User Manual
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1. gt Pre warm the fully supplemented complete OriCell ADSC Growth Medium to 57 2 Add 9 mL of OriCell ADSC Growth Medium to a 15 mL conical tube Remove the cryovial of OriCell SD Rat ADSCs from liquid nitrogen Quickly thaw the cryovial in 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 vial with 70 v v ethanol 6 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 7 Rinse the vial with 1 mL of the medium to reduce cell loss Subsequently transfer this 1 mL of cell suspension to 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 ADSC Growth Medium pre warmed to 37 C 11 Gently resuspend the cells in OriCell ADSC Growth Medium 12 Seed the cells into a T25 f2. neural and orthopedic disease OriCell SD Rat 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 OriCell SD Rat ADSCs as a backup l Note The OriCell SD RAT ADSCs can be frozen thawed at least one times 3 For general maintenance of cells we recommend the seeding density to be 1 5 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 medium appears yellow In general change the growth medium every three days 6 Do not let OriCellTM SD Rat ADSCs overgrow as it will result in contact inhibition When the cells are 80 90 confluent subculturing the cells is strongly recommended 4 Note We strongly recommend the use of OriCell culture media and other related reagents for optimal results THAWING AND ESTABLISHING OriCell SD RAT ADSCs Materials Required e OriCell Adipose derived Stem Cell Growth Medium Cat No GUXMD 90011 Thawing and Establishing SD Rat ADSCs IMPI0021A2 RASMD 01001 Page 4 of 14 a DS PHARMA BIOMEDICAL OH C y d 6 EK
3. wells and add 2 mL of OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation medium A induction medium per well Three days later change the medium to OriCellTM 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 maintenance three times IMPI0021A2 RASMD 01001 Page 9 of 14 a DS PHARMA VY BIOMEDICAL OH P y d H e H 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 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 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 Fig 4 OriCell SD Rat ADSCs are differentiated into adipocytes and are stained with oil red O C
4. Le DS PHARMA J Y BIOMEDICAL E OH CVA D 6 Il We help goa discover life OriCell Sprague Dawley SD Rat Adipose Derived Mesenchymal Stem Cells ADSCs Cat No RASMD 01001 a DS PHARMA VY BIOMEDICAL OH C y d H 6 n Table of Contents Contents and EH e Ve Le TEE 3 Product Introduction siena i ee ro 3 Cell Characteristics and Identity ini ii 3 Product ADDIICALIONS sini 4 General Handling PrincipleS ssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 4 Culturing OriCell SD Rat ADSCs Thawing and Establishing OriCell SD Rat ADSCS ccccscccsseceeueeseeeeeeeeeueeeeueuseneesaneesees 4 Passaging Cyagen OriCell SD Rat ADSCS 000 rin 6 Differentiation of OriCell SD Rat ADSCS 1 1000 iii 8 Cryopreservation of OriCell SD Rat ADSCS rr 13 APENE sn RR 14 TrOUDIESNOOEMO TN 14 Related Products EE 15 References i cisurian iaia ine 15 a DS PHARMA VY BIOMEDICAL OH C y d H 6 n CONTENTS AND STORAGE Sprague Dawley SD Rat Adipose derived p N roduct Name Mesenchymal Stem Cells Catalog No RASMD 01001 Amount per Vial 1x10 Cells Cryopreserved At Second 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 Adipose derived mesenchymal stem cell
5. SCs 3 Add 1xPBS 6 mL for T75 flask 3 mL for T25 flask Be careful not to disturb the monolayer Rinse the monolayer by gently rocking the flask back and forth 4 Aspirate 1x PBS off 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 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 7 After the cells are visibly detached immediately add the pre warmed OriCell IMPIO021A2 RASMD 01001 Page 6 of 14 a DS PHARMA WY BIOMEDICAL OH C y a H e H 10 11 13 14 ADSC 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 conic
6. al 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 ADSC Growth Medium to the conical tube and gently resuspend the cells thoroughly Plate the cells into appropriate flasks OriCell SD Rat ADSCs 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 bubble 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 SD Rat ADSCs 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 er na SIAL AGL Geri Se 7 f n r E gm f Passage 3 at 40x Passage 3 at 100x Fig 2 Images of OriCell SD Rat ADSCs at passage 3 IMPI0021A2 RASMD 01001 Page 7 of 14 a DS PHARMA VY BIOMEDICAL OH C y d H 6 n OriCell SD RAT ADSCs DIFFERENTIATION USING OriCell DIFFERENTIATION MEDIA gt OriCell SD Rat ADSCs ca
7. ate 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 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 14 fA DS PHARMA CAN ere OH P y d e H RELATED PRODUCTS Product Catalog Number OriC
8. ell Mesenchymal Stem Cell Growth Medium GUXMX 90011 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 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 ABMERRRT IL 7 DS IP VINA XTD LIRR rt T564 0063 Zeie Dep IRET B1B435 KYUHOiTIRE JL Efe FE IE AREA HAS TEL 06 6990 8051 FAX 06 6325 6058 F9 DI HH TEL 072 636 8160 FAX 072 634 7222 http www dspbio co jp dspb ls bio ds pharma co jp IMPI0021A2 RASMD 01001 Page 14 of 14
9. em 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 Note Change the culture medium with fresh growth medium 24 hours before A 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 After 24 hours transfer the frozen vials to liquid nitrogen for long term preservation IMPI0021A2 RASMD 01001 Page 12 of 14 fA DS PHARMA Ska BIOMEDICAL Cyagen The table below lists some potential problems and solutions for culturing ADSCs Problem Low cell recovery rate Slow cell growth Cell aging IMPI0021A2 RASMD 01001 Cause The storage condition does not meet the requirements Thawing of the cells takes too long Cells are incompletely recovered after thawing Cells are handled roughly Medium is not pre warmed Mycoplasma contamination Over digestion Plating density is too low Inappropri
10. hondrogenic Differentiation Materials Required OriCell Mesenchymal Stem Cell Chondrogenic Differentiation Medium Cat No GUXMX 90041 Chondrogenesis Protocol 1 Calculate the total number of 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 3 Resuspend the ADSCs in Complete Chondrogenic medium to a concentration of IMPI0021A2 RASMD 01001 Page 10 of 14 fA DS PHARMA Ska RIOMEDICAL OH C y a H e H 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 one half turn 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 replacing the medium in each tube to avoid aspirating the pellets when aspirating the medium attach a sterile 1 200uUL pi
11. lask and add a sufficient amount of OriCell ADSC Growth Medium Gently rock the culture flask to evenly distribute the cells 13 Incubate the flask at 37 C in 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 two days until the cells are 80 confluent thereafter 16 When the cells are approximately 80 90 confluent they can be dissociated with 0 25 Trypsin 0 04 EDTA and passaged Note Changing Medium 1 2 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 IMPI0021A2 RASMD 01001 Page 5 of 14 a DS PHARMA 5 en J yy BIOMEDICAL OH 6 y a y Figure 1 OriCell SD Rat ADSCs are established Passaging OriCell SD Rat ADSCs Materials Required e 0 25 Trypsin 0 04 EDTA Cat No TEDTA 10001 e Phosphate Buffered Saline 1x PBS Cat No PBS 10001 e OriCellTM ADSC Growth Medium Cat No GUXMD 90011 Passaging SD Rat ADSC 1 Pre warm the OriCell ADSC Growth Medium 1xPBS and 0 25 Trypsin 0 04 EDTA solution to 37 C 2 Carefully aspirate the spent medium from the 80 90 confluent monolayer of SD Rat AD
12. n 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 SD Rat ADSCs in OriCell ADSC 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 ADSCs in growth medium at 3 x 10 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 CO2 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 3 days for 2 3 weeks by completely replace 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 stained with alizarin red S 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 differentiatio
13. n 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 IMPI0021A2 RASMD 01001 Page 8 of 14 a DS PHARMA EK BIOMEDICAL OH C y d 6 Fig 3 OriCell SD Rat ADSCs 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 SD Rat ADSCs in the OriCell 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 oTrypsin 0 04 EDTA 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 aspirate off the spent growth medium from the
14. pette 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 SD Rat ADSCs are differentiated into chondrocytes and are stained with alcian blue IMPIO021A2 RASMD 01001 Page 11 of 14 fA DS PHARMA Ska RIOMEDICAL OH P y d e H 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 th
15. s ADSCs are multipotent stem cells that can differentiate into a variety of cell types including osteocytes adipocytes and chondrocytes These cells have wide applications in tissue engineering cell therapy and gene therapy OriCell SD Rat ADSCs are derived from the adipose tissue of qualified SD rat inguen 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 e Multipotentdifferentiation ability along osteogenic chondrogenic and adipogenic lineages IMPI0021A2 RASMD 01001 Page 3 of 14 fA DS PHARMA Ska RIOMEDICAL OH P y a e H e Positive for CD44 CD90 and CD29 gt 70 negative for CD34 CD11b and CD45 lt 5 in flow cytometry analysis PRODUCT APPLICATIONS ADSCs have become a popular research target for their potential use in regenerative medicine and tissue engineering in areas such as cardiovascular
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