User Manual - Cyagen Biosciences
Contents
1. Cat No MUXEF 90011 Thawing and Establishing MEFs Pre warm the OriCell MEF Growth Medium to 37 C Add 9 mL of OriCell MEF Growth Medium to a 15 mL conical tube Remove the cryovial of OriCell Strain ICR MEFs from liquid nitrogen Quickly thaw the vial 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 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 15 mL conical tube containing OriCell MEF Growth Medium inside a biosafety cabinet Be careful not to introduce any bubbles during the transfer process Rinse the vial with 1 mL of medium to reduce cell loss Subsequently transfer this 1mL of cell suspension to 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 3 mL of fresh OriCell MEF Growth Medium pre warmed to 37 C 10 Gently resuspend the cells in OriCell MEF Growth Medium IMPIO024A3 MUBES 01001 Page 5 o2. Remove the cryovial of OriCell Strain C57BL 6 Mouse ESCs from liquid nitrogen 4 Quickly thaw the vial in 37 C water bath until the last ice piece 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 In a laminar flow hood use pipette to transfer the cells to the conical tube containing OriCell Mouse ESC Growth Medium A Note Be careful not to introduce any bubbles during the transfer process 7 Rinse the vial with 1 ml of medium to reduce the loss of cell and then transfer the 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 suspensions at 250 x g for 5 minutes 10 Carefully aspirate as much of the supernatant as possible and add 3 mL of fresh OriCell Mouse ESC Growth Medium pre warmed to 37 C 11 Gently re suspend the cells in OriCell Mouse ESC Growth Medium 12 Plate the cells into TWO T25 flasks and add sufficient OriCell Strain C57BL 6 Mouse ESCs Gently rock the culture flask to evenly distribute the cells 13 Incubate at 37
3. 9 of 14 o eyagen exists and gap juncitons may be established Protocol il 10 11 12 Dissociate OriCell Strain C57BL 6 Mouse ESCs by incubating the cells with trypsin solution at 37 C for 1 2 min Add an appropriate volume of Cyagen OriCell EB Formation Medium e g 3 mL for each well of six well plate to stop reaction and gently pipette up and down until cells in colonies become single cells Transfer cell suspension into a 15 ml conical tube and centrifuge at 250 X g for 5 minutes to pellet the cells Carefully aspirate as much of the supernatant as possible Add appropriate amount of Cyagen OriCell EB Formation Medium to the conical tube and gently resuspend the cells Plate cell suspension in 100 mm adherent dishes Incubate the adherent dishes in a 37 C incubator for 30 40 minutes to separate Mouse Embryonic Fibroblasts from OriCell Strain C57BL 6 Mouse ESCs Carefully collect the suspending OriCell Strain C57BL 6 Mouse ESCs and adjust the cell concentration to 5 x 10 cells mL with OriCell EB Formation Medium Plate 10mL cell suspension in one 100 mm non adherent petri dish Incubate the cells at 379C in a 5 CO humidified incubator for 5 days to form EB and change the medium every other day Plate EB into adherent surface of gelatin coated tissue culture vessels in Cyagen OriCell EB Formation Medium Incubate the EB at 379C in a 5 CO humidified incubator for about 14 days Cha
4. C in a 5 CO humidified incubator 14 The next day change the medium with fresh OriCell Strain C57BL 6 Mouse ESCs pre warmed to 37 C A Note Changing Medium 1 Warm an appropriate amount of OriCell Mouse ESC Growth Medium to 37 C in a sterile container Remove the spent medium and replace it with the warmed fresh medium and return the flask to the incubator 2 Avoid repeated warming and cooling of the medium If the entire contents are not needed for a single procedure transfer only the required volume to a sterile secondary container IMPIO024A3 MUBES 01001 Page 7 of 14 O Cyagen ANA rs i Oe D vre Wy Fig 4 Image of OriCell Stain C57BL 6 Mouse Embryonic Stem Cells at P21 cultured on OriCell ICR Mouse Embryonic Fibroblasts Irradiated feeder cells PASSAGING OriCell STRAIN C57BL 6 Mouse EMBRYONIC STEM CELLS ESCs Materials Required e OriCell Mouse Embryonic Stem Cell Growth Medium Cat No MUXES 90011 e Vessels plated with MEF Passaging C57BL 6 Mouse ESCs 1 Pre warm OriCell Mouse ESC Growth Medium 1xPBS Trypsin EDTA solution to Sz 2 Aspirate the spent medium from the OriCell ICR Mouse Embryonic Fibroblasts MEF Rinse MEF with 1xPBS 3 mL for one well of six well plate Aspirate the 1xPBS from the flask and discard Repeat step 3 4 once or twice Add the pre warmed OriCell Mouse ESC Growth Medium Return the MEF to the 5 CO humidified incubator D E E
5. Note Be careful not to disturb the monolayer of MEF during step 2 6 Carefully aspirate off spent medium from OriCell Strain C57BL 6 Mouse ESCs Rinse the cells with 1xPBS 3 mL for one well of six well plate Aspirate the 1xPBS from the flask and discard 10 Repeat the step 8 9 two or three times 11 Add Trypsin EDTA solution 200 uL for one well of six well plate and incubate for 1 2 minutes until the OriCell Strain C57BL 6 Mouse ESCs are dissociated At this point gently tap the side of the flask to release the majority of cells from the culture surface IMPIO024A3 MUBES 01001 Page 8 of 14 Cyagen 12 Add OriCell Mouse ESC Growth Medium 3 mL for one well of six well plate and gently pipette up and down until colonies become dissociated to single cells Note Be careful not to introduce any bubbles 13 Transfer the dissociated cells into a 15 mL conical tube 14 Centrifuge the tube at 250 x g for 5 minutes to pellet the cells 15 Carefully aspirate off as much of the supernatant as possible 16 Add 2 mL of OriCell Mouse ESC Growth Medium to the conical tube and re suspend the cells thoroughly but gently 17 Plate the cells into flasks containing the MEF Split ratios for OriCell Strain C57BL 6 Mouse ESCs can vary from 1 6 to 1 10 Do not exceed 1 10 18 Add sufficient medium 19 Incubate the cells at 37 C in a 5 CO humidified incubator until it is time to split again We typically split Ori
6. eit eee 2 Cyagen CONTENTS AND STORAGE Product Name Strain C57BL 6 Mouse Embryonic Stem Cells Catalog No MUBES 01001 Amount per Vial 1x10 Cells Cryopreserved At Twentieth Passage Storage Condition Liquid 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 Embryonic Stem Cells ESCs are pluripotent cells derived from the inner cell mass of blastocysts These cells are able to differentiate into all derivatives of the primary germ layers including ectoderm endoderm and mesoderm thus generating every cell type in the body Different from most other stem cells ESCs are capable of self renewal indefinitely Because of their plasticity and potentially unlimited capacity for self renewal ES cell therapies have been proposed for regenerative medicine and tissue replacement OriCell Strain C57BL 6 Mouse ESCs maintain diploid karyotype after extended passages in vitro These cells express specific clusters of different proteins for ESCs and are capable of forming embryoid bodies in vitro and developing teratomas in nude mice Cyagen OriCell Strain C57BL 6 Mouse ESCs are derived from the inner cell mass of strain C57BL 6 Mouse blastocysts at 3 5 dpc and cultured on y ray irradiated mouse embryonic fibroblasts as feeder cells in OriCell Mouse ESC Growth Medium In addition
7. of the cell suspension into cryogenic storage vials that are properly labeled 5 Place the vials directly in a 809C freezer After 24 hours transfer the frozen vials to liquid nitrogen for long term preservation IMPIO024A3 MUBES 01001 Page 11 of 14 Cy ager 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 finally cell aging Cell aging Plating density is too low The table below lists some potential problems and solutions for culturing OriCell Strain C57BL 6 Mouse Embryonic Stem Cells Problem Cause Solution The storage condition does Purchase a replacement and store in liquid not meet the requirements nitrogen for long term preservation Thawing the cells takes too 5 Thaw cells for no more than 3 minutes long time Cells reper eral After aspirating off medium wash the tube Low cell recovery F v 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 warmed Warm medium to 37 C before recovery Discard the cells in question and disinfect the Mycoplasma contamination laboratory environment before recovering the next batch of cells Wash the cells with PBS 2 3 times to remo
8. CellTM Strain C57BL 6 Mouse ESCs every other day A Note 1 OriCell Strain C57BL 6 Mouse ESCs should be plated at a density that provides an even distribution of colonies over the surface but does not result in contact between the colonies Differentiation can occur if the colonies are plated too densely or too sparsely 2 OriCell Strain C57BL 6 Mouse ESCs should not be over subcultured minimize the number of passages and the length of time the cells are kept in culture This will ensure enhanced and reproducible experimental results L Hints Time to Split Strain OriCell C57BL 6 Mouse Embryonic Stem Cells Passage the cells before the colonies become too large and dense When plated at the optimum density OriCell Strain C57BL 6 Mouse ESCs should be passaged every 48 hours DIFFERENTIATION OF OriCell STRAIN C57BL 6 Mouse EMBRYONIC STEM CELLS ESCs OriCell Strain C57BL 6 Mouse ESCs are capable of forming embryoid bodies in vitro and teratomas in nude mice Materials Required OriCell Embryoid Body EB Formation Medium Cat No MUXES 90051 The formation of embryoid body EB is the principal step in the differentiation of ES cells When maintained in the EB formation medium and in the absence of MEF feeder layers ES cells differentiate spontaneously and then form three dimensional aggregates This structure facilitates multicellular interactions in which cell cell contact IMPIO024A3 MUBES 01001 Page
9. Free Cryopreservation Medium NCPF 10001 References G R Martin 1981 Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells PNAS 78 7634 7638 T M Magin J McWhir and D W Melton 1992 A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency Nucleic Acids Research 20 14 3795 3796 J A Thomson J Kalishman and T G Golos 1995 Isolation of a primate embryonic stem cell line PNAS 92 7844 7848 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 IMPIO024A3 MUBES 01001 Page 14 of 14
10. ae Cyd J 6 We help gou discover life OriCell Strain C57BL 6 Mouse Embryonic Stem Cells ESCs Cat No MUBES 01001 cp Cyagen Table of Contents Contents and StOrAG sssrinds aranana EVU E US din dO UR OE RR daan en 3 Product TNEFOGUCEION 5 545 sen sac monaco RENE PUN dk messe cns suisses S ZVEMF ANDRE EEEE 3 Cell Characteristics and Identity iiia innkaniicics ic a ORO RE RE Rb CER Rr edn 3 Product ABDIICatIOnS aante VR a LUNA REEF dde oder tete non in ciao 4 General Handling Principles 5 5 isss uasasexPaNREn RRERVENRRNEENERNEREREEEREERERFSEEAEFAEFRENEFERFVRTRECENKEFRFEEE 4 Gelatin Coating of Tissue Culture Vessels for MEFS errem nn 4 Culturing OriCell Strain C57BL 6 Mouse ESCs Thawing and Establishing of MEF Feeder Cells nnn nnnm nnn 5 Thawing and Establishing of OriCell Strain C57BL 6 Mouse ESCS sn 6 Passaging OriCell Strain C57BL 6 Mouse ESCS eeee enne nere rina nan nn a an 8 Differentiation of OriCell Strain C57BL 6 Mouse ESCS eennne nemen 9 Cryopreservation of OriCell Strain C57BL 6 Mouse ESCSs ernnmm vennen enn 11 AARDS NO ne D TrOUDIESNOOUING csenkrsax diitun amp kki dva dis ERR GU M VEI CRYRWIDOER Vari aci RO cU wl an Ran RO n EN ER EU RR RR Wr 12 Related BFOGOUCES immens D NR NA VR ee E Vira RUE KR bend 14 Refenrlences arianen ennn COR dO R ee GOGH ACORN anna aen OO I UC RON GF RC GGG 14 Techno BUD DD wennen
11. f 14 cyagen 11 Seed the cells into 6 well plates pre coated with Gelatin Solution or other appropriate flasks and add sufficient OriCell MEF Growth Medium Gently rock the culture plate to evenly distribute the cells A Note We recommend the seeding density of MEFs to be 2 0 3 0x 10 cells cm 12 Incubate at 37 C in a 5 CO humidified incubator 13 The next day change the medium with fresh OriCell MEF Growth Medium pre warmed to 37 C A Note 1 If the next day thawing of the embryonic stem cells is performed the medium can be changed directly to embryonic stem cell growth medium 2 Thawing the feeder cells should be performed at least one day before thawing embryonic stem cells 3 The feeder cells should be used as soon as possible once thawed Fig 1 Cyagen OriCell Mouse Embryonic Fibroblasts Irradiated plated on culture vessels coated with 0 1 gelatin THAWING AND ESTABLISHING OriCell STRAIN C57BL 6 Mouse EMBRYONIC STEM CELLS ESCs Materials Required e Gelatin Solution Cat No GLT 11301 e OriCell Strain C57BL 6 Mouse Embryonic Stem Cells Cat No MUAES 01001 e OriCell Mouse Embryonic Stem Cell Growth Medium Cat No MUXES 90011 Thawing and Establishing Strain C57BL 6 Mouse ESCs 1 Pre warm the OriCell Mouse ESC Growth Medium and 1xPBS to 37 C IMPIO024A3 MUBES 01001 Page 6 of 14 Cyagen Add 9 mL of OriCell Mouse ESC Growth Medium to a 15 mL conical tube
12. ibroblasts MEFs feeder layers We recommend using Cyagen OriCell Strain ICR MEFs Irradiated for culturing mouse ESCs 4 For general maintenance of cells we recommend the seeding density to be 2 0 2 5 x 10 cells cm 5 Do not let OriCell C57BL 6 Mouse ESCs overgrow as it will result in contact between the colonies We recommend that the Mouse ESCs are routinely passaged every 48 hrs Gelatin Coating of Tissue Culture Vessels for Mouse Embryonic Fibroblasts MEFs Materials Required e Gelatin Solution Cat No GLT 11301 Gelatin Coating of Tissue Culture Vessels 1 Add sufficient Gelatin Solution into the culture vessel to completely cover its base 2 Swirl until Gelatin Solution coats the entire base of vessel Let it sit for at least 30 minutes at room temperature IMPIO024A3 MUBES 01001 Page 4 of 14 A cyagen Aspirate off all of the Gelatin Solution and allow the residual amount to evaporate by leaving the vessel sitting open in the laminar flow hood biological safety cabinet for no more than 30 minutes Enclose the culture vessel once it has dried Note Gelatinized dishes or flasks can be stored at 4 C for no more than 2 weeks provided they remain sterile THAWING AND ESTABLISHING MOUSE EMBRYONIC FIBROBLASTS MEFs Materials Required Gelatin Solution Cat No GLT 11301 OriCell Strain ICR Mouse Embryonic Fibroblasts Cat No MUIEF 01002 OriCell Mouse Embryonic Fibroblast Growth Medium
13. nge media every other day Stain the differentiated cells with antibodies against endodermal mesodermal and ectodermal markers at day 14 after EB differentiation Fig 6 Image of embryoid bodies derived from OriCell Strain C57BL 6 Mouse Embryonic Stem Cells IMPIO024A3 MUBES 01001 Page 10 of 14 Cyagen CRYOPRESERVATION OF OriCell STRAIN C57BL 6 Mouse EMBRYONIC STEM CELLS ESCs USING OriCell NCR PROTEIN FREE 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 A slow programmed freeze this product allows the cells to be directly frozen at 80 C mm Cryopreservation 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 at a cell density of 10 10 cells mL NCR Protein Free Cryopreservation Medium 4 Dispense aliquots
14. 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 IMPIO024A3 MUBES 01001 Page 3 of 14 Cyagen CELL CHARACTERISTICS AND IDENTITY e Ability to differentiate into all derivatives of the three primary germ layers e Reproduce indefinitely under proper conditions e Positive for pluripotent stem cell markers Oct4 SSEA 1 and Nanog 2 90 negative for SSEA 3 and SSEA 4 x 5 PRODUCT APPLICATIONS OriCell Strain C57BL 6 Mouse Embryonic Stem Cells ESCs are potent tools for basic and applied research in diverse fields including basic mechanism involved in developmental procedure and disorder regenerative biology and potential therapies Specially ESCs are a valuable utility to make genetically modified mice by introducing mutations into the mouse germ line 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 C57BL 6 Mouse ESCs as a backup 3 Establish and maintain Cyagen OriCell Strain C57BL 6 Mouse Embryonic Stem Cells on mouse embryonic f
15. ve serum prior to trypsinization serum will Slow cell growth Over digestion inhibit the function of trypsin Control the digestion time Plating density is too low Increase the plating density MEFs have been cultured for MEFs should be used up in 5 7days after too long recovery Use Cyagen tailor made culture media If Inappropriate serum and other serum and media products are used medium please perform validation to ensure compatibility Cell aging Dead cells are not removed Change the medium the next day after promptly recovery to ensure removal of all dead cells Discard the cells in question and disinfect Cell Contamination the experimental environment before recovering IMPI0024A3 MUBES 01001 Page 12 of 14 cyagen Control the digestion time Wash the cells with pre warmed medium 2 3 times during recovery Lower plating density Use Cyagen tailor made differentiation medium IMPIO024A3 MUBES 01001 Page 13 of 14 Cyagen Related products Product Catalog Number Gelatin Solution GLT 11301 OriCell Mouse Embryonic Fibroblasts MUIEF 01002 OriCell Mouse Embryonic Fibroblast Growth MUXEF 90011 Medium OriCell Strain C57BL 6 Mouse Embryonic Stem MUAES 01001 Cells OriCell Mouse Embryonic Stem Cell Growth MUXES 90011 Medium Phosphate Buffered Saline 1xPBS PBS 10001 Trypsin EDTA TEDTA 10001 OriCellTM Embryoid Body EB Formation Medium MUXES 90051 OriCell NCR Protein
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