STEMCELL Quality Control Kits

Transcription

1 [Type text] TECHNICAL MANUAL STEMCELL Quality Cntrl Kits

2

3 i Table f Cntents 1.0 Intrductin Thawing Cells, Plating, and Clny Cunting Supplies and Reagents Included in the QC Kit Additinal Reagents and Equipment Required Prduct Strage Methd Definitins Thawing Cells Perfrming Cell Cunts Diluting Cell Stck Cunting Clnies CD34 + Cell Cunting (Optinal) Wrksheet... 9

4

5 1 1.0 Intrductin Clngenic assays are dependent n the ability f single hematpietic prgenitr cells t divide and differentiate, frming clusters f cells (clnies) in semi-slid media cntaining apprpriate grwth factrs. Since their intrductin mre than 40 years ag, clny assays have been used extensively fr research and clinical applicatins including identificatin f stimulatry and inhibitry grwth factrs, supprtive diagnstic assays f myelprliferative disrders and leukemias, and evaluatin f the hematpietic ptential f bne marrw, leukapheresis, and crd bld cell preparatins fr clinical transplantatin. The clny assay is the benchmark functinal assay t assess the ability f varius hematpietic cell surces t divide and differentiate, especially fllwing ex viv manipulatins including T cell depletin, vlume reductin, CD34 + cell enrichment, crypreservatin, lng-term strage, thawing, and washing. It is imprtant t maintain a high degree f cnsistency in the prgenitr assay setup and readut (clny cunting) within a given labratry. The STEMCELL Quality Cntrl Kits enable labratries t mnitr their cnsistency n a regular basis. Clny-frming unit (CFU) assays that are set up each mnth using crypreserved samples f the same human cell preparatin supplied with each STEMCELL Quality Cntrl Kit generate data that will prvide a recrd f the labratry s r individual technlgist s reprducibility at setting up, culturing, and cunting hematpietic clnies ver the curse f ne year. Variatins in the numbers f clnies cunted in successive mnthly assays can reveal incnsistencies in lab perfrmance that may need t be addressed. In additin, large variatins in clny numbers may highlight equipment malfunctin (e.g. freezer r incubatr) that may therwise have gne undetected. STEMCELL Quality Cntrl Kits are available with human cells frm bne marrw (STEMCELL QC-BM) r crd bld (STEMCELL QC-CB) t allw assessment f the cell type mst apprpriate t a given labratry s applicatins. PRODUCT CATALOG # CELL TYPE PROVIDED IN KIT STEMCELL QC-BM Human Bne Marrw STEMCELL QC-CB Human Crd Bld

6 2 2.0 Thawing Cells, Plating, and Clny Cunting 2.1 Supplies and Reagents Included in the QC Kit 12 frzen vials f pre-tested cells The surce f the cells will vary depending n the catalg number rdered: Catalg #00650: Frzen Human Bne Marrw, mnnuclear cells Catalg #00651: Frzen Human Crd Bld, mnnuclear cells 24 tubes f MethCult H4034 Optimum (3 ml per tube) Nte: Only 12 tubes f MethCult are required fr use with the QC Kit. Use the remaining tubes as desired. Prduct cntains the fllwing: Methylcellulse in Iscve s Mdified Dulbecc s Medium (IMDM) Fetal bvine serum (FBS) Bvine serum albumin (BSA) 2-Mercaptethanl Recmbinant human (rh) stem cell factr (SCF) rh Granulcyte macrphage clny-stimulating factr (GM-CSF) rh Interleukin 3 (IL-3) rh Granulcyte clny-stimulating factr (G-CSF) rh Erythrpietin (EPO) Supplements 15 sterile 16 gauge blunt-end needles 15 sterile 3 ml syringes 40 x 35 mm tissue culture dishes 5 x 60 mm gridded scring dishes 15 x 100 mm dishes 12 x 100 ml bttles f IMDM + 2% FBS Trypan Blue (20 ml bttle) Atlas f Human Hematpietic Clnies Instructin manual Letter, with lt-specific infrmatin that references the 10X Plating Density

7 3 2.2 Additinal Reagents and Equipment Required Bihazard safety cabinet certified fr Level II handling f bilgical materials Lw-speed centrifuge equipped with bihazard cntainers fr handling human cells 37 C incubatr with humidity and gas cntrl t maintain > 95% humidity and an atmsphere f 5% CO 2 in air Vrtex mixer Inverted micrscpe with flat field bjectives and eyepieces t give ttal bject magnificatin f apprximately 20-30X, 40-63X, and X. Nte: Ttal bject magnificatin = eyepiece x bjective i.e. 25X = 2.5 x 10 Hemcytmeter (e.g. Neubauer) 70% ethanl r isprpanl 3% Acetic Acid with Methylene Blue (Catalg #07060) Rutine light micrscpe fr hemcytmeter cell cunts 14 ml plystyrene tube (e.g. Catalg #38008) 5 ml rund-bttm plystyrene tube (e.g. Catalg #38007) 50 ml cnical tube (e.g. Catalg #38010) 1 ml, 2 ml, and 10 ml serlgical pipettes (e.g. Catalg #38001, 38002, and 38004) Sterile distilled water Hand tally cunter 2.3 Prduct Strage Stre bne marrw and crd bld cells at -135 C r clder, r in liquid nitrgen, fr up t 2 years. Stre MethCult H4034 Optimum and IMDM + 2% FBS at -20 C (-25 C t -15 C). D nt exceed expiry date (EXP) as indicated n label. Stre all ther materials and reagents at rm temperature (15-25 C).

8 4 2.4 Methd Definitins Cell Stck: The neat mnnuclear cell sample washed with IMDM with 2% FBS Viable Cell Cncentratin (cells per ml): Cell Stck Cncentratin (sectin step A6) x % Viability 10X Plating Density (cells per ml): The viable cell cncentratin f the Cell Stck which has been diluted in IMDM with 2% FBS t ten times (10X) the final cell plating density Final Plating Density (cells per ml): The number f viable cells per vlume f semi-slid culture medium per well Thawing Cells 1. Thaw cells quickly (within 2 minutes) in a 37 C water bath by gently swirling. D nt vrtex cells at any time during the thawing prcedure. 2. When the cells are almst cmpletely thawed, wipe the utside f the vial with 70% ethanl r isprpanl. 3. Gently transfer cells t a 14 ml plystyrene tube. 4. Slwly (drpwise) add 10 ml f IMDM + 2% FBS while gently swirling the tube (apprximately 1-2 minutes). 5. Gently invert tube t mix. 6. Centrifuge cells at 300 x g fr 10 minutes at rm temperature (15-25 C). 7. Carefully remve the supernatant, taking care nt t disldge the cell pellet. D nt pur ff. Gently flick the tube t resuspend the cell pellet. 8. Add 2 ml f IMDM + 2% FBS t the tube. This is the Cell Stck Perfrming Cell Cunts The cell cunting prcedures utlined belw are suggestins. Use the prcedures that have been validated in yur institutin. A) Manual Nucleated Cell Cunt 1. Clean cverslip and hemcytmeter thrughly with alchl. Dry cverslip and hemcytmeter with lint-free tissue befre using. Place the cverslip n the hemcytmeter s that it is centered ver bth chambers. 2. Dilute Cell Stck fr a nucleated cell cunt accrding t yur labratry standards. Mix the diluted sample. Example: Place 20 µl f Cell Stck int 380 µl f 3% Acetic Acid with Methylene Blue t achieve a 1 in 20 dilutin. 3. Draw up an aliqut f diluted sample using a micrpipettr r capillary tube. 4. Fill bth chambers f the hemcytmeter using a micrpipettr r capillary tube. D nt verfill r underfill the chambers.

9 5 5. Starting with ne chamber f the hemcytmeter, cunt all the nucleated cells in at least tw f the majr crner 1 mm squares using a hand tally cunter r ther similar device. Cunt the same number f squares in the ppsite chamber. Keep a ttal cunt f the cells and establish the average number f cells per square. If the cell cunt is less than 10 cells per square, a mre cncentrated suspensin shuld be prepared (i.e. 20 µl f sample int 180 µl f 3% Acetic Acid with Methylene Blue t achieve a 1 in 10 dilutin). 6. Determine the Cell Stck Cncentratin as fllws: Each f the 9 majr squares f the hemcytmeter, with cverslip in place, represents a ttal vlume f 0.1 mm 3 (r 10-4 cm 3, which is equivalent t 10-4 ml). The Cell Stck Cncentratin and ttal number f cells can be determined using the fllwing calculatins: Cell Stck Cncentratin (cells per ml) = average cunt per square x dilutin factr x 10 4 TOTAL CELLS = Cell Stck Cncentratin x riginal start vlume B) Viable Cell Cunt (Trypan Blue Exclusin Test) 1. Clean cverslip and hemcytmeter thrughly with alchl. Dry cverslip and hemcytmeter with lint-free tissues befre using. Place the cverslip n the hemcytmeter s that it is centered ver bth chambers. 2. Dispense 100 µl f Cell Stck int a 12 x 75 mm tube. 3. Dispense 100 µl f Trypan Blue int the same tube. 4. Agitate gently and let tube stand undisturbed fr 2 minutes. D nt let the mixture stand fr lnger than 5 minutes as viable cells may begin t take up the stain as well. 5. Draw up an aliqut f the diluted sample using a pipettr r capillary tube. 6. Fill bth chambers f the hemcytmeter using a pipettr r capillary tube. D nt verfill r underfill the chambers. 7. Using a multi-channel cunter r tw hand tally cunters, cunt each viable, clear (nn-blue) nucleated cell and each nn-viable, blue nucleated cell (cells with damaged membranes) separately. Cntinue t scre squares in the hemcytmeter until yu have a cunted a minimum f 100 cells. 8. Calculate the % Viability using the fllwing frmula: % = ( ) 100%

10 Diluting Cell Stck 1. Thaw a tube f MethCult H4034 Optimum at rm temperature (15-25 C) r vernight at 2-8 C. 2. Refer t the lt-specific infrmatin letter (Dcument #29115 r 29116) included in the QC Kit. The letter references a cell cncentratin called 10X Plating Density t be used t set up the CFU assay with a predetermined cell number. The fllwing steps utline hw t dilute the Cell Stck t prepare the 10X Plating Density, which is then diluted 10-fld in the semi-slid culture medium t generate the Final Plating Density. 3. Use this frmula t calculate the Viable Cell Cncentratin f the Cell Stck: Example: Viable Cell Cncentratin = Cell Stck Cncentratin X (sectin step A6) Viable Cell Cncentratin = 3.8 x 10 6 cells per ml x 92% = 3.5 x 10 6 cells per ml % Viability (sectin step B8) 4. Use this frmula t calculate the Vlume f Cell Stck and Vlume f IMDM + 2% FBS required t prepare 1 ml f the 10X Plating Density: ( )= ( ; # ) ( )(.. ) Vlume f (1 IMDM ml) = (1 ml) -(Vlume f Cell Stck [ml]) + 2% FBS (ml) 5. Gently mix the Vlume f Cell Stck needed + Vlume f IMDM with 2% FBS (as calculated abve) t prepare the 10X Plating Density fr CFU assay setup. 6. Add 0.3 ml f the 10X Plating Density (step 5) t the 3 ml tube f MethCult H4034 Optimum t btain the Final Plating Density. 7. Vrtex the tube vigrusly fr at least 4 secnds. After vrtexing, let the tube stand fr at least 5 minutes t allw all bubbles t rise t the surface. 8. Prepare 35 mm dishes by placing them in pairs inside a 100 mm dish. Be sure t add a third 35 mm dish (withut its lid) fr a water dish. The purpse f the water dish is t ensure that maximum humidity is maintained during incubatin. The 35 mm dishes used fr the assay cultures have been pre-tested fr ptimal clny grwth and d nt supprt grwth f anchrage-dependent cells. 9. The package f 10 x 35 mm dishes shuld be resealed fr future assays. 10. T plate the MethCult /cell mixture int the sterile dishes, attach a 16 gauge blunt-end needle t a 3 ml syringe. Draw up the MethCult /cell mixture t the 1.0 ml mark and slwly dispense this initial vlume back int the tube in rder t remve the large air bubble that is present in the syringe and needle. Draw up the mixture again t the 2.6 ml mark. Dispense 1.1 ml int a labeled 35 mm dish (plunger nw at 1.5 ml mark). Dispense anther 1.1 ml int the secnd labeled dish (plunger nw at 0.4 ml mark). 11. Rtate and tilt each dish t spread the viscus mixture evenly acrss the surface f each dish. 12. Add 3 ml f sterile water t the water dish. Place the cultures n a level tray in a 37 C humidified incubatr with 5% CO 2 in air. It is imprtant that the crrect temperature, CO 2, and humidity (> 95%) are maintained in the incubatr during the entire culture perid.

11 7 13. Incubate fr 14 days at 37 C, 5% CO 2 in air, and > 95% humidity Cunting Clnies 1. Prepare a 60 mm gridded scring dish by drawing tw perpendicular lines acrss the center f the dish using a permanent fine felt marker n the bttm f the dish (refer t page 4 f the Atlas f Human Hematpietic Clnies). This scring dish can be used again t scre ther culture dishes. 2. Remve cultures frm the incubatr and place them (with the lid still n), ne at a time, inside the 60 mm gridded tissue culture dish t cunt the clnies in situ using an inverted micrscpe. Cunting is usually easier if clnies are cunted in vertical rws by mving the micrscpe stage up and dwn (rather than acrss) the dish. 3. Cunts at day shuld include the smaller erythrid clnies, derived frm the mst mature types f erythrid clny-frming cells (i.e. frm CFU-E); the larger erythrid clnies (frm primitive BFU-E); all granulpietic clnies (frm CFU-GM); and clnies cntaining multiple lineages f cells (frm CFU-GEMM). Fr detailed assistance in the recgnitin f varius clny types, refer t the Atlas f Human Hematpietic Clnies prvided. CFU-E BFU-E CFU-GM CFU-GEMM Clny-frming unit-erythrid f 1-2 small clusters cntaining erythrblasts Burst-frming unit-erythrid cntaining greater than 200 erythrblasts (may cntain greater than 2 clusters) Clny-frming unit-granulcyte, macrphage cntaining 40 r mre cells f the granulcyte and/r macrphage lineage Clny-frming unit-granulcyte, erythrid, macrphage, megakarycyte cntaining erythrid cells and 20 r mre granulcyte, macrphage, erythrid, and megakarycyte cells Nte: A blue filter enhances the red clr f the erythrid clnies and may help in the identificatin f bth CFU-E and BFU-E. 4. Scan the entire dish at a magnificatin f 20-30X t ensure that the plating efficiency is representative f the entire dish. Scring r cunting the number and types f clnies is best dne using an inverted micrscpe equipped with high quality flatfield bjectives and eyepieces t give ttal bject magnificatin f apprximately 20-30X, 40-63X, and X. Ttal bject magnificatin = eyepiece magnificatin x bjective magnificatin Example Ttal bject magnificatin = (12.5X) x (5X) = 62.5X 5. Scre the CFU-E using the ttal bject magnificatin 40-63X. Once cmpleted, scre the remaining BFU-E, CFU-GEMM and CFU-GM clnies using the 20-30X ttal bject magnificatin. Use a higher magnificatin t cnfirm clny type if uncertain. 6. Recrd the number f clnies in each f the culture dishes n the wrksheet prvided in sectin 4.0. Calculate the average number f each type f prgenitr detected in the clny assay by dividing the sum f the number f clnies in the tw dishes by tw. Keep a cpy f this Wrksheet in yur files fr future use.

12 8 3.0 CD34 + Cell Cunting (Optinal) Materials and recmmendatins fr CD34 + cell cunting have nt been supplied with this kit. Hwever, the cell suspensin may be tested t assess the cnsistency f CD34 + cell cunting by flw cytmetry using the standard perating prcedures and antibdies emplyed in yur labratry. The CD34 + cell frequency determined fllwing incubatin f cnjugated antibdies and flw cytmetry analysis shuld be recrded fr each test. Plts generated frm the mnthly analysis f CD34 + cell frequency will demnstrate the reprducibility f yur prtcl in cunting the CD34 + cell cntent f a given sample.

13 9 4.0 Wrksheet STEMCELL QC LOT# CFU-E BFU-E CFU-GM CFU-GEMM TOTAL CFU Test 1 Test 2 Test 3 Test 4 Test 5 Test 6 Test 7 Test 8 Test 9 Test 10 Test 11 Test 12 Cpyright 2018 by STEMCELL Technlgies Inc. All rights reserved, including graphics and images. STEMCELL Technlgies & Design, STEMCELL shield, Scientists Helping Scientists, and MethCult are trademarks f STEMCELL Technlgies Canada Inc. All ther trademarks and registered trademarks are prperty f their respective hlders. While STEMCELL has made all reasnable effrts t ensure that the infrmatin prvided by STEMCELL and its suppliers is crrect, it makes n warranties r representatins as t the accuracy r cmpleteness f such infrmatin.

14

15

16 TECHNICAL MANUAL STEMCELL Quality Cntrl Kits TOLL-FREE PHONE PHONE INFO@STEMCELL.COM TECHSUPPORT@STEMCELL.COM FOR GLOBAL CONTACT DETAILS VISIT DOCUMENT #28465 VERSION NOV 2018