StemPro® Alk Phos-expressing Rat
Contents
1. Follow the general guidelines below to grow and maintain StemPro Alk Phos expressing Rat Mesenchymal Stem Cells e All solutions and equipment that come in contact with the cells must be sterile Always use proper aseptic 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 mid 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 As with other mammalian cell lines when working with MSCs handle as potentially biohazardous material under at least Biosafety Level 1 BL 1 containment For more information on BL 1 guidelines refer to Biosafety in Microbiological and Biomedical Laboratories 4 ed published by the Centers for Disease Control or see the following website www cdc gov od ohs biosfty bmbl4 bmbl4toc htm It is very important to strictly follow the guidelines for culturing StemPro Alk Phos expressing Rat Mesenchymal Stem Cells in this manual to2. Bony C Tropel P Apparailly F Sany J Noel D and Jorgensen C 2003 Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals Blood 102 3837 3844 Gojo S Gojo N Takeda Y Mori T Abe H Kyo S Hata J and Umezawa A 2003 In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells Exp Cell Res 288 51 59 Han S S Kang D Y Mujtaba T Rao M S and Fischer I 2002 Grafted lineage restricted precursors differentiate exclusively into neurons in the adult spinal cord Exp Neurol 177 360 375 Han S S Liu Y Tyler Polsz C Rao M S and Fischer I 2004 Transplantation of glial restricted precursor cells into the adult spinal cord survival glial specific differentiation and preferential migration in white matter Glia 45 1 16 Houghton J Stoicov C Nomura S Rogers A B Carlson J Li H Cai X Fox J G Goldenring J R and Wang T C 2004 Gastric cancer originating from bone marrow derived cells Science 306 1568 1571 Kinnaird T Stabile E Burnett M S Shou M Lee C W Barr S Fuchs S and Epstein S E 2004 Local delivery of marrow derived stromal cells augments collateral perfusion through paracrine mechanisms Circulation 109 1543 1549 Kisseberth W C Brettingen N T Lohse J K and Sandgren E P 1999 Ubiquitous expression of marker transgenes in mice and rats D
3. allow the cells to drain Add the equivalent of 2 volumes twice the volume used for the TrypLE Express of pre warmed complete a MEM 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 300 x 2 for 5 minutes at room temperature Aspirate the medium used for washing the cells step 7 Resuspend the cell pellet in a minimal volume of pre warmed complete a MEM medium and remove a sample for counting Determine the total number of cells using your method of choice 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 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 Differentiation Media Introduction Mesenchymal Stem Cell Basal Medium Osteogenic Differentiation Medium One critical hallmark of MSCs is their ability to differentiate into three or more mature cell types Traditional and modern bioassays are used to demonstrate the multipotency of MSC
4. at least Biosafety Level 1 BL 1 containment This product contains Dimethyl Sulfoxide DMSO a hazardous material Review the Material Safety Data Sheet MSDS before handling Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds StemPro Alk Phos expressing Rat Mesenchymal Stem Cells are genetically modified and carry a chromosomal human Alkaline Phosphatase gene As a condition of sale this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms Additional Products Additional The products listed in this section may be used with StemPro Products Alk Phos expressing Rat Mesenchymal Stem Cells For more information refer to our website www invitrogen com or contact Technical Support see page 21 Ttem Quantity Cat no Minimum Essential Medium MEM a Medium 1X with GlutaMAX I ribonucleosides and deoxyribonucleosides m 32571 036 GlutaMAX I Supplement 100 ml 35050 061 Fetal Bovine Serum FBS MSC Qualified 100 ml 12662 011 500 ml 12662 029 StemPro Adipogenesis Differentiation Kit 100 ml A10070 01 StemPro Chondrogenesis Differentiation Kit 100 ml A10071 01 StemPro Osteogenesis Differentiation Kit 100 ml A10072 01 Gentamicin 10 mg ml 10 ml 15710 064 Dulbecco s Phosphate Buffered Saline DPBS containing 500 ml 14190 14
5. 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 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com 23 References Anjos Afonso F Siapati E K and Bonnet D 2004 In vivo contribution of murine mesenchymal stem cells into multiple cell types under minimal damage conditions J Cell Sci 117 5655 5664 Bruder S P Jaiswal N and Haynesworth S E 1997 Growth kinetics self renewal and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation J Cell Biochem 64 278 294 De Ugarte D A Morizono K Elbarbary A Alfonso Z Zuk P A Zhu M Dragoo J L Ashjian P Thomas B Benhaim P Chen I Fraser J and Hedrick M H 2003 Comparison of multi lineage cells from human adipose tissue and bone marrow Cells Tissues Organs 174 101 109 Deng W Obrocka M Fischer I and Prockop D J 2001 In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP Biochem Biophys Res Commun 282 148 152 Djouad F Plence P
6. remove the medium Replace with an equal volume of complete a MEM 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 ordering Needed information e Culture vessels containing StemPro Alk Phos expressing Rat MSCs e a MEM 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 e Hemacytometer cell counter and Trypan Blue LIVE DEAD Cell Vitality Assay Kit or the Countess Automated Cell Counter e Sterile freezing vials Guidelines When freezing MSCs we recommend the following e Freeze cells at a density of 1 2 x 10 viable cells ml e Usea freezing medium composed of final concentrations of 20 MSC Qualified FBS 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 10 cells ml see the freezing procedure below 1 In a sterile 15 ml tube mix toget
7. within 2 minutes for complete detachment of the cells Gently tap the vessel to expedite cell detachment Spin for 5 minutes at 300 x g at room temperature While the cells are spinning perform a viable cell count using your method of choice note total cell number Calculate required amount of MSC basal medium to obtain the appropriate seeding concentration see below Resuspend cells in the appropriate amount of MSC basal medium Dispense cell solution according to differentiation condition being tested see protocols below Osteogenic Follow the protocol below to differentiate your StemPro Alk Differentiation Phos expressing Rat MSCs into an osteogenic phenotype Protocol 1 Seed the MSCs into culture vessels at 1 9 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 To six wells of a 12 well plate add 1 ml of cell solution per well and let attach in the 37 C 5 CO incubator for a minimum of two hours Replace three wells with MSC basal medium as negative controls and other three wells with fresh OD medium Let culture at 37 C with 596 CO Refeed cultures every 2 3 days with media prepared at initiation of differentiation MSCs will continue to expand as they differentiate under osteogenic conditions After s
8. 0 11 12 13 14 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 gently to expedite cell detachment 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 TrypLE Express of pre warmed complete a MEM medium Disperse the medium by pipetting over the cell layer surface several times Transfer the cells to a 50 ml conical tube and centrifuge at 300 x g for 5 minutes at room temperature Aspirate and discard the medium Resuspend the cell pellet in a minimal volume of pre warmed complete a MEM medium and remove a sample for counting Determine the total number of cells and percent viability using your method of choice If necessary add complete a MEM 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 a MEM 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 3 4 days after seeding completely
9. 4 no calcium magnesium or phenol red TrypLE Express Dissociation Enzyme without Phenol 100 ml 12604 013 Red 20 x 100 ml 12604 039 Antibiotic Antimycotic 100X liquid 100 ml 15240 062 Gentamycin Reagent Solution 10 mg ml liquid 10 ml 15710 064 Gentamycin Reagent Solution 50 mg ml liquid 10 ml 15750 060 Trypan Blue Stain 100 ml 15250 061 LIVE DEAD Cell Vitality Assay Kit 1000 assays 134951 Countess Automated Cell Counter includes 50 1 unit C10227 Countess cell counting chamber slides and 2 ml of Trypan Blue Stain ELF 97 Endogenous Phosphatase Detection Kit 1 kit E6601 CultureWell chambered coverglass 16 wells per coverglass set of 8 Test C3000 vi Introduction Introduction StemPro Alk Phos expressing Rat Mesenchymal Stem Cells MSCs are produced from bone marrow isolated from transgenic Fischer 344 rats expressing the human placental alkaline phosphatase hPAP gene linked to the ubiquitously active ROSA26 R26 gene promoter Kisseberth et al 1999 Mujtaba et al 2002 The cells were isolated under sterile conditions and cryopreserved from primary cultures Before cryopreservation the MSCs are expanded for three passages in a MEM medium supplemented with 10 MSC Qualified FBS and antibiotic antimycotic solution The freezing medium consisted of 70 a MEM 20 MSC Qualified FBS and 10 DMSO Each vial of MSCs contains cells that can differentiate
10. 955 6288 Tel 813 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail E mail tech supportGinvitrogen com jpinfo invitrogen com 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 Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is applicable unless all product components are stored in accordance with instructions In
11. Adipocyte Differentiation Basal Media differentiation appears to be more efficient with a MEM as the basal media Store the AD medium at 4 C in the dark up to four weeks Component Final Conc For100 ml a MEM medium with 1X 90 ml GlutaMAX I StemPro Adipogenesis 1X 10 ml Supplement Gentamicin 10 mg ml 5 pg ml 50 ul To prepare chondrogenic differentiation CD medium combine the following in a sterile flask Although you may use the StemPro Osteocyte Chondrocyte Differentiation Basal Media differentiation appears to be more efficient with a MEM as the basal media Store the CD medium at 4 C in the dark up to four weeks Component Final Conc For100 ml a MEM medium with 1X 90 ml GlutaMAX I StemPro Chondrogenesis 1X 10 ml Supplement Gentamicin 10 mg ml 5 pg ml 50 ul Differentiating StemPro Alk Phos expressing Rat MSCs Introduction This section provides guidelines and instructions for inducing StemPro Alk Phos expressing Rat MSCs to differentiate into osteogenic adipogenic and chondrogenic cell types Materials The following materials are required see page vi for ordering Needed information Culture vessels containing your MSCs Tissue culture treated flasks plates or dishes MSC Basal Medium prewarmed to 37 C see page 13 Appropriate Differentiation Medium pre warmed to 37 C see pages 13 14 Dulbecco s Phosphate Buf
12. MSCs has not been definitely established but long term culture and high cell density are implicated in the loss of differentiation potential Meirelles Lda amp Nardi 2003 Continued on next page Introduction continued Alkaline In vivo tracking of implanted MSCs in cell and gene therapy Phosphatase protocols is very important as the success of these therapies Expression depends on MSCs engraftment abilities especially after systemic infusion Meirelles Lda amp Nardi 2003 Further it has been shown that MSCs can fuse with other cells and acquire their characteristics Spees et al 2003 The StemPro Alk Phos expressing Rat MSCs allow the user to track the implanted cells Han et al 2002 Han et al 2004 Mujtaba et al 2002 using a simple fluorescence based enzymatic assay where the removal of the phosphate from the substrate provided in the assay kit causes an intense yellow green fluorescence ELF 97 Endogenous Phosphatase Detection Kit see page vi for ordering information LES S i PA q z Bright field images 10X of StemPro Alk Phos expressing Rat MSCs at P4 that have been in culture for 14 days Fluorescence images 20X and 40X of StemPro Alk Phos expressing Rat MSCs at P4 that have been in culture for 5 days Alkaline phosphatase expression is detected using the ELF 97 Endogenous Phosphatase Detection Kit Methods General Information General Cell Handling Important
13. c antimycotic or gentamycin to 37 C Remove the cells from liquid nitrogen storage and wipe the cryovial with ethanol or 70 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 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 15 ml sterile tube and add pre warmed complete a MEM medium dropwise up to 10 ml Centrifuge cells for 5 minutes at 300 x g Aspirate supernatant and resuspend cells in 2 ml of complete a MEM medium Determine the viable cell count using your method of choice and plate the resuspended cells at a seeding density of 5 000 cells per cm If necessary add complete a MEM medium to the cells to achieve the desired cell concentration and recount the cells Incubate at 37 C 5 CO and 90 humidity and allow cells to adhere for at least 24 hours The next day replace the medium with an equal volume of fresh pre warmed complete a MEM medium Change the medium every 3 4 days Subculturing Cells Introduction Materials Needed Passaging Cells Follow the protocol below to culture StemPro Alk Phos expressing Rat MSCs Subculture cells when needed before colon
14. ev Biol 214 128 138 Continued on next page 24 References continued Krampera M Glennie S Dyson J Scott D Laylor R Simpson E and Dazzi F 2003 Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen specific T cells to their cognate peptide Blood 101 3722 3729 Meirelles Lda S and Nardi N B 2003 Murine marrow derived mesenchymal stem cell isolation in vitro expansion and characterization Br J Haematol 123 702 711 Moscoso I Centeno A Lopez E Rodriguez Barbosa J I Santamarina I Filgueira P Sanchez M J Dominguez Perles R Penuelas Rivas G and Domenech N 2005 Differentiation in vitro of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation Transplant Proc 37 481 482 Mujtaba T Han S S Fischer I Sandgren E P and Rao M S 2002 Stable expression of the alkaline phosphatase marker gene by neural cells in culture and after transplantation into the CNS using cells derived from a transgenic rat Exp Neurol 174 48 57 Oh J Y Kim M K Shin M S Lee H J Ko J H Wee W R and Lee J H 2008 The anti inflammatory and anti angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury Stem Cells 26 1047 1055 Olivares E L Ribeiro V P Werneck de Castro J P Ribeiro K C Mattos E C Goldenberg R C Mill J G Do
15. fered Saline DPBS containing no calcium magnesium or phenol red Disposable sterile 50 ml tubes 37 C incubator with humidified atmosphere of 5 CO TM TrypLE Express pre warmed to 37 C Hemacytometer cell counter and Trypan Blue LIVE DEAD Cell Vitality Assay Kit or the Countess Automated Cell Counter Harvesting Follow the protocol below to harvest your StemPro Alk Phos MSCs expressing Rat MSCs for differentiation experiments We recommend that you expand your cells to x 70 confluency in a tissue culture treated T 225 flask and prepare the appropriate differentiation medium ahead of time 1 4 Aspirate complete a MEM medium from the flask and rinse the surface with DPBS without Ca and Mg 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 Aspirate the DPBS and discard To detach the cells add a sufficient volume of TM pre warmed TrypLE Express to cover the cell layer approx 0 5 ml 10 cm Incubate at 37 C for approximately 5 8 minutes Procedure continued on next page Continued on next page 15 Differentiating StemPro Alk Phos expressing Rat MSCs continued Harvesting Procedure continued from previous page MSCs 5 continued Observe the cells under a microscope If the cells are less than 90 detached continue incubating and observe
16. ferentiation Potential e Are prepared from low passage passage 3 adherent rat primary cell cultures e Express a flow cytometry cell surface protein profile positive for CD29 CD73 and CD90 gt 70 and negative for CD45 10 e Stain positive for Alkaline Phosphatase gt 80 e Contain cells characteristic of at least tri potential differentiation that can differentiate into osteogenic adipogenic and chondrogenic lineages StemPro AIk Phos expressing Rat MSCs are extracted from the hind leg bones of alkaline phosphatase transgenic Fischer 344 rats through mechanical and enzymatic digestion Cells are expanded using a MEM medium supplemented with 10 MSC Qualified FBS and antibiotic antimycotic solution which supports a cell doubling time of 30 5 hours The in vitro growth capacity of MSCs has not been definitely established and can vary greatly depending on the culture conditions such as seeding density and growth factors used but the cells can be expected to expand for at least 30 population doublings before their growth rate decreases significantly Bruder et al 1997 Meirelles Lda amp Nardi 2003 Multiple investigators have demonstrated that MSCs can be differentiated towards multiple mature cell phenotypes In addition to traditional mesenchymal lineages MSCs have been differentiated towards cardiomyocytic and neuronal phenotypes using specialized media The in vitro differentiation potential of
17. fferentiated conditions not StemPro Alk Phos expressing Rat MSCs correct Follow thawing instructions page 6 and subculture procedures page 8 exactly Cells too old MSCs above passage 5 may become differentiated Continued on next page 19 Troubleshooting continued Culturing The table below lists some potential problems and solutions that Cells help you troubleshoot your cell culture problems continued Problem Cause Solution Cells not Used serum Be sure to prepare your culture medium adherent after other than MSC using MSC Qualified FBS see page vi for initial thaw Qualified FBS ordering information Cannot detect expression of Assay system not sensitive enough Use ELF 97 Endogenous Phosphatase Detection Kit see page vi for ordering alkaline information phosphatase Differen The table below lists some potential problems and solutions that tiating Cells help you troubleshoot your cell culture problems Problem Cause Solution Cells fail to Used StemPro Although you may use the StemPro differentiate Osteocyte Chon Osteocyte Chondrocyte or Adipocyte drocyte or Differentiation Basal Media for your Adipocyte differentiation studies we have observed Differentiation that differentiation is more efficient with Basal Media a MEM as the basal media Repeat your differentiation studies using a MEM as the basal media Initial spo
18. her the following reagents for every 1 ml of Freezing Medium A needed a MEM medium 0 6 ml FBS 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 a MEM 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 rm Aspirate complete a MEM medium from the flask well or dish Rinse the surface with DPBS without Ca and Mg 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 Aspirate the DPBS and discard To detach the cells add a sufficient volume of pre warmed TrypLE Express to cover the cell layer approximately 0 5 ml 10 cm Incubate at 37 C for approximately 5 8 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 Gently 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 When 290 of the cells have detached tilt the vessels on end for a minimal length of time to
19. hmamn H F dos Santos R R de Carvalho A C and Masuda M O 2004 Bone marrow stromal cells improve cardiac performance in healed infarcted rat hearts Am J Physiol Heart Circ Physiol 287 H464 470 Orlic D Kajstura J Chimenti S Jakoniuk I Anderson S M Li B Pickel J McKay R Nadal Ginard B Bodine D M Leri A and Anversa P 2001 Bone marrow cells regenerate infarcted myocardium Nature 410 701 705 Phinney D G Kopen G Isaacson R L and Prockop D J 1999 Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice variations in yield growth and differentiation J Cell Biochem 72 570 585 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 Singh S K Hawkins C Clarke I D Squire J A Bayani J Hide T Henkelman R M Cusimano M D and Dirks P B 2004 Identification of human brain tumour initiating cells Nature 432 396 401 Continued on next page 25 References continued Spees J L Olson S D Ylostalo J Lynch P J Smith J Perry A Peister A Wang M Y and Prockop D J 2003 Differentiation cell fusion and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow
20. ials are required see page vi for Needed ordering information e StemPro Alk Phos expressing Rat MSCs stored in liquid nitrogen e Ethanol or 70 isopropanol e a MEM medium with GlutaMAX I containing 10 MSC Qualified FBS plus antibiotic antimycotic or gentamycin pre warmed to 37 C e Disposable sterile 15 ml tubes e 37 C water bath e 37 C incubator with a humidified atmosphere of 5 CO e Microcentrifuge e Tissue culture treated flasks plates or dishes e Hemacytometer cell counter and Trypan Blue LIVE DEAD Cell Vitality Assay Kit or the Countess Automated Cell Counter Invitrogen s Countess Automated Cell Counter is a benchtop counter designed to measure cell count and viability live dead and total cells accurately and precisely in less than a minute per sample using the standard Trypan Blue technique see page vi for ordering information Note Using the same amount of sample that you currently use with the hemocytometer the Countess Automated Cell Counter takes less than a minute per sample for a typical cell count and is compatible with a wide variety of eukaryotic cells and provides information on cell size Continued on next page Thawing and Establishing Cells continued Thawing To thaw and establish StemPro Alk Phos expressing Rat Procedure MSCs continued 1 Pre warm the prepared a MEM medium with 10 GlutaMAX I containing 10 MSC Qualified FBS and antibioti
21. ies start contacting each other typically every 7 10 days The following materials are required see page vi for ordering information Culture vessels containing StemPro Alk Phos expressing Rat MSCs Tissue culture treated flasks plates or dishes a MEM medium with GlutaMAX I supplemented with 10 MSC Qualified FBS and containing antibiotic antimycotic or gentamycin pre warmed to 37 C Disposable sterile 50 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 pre warmed to 37 C Hemacytometer cell counter and Trypan Blue LIVE DEAD Cell Vitality Assay Kit or the Countess Automated Cell Counter 5 Aspirate the complete a MEM medium from the cells Rinse the surface of the cell layer with DPBS without Ca and Mg 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 Aspirate the DPBS and discard To detach the cells add a sufficient volume of pre warmed TrypLE Express to cover the cell layer approximately 0 5 ml 10 cm Incubate at 37 C for approximately 5 8 minutes Procedure continued on next page Continued on next page Subculturing Cells continued Passaging Procedure continued from previous page Cells 6 continued 1
22. into multiple mature cell phenotypes in vitro including adipocytes osteocytes and chondrocytes De Ugarte et al 2003 Meirelles Lda amp Nardi 2003 Pittenger et al 1999 Wu et al 2002 In vitro differentiation into non mesenchymal cell types such as neuronal and myogenic cells have also been described Anjos Afonso et al 2004 Deng et al 2001 Han et al 2002 Han et al 2004 Moscoso et al 2005 Phinney et al 1999 Wakitani et al 1995 In addition MSCs are shown to be involved in certain types of cancers Houghton et al 2004 Singh et al 2004 and are known to secrete immunomodulatory anti angiogenic anti inflammatory pro cardiovasculogenic and pro arteriogenic factors Djouad et al 2003 Gojo et al 2003 Houghton et al 2004 Kinnaird et al 2004 Krampera et al 2003 Oh et al 2008 Olivares et al 2004 Orlic et al 2001 StemPro Alk Phos expressing Rat MSCs can be used for studies of adult stem cell differentiation tissue engineering cell and genetic therapy and potential future clinical applications These cells can also be used in transplant studies to track transplanted cells as they differentiate into mature phenotypes We recommend that you use a MEM with GlutaMAX I and MSC Qualified FBS see page vi for optimal growth and expansion Continued on next page Introduction continued Characteristics of StemPro Alk Phos Expressing MSCs Isolation and Expansion Dif
23. invitrogen StemPro Alk Phos expressing Rat Mesenchymal Stem Cells Catalog no R7789 120 Version A 14 November 2008 A10855 ii Table of Contents Contents add ui tas v Additional Products vi Introduction ar 1 MOS aida 4 General Information enrnrernnvnrerevrerevvnrennnverevavrereventvnnnenserevesssrererevennsnsavssssrevenene 4 Thawing and Establishing Cells sss 6 Subc lt ring Cells ite Roe et qe lich tet ded ce 8 Freezing Cells lirios 10 Differentiation Media oreet ihe Oe ee CR Ee 8g 13 Differentiating StemPro Alk Phos expressing Rat MSCs 15 Appendix A A a oe SE oo 19 Troubleshooting icici erar e 19 Technical SUP iii ett enit game 21 Purchaser Notification srenvorevrorevonrernnenvevevrevererennnnenvereveesereverennnnensavsssererevense 22 RES a ett een DR e EE do eres 24 iii iv Contents and Storage Shipping Contents and StemPro Alk Phos expressing Rat Mesenchymal Stem Cells are shipped on dry ice Contents and storage conditions for StemPro Alk Phos Storage expressing Rat Mesenchymal Stem Cells are listed in the table below For components of the freezing medium see page 11 Product Amount Storage StemPro Alk Phos expressing Rat 1ml Liquid nitrogen freezing medium Mesenchymal Stem Cells 1 x 10 cells ml in Information for European Customers Handle cells as potentially biohazardous material under
24. keep them undifferentiated Continued on next page General Information continued Media Requirements Important We recommend using Minimum Essential Medium MEM a medium a MEM medium with GlutaMAX I and supplemented with 10 MSC Qualified Fetal Bovine Serum FBS for optimal growth and expansion of StemPro Alk Phos expressing Rat MSCs and to keep them undifferentiated see page vi for ordering information e Prepare your growth medium prior to use e When thawing or subculturing cells transfer cells into pre warmed medium at 37 C e You may store the complete growth medium in the dark at 4 C for up to four weeks e Avoid repeated freeze thaw cycles of MSC Qualified FBS We have observed that StemPro Alk Phos expressing Rat MSCs adhere poorly when plated on media other than a MEM medium supplemented with 10 MSC Qualified FBS after their initial thaw Although they recover and adhere well after their first passage we suggest that you use the recommended media If you prefer to culture your MSCs on growth media other than the recommended we advise you to optimize your growth conditions and treat the your cells gently i e do not vortex bang the flasks to dislodge the cells or centrifuge the cells at high speeds Thawing and Establishing Cells Introduction To thaw StemPro Alk Phos expressing Rat MSCs and to initiate cell culture follow the protocol below Materials The following mater
25. onents 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 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
26. pecific 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 For long term culture gt 21 days we recommend that you reduce the seeding density by half 9 5 x 10 cells cm to prevent overgrowth and cell detachment 16 Continued on next page Differentiating StemPro Alk Phos expressing Rat MSCs continued Adipogenic Follow the protocol below to differentiate your StemPro Alk Differentiation Phos expressing Rat MSCs into an adipogenic phenotype Protocol 1 Seed the MSCs into culture vessels at 7 6 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 2 Tosix wells of a 12 well plate add 1 ml of cell solution per well and let attach in the 37 C 5 CO incubator for a minimum of two hours 3 Replace three wells with MSC basal medium as negative controls and other three wells with fresh AD medium Let culture at 37 C and 596 CO 4 Refeed cultures every 3 4 days with media prepared at initiation of differentiation MSCs will continue to undergo limited expansion as they differentiate under adipogenic conditions 5 After specific periods of cultivation adipogenic cult
27. roubleshooting Culturing 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 10 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 Alk Phos expressing Rat MSCs Thawing Use pre warmed complete a MEM medium medium not prepared as described on page 5 Be sure to correct use MSC Qualified FBS Cells too diluted Generally we recommend thawing one vial at a density of 5 000 cells per cm Cell not handled StemPro Alk Phos expressing Rat MSCs are gently fragile treat your cells gently do not vortex bang the flasks to dislodge the cells or centrifuge the cells at high speeds Cells grow Growth medium Use prewarmed complete a MEM medium slowly not correct Cells too old Use healthy MSCs under passage 5 do not overgrow Cells Culture Thaw and culture fresh vial of new di
28. s to differentiate along the osteogenic adipogenic and chondrogenic lineages This section provides guidelines for preparing media that are used for inducing StemPro Alk Phos expressing Rat MSCs to differentiate into osteogenic adipogenic and chondrogenic cell types MSC basal medium is used a cell attachment medium and as a negative control during differentiation experiments It consists of a MEM medium with GlutaMAX I containing 10 MSC Qualified FBS and 5 ul ml gentamicin see page vi Component Final Conc For 500 ml a MEM medium with 1X 450 ml GlutaMAX I FBS MSC Qualified 10 50 ml Gentamicin 10 mg ml 5 pg ml 250 pl To prepare osteogenic differentiation OD medium combine the following in a sterile flask Although you may use the StemPro Osteocyte Chondrocyte Differentiation Basal Media differentiation appears to be more efficient with a MEM as the basal media Store the OD medium at 4 C in the dark up to four weeks Component Final Conc For100 ml a MEM medium with 1X 9 ml GlutaMAx I StemPro Osteogenesis 1X 10 ml Supplement Gentamicin 10 mg ml 5 pg ml 50 pl Continued on next page 13 Differentiation Media continued Adipogenic Differentiation Medium Chondrogenic Differentiation Medium 14 To prepare adipogenic differentiation AD medium combine the following in a sterile flask Although you may use the StemPro
29. stroma Proc Natl Acad Sci US A 100 2397 2402 Wakitani S Saito T and Caplan A I 1995 Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5 azacytidine Muscle Nerve 18 1417 1426 Wu Y Y Mujtaba T Han S S Fischer I and Rao M S 2002 Isolation of a glial restricted tripotential cell line from embryonic spinal cord cultures Glia 38 65 79 2008 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 26 Notes 27 28 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
30. tting Tf this step is not performed under high step not humidity conditions the spots may performed under dehydrate and the formation of high humidity if chondrogenic plates inhibited Repeat the differentiating initial spotting step at 37 C 5 CO and into 90 humidity chondrocytes Cells have Initial seeding For long term culture gt 21 days we overgrown density too high recommend that you seed at a lower cell the culture density of 3 x 10 cells cm to prevent plates and overgrowth and cell detachment have detached 20 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete Technical Support 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 website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan Minato ku 3 Fountain Drive Tel 1 760 603 7200 Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800
31. ures can be processed for Oil Red O or LipidTOX staining beginning at 7 14 days gene expression analysis or protein detection Continued on next page 17 Differentiating StemPro Alk Phos expressing Rat MSCs continued Chondrogenic Follow the protocol below to differentiate your StemPro Alk Differentiation Phos expressing Rat MSCs into a chondrogenic phenotype Protocol 1 Important Detach cells using TrypLE Express and perform a cell count as described in Harvesting MSCs pages 15 16 through Step 6 Resuspend the cells in MSC basal medium to a concentration of 8 x 10 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 5 CO and 90 humidity Note If this step is not performed under high humidity conditions the spots may dehydrate and the formation of chondrogenic pellets inhibited To three of the spotted wells add 1 ml of MSC basal medium as a negative control To the other three wells add 1 ml of CD medium Incubate at 37 C 5 CO and 90 humidity Refeed cultures every 2 3 days with same media prepared at the initiation of differentiation Check for chondrogenesis after a set period of cultivation You may perform alcian blue staining on the pellets to detect glycosaminoglycans after 14 days or paraffin section of pellets for collagen 2a immunohistological staining after 21 days 18 Appendix T
32. vitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification continued Limited Use Label License No 5 Invitrogen Technology 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 comp
Download Pdf Manuals
Related Search