Manual Cryostorage:

Products, Applications

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Cryostorage & Cryogenic Preservation

What?

• The storage of biological samples at very low temperatures

Why?

• Avoid sample degradation over time (research / reference / analysis)

• Preserve genetic & phenotypic stability of cells / Avoid cell differentiation & senescence

How ?

• Cryogenic temperatures : below -135°C (re-crystallization temperature of water: guarantee all of the liquid water will be frozen) : all biological activity – incl. biochemical reactions - is effectively stopped

• For biological samples, storage at -80°C has been shown to effectively preserve all sample characteristics (DNA, Biomarkers, solutes, blood cell integrity, etc.)

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Cryostorage Workflow

Identify and

Store

Retrieve

Analyze or Grow

Collect

Fractionate or Aliquot

Record info

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Cryopreservation of Cells—Types of Freezers

Vapor Phase

Liquid Phase

Dewars (-178°C)

• Can be cumbersome (refill/monitor)

• Low cost/investment to purchase

• Low energy costs

Mechanical Freezers (-150°C)

• Easy installation/operation

• Medium cost/investment to

purchase

• High cost to operate

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Question 1

What happens with the cells after cryopreservation?

a. These cells are analyzed

b. These cells are put in culture again

c. These cells are thrown away, as they are no longer needed

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Cell’s Reaction to Cryopreservation

Ice crystals inside & outside cells:

Less osmotic imbalance

Less dehydration

More intracellular ice : cell death upon recovery

Less intracellular ice

More osmotic imbalance

More dehydration / shrinkage :

irreparable membrane loss

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Cryopreservation of Cells - Important Factors

Growth phase/stage of the cell cycle • Adherent cultured cells: near confluency

• Bacteria’s growth stage important—most optimum being late log phase

Number and concentration of cells • Generally store between 106 – 107 cells per ml

• Intially prepare pellet with 2x this concentration, then dilute with cryoprotectant and media

Cryoprotective agent • Equilibrate cells with cryoprotectant for 15-30 minutes to

allow penetration – will avoid osmotic imbalance

Freezing process • Ideally freeze at ~1°C/ min.; important for good recovery of

the frozen cells

• Avoid under-cooling at or around 0°C

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Controlled freezing using Thermo Scientific™ Nalgene™ “Mr. Frosty”

• Accurate & reproducible cooling rates : better than beakers & foam boxes to

obtain the “ideal” -1°C/minute cooling rate

• Economical: less expensive than controlled rate freezers – requires only IPA

• Flexible: Options for 1 to 5 ml cryotubes (cat. n° 5100-0001, -0036, -0050)

• Easy to Use: instructions printed on its side

• Fill to line with 100% IPA

• Replace insert

• Insert tubes and cover

• Place (overnight) into -70 to -80°C freezer

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Liquid Nitrogen caution: Do not store in liquid phase

No plastic vial is truly leakproof in the

liquid phase of LN2

Warning: Do not use plastic vials for freezing in

liquid nitrogen (liquid phase storage) unless

correctly sealed in Thermo Scientific Nunc™

CryoFlex Tubing, as this poses a safety hazard.

Improper use may cause entrapment of liquid

nitrogen inside the vial and lead to pressure

build-up resulting in a possible explosion or

biohazard release.

Use appropriate safety procedures when

handling and disposing of vials

Always store sample containers in the

vapor phase of liquid nitrogen tanks.

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Use of Nunc CryoFlex Tubing (cat. n° 343958)

• Crimp or squeeze ends with heated forceps or hemostat

• Trim off excess Nunc CryoFlex tubing

• Store in canes or consult compatibility charts for available

cryoboxes that will fit Nunc CryoFlex-sealed tubes

• See demo on www.thermoscientific.com/storagevideos

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Thawing of Frozen Cells

Thawing requires the opposite temperature profile of freezing:

• Rapid thawing is preferred

• Rapid removal of cryoprotectant agent is important

• The frozen vial can be swirled in a 37° C water bath to speed thawing

• Thaw the vial just until the last piece of ice melts, vial contents will be

~0° C

• Disinfecting the outside of the vial with 70% alcohol prior to removing

the contents is recommended

• Remove cryoprotective agents as quickly as possible, before putting

cells back into culture.

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Question 2

To improve recovery after cryopreservation, cells must

be frozen …

• a. quickly, using Nunc Cryoflex & cryoprotective agent

• b. slowly, using Nunc Cryoflex & Nalgene Mr Frosty

• c. slowly, using Nalgene Mr Frosty & cryoprotective agent

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Thermo Scientific Cryogenic Consumables

• Nalgene & Nunc cryogenic tubes & boxes:

strong brands, market leaders in Cryoware

• Mainly sold through Channel Partners in

Europe & Emerging Markets

• Unique accessories : Nalgene Mr Frosty,

Labtop Coolers, holders for one-handed

operation : great promotional possibilities

• Thermo Scientific’s unique value proposition:

Only manufacturer of a full line of cryo

Consumables and Equipment:

ideal for joint marketing actions

ideal for workflow selling solutions:

Value (tubes) = 2 – 3 x Value (freezer)

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Cryotubes: External vs Internal Threads

External

Nalgene & Nunc Internal

Nunc only

Both rated for liquid nitrogen storage (vapor phase)

• External: good for benchtop operation (hand pipetting)

• Minimize potential thread contamination

• Internal thread (gasket!) more automation-friendly, reduced space requirement (10 x 10 in standard 2” cryobox)

• Really just customer preference

• Universal cap will support customer

flexibility

• Move from cryo to storage tracking

• Move from cap to cap (int/ext)

• Be able to use same decappers,

• equipment

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Thermo Scientific Nunc CryoTubes - Overview

• Internal thread: 1.0 – 1.8 – 3.6 – 4.5 ml

• (Frozen Working Volume)

• PP screw cap with O-ring

• External thread: 1.0 – 1.8 – 4.5 ml

• HDPE screw cap without O-ring

• Sterile (50/ zip bag in cartons)

• Round or conical bottom

• Round, skirted or starfoot base

• Non-sterile (bulk): External, separate cap

• Barcoded Nunc CryoTubes & scanner

NUNC

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Features / Benefits Nunc CryoTubes

• Externally & internally

threaded tubes: leakproof per

IATA regulations (95 kPa)

• Virgin plastic with low cell

adhesion and low protein

binding

• Round, skirted or starfoot base

• White marking area & graduations or bar-coding

• Certifications:

Sterile (SAL10-6)

Non-toxic & non-pyrogenic.

CE-marked (IVD directive)

RNase/DNase-free

5 year shelf life

• Zip-lock bag + cat. n° & lot number information

• No sample loss during storage

or transport; meet air

transportation regulations for

biological samples

• Maximize cell recovery after

thawing; no loss of bound

proteins

• Complete selection to meet

customer’s preference; starfoot

allows one-handed operation.

• Easy hand-written or

mechanical (ID scribe)

tube/sample identification , or

error-free pre-labelling of tube

• Certificate in each box

guarantees highest quality

tubes for each lot, to give

customers’ peace of mind

• Resealable bag keeps unused

tubes sterile

Feature Benefit

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Question 3

Internally-threaded Nunc CryoTubes are more suitable to …

a. reduce sample space requirement in the freezer

b. increase the recovery rate of frozen cells

c. make it easy to pipet the sample in & out of the tube

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Thermo Scientific Nalgene Cryovials – Overview

Standard vials (5000-series)

• 1.2 - 2.0 - 5.0ml (Max RT Volume)

• Sterile (25 or 10/sealed pack – 500 or 250/case)

• non-sterile and/or bulk packed

• Standard dimensions (9 x 9 vials / Cryobox)

SYSTEM 10 vials with PP closure & silicone gasket

• 1.0 and 1.5 ml

• Smaller OD than Standard range: spin in centrifuge

(8000 x g)

• 10 x 10 vials in System100 Cryobox

Bar-coded vials available for both systems:

Standard: code 128 --- Custom BC: on request

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Features / Benefits Cryovials

• Virgin, medical-grade plastics

for tubes & closure

• Skirted, self standing vials

with interlocking base

• White marking area & graduations or bar-coded vials

• Certifications:

Sterile (SAL10-6)

Non-toxic & non-pyrogenic.

CE-marked (IVD directive)

RNase/DNase-free

5 year shelf life

• No risk of leachables &

extractables found in sample,

potentially hazardous to cells.

• Interlocking base allows one-

handed operation when placed

in NALGENE vial holder

• Easy hand-written or

mechanical (ID scribe)

tube/sample identification or

error-free pre-labeled tube

• Certificate in each box

guarantees highest quality

tubes for each lot

Feature Benefit

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FisherBrand Cryovials – Product Offering

External Thread

• Same as Nalgene 1.2 - 2.0 - 5.0ml

• 1000/case

• Sterile & bulk pack.

• No certification

Internal Thread

• Same as Nunc 1 – 1.8 ml – 4.5 ml

• Sterile, starfoot version

• 1000/cs

• No certification

• Cost : less than 50 % of branded products : “fighter” brand when not

possible to offer branded product

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Question 4

System 100 Cryovials are…

a. not available as bar-coded vials

b. not available as FisherBrand cryovials

c. not suitable for liquid nitrogen (vapor phase) storage

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Other General Accessories

Many are universal

• Boxes

• Canes

• Racks

• Labtop coolers

• Dewars

• Labels

• Pens

Some accessories are only

compatible with Nalgene or

Nunc Cryotubes:

• Tube holders

• Color coders for tubes

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Cryostorage - Positioned for Customer Application

Thermo Scientific Nunc

• Regulated applications

• Irreplaceable high value content storage

• Tissue, cells, blood storage for biobanking

• Peace of mind when storing down to VPLN

Thermo Scientific Nalgene

• High value content storage

• Genomic material storage

• General purpose storage applications

FisherBrand (in NA & EU)

• Quality solution for the price conscious researcher

• General purpose storage applications

Brands specifically tailored to storage applications

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Question 5

Which brand cryovials should be offered to a

pharmaceutical company who wants to ship samples

from their cell bank to a lab in a different country?

a. Thermo Scientific Nunc CryoTubes

b. Thermo Scientific Nalgene Cryovials

c. Both brands are suitable

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Further Information on Cryostorage

• www.thermoscientific.com/samplestorage

• Products brochure

• Cryopreservation Guide

• LSP catalogue p. 562 – 607 : Sample Storage (Cryoware + 2D

Storage)