INSTRUCTION MANUAL
Contents
1. CELLYARD beads 2 CELLYARD beads 37 C 5 CO CELLYARD beads 3 CELLYARD beads 4 5 CELLYARD beads 5 1 01 1 05 g cm ALE 200 um 50um D gt amp RE 0 55 0 67 g cm 121C 1bar2. 2 4 2 CELLYARD beads 10 ml 6 we0 1600 5 PBS 2 ESE LED CELLYARD beads MEM 10 ml ES Ss DRR 01600 5 Cy eS 7 CELLYARD beads 3 10 10 ml CELLYARD beads 7 8 3 1 0 X 107 10 20 ml 8 A 9 CELLYARD beads xa Q
3. Surface activated cell carrier beads for high density cell culture CELLYARD beads 20 1 CELLYARD beads 2 CELLYARD beads 1 CELLYARD beads 1 200 um Caio PO4e OH 2 Diut CELLYARD beads 2 CELLYARD beads 3 CELLYARD beads CELLYARD beads BIA 2 CELLYARD beads 2
4. pH5 EDTA 6 1 A Tiss Cult Res Commun 18 235 244 1999 2 231 1992 3 Sugo K Kato M Ishikawa T Yamamoto A and Ogawa T Hydroxyapatite microcarrier 1 Tiss Cult Res Commun 25 105 111 2006 4 Sugo K Kato M Ishikawa T Yamamoto A and Ogawa T Hydroxyapatite microcarrier 2 Tiss Cult Res Commun 25 113 118 2006 5 Sugo K and Ogawa T Three dimensional Culture of Rat Bone Marrow Cells Using Hydroxyapatite Microcarrier Tiss Cult Res Commun 26 125 131 2007 6 Sugo K Nakayama M and Ogawa T Japanese Encephalitis Virus Production Using Hydroxyapatite Microcarrier Medicine and Biology 151 7 237 243 2007 HOYA PENTAX T 174 8639 2 36 9 E200804 TEL 03 3960 1290
5. 3 Vero 3 _ 2 121 C 20 1 2 3 4 FODRET SRL OT is n n mr 6 500um CELLYARD beads B CELLYARD beads200 CELLYARD beads500 _ e nE ooa 1 01 1 05 g cm 1 01 1 05 glom ___ Al 200um 50um 500 um 75 um 0 55 0 67 gleme 0 55 0 67 gicm 121 C 1 baric SMERRMBMATAE TT pH 5 EDIA
6. CELLYARD beads200 CELLYARD beads500 ogg zai CELLYARD beads200 SE me r LY r 2 AN a oe CELLYARD beacds500 SEM Vero 3 ra 2200350 _ _ _ Ld F F 2200352 CELLYARD beads200 500 g 120 000 2200358 CELLYARD beads500 50 g 18 000 Tee EES ae aE ls eee aes eee el LOO rere llr ree rll rE aS ee ee ee GG ee ee ee E E E 2200357 CELLYARD beads500 500 g 120 000 Sugo K Kato M Ishikawa T Yamamoto A and Ogawa T HYDROXYAPATITE MICROCARRIER 1 Tiss Cult Res Commun 2006 25 105 111 2 Sugo K Kato M Ishikawa T Yamamoto A and Ogawa T HYDROXYAPATITE MICROCARRIER 2 Tiss Cult Res Commun 2006 25 113 118 3 Sugo K and Ogawa T Three dimensional Culture of Rat Bone Marrow Cells using Hydroxyapatite Microcarrier Tiss Cult Res Commun 2007 26 125 131 4 Sugo K Nakayama M and Ogawa T Japanese Encephalitis Virus Production using Hydroxyapatite Microcarrier Medicine and Biology 2007 131 7 237 243 PENTAX HOYA PENTAX 174 8639 2 36 9 TEL 03 3960 1290 FAX 03 3960 2681 E100401 2008 04
7. 7 3 CELLYARD beads SEM 3 CELLYARD beads 1 03 gcm3 HEU E tk CELLYARD beads 4 CELLYARD beads MAR L 4 CELLYARD beads C6 36 PENTAX 3 CELLYARD beads CELLYARDS beads Vero 100 ml 1 CELLYARD beads CELLYARD beads 50 g PBS 500 ml Ra L rH ArT ORE LES 121 C 2045 CELLYARD beads PBSC 100 gD CELLYARD beads 5
8. FAX 03 3960 2681 4
9. stood for 3 hours the adhesion stage of the cell from the culture start without connected agitation Hig 10 In the time please do 1 minute agitation at 37 rpm speed every 60 minute In this time agitation speed you do not mind at the speed in the case of the connected agitation in the vegetation stage of the cell 3 hours passed and MEM medium 10 FCS is added in the clean bench to the final volume 100 mL and the connected agitation is started Fig 11 The aspect of the cell which passed of culture Fig 12 The CELLYARD beads in which the Vero EDO DOun ae Oe TOT cell adhered The culture start post 3 Fig 1 2 and Fig 13 i hour course Fig 13 The CELLYARD beads that the surface is perfectly covered by the Vero cell The culture start post 3 day course www cosmobio co jp COSMO BIO CO LTD Inspiration for Life Science gt 4 ATTENTION IN THE HANDLING 1 By seeding cell standing time in the adhesion stage must be adjusted in the about 8 6 hour And there is the case in which the connected agitation should be done by adding the medium of the final volume right after the seeding of the cell and the agitation should not be completely done while it is being stood in the adhesion stage By adjusting optimum culture condition of seeding cell please use the CELLYARD beads 2 It is recommended that the medium used for washing of the CELLYARD Beads and culture are beforehand equ
10. 00 ml CELLYARD beads DRJ 10 ml 20 ml 30 ml 173 9 5 4 37C 5 CO a 3 FRR GRE OTEN EA gy ie 10 60 10 1 87 rpm HETA TOL DAPRE EA ER A 3 10 100 ml ECM 11 3 12 3 13 11 12 Vero CELLYARD beads 13 Vero 3 CELLYARD beads 3 H H A 1 8 6
11. 6 JAPAN http www cosmobio co jp e mail export cosmobio co jp Phone 81 3 5632 9617 FAX 81 3 5632 9618 hy 01 8 1 m ty z M Do 7 4 nee i a F I pm nt TU E ona gt i u 7 i 1 q 1 i al 1 mh 1 Y x J ir i 1 f ov ii i x i fie 4 ih h Surface activated cell carrier beads a for high density cell culture CELLYARD CELLYARD beads 200 m Caio P04 0H ff Vero
12. COSMO BIO CO LTD Inspiration for Life Science CELLYARD beads 200 i ys INSTRUCTION MANUAL 1 USE The high density mass culture of various attachment dependent cell 2 MERIT 1 The CELLYARD beads Fig 1 is composed of microspheres that hydroxyapatite Caio PO4 6 OH 2 fine particles cover the surface of the nylon particles of average particle size 200 u m Hydroxyapatite is one of calcium phosphate system compounds has excellent biocompatibility and has already been used as artificial bone bone prosthetic material and so on In comparison with surface of the nylon particle Fig 2 and surface of the CELLYARD beads Fig 3 the structure of the nylon which exists on the surface of the nylon particle is not completely observed in the surface of the CELLYARD beads This means that the particle surface of the CELLYARD beads is covered with hydroxyapatite nearly completely Therefore the CELLYARD beads has the capability to SA adsorb various protein a cell etc like oh a RY gt hydroxyapatite Fig 4 4 ae saan gk V S58 ORNS gn x se k Ta a Fig 2 SEM photograph of the surface of nylon particle E ee 2 The particle size of the CELLYARD beads is oe rR A I suitable for adhesion and multiplication of cells 3 The specific gravity of the CELLYARD beads 1 01 1 05 g cm3 leads suspension in calm agitation and spontaneous sedimentation in standing con
13. dition and is suitable for recovery of the CELLYARD beads or medium i 4 The CELLYARD beads do not swell in solution Fig 4 The C6 36 cells adhering to the surface of CELLYARD beads www cosmobio co jp COSMO BIO CO LTD Inspiration for Life Science 3 USAGE The CELLYARD beads is suitable for high density culture of various attachment dependent cell but optimum condition such as capacity of culture flask form and agitation speed change with kind of cell Therefore this instruction manual explains the method for culture of the Vero cell the African green monkey kidney origin cell using spinner flask for 100 mL 1 STERILIZATION 50g the CELLYARD beads suspended to 500 mL PBS are carried out the autoclave sterilization 121 C 20 min The CELLYARD beads is finely dispersed after sterilization although it may be hard to disperse to PBS at first Sterilized suspension 100 g l is conserved at room temperature Fig 5 X Please perform operation of following 2 4 within the clean bench of aseptic condition A TI ria ie iF my 2 WASHING 10 mL of suspension of the CELLYARD beads prepared in 1 is moved to another centri fugal tube which sterilized Fig 6 and supernatant is discarded by centrifugation TL TLE Fig 6 The suspension of CELLYARD beads is moved to centrifugal tube 1600 rpm 5 min You may carry ou
14. ilibrated in incubator at 37C and 5 COQOz2 So the CELLYARD beads can be used more effectively 3 Please do not dip the CELLYARD beads in acid solution and solution including chelating reagent and organic solvent such as acetone xylene and so on The performance may deteriorate 4 There is no problem on the performance though the particle of black or brown may have been mixed in the product 5 Please dispose of the CELLYARD beads as an incombustible refuse after the use 5 REFERENCE 1 Yamamoto A Nakajima T Tominaga Y and Ogawa T Porus hydroxyapatite ceramics Tiss Cult Res Commun 18 235 244 1999 2 Shibata T The world of the bioreactor the base and application for the practice person Hario Laboratory Co Ltd 231 1992 3 Sugo K Kato M Ishikawa T Yamamoto A and Ogawa T Hydroxyapatite microcarrier 1 Tiss Cult Res Commun 25 105 111 2006 4 Sugo K Kato M Ishikawa T Yamamoto A and Ogawa T Hydroxyapatite microcarrier 2 Tiss Cult Res Commun 25 113 118 2006 5 Sugo K and Ogawa T Three dimensional Culture of Rat Bone Marrow Cells Using Hydroxyapatite Microcarrier Tiss Cult Res Commun 26 125 131 2007 6 Sugo K Nakayama M and Ogawa T Japanese Encephalitis Virus Production Using Hydroxyapatite Microcarrier Medicine and Biology 151 7 237 2438 2007 200804 BSI gt COSMO BIO Co LTD Inspiration for Life Science TOYO 2CHOME KOTO KU TOKYO 135 001
15. t spon taneous sedimentation without using centrifuge 10 mL of the MEM medium FCS free is added to the pellet of the CELLYARD beads and is washed Then centrifugation 1600 rpm 5 min or spontaneous sedimentation is carried out and supernatant is Fig 7 CELLYARD beads is suspended to the medium discarded Please repeat this operation 3 times 10 mL of MEM medium 10 FCS is newly added to the pellet of the CELLYARD beads in order to prepare the suspension of the CELLYARD beads ee Please use a pipette with the large path of Fig 8 The recovery of the cell the hole at a tip www cosmobio co jp COSMO BIO CO LTD Inspiration for Life Science 3 PREPARATION OF THE CELL The cell is recovered at 20 mL of MEM medium 10 FCS in order to prepare the suspension of the cell so that the concentration of seeding cell may consist as 1 0 X 107 cells Fig 8 Fig 9 Suspension of the CELLYARD beads and the cell are added to the spinner flask 4 SEEDING OF THE CELL In sterilized spinner flask for 100 mL 10 mL of suspension of the CELLYARD beads prepared in 2 and 20 mL suspension of the cell prepared in 3 are added and the whole volume is done at about 30 mL 1 3 of the final volume Fig 9 Fig 10 Itis stood in the sanction stage of 5 CULTURE START the cell without agitation The spinner flask is stood in incubator 37 C 5 CO2 in the condition of 4 It is
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