Isolation of Human Mononuclear Cells from Synovial Fluid

Transcription

1 UCANU 0008 Version 01, November 2011 Page 1 of 8 CMCI (Center for molecular and Cellular Intervention) University Medical Center Utrecht Written By Name Function Date Signature Mark Klein Lab manager Conformation Name Function Date Signature Prof.dr. A.B.J. Prakken CoChair CMCI Changes from last version Date of version Paragraphs Date of version Paragraphs

2 UCANU 0008 Version 01, November 2011 Page 2 of 8 Content 1. Subject Application Definitions and Abbreviations Principle Safety precautions Reagents Chemicals Basic Culture Medium Wash Medium Freezing Medium % AB medium Equipment and Accessories tools Equipment Accessories Samples Sample Collection Sample Processing Procedure Processing of the Results Calculation of the counted cells Documentation of cells... Error! Bookmark not defined Errors Documentation... Error! Bookmark not defined. 12. Accuracies and Precision Quality Control Accompanied Forms/ Attachments/ Document... Error! Bookmark not defined. 15. Remarks Literature... 8 Deleted: 3 Deleted: 5 Deleted: 5 Deleted: 7 Deleted: 7 Deleted: 7 Deleted: 7 Deleted: 8

3 UCANU 0008 Version 01, November 2011 Page 3 of 8 1. Subject This Standard Operation Procedure (SOP) describes a method to isolate plasma and mononuclear cells from synovial fluid using gradient centrifugation. 2. Application Isolation of lymphocytes and monocytes from synovial fluid using density centrifugation. 3. Definitions and Abbreviations SFMC FBS RT P/S Glu v/v rpm ml DMSO g/l = Synovial fluid mononuclear cells = foetal bovine serum ( foetal calf serum) = room temperature = PenicillinStreptomycin = Lglutamine = volume/volume = rotations per minute = millilitre = dimethylsulfoxide = gram per litre 4. Principle Defibrinated or anticoagulant treated blood or synvial fluid is layered on ficoll solution (figure 1A) and centrifuged for a short period of time. Differential migration during centrifugation results in the formation of layers containing different cell types. The bottom layer contains erythrocytes and debris which have been aggregated by the ficoll and, therefore, sediment completely through the ficoll solution. The layer immediately above the erythrocyte layer contains mostly granulocytes which at the osmotic pressure of the ficoll solution attain a density great enough to migrate through the ficoll layer (figure 1B). Because of their lower density, the lymphocytes are found at the interface between the plasma and the ficoll with other slowly sedimenting particles (platelets and monocytes). The lymphocytes are recovered from the interface and are subjected to washing steps to remove any platelets, ficoll and plasma residues (1). A Sample Ficoll B Plasma Lymphocytes Ficoll Granulocytes / Erythrocytes Figure 1. Cell separation with Ficoll. A) Before centrifugation. B) After centrifugation

4 UCANU 0008 Version 01, November 2011 Page 4 of 8 5. Safety precautions Treed every blood and synovial fluid samples as infectious material. Wear disposable gloves. SumaTab contains sodiumdichloroisocynate Turk solution Trypan blue Xn, Harmful Xn, Harmful T, Toxic 6. Reagents 6.1 Chemicals Reagents Formula Supplier order number Store at ( C) FicollPaque PLUS GE Lifesciences Hyalorunidase 20 RPMI Lglutamine PenicillinStreptomycin Foetal Bovine Serum RT Turck solution (CH 3 ) 2 SO RT Trypan Blue C 34 H 28 N 6 O 14 S RT Dimethylsulfoxide Sanquin Bloodbank 20 Human AB serum 6.2 Basic Culture Medium RPMI 1640 supplemented with 1% v/v P/S (= 5ml) and 1% v/v glu (=5ml) Store at 4 C up to 1 month 6.3 Wash Medium RPMI 1640 supplemented with 1 v/v% P/S (= 5ml), 1 v/v% glu (=5ml) and 2% v/v FBS (=10 ml) FBS should be heat inactivated (1 hr at 56 C) and filtered through a 0.20 µm sterile filter using a 10 ml sterile syringe before usage. Store at 4 C up to 1 month.

5 UCANU 0008 Version 01, November 2011 Page 5 of Freezing Medium Mix 10 v/v% DMSO (5 ml) with 90 v/v% FBS in a 50 ml sterile tube. Filter through a 0.20 µm filter using a 50 syringe in to a new 50 ml sterile tube. Store at 4 C up to 1 month % AB medium Thaw 5 ml heat inactivated (1 hr at 56 C) human serum and filter through a 0.20 µm sterile filter using a 10 ml sterile syringe. Add 20 ml basic culture medium with a sterile 25 ml pipet and mix. Store at 4 C up to 1 week. 7. Equipment and Accessories tools 7.1 Equipment Centrifuge Hettich Rotanta 46 (UMC# ) Reichert brightline hemacytometer (Hausser Scientific Company, Horsham PA, USA) Easypet pipet (Eppendorf, Germany, ) Pipets µl (Gilson, The Hague, The Netherlands) 7.2 Accessories 1.8 ml sterile polypropylene Nunc crytubes (Nalge Nunc, Roskilde, Denmark, ml sterile polypropylene conical tubes (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) 50 ml sterile polypropylene conical tubes (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) Cell Strainer 70 µm (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) Minisart 0.20 µm single use sterile filter (Sartorius, Hanover, Germany, 16534) 10 ml sodiumheparin vacutainer blood collection tubes (Becton Dickinson, Erembodegem, Belgium, ) Sterile 10 ml syringe (Becton Dickinson, Erembodegem, Belgium) Sterile 50 ml syringe (Tyco Healthcare, Gosport, Northern Ireland, ) 2 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) 5 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) 10 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) 25 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) Sterile Pasteur pipettes Disposable gloves (Kimberly Clark, Zaventem, Belgium) Easyload 200 µl (741035) and 1000 µl (741000) sterile pipettips (Greiner Bioone, Germany) 8. Samples 8.1 Sample Collection Synovial fluid should be collected in a syringe. The synovial fluid should be transferred to

6 UCANU 0008 Version 01, November 2011 Page 6 of 8 sodium heparin tubes using a sterile needle that is pushed through the rubber stopper of the collection tube. 8.2 Sample Processing All handlings of the sample should be done in a biohazard safety cabinet Take ml of the synovial sample and place in a 15 ml sterile tube. Centrifuge the sample samples 10 minutes at 980 x g (= 3000 rpm). Collect the supernatant in a 1.8 ml properly labeled nunc tube and store at 20 C. Trash the 15 ml tube with the synovial debris.. 9. Procedure 1 Transfer the synovial fluid to a sterile tube and incubate 30 minutes at 37 C with Hyalorunidase (10 µl/ml synovial fluid, 20 U/ml) 2 Take ml of the synovial sample (depending on the amount of synovial fluid) and place in a 15 ml sterile tube. Centrifuge the sample samples 10 minutes at 980 x g (= 3000 rpm). Collect 150 µl of the supernatant in a 1.8 ml properly labeled nunc tube and store at 20 C. Collect the rest of the supernatant also in properly labeled nunc tube (1.5 ml/vial) and store at 20 C.Trash the 15 ml tube with the synovial debris. 3 Fill a 15 ml sterile tube with 4 ml ficoll (density = 1077 g/l) 4 Transfer the synovial fluid from the blood collection tube in to a sterile 50 ml tube. 5 Dilute the samples with a double volume of basic culture medium (20 ml synovial fluid with 40 ml basic culture medium) 6 Run the diluted synovial fluid through a 0.70 µm cell strainer filter that is placed above a sterile 50 ml tube to remove large chunks of debris 7 Slowly layer the diluted samples with a 10 ml sterile pipet onto the ficoll. Be careful not to disturb or mix the ficoll sample interface. Note that a minimum of 4 ml sample is needed that can be layered onto 4 ml ficoll. 8 Centrifuge the 15 ml tubes 20 minutes at 580 x g (= 2300 rpm) at RT with a rampup of 9 and a break of maximum 3. 9 Fill a 50 ml with 10 ml wash medium 10 Harvest the interface (SFMC fraction) of the 15 ml ficoll tube using a sterile Pasteur pipet into the 50 ml tube containing the wash medium. Collect as little plasma and ficoll as possible. 11 Fill the 50 ml tube containing the SFMC up to 50 ml with wash medium 12 Centrifuge 10 minutes at RT at 280 x g (1600 rpm), maximum rampup and brake 13 Remove the supernatant and resuspend the cells with a 2 ml sterile pipet 14 Remove cellular debris within the 2 ml pipet by keeping it at an angel of 90 and empty slowly 15 Fill the 50 ml tube up to 50 ml with wash medium 16 Centrifuge 10 minutes at RT at 280 x g (1600 rpm), maximum rampup and brake 17 Remove the supernatant of the 50 ml 18 Resuspend the cells with a sterile 2 ml pipet a) in 2 ml wash medium when cells are frozen b) in 2 ml 20% AB medium when cells are cultured 19 Dilute 10 µl cell suspension with 90 µl counting solution

7 UCANU 0008 Version 01, November 2011 Page 7 of 8 a) Use Turk solution when cells are from freshly isolated b) Use Trypanblue when viability has to be assed 20 Count cells a) Add approximately 10 µl countingcell solution to the counting chamber b) Do not over or under fill and leave the counting chamber for one minute at RT so the cells can settle c) Count the center 1 cubic millimeter square (each cubic millimeter equals the 25 groups of 16 squares, see figure 2). d) Count total cells per ml when using Turk solution (dark colored cells are cells with a nucleus which is stained with Turk, erythrocytes are lysed in this solution) e) Count total live and dead cells per ml when using Trypan blue (Trypan blue is a viable dye: living cells with an intact cellular membrane are capable of excluding the dye. Dead cells are stained dark blue. Note that erythrocytes are visible.) f) Count a clump of cells as one. If there are many clumps resuspend the original sample additionally by pipetting and recount the sample) Figure 2. Layout of Reichert brightline hemacytometer 21 Adjust the cell concentration to 2*106 per ml with 20% AB medium when cells are being cultured 22 When cells are frozen, fill the 50 ml tube containing the cell suspension up to 50 ml with wash medium 23 Centrifuge 10 minutes at RT at 280 x g (1600 rpm), maximum rampup and brake 24 Label sterile1.8 ml nunc cryovials 25 Remove the supernatant of the 50 ml 26 Resuspend the cells in a concentration of 10*106 per 1.5 ml in to cold (+4 C) freezing medium (the ideal number is 10*106 per 1.5 ml. More and less cells can be frozen in the same volume but do not exceed the limit of 4 15 *106 cells per Place the Nunc 1.8 ml cryovials on ice 28 Place the Nunc 1.8 ml cryovials on ice 29 Aliquot the cell suspension at 1.5 ml per nunc cryovial 30 Place cells in 80 C freezer within 15 minutes after resuspending with freezing medium 31 Transfer the cryovials within one week from the 80 C into the liquid nitrogen tank Processing of the Results Calculation of the counted cells Number of cells per ml = Counted cells per mm2 (25 squares) * dilution (10) * = Counted cells * 105

8 UCANU 0008 Version 01, November 2011 Page 8 of Errors The Isolation of cells can be disturbed by a number of factors Too low temperature of the reagents (ficoll) or sample (<16 C) Wrong layering of the sample (to fast) Wrong centrifugation speed Full break after centrifugations Not careful collection of the interphase Furthermore clotted blood can disturb the interphase when clots are sinned through the ficoll. 11. Accuracies and Precision Accuracy is depending on the sample and the temperature of the sample and the reagents. Ideal temperature is between 16 and 22 C. The recovery of cells when the procedure is followed correctly: Blood Synovial Fluid 12. Quality Control ± 1.0 *10 6 /ml depending on health status of each individual 95 ± 5% of cells present in fraction are mononuclear cells 60 ± 10% lymphocytes from original sample > 90% vitality 3 ± 2% granulocytes 5 ± 2% erythrocytes N/A The mononuclear fraction can be checked for purity by staining for CD15 which is a granulocyte marker. The percentage of CD15 positive cells should be less than 5%. 13. Remarks When closing 1.8 ml cryotubes do not close them too tight, as this will break the inner seal. Screw lids firmly but stop when a resistance of the lid is felt. 14. Literature (1) Boyum A. Separation of White Blood Cells. Nature 1964; 204:7934.: ***END***