User Manual - Cyagen Biosciences
Contents
1. 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 cyagen 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 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 809C Cryopreservation A 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 20 C 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 80 C freezer A2. 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 1000 3 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 4 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 6 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 7 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 10 After three to five cycles of induction and maintenance culture the cells in OriCell 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 6 cyagen the medium every three d
3. If other serum and media products are used Inappropriate serum and please perform validation to ensure medium compatibility Ball aoce eae Change the medium the next day after Cell aging recovery to ensure removal of all dead ee cells Discard the cells in question and disinfect the laboratory environment before Cell Contamination recovering the next batch of cells IMPI0020A2 MUBMD 01001 Page 13 of 14 6 cyagen 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 OriCcell 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 ISCT 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 ada
4. adipogenic lineages e Positive for CD29 CD44 and Sca 1 gt 70 and negative for CD117 lt 5 in flow cytometry assays PRODUCT APPLI CATI ONS 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 HANDLI NG 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 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 Mouse ADSCs overgrow as it will result in contact inhibition When the cells are 80 90 confluent subculturing the cells
5. is strongly recommended l 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 Q cyagen OriCell Mouse Adipose Derived Mesenchymal Stem Cell Growth Medium Cat No MUXMD 90011 Thawing and Establishing Mouse ADSCs ANTT p 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 yN Note Results will be less than optimal if the cells are thawed for more than 3 minutes 10 Ii 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 OriCell Mouse ADSC Growth Medium inside a biosafety cabinet Be careful not to introduce any bubbles during the transfer process Rinse the vial
6. After the cells are visibly detached immediately add the pre warmed OriCell Mouse IMPI0020A2 MUBMD 01001 Page 6 of 14 6 cyagen 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 gently 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 37 C inside a 5 CO humidified incubator A 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 thre
7. D cyd i 0 We help gou discover life OriCell Strain C57BL 6 Mouse Adipose Derived Mesenchymal Stem Cells ADSCs Cat No MUBMD 01001 Table of Contents Contents and Storage ri ia RR a GY NS A Y A Product IntroductlOll sai RYG y wi MR RD WR RAN GW RR DR RR Y RW RF FR RR RR GOD YD WW WR Y Cell Characteristics and Identity si iu awA wb niw i R GN UR Yw sd wM A n NR nnn Product ADDICaEIOD S5 aiii GY I i nw O YA A AS CY ANAW GR 0 Y General Handling PrincipleS uii NWR GRWN RW iu NN GWM G WW WU ARAN W nM u w A MA RA WRN Ri WR ADO YRR RO A WA Culturing OriCell Strain C57BL 6 Mouse ADSCs Thawing and Establishing OriCell Strain C57BL 6 Mouse ADSCS ccscsseeeeceeeneneneneees Passaging Cyagen OriCell Strain C57BL 6 Mouse ADSCS y dnn Differentiation of OriCell Strain C57BL 6 Mouse ADSCs SW Y Yny ynan Cryopreservation of OriCell Strain C57BL 6 Mouse ADSCS e iun BONG societies cee TE OUDICS OO EII1 iia cui Gun Ri AY nie see Co RF CR NS ffa FYR EEEE E neds Related Products iei YG RY A A Gn Cyn OC DD dT ARON OND OU hU AFF Y References EUUHHHHEHEHHHUHHEHHHHHHEHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHEHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHEHHHEUHH 6 cyagen 12 13 13 14 14 6 cyagen CONTENTS AND STORAGE Strain C57BL 6 Mouse Adipose Product Name j Derived Mesenchymal Stem Cell Catalog No MUBMD 01001 Amount per Vial 1x10 Ce
8. TM 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 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 9 cyagen 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 1 Culture the OriCell Strain C57BL 6 Mouse ADSCs in the OriCell Mouse ADSC Growth Medium at 37 C in a 5
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10. ays 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 3 Rinse wells 2 3 times with 1x PBS 4 Cells can now be visualized and analyzed under a microscope Fig 4 OriCell Strain 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 cell
11. d e 0 2590Trypsin O 0496EDTA Cat No TEDTA 10001 e Phosphate Buffered Saline 1x PBS Cat No PBS 10001 e OriCell Strain C57BL 6 Mouse Adipose Derived Mesenchymal Stem Cells Cat No MUBMD 01001 e OriCell Mouse Adipose Derived Mesenchymal Stem Cell Growth Medium Cat No MUXMD 90011 Passaging Mouse ADSC 1 Pre warm the OriCell Mouse ADSC Growth Medium 1xPBS and 0 25 Trypsin 0 04 EDTA solution to 37 C 2 Carefully aspirate spent medium from the 80 90 confluent monolayer of OriCell Strain C57BL 6 Mouse ADSCs 3 Add 1xPBS 6 mL for T25 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 1x PBS and discard Repeat the step 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 0 25 Trypsin 0 04 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 release the majority of cells from the culture surface Important Avoid leaving cells exposed to the trypsin longer than necessary no A 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 7
12. e 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 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 qcyagen 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 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 OriCell
13. fter 24 hours transfer the frozen vials to liquid nitrogen for long term preservation IMPI0020A2 MUBMD 01001 Page 12 of 14 Cyagen Some stem cells can secrete factors to tan support cell growth Therefore a certain Plating density is too low degree of plating density must be Cell aging maintained otherwise it will lead to cell The table below lists some potential problems and solutions for culturing ADSCs Problem Cause Solution Purchase a replacement and store in liquid The storage condition does nitrogen for long term preservation not meet the requirements Thawing of the cells takes too 8 Thaw cells for no more than 3 minutes long EA Renee After aspirating off medium wash the Low cell recovery a a tube with culture medium twice and transfer all of the cells to the dish rate Care should be taken to avoid introducing bubbles during pipetting Also avoid Cells are handled roughly vortexing and high speed centrifugation Medium is not pre warmed Warm medium to 37 C before recovery Discard the cells in guestion and disinfect the laboratory environment before Mycoplasma contamination recovering the next batch of cells Wash the cells with PBS 2 3 times to remove serum prior to trypsinization serum Slow cell growth 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
14. lls Cryopreserved At Sixth Passage Storage Condition Liquid Nitrogen CAUTI ON Please handle this product as a potentially biohazardous material This product contains Dimethyl Sulfoxide DMSO a hazardous material in the freezing medium PRODUCT I NTRODUCTI ON 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 C57BL 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 IMPI0020A2 MUBMD 01001 Page 3 of 14 qj cyagen e Multipotent differentiation ability along osteogenic chondrogenic and
15. pted for other purposes without written permission from Cyagen Biosciences IMPI0020A2 MUBMD 01001 Page 14 of 14
16. s mL 4 Aliquot 0 5 mL 2 5x10 cells of the cell suspension into 15 mL polypropylene IMPI0020A2 MUBMD 01001 Page 10 of 14 O cyagen culture tubes Centrifuge the cells at 150 x g for 5 minutes at room temperature y Note DO NOT aspirate the supernatant or resuspend the pellet 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 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 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 379C incubator 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 1 The tissue sample should be formalin fixed and paraffin embedded already 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
17. 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 37 C 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 secondary container IMPI0020A2 MUBMD 01001 Page 5 of 14 6 cyagen Fy CD pa t ay hen df nm y SSS AY YN ies A Ag a 1 rd te
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