User Manual
Contents
1. Product Name S Derived Mesenchymal Stem Cell Catalog No MUBMD 01001 Amount per Vial 1x10 Cells Cryopreserved At Sixth Passage Storage Condition Liquid Nitrogen CAUTION Please handle this product as a potentially biohazardous material This product contains Dimethyl Sulfoxide DMSO a hazardous material in the freezing medium PRODUCT INTRODUCTION Adipose Derived Mesenchymal Stem Cells ADSCs are multipotent stem cells that can differentiate into a variety of cell types including osteocytes adipocytes and chondrocytes In addition ADSCs have wide applications in tissue engineering cell therapy and gene therapy Cyagen OriCell Strain C57BL 6 Mouse ADSCs are derived from adipose tissue at inguen of qualified strain Cb7BL 6 mice These cells express clusters of proteins specific for ADSCs and have a strong capacity for self renewal while maintaining multipotency In addition these cells have been tested for e Exogenous Factors bacterial fungal contamination mycoplasma contamination and endotoxin contamination e Characteristics post thaw viability cell cycle verification of undifferentiated state and differentiation potential This product is intended for laboratory research use only It is not intended for diagnostic therapeutic clinical household or any other applications CELL CHARACTERISTICS AND IDENTITY e Strong capacity to expand Can be passaged at least 3 times IMPIO020A2 MUBMD 01001 Page 3 o2. incubator Additional Tips Time to Split OriCell Strain C57BL 6 Mouse Neural Stem Cells With GFP When the neurospheres have a dark clump inside or ruffling on the outside of the neuro sphere it is recommended to split the cells We typically split OriCell Strain C57BL 6 Mouse Neural Stem Cells With GFP every two days OriCell Strain C57BL 6 Mouse NSCs GFP DIFFERENTIATION Neural Stem Cells NSCs GFP are maintained in serum free culture medium supplemented with the mitogenes epidermal growth factor EGF and fibroblast growth factor 2 FGF2 Removal of the mitogenes results in spontaneous differentiation of NSCs GFP into neurons astrocytes and oligondendroytes Besides spontaneous differentiation NSCs GFP can be directly differentiated into those lineages in defined conditions IMPIO065A1 MUBNF 01101 Page 6 of 10 Fy Jd DS PHARMA i P BIOMEDICAL D C V d Ju er ADSC Growth Medium 6 ml for T75 flask 3 ml for T25 flask to neutralize the trypsinization 8 Gently pipet the medium over the cells to dislodge and resuspend the cells Repeat 5 6 times until all the cells are dissociated from the flask and evenly dispersed into a single cell suspension 9 Transfer the dissociated cells into a 15 mL conical tube 10 Centrifuge at 250 x g for 5 minutes to pellet the cells 11 Carefully aspirate off as much of the supernatant as possible 12 Add 2 ml of OriCell Mouse ADSC Growth Medium to the conical tube and gent
3. saline PBS Fix cells with 2 mL of 4 formaldehyde solution for 30 minutes 2 Rinse wells twice with 1x PBS Stain the cells with 1 mL alizarin red S working solution for 3 5 minutes Rinse wells 2 3 times with 1x PBS 4 Cells can now be visualized and analyzed under a microscope IMPI0020A2 MUBMD 01001 Page 8 of 14 A DS PHARMA GY BIOMEDICAL D C y al lj e Fig 3 OriCell Strain C57BL 6 Mouse Adipose derived Mesenchymal Stem Cells are differentiated into Osteocytes and are stained with Alizarin Red S Adipogenic Differentiation Materials Required OriCell Mesenchymal Stem Cell Adipogenic Differentiation Medium Cat No GUXMX 90031 Adipogenesis Protocol A Note The protocol listed below is for 6 well tissue culture plates 10 Culture the OriCell Strain C57BL 6 Mouse ADSCs in the OriCell Mouse ADSC Growth Medium at 37 C in a 5 CO humidified incubator When cells are approximately 80 90 confluent they can be dissociated with 0 25 Trypsin 0 04 EDTA Cat No TEDTA 1000 Reseed the ADSCs in growth medium at 2x10 cells cm in a 6 well tissue culture plate with a medium volume of 2 mL per well Incubate the cells at 37 C in a 5 CO humidified incubator Feed the cells every three days until they are 100 confluent or post confluent Induction of adipogenic differentiation at post confluency is strongly recommended When the cells are 100 confluent or post confluent carefully aspira
4. C57BL 6 Mouse Adipose derived Mesenchymal Stem Cells are differentiated into adipocytes and are stained with Oil Red O Chondrogenic Differentiation Materials Required OriCell Mesenchymal Stem Cell Chondrogenic Differentiation Medium Cat No GUXMX90041 Chondrogenesis Protocol 1 Calculate the total number of ADSC pellet cultures required for your experiment 2 5x10 ADSCs are needed to form each chondrogenic pellet Transfer this amount of cells into an appropriate culture tube 2 Wash the ADSCs with Incomplete Chondrogenic Medium Centrifuge the cells at 150 x g for 5 minutes at room temperature and then aspirate off the supernatant Resuspend the cells in 1 mL of Incomplete Chondrogenic Medium per 7 5x105 cells Centrifuge again at 150 x g for 5 minutes and then aspirate off the medium 3 Resuspend the ADSCs in Complete Chondrogenic medium to a concentration of 5 0x10 cells mL 4 Aliquot 0 5 mL 2 5x10 cells of the cell suspension into 15 mL polypropylene IMPI0020A2 MUBMD 01001 Page 10 of 14 DS PHARMA JY BIOMEDICAL D C y n Ij e culture tubes Centrifuge the cells at 150 x g for 5 minutes at room temperature A Note DO NOT aspirate the supernatant or resuspend the pellet 5 Loosen the caps of the tubes in order to allow gas exchange and incubate the tubes at 37 C in a humidified atmosphere of 5 CO2 Do not disturb the pellets for 24 hours 6 Feed the cell pellets every 2 3 days by completely repl
5. Mouse ADSCs overgrow as it will result in contact inhibition When the cells are 80 90 confluent subculturing the cells is strongly recommended i Note We strongly recommend the use of OriCell culture media and other related reagents for optimal results THAWING AND ESTABLISHING OriCell STRAIN C57BL 6 MOUSE ADSCs Materials Required e OriCell Strain C57BL 6 Mouse Adipose Derived Mesenchymal Stem Cells Cat No MUBMD 01001 IMPI0020A2 MUBMD 01001 Page 4 of 14 DS PHARMA VY BIOMEDICAL D C y d i B ll OriCell M Mouse Adipose Derived Mesenchymal Stem Cell Growth Medium Cat No MUXMD 90011 Thawing and Establishing Mouse ADSCs D m Pre warm OriCell Mouse ADSC Growth Medium to 37 C Add 9 mL of OriCell Mouse ADSC Growth Medium to a 15 mL conical tube Remove the cryovial of OriCell Strain C57BL 6 Mouse ADSCs from liquid nitrogen Quickly thaw the cryovial in a 37 C water bath until the last ice crystal disappears Be sure to finish the thawing procedure within 3 minutes Be careful not to submerge the entire vial Maximum cell viability is dependent on the rapid and complete thawing of frozen cells A Note Results will be less than optimal if the cells are thawed for more than 3 minutes 10 LE 12 13 14 15 16 As soon as the cells are completely thawed disinfect the outside of the vial with 70 v v ethanol Use a pipette to transfer the cells to the conical tube containing OriCe
6. Osteogenic Differentiation Materials Required OriCell Mesenchymal Stem Cell Osteogenic Differentiation Medium Cat No GUXMX 90021 Osteogenesis Protocol A Note The protocol listed below is for 6 well tissue culture plates 1 Culture the OriCell Starin C57BL 6 Mouse ADSCs in OriCell Mouse ADSC Growth Medium at 37 C in a 5 CO2 humidified incubator 2 When cells are approximately 80 90 confluent they can be dissociated with 0 25 Trypsin 0 04 EDTA Cat No TEDTA 10001 3 Reseed the OriCell Starin C57BL 6 Mouse ADSCs in the growth medium at 3x 10 cells cm in a 6 well tissue culture with a medium volume of 2 mL per well 4 Incubate the cells at 37 C in a 5 CO humidified incubator When cells are approximately 60 70 confluent carefully aspirate off the growth medium from each well and add 2 mL of OriCellTM Mesenchymal Stem Cell Osteogenic Differentiation Medium pre warmed to 379C 6 Feed cells every 3 days for 2 3 weeks by completely replace the medium with fresh OriCell Mesenchymal Stem Cell Osteogenic Differentiation Medium 7 After 2 3 week differentiation cells can be fixed and stained with Alizarin Red S A Note To prevent osteoblasts from detaching it is recommended to change half of the medium every two days before analysis Alizarin Red Stain Analysis 1 After the cells have differentiated remove the osteogenic differentiation medium from the wells and rinse with 1x phosphate buffered
7. P y e n proliferation slow down and cell aging Control the digestion time Wash the cells with pre warmed medium 2 3 times during recovery Use cells at a low original passage number Related Products Product Catalog Number OriCell Mouse Adipose Derived Mesenchymal MUXMD 90011 Stem Cell Growth Medium OriCell Mesenchymal Stem Cell Osteogenic GUXMX 90021 Differentiation Medium OriCell Mesenchymal Stem Cell Adipogenic GUXMX 90031 Differentiation Medium OriCell Mesenchymal Stem Cell Chondrogenic GUXMX 90041 Differentiation Medium 0 25 Trypsin 0 04 EDTA TEDTA 10001 Phosphate Buffered Saline 1xPBS PBS 10001 OriCell NCR Protein Free Cryopreservation Medium NCPF 10001 References JM Gimble and F Guilak 2003 Adipose derived adult stem cells isolation characterization and differentiation potential JSCT 5 362 369 Cyagen Biosciences reserves all rights on the technical documents of its OriCell cell culture products No part of this document may be reproduced or adapted for other purposes without written permission from Cyagen Biosciences KAAERBET IL 7 DS IP VINAAATAAILARWNSA 7564 0063 AMARA MPILIRA2T H1845 KYUHOI IRE JLB KE EE HA 33K TEL 06 6990 8051 FAX 06 6325 6058 FI AILYAR k TELO072 636 8160 FAX 072 634 7222 http www dspbio co jp dspb ls bio ds pharma co jp IMPI0020A2 MUBMD 01001 Page 14 of 14
8. acing the medium in each tube to avoid aspirating the pellets when aspirating the medium attach a sterile 1 200uL pipette tip to the end of the aspirating pipette Add 0 5 mL of freshly prepared Complete Chondrogenic Medium to each tube 7 After replacing the medium flick the bottom of the tube to ensure that the pellet is free floating Loosen the caps and return the tubes to the 37 C incubator 8 Chondrogenic pellets should be harvested after 14 28 days in culture Pellets may be formalin fixed and paraffin embedded for alcian blue stain analysis Alcian Blue Staining Procedure The tissue sample should be formalin fixed and paraffin embedded already 2 Staining procedure a Deparaffinize slides and hydrate to distilled water b Stain in alcian blue solution for 30 minutes c Wash in running tap water for 2 minutes d Rinse in distilled water e Visualize under a light microscope and capture images for analysis Blue staining indicates synthesis of proteoglycans by chondrocytes Fig 5 OriCell Strain C57BL 6 Mouse Adipose Derived Mesenchymal Stem Cells are differentiated into chondrocytes and are stained with Alcian Blue IMPI0020A2 MUBMD 01001 Page 11 of 14 DS PHARMA JY BIOMEDICAL D C y a e CRYOPRESERVATION OF OriCell STRAIN C57BL 6 MOUSE ADSCs OriCell NCR Protein Free Cryopreservation Medium Cat No NCPF 10001 is a protein free ready to use freezing medium Its chemically defined and protei
9. f 14 DS PHARMA JY BIOMEDICAL D C y a e e Multipotent differentiation ability along osteogenic chondrogenic and adipogenic lineages e Positive for CD29 CD44 and Sca 1 gt 70 and negative for CD117 lt 5 in flow cytometry assays PRODUCT APPLICATIONS Adipose Derived Mesenchymal Stem Cells ADSCs have become a hot research target for their potential use in regenerative medicine and tissue engineering in areas such as cardiovascular neural and orthopaedic disease Cyagen OriCell Strain C57BL 6 Mouse ADSCs can be used as cell models to evaluate the immunoreactions proliferation immigration and differentiation of ADSCs both in vivo and in vitro GENERAL HANDLING PRINCIPLES 1 Aseptic handling of the product is necessary throughout 2 Once the cells have been established always freeze up several vials of Adipose Derived Mesenchymal Stem Cells ADSCs as a backup A Note The OriCell Strain C57BL 6 Mouse ADSCs can be frozen thawed at least one times 3 For general maintenance of cells we recommend the seeding density to be 1 0 2 0x10 cells cm 4 For all studies it is strongly recommended to use cells that are at or under an original passage number of 10 5 For general maintenance of cells we recommend that the medium is changed if it becomes acidic the pH indicator in culture medium appears yellow In general change the growth medium every three days 6 Do not let Strain C57BL 6
10. fA DS PHARMA VY BIOMEDICAL C y d y 0 I We help You discover life OriCell Strain C57BL 6 Mouse Adipose Derived Mesenchymal Stem Cells ADSCs Cat No MUBMD 01001 DS PHARMA VY BIOMEDICAL D P y i e n Table of Contents Contents and Storage useconaieediantkandunrdn uiu d Ue REESE NER Gra CN C SUR RC RID RC RD AL PRG CR NE EU 3 Product IDtrOOHCEIODI see ui YCS DR UST uEN IINE CG ns EIE NS Y SECUN EENIV SRM ND ERW rU KDE 3 Cell Characteristics and Identity inii sii wa Cni RD RW TER RAN WYW RW ANCR WD COC RC RD RUTRUM 3 Product AppHlIcatlOlS uni Yni A o RC C DC E ba A N Wi FR 4 General Handlllig PEHIBCIDIGS eiii dci o Ron inii ROGER CR RERO CR CER WN Wa RR Rr 4 Culturing OriCell Strain C57BL 6 Mouse ADSCs Thawing and Establishing OriCell Strain C57BL 6 Mouse ADSCS ernnn nenne 4 Passaging Cyagen OriCell Strain C57BL 6 Mouse ADSCS ssssssssnssnnnnnnnnnnnnnnnnnnnn 6 Differentiation of OriCell Strain C57BL 6 Mouse ADSCS ennemi 8 Cryopreservation of OriCell Strain C57BL 6 Mouse ADSCS eene 12 iio y lb si O O O O HR ERAN ee REDE REN USUS 13 TFOUDICSNOOUING ET a D EET R E RAE EE RE ME 13 Related ProducLS sc O dineu au IA OR WR RYS ME S REI NG aan EAS RONA UO SRG AU SR 14 FA MS MERE EE HNN RH RAW PN Y m 14 DS PHARMA VY BIOMEDICAL D C y d J 0 I CONTENTS AND STORAGE Strain C57BL 6 Mouse Adipose
11. ge condition does Purchase a replacement and store in liquid not meet the requirements nitrogen for long term preservation Thawing of the cells takes too 5 Thaw cells for no more than 3 minutes long MR TE After aspirating off medium wash the Low cell recovery Ern Pec tube with culture medium twice and rate 5 transfer all of the cells to the dish Care should be taken to avoid introducing Cells are handled roughly bubbles during pipetting Also avoid vortexing and high speed centrifugation Medium is not pre warmed Warm medium to 37 C before recovery Discard the cells in question and disinfect Mycoplasma contamination the laboratory environment before recovering the next batch of cells Wash the cells with PBS 2 3 times to Slow cell growth l remove serum prior to trypsinization serum Over digestion will inhibit the function of trypsin Control the digestion time Plating density is too low Increase the plating density Use Cyagen tailor made culture media If Inappropriate serum and other serum and media products are used medium please perform validation to ensure compatibility Change the medium the next day after recovery to ensure removal of all dead cells Cell aging Dead cells are not removed promptly Discard the cells in question and disinfect Cell Contamination the laboratory environment before recovering the next batch of cells IMPI0020A2 MUBMD 01001 Page 13 of 14 DS PHARMA JY MOMEDICAL D
12. ll Mouse ADSC Growth Medium inside a biosafety cabinet Be careful not to introduce any bubbles during the transfer process Rinse the vial with 1 mL of the medium to reduce cell loss Subsequently transfer this 1 mL of cell suspension into the conical tube Gently mix the cell suspension by slowly pipetting up and down Be careful not to introduce any bubbles Centrifuge the cell suspension at 250 x g for 5 minutes Carefully aspirate off as much of the supernatant as possible and add 2 3 mL of fresh OriCell Mouse ADSC Growth Medium pre warmed to 37 C Gently re suspend the cells in OriCell Mouse ADSC Growth Medium Plate the cells into a T25 flask and add a sufficient OriCell Mouse ADSC Growth Medium Gently rock the culture flask to evenly distribute the cells Incubate at 37 C in a 5 CO humidified incubator The next day change the medium with fresh growth medium pre warmed to 379C Change the growth medium every three days thereafter When the cells are approximately 80 90 confluent they can be dissociated with 0 25 Trypsin 0 04 EDTA and passaged A Note Changing Medium Warm an appropriate amount of medium to 37 C in a sterile container Replace the spent medium with the pre warmed fresh medium Once completed return the flask to the incubator Avoid repeated warming and cooling of the medium If the entire content is not needed for a single procedure transfer only the required volume to a sterile
13. ly re suspend the cells thoroughly 13 Plate the cells into appropriate flasks OriCell Strain C57BL 6 Mouse ADSCs can be split at 1 2 or other appropriate ratio 14 Add an appropriate amount of medium to the cells Incubate the cells at 379C inside a 5 CO humidified incubator Note Care should be taken to avoid introducing bubbles during pipetting Additional Tips Time to Change Medium It is recommended to change the culture medium if there are too many dead cells after passaging It is recommended to change the culture medium whenever the medium becomes acidic even if the cells do not reach 80 90 confluency The pH indicator in the culture medium will appear yellow when acidic In general change the growth medium every three days Time to Subculture When OriCell Strain C57BL 6 Mouse ADSCs are 80 90 confluent it is recommended that the cells be subcultured Do not let OriCell Strain C57BL 6 Mouse ADSCs overgrow as it will result in contact inhibition 3 yf dyg ON met ng S af Passage 9 40x Passage 9 100x Fig 2 Images of OriCell Strain C57BL 6 Mouse Adipose Derived Mesenchymal Stem Cells at passage 9 IMPI0020A2 MUBMD 01001 Page 7 of 14 DS PHARMA JY BIOMEDICAL D C y a er OriCell STRAIN C57BL 6 MOUSE ADSCs DIFFERENTIATION USING OriCell DIFFERENTIATION MEDIA Cyagen OriCell ADSCs can differentiate into a variety of cell types including osteocytes adipocytes and chondrocytes
14. n free formulation has been optimized to stem cells and primary cells thus greatly enhancing the viability and integrity of these cells by protecting them from damage during the one step freeze thaw procedure Unlike other conventional freezing media which require a slow programmed freeze this product allows the cells to be directly frozen at 809C Cryopreservation Note Change the culture medium with fresh growth medium 24 hours before freezing 1 Collect cells that are in the logarithmic growth phase Perform a cell count to determine the viable cell density 2 Centrifuge the cells for 3 5 minutes at 250 x g and 209C Remove and discard the supernatant using a pipette 3 Resuspend the cell pellet in the OriCellTM NCR Protein Free Cryopreservation Medium at a cell density of 10 10 cells mL 4 Dispense aliquots of the cell suspension into cryogenic storage vials that are properly labeled 5 Place the vials directly in a 809C freezer After 24 hours transfer the frozen vials to liquid nitrogen for long term preservation IMPI0020A2 MUBMD 01001 Page 12 of 14 WA DS PHARMA Jy BIOMEDICAL P y i e n Some stem cells can secrete factors to support cell growth Therefore a certain degree of plating density must be maintained otherwise it will lead to cell Cell aging Plating density is too low The table below lists some potential problems and solutions for culturing ADSCs Problem Cause Solution The stora
15. secondary container IMPI0020A2 MUBMD 01001 Page 5 of 14 DS PHARMA JY BIOMEDICAL C V a U e n PASSAGING OriCell Strain C57BL 6 Mouse NSCs GFP Materials Required e Trypsin Like Enzyme Solution e Phosphate Buffered Saline 1xPBS Cat No PBS 10001 e OriCell Strain C57BL 6 Mouse Neural Stem Cells With GFP Cat No MUBNF 01101 e OriCell Neural Stem Cell Growth Medium Cat No GUXNX 90011 Passaging Strain C57BL 6 Mouse NSCs GFP IM 1 Pre warm OriCell Neural Stem Cell Growth Medium 1xPBS Trypsin Like Enzyme solution to 379C 2 Transfer the media containing the floating neurospheres to a 15 mL conical tube Centrifuge at 250 x g for 5 minutes 4 Aspirate and discard all the supernatant and add 2mL of 1xPBS and 200uL of Trypsin Like Enzyme to the conical tube and resuspend with a fire polished glass pipette 5 Mechanically dissociate the neurospheres by gently pipetting up and down 8 10 times with a fire polished glass pipette be careful not to introduce bubbles in the suspension Add 10 mL 1xPBS to the conical tube and mix well Centrifuge at 250 x g for 5 minutes Carefully aspirate off as much of the supernatant as possible i m Re suspend the cells in 3 mL of OriCell Neural Stem Cell Growth Medium pre warmed to 379C 10 Plate cells into two or three T25 flasks and add sufficient OriCell Neural Stem Cell Growth Medium 11 Incubate the cells at 37 C in a 5 CO humidified
16. te off the spent growth medium from the wells and add 2 mL of OriCell Mesenchymal Stem Cell Adipogenic Differentiation medium A induction medium per well Three days later change the medium to OriCell Mesenchymal Stem Cell Adipogenic Differentiation medium B maintenance medium by completely replacing the spent medium A 24 hours later change the medium back to MSC Adipogenic Differentiation medium A To optimally differentiate ADSCs into adipogenic cells repeat the cycle of induction and maintenance three times After three to five cycles of induction and maintenance culture the cells in OriCell M Mesenchymal Stem Cell Adipogenic Differentiation medium B for an additional 4 7 days until the lipid droplets are big round enough During these days period change IMPI0020A2 MUBMD 01001 Page 9 of 14 DS PHARMA Y BIOMEDICAL 9D C y e n the medium every three days Oil Red O Stain Analysis 1 After the cells have differentiated remove the MSC Adipogenic Differentiation Medium from the wells and rinse with 1x phosphate buffered saline PBS Fix cells with 2 mL of 4 formaldehyde solution for 30 minutes 2 Rinse wells twice with 1x PBS and stain cells with 1 mL of oil red O working solution 3 2 dilution with distilled water and filter with filter paper for 30 minutes Rinse wells 2 3 times with 1x PBS 4 Cells can now be visualized and analyzed under a microscope tex Fig 4 OriCell Strain
Download Pdf Manuals
Related Search