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Manual Cryostorage:
Products, Applications
2
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.)
3
Cryostorage Workflow
Identify and
Store
Retrieve
Analyze or Grow
Collect
Fractionate or Aliquot
Record info
4
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
7
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
11
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)
14
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
15
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
16
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
17
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
18
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
19
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
20
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
21
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
22
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
23
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
24
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
25
Further Information on Cryostorage
• www.thermoscientific.com/samplestorage
• Products brochure
• Cryopreservation Guide
• LSP catalogue p. 562 – 607 : Sample Storage (Cryoware + 2D
Storage)