Human Adult Stem Cell Manual - Zen
Contents
1. Seo et al Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo Biochem Biophys Res Comm 2005 328 258 264 Rodriguez RV et al Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells Proc Natl Acad Sci USA 2006 103 32 12167 12172 Wouter JFM et al Effect of tissue harvesting site on yield of stem cells derived from adipose tissue implications for cell based therapies Cell Tissue Res 2008 332 3 415 426 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 Rev Oct 2010 Page 13 of 13 US PATENT 6 555 3742. 555 374 Table 3 Osteogenesis Feeding Volumes Format Volume per well 96 well plate 150 ul well 48 well plate 300 ul well 24 well plate 1 0 ml well 12 well plate 2 0 ml well 6 well plate 3 0 ml well T 75 flask 12 ml flask T 25 flask 7 mi flask Figure 2 Osteoblasts 20X Alizarin red staining of 14 day old cells shows significant mineralization Rev Oct 2010 Page 9 of 13 ZenBio Inc US PATENT 6 555 374 CHONDROGENESIS PROCEDURE Differentiation of Adult Stem Cells into Chondrocytes 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 Preadipocyte Medium cat PM 1 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 1 0 or 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 PM 1 dilute an aliquot in 0 4 trypan blue solution We suggest withdrawing an aliquot of 50 ul of cells and mixing with 10
3. 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 Growth Medium Preadipocyte Medium PM 1 3 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 4 Place approximately 6 7 X 10 cells in T 75 culture flasks using PM 1 5 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 page 8 for picture of 100 confluent cells Cells will need to be fed every other day with PM 1 6 Aspirate medium and wash adult stem cells 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 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 7 Neutralize the trypsin using 7 ml 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 8 Count the cells and plate in desired format Ensure cells are evenly suspended when plating large numbers of plates or flas
4. 0 ul of the trypan blue solution resulting in a dilution factor of 3 Count live unstained cells on a hemacytometer 4 Remove the supernatant and resuspend the cell pellet in 1 2 alginate solution made in 150mM NaCl at 4 x 10 cells ml Pipette up and down without creating bubbles to mix thoroughly Draw the cell seeded alginate suspension into a sterile 10 cc syringe using a 22 gauge needle 6 Add 3 ml of CaCl 102 mM into one well of a 6 well culture plate Carefully and slowly dispense equal sized droplets of the cell seeded alginate solution into the CaCl solution dispense10 30 beads per well taking care to avoid clumping the beads Cure the beads in the CaCl solution for 10 minutes at room temperature 8 Using a glass pipette aspirate the CaCl solution off the beads Be careful not to aspirate the beads with the solution 9 Wash the cell seeded alginate beads three times with NaCl 150 mM and then one more time with DMEM HG 10 Add 3 ml of the Chondrogenic Differentiation Medium cat CM 1 11 Incubate at 37 C 5 0 COs and 95 relative humidity for the duration of the experiment 12 Change media every three days eo E Q a P 4 A S Sho o p 9 Figure 3 Differentiated cells stained F D 48 oa i Se 8 2 amp for collagen production E Don pieo S y 00 6 G0 bs Sin N pP T apt CR a oe Bn 20 6 Sq Fe a a2 a Rev Oct 2010 Page 10 of 13 US PATENT 6 555 374 TROUBLESHOOTING GU
5. IDE Observation Possible causes Suggestions Adult stem cells do not differentiate Cells have been passaged too many times Use cells of a lower passage number Differentiation conditions not 2 Use our defined differentiation optimal medium Cells were 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 brand adipocytes cultureware for all Adipogenesis cell culture applications Differences in cultureware 5 Verify the surface area for the brand surface area may affect cultureware brand you are using plating density in unknown Adult stem cells do not Cells have been passaged 1 Use cells of a lower passage grow too many times number Cells expanded too high 2 Do not exceed 1 6 expansion ratio Cultureware used not 3 Use only Costar Nunc or Greiner optimal for human primary cultureware adult stem cells Edge effects 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 Adipogenesis Medium was completely 1 Make sure to follow instructions Adipocytes appear removed during feeding listed in Table 1 Feeding Volumes tcl 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 Cells placed omuneven well
6. ace 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 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 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 PER WELL VOLUME PER 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 mil 7 0 ml We recommend preparing slightly larger volu
7. ells 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 30 000 cells cm using the media volumes from the table below Refer to the manufacturer s specifications for the specific cultureware brand you are using FORMAT VOLUME TOTAL VOLUME PER NUMBER CELLS PER PER WELL FORMAT FORMAT 96 well plate 150 ul 15 ml plate 0 93 X 10 cells 15 ml 48 well plate 500 ul 24ml plate 1 25 X 10 cells 25 ml 24 well plate 1 ml 25ml plate 1 44X 10 cells 26ml 12 well plate 2 ml 25ml plate 1 5 X 10 cells 26ml 6 well plate 3 ml 18 ml plate 1 8 X 10 cells 20ml 10 cmdish 15 ml 15ml dish 2 25 X 10 cells 15 ml T 75 flask 20 ml 20 mi flask 2 25 X 10 cells 20ml T25 flask 7 ml 7 ml flask 0 75 X 10 cells 7ml 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 aspirate the entire volume of Preadipocyte Medium from all wells Add the appropriate volume of Osteoblast Differentiation Medium catalog OB 1 to the wells see Table 3 Osteogenesis Feeding Volumes Incubate cells at 37 C and 5 COs Feed cells every 3 days with Osteoblast Differentiation Medium catalog OB 1 Rev Oct 2010 Page 8 of 13 US PATENT 6
8. gonist Streptomycin Penicillin Amphotericin B Streptomycin Amphotericin B Adipocyte Basal Osteoblast Differentiation Medium Chondrocyte Differentiation Medium Cat OB 1 Medium Cat BM 1 Cat CM 1 DMEM Ham s F 12 1 1 v v DMEM Ham s F 12 1 1 v v DMEM high glucose HEPES pH 7 4 Fetal Bovine serum Fetal bovine serum Biotin B glycerophosphate Transforming growth factor B TGF B1 Pantothenate Ascorbate 2 phosphate Ascorbate 2 phosphate Dexamethasone 1 25 OH Vitamin D3 Penicillin Streptomycin NOTE Dexamethasone Insulin transferrin selenium plus ITS Penicillin Streptomycin All media except Chondrocyte Medium contains 3 15g L 17 5mmol L D glucose Chondrocyte Differentiation Medium contains 4 5g L 25 0mmol L D glucose All media are also available as phenol red free and or without serum Please inquire for custom media requests MEDIA EXPIRATION DATES If placed at 4 C upon arrival the media is stable until the expiration date on the bottle label 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 13 US PATENT 6 555 374 PLATING AND EXPANSION PROCEDURES Cryopreserved Adult Stem Cells 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
9. ks Do not agitate plates and flasks after plating Place ina humidified incubator at 37 C and 5 COs making sure the surface is level for even cell distribution 9 OPTIONAL Cryopreserve stem cells after counting Centrifuge at 280 x g 20 C 5 minutes Suspend in cold cryopreservation medium Cat FM 1 100 at a concentration of 1X10 cells ml Do not exceed a 6 1 ratio of cells per million volume cryopreservation medium per ml Remember to account for the volume of the cell pellet before adding the volume of cryopreservation medium necessary for cell suspension 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 For best results we recommend transferring the vials to the vapor phase of a liquid nitrogen storage facility as soon as possible after the cells have reached 80 C Rev Oct 2010 Page 5 of 13 US PATENT 6 555 374 ADIPOGENESIS PROCEDURE Differentiation of Adult Stem Cells into Adipocytes 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 1 Remove cells from liquid nitrogen and pl
10. mes 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 COs 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 INDUCE DIFFERENTIATION differentiation will be poor Contact Zen Bio immediately Rev Oct 2010 Page 6 of 13 US PATENT 6 555 374 7 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 Incubate plate for 7 days at 37 C and 5 CO 8 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 9 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 Adipogenesis Feeding Volumes SS a a a a A 100 Confluent B 1 week old adipocytes C 2 week old adipocytes Adul
11. nski protocol You can expect approximately 20 ug total RNA from a 10 cm dish of adult stem cells What donor information do I receive The donor s age gender and BMI are provided in the certificate of analysis that accompanies each lot of cells What is the formulation of Zen Bio s serum free media Zen Bio s serum free media are not enhanced to supplement the absence of serum These media are available for assay procedures where cells are rested from serum Do not differentiate adult stem cells into adipocytes using medium without serum What quality control testing is conducted on the cells We do confirm the presence of several cell surface markers indicative of stem cells via flow cytometry The adult stem cells stain gt 99 positive for CD105 and CD44 negative for CD31 and CD45 Our quality control for adipocytes is lipid staining total triglyceride content and functional lipolysis for osteoblasts it is measurement of degree of mineralization as assessed by Alizarin Red staining for chondrocytes it is collagen staining Rev Oct 2010 Page 12 of 13 US PATENT 6 555 374 gt Can the adult stem cells be differentiated into any additional cell types Other researchers have differentiated adult stem cells into smooth muscle neuronal and hepatocyte lineages See selected references below Mizuno et al Myogenic differentiation by human processed lipoaspirate cells Plastic and Reconstructive Surgery 2002 109 199 209
12. riangle 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 1 of 13 US PATENT 6 555 374 CONTENTS PAGE Introduction 3 Materials Provided for Each Catalog Item 3 Media Compositions 4 Plating and Expansion of Cryopreserved Adult Stem Cells 5 Adipogenesis Protocol 6 Osteogenesis Protocol 8 Chondrogenesis Protocol 10 Troubleshooting 11 Frequently Asked Questions 12 Pathogen testing 13 Rev Oct 2010 Page 2 of 13 US PATENT 6 555 374 INTRODUCTION The adipose derived adult stem cells are isolated from subcutaneous adipose tissue of healthy non diabetic donors between 18 and 60 years old undergoing elective surgery The cells are isolated by centrifugal force after collagenase treatment Adult stem cells can be differentiated into various lineages using Zen Bio media formulations and protocols This instruction manual describes procedures to induce human adipose derived adult stem cells ASC to differentiate into 1 mature adipocytes 2 osteoblasts and 3 chondrocytes The process of differentiating human adipose tissue derived adult stem cells 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 diagno
13. s and slowly release the surface in the incubator mecina 3 Place cultureware are on a level surface in the incubator to ensure cells attach evenly Rev Oct 2010 Page 11 of 13 US PATENT 6 555 374 FREQUENTLY ASKED QUESTIONS gt Can I pass the cells Adult Stem Cells can be trypsinized and replated several times They grow slower with each passage and undergo adipogenesis poorly after passage 4 All cells are shipped at Passage 2 3 How fast do the cells replicate The average doubling time is 48 84 hours However keep in mind that the replication rate for human adult stem cells varies slightly 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 All Zen Bio media contain antibiotics and or anti fungal agents except Adipocyte Basal Medium cat BM 1 Where are the cells obtained The adult stem cells are isolated from human subcutaneous adipose tissue 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 How do I obtain RNA from the cells How much RNA can expect Use RNA Tri reagent Molecular Products RNeasy kit Qiagen or a guanidine thiocyanate solution Chomzy
14. stic 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 adult stem cell 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 Cryopreserved Human Adipose Derived Adult Stem Cells catalog ASC F Frozen vial containing either 1 0 or 2 0 x10 viable adult stem cells store in liquid nitrogen upon receipt 50 ml Preadipocyte Medium NOTE this medium is suitable as a plating medium for the adipose derived adult stem cells Rev Oct 2010 Page 3 of 13 US PATENT 6 555 374 MEDIA COMPOSTIONS Preadipocyte Medium Adipocyte Differentiation Adipocyte Maintenance cat PM 1 Medium Medium cat DM 2 cat AM 1 DMEM Ham s F 12 1 1 DMEM Ham s F 12 1 1 v v DMEM Ham s F 12 1 1 v v viv HEPES pH 7 4 HEPES pH 7 4 HEPES pH 7 4 Fetal bovine serum Fetal bovine serum Fetal bovine serum Biotin Biotin Penicillin Pantothenate Pantothenate Streptomycin Human insulin Human insulin Amphotericin B Dexamethasone Dexamethasone Isobutylmethylxanthine Penicillin PPARy a
15. t stem cells 1 wk post differentiation 2 wks post differentiation PREADIPOCYTE MATURE ADIPOCYTE nucleus Figure 1 Photographs of 100 confluent Adult stem cells ASC A 1 week old post differentiation cultured adipocytes B and mature 2 weeks post differentiation cultured Adipocytes C These are unstained photographs of human adult stem cell morphology 20X The cells should appear comparable in appearance to these pictures Rev Oct 2010 Page 7 of 13 US PATENT 6 555 374 OSTEOGENESIS PROCEDURE Differentiation of Adult Stem Cells into Osteoblasts de 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 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 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 c
16. zenbio gt Human Adult Stem Cell Manual Differentiation protocols for human adipose derived adult stem cells INSTRUCTION MANUAL 2ZBM0015 03 SHIPPING CONDITIONS Human Adult Stem Cells ASC 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 adult stem cells 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 Research T
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