The document provides guidance on harvesting cells grown on BIONOC II carriers. BIONOC II carriers are made of multiple layers of PET fibers that form a mesh-like structure for cell growth. Cells grow along and between the fibers and produce extracellular matrix (ECM). To harvest cells, the ECM must be fully digested using enzymatic treatment and tapping or agitation of the carriers to generate a counterforce that releases the cells from the fibers and ECM. A multi-step process including washing, trypsinization, tapping, and repeating is recommended to efficiently harvest the cells.

1. 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

2. 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 SIDE VIEW OF CELL
STRUCTURE ON BIONOC II STRUCTURE ON BIONOC II

3. 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.
COUNTERFORCE
FORCE ON
GENERATED FOR
IF ECM IS NOT WELL
DIGESTED, ECM WILL
CATCH CELLS.

4. 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.

5. GENERAL GUIDE FOR CELL HARVEST FROM BELLOCELL BOTTLES
1. Discard culture medium
2. Wash with 500 ml PBS/5mM EDTA twice (critical)
3. Compress the bottle with Cell Dissociation Supporter
4. Add 200 ml trypsin/EDTA (0.25%/0.5mM)
5. Incubate for 10 mins
Harvest cells
6. Discard trypsin/EDTA
directly from the
7. Incubate for another 10 mins
bottles
8. Tap the bottles against your palm or a soft edge for 3
Note
mins
1. Users could selectively test different cell dissociation reagents
9. Wash off the cells with PBS or culture medium
Yes such as Accutase, AccuMax/EDTA, or other commercial reagents
10. Repeat step 8 and 9 three times to harvest most of
in order to optimize the cell harvest procedure.
the cells
2. The remaining dead cells on the carriers will release DNA and
Cell Culture Sterility
Cell Dissociation Supporter may form viscous mass or gel to besiege other alive cells if over-
Complete trypsinized. When this situation occurs, it increases the difficulty to
harvest cells from the carriers. In order to alleviate this situation,
1. Wash with PBS/5mM EDTA twice (critical)
wash the carriers with PBS/EDTA twice prior to trypsinization, and
2. Add trypsin/EDTA (0.25%/0.5mM) to submerge the
No avoid trypsinize to over 20 mins. Alternatively, supply DNAse (1
carriers mg/ml) after trypsinization, or use AccuMax+ 0.5mM EDTA for the
3. Incubate for 10 mins whole process instead of trypsin.
Dismatle the bottles, collect
Yes 4. Discard trypsin/EDTA
carriers In a separated PE
Want cell mass 5. Incubate for another 10 mins
bottle and harvest cells from
6. Tap the bottles against your palm or a soft edge for 3
the PE bottle
mins
7. Wash off the cells with PBS or culture medium
Bottle Opener 8. Repeat step 6 and 7 three times to harvest most of the
No cells
Dismantle the bottles, collect 1. Directly exact the protein inside or on the membrane of the cells
carriers and extract target with ultrasonic, freeze-and-thaw, or by adding lysis buffer. Follow
products from the carriers generatl protocol for those cell lysis method.
Bottle Opener
Meaning of the Protocol
Route of Note
Tool required
symbols: purpose