User Manual - Cyagen Biosciences
Contents
1. Osteogenic Differentiation Materials Reguired 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 Human MSCs in OriCell Human MSC 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 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 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 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 PBS Fix cells with 2 mL of 4 formald2. directly frozen at 809C Cryopreservation l Note Change the culture medium with fresh growth medium 24 hours before N 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 IMPI0054A2 HUXMA 01001 Page 12 of 15 Cyagen The table below lists some potential problems and solutions for culturing MSCs Problem Cause Solution The storage condition does Purchase a replacement and store in liquid not meet the requirements nitrogen for long term preservation Thawing of the cells takes too 8 Thaw cells for no more than 3 minutes long Gallearauncemplctal After aspirating off medium wash the tube LON cE Eeevely P ol with culture medium twice and transfer all of rate recovered after thawing 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 warm
3. 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 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 Mesenchymal Stem Cell Adipogenic Differentiation medium B for an additional 4 7 IMPI0054A2 HUXMA 01001 Page 9 of 15 6 cyagen 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 Cells can now be visualized and analyzed under a microscope Fig 4 OriCell Human MSCs are differentiated into adipocytes and are stained with oil red O Chondrogenic Differentiation Materials Required OriCell Mesenchymal Stem Cell Chondrogenic
4. 7 After the cells are visibly detached immediately add the pre warmed OriCell IMPI0054A2 HUXMA 01001 Page 6 of 15 6 cyagen Human MSC Growth Medium 6 mL for T75 flask 3 mL for T25 flask 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 Human MSC Growth Medium to the conical tube and gently resuspend the cells thoroughly 13 Plate the cells into appropriate flasks OriCell Human MSCs can be split at 1 2 or other appropriate ratios 14 Add an appropriate amount of medium to the cells Incubate the cells at 379C inside a 5 CO gt 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 Time to Subculture When OriCell Human MSCs are 80 90 confluent it is recomme
5. 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 Luis A Solchaga Brian Johnstone and Jung U Yoo 1999 High variability in Human bone marrow derived mesenchymal cell preparations Cell transplantation 8 511 519 C Y Charles Huang Kristen L Hagar and Lauren E Frost 2004 Effects of Cyclic Compressive Loading on Chondrogenesis of Human Bone Marrow Derived Mesenchymal Stem Cells Stem Cells 22 313 323 IMPI0054A2 HUXMA 01001 Page 14 of 15 6 cyagen 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 IMPI0054A2 HUXMA 01001 Page15of 15
6. 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 Human MSC Growth Medium pre warmed to 37 C Gently resuspend the cells in OriCell Human MSC Growth Medium Seed the cells into an appropriate flask such that the density is 2 0 2 5x10 alive cells cm and add a sufficient amount of OriCell Human MSC Growth Medium Gently rock the culture flask to evenly distribute the cells Incubate the cells at 37 C inside 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 two days until the cells are 80 confluent thereafter 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 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 IMPI0054A2 HUXMA 01001 Page 5 of 15 6 cyagen Fig 1 OriCell Human Mesenchyma
7. 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 Human MSCs are differentiated into cartilages and are stained with alcian blue IMPI0054A2 HUXMA 01001 Page 11 of 15 6 cyagen 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
8. Differentiation Medium Cat No GUXMX 90041 Chondrogenesis Protocol 1 Calculate the total number of MSC pellet cultures required for your experiment 2 5x10 MSCs are needed to form each chondrogenic pellet Transfer this amount of cells into an appropriate culture tube 2 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 3 Resuspend the MSCs in Complete Chondrogenic medium to a concentration of 5 0x10 cells mL IMPI0054A2 HUXMA 01001 Page 10 of 15 6 cyagen 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 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 1 200 uL pipette tip to the end of the aspirating pipette Add 0 5 mL of freshly prepared Complete Chondrogenic Medium to each tube 7 After
9. NTS AND STORAGE Product Name Human Mesenchymal Stem Cells Catalog No HUXMA 01001 Amount per Vial 1x10 Cells Cryopreserved At Second Passage Storage Condition Liguid 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 Mesenchymal stem cells MSCs are multipotent stem cells that can differentiate into a variety of cell types including osteocytes adipocytes and chondrocytes MSCs proliferate guickly 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 Human Mesenchymal Stem Cells are derived from the bone marrow of New Zealand Humans 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 or any other applications CELL CHARACTERISTICS AND IDENTITY e Strong capacity to expand Can be passaged at least 5 times e M
10. ae cya J 0 n We help gou discover life OriCell Human Mesenchymal Stem Cells MSCs Cat No HUXMA 01001 6 cyagen Table of Contents Contents and Storddg inii WR YY YRS DAWN WAY WORN MM WAWR NA N WW MU UWYD RYW A YN CC GT 3 Product IntroductilOPM eiii w n GR wn GWGAN a n NON Wn Ai a i Fa Ro nd E 3 Cell Characteristics and Identity inii YH Y YN RY GRWN RWY WD ARR WNAWN WAW RH UR SN UDA RGC MR 3 Product ADDICAUION iii iran HWNN NW NR Y n n WWW RWYD WN A N AU MG A N CR aW O WT AC YS MG NS 4 General Handling Principles sssssiicscccssccsscccssicarcancsascnreedcssadedsauseusescquvessacssaneseestversssunveus 4 Culturing OriCell Human MSCs Thawing and Establishing OriCell Human MSCS sssssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn Hu 4 Passaging Cyagen OriCell Human MSCS cccccccececcececucueeneeeeeueueueueaveveeeueueeneveueuaneusenenes 6 Differentiation of OriCell Human MSCS csccsccccceeeeeseeseceeeeeeeeeeeueeeeeseueeuecneaeeueaueensseaneaes 8 Cryopreservation of OriCell Human MSCS ccccsccecsccceeeeecceeeeueueaeeeeeeueueueeveeeeaueusanenenans 12 Tajo ils Geen enter O AO A RE UE RH ere errr ere rece re rrr errr er err er rece rrr TT 13 Troubleshooting seinir yn NY NWYN RR RC CS WC CAR CW YS 13 Related PI OdHcCLS aei YU A A NAWR ST DU S UNO N CN G WR MAN Mi Sn Y A 14 ReTer6nce uei aieu su Gu OR wdn ST WD UR DC ATRA Yn A UR RY 14 Tecnni lt cai SUnDDDI U seen GR dr GY ieee 15 6 cyagen CONTE
11. ed Warm medium to 37 C before recovery Discard the cells in guestion and disinfect the Mycoplasma contamination laboratory environment before recovering the next batch of cells Wash the cells with PBS 2 3 times to remove Slow cell growth serum prior to trypsinization serum will inhibit Over digestion 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 Dead cells are not removed Change the medium next day after recovery promptly to ensure removal of all dead cells Discard the cells in question and disinfect the Cell aging Cell Contamination laboratory environment before recovering the next batch of cells Some stem cells can secrete factors to support cell growth Therefore a certain Plating density is too low degree of plating density must be maintained otherwise it will lead to cell proliferation slow down and cell aging IMPI0054A2 HUXMA 01001 Page 13 of 15 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 OriCell Mesenchymal Stem Cell Growth Medium GUXMX 90011 OriCell Mesenchymal Stem Cell Osteogenic GUXMX 90021 Differentiation Medium
12. ehyde 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 IMPIOO54A2 HUXMA 01001 Page 8 of 15 6 cyagen Fig 3 OriCell Human MSCs 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 Human MSCs 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 MSCs 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
13. l Stem Cells are established PASSAGING OriCell HUMAN MSCs Materials Required 0 2596Trypsin O 0496EDTA Cat No TEDTA 10001 e Phosphate Buffered Saline 1xPBS Cat No PBS 10001 e OriCell Human Mesenchymal Stem Cells Cat No HUXMA 01001 e OriCell Human Mesenchymal Stem Cell Growth Medium Cat No HUXMX 90011 Passaging OriCell Human MSCs 1 Pre warm the OriCell Human MSC 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 MSCs 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 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 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
14. nded that the cells be subcultured Do not let the cells overgrow as it will result in contact inhibition I NY t NAY OR CE f RYAN KA N A LE en ty ene Wigs NESS Set cr i MEN E ZAI SE SIN OO SS A NN IMs See NEE AS Tse AY AYES Wd at 21 fal DAS AN EES SS Hh Hes INN ison EN dd h i PENOLA UE SST UN Ge cee eee orem I IL CESS AALS SSS YN ANAN NHS A INN _ eri 5 lf We ENN te NN SN SS DY A y NE Wr YN A FAU Me lt me ae r Sh NS YD i NN ue AW LA LY Fay gi 5y A q Ap IAN ONN AH UF APT Le Nis Y SN AES y YDYM AC SS Ais Ui fed i A ae N NRE OA ie Z aif aaa A cs ii Se MA ANI A A A SD MWD Mani ARSC ar z my WNCUD E i yy aM MAES U SW MEDU MW mA BW NWN iin NY usa Ly is a Rea SN AY i 3 ee ws sehen ya ddd YU oh Warne AAS ay ied f TIN E WN SN y WIB 00 YD a WN OY SS o hs KK DA FD el WE AW i ARR O Li AHA Sie EEA oe Rr NIAD MNE TN oes NW Wises PT FFAN N N ee Ne WNW NN SCZ NWN iS I NN SS NN NN WN x NW LEGGE If ZEAE u Passage 3 at 40x Passage 3 at 100x Fig 2 Images of OriCell Human Mesenchymal Stem Cells are 90 confluent at passage 3 IMPI0054A2 HUXMA 01001 Page 7 of 15 6 cyagen OriCell HUMAN MSC DIFFERENTIATION USING OriCell M DIFFERENTIATION MEDIA OriCell Human MSCs can differentiate into a variety of cell types including osteocytes adipocytes and chondrocytes
15. ring the cells is strongly recommended Note We strongly recommend the use of OriCell culture media and other related reagents for optimal results THAWING AND ESTABLISHING OriCell HUMAN MSCs Materials Required e OriCell Human Mesenchymal Stem Cell Growth Medium Cat No HUXMX 90011 Thawing and Establishing Human MSCs IMPIOO54A2 HUXMA 01001 Page 4 of 15 oS SS 6 cyagen Pre warm the fully supplemented complete OriCell MSC Growth Medium to 37 C Add 9 mL of OriCell MSC Growth Medium to a 15 mL conical tube Remove the cryovial of OriCell Human 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 minutes l Note Results will be less than optimal if the cells are thawed for more than 3 10 11 12 13 14 15 16 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 Human MSC 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
16. ultipotent differentiation ability along the osteogenic chondrogenic and IMPI0054A2 HUXMA 01001 Page 3 of 15 adipogenic lineages e Positive for CD29 CD44 and CD105 gt 70 and negative for CD34 and CD45 lt 5 in flow cytometry assays PRODUCT APPLICATIONS Human 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 Human MSCs can be used as cell models to evaluate the immunoreactions proliferation immigration and differentiation of MSCs both n 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 several vials of OriCell Human MSCs as a backup Note The OriCell Human MSCs can be frozen thawed at least one 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 1 0 2 0x10 cells cm 5 For general maintenance of cells we recommend that the medium is changed if it becomes acidic the pH indicator in the medium appears yellow In general change the growth medium every three days 6 Do not let OriCell Human MSCs overgrow as it will result in contact inhibition When the cells are 80 90 confluent subcultu
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