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1. 3 Reseed the MSCs in the growth medium at 3x 10 cells cm in a 6 well tissue culture plate pre coated with 0 1 gelatin solution 4 Incubate the cells at 37 C inside 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 OriCell Mesenchymal Stem Cell Osteogenic Differentiation Medium 6 Feed cells every three days for 2 4 weeks by completely replacing the medium with fresh OriCell Mesenchymal Stem Cell Osteogenic Differentiation Medium pre warmed to 37 C 7 After 2 4 weeks of differentiation cells can be fixed and stained with alizarin red S Note To prevent osteoblasts from detaching it is recommended to change half of the A medium every two days before analysis Alizarin Red S Staining Analysis 1 After the cells have differentiated remove the osteogenic 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 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 IMPIOO30A2 MUBMX 01001 Page 8 of 15 Jd SE 05 piama 9 Cyagen is Poa 4 o cds lt re d e ag ay Se e rat ADAM VT dorm RUGAS ee AN gt o os P 4 od j rei s 5 go T 2 Ee Fig2. 3 OriCell Strain C57BL 6 Mouse MSCs 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 Note The protocol listed below is for 6 well tissue culture plates Culture the OriCell STRAIN C57BL 6 MOUSE MSCs in the OriCell Mesenchymal Stem Cell 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 MSCs 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 aspirate 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 T
3. Protein Free Cryopreservation Medium References IMPIOO30A2 MUBMX 01001 Control the digestion time Use cells at a low original passage number O cyagen Wash the cells with pre warmed medium 2 3 times during recovery Catalog Number MUXMX 90011 GUXMX 90021 GUXMX 90031 GUXMX 90041 TEDTA 10001 PBS 10001 NCPF 10001 Alexandra Peister Jason A Mellad and Benjamin L Larson 2004 Adult stem cells from bone marrow MSCs isolated from different strains of inbred mice vary in surface epitopes rates of proliferation and differentiation potential BLOOD 1 1662 1668 Masoud Soleimani and Samad Nadn 2009 A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow NETURE 4 102 106 Page 14 of 15 fA DS PHARMA AN BIOMEDICAL C y a l C Philippe Tropel Daniele Noel and Nadine Platet 2004 Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow Experimental Cell Research 295 395 406 Lindolfo da Silva Meirelles Pedro Cesar Chagastelles and Nance Beyer Nardi 2006 Mesenchymal stem cells reside in virtually all post natal organs and tissues Jourmal of Cell Science 119 2204 2213 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 KAAERHETI F D
4. Transfer this amount of cells into an appropriate culture tube Wash the MSCs 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 5x10 cells Centrifuge again at 150 x g for 5 minutes and then aspirate off the medium Resuspend the MSCs in Complete Chondrogenic medium to a concentration of 5 0x 10 cells mL 4 Aliquot 0 5 mL 2 5x10 cells of the cell suspension into 15 mL polypropylene IMPIOO30A2 MUBMX 01001 Page 10 of 15 fA DS PHARMA IY oN O C y a e culture tubes Centrifuge the cells at 150 x g for 5 minutes at room temperature DO NOT aspirate the supernatant or resuspend the pellet 5 Loosen the caps of the tubes one half turn in order to allow gas exchange and incubate the tubes at 37 C in a humidified atmosphere of 5 CO Do not disturb the pellets for 24 hours 6 Feed the cell pellets every 2 3 days by completely replacing 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 h
5. 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 Time to Subculture When OriCell Strain C57BL 6 Mouse MSCs are 80 90 confluent it is recommended that the cells be subcultured Do not let the cells overgrow as it will result in contact inhibition Passage 7 at 40x Passage 7 at 100x TM Fig 2 Images of OriCell Strain C57BL 6 Mouse Mesenchymal Stem Cells at passage 7 IMPIOO30A2 MUBMX 01001 Page 7 of 15 AA DS PHARMA IY BIOMEDICAL P y a I e n OriCell STRAIN C57BL 6 MOUSE MSC DIFFERENTIATION USING Oricell DIFFERENTIATION MEDIA OriCell Strain C57BL 6 Mouse MSCs can differentiate into a variety of cell types including osteocytes adipocytes and chondrocytes 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 STRAIN C57BL 6 MOUSE MSCs in OriCell Mesenchymal Stem Cell Growth Medium at 37 C in a 5 CO 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
6. pH indicator in the medium appears yellow In general change the growth medium every three days 6 Do not let OriCell Strain C57BL 6 Mouse MSCs overgrow as it will result in contact inhibition When the cells are 80 90 confluent subculturing the cells is strongly recommended pr 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 MSCs Materials Required e OriCell Mouse Mesenchymal Stem Cell Growth Medium Cat No MUXMX 90011 IMPIOO30A2 MUBMX 01001 Page 4 of 15 AA DS PHARMA AN BIOMEDICAL C y a l C Thawing and Establishing Strain C57BL 6 Mouse MSCs Pre warm the fully supplemented complete OriCell Mouse MSC Growth Medium to 37 C Add 9 mL of OriCell Mouse MSC Growth Medium to a 15 mL conical tube Remove the cryovial of OriCell Strain C57BL 6 Mouse MSCs from liquid nitrogen Quickly thaw the cryovial in a 37 C water bath until the last ice crystal disappears For optimal results 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 Note Results will be less than optimal if the cells are thawed for more than 3 minutes 5 As soon as the cells are completely thawed disinfect the outside of the cryovial with 70 v v ethanol 6
7. DONE aapusininisesidr and ir einstein 15 fA DS PHARMA avy ROMEDICAL P y a I e n CONTENTS AND STORAGE Strain C57BL 6 Mouse Product Name Mesenchymal Stem Cells Catalog No MUBMX 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 A product contains dimethyl sulfoxide DMSO a hazardous material in the freezing medium PRODUCT INTRODUCTION Mesenchymal stem cells MSCs are multipotent stem cells that can differentiate into a variety of cell types including osteocytes adipocytes and chondrocytes MSCs proliferate quickly and are capable of generating a local immunosuppressive microenvironment thus contributing to their wide application potentials in tissue engineering cell therapy and gene therapy OriCell Strain C57BL 6 Mouse Mesenchymal Stem Cells are derived from the bone marrow of Strain C57BL 6 mice They have a strong capacity for self renewal while maintaining their 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
8. S TIP VINAAATAAIVANBA 7564 0063 ABRAFERATHAILIRET ZT B1B435 KYUHO TIRE JL 8 EE ANA HSA TEL 06 6990 8051 FAX 06 6325 6058 FO AILYAR k TEL 072 636 8160 FAX 072 634 7222 http www dspbio cojp dspb ls bio ds pharma co jp IMPIOO30A2 MUBMX 01001 Page 15 of 15
9. Use a pipette to transfer the cells to the 15 mL conical tube containing OriCell Mouse MSC Growth Medium inside a biosafety cabinet Be careful not to introduce any bubbles during the transfer process 7 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 8 Gently mix the cell suspension by slowly pipetting up and down Be careful not to introduce any bubbles 9 Centrifuge the cell suspension at 250 x g for 5 minutes 10 Carefully aspirate off as much of the supernatant as possible and add 2 3 mL of fresh OriCell Mouse MSC Growth Medium pre warmed to 37 C 11 Gently resuspend the cells in OriCell Mouse MSC Growth Medium 12 Seed the cells into a T25 flask and add a sufficient amount of OriCell Mouse MSC Growth Medium Gently rock the culture flask to evenly distribute the cells 13 Incubate the flask at 37 C inside a 5 CO humidified incubator 14 The next day change the medium with fresh growth medium pre warmed to 37 C 15 Change the growth medium every three days thereafter 16 When the cells are approximately 80 90 confluent they can be dissociated with Trypsin EDTA and passaged A Note Changing Medium 1 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 co
10. arvested after 14 28 days in culture Pellets may be formalin fixed and paraffin embedded for alcian blue stain analysis Alcian Blue Staining Procedure 1 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 MSCs are differentiated into cartilages and are stained with alcian blue IMPIOO30A2 MUBMX 01001 Page 11 of 15 fA DS PHARMA ay SOMEDICAL P y a I e CRYOPRESERVATION OF CELLS USING OriCell CRYOPRESERVATION MEDIA OriCell NCR Protein Free Cryopreservation Medium Cat No NCPF 10001 is a protein free ready to use freezing medium Its chemically defined and protein 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 80 C Cryopreservation A Note Change the culture medium with fresh growth medium 24 hours before freezing 1 Col
11. fA DS PHARMA J Y BIOMEDICAL aaa O Cya e We help You discover life OriCell Strain C57BL 6 Mouse Mesenchymal Stem Cells MSCs Cat No MUBMX 01001 AA DS PHARMA AN BIOMEDICAL D C y d C Table of Contents Contents and StOlaGe aanianapinnsa dis sd den a Di na 3 PFOGUCE INTFOGUCEION ainasasicarsarissdesacicdsdosai A A A dani dai did pass dinda nadar 3 Cell Characteristics and Identity sssssss2225 255225222222202u202u220u20u200uunnnnnnnnnnnnnnnnnnnnnnnnnnnnn 3 Product APDIICATION iiidanciinadad a dad O da ad mad 4 General Handling Principl s svsccisiedvensdnccesccrrsnssendvesventcnedenssaagns NAAR 4 Culturing OriCell Strain C57BL 6 Mouse MSCs Thawing and Establishing OriCell Strain C57BL 6 Mouse MSCS sssssssssssnnnnnnnnnnnnnnnnnn 4 Passaging Cyagen OriCell Strain C57BL 6 Mouse MSCS vcccccsceseseeeucueeveveeeeeueeeenaraeas 6 Differentiation of OriCell Strain C57BL 6 Mouse MSCS s asssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 8 Cryopreservation of OriCell Strain C57BL 6 Mouse MSCS cccssececeecueceeveveeeeeueeneeeunaras 11 APPENA Qe er rene Tre re TT ter tr Tr ery rere er ere hr rr rr rrr TT rrr 13 TFOUDICSNOOUNG jossesisainiiisniiiisienies ini di did 13 Related Products assnsusmesssisaronicaasdvrsisisisdideriniodadncirecias ia dini disc ndid nisi idcadiis ada ic idas an 14 ROTOFONCOS ausigasiniasiasidasnad agendada Do dd aa dd dd ad ad EREE Rd a ed eaencens 14 TECHNICAL SUD
12. lease the majority of cells from the culture flask surface Important Avoid leaving cells exposed to the trypsin longer than necessary no more than two minutes if using Cyagen s trypsin EDTA solution Care should also be taken that the cells are not forced to detach prematurely as this may result in clumping IMPIOO30A2 MUBMX 01001 Page 6 of 15 fA DS PHARMA BIOMEDICAL C y a e o Jy 10 11 13 14 After the cells are visibly detached immediately add the pre warmed OriCell Mouse MSC Growth Medium 6 mL for T75 flask 3 mL for T25 flask to neutralize the trypsinization Gently pipette 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 Transfer the dissociated cells into a 15 mL conical tube Centrifuge at 250 x g for 5 minutes Carefully aspirate off as much of the supernatant as possible 12 Add 2 mL of OriCell Mouse MSC Growth Medium to the conical tube and gently resuspend the cells thoroughly Plate the cells into appropriate flasks OriCell Strain C57BL 6 Mouse MSCs can be split at 1 2 or other appropriate ratios Add an appropriate amount of medium to the cells Incubate the cells at 37 C inside a 5 CO humidified incubator Note Care should be taken to avoid introducing bubbles during pipetting Additional Tips Time to Change Medium It
13. lect 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 20 C Remove and discard the Supernatant using a pipette 3 Resuspend the cell pellet in the OriCell 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 80 C freezer After 24 hours transfer the frozen vials to liquid nitrogen for long term preservation IMPIOO30A2 MUBMX 01001 Page 12 of 15 fA DS PHARMA avy BIOMEDICAL Cyagen The table below lists some potential problems and solutions for culturing MSCs Problem Cause The storage condition does not meet the requirements Thawing of the cells takes too long Cells are incompletely Low cell recovery recovered after thawing rate Cells are handled roughly Medium is not pre warmed Mycoplasma contamination Slow cell growth Over digestion Plating density is too low Inappropriate serum and medium Dead cells are not removed promptly Cell aging Cell Contamination Plating density is too low IMPIOO30A2 MUBMX 01001 Solution Purchase a replacement and store in liquid nitrogen for long term preservation Thaw cells for no more than 3 minutes After aspirating off medium wash the tube with culture medium twice and tra
14. nsfer all of the cells to the dish Care should be taken to avoid introducing bubbles during pipetting Also avoid vortexing and high speed centrifugation Warm medium to 37 C before recovery Discard the cells in question and disinfect the laboratory environment before recovering the next batch of cells Wash the cells with PBS 2 3 times to remove serum prior to trypsinization serum will inhibit the function of trypsin Control the digestion time Increase the plating density Use Cyagen tailor made culture media If other serum and media products are used please perform validation to ensure compatibility Change the medium next day after recovery to ensure removal of all dead cells Discard the cells in question and disinfect the laboratory environment before recovering the next batch of cells 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 proliferation slow down and cell aging Page 13 of 15 fA DS PHARMA VY BIOMEDICAL Related Products Product OriCell Mouse Mesenchymal Stem Cell Growth Medium OriCell Mesenchymal Stem Cell Osteogenic Differentiation Medium OriCell Mesenchymal Stem Cell Adipogenic Differentiation Medium OriCell Mesenchymal Stem Cell Chondrogenic Differentiation Medium 0 25 Trypsin 0 04 EDTA Phosphate Buffered Saline 1xPBS OriCell NCR
15. o optimally differentiate MSCs into adipogenic cells repeat the cycle of induction and maintenance at least three times 10 After three to five cycles of induction and maintenance culture the cells in OriCell IMPIOO30A2 MUBMX 01001 Page 9 of 15 AA DS PHARMA AN BIOMEDICAL C y a y C 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 the medium every three days Oil Red O Stain Analysis 1 After the cells have differentiated remove the MSC maintenance medium from the wells and rinse with 1x phosphate buffered saline PBS Fix cells with 2 mL of 4 formaldehyde solution for 30 minutes 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 Cells can now be visualized and analyzed under a microscope EREN EDES 4 o J mo TASS ad Fig 4 OriCell Strain C57BL 6 Mouse MSCs are differentiated into adipocytes and are stained with oil red O Chondrogenic Differentiation Materials Required OriCell Mesenchymal Stem Cell Chondrogenic Differentiation Medium Cat No GUXMX 90041 Chondrogenesis Protocol il Calculate the total number of MSC pellet cultures required for your experiment 2 5x10 MSCs are needed to form each chondrogenic pellet
16. oling of the medium If the entire content is not needed for a single procedure transfer only the required volume to a sterile secondary container IMPIOO30A2 MUBMX 01001 Page 5 of 15 fA DS PHARMA Y Scyagen BIOMEDICAL AY Fig 1 OriCell Strain C57BL 6 Mouse Mesenchymal Stem Cells are established PASSAGING OriCell STRAIN C57BL 6 MOUSE MSCs Materials Required e 0 25 Trypsin 0 04 EDTA Cat No TEDTA 10001 e Phosphate Buffered Saline 1xPBS Cat No PBS 10001 e OriCell STRAIN C57BL 6 MOUSE Mesenchymal Stem Cells Cat No MUBMX 01001 e OriCell Mouse Mesenchymal Stem Cell Growth Medium Cat No MUXMX 90011 Passaging OriCell STRAIN C57BL 6 MOUSE MSCs 1 Pre warm the OriCell Mouse MSC Growth Medium 1xPBS and Trypsin EDTA solution to 37 C 2 Carefully aspirate the spent medium from the 80 90 confluent monolayer of MSCs Add 1xPBS 6 mL for T75 flask 3 mL for T25 flask Be careful not to disturb the monolayer Gently rock the flask back and forth to rinse the monolayer 4 Aspirate 1xPBS off and discard Repeat steps 3 4 two or three times 6 Add 0 25 Trypsin 0 04 EDTA solution 2 3 mL for T75 flask 1 mL for T25 flask Gently rock the flask back and forth to ensure that the entire monolayer is covered with the Trypsin EDTA solution Allow trypsinization to continue until the majority of the cells approximately 80 are rounded up At this point gently tap the side of the flask to re
17. or any other applications CELL CHARACTERISTICS AND IDENTITY e Strong capacity to expand Can be passaged at least 5 times e Multipotent differentiation ability along the osteogenic chondrogenic and IMPIOO30A2 MUBMX 01001 Page 3 of 15 Jd SSE ps parma 9 Cyagen adipogenic lineages e Positive for CD29 CD44 CD31 and Sca 1 gt 70 and negative for CD117 lt 5 in flow cytometry assays PRODUCT APPLICATIONS Strain C57BL 6 Mouse MSCs have become a popular research target due to their potential use in regenerative medicine and tissue engineering in areas such as cardiovascular neural and orthopedic disease OriCell Strain C57BL 6 Mouse MSCs can be used as cell models to evaluate the immunoreactions proliferation immigration and differentiation of MSCs both in vivo and in vitro GENERAL HANDLING PRINCIPLES A 1 Aseptic handling of the product is necessary throughout 2 Once the cells have been established always freeze several vials of OriCell Strain C57BL 6 Mouse MSCs as a backup Note The OriCell Strain C57BL 6 Mouse MSCs can be frozen thawed at least two times 3 For all studies it is strongly recommended to use cells that are at or under an original passage number of 10 4 For general maintenance of cells we recommend the seeding density to be 2 0 3 0x 10 cells cm 5 For general maintenance of cells we recommend that the medium is changed if it becomes acidic the
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