StemPro ADSCs - Thermo Fisher Scientific
Contents
1. e TrypLE Express without phenol red e Reagents and equipment to determine viable and total cell counts e g Trypan Blue hemacytometer Coulter Counter or the Countess automated cell counter Passaging 1 Aspirate the Complete MesenPRO RS Medium from the Cells cells 2 Rinse the surface of the cell layer with DPBS approximately 2 ml DPBS per 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 3 Remove the DPBS by aspiration and discard 4 To detach the cells add a sufficient volume of prewarmed TrypLE Express without phenol red to cover the cell layer approximately 0 5 ml per 10 cm 5 Incubate at 37 C for approximately 7 minutes 6 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 Cells continued Procedure continued from previous page 7 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 Medium2. available separately for catalog no R7788 115 see page vi for ordering information The STEMPRO Osteogenesis Differentiation Kit Chondrogenesis Differentiation Kit and Adipogenesis Differentiation Kit provide specialized media and reagents to promote pathway specific differentiation of human MSC like cells including ADSCs in tissue culture vessels Each kit contains media and reagents for inducing MSCs to be committed to the osteogenic chondrogenic 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 chondrocytes or lipid vesicle forming adipocytes Methods General Information General Cell Follow the general guidelines below to grow and maintain Handling STEMPRO Human Adipose Derived Stem Cells e 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 e Before starting experiments ensure cells have been established at least 1 passage and also have some frozen stocks on hand e For differentiation studies and other experiments we recommend using cells below passage 5 e 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 m
3. complete medium in the dark at 2 to 8 C and use within 15 days 1 2 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 Materials Needed Thawing Procedure Follow the protocol below to thaw STEMPRO ADSCs to initiate cell culture The following materials are required see page vi for 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 15 ml conical tubes 37 C water bath 37 C incubator with a humidified atmosphere of 5 CO Tissue culture treated 35 mm dish To thaw and establish STEMPRO ADSCs 1 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 Remove the cells immediately when the last bit of ice has melted typically lt 2 minutes Do not submerge the vial
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6. C in the dark Component Final Conc For 100 ml STEMPRO Osteocyte Chondrocyte 1X 90 ml Differentiation Basal Medium STEMPRO Chondrogenesis Supplement 1X 10 ml Gentamicin 10 mg ml 5 pg ml 50 pl 18 Continued on next page Chondrogenic Differentiation Media and Methods continued MSC If growing your cells in StemPro MSC SEM or standard Attachment growth medium DMEM 10 FBS prepare MSC Medium Attachment Medium as below MSC Attachment Medium Final Conc For 100 ml DMEM low glucose 89 mL MSC qualified FBS 10 10 mL GLUTAMAX I 200 mM 2mM 1 mL Gentamicin 10 mg mL 5 pg mL 50 ul Preparing a Chondrogenic Cell Culture Observe cell monolayer from basal cultures expanded in StemPro MSC SFM MesenPRO RS medium or standard growth medium DMEM 10 FBS to ensure mid log growth phase confluence 60 to 80 Aspirate medium and floating cells from culture flask and discard Add 5 to 10 mL DPBS Gently rinse cell monolayer Remove DPBS add 5 to 7 mL of pre warmed TrypLE Express to flask and completely coat the culture surface Incubate for 5 to 8 minutes at 36 to 38 C or until cells have fully detached Gently pipet detached cells into a single cell solution and verify on inverted microscope Remove cell suspension from flask transfer into a centrifuge tube and pellet cells at 100 x g for 5 to 10 minutes Determine cell viability and total cel
7. 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 cell counter and Trypan Blue exclusion or the Countess automated cell counter If necessary 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 CO 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 Complete MesenPR
8. O 0 2 ml 3 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 Aspirate Complete MesenPRO RS Medium from the flask well or dish 2 Rinse the surface with DPBS approximately 2 ml DPBS per 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 3 Remove the DPBS by aspiration and discard 4 To detach the cells add a sufficient volume of prewarmed TrypLE Express without phenol red to cover the cell layer approximately 0 5 ml per 10 cm 5 Incubate at 37 C for approximately 7 minutes 6 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 12 Procedure continued from previous page 7 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
9. O 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 e TrypLE Express without phenol red e Reagents and equipment to determine viable and total cell counts e g Trypan Blue hemacytometer Coulter Counter or the Countess automated cell counter e Sterile freezing vials Guidelines When freezing ADSCs we recommend the following e Freeze cells at a density of 1 x 10 2 x 10 viable cells ml e Usea freezing medium composed 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 x 10 2 x 10 cells ml see the freezing procedure below 1 Ina 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 2 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 DMS
10. PRO Adipogenesis Differentiation Kit 1 kit A1007001 Gentamicin 10 mg ml 10 ml 15710 064 Dulbecco s Phosphate Buffered Saline DPBS containing no 500 ml 14190 144 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 sodium 500 ml 11054 020 pyruvate without L glutamine and phenol red Dulbecco s Modified Eagle Medium DMEM 1X high glucose with 4 5 g l D glucose and sodium pyruvate 500 ml 10313 021 without L glutamine L glutamine 200 mM liquid 100 ml 25030 081 Countess Automated Cell Counter 1 unit C10227 Continued on next page vi Additional Products continued Antibodies A variety of antibodies for characterizing ADSCs are available from Invitrogen The following table lists purified antibodies only For labeled antibodies or additional information refer to our website www invitrogen com or contact Technical Support see page 22 Item Quantity Cat no CD 31 Mouse Anti Human Purified 100 pg MHCD3100 CD 90 Purified MS X HU BioSource 100 ug AHU0051 CD 29 Mouse Anti Human Purified 100 ug CD2900 CD 14 Mouse Anti Human Purified 100 ug MHCD1400 CD 105 Mouse Anti Hu
11. ating 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 electronically using the Countess automated cell counter or a Coulter Counter or manually using a hemacytometer and an inverted microscope 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 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 6 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
12. 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 electronically using the Countess automated cell counter or a Coulter Counter or manually using a hemacytometer and an inverted microscope Gently aspirate media from the vessel and resuspend the cells to a concentration of 4 x 10 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 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 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 informatio
13. completely Do not thaw the cells for longer than 2 minutes When thawed immediately transfer cells into a 15 ml sterile conical tube and dropwise add 1 ml of prewarmed Complete MesenPRO RS Medium with gentle mixing Plate the cells 2 ml on a tissue culture treated 35 mm dish The recommended seeding density for Adipose Derived Stem Cells is 5 000 cells per cm Procedure continued on next page Continued on next page Thawing and Establishing Cells continued Thawing Procedure continued from previous page Procedure 6 Incubate at 37 C 5 CO and 90 humidity and allow continued cells to adhere for several hours or overnight 7 When the cells have attached to the growth surface replace the medium with an equal volume of fresh prewarmed Complete MesenPRO RS Medium 8 Change the medium every 34 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 e Culture vessels containing ADSCs e Tissue culture treated flasks plates or dishes e Complete MesenPRO RS Medium prewarmed to 37 C e Disposable sterile 15 ml tubes e 37 C incubator with humidified atmosphere of 5 CO e Dulbecco s Phosphate Buffered Saline DPBS containing no calcium magnesium or phenol red
14. d 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 pg 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 Aspirate 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 inver
15. 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 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 with humidified atmosphere of 5 CO e Reagents and equipment to determine viable and total cell counts e g Trypan Blue hemacytometer Coulter Counter or the Countess automated 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 an
16. edium is recommended for use with these cells for optimal growth and expansion Characteristics of STEMPRO ADSCs e Are prepared from low passage passage 1 adherent human adipose derived primary cell cultures e Express a flow cytometry cell surface protein profile positive for CD29 CD44 CD73 CD90 CD105 and CD166 gt 95 and negative for CD14 CD31 CD45 and Lin1 lt 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 SD a A ADSCs ind
17. g any warranty of merchantability or fitness for a particular purpose 23 Purchaser Notification continued Limited Use Label License No 5 Invitrogen Technology 24 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the
18. ick M H 2006 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 Inmunomodulatory 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 cli
19. id logarithmic phase prior to subculturing e When thawing or subculturing cells transfer cells into pre warmed medium e Antibiotic antimycotic containing penicillin streptomycin and amphotericin B may be used if required see page vi for ordering information It is very important to strictly follow the guidelines for culturing ADSCs in this manual to keep them Im n partant 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 Do not store the prepared complete MesenPRO RS Medium longer than 15 days Prepare Complete MesenPRO RS Medium with MesenPRO TM RS Growth Supplement and L glutamine prior to use as follows Store the
20. invitrogen STEMPRO Human Adipose Derived Stem Cells Catalog nos R7788 110 and R7788 115 A10296 Version D 13 February 2009 ii Table of Contents Contents and SOTABE cinc v Additional Produc iia A as vi Introduction cia rennen 1 MethodS umssvnnanvennnnvnnnnnnnnnnennnnvnnnnnnnnnnnnnnnnvennnnnnnnnnnnnnnnnnnnvnnnnnnnnnnnnnnnneer 4 General Information momia 4 Preparing Complete MesenPRO RS Medium uenenennennensensnnen 5 Thawing and Establishing Cells orersererrvrererrererrererrrrerrrsererrarersrsererserersesersssene 6 Subeulturing Cells cuidad dira 8 Freezing Cll iii cido 10 Osteogenic Differentiation Media and Methods enenneneenee 13 Adipogenic Differentiation Media and Methods neeeeeneene 15 Chondrogenic Differentiation Media and Methods 18 A A beozei 21 Troubleshooting iii meer 21 Technical Support cirio iii iii at aSa 22 Purchaser Notification ii ii 23 Referido ibi 25 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 below Cells 1 x 10 cells ml in freezi
21. l density electronically using the Countess automated cell counter or a Coulter Counter or manually using a hemacytometer and an inverted microscope For MesenPRO RS expansion cultures resuspend pellet in an appropriate volume of pre warmed MesenPRO RS media to generate a cell solution of 1 6 x 10 viable cells ml For STEMPRO MSC SFM or standard growth medium use MSC Attachment Medium see above to generate a cell solution of 1 6 x 10 viable cells ml Procedure continued on the next page Continued on next page 19 Chondrogenic Differentiation Media and Methods continued Preparing a Chondrogenic Cell Culture continued 20 Procedure continued from the previous page 8 10 11 Generate micromass cultures by seeding 5 ul droplets of cell solution in the center of multi well plate wells for classical stain or 100 mm Petri dish for gene expression analysis protein detection or immunohistochemistry After cultivating micromass cultures for 2 hours under high humidity conditions add warmed chondrogenesis media to culture vessels and incubate in 37 C incubator with 5 CO Refeed cultures every 2 to 3 days After specific periods of cultivation chondrogenic pellets can be processed for Alcian Blue or Safranin O staining gt 14 days gene expression analysis protein detection or immunohistochemistry Appendix Troubleshooting Culturing The table below lists some potential
22. man 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 vii Introduction Introduction STEMPRO Human Adipose Derived Stem Cells ADSCs are isolated 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 M
23. n 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 Reagents and equipment to determine viable and total cell counts e g Trypan Blue hemacytometer Coulter Counter or the Countess automated 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 C water bath and prepare Medium as below Store complete medium at 2 8 C in the dark Component Final Conc For 100 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 flo
24. ng medium R7788 110 Amount Storage STEMPRO Human Adipose Derived Stem 1ml Liquid nitrogen Cells 1 x 10 cells ml in freezing medium MesenPRO RS Basal Medium 500 ml 2 to 8 C in the dark MesenPRO RS Growth Supplement 10 ml 5 to 20 C in the dark R7788 115 Amount Storage STEMPRO Human Adipose Derived Stem 1ml Liquid nitrogen 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 website www invitrogen com or contact Technical Support see page 22 Item Quantity Cat no ere Medium includes Basal Medium and Growth 1 kit 12746 012 GlutaMAX 1 Supplement 100 ml 35050 061 STEMPRO MSC SFM 1 kit A10332 01 STEMPRO Osteogenesis Differentiation Kit 1kit A1007201 STEMPRO Chondrogenesis Differentiation Kit 1 kit A1007101 STEM
25. nical implications 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 Garcia Castro J Colmenero I Ramirez 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 2009 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 25 26 Notes 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
26. 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 x 10 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 correct page 6 and subculture procedures page 8 exactly Cells too old ADSCs above passage 5 may become differentiated 21 Technical Support Web Visit the Invitrogen
27. 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 terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies 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 Life Technologies 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 Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com 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 S33 37 Fraser J K Wulur I Alfonso Z and Hedr
28. s gene expression analysis or protein detection 17 Chondrogenic Differentiation Media and Methods Introduction Materials Needed STEMPRO Chondro genesis Differentiation Kit This section provides media preparation guidelines and a protocol for inducing STEMPRO ADSCs to differentiate towards chondrocytes using the STEMPRO Chondrogenesis Differentiation Kit The following materials are required see page vi for ordering information e STEMPRO Chondrogenesis 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 Reagents and equipment to determine viable and total cell counts e g Trypan Blue hemacytometer Coulter Counter or the Countess automated cell counter The STEMPRO Chondrogenesis Differentiation Kit provides specialized media and reagents for chondrogenic differentiation of ADSCs in tissue culture vessels See the insert provided with the kit for detailed information and protocols Complete To prepare Complete STEMPRO Chodrogenesis Differentiation Chondro Medium thaw the STEMPRO Chondrogenesis Supplement at genesis 4 C room temperature or in a 37 C water bath and prepare Differentiation as below Medium Store complete medium at 2 8
29. ted 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 electronically using the Countess automated cell counter or a Coulter Counter or manually using a hemacytometer and an inverted microscope 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 6 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 Adipogenesis Cell Culture Differentiation Medium and continue incubation ADSCs continued 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 day
30. uced 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 red 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
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