Cryobanking of animal cells

Shiaw-Min HwangBioresource Collection and Research Center (BCRC)

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Outline

Why cryobanking the cells?How to do cell banking?Quality control/assurance of cellsManagement the cryobanksCryo-processing

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Reasons for cryobanking cells (I)Obvious reasons (SAVE):

Save work, including time and moneyAssurance of self-cell supplyValidated cells, tested and qualified Emergency, a back up supply

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Reasons for cryobanking cells (II)Less obvious reasons (another SAVE):

Shipping, Aging, Variation, Evolution,

Keeps your cells young and performing

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Preparation of Cell Banking

This works if you remember:Two stocks: M & W (master & working cell bank)

Store cultures appropriately

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How to do it - Step 1 (MMaster stock)

Harvest, pool and freeze

down 20 vials

No antibiotics!

(master stock, passage:N)

p:N p:N

p:Np:N

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How to do it - Step 2 (expansion)

Harvest, pool and freeze

down 20 vials

Master stock

No antibiotics!

Thaw 1 vial and check recovery

Expansion

No antibiotics!

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How to do it - Step 3 (WWorking stock)

Harvest, pool and freeze

down 20 vials

(Working stock, passage:N+2)Cells from

seed stock

p:N+2

p:N+2

p:N+2

p:N+2

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How to do it - Step 4 (check)

Harvest, pool and freeze

down 20 vials

Cells from seed stock

Thaw I vial, check viability and test for contamination

Once tested, a new vial is used every 3 months or 10p

working stock

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Simple calculations

So how many vials you have now:Master stock (20-1) plus Working stock (20-1)

Total vials you will have at the same passage number (p:N+2):

20 (Master) x 20 (working) = 400 (vials)

How long can you handle this cell line? (if: 1 vial for 3 months)

3 month x 400 = 1200 months (100 years!!!)

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M&M detection:Microbials and Mycoplasma

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QA managementAuthentication: Origin & Species

Isoenzyme analysis

Karyotype analysis

STR DNA fingerprinting

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Management of CryoBank

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60001

60003

6000460005

6000660007

600086000960010

60002

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Availability of Tanks

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中央系統液態氮之充填

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線上溫度監視系統

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第一道門禁系統

庫房外的走廊 第二道門禁系統

庫房

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ISO 9001:2000

BCRC is granted ISO certification since 2001

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TAF Certificate

Oct. 24, 2007BCRC accredited

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Key steps for freezing cellsStart with healthy, actively growing cells

Use slow freezing to prevent intracellular ice crystals

Use cryoprotectants to minimize dehydration effects

Store cells below -150°C to stop biological time Use healthy,

happy cells

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Glass ampules:

Not practical or recommended for most labs

Labeling problems

Open problems

Hard to seal correctly by hand

May leak (pinhole) in liquid nitrogen! – can explode violently

Selecting a storage vessel: glass or plastic Selecting a storage vessel: glass or plastic

Selecting a storage vessel: glass or plasticPlastic vials:

Several cap designs with or without o-rings or gaskets

1 to 5mL sizes

Easy to label

May leak in liquid nitrogen – can explode!

Accessories

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2 types of LN2 storage

In Vapor Phase

In Liquid Phase

• Lower than -150°C

• Very safe• Shorter holding

time • Reduced vial

capacity

• -196°C• Longer holding

time • Greater vial

capacity •Danger of vials

exploding! • Contamination

issues!

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Plastic vialsWARNING! Do not use cryogenic vials for storage in the liquid phase of liquid nitrogen. Such use may cause entrapment of liquefied nitrogen inside the vial and lead to pressure build-up resulting in possible explosion or biohazard release. –Nalgene

--Corning

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Wanted:

SubmergedConstant ultra-low temp, -196oCLargest capacity

Plastic vialFriendly & easy to use

Non-explosive, no contaminationStop LN2 invasion

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CryoflexTM

Disadvantages:--not easy to use--can’t fit commercial storage box--viability low--expensive

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Partial sealing

Cryoflex TM

cryovial

If works, then•Min. Cryoflex usage•Fit storage box•No heat to cells•No explosion•Automation

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Cryosealer 冷凍封管機

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Publication

Cryobiology (2006)53:283-287Cryovial with partial membrane

sealing can prevent liquid nitrogen penetration in submerged storage

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Thawing (I)Always use appropriate safety equipment:

full face shield heavy gloves lab coat

If the stored materials contain biohazards additional precautions must be taken

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Thawing (II)

Rapid thawing (60 to 90 seconds at 37°C)

Double check label to make sure it is the right vial!

Wipe off vial with 70% ethanol before opening

Check viability –

Trypan Blue may overestimate actual survival after thawing.

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報告完畢報告完畢敬請指教敬請指教

Cryobanking of �animal cellsOutlineReasons for cryobanking cells (I)投影片編號 4Preparation of Cell BankingHow to do it - Step 1 (Master stock)How to do it - Step 2 (expansion)How to do it - Step 3 (Working stock)How to do it - Step 4 (check)Simple calculations投影片編號 11QA management投影片編號 13投影片編號 14投影片編號 15投影片編號 16投影片編號 17投影片編號 18線上溫度監視系統投影片編號 20投影片編號 21ISO 9001:2000 TAF Certificate投影片編號 24投影片編號 25投影片編號 26投影片編號 27投影片編號 28Key steps for freezing cellsSelecting a storage vessel: glass or plasticSelecting a storage vessel: glass or plastic2 types of LN2 storage投影片編號 33Wanted:CryoflexTMPartial sealingCryosealer 冷凍封管機 Publication投影片編號 39Thawing (I)Thawing (II)投影片編號 42