User Manual - Cyagen Biosciences
Contents
1. cells from the culture flask surface IMP10082A1 HUXUC 01001 Page 7 of 17 Cy cyagen 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 n clumping 7 After the cells are visibly detached immediately add the pre warmed OriCell 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 Umbilical Cord Blood MSCs can be split at 1 3 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 1 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 many2. 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 Human Umbilical Cord 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 IMP10082A1 HUXUC 01001 Page 8 of 17 O eyagen VES j Passage 7 40x Passage 7 100x Fig 2 Images of OriCell Human Umbilical Cord MSCs at passage 7 OriCell HUMAN UMBILICAL CORD MSC DIFFERENTIATION USING OriCell DIFFERENTIATION MEDIA OriCell Human Umbilical Cord 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 Human Umbilical Cord 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 3 Reseed the UC MSCs in the growth medium at 2x10 cells cm in a 6 w
3. 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 Fig 1 OriCell Human Umbilical Cord Mesenchymal Stem Cells are established PASSAGING OriCell Human Umbilical Cord 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 Human Umbilical Cord Mesenchymal Stem Cells Cat No HUXUC 01001 e OriCell Human Mesenchymal Stem Cell Growth Medium Cat No HUXMX 90011 Passaging OriCell Human Umbilical Cord MSCs 1 Pre warm the OriCell Human MSC Growth Medium 1xPBS and 0 25 Trypsin 0 0490EDTA solution to 379C 2 Carefully aspirate the spent medium from the 80 90 confluent monolayer of Human Umbilical Cord 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 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
4. Too 9 cya n We help you discover fe OriCell Human Umbilical Cord Mesenchymal Stem Cells Cat No HUXUC 01001 C3 cyagen Table of Contents Contents and Storage wrcccecccceeececeeveeeeeeeeeeeeaeeueeeeeeeeuaueuauauavauagaeageeeeueeeueueuauaeavauaeageseeuaeanay 3 Product Introduction nui YY KNA NA ALL ALAR ANA AA NAA NK NANANA aa NANANA NANANA KAKANAN 3 Cell Characteristics and Identity 2202242 00000004 G ANAKAN ANAK AKEN ANN NNN AA KAKANAN NANANA ANAKAN ANAKAN ANAKAN 3 Product Applications 22ANG AANI NAG 4 General Handling Principles 1 1 a 4 Culturing OriCell Human Umbilical Cord MSCs Thawing and Establishing OriCell Human Umbilical Cord MSCS sssssssssssssnsnsnnnnnnnnnnnnnn 5 Passaging Cyagen OriCell Human Umbilical Cord MSCS un 6 Differentiation of OriCell Human Umbilical Cord MSCS sccccsssseeseeeeeseeeneeeeseneeeees 8 Cryopreservation of OriCell Human Umbilical Cord MSCS ccccccssssssssessusseeeaeeseneeeens 12 Appendix HITHEU Troubleshooting EHHHHHHHHH GGH FR FERHRHEHRHHEFHRHFHRHEN 14 Related Products LWY RL NL ALLAH RL LRE ARR sae aeeaeaceeeae see aeaaeaaeesae EDR REN REN REN Eu 15 References Hn 15 6 cyagel CONTENTS AND STORAGE Product Name Human Umbilical Cord Mesenchymal Stem Cells Catalog No HUXUC 01001 Amount per Vial 1x10 Cells Cryopreserved At Second Passage Storage Condition Liguid Nitrogen CAUTION Ple
5. When the cells are 80 90 confluent subculturing the cells is strongly recommended 1 Note We strongly recommend the use of OriCell culture media and other related reagents for optimal results IMP10082A1 HUXUC 01001 Page 4 of 17 C3 cyagen THAWING AND ESTABLISHING OriCell Human Umbilical Cord MSCs Materials Required OriCell Human Umbilical Cord Mesenchymal Stem Cells Cat No HUXUC 01001 OriCell Human Mesenchymal Stem Cell Growth Medium Cat No HUXMX 90011 Thawing and Establishing Human Umbilical Cord MSCs 1 Pre warm the fully supplemented complete OriCell UC MSCs Growth Medium to 37 C 2 Add 9 mL of OriCell Human MSC Growth Medium to a 15 mL conical tube Remove the cryovial of OriCell Human Umbilical Cord MSCs from liquid nitrogen 4 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 A 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 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 an
6. ase handle this product as a potentially biohazardous material This A product contains Dimethyl Sulfoxide DMSO a hazardous material in the freezing medium PRODUCT INTRODUCTION Human Umbilical Cord Mesenchymal Stem Cells are multipotent stem cells that can differentiate into a variety of cell types including osteocytes adipocytes and chondrocytes Human umbilical cord 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 Human Umbilical Cord MSCs are derived from the Wharton s Jelly in umbilical cord which collected after normal full term delivery of healthy pregnant women They 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 5 times IMP10082A1 HUXUC 01001 Page 3 of 17 Cy cyagen e Multipotent differentiation ability along the
7. c 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 Umbilical Cord MSCs in the OriCell Human MSC 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 IMP10082A1 HUXUC 01001 Page 10 of 17 6 cyagen 3 Reseed the Human UC 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 5 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 MSCs into adipogenic cells repeat the cycle of induction and maintenance three times 10 After thre
8. 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 will lead to cell proliferation slow down and cell aging Page 15 of 17 Cp 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 Human Umbilical Cord Mesenchymal Stem Cells HUXUC 01001 OriCell Human Mesenchymal Stem Cell Growth Medium HUXMX 90011 OriCell Mesenchymal Stem Cell Osteogenic Differentiation GUXMX 90021 Medium OriCell Mesenchymal Stem Cell Adipogenic Differentiation GUXMX 90031 Medium OriCell Mesenchymal Stem Cell Chondrogenic GUXMX 90041 Differ
9. e 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 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 Human Umbilical Cord Mesenchymal Stem Cells are differentiated to adipocytes and are stained with Oil Red O IMP10082A1 HUXUC 01001 Page 11 of 17 Cy cyagen Chondrogenic Differentiation Materials Required OriCell Mesenchymal Stem Cell Chondrogenic 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 te
10. ell tissue culture plate pre coated with 0 1 gelatin solution 4 Incubate the cells at 37 C in a 5 CO humidified incubator 5 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 replacing the medium with fresh OriCell Mesenchymal Stem Cell Osteogenic Differentiation Medium pre warmed to 37 C IMP10082A1 HUXUC 01001 Page 9 of 17 6 cyagen 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 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 Fig 3 OriCell Human Umbilical Cord Mesenchymal Stem Cells are differentiated to Osteocytes and are stained with Alizarin Red S Adipogenic Differentiation Materials Required OriCell Mesenchymal Stem Cell Adipogeni
11. entiation Medium OriCell NCR Protein Free Cryopreservation Medium NCPF 10001 References Hwai Shi Wang Shih Chieh Hung and Shu Ting Peng 2004 Mesenchymal Stem Cells in the Wharton s Jelly of the Human Umbilical Cord Stem Cells 22 1330 1337 Rahul Sarugaser David Lickorish and Dolores Baksh 2005 Human Umbilical Cord Perivascular HUCPV Cells A Source of Mesenchymal Progenitors Stem Cells 23 220 229 Sercin Karahuseyinoglu Ozgur Cinar and Emine Kilic 2007 Biology of Stem Cells in Human Umbilical Cord Stroma In Situ and In Vitro Surveys Stem Cells 25 319 331 IMP10082A1 HUXUC 01001 Page 16 of 17 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 IMP10082A1 HUXUC 01001 Page17of17
12. fin embedded already 2 Staining procedure a b c d e Deparaffinize slides and hydrate to distilled water Stain in alcian blue solution for 30 minutes Wash in running tap water for 2 minutes Rinse in distilled water Visualize under a light microscope and capture images for analysis Blue staining indicates synthesis of proteoglycans by chondrocytes Fig 5 OriCell Human Umbilical Cord MSCs are differentiated to chondrocytes and are stained with Alcian Blue 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 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 IMP10082A1 HUXUC 01001 Page 13 of 17 Cy cyagen 3 Resuspend the cell pel
13. let in the OriCell NCR Protein Free Cryopreservation Medium at a cell density of 10 10 cells mL supernatant using a pipette 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 IMP10082A1 HUXUC 01001 Page 14 of 17 Cyagen The table below lists some potential problems and solutions for culturing Human UC MSCs Problem Low cell recovery rate Slow cell growth Cell aging IMP10082A1 HUXUC 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 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
14. mperature 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 0x 10 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 nor 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 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 harvested after 14 28 days in culture Pellets may be formalin fixed and paraffin embedded for alcian blue stain analysis IMP10082A1 HUXUC 01001 Page 12 of 17 C3 cyagen Alcian Blue Staining Procedure 1 The tissue sample should be formalin fixed and paraf
15. osteogenic chondrogenic and adipogenic lineages e Positive for CD29 CD44 CD73 CD105 and CD166 and negative for CD11a CD34 and CD45 in flow cytometry assays PRODUCT APPLICATIONS Umbilical Cord Mesenchymal Stem Cells UC 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 orthopaedic disease OriCell Umbilical Cord Mesenchymal Stem Cells can be used as cell models to test and evaluate the immunoreactions proliferation immigration and differentiation of UC MSCs 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 Umbilical Cord Mesenchymal Stem Cells UC MSCs as a backup A Note The OriCell Human Umbilical Cord MSCs can be frozen thawed at least one ZZ 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 Human Umbilical Cord MSCs overgrow as it will result in contact inhibition
16. y 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 Human MSC Growth Medium pre warmed to 37 C 11 Gently resuspend the cells in OriCell Human MSC Growth Medium 12 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 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 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 IMP10082A1 HUXUC 01001 Page 5 of 17 6 cyagei IMP10082A1 HUXUC 01001 Page 6 of 17 Note Changing Medium A 1 Warm an appropriate amount of medium to 37 C in a sterile container Replace the f spent medium with the pre warmed fresh medium Once completed return the flask to the incubator 2
Download Pdf Manuals
Related Search