentiation efficiency varies depending on the donor The patient information provided on the product sheet is limited to sex age and BMI of the donor Please see the Certificate of Analysis that came with your order for information specific to the cells you are using Rev Oct 2010 Page 5 of 14 US PATENT 6 153 432 MAINTENANCE OF 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 w
3. 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 e Do the cells express the uncoupling proteins Which ones The UCP1 mRNA can be detected by PCR and bDNA after stimulating the cells with PPARy agonist UCP2 mRNA is expressed We do not know if UCP3 is expressed or not e Can order visceral adipose Yes Please see our online pricelist for visceral adipose tissue derived cells and related reagents Please contact Zen Bio Inc for special procurement or contract assay services e How do obtain RNA from the cells How much RNA can I expect Use RNA Tri reagent Molecular Products RNeasy kit Qiagen or a guanidine thiocyanate solution Chomzynski protocol You can expect approximately 20 ug total RNA from a 10 cm dish of preadipocytes and 40 60 ug of RNA from a 10 cm dish of adipocytes 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 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 want to test my own compounds in differentiation Please call or fax special media requests e What is the formulation of Zen Bi
4. 100 confluent Preadipocytes A 1 week old post differentiation cultured adipocytes B and mature 2 weeks post differentiation cultured Adipocytes C These are unstained photographs of human preadipocyte morphology 20X The cells should appear comparable in appearance to these pictures The preadipocytes should be confluent 24 48 hours after plating for differentiation If they are not 100 confluent the cells will not differentiate well Please see the Troubleshooting guide for any problems Rev Oct 2010 Page 8 of 14 US PATENT 6 153 432 PLATING PROCEDURE Cryopreserved Subcutaneous Preadipocytes Catalog SP 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 tissue 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 Preadipocyte Medium cat PM 1 Centrifuge 1 200 rom 282 X g 20 C 5 minutes Aspirate t
5. UCE DIFFERENTIATION differentiation will be poor Contact Zen Bio immediately To differentiate the cells please see the protocol on page 7 starting at step 2 Rev Oct 2010 Page 9 of 14 US PATENT 6 153 432 EXPANSION PROCEDURE Cryopreserved Subcutaneous Preadipocytes Catalog SP F 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 Upon the thawing add the cells to a sterile conical bottom centrifuge tube containing 10 ml of Preadipocyte Medium PM 1 Centrifuge at 280 x g 20 C 5 minutes Aspirate the medium and resuspend cells in a volume of PM 1 appropriate for counting the cells Count using a hemacytometer Place approximately 6 7 X 10 cells in T 75 culture flasks using Preadipocyte Medium PM 1 Incubate cells until they are 85 90 confluent in about 4 5 days Do not let the cells become 100 confluent see Figure 1 A for picture of 100 confluent cells Cells will need to be fed every other day with PM 1 Aspirate medium and wash preadipocytes 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 or 6 ml T 225 flask
6. 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 2010 Page of 14 US PATENT 6 153 432 CONTENTS Introduction Materials Provided for Each Catalog Item Media Compositions Maintenance of Preadipocytes Maintenance of Adipocytes Differentiation of Preadipocytes into Adipocytes Plating Procedure for Cryopreserved Subcutaneous Preadipocytes Expansion Procedure for Cryopreserved Subcutaneous Preadipocytes Troubleshooting Frequently Asked Questions Pathogen Testing Rev Oct 2010 Page 2 of 14 PAGE N OF fF W 10 11 12 14 US PATENT 6 153 432 INTRODUCTION Cultured human adipocytes The adipocyte precursor cells preadipocytes are isolated from subcutaneous adipose tissue of healthy non diabetic donors between 18 and 60 years old undergoing elective surgery The preadipocytes are isolated by centrifugal force after collagenase treatment 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 to induce human preadipocytes to differentiate into mature adipocytes as well as culturing methods for human preadipocytes and adipocytes The process of differentiating preadipo
7. ations 5 Differences in cultureware 5 Verify the surface area for the brand surface area may ies cultureware brand you are using affect plating density if unknown Preadipocytes do not 1 Cells have been passaged 1 Use cells of a lower passage attach well or do not too many times number grow 2 Cells expanded too high 2 Do not exceed 1 6 expansion ratio 3 Cultureware used not 3 Use only Costar Nunc or Greiner optimal for human primary cultureware adipocytes Edge effects 1 Medium in outside wells 1 Ensure a saturated humidity in the evaporated incubator Make sure multiple plates are stacked no more than 3 plates high Adipocytes appear 1 Medium was completely 1 Make sure to follow instructions uneven in each well removed during feeding listed in Table 1 Feeding Volumes 2 Fresh medium was added 2 Add media slowly to each well too quickly Position the pipet tips halfway down pressing on the side of the wells and slowly release the medium 3 Cells placed on uneven 3 Place cultureware are on a level surface in the incubator surface in the incubator to ensure cells attach evenly Rev Oct 2010 Page 11 of 14 US PATENT 6 153 432 FREQUENTLY ASKED QUESTIONS e When do the cells differentiate Oil droplets should appear within 4 7 days after differentiation is induced They look extremely small initially Lipid accumulation continues throughout the first two weeks The oil droplets gradually fuse to s
8. cytes to adipocytes has been patent protected by Zen Bio under US patent number 6153432 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 observed 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 Human Subcutaneous Preadipocytes Cat SP 2096 SP 2048 SP 2024 SP 2012 SA 2006 SP 75 SP 25 e Approximately 100 confluent Human Subcutaneous Adipocytes Cat SA 1096 SA 1048 SA 1024 SA 1012 SA 1006 SA 75 SA 25 Cryopreserved subcutaneous preadipocytes catalog SP F Frozen vial containing at least 2 x10 preadipocytes store in liquid nitrogen upon receipt 50 ml Preadipocyte medium Rev Oct 2010 Page 3 of 14 US PATENT 6 153 432 MEDIA COMPOSTIONS Preadipocyte Medium Adipocyte Differentiation Medium cat PM 1 ca
9. e purchase order is confirmed Please inquire as to the availability of the adipocytes when ordering A limited number of 96 well plates are kept in inventory Special requests or other formats require 2 weeks to prepare e Is the B3 receptor present on these cells Yes to a small extent The subcutaneous adipocytes express B81 adrenoceptor 35 and 2 adrenoceptor 65 The mRNA for the B3 receptor is not detectable by Northern analysis e Do the cells express leptin How do you measure it Yes Mature adipocytes greater than 2 weeks post differentiation do secrete leptin Commercial leptin ELISA kits are available from many venders R amp D Systems Diagnostic Laboratories e Can get brown fat No Brown fat is unavailable at this time Rev Oct 2010 Page 12 of 14 US PATENT 6 153 432 e Do the cells respond to insulin treatment Yes Insulin does stimulate both glucose uptake and lipid accumulation 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 have cDNA libraries Zen Bio and Stratagene Inc collaborated to make cDNA libraries of preadipocytes and adipocytes The libraries can be ordered through Stratagene cat no For adipocyte library is 937249 and preadipocyte libraries are 939234 and 937247 e Do you test for pathogens Which ones Yes Samples from
10. everal big locules See Figure 1 e Can l pass the cells Adipocytes cannot be passed since they float after trypsinization Preadipocytes can be trypsinized and replated several times Preadipocytes grow slower with each passage and differentiate poorly after passage 4 Cells are shipped at Passage 2 3 e How fast do the cells replicate The average doubling time is 48 84 hours However keep in mind that the replication rate for human preadipocytes varies slightly from patient to patient e How long do the cells last in culture Adipocytes retain similar morphology and express adipocyte specific genes for at least 4 weeks after induction of differentiation 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 Basal Medium BM 1 e Where are the cells from The preadipocytes are isolated from human subcutaneous 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 cells to domestic locations on Fridays In general preadipocytes in culture or cryopreserved can be shipped the second day after th
11. he supernatant TAKE CARE TO NOT ASPIRATE ANY OF THE CELL PELLET The cell vial contains a minimum of 2 0 x 10 viable cells however we recommend performing a cell count to determine a more exact number of cells Resuspend the cell pellet in 2 ml Preadipocyte 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 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 FORMAT VOLUME TOTAL VOLUME PER 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 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 IND
12. ith 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 2 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 3 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 4 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 to determine medium volumes to remove from each well Cultureware Total shipping volume per well Removal volume per well 96 well pla
13. o s serum free media Zen Bio s serum free media are not enhanced to supplement the absence of serum They are simply prepared in the absence of fetal bovine serum These media are available for assay procedures where cells are rested from serum Do not differentiate preadipocytes in serum free medium Rev Oct 2010 Page 13 of 14 US PATENT 6 153 432 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 ZenBio 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 o 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 14 of 14 US PATENT 6 153 432
14. 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 Neutralize the trypsin using 7 ml Preadipocyte Medium cat PM 1 per T 75 flask or 21 ml per T 225 flask Check the flask under a microscope to ensure all cells are free of the flask bottom Count the cells and plate in desired format see page 9 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 COs making sure the surface is level for even cell distribution Follow the differentiation protocol as outlined on page 7 We DO NOT recommend differentiating preadipocytes that are older than Passage 4 Cells will arrive at Passage 2 or 3 Rev Oct 2010 Page 10 of 14 US PATENT 6 153 432 TROUBLESHOOTING GUIDE Observation Possible causes Suggestions Preadipocytes do not 1 Cells have been passaged 1 Use cells of a lower passage differentiate too many times number 2 Use our defined differentiation 2 Differentiation conditions not eda Nae ive hanee Are optimal confluent BEFORE initiating differentiation a PRE IE plated at a low 3 Use the cell density recommended density in our manual Cultureware used not 4 Zen Bio does not recommend the optimal for human primary use of Falcon or Sarstedt adipocytes cultureware for all cell culture applic
15. omised 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 2 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 3 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 4 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 to determine medium volumes to remove from each well Cultureware Total shipping volume per well Removal volume per well 96 well plates 300 pl well 150 ul 48 well plates 1 3 ml well 0 8 ml
16. t DM 2 DMEM Ham s F 12 1 1 v v DMEM Ham s F 12 1 1 v v HEPES pH 7 4 HEPES pH 7 4 Fetal bovine serum Fetal bovine serum Penicillin Biotin Streptomycin Pantothenate Amphotericin B Human insulin Dexamethasone Isobutylmethylxanthine PPARy agonist Penicillin Streptomycin Amphotericin B Adipocyte Maintenance Medium Adipocyte Basal Medium cat AM 1 Cat BM 1 DMEM Ham s F 12 1 1 v v DMEM Ham s F 12 1 1 v v HEPES pH 7 4 HEPES pH 7 4 Fetal bovine serum Biotin Biotin Pantothenate Pantothenate Human insulin Dexamethasone Penicillin Streptomycin Amphotericin B All media contain 3 15g L 17 5 mmol L D glucose All media are also available as without serum and or phenol red free 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 Page 4 of 14 US PATENT 6 153 432 MAINTENANCE OF PREADIPOCYTES Your 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 compr
17. tes 300 pl well 150 ul 48 well plates 1 3 ml well 0 8 ml 24 well plates 3 0 ml well 2 0 ml 12 well plates 5 8 ml well 3 8 ml 6 well plates 8 8 ml well 5 8 ml 75cm flask 260ml flask 240 ml 25cm flask 72 ml flask 65 ml 5 Keep the plates at 37 C with 5 CO in a humidified incubator until ready for use 6 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 four weeks after induction of differentiation Unless otherwise stated on the plate cultured adipocytes will be 2 3 weeks old upon receipt Different lots will vary due to patient variation We recommend doing one whole set of experiments using cells from the same lot number When large numbers of plates are needed please contact Zen Bio to reserve a lot for any specific orders Rev Oct 2010 Page 6 of 14 US PATENT 6 153 432 DIFFERENTIATION OF PREADIPOCYTES INTO ADIPOCYTES 1 Preadipocytes are plated confluent in Preadipocyte Medium cat PM 1 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 2 To start the process aspirate the entire volume of Preadipocyte Medium from all wells Add the appropriate volume of Adipocyte Differentiation Medium catalog DM 2 to the wells see Table 1 Feeding Volumes Inc
18. ubate plate for 7 days at 37 C and 5 COs 3 After 7 days cells should be fed by removing some of the media and replacing with fresh Adipocyte Medium catalog AM 1 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 4 Two 2 weeks after the initiation of differentiation cells should appear rounded with large lipid droplets apparent in the cytoplasm see Figure 1 C Cells are now considered mature adipocytes and are suitable for most assays Table 1 Feeding Volumes Format J Peina Charge PFT DN Grange DIF AIT Changs ATT ANT ES i a 24 well plate 1 0 ml well 1 0 ml well 1 0 ml well 0 6 ml well 0 8 ml well 0 6 ml well 0 6 ml well 12 well plate 2 0 ml well 2 0 ml well 2 0 ml well 1 2 ml well 1 6 ml well 1 2 ml well 1 2 ml well 6 well plate 3 0 ml well 3 0 ml well 3 0 ml well 1 8 ml well 2 4 ml well 1 8 ml well 1 8 ml well T 75 flask 20 mi flask 20 ml flask 20 ml flask 12 ml flask 16 ml flask 12 ml flask 12 ml flask T 25 flask 7 mi flask 7 mi flask 7 mi flask 4 2ml flask 5 6ml flask 4 2 ml flask 4 2 ml flask Rev Oct 2010 Page 7 of 14 US PATENT 6 153 432 A 100 Confluent B 1 week old adipocytes C 2 week old adipocytes preadipocytes 1 wk post differentiation 2 wks post differentiation PREADIPOCYTE gt MATURE ADIPOCYTE nucleus Figure 1 Photographs of
19. zenbio gt Subcutaneous Human Adipocytes Maintenance and Differentiation from Preadipocytes to Adipocytes INSTRUCTION MANUAL 2ZBM0001 03 SHIPPING CONDITIONS Human 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 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 preadipocytes are assured to be viable when thawed and maintained according to Zen Bio protocols ORDERING INFORMATION AND TECHNICAL SERVICES ZenBio Inc 3200 Chapel Hill Nelson Blvd Suite 104 PO Box 13888 Rese
Download Pdf Manuals
Related Search