User Manual - Cyagen Biosciences
Contents
1. M Fig 2 Images of OriCell Dog Adipose Derived Mesenchymal Stem Cells at passage 5 IMPIO053A2 CAXMD 01001 Page 7 of 14 qpcyagen OriCell DOG ADSC DIFFERENTIATION USING OriCellI DIFFERENTIATION MEDIA OriCell Dog 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 Note The protocol listed below is for 6 well tissue culture plates 1 Culture the OriCell Dog ADSCs 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 3 Reseed the MSCs in the growth medium at 2x10 cells cm in a 6 well tissue culture plate pre coated with 0 1 gelatin solution 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 Mesenchymal Stem Cell Osteogenic Differentiation Medium 6 Feedcells every 3 days for 2 3 weeks by completely replacing the medium with fresh OriCell Mesenchymal Stem Cell Osteogenic Differentiation Medium pre warmed to 37 C 7 After 2 3 weeks of differentiation cells can be fixed and st2. 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 Fig 4 OriCell Dog ADSCs 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 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 5x10 cells Centrifuge again at 150 x g for 5 minutes and then aspirate off the medium IMPIO053A2 CAXMD 01001 Page 10 of 14 K cyagen 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 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 in order to allow gas exchange and incubate the tubes at 37 C in a humidified atmosphere o
3. No CAXMD 01001 Amount per Vial 1x10 Cells Cryopreserved At Second 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 A 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 ADSCs 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 Dog Adipose Derived Mesenchymal Stem Cells are derived from the adipose tissue at inguen of newborn healthy Beagle dogs These cells 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 or any other applications CELL CHARACTERISTICS AND IDENTITY e Strong capacity to expand Can be passaged at least 5 times e Mu
4. related reagents for optimal results THAWING AND ESTABLISHING OriCell DOG ADSCs Materials Required OriCell Adipose Derived Stem Cell Growth Medium Cat No GUXMD 90011 Thawing and Establishing Dog ADSCs Pre warm the fully supplemented complete OriCell ADSC Growth Medium to IMPI0053A2 CAXMD 01001 Page 4 of 14 cyagen Add 9 mL of OriCell ADSC Growth Medium to a 15 mL conical tube Remove the cryovial of OriCell Dog ADSCs 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 10 11 12 13 14 15 16 A As soon as the cells are completely thawed disinfect the outside of the cryovial with 70 v v ethanol Use a pipette to transfer the cells to the 15 mL conical tube containing OriCell 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 bu
5. ED UM o zzQilll o C yd e We help You discover life OriCell Dog Adipose Derived Mesenchymal Stem Cells ADSCs Cat No CAXMD 01001 id 3 P we 2 si es C cyagen Table of Contents Contents aBd StOldU6 sisiiccncstsanscrnsdessesescancnd disses WNA R NA nin A SYRIA ONIS Y SCA Sn SERV Y ES SERES IMS 8 Product IntroduCcLtlOfi iesin aka E Rr dw dA ul iow a darn woo RW WA AW KE Cell Characteristics and Identity ini Yi YW YR sus EE Eua ERE ERE ER EE a BN cAn nw DAMES D Product ADDIICATIONS axeeaxuxxunsdf uiri b nb o UNDE OD RRKUEDS Su abb copo ee CS General Handlung PrincIDl S eidi Duu sus GR GN a HWNNA EE FEE a Enw Ra ECKE A Culturing OriCell Dog ADSCs Thawing and Establishing OriCell Dog ADSCS u uw nnn WW NL Y nun nun An 4 Passaging Cyagen OriCell Dog ADSCS enne nennen nnn nnne WAL NENN NY uu uu Differentiation of OriCell Dog ADSCs eene nennen nni nnnm uua h una nean YNN nun Yna nuu 8 Cryopreservation of OriCell Dog ADSCS eroe enne nennen WL WAY ARY aa a au n 12 APPEND dd O IH one Sad TFOUDIGSMOOUUNG aneis _ RH HER O FETH ANT CE RD RF HF PE HN AA oS Related Products c nun ARR EN RR HNN EENEEEEEENNNENNEEEEEENEENNNENNNEEEEEENNENNNENNEEEEEEENEEEEEau LG 6 cyagen CONTENTS AND STORAGE Product Name Dog Adipose derived Mesenchymal Stem Cells Catalog
6. ained 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 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 3 Rinse wells 2 3 times with 1x PBS 4 Cells can now be visualized and analyzed under a microscope IMPIO053A2 CAXMD 01001 Page 8 of 14 6 cyagen Fig 3 OriCell Dog ADSCs are differentiated into Osteocytes and are stained with Alizarin Red S Adipogenic Differentiation Materials Reguired 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 1 Culture the OriCell Dog ADSCs in the 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 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 incu
7. bator 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 8 24 hours later change the medium back to MSC Adipogenic Differentiation medium A 9 To optimally differentiate ADSCs into adipogenic cells repeat the cycle of induction and maintenance three times IMPIO053A2 CAXMD 01001 Page 9 of 14 o cyagen 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 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
8. bbles 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 ADSC Growth Medium pre warmed to 37 C Gently resuspend the cells in OriCell ADSC Growth Medium Seed the cells into a T25 flask and add a sufficient amount of OriCell ADSC Growth Medium Gently rock the culture flask to evenly distribute the cells Incubate the flask at 37 C inside a 5 CO humidified incubator The next day change the medium with fresh growth medium pre warmed to 379C Change the growth medium every two days until the cells are 80 confluent thereafter When the cells are approximately 80 90 confluent they can be dissociated with 0 25 Trypsin 0 04 EDTA and passaged Note Changing Medium Warm an appropriate amount of medium to 37 C ina 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 IMPI0053A2 CAXMD 01001 Page 5 of 14 o cyagen Fig 1 Oricell Dog Adipose Derived Mesenchymal Stem Cells are established PASSAGI NG OriCell DOG ADSCs Materials Reguired e 0 25 Trypsin 0 04 EDTA Cat No TEDTA 10001 e Phosphate Buffered Saline 1xPBS Cat No PBS 10001 e O
9. f 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 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 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 Dog ADSCs are differentiated into chondrocytes and are stained with Alcian Blue IMPIO053A2 CAXMD 01001 Page 11 of 14 cyagen CRYOPRESERVATION OF CELLS USING OriCell CRYOPRESERVATION MEDI A OriCell NCR Protein Free Cryopreservation Medium Cat No NCPF 10001 is a protein free ready to use freez
10. ing 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 I 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 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 IMPI0053A2 CAXMD 01001 Page 12 of 14 Cyagen The table below lists some potential problems and solutions for culturing ADSCs Problem Low cell recovery rate Slow cell growth Cell aging IMPIO053A2 CAXMD 01001 Cause The storage condition does not meet the requirements Thawing of the cells takes too long Cells are incompletely rec
11. ltipotent differentiation ability along the osteogenic chondrogenic and IMPIO053A2 CAXMD 01001 Page 3 of 14 5 cyagen adipogenic lineages PRODUCT APPLICATIONS Dog ADSCs 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 Dog 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 PRI NCI PLES A 1 Aseptic handling of the product is necessary throughout 2 Once the cells have been established always freeze up several vials of OriCell Dog ADSCs as a backup Note The OriCell DOG ADSCs can be frozen thawed at least twice For general maintenance of cells we recommend the seeding density to be 2 0 2 5x10 cells cm For all studies it is strongly recommended to use cells that are at or under an original passage number of 10 For general maintenance of cells we recommend that the medium is changed if it becomes acidic the pH indicator in medium appears yellow In general change the growth medium every three days Do not let OriCell DOG ADSCs overgrow as it will result in contact inhibition When the cells are 80 90 confluent subculturing the cells is strongly recommended Note We strongly recommend the use of OriCell culture media and other
12. overed after thawing Cells are handled roughly Medium is not pre warmed Mycoplasma contamination Over digestion Plating density is too low Inappropriate serum and medium Dead cells are not removed promptly Cell Contamination Plating density is too low 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 transfer 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 the 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
13. riCell Dog Adipose Derived Stem Cells Cat No CAXMD 01001 e OriCell Adipose Derived Stem Cell Growth Medium Cat No GUXMD 90011 Passaging OriCell Dog ADSCs 1 Pre warm the OriCell ADSC Growth Medium 1xPBS and 0 25 Trypsin 0 04 EDTA solution to 379C 2 Carefully aspirate the spent medium from the 80 90 confluent monolayer of ADSCs 3 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 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 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 flask surface I mportant 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 A IMPIO053A2 CAXMD 01001 Page 6 of 14 o cyagen 7 After the cells are visibly detached immediately add the pre warmed OriCell M ADSC Growth Medium 6 mL for T75 flask 3 mL for T25 fla
14. sk to neutralize the trypsinization 8 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 9 Transfer the dissociated cells into a 15 mL conical tube 10 Centrifuge at 250 x g for 5 minutes 11 Carefully aspirate off as much of the supernatant as possible 12 Add 2 mL of OriCell ADSC Growth Medium to the conical tube and gently resuspend the cells thoroughly 13 Plate the cells into appropriate flasks OriCell Dog ADSCs can be split at 1 2 or other appropriate ratios 14 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 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 Dog 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 md re ae LA Passage 5 40x Passage 5 40x
15. will lead to cell proliferation slow down and cell aging Page 13 of 14 6 cyagen 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 0 25 Trypsin 0 04 EDTA TEDTA 10001 Phosphate Buffered Saline 1xPBS PBS 10001 OriCell Dog Adipose Derived Mesenchymal Stem Cells CAXMD 01001 OriCell Dog Adipose Derived Mesenchymal Stem Cell GUXMD 90011 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 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 adapted for other purposes without written permission from Cyagen Biosciences IMPIO053A2 CAXMD 01001 Page 14 of 14
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