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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