instructions for use · 2019-04-30 · 96-well bioprinting kit black clear® plates - instructions...
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Greiner Bio-One GmbHMaybachstr.272636FrickenhausenGermany
Instructions For Use
96-Well Bioprinting Kit (black plates)
REF 655841
Revision01:April2019-U072109
96-WellBioprintingKitblackµClear®plates-InstructionsForUse
TABLE OF CONTENT
1. MATERIALS AND SUPPLIES................................................................... 3
2. INSTRUCTIONS........................................................................................4
2.1 TreatingCellswithNanoShuttle™-PL...............................................4
2.2 CellDetachment.................................................................................5
2.3 SpheroidPrinting...............................................................................6
2.4 Post-CultureHandling........................................................................6
3. TROUBLESHOOTING................................................................................7
4. CELL TYPES..............................................................................................8
5. REFERENCES...........................................................................................8
96-Well Bioprinting Kit Instructions For UseThank you for purchasing thisGreinerBio-One product. The 96-Well BioprintingKit usesNanoShuttle™-PL,ananoparticleassemblyconsistingofgold,ironoxide,andpoly-L-lysinetomagnetisecells,atwhichpointtheycanbemagneticallydirected.Inthiskit,cellsina96-wellplateareprintedintospheroidsusingamagneticdrivetoaggregatecellsatthebottomofthewell.NanoShuttle™-PLhastobestoredsterileatroomtemperature.
The magnets in this kit are strong, can damage electronics, and cause injury if not handled correctly. DO NOT remove the magnets from the protective covers. DO NOT autoclave. DO NOT store near metal surfaces. Read the attached instructions carefully on how to handle the magnets.
Product UseThe96-WellBioprintingKitisforresearchuseonly.Itisnotapprovedforhumanoranimaluse.
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96-Well Bioprinting Kit black µClear® plates - Instructions For Use
1. MATERIALS AND SUPPLIES
Materials and Supplies Needed to Make Spheroids
96-WellBioprintingKit,whichincludes:NanoShuttle™-PL(3x600µLvials);SpheroidDrive(1);HoldingDrive(1);Cell-Repellent96-WellPlates,black,µClear®(2).
Other Materials Provided by User:
70%Ethanol
PhosphateBufferedSaline(PBS,CalciumandMagnesiumfree)
0.25%Trypsin/EDTASolutionortherecommendeddetachingsolutionforyourcelltype
Pipettes,flasks,othergeneraltissueculturesuppliesandtools
Cells(insuspensionormonolayer)
Media(usetypicalmediafor2Dculture,ifserum-free,usetrypsinneutralisationsolutiontoinactivatetrypsin)
Microscope
Anyadditionalsuppliesforthespecificcelltypeandapplication
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96-Well Bioprinting Kit black µClear® plates - Instructions For Use
2. INSTRUCTIONSInstructions for forming spheroids in the 96-Well Bioprinting Kit
Overview:600µLofNanoShuttle™-PLwilltreatoneT-75flaskofcellsat80%confluence(approximately6millioncells).At50,000cells/spheroid,thisisenoughtoform120spheroids.Spheroidstobeparaffin-embeddedmayrequiremorecellsperspheroid.The96-wellBioprintingKitworksonlywithCELLSTAR®Cell-Repellent96-WellPlates,black,µClear®(REF655976-SIN,GreinerBio-One,includedinthekit).
! Optimisation may be required for different cell types or specific experimental aims.
2.1 Treating Cells with NanoShuttle™-PL
1.Culturecellsto80%confluenceinaT-25,T-75,orT-150cultureflaskusingstandardproceduresinyourlaboratoryforyourspecificcelltype.
2.TreatcellswithNanoShuttle™-PLasfollows: a)HomogeniseNanoShuttle™-PLinitsvialbypipettingitupanddownatleast10times. b)ForaT-25flaskadd200µLNanoShuttle™-PL,or foraT-75flaskadd600µLNanoShuttle™-PL,or foraT-150flaskadd1,200µLNanoShuttle™-PLdirectlytothemedia. c)IncubatecellswithNanoShuttle™-PLovernight.
!The amount of NanoShuttle™-PL added can be optimised to use more or less volume for specific cell types. Optimise the volume before experimentation by forming 3D cultures with more or less NanoShuttle™-PL added. A benchmark concentration is 1 µL/10,000 cells.
! NanoShuttle™-PL is brown in color. After incubation, the cells will appear peppered with the brown NanoShuttle™-PL (Figure 1).
Figure 1: After incubation with NanoShuttle™-PL, cells will appear peppered with the brown nanoparticles, as demonstrated by primary human pulmonary fibroblasts. Scale bar = 100 µm1
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96-Well Bioprinting Kit black µClear® plates - Instructions For Use
2.2 Cell Detachment
3.Afterincubation,warm/thawTrypsin/EDTAsolution,PBS,andmediainawaterbathto37ºC.
4.Inasterilehood,aspirateallmedia(includingexcessNanoShuttle™-PL)fromtheflask.
5.WashcellstoremoveanyremainingmediaandexcessNanoShuttle™-PLbyaddingPBStotheflaskandgentlyagitating.Werecommend2mLofPBSforaT-25flask,5mLforaT-75flask,and10mLforaT-150flask.
6.AspiratePBSandaddTrypsin/EDTAsolutiontotheflask.AddenoughTrypsin/EDTAsolutiontocoverthecellmonolayer,about1mLtoaT-25flask,2mLtoaT-75flask,or4mLtoaT-150flask.
Followyourlaboratory’scell-specificdetachmentprotocols.
7.Placetheflaskinanincubatorforapproximately3-5minutesorforatimeprescribedbyyourstandardprotocolfordetachingcells.Checkfordetachmentunderamicroscope.
8.Whilewaitingforcellstodetach,cleanthemagneticdrivesthatyouwillusebywipingthemwith70%ethanol.Keepthemagneticdrivessterile.
! Do not soak drives in ethanol. Lightly spray and wipe to sterilise.
9.Removeflaskfromincubatorandcheckunderamicroscopethatthecellsaredetachedfromthesurface.ExcessexposuretoTrypsin/EDTAwilladverselyaffectcellhealth,soproceedtothenextstepquickly.
10.DeactivateTrypsin/EDTAbyadding37°Cmediawithserum.TheamountofmediawithserumaddedshouldatleastmatchtheoriginalvolumeofTrypsin/EDTAadded.Ifcellsaresensitivetoserum,eitherusetrypsinneutralisingsolution,orimmediatelycentrifugecells(atleast 100Gfor5min)andaspiratethetrypsin.
11.CountthecellsusingahemacytometerorCoultercounter.Centrifugecellsandresuspendthemintherequiredamountofmedia(150µLperspheroid).
!We recommend forming spheroids with 50,000 cells each (333,333 cells/mL), but the number of cells per spheroid can be different. Cultures have successfully been formed with cell numbers from 100,000 to 20,000. Optimise the number of cells per spheroid by forming spheroids with less cells.
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96-Well Bioprinting Kit black µClear® plates - Instructions For Use
2.3 Spheroid Printing
12.Placeacell-repellent96-wellplateatopthespheroiddrive(Figure 2).
13.Dispensethecellsintotheplatewith150µLofsolutionperwellandclosetheplate.Thecellswithinthesolutionwillaggregateatthebottomofthewellplateintheshapeofthemagnet.Leavetheplateonthedriveintheincubatorfor15mintoafewhourstoyieldacompetentspheroid.
!Longer printing times, although possible, may not be necessary, as the magnet will aggregate cells very quickly. Optimise the printing time for your specific experiment so that the resulting spheroid can be removed from the magnet and still maintain its structure.
Figure 2: Take the spheroid drive (a) and place a cell-repellent 96-well plate (b) atop the spheroid drive to print the cells into a spheroid (c).
14.Afterprinting,removetheplateoffthedriveandtransferitbacktotheanincubatorforthelengthoftheexperiment.Thespheroidscanbeculturedupto3weeks.Ifnecessary,replacethemediainthewellsafter2-3daysofculture.Usetheholdingdrivetoholdthespheroidsdownwhileaspiratingsolutionstopreventunwantedcellloss(Figure 3).
2.4 Post-Culture Handling
Afterculturing,standardtissueprocessingtechniquescanbeperformed,suchasfixation,paraffinembeddingforimmunohistochemistry,orRNAisolationforqRT-PCR.Usetheholdingdrivetoholdcellsdownwhileaddingandremovingliquids(Figure 3).
Figure 3: Use the holding drive to hold 3D cultures as you add and remove liquids
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A B C
96-Well Bioprinting Kit black µClear® plates - Instructions For Use 7
3. TROUBLESHOOTING
Problem Probable Cause Solution
NanoShuttle™-PLappearsseparated
NanoShuttle™-PLhassett-ledatthebottomofthevial
HomogenisetheNano Shuttle™-PLbeforeusebypipettingupanddown10X
NanoShuttle™-PLdonotap-peartofullybindwithcells,floatinginmedium
BindingwithNanoShuttle™-PLvariesinefficiencyamongcelltypes
NanoShuttle™-PLwillappearpepperedoncellsandsomewillfloat,butthecellsarestillmagnetised.AddlessNanoShuttle™-PLiftooexcessive
CellswereincubatedwithNanoShuttle™-PLtoolong
Incubatecellswith NanoShuttle™-PLovernightatmost
Cellstakinglongerthanusualtodetach
Cellsstronglyadheredtosubstrate
Beforeaddingtrypsin,washflaskwithPBS1-2X
NanoShuttle™-PLsparselyattachedtocells
Too many cells
IncreaseNanoShuttle™-PLvolumeaddedtoeachwelltoyieldanidealconcentrati-onof1µL/10,000cells
CellsaresensitivetoserumCellsmayundergounwanteddifferentiationwithserum
Useatrypsin-neutralisingsolutioninlieuofserum-con-tainingmediatostoptrypsinactivity.Centrifugecellsimmediatelyafterandremovetrypsinsolution
Magnetisedcellsattachingtobottomoftheplate
Magnetisedcellsareweaklyornotboundto NanoShuttle™-PL
Usecell-repellentplatestopreventcellsfromadheringandcollectweaklymagnetisedcells
SpheroidappearsspreadoutCellshavenotbeenbioprintedforenoughtime
Printthecellslongerandca-refullymonitortheformationofthe3Dculture
3Dculturesarelostorbro-kenwhenremovingliquids
3Dcultureisnothelddownwhileliquidsaretransferred
Usetheholdingdrivetoholddowncultureswhileaddingandremovingliquids
96-Well Bioprinting Kit black µClear® plates - Instructions For Use 8
4. CELL TYPESCelltypesthathavebeensuccessfullyculturedusingtheprocedureinclude:
Cell lines• MurineEndothelialCells• MurineEmbryonicFibroblasts,pre-adipocytes(3T3)• MurineAdipocytes• Murine Melanoma• MurineNeuralStemCells• RatHepatoma• HumanAstrocytes• HumanGlioblastomaMultiforme(LN229)• HumanEmbryonicKidneyCells(HEK293)• RatVascularSmoothMuscleCells(A10)• HumanHepatocellularCarcinoma(HepG2)• HumanLungAdenocarcinoma(A549)• HumanColorectalCarcinoma(HCT116)• HumanPancreaticEpithelioidCarcinoma(PANC-1)
Primary cells• HumanPulmonaryMicrovascularEndothelialCells• HumanTrachealSmoothMuscleCells• HumanSmallAirwayEpithelialCells• HumanPulmonaryFibroblasts• HumanMesenchymalStemCells• HumanBoneMarrowEndothelialCells• HumanUmbilicalVeinEndothelialCells• HumanAorticVascularSmoothMuscleCells• HumanNeonatalDermalFibroblasts• MurineChondrocytes
5. REFERENCES1 Haisler, W. L. et al. Three-dimensional cell culturing by magnetic levitation. Nat. Protoc. 8, 1940–9 (2013).
96-Well Bioprinting Kit black µClear® plates - Instructions For Use 9
NOTES
96-Well Bioprinting Kit black µClear® plates - Instructions For Use
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96-Well Bioprinting Kit black µClear® plates - Instructions For Use
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