StemPro ADSCs - Thermo Fisher Scientific
Contents
1. C water bath and prepare Medium as below Store complete medium at 2 8 C in the dark Component Final Conc For100 ml STEMPRO Osteocyte Chondrocyte 1X 90 ml Differentiation Basal Medium STEMPRO Osteogenesis Supplement 1X 10 ml Gentamicin 10 mg ml 5 pg ml 50 pl Continued on next page 13 Osteogenic Differentiation Media and Methods continued Preparing an Osteogenic Cell Culture 14 10 11 Observe the ADSC monolayer to ensure mid log growth phase confluence 60 80 Aspirate the medium and floating cells from the culture flask and discard Add 5 10 ml DPBS to the flask Gently rinse the cell monolayer Remove DPBS and add 5 7 ml of pre warmed TrypLE Express to the flask and completely coat the culture surface Incubate for 5 8 minutes at 36 38 C or until cells have fully detached Gently pipet detached cells into a single cell solution and verify on inverted microscope Remove the cell suspension from the flask transfer into a centrifuge tube and pellet cells at 100 x g for 5 10 minutes Determine cell viability and total cell density using Trypan Blue Stain and electronic i e Coulter Counter or manual i e hemocytometer cell counting method Resuspend the pellet in an appropriate volume of pre warmed Complete MesenPRO RS Medium Seed the ADSCs into culture vessels at 5 x 10 cells cm For classical stain differentiation assays seed into a 12 well pla2. in tissue culture vessels Each kit contains media and reagents for inducing MSCs to be committed to the osteogenic or adipogenic pathway Using STEMPRO Differentiation Kits in combination with MesenPRO RS Medium or STEMPRO MSC SEM provides a standardized culture workflow solution for MSC isolation expansion and differentiation into matrix forming osteoblasts or lipid vesicle forming adipocytes Methods General Information General Cell Handling Important Follow the general guidelines below to grow and maintain STEMPRO Human Adipose Derived Stem Cells All solutions and equipment that come in contact with the cells must be sterile Always use proper sterile technique and work in a laminar flow hood Before starting experiments ensure cells have been established at least 1 passage and also have some frozen stocks on hand For differentiation studies and other experiments we recommend using cells below passage 5 For general maintenance of cells cell confluency should be 60 80 cell viability should be at least 90 and the growth rate should be in mid logarithmic phase prior to subculturing When thawing or subculturing cells transfer cells into pre warmed medium Antibiotic antimycotic containing penicillin streptomycin and amphotericin B may be used if required see page vi for ordering information Itis very important to strictly follow the guidelines for culturing ADSCs in this manual
3. Adipogenesis Cell Culture Differentiation Medium and continue incubation continued ADSCS will continue to undergo limited expansion as they differentiate under adipogenic conditions Refeed cultures every 3 4 days 11 After specific periods of cultivation adipogenic cultures can be processed for Oil Red O or LipidTOX staining beginning at 7 14 days gene expression analysis or protein detection 17 Chondrogenic Differentiation Media and Methods Introduction This section provides media preparation guidelines and a protocol for inducing STEMPRO ADSCs to differentiate towards chondrocytes using published recipes Mackay et al 1998 Pittenger et al 1999 Materials The following materials are required see page vi for Needed ordering information Chondrogenic differentiation medium see below Culture vessels containing ADSCs DPBS without Ca and Mg TrypLE Express without phenol red Tissue culture treated vessels Disposable sterile 15 ml tubes 37 C incubator with humidified atmosphere of 5 CO Hemacytometer or cell counter Chondrogenic Chondrogenic Differentiation Medium is prepared as Differentiation follows Store the prepared medium in the dark at 2 to 8 C CD Medium Component Source Cat no Volume Conc DMEM low glucose without Invitrogen 11054 020 100 ml L glutamine and phenol red 200 mM L glutamine Invitrogen 25030 081 1
4. The next day transfer the frozen vials to a liquid nitrogen tank for long term storage Note You may check the viability and recovery of frozen cells 24 hours after storing cryovials in liquid nitrogen by following the procedure outlined in Thawing and Establishing Cells page 6 12 Osteogenic Differentiation Media and Methods Introduction Materials Needed StemPro Osteogenesis Differentiation Kit This section provides media preparation guidelines and a protocol for inducing STEMPRO ADSCs to differentiate towards osteoblasts using the STEMPRO Osteogenesis Differentiation Kit The following materials are required see page vi for ordering information e STEMPRO Osteogenesis Differentiation Kit e Gentamicin 10 mg ml e Culture vessels containing ADSCs e DPBS without Ca and Mg e TrypLE Express without phenol red e Tissue culture treated vessels e Disposable sterile 15 ml tubes e 37 C incubator with humidified atmosphere of 5 CO e Hemacytometer or cell counter The STEMPRO Osteogenesis Differentiation Kit provides specialized media and reagents for osteogenic differentiation of ADSCs in tissue culture vessels See the insert provided with the kit for detailed information and protocols Preparing To prepare Complete STEMPRO Osteogenesis Differentiation Complete Medium thaw the STEMPRO Osteogenesis Supplement at Differentiation 4 C room temperature or in a 37
5. for CD29 CD44 CD73 CD90 CD105 and CD166 gt 95 and negative for CD14 CD31 CD45 and Lin1 2 e Contain cells characteristic of at least bi potential differentiation Continued on next page Introduction continued Isolation and Expansion Differentiation Potential Differentiation into Mesenchymal Cell Types ADSCs are extracted from human adipose tissue through mechanical and enzymatic digestion Cells are expanded using MesenPRO RS Medium which supports a much shorter cell doubling time 36 4 hours than traditional medium DMEM 10 FBS resulting in a cell doubling time of 54 4 hours ADSCs can be expanded to 4 5 passages before they lose their ability to grow or differentiate into all potential phenotypes Multiple investigators have demonstrated that ADSCs can be differentiated towards multiple mature cell phenotypes In addition to traditional mesenchymal lineages ADSCs have been differentiated towards cardiomyocytic pancreatic epithelial and other phenotypes using specialized media The images below show the differentiation of ADSCs into mesenchymal cell types A ADSCs induced to differentiate towards chondrocytes for 29 days and then stained with safranin orange dye pellet cross sectional staining for proteoglycan content image captured using 4x objective lens B ADSCs induced to differentiate towards osteoblasts for 29 days and then stained with alizarin r
6. to keep them undifferentiated As with other human cell lines when working with ADSCs handle as potentially biohazardous material under at least Biosafety Level 1 containment Preparing Complete MesenPRO RS Medium Introduction Materials Needed Note Preparing Complete MesenPRO RS Medium Follow the instruction in this section for preparing Complete MesenPRO RS Medium The following materials are required MesenPRO RS Basal Medium and MesenPRO RS Growth Supplement included with catalog no R7788 110 and available separately for catalog no R7788 115 see page vi for ordering information L glutamine 200 mM liquid see page vi for ordering information Store all media components in the dark Thaw MesenPRO RS Growth Supplement at 2 to 8 C prior to use Avoid repeated freeze thaw cycles of the supplement TM Do not store the prepared complete MesenPRO RS Medium longer than 15 days Prepare Complete MesenPRO RS Medium with MesenPRO RS Growth Supplement and L glutamine prior to use as follows Store the complete medium in the dark at 2 to 8 C and use within 15 days 1 Aseptically add 10 ml of MesenPRO RS Growth Supplement to 500 ml of MesenPRO RS Basal Medium Aseptically add L glutamine to the medium to a final concentration of 2 mM e g add 5 ml of 200 mM L glutamine stock to 500 ml of medium Thawing and Establishing Cells Introduction Follow the protocol be
7. with humidified atmosphere of 5 CO e Hemacytometer or cell counter The STEMPRO Adipogenesis Differentiation Kit provides specialized media and reagents for adipogenic differentiation of ADSCs in tissue culture vessels See the insert provided with the kit for detailed information and protocols To prepare the complete medium thaw the supplement in a 37 2 C water bath swirl and warm the supplement to promote dissolution of the precipitate see Note on the following page and prepare the medium as described in the table below Store complete medium at 2 8 C in the dark Component Final Conc For 100 ml STEMPRO Adipocyte Differentiation 1X Basal Medium 90 ml STEMPRO Adipogenesis Supplement 1X 10 ml Gentamicin 10 mg ml 5 ug ml 50 ul Continued on next page 15 Adipogenic Differentiation Media and Methods continued Note Preparing an Adipogenic Cell Culture 16 It is normal to see a precipitate formed in the supplement after thawing To promote dissolution of the precipitate warm the supplement with swirling for no more than 30 minutes prior to preparing complete media Any remaining precipitate should be suspended in solution before it is added to STEMPRO Adipocyte Differentiation Basal Medium and will dissolve completely when mixed with the Basal Medium and warmed Observe the ADSC monolayer to ensure mid log growth phase confluence 60 80 Aspira
8. 0 cm Incubate at 37 C for approximately 7 minutes Observe the cells under a microscope If the cells are less than 90 detached continue incubating and observe within 2 minutes for complete detachment of the cells Tap the vessel to expedite cell detachment Procedure continued on next page Continued on next page 11 Freezing Cells continued Freezing Cells Procedure continued from previous page Procedure 7 continued 10 11 12 13 14 15 When 290 of the cells have detached tilt the vessels on end for a minimal length of time to allow the cells to drain Add the equivalent of 2 volumes twice the volume used for the TrypLE Express of temperature equilibrated Complete MesenPRO RS Medium to each vessel Disperse the medium by pipetting over the cell layer surface several times Transfer the cells to a 15 ml conical tube and centrifuge at 210 x g for 5 minutes at room temperature Resuspend the cell pellet in a minimal volume of temperature equilibrated Complete MesenPRO RS Medium and remove a sample for counting Determine the total number of cells using a hemacytometer or cell counter Gently aspirate media from the vessel and resuspend the cells to a concentration of 4 x 106 cells ml in Freezing Medium A Add the same volume of Freezing Medium B to cells in a dropwise manner Aliquot 1 ml to each freezing vial and store at 80 C overnight in an isopropanol chamber
9. 6 Fat tissue an underappreciated source of stem cells for biotechnology Trends Biotechnol 24 150 154 Mackay A M Beck S C Murphy J M Barry F P Chichester C O and Pittenger M F 1998 Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow Tissue Eng 4 415 428 Pittenger M F Mackay A M Beck S C Jaiswal R K Douglas R Mosca J D Moorman M A Simonetti D W Craig S and Marshak D R 1999 Multilineage potential of adult human mesenchymal stem cells Science 284 143 147 Puissant B Barreau C Bourin P Clavel C Corre J Bousquet C Taureau C Cousin B Abbal M Laharrague P Penicaud L Casteilla L and Blancher A 2005 Immunomodulatory effect of human adipose tissue derived adult stem cells Comparison with bone marrow mesenchymal stem cells Br J Haematol 129 118 129 Rehman J Traktuev D Li J Merfeld Clauss S Temm Grove C J Bovenkerk J E Pell C L Johnstone B H Considine R V and March K L 2004 Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells Circulation 109 1292 1298 Safford K M and Rice H E 2005 Stem cell therapy for neurologic disorders Therapeutic potential of adipose derived stem cells Current Drug Targets 6 57 62 56 Sch ffler A and B chler C 2007 Concise review adipose tissue derived stromal cells basic and clinical implication
10. 90 144 no calcium magnesium or phenol red Fetal Bovine Serum MSC Qualified 100 ml 12662 011 TrypLE Express without phenol red 100 ml 12604 013 Antibiotic Antimycotic 100X liquid 100 ml 15240 062 Dulbecco s Modified Eagle Medium DMEM 1X low glucose with 1 000 mg l D glucose and 110 mg l 500 ml 11054 020 sodium pyruvate without L glutamine and phenol red L glutamine 200 mM liquid 100 ml 25030 081 Dulbecco s Modified Eagle Medium DMEM 1X high glucose with 4 5 g 1 D glucose and sodium pyruvate 500 ml 10312 021 without L glutamine Antibodies A variety of antibodies for characterizing ADSCs are available from Invitrogen The following table lists catalog numbers for purified antibodies only For labeled antibodies or additional information refer to our Web site www invitrogen com or contact Technical Support see page 21 Item Quantity Catalog no CD 31 Mouse Anti Human Purified 100 pg MHCD3100 CD 90 Purified MS X HU BioSource 100 ug AHUO051 CD 29 Mouse Anti Human Purified 100 ug CD2900 CD 14 Mouse Anti Human Purified 100 ug MHCD1400 CD 105 Mouse Anti Human Purified 100 ug MHCD10500 CD 44 Mouse Anti Human Purified 100 ug MHCD4400 CD 45 Mouse Anti Human Purified 100 ug MHCD4500 CD 73 Host Mouse Clone 7G2 100 ug 41 0200 vi Introduction Introduction STEMPRO Human Adipose Derived Stem Cells ADSCs are isol
11. ated from human adipose tissue collected during liposuction procedures and cryopreserved from primary cultures Before cryopreservation the ADSCs are expanded for one passage in MesenPRO RS Medium Each lot of ADSCS originates from a single donor of human lipoaspirate tissue Each vial of ADSCs contains cells that can differentiate into multiple mature cell phenotypes in vitro including adipocytes osteoblasts and chondrocytes Fraser amp Schreiber et al 2006 Fraser amp Wulur et al 2006 Sch ffler amp B chler 2007 Strem et al 2005 In vitro differentiation into non mesenchymal cell types such as neuronal and glial progenitors hepatocytes and vascular endothelial progenitors have also been described Rehman et al 2004 Safford amp Rice 2005 Strem et al 2005 In addition ADSCs are known to secrete pro angiogenic immunomodulatory and anti apoptotic factors Puissant et al 2005 Rehman et al 2004 Yafiez et al 2006 ADSCs can be used for studies of adult stem cell differentiation tissue engineering and potential future clinical applications They may also be used for the delivery of recombinant DNA constructs MesenPRO RS Medium is recommended for use with these cells for optimal growth and expansion Characteristics of STEMPRO ADSCs Are prepared from low passage passage 1 adherent human adipose derived primary cell cultures e Express a flow cytometry cell surface protein profile positive
12. d of final concentrations of 20 Fetal Bovine Serum MSC Cell qualified and 10 DMSO e Bring the cells into freezing medium in two steps as described in this section Continued on next page 10 Freezing Cells continued Preparing Freezing Media Freezing Cells Procedure Prepare Freezing Medium A and B immediately before use You will need enough of each freezing medium to resuspend cells at a density of 1 2 x 106 cells ml see the freezing procedure below 1 In a sterile 15 ml tube mix together the following reagents for every 1 ml of Freezing Medium A needed Complete MesenPRO RS Medium 0 6 ml Fetal Bovine Serum MSC Qualified 0 4 ml In another sterile 15 ml tube mix together the following reagents for every 1 ml of Freezing Medium B needed Complete MesenPRO RS Medium 0 8 ml DMSO 0 2 ml Place tube with Freezing Medium B on ice until use leave Freezing Medium A at Room Temperature Note Discard any remaining freezing medium after use m Aspirate Complete MesenPRO RS Medium from the flask well or dish Rinse the surface with DPBS approximately 2 ml DPBS 10 cm culture surface area by adding the DPBS to the side of the vessel opposite the attached cell layer and rocking back and forth several times Remove the DPBS by aspiration and discard To detach the cells add a sufficient volume of prewarmed TrypLE Express without phenol red to cover the cell layer approx 0 5 m1 1
13. de of the vessel opposite the attached cell layer and rocking back and forth several times Remove the DPBS by aspiration and discard To detach the cells add a sufficient volume of prewarmed TrypLE Express without phenol red to cover the cell layer approx 0 5 ml 10 cm Incubate at 37 C for approximately 7 minutes Observe the cells under a microscope If the cells are less than 90 detached continue incubating and observe within 2 minutes for complete detachment of the cells Tap the vessel to expedite cell detachment Procedure continued on next page Continued on next page Subculturing Cells continued Passaging Procedure continued from previous page Cells 7 continued 10 11 12 13 14 15 When gt 90 of the cells have detached tilt the vessel for a minimal length of time to allow the cells to drain Add the equivalent of 2 volumes twice the volume used for the TrypLE Express of temperature equilibrated Complete MesenPRO RS Medium Disperse the medium by pipetting over the cell layer surface several times Transfer the cells to a 15 ml conical tube and centrifuge at 210 x g for 5 minutes at room temperature Resuspend the cell pellet in a minimal volume of temperature equilibrated Complete MesenPRO RS Medium and remove a sample for counting Determine the total number of cells and percent viability using a hemacytometer or cell counter and Trypan Blue exclusion If nece
14. ed dye which stains mineralized extracellular matrix image captured using 4x objective lens C ADSCs induced to differentiate towards adipocytes for 14 days and then stained with oil red O which stains lipid vacuoles and counterstained with hematoxylin image captured using 10x objective lens Continued on next page Introduction continued MesenPRO RS Medium STEMPRO Differentiation Kits MesenPRO RS Basal Medium and Growth Supplement have been developed for the growth and expansion of human mesenchymal stem cell like cells including ADSCs in tissue culture vessels Complete MesenPRO RS Medium is a reduced serum medium 2 FBS for reduced MSC doubling times improved MSC expansion and improved multilineage differentiation capability Complete MesenPRO RS Medium provides the following advantages for culturing human ADSCs e Consistently improves expansion compared to traditional medium DMEM 10 FBS e Maintains multilineage differentiation capabilities e Eliminates time and money spent pre qualifying FBS lots MesenPRO RS Basal Medium and Growth Supplement are included with catalog no R7788 110 and are available separately for catalog no R7788 115 see page vi for ordering information The STEMPRO Osteogenesis Differentiation Kit and Adipogenesis Differentiation Kit provide specialized media and reagents to promote pathway specific differentiation of human MSC like cells including ADSCs
15. elow STEMPRO Human Adipose Derived Stem Cells 1 x 106 cells ml in freezing medium dark MesenPRO RS Growth Supplement 10 ml 5 to 20 C in the dark R7788 115 Amount Storage STEMPRO Human Adipose Derived Stem 1 ml Liquid nitrogen Cells 1 x 106 cells ml in freezing medium Handle cells as potentially biohazardous material under at least Biosafety Level 1 containment This product contains Dimethyl Sulfoxide DMSO a hazardous material Review the Material Safety Data Sheet MSDS before handling Product The Certificate of Analysis provides detailed quality control Qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Additional Products Additional The products listed in this section may be used with STEMPRO Products Human Adipose Derived Stem Cells For more information refer to our Web site www invitrogen com or contact Technical Support see page 21 Item Quantity Catalog no Eu c includes Basal Medium and l kit 12746 012 L glutamine 200 mM liquid 100 ml 25030 081 STEMPRO Osteogenesis Differentiation Kit 1 kit A10072 01 STEMPRO Adipogenesis Differentiation Kit 1 kit A10070 01 Gentamicin 10 mg ml 10 ml 15710 064 Dulbecco s Phosphate Buffered Saline DPBS containing 500 ml 141
16. invitrogen STEMPRO Human Adipose Derived Stem Cells Catalog nos R7788 110 and R7788 115 A10296 Version C 25 July 2008 ii Table of Contents Contents and Storage csetera nace rete thee ten v Additional Products iet RH vi Introduction hti n tee a Ee eie A anu denies 1 Methods P C 4 General Information 2 n iterare ree iter o de tee eod 4 Preparing Complete MesenPRO RS Medium sss 5 Thawing and Establishing Cells seen 6 Subc lturing Cells rrt tette tice cre teet ner 8 Freezing Cells teet eerte tts 10 Osteogenic Differentiation Media and Methods sss 13 Adipogenic Differentiation Media and Methods sss 15 Chondrogenic Differentiation Media and Methods 18 aale e 112 quc PE 20 Troubleshooting eye e ROO RI AR HR ERRARE tees 20 Technical Support hotte teen eene tette tbe 21 Purchaser Notification ec Re iie i etes 22 References ctt tei rU C e EBD cec El Cedere tovt 24 iv Contents and Storage Kit Catalog no R7788 110 includes cells plus media Configurations Catalog no R7788 115 includes cells only Shipping STEMPRO Human Adipose Derived Stem Cells and MesenPRO RS Growth Supplement are shipped on dry ice MesenPRO RS Basal Medium is shipped at room temperature Kit Contents Kit components and storage conditions for R7788 110 and and Storage R7788 115 are listed in the table b
17. l Support Web Resources Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters European Headquarters Invitrogen Corporation Invitrogen Ltd 5791 Van Allen Way Inchinnan Business Park Carlsbad CA 92008 USA 3 Fountain Drive Tel 1 760 603 7200 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Tech Fax 44 0 141 814 6117 E mail tech_support Invitrogen com E mail eurotech invitrogen com Material Safety Data Sheets MSDSs Certificate of Analysis MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds The Certificate of Analysis provides detailed quality control information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box 21 Purchaser Notification Limited Warranty 22 In
18. low to thaw STEMPRO ADSCS to initiate cell culture Materials The following materials are required see page vi for Needed ordering information STEMPRO Human Adipose Derived Stem Cells stored in liquid nitrogen Ethanol or isopropanol Prepared Complete MesenPRO RS Medium see previous page prewarmed to 37 C Disposable sterile 50 ml tubes 37 C water bath 37 C incubator with a humidified atmosphere of 5 CO Microcentrifuge Tissue culture treated 35 mm dish Thawing To thaw and establish STEMPRO ADSCs Procedure 1 9 6 Prewarm prepared Complete MesenPRO RS Medium to 37 C Remove the cells from liquid nitrogen storage and wipe the cryovial with ethanol or isopropanol before opening In an aseptic field briefly twist the cap a quarter turn to relieve pressure and then retighten Do not expose cells to air before thawing Quickly thaw the vial of cells by swirling it in a 37 C water bath and removing it when the last bit of ice has melted typically lt 2 minutes Do not submerge the vial completely Do not thaw the cells for longer than 2 minutes When thawed immediately transfer cells into a 50 ml sterile tube and add prewarmed Complete MesenPRO TM RS Medium dropwise up to 10 ml Centrifuge cells for 5 minutes at 210 x g Aspirate supernatant Procedure continued on next page Continued on next page Thawing and Establishing Cells continued Thawing Procedure conti
19. lturing The table below lists some potential problems and solutions that Cells help you troubleshoot your cell culture problems Problem Cause Solution No viable Stock not stored Order new stock and store in liquid cells after thawing stock correctly nitrogen Keep in liquid nitrogen until thawing Home made stock not viable Freeze cells at a density of 1 2 x 106 viable cells ml Use low passage cells to make your own stocks Follow procedures in Freezing Cells page 10 exactly Slow freezing and fast thawing is the key Add Freezing Medium B drop wise manner slowly At time of thawing thaw quickly and do not expose vial to the air but quickly change from nitrogen tank to 37 C water bath Obtain new STEMPRO ADSCs Thawing medium not correct Use prewarmed Complete MesenPRO RS Medium prepared as described on page 5 Cells too diluted Generally we recommend thawing one vial in a 35 mm dish at a density of 5 000 cells per cm Cells grow Growth medium Use prewarmed Complete MesenPRO RS slowly not correct Medium Cells too old Use healthy ADSCs under passage 5 do not overgrow Cells Culture Thaw and culture fresh vial of STEMPRO differentiated conditions not ADSCs Follow thawing instructions page correct 6 and subculture procedures page 8 exactly Cells too old ADSCs above passage 5 may become differentiated 20 Technica
20. ml 2mM Insulin Transferrin Selenium BD 354352 1ml 1X Plus Biosciences 50 mM L ascorbic acid Sigma A8960 5G 100 ul 50 uM 40 mg ml L proline Sigma P5607 100ul 40 ug ml 100 uM dexamethasone Sigma D8893 100 ul 0 1 uM 10 ug ml recombinant human Invitrogen PHG9304 100 ul 10 ng ml TGF beta 3 BioSource 10 mg ml gentamicin Invitrogen 15710 064 50 ul 5 ug ml 18 Continued on next page Chondrogenic Differentiation Media and Methods continued Preparing a Chondrogenic Cell Culture 1 Detach cells using TrypLE Express and perform a cell count as described in Passaging Cells pages 8 9 through Step 10 Resuspend the cells in DMEM low glucose with 10 MSC qualified FBS to a concentration of 8 x 106 cells ml To six wells in a 12 well tissue culture dish spot 10 ul of cells per well Incubate for two hours at 37 C 596 C0 and 9096 humidity To three of the spotted wells add 1 ml of DMEM low glucose with 10 MSC qualified FBS To the other three wells add 1 ml of Chondrogenic Differentiation Medium prepared as described above Incubate at 37 C 5 C0 and 90 humidity Refeed cultures every three to four days with same media prepared at the initiation of differentiation Check for chondrogenesis after a set period of cultivation You can perform alcian blue staining to detect glycosaminoglycans or collagen 2a immunostaining after 28 days 19 Appendix Troubleshooting Cu
21. nued 7 10 Procedure continued from previous page TM Resuspend cells in Complete MesenPRO RS Medium 2 ml for a 35 mm dish and plate the resuspended cells The recommended seeding density for Adipose Derived Stem Cells is 5 000 cells per cm Incubate at 37 C 5 C0 and 90 humidity and allow cells to adhere for several hours or overnight When the cells have attached to the growth surface replace the medium with an equal volume of fresh TM prewarmed Complete MesenPRO RS Medium Change the medium every 3 4 days Subculturing Cells Introduction Follow the protocol below to culture ADSCs Subculture cells when needed before colonies start contacting each other typically every 10 14 days Materials The following materials are required see page vi for Needed ordering information Culture vessels containing ADSCs Tissue culture treated flasks plates or dishes Complete MesenPRO RS Medium prewarmed to 37 C Disposable sterile 15 ml tubes 37 C incubator with humidified atmosphere of 5 CO Dulbecco s Phosphate Buffered Saline DPBS containing no calcium magnesium or phenol red TrypLE Express without phenol red Hemacytometer or cell counter Trypan blue Passaging 1 Cells 2 5 Aspirate the Complete MesenPRO RS Medium from the cells Rinse the surface of the cell layer with DPBS approximately 2 ml DPBS 10 cm culture surface area by adding the DPBS to the si
22. prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the most restrictive terms apply Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com 23 References Fraser J K Schreiber R Strem B Zhu M Alfonso Z Wulur I and Hedrick M H 2006 Plasticity of human adipose stem cells toward endothelial cells and cardiomyocytes Nat Clin Pract Cardiovasc Med 3 533 37 Fraser J K Wulur I Alfonso Z and Hedrick M H 200
23. s for novel cell based therapies Stem Cells 25 818 827 Strem B M Hicok K C Zhu M Wulur I Alfonso Z Schreiber R E Fraser J K and Hedrick M H 2005 Multipotential differentiation of adipose tissue derived stem cells Keio J Med 54 132 141 Yafiez R Lamana M L Garc a Castro J Colmenero I Ram rez M and Bueren J A 2006 Adipose tissue derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft versus host disease Stem Cells 24 2582 2591 2007 2008 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 24 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
24. ssary add Complete MesenPRO RS Medium to the cells to achieve the desired cell concentration and recount the cells Determine the total number of vessels to inoculate by using the following equation Number of vessels Number of viable cells growth area of vessel in cm x 5 000 cells per cm recommended seeding density Add Complete MesenPRO RS Medium to each vessel so that the final culture volume is 0 2 0 5 ml per cm Add the appropriate volume of cells to each vessel and incubate at 37 C 5 C0 and 90 humidity Three to four days after seeding completely remove the medium Replace with an equal volume of Complete MesenPRO RS Medium Freezing Cells Introduction Guidelines and procedures for preparing freezing medium and freezing cells are provided in this section Materials The following materials are required see page vi for Needed ordering information e Culture vessels containing ADSCs e Complete MesenPRO RS Medium e Fetal Bovine Serum MSC Qualified e DMSO use a bottle set aside for cell culture open only in a laminar flow hood e Disposable sterile 15 ml conical tubes e DPBS containing no calcium magnesium or phenol red TM e TrypLE Express without phenol red e Hemacytometer or cell counter e Sterile freezing vials Guidelines When freezing ADSCs we recommend the following e Freeze cells at a density of 1 2 x 10 viable cells ml e Use a freezing medium compose
25. te For gene expression profile studies seed into a T 75 flask For immunocytochemistry studies seed into a 16 well CultureWell chambered coverglass or 96 well plate Incubate in Complete MesenPRO RS Medium at 36 38 C ina humidified atmosphere of 46 CO for a minimum of 2 hours up to 4 days Replace media with pre warmed Complete STEMPRO Osteogenesis Differentiation Medium and continue incubation ADSCs will continue to expand as they differentiate under osteogenic conditions Refeed cultures every 3 4 days After specific periods of cultivation osteogenic cultures can be processed for alkaline phosphatase staining 7 14 days or Alizarin Red S staining gt 21 days gene expression analysis or protein detection Adipogenic Differentiation Media and Methods Introduction Materials Needed StemPro amp Adipogenesis Differentiation Kit Complete Adipogenic Differentiation Medium This section provides media preparation guidelines and a protocol for inducing STEMPRO ADSCs to differentiate towards adipocytes using the STEMPRO Adipogenesis Differentiation Kit The following materials are required see page vi for ordering information e STEMPRO Adipogenesis Differentiation Kit e Gentamicin 10 mg ml e Culture vessels containing ADSCs e DPBS without Ca and Mg e TrypLE Express without phenol red e Tissue culture treated vessels e Disposable sterile 15 ml tubes e 37 C incubator
26. te the medium and floating cells from culture flask and discard Add 5 10 ml DPBS Gently rinse the cell monolayer Remove the DPBS and add 5 7 ml of pre warmed TrypLE Express to the flask and completely coat the culture surface Incubate for 5 8 minutes at 36 38 C or until cells have fully detached Gently pipet the detached cells into a single cell solution and verify on inverted microscope Remove the cell suspension from the flask transfer into a centrifuge tube and pellet cells at 100 x g for 5 10 minutes Determine cell viability and total cell density using Trypan Blue Stain and electronic i e Coulter Counter or manual i e hemocytometer cell counting method Resuspend the pellet in an appropriate volume of pre warmed Complete MesenPRO RS Medium Seed the ADSCs into culture vessels at 1 x 10 cells cm For classical stain differentiation assay seed into a 12 well plate For gene expression profile studies seed into a T 75 flask For immunocytochemistry studies seed into a 16 well CultureWell chambered coverglass or 96 well plate Incubate in Complete MesenPRO RS Medium at 36 38 C in a humidified atmosphere of 4 676 CO for a minimum of 2 hours up to 4 days Procedure continued on the next page Continued on next page Adipogenic Differentiation Media and Methods continued Preparing an Procedure continued from the previous page Adipogenic 10 Replace media with pre warmed Complete
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