Protocol A Simple and Reliable Protocol for the Preparation and Culturing of Fresh Surgically Resected Human Glioblastoma Tissue
Liyen Katrina Kan 1,2,3, Katharine J Drummond 4,5, Martin Hunn 6, David A Williams 3, Terence J O’Brien 1 and Mastura Monif 1,2,3,4,*
1 Department of Neuroscience, Monash University, Melbourne, VIC 3004, Australia; [email protected] (L.K.K.); [email protected] (T.J.O.) 2 Department of Neurology, Royal Melbourne Hospital, Melbourne, VIC 3050, Australia 3 Department of Physiology, The University of Melbourne, Melbourne, VIC 3010, Australia; [email protected] 4 Department of Surgery, The University of Melbourne, Melbourne, VIC 3010, Australia; [email protected] 5 Department of Neurosurgery, Royal Melbourne Hospital, Melbourne, VIC 3050, Australia 6 Department of Neurosurgery, Alfred Health, Melbourne, VIC 3004, Australia; [email protected] * Correspondence: [email protected]
Received: 8 December 2019; Accepted: 21 January 2020; Published: 22 January 2020
Abstract: Glioblastoma is a heterogeneous glial cell malignancy with extremely high morbidity and mortality. Current treatment is limited and provide minimal therapeutic efficacy. Previous studies were reliant on cell lines that do not accurately reflect the heterogeneity of the glioma microenvironment. Developing reliable models of human glioblastoma is therefore essential. Direct culture of human brain tumours is often difficult and there is a limited number of protocols available. Hence, we have developed an effective method for the primary culture of human glioblastoma samples obtained during surgical resection. Culturing tumour tissue direct from human brain is advantageous in that cultures (1) more closely resemble true human disease, relative to the use of cell lines; (2) comprise a range of cellular components present in the natural tumour microenvironment; and (3) are free of added antibodies and reagents. Additionally, primary glioblastoma cultures are valuable in studies examining the effects of anti-cancer pharmaceuticals and therapeutic agents, and can be further used in live cell imaging, immunocytochemistry, flow cytometry and immunoassay experiments. Via this protocol, cells are maintained in supplemented medium at 37 ◦C (5% CO2) and are expected to achieve sufficient confluency within 7 days of initial culture.
Keywords: brain tumour; glioma; glioblastoma; cell culture; tissue culture; primary cell culture
1. Introduction Glioblastoma is the most predominant and aggressive central nervous system malignancy, accounting for over 60% of all brain tumours in adults [1]. Current treatments are limited in range and efficacy, with patients surviving for a median of only 14 to 15 months post-diagnosis [1]. Hence, a deeper understanding of glioblastoma pathology and the mechanisms of tumour pathogenesis is pivotal for research progression. The heterogeneity of glioblastoma is reflected by the crosstalk between tumour cells, microglia and a multitude of invading immune cells and cytokines [2,3]. This presents as a hurdle to overcome when developing reliable models of glioblastoma. Conventionally, glioblastoma research has been based on data obtained from homogeneous, murine and patient-derived cell lines. While cell lines provide a rapid and reproducible means
Methods Protoc. 2020, 3, 11; doi:10.3390/mps3010011 www.mdpi.com/journal/mps Methods Protoc. 2020, 3, 11 2 of 7 Methods Protoc. 2019, 2, x FOR PEER REVIEW 2 of 7 ofvitroin vitroexperimentationexperimentation [4], they[4], they do donot not accurately accurately mimic mimic the the heterogeneity heterogeneity of of tumours tumours inin vivo vivo.. Glioblastoma cell cell lines lines have have been been demonstrated demonstrated to express to express markedly markedly different different gene expression gene expression profiles whenprofiles compared when compared to primary to primary tumours tumour [5,6] ands [5,6] are susceptible and are susceptible to genetic to drift genetic across drift passages across passages that alter experimentalthat alter experimental reproducibility reproducibility [4]. [4]. Importantly, surgically resected human glioblastoma tissue retains the molecular molecular and cellular cellular characteristicscharacteristics of of the original tumour mass. These samples have also been demonstrateddemonstrated to expressexpress microglia, the largest immune cell infiltratesinfiltrates of the glioblastoma microenvironment [[7,87,8].]. Other immune cells present present may may include include lymphocytes, lymphocytes, neutrophils, neutrophils, monocytes monocytes/macrophages/macrophages and and myeloid-derived myeloid-derived suppressor cells [[99–12]12].. Cellular infiltrates infiltrates serve serve a a range of functions that disparately aaffectffect tumour growth. Hence, Hence, relative relative to to cell cell lines, lines, the the culturing of samples directly from surgically surgically resected resected glioblastoma more closely resembles true true disease and takes into account the eeffectffect of infiltrating infiltrating immune cells. Existing protocols for for the culture of primary glioblastomaglioblastoma tissue are limited and have limited growth growth efficacy efficacy [13] [13].. In In light light of of this this,, we we demonstrate demonstrate a simple a simple,, reliable reliable and and efficient efficient protocol protocol for forthe thedirect direct culture culture of human of human glioblastoma glioblastoma tissue. tissue. Importantly, Importantly, this protocol this protocol processes processes tumour samples tumour samplesfor culturing for culturing immediately immediately after after surgical surgical resection resection,, which which minimizes minimizes potential potential environmental disruptions that may significantlysignificantly aaffectffect thethe tumourtumour microenvironment.microenvironment. This method is particularlyparticularly useful for for drug drug testing testing inin vitro vitro, ,and and can can be be used used to toassess assess the the presence presence of various of various cell cellpopulations populations and andbiomarkers biomarkers within within the tumour the tumour microenvironment microenvironment via immunocytochemistry, via immunocytochemistry, flow flow cytometry cytometry and andimmunoassays. immunoassays.
2. Experimental Design
EnsureEnsure ALLALL materialsmaterials andand equipmentequipment areare sterilesterile priorprior toto usageusage..
2.1. Materials
2.1.1. Poly Poly-D-Lysine-D-Lysine Plate Coating Solution Poly-D-lysine hydrobromide powder, 5 mg (Merck, Australia; Cat. no.: P6407) • Poly-D-lysine hydrobromide powder, 5 mg (Merck, Australia; Cat. no.: P6407) Sterile distilled water • Sterile distilled water
2.1.2. Enzymatic Tissue DissociationDissociation Solution Papain from papaya latex (Merck, Australia; Cat. no.: P3125) • Papain from papaya latex (Merck, Australia; Cat. no.: P3125) Earle’s Balanced Salt Solution (EBSS; Thermo Fisher Scientific, Australia; Cat. no.: 14155063) • Earle’s Balanced Salt Solution (EBSS; Thermo Fisher Scientific, Australia; Cat. no.: 14155063)
2.1.3. Culture Medium Minimum Essential Medium, 1X, 500 mL (Thermo Fisher Scientific, Australia; Cat. no.: 10370021) • Minimum Essential Medium, 1X, 500 mL (Thermo Fisher Scientific, Australia; Cat. no.: 10370021) D-glucoseD-glucose (Merck, Australia;Australia; Cat.Cat. no.: G7021) • L-glutamineL-glutamine (Thermo Fisher Scientific,Scientific, Australia;Australia; Cat.Cat. no.: 25030081) • Penicillin-streptomycinPenicillin-streptomycin (Thermo(Thermo FisherFisher Scientific,Scientific, Australia;Australia; Cat.Cat. no.:no.: 15070063)15070063) • Heat-inactivatedHeat-inactivated fetal bovinebovine serumserum (Thermo(Thermo FisherFisher Scientific,Scientific, Australia;Australia; Cat.Cat. no.:no.: 10100147)10100147) • CorningCorning®® MITOMITO++ Serum Extender (Merck, Australia; Cat.Cat. no.: DLW355006) • 2.2. Equipment Class II biological safety cabinetcabinet • 12-well12-well cell culture platesplates (Merck,(Merck, Australia;Australia; Cat.Cat. no.: SIAL0512) • 18 mm glass coverslips (Thermo(Thermo FisherFisher Scientific,Scientific, Australia;Australia; Cat.Cat. no.: CB00180RA020MNT0)CB00180RA020MNT0) • ParaParaffinffin • Surgical tweezer Surgical tweezer • 50 mL syringe (Merck, Australia; Cat. no.: Z683698) 50 mL syringe (Merck, Australia; Cat. no.: Z683698) • Syringe filter, 0.2 µm pore (Merck, Australia; Cat. no.: CLS431229) 50 mL centrifuge tubes (Merck, Australia; Cat. no.: CLS430828) Petri dish (Merck, Australia; Cat. no.: P5481)
Methods Protoc. 2020, 3, 11 3 of 7
Syringe filter, 0.2 µm pore (Merck, Australia; Cat. no.: CLS431229) • Methods50 Protoc. mL centrifuge 2019, 2, x FOR tubes PEER (Merck, REVIEW Australia; Cat. no.: CLS430828) 3 of 7 Methods• Protoc. 2019, 2, x FOR PEER REVIEW 3 of 7 MethodsMethodsPetri Protoc. Protoc. dish 2019 2019 (Merck,, ,2 2, ,x x FOR FOR Australia; PEER PEER REVIEW REVIEW Cat. no.: P5481) 33 of of 7 7 • SurgicalSurgical scalpelscalpel Surgical scalpel • SurgicalPiSurgicalpette Boy scalpelscalpel (Eppendorf, Australia; Cat. no.: 4430000018) PipettePipette Boy (Eppendorf, Australia; Cat. no.: 4430000018) • Pi10Pipettepette mL pipettes BoyBoy (Eppendorf,(Eppendorf, (Sterilin, Australia;Australia; Cat.Cat. no.:no.: 4430000018)47510)4430000018) 10 mLmL pipettes (Sterilin,(Sterilin, Australia; Cat. no.: 47510) • 10Pasteur10 mmLL pipettespipettes pipette (Sterili(Steriliwith rubbern,n, Australia;Australia; bulb Cat.Cat. no.:no.: 47510)47510) Pasteur pipette with rubber bulb • PasteurBunsenPasteur pipetteburnerpipette withwith rubberrubber bulbbulb Bunsen burner • BunsenWaterBunsen bath burnerburner set at 37 °C Water bathbath setset atat 3737 °CC • WaterAutomatedWater bathbath setsetcell atat counter 3737 ◦ °°CC Automated cellcell countercounter • HumidifiedAutomated cell5% COcounter2/95% O2 incubator, 37 °C (Panasonic; Model no.: MCO-170AICUV-PE) 2 2 HumidifiedHumidified 5%5% COCO22/95% O 2 incubator,incubator,incubator, 37 37 °◦C (Panasonic;(Panasonic; Model Model no. no.:no.:: MCO MCO-170AICUV-PE)--170AICUV--PE) • Humidified 5% CO2/95% O2 incubator, 37 °C (Panasonic; Model no.: MCO-170AICUV-PE) 3. Procedure 3. Procedure Procedure 3.3. ProcedureProcedure Ensure ALL experiments are completed under sterile conditions with appropriate aseptic Ensure ALL experiments are completed under sterile conditions with appropriate aseptic technique EnsureEnsures ALLto ALL minimize experiments experiments sample are are contamination completed completed under under and exposure sterile sterile conditions conditionsto human tissue. with with appropriate appropriate aseptic aseptic techniques to minimize sample contamination and exposure to human tissue. techniquestechniquetechniquess toto minimizeminimizeminimize samplesamplesample contaminationcontaminationcontamination and andand exposure exposureexposure to toto human humanhuman tissue tissue.tissue.. 3.1. Poly-D-Lysine Plate Coating Time for Completion: 3 Days 3.1. Poly Poly-D-Lysine-D-Lysine Plate Coating Time for Completion: 3 Days 3.1.3.1. PolyPoly--DD--LysineLysine PlatePlate CoatingCoating TimeTime forfor Completion:Completion: 33 DaysDays 1. Add 50 mL of autoclaved distilled water into 5 mg stock poly-D-lysine powder using a 50 mL 1. AddAdd 50 50 mL mL of of autoclaved autoclaved distilled distilled water water into into 5 5 mg mg stock poly poly-D-lysine-D-lysine powder powder using using a a 50 mL 1.1. AddsyringeAdd 5050 mLwithmL ofof a autoclaved0.2autoclaved µm pore distilleddistilled syringe waterfilter.water Reintointo-cap 55 mg mgthe stock stock polypolybottle--DD and--lysinelysine shake powderpowder lightly. usingusing aa 5050 mLmL syringesyringe with with a a 0.2 0.2 µmµm pore pore syringe syringe filter. filter. Re Re-cap-cap the stock bottle and shake lightly. 2. syringeTransfersyringe withwith one aa 0.20.2 18 µmµm mm porepore glasssyringesyringe coverslip filter.filter. ReRe --capcap per thethe well stockstock ontobottlebottle and 12and-well shakeshake cell lightly.lightly. culture plates. 2. Transfer one 18 mm glass coverslip per well onto 12-well cell culture plates. 2.2. TransferNOTE:Transfer A one onesingle one 18 18mm 1850 mL mm glassmm poly coverslipglass glass-D- lysine coverslip coverslip per solution well per ontoper can well12-well wellbe used onto onto cell to cultureprepare 12 12--wellwell plates. approximately cell cell NOTE: culture culture A thirteen plates. singleplates. NOTE: A single 50 mL poly-D-lysine solution can be used to prepare approximately thirteen NOTE:poly50NOTE: mL-D poly-D-lysine - AlysineA singlesingle-coated 5050 mLmL solution 12 -polypolywell- can-DcellD--lysinelysine beculture used solutionsolution toplates. prepare can can approximatelybebe usedused toto prepareprepare thirteen approximatelyapproximately poly-D-lysine-coated thirteenthirteen poly-D-lysine-coated 12-well cell culture plates. 3. poly12-wellAddpoly- -D300D--lysine celllysine µL cultureof--coated coatedpoly plates.-D 1212-lysine--wellwell cellsolutioncell cultureculture per plates.plates. well and leave at room temperature (25 °C) for 2 h. 3. Add 300 µL of poly-D-lysine solution per well and leave at room temperature (25 °C) for 2 h. 3.4.3. AddAspirateAdd 300 300 µLµµLresidualL of of poly poly-D-lysinepoly poly--DD--lysine-lysineD-lysine solution solution solution perper wellinwell each andand well leaveleave and atat roomroomleave temperature temperaturein sterile conditions (25(25 ◦ °°C)C) forfor at 2 2room h.h. 4. Aspirate residual poly-D-lysine solution in each well and leave in sterile conditions at room 4.4. AspiratetemperatureAspirate residual residual for 48 poly poly-D-lysinepoly h or-- DuntilD--lysinelysine the solutionwells solution have in in dried. each each well well and and leave leave in in sterilesterile conditionsconditions at at roomroom temperature for 48 h or until the wells have dried. 5. temperaturetemperature PAUSE STEP for forfor 48 4848: Seal h hh or oror plates until untiluntil the thethewith wells wells paraffin have have and dried. dried. store at 2–8 °C until usage. 5. PAUSE STEP:: Seal plates with paraffin and store at 2–8 °C until usage. 5.5. PAUSEPAUSE STEP:STEP: SealSeal platesplates withwith paraparaffinffin andand storestore atat 2–82–8◦ °CC untiluntil usage.usage. 3.2. Sample Preparation and Tissue Culture. Time for Completion: 75 Min for Sample Preparation. 7 Days to 3.2. Sample Preparation and Tissue Culture. Time for Completion: 75 Min for Sample Preparation. 7 Days to 3.2.R3.2.each SampleSample 80% CPreparationPreparationonfluency. andand TissueTissue Culture.CultureCulture.. TimeTime forfor Completion:CompletCompletion:ion: 7575 MinMMinin forfor SampleSSampleample Preparation.PPreparation.reparation. 77 DDaysDaysays toto Reach 80% Confluency. ReachRReacheach 80%80% ConfluencyCConfluency.onfluency. 6. Pre-warm 10 mL of EBSS in a 50 mL centrifuge tube and culture medium at 37 °C. 6. Pre-warm 10 mL of EBSS in a 50 mL centrifuge tube and culture medium at 37 °C. 6.7.6. PreImmediatelyPre-warmPre--warmwarm 10 10 mLafter mLmL ofof collection EBSSEBSS inin aaof 5050 the mLmL fresh centrifugecentrifuge sample, tubetube add andand 200 culturecultureunits of mediummedium papain from atat 3737 papaya ◦°°C.C. latex into 7. Immediately after collection of the fresh sample, add 200 units of papain from papaya latex into 7.7. ImmediatelywarmImmediately EBSS. after afterafter collection collectioncollection of ofof the thethe fresh freshfresh sample, sample, add add 200 200 unitsunits ofof pappapainpapainain fromfrom papayapapaya latexlatex intointo warm EBSS. warmNOTE:warm EBSS. EBSS. This NOTE: concentrationThis concentration of papain applies of papain to aapplies sample to of a sample approximately of approximately 5 mm2. Increase 5 mm2. NOTE: This concentration of papain applies to a sample of approximately 5 mm22. Increase NOTE:concentrationIncreaseNOTE: ThisThis concentration concentration concentrationwith larger with samples. of largerof papain papain samples. applies applies to to a a sample sample of of approximately approximately 5 5 mm mm2.. Increase Increase concentration with larger samples. 8.8. concentrationconcentration CRITICALCRITICAL withwith STEP:STEP largerlarger: TransferTransfer samples.samples. thethe tumourtumour tissuetissue intointo aa petripetri dishdish andand gentlygently fragmentfragment thethe tissuetissue 8. CRITICAL STEP: Transfer the tumour tissue into a petri dish and gently fragment the tissue 8.8. with CRITICALCRITICAL a surgical scalpelSTEPSTEP:: Transfer Transfer (Figure1 1). the).the tumourtumour tissuetissue intointo aa petripetri dishdish andand gentlygently fragmentfragment thethe tissuetissue with a surgical scalpel (Figure 1). withwith aa surgicalsurgical scalpelscalpel (Figure(Figure 1).1).
(a) (b) (a) (b) ((aa)) ((bb)) FigureFigure 1.1. Fresh surgically resectedresected humanhuman glioblastomaglioblastoma tissuetissue ((aa)) priorprior toto andand ((bb)) afterafter fragmentationfragmentation Figure 1. Fresh surgically resected human glioblastoma tissue ((a)) prior to and (b)) afterafter fragmentationfragmentation withwithFigure aa surgical surgical1. Fresh scalpel. scalpelsurgically. resected human glioblastoma tissue (a) prior to and (b) after fragmentation with a surgical scalpel. withwith aa surgicalsurgical scalpelscalpel.. 9. Transfer fragmented sample into the warm EBSS and papain solution and place in a water bath 9. Transfer fragmented sample into the warm EBSS and papain solution and place in a water bath 9.9. TransferatTransfer 37 °C for fragmentedfragmented 40 min. samplesample intointo thethe warmwarm EBSSEBSS andand papainpapain solutionsolution andand placeplace inin aa waterwater bathbath at 37 °C for 40 min. 10. atGentlyat 3737 °°CC aspirate forfor 4040 min.min. sample with a 10 mL pipette and transfer into a new 50 mL centrifuge tube with 10. Gently aspirate sample with a 10 mL pipette and transfer into a new 50 mL centrifuge tube with 10.10. Gently3Gently mL culture aspirateaspirate medium. samplesample withWashwith aa 10in10 triplicatemLmL pipettepipette with andand culture transfertransfer medium, intointo aa newnew topping 5050 mLmL up centrifugecentrifuge with new tubetube culture withwith 3 mL culture medium. Wash in triplicate with culture medium, topping up with new culture 3medium3 mLmL cultureculture after medium. medium.each wash. WashWash inin triplicatetriplicate withwith cultureculture medium,medium, toppingtopping upup withwith newnew cultureculture medium after each wash. mediummedium afterafter eacheach wash.wash.
Methods Protoc. 2020, 3, 11 4 of 7
9. Transfer fragmented sample into the warm EBSS and papain solution and place in a water bath at 37 ◦C for 40 min. 10. Gently aspirate sample with a 10 mL pipette and transfer into a new 50 mL centrifuge tube with Methods Protoc. 2019, 2, x FOR PEER REVIEW 4 of 7 Methods3 mLProtoc. culture 2019, 2, medium.x FOR PEER WashREVIEW in triplicate with culture medium, topping up with new culture4 of 7 11. medium CRITICAL after each STEP: wash. With a gentle up and down pipetting motion, use a Pasteur pipette with 11.11. a rubber CRITICALCRITICAL bulb to STEP:STEP disassociate:With With a a gentlethe gentle cells, up up creating and and down down a single pipetting pipetting cell suspension. motion, motion, use use a a Pasteur Pasteur pipette pipette with with a TIP:arubber rubber Soften bulb bulb the to to disassociate edges disassociate of the the thePasteur cells, cells, creating pipette creating a with single a single a cellBunsen cell suspension. suspension. burner TIP:to minimizeSoften the disruption edges of the to cellularTIP:Pasteur Soften integrity. pipette the withedges a Bunsenof the P burnerasteur pipette to minimize with disruptiona Bunsen burner to cellular to minimize integrity. disruption to 12.12. CountcellularCount the the integrit cells cells usingy. using an automated automated cell counter and top up accordingly with culture medium to 5 12. makeCountmake up the 5 cells× 1010 5using viableviable an (live) (live) automated cells/mL. cells/mL. cell Alternativel Alternatively, counter andy, topa a haemocytometer haemocytometer up accordingly canwith can be be culture used. used. medium to × 5 13.13. HomogenizemakeHomogenize up 5 × 10the the viablecell solution (live) cells/mL. by pipetting Alternatively, up an andd down a haemocytometer and transfer 1 mL can per be wellused. onto 12-well 13. cellHomogenizecell culture culture plates plates the cellpre-coated pre-coated solution with by with pipetting poly-D-lysine. poly-D-lysine. up and Maintain down Maintain and cells transfer cells in a inhumidified 1 a mL humidified per incubatorwell incubatoronto at12 37-well °C at cell culture plates pre-coated with poly-D-lysine. Maintain cells in a humidified incubator at 37 °C with37 ◦C 5% with CO 5%2 for CO 7 2daysfor 7 or days until or cells until are cells 80% are confluent. 80% confluent. NOTE: For optimal growth, replace NOTE:withthe culture 5% For CO mediumoptimal2 for 7 days growth, every or until 3–4 replace days.cells arethe 80%culture confluent. medium every 3–4 days. NOTE: For optimal growth, replace the culture medium every 3–4 days. 4. Expected Results 4. Expected Results It isis importantimportant to notenote thatthat duedue toto existingexisting heterogeneity between each tumour, there may be a didifferencefferenceIt is important inin proliferation proliferation to note rate ratethat per dueper culture. culture.to existing The Th time heterogeneitye time taken taken to reach tobetween 80%reach confluency each80% tumour,confluency might there vary might may between varybe a eachbetweendifference culture; each in we culture; proliferation have previouslywe have rate previously per cultured culture. cultured 20 tumours The time 20 tumours using taken this tousing protocol, reach this 80% protocol, of which confluency of 17 which (85%) might 17 reached (85%) vary 80%reachedbetween confluency 80% each confluency culture; within we a within time have span previously a time of 7 span days. culture of 7 days.d 20 tumours using this protocol, of which 17 (85%) reachedSurgically 80% confluency resectedresected human withinhuman glioblastoma a glioblastomatime span tissueof 7tissue days. cultured cultured via thisvia protocolthis protocol closely closely reflect thereflect cellular the heterogeneitycellularSurgically heterogeneity of resectedthe original of the human tumour.original glioblastoma tumour. In particular, In tissueparticular, glioma cultured cells glioma as via well cells this as as microglia protocol well as microglia and closely other reflect and immune other the cellsimmunecellular are expectedheterogeneity cells are to expected contribute of the originalto to contribute the vast tumour. majority to Inthe particular, ofvast the majority cellular glioma architecture of cellsthe ascellular well [7,8 ].as architecture In microglia Figure2, and we [7,8]. show other In theFigureimmune presence 2, cellswe ofshow are glioma expectedthe cellspresence marked to contribute of byglioma glial tocells fibrillary the ma vastrked acidic majority by protein glial offibrillary (GFAP), the cellular acidic as well architecture protein as CD11b-positive (GFAP), [7,8]. asIn immunewellFigure as 2,CD11b-positive cellwe infiltratesshow the inpresence immune culture. ofcell CD11b glioma infiltrates is cells known inmarked culture. to be by expressed CD11b glial fibrillary is on known the surfaceacidic to be protein ofexpressed many (GFAP) immune on ,the as cells,surfacewell as including CD11bof many- tumour-associatedpositive immune immune cells, includingcell microglia infiltrates /tumomacrophages, inur-associated culture. monocytes,CD11b microglia/ma is known neutrophils crophages,to be andexpressed natural monocytes, on killer the cellsneutrophilssurface [14 – of16 ]. manyand Cells natural immune in culture killer cells, werecells including [14–16]. fixed at Cells 80% tumour confluency,in culture-associ atedwere stained microglia/macrophages, fixed via at immunocytochemistry80% confluency, monocytes,stained and via visualizedimmunocytochemistryneutrophils using and natural fluorescence and killer visualized confocalcells [14 – microscopy.usin16].g Cells fluorescence in culture confocal were fixed microscopy. at 80% confluency , stained via immunocytochemistry and visualized using fluorescence confocal microscopy.
(a) (b) (c) (a) (b) (c) Figure 2.2. Immunocytochemical staining and fluorescencefluorescence confocal microscopy of primaryprimary humanhuman glioblastomaFigure 2. Immunocytochemical culture fixedfixed at 80%80% staining confluency.confluency. and fluorescence GliomaGlioma cells confocal were stained microscopy with a of primaryprimary primary anti-glialanti-glial human fibrillaryfibrillaryglioblastoma acidic culture protein fixed (GFAP) at 80% antibody confluency and secondary. Glioma cells Texas were RedRed stained X.X. CellCell with nucleinuclei a werewereprimary stainedstained anti - withwithglial DAPI.fibrillary (a) acidic Stained protein cultures (GFAP) at 40 antibodyobjective. and ( bsecondary) Stained culturesTexas Red at X. 63 Cellobjective. nuclei were (c) Immunestained with cell DAPI. (a) Stained cultures at 40 ×× objective. (b) Stained cultures at 63 ×× objective. (c) Immune cell stainedDAPI. ( withwitha) Stained anti-CD11banti-CD11b cultures (red)(red) at presentpresent40 × objective. withinwithin aa(b glioblastomag)lioblastoma Stained cultures cultureculture at imagedimaged 63 × objective. withwith aa 40X40X (c) objective.objective.Immune cell stained with anti-CD11b (red) present within a glioblastoma culture imaged with a 40X objective. Troubleshooting 4.1. Troubleshooting TroubleshootingPotential issues arising and their respective troubleshooting solutions are presented in Table1. Potential issues arising and their respective troubleshooting solutions are presented in Table 1. Potential issues arising and their respective troubleshooting solutions are presented in Table 1.
Methods Protoc. 2020, 3, 11 5 of 7
Table 1. Potential issues arising and respective troubleshooting.
Step Issue Causes Suggestions 1. Ensure the tissue is 1. The tissue sample was fragmented into smaller not sufficiently (<2 mm) pieces in Step 8 fragmented in Step 8 2. Ensure proper storage of 2. The quality/strength of papain solution when not in use. Ensure that the Tissue does not easily the papain enzyme 11 papain solution has not dissociate when pipetted solution might be compromised expired. Increase the 3. The tissue was not left in concentration of papain papain solution for with larger samples. long enough 3. Leave sample in papain solution for an additional 5–10 min 1. Follow the suggestions for Step 11 (above) to ensure tissue is properly dissociated. Larger chunks of tissue 2. Use a 10 mL pipette to mix are present in Tissue was not properly cell solution and allow 13 homogenized cell dissociated in Step 11 larger pieces of tissue to settle at the bottom. solution Exclude the larger pieces of tissue when transferring onto 12-well culture plates. Change culture medium after 3 days. 1. Ensure tissue is 1. The tissue was damaged fragmented gently in by over-fragmentation in Step 8 Step 8 2. Smoothen the tip of the 2. The tip of the Pasteur Pasteur pipette with a pipette was broken or Bunsen burner prior to Cells are not becoming usage. Ensure tip is 13 too sharp, which confluent damaged cellular not damaged. integrity when pipetted 3. Ensure proper storage of 3. The quality of the culture medium when not culture medium has in use and that the been compromised medium or any component of the medium has not expired.
5. Reagents Setup
5.1. Poly-D-Lysine Plate Coating Solution Poly-D-lysine hydrobromide powder, 5 mg • 50 mL sterile distilled water • 5.2. Enzymatic Tissue Dissociation Solution 200 units papain from papaya latex (approximately 155 µL in 10 mL EBSS) • 10 mL EBSS • 5.3. Culture Medium Minimum Essential Medium, 1 , 500 mL • × Methods Protoc. 2020, 3, 11 6 of 7
1 mM D-glucose • 2 mM L-glutamine • 50 units/mL penicillin-streptomycin • 10% heat-inactivated foetal bovine serum • Corning®MITO+ Serum Extender (Merck, Australia; Cat. no.: DLW355006) •
Author Contributions: Methodology, M.M.; writing—original draft preparation, L.K.K.; writing—review and editing, L.K.K. and M.M.; supervision, M.M.; project administration, M.M.; overall project design and intellectual input, K.J.D., M.H., T.J.O. and D.A.W.; resection of human brain tumours, K.J.D. and M.H.; All authors have read and agreed to the published version of the manuscript. Funding: This research has been funded by Brain Foundation (Australia). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
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