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CELL HARVEST FROM BIONOC II CARRIERS
– PRINCIPLE AND STRATEGY –
GEOMETRY BIONOC II IS NON-WOVEN FABRIC MADE BY 20~25 LAYERS OF PET FIBERS WITH 300~400 UM THICKNESS. EACH LAYER IS LIKE
MESH WHICH RANDOMLY FORMS GRIDS WITH 100~1000 × 100~1000 UM
DIAMETER. THERE IS NO CHANNEL IN THE CARRIERS AS OTHER
CONVENTIONAL MACROPOROUS MICROCARRIERS. CELLS GROWN IN
THE FIBERS CAN BE HARVESTED DUE TO THE OPENING OF THE
FABRIC STRUCTURE.
SEVERAL LAYERS OF
NETTING FIBERS FORM
BIONOC II CARRIERS
FRONT VIEW OF SIDE VIEW OF
CELL GROWTH CELLS IN BIONOC II CARRIERS GROW ALONG THE FIBERS, AND THEN PILE UP TO FILL THE SPACE IN THE NET. A LOT OF
EXTRACELLULAR MATRIX (ECM) WILL BE PRODUCED IN ORDER TO
FORM 3-D TISSUE STRUCTURE IN THE CARRIERS.
FRONT VIEW OF CELL
STRUCTURE ON BIONOC II
SIDE VIEW OF CELL
STRUCTURE ON BIONOC II
PRINCIPLE TO HARVEST CELLS PRINCIPALLY THERE ARE TWO MAJOR FACTORS TO AFFECT HARVESTING CELLS FROM BIONOC II: ONE IS FABRIC; THE OTHER IS
ECM. THE FABRIC WILL PROTECT CELLS FROM SHEAR STRESS AND
LIQUID FLOW BUT IT WOULD BE DIFFICULT TO RELEASE CELLS BY
DISTURBING THE CULTURE MEDIUM ONLY. IT IS A WAY TO RELEASE
CELLS FROM THE CARRIERS BY PUTTING A FORCE ON THE CARRIER
TO PRODUCE A REACTING ONE- THIS IS WHY WE NEED TO TAP THE
BELLOCELL BOTTLE IN ORDER TO RELEASE THE CELLS.
FORCE ON COUNTERFORCE
GENERATED FOR
IF ECM IS NOT WELL
DIGESTED, ECM WILL
CATCH CELLS.
STRATEGY STRATEGY TO HARVEST CELLS FROM BIONOC II CARRIERS
EFFICIENTLY:
ONE IS TO DIGEST THE ECM COMPLETELY; THE OTHER IS TO
GENERATE A COUNTERFORCE ON THE CARRIERS TO RELEASE CELLS.
THE FORMER ONE IS THE MOST IMPORTANT. THE TIPS ARE LISTED
AS FOLLOWS.
1. IF THE DIGESTION IS NOT COMPLETE, FEW CELLS COULD BE
RELEASED NO MATTER HOW TAPPING WORK IS DONE.
2. PRE-WASH WITH PBS/5 MM EDTA 10~30 MINS COULD HELP FOR
EFFICIENT DIGEST BY THE LATER ENZYMATIC TREATMENT.
3. 20~30 MINS ENZYMATIC DIGESTION (SOAK10 MINS AND THEN
INCUBATE AT 37 FOR ℃ 10~20 MINS ) MAY BE NEEDED FOR MOST
CASES.
4. TAPPING AND WASHING 3 TIMES COULD RELEASE MOST CELLS
FROM THE CARRIERS.
5. IF TAPPING BY PALM IS UNCOMFORTABLE, USERS MAY TAP THE
BOTTLE BY HITTING THE EDGE OF A BENCH.
6. VIABILITY OF CELLS WILL DROP AS THE TAPPING/WASHING CYCLE
IS INCREASED. 3~4 CYCLES ARE ENOUGH FOR MOST CASES.
7. PLEASE READ THE GENERAL GUIDE FOR CELL HARVEST IN THE
FOLLOWING PAGE.
8. FOR DETAIL CELL HARVEST PROTOCOL, PLEASE CHECK THE
STEP-BY-STEP PROTOCOL IN BELLOCELL USER’S INSTRUCTION
MANUAL.
GENERAL GUIDE FOR CELL HARVEST FROM BELLOCELL BOTTLES
Cell Culture Complete
Sterility
Harvest cells directly from the
bottles
Dismatle the bottles, collect carriers In a separated PE
bottle and harvest cells from the PE bottle
Yes
No
Dismantle the bottles, collect carriers and extract target products from the carriers
Want cell massYes
No
1. Discard culture medium2. Wash with 500 ml PBS/5mM EDTA twice (critical)3. Compress the bottle with Cell Dissociation Supporter4. Add 200 ml trypsin/EDTA (0.25%/0.5mM)5. Incubate for 10 mins6. Discard trypsin/EDTA7. Incubate for another 10 mins8. Tap the bottles against your palm or a soft edge for 3 mins9. Wash off the cells with PBS or culture medium10. Repeat step 8 and 9 three times to harvest most of the cells
1. Wash with PBS/5mM EDTA twice (critical)2. Add trypsin/EDTA (0.25%/0.5mM) to submerge the carriers3. Incubate for 10 mins4. Discard trypsin/EDTA5. Incubate for another 10 mins6. Tap the bottles against your palm or a soft edge for 3 mins7. Wash off the cells with PBS or culture medium8. Repeat step 6 and 7 three times to harvest most of the cells
Bottle Opener
Bottle Opener
Cell Dissociation Supporter
Note1. Users could selectively test different cell dissociation reagents such as Accutase, AccuMax/EDTA, or other commercial reagents in order to optimize the cell harvest procedure.
2. The remaining dead cells on the carriers will release DNA and may form viscous mass or gel to besiege other alive cells if over- trypsinized. When this situation occurs, it increases the difficulty to harvest cells from the carriers. In order to alleviate this situation, wash the carriers with PBS/EDTA twice prior to trypsinization, and avoid trypsinize to over 20 mins. Alternatively, supply DNAse (1 mg/ml) after trypsinization, or use AccuMax+ 0.5mM EDTA for the whole process instead of trypsin.
1. Directly exact the protein inside or on the membrane of the cells with ultrasonic, freeze-and-thaw, or by adding lysis buffer. Follow generatl protocol for those cell lysis method.
Tool requiredProtocolNoteRoute of
purposeMeaning of the
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