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Liquidus Tracking System & Software from Planer, 115 V

Liquidus Tracking System & Software from Planer

Gradually increases the concentration of DMSO as the temperature decreases and its toxicity decreases
Uses PT100 sensing equipment
Stores up to 10 profiles (32 steps per profile)
Easy to use 3 button controller containing PID engine
Alarm outputs
Runs saved to USB (if connected to controller)
USB port to connect to PC
Has Planer's Delta T software management option
Liquidus tracking achieves vitirifcation without the fast rates
Avoids high concentrations of DMSO at the start of the process
Gradually increases the concentration of DMSO as the temperature decreases and its toxicity decreases
Cooling rates: 0.01° to 50 °C/min
Heating rates: 0.01° to 10 °C/min
Temperature range: +40° to -180 °C

Liquidus tracking is a cryopreservation technique developed to cope with samples that are unsuitable for preservation by conventional means such as controlled-rate freezing and ultra-fast vitrification.

The preservation of biological matter is medically extremely important. Human and animal tissue is stored for research and regenerative medical purposes – bone marrow for cancer treatment, blood products, embryos, skin, bone and cell lines etc. are all frozen for later use. Whilst extra cold deep freezers (similar to domestic ones) can be used, generally the ultra cold of liquid nitrogen at -196°C is required for the greatest storage stability. Current Planer equipment allows some ‘difficult to freeze’ specimens to be cryopreserved with high post-thaw survival of living cells: cartilage is an example of a tissue that cannot currently be preserved with the survival of living cells.

Liquidus Tracking In tissues, for example, extra-cellular ice can form during conventional freezing. In contrast to cell suspensions, this extra-cellular ice can be damaging as it is contained within the system being preserved.

Conventional controlled-rate freezing only tackles the problem of inter-cellular ice, and although conventional vitrification inherently prevents the formation of ice, it typically relies on ultra-fast cooling rates of around 20,000°C/min; these fast rates are unachievable with large samples.

By slowly increasing the concentration of cryoprotectant during the preservation process, the Liquidus Tracker prevents the formation of ice crystals within the samples, allowing vitrification without the necessity for fast cooling rates. This slow vitrification is achieved by increasing the concentration of cryoprotectant while the sample is cooled; the cytotoxicity of the cryoprotectant decreases with temperature and as such higher concentrations can be tolerated at the lower temperatures.

The Liquidus Tracker is a system designed to allow research into the use of liquidus tracking as a means of cryopreserving biological samples that are not suitable for conventional slow-rate freezing or ultra-fast vitrification.

Dual Solution Mode

Two peristaltic pumps feed two solutions to the sample
First pump feeds a high concentration DMSO solution to the sample container c. 80%
Second pump feeds a 0% solution
Pump speeds are adjusted to control the concentration of cryoprotectant surrounding the sample while maintaining a constant flow rate
Appropriate for small sample containers
Simple mixing before sample
Aim to replace volume every minute
For run from 0 to -70° C at 0.3° C/minute, container volume of Vs uses 233 x Vs of DMSO
Impractical iv Vs is large
320 ml sample container would use 75 L of DMSO

Dual Solution Target Concentration

Required concentration determined from look-up table
Table clipped between TL and TU
Concentration determined from current demand temperature
Concentration table is %v/v - not %w/w

Single Solution Mode

Two peristaltic pumps used
First pump feeds a solution at high DMSO concentration into the sample container
Second pump extracts the solution from the sample container
Constant volume maintained in the container
Flow rate into the sample container is adjusted to obtain the correct concentration
Single solution mode suitable for large sample containers
Mixing more complex as it needs to be done in the sample container
Injecting 80% DMSO uses 2.3 x Vs of DMSO to reach concentration of 72%
320 ml container uses 736 ml DMSO

Single Solution Target Concentration

Dual solution has two pumps running together
Single solution has two pumps that alternate
One injects and one extracts
Pumps do not run at the same time
Uses same look-up table as dual solution algorithm
Does not use current demand
Predicts demand at the end of the cycle
Prediction simply based on demand at start of cycle and rate at start of cycle
Does not allow for rate changes mid-cycle

Volume required in Single Solution Mode

Let Vs = sample container volume
V = volume introduced
Ksol = concentration DMSO
Kt = final target concentration
V = Vs.In(Ksol/(Ksol = Kt)

Single Solution Pump Timings

Pumps run in a 3 phase cycle
Phase 1: Pump #1 injects solution for Tstep seconds
Phase 2: Both pumps off and solution left to stand for Tstand seconds to ensure mixed
Phase 3: Pump 2 runs for Tstep seconds to bring volume back to original value

What happens?

Liquidus Tracking aims to prevent the formation of ice crystals within the sample being preserved by vitrification
Vitrification normally requires ultra-fast cooling rates 20K°C/min

Vitrification normally requires small samples to achieve rates

Both systems increase concentration

The end result is the same
As the temperature of the sample decreases the concentration of the surrounding cryoprotectant increases
Dual solution mode continuous
Single solution runs in small steps

Dual Solution Fluid Path

DMSO
Pump 1
0%
Pump 2
Y-piece
Sample carrier
Waste collection
Filtered exhaust
Filtered inlet
Injection line 1
Injection line 2

Dual Solution Sterile Assembly

Sample carrier and fluids assembled using aseptic technique
Completed assembly can then be taken to the liquidus tracker unit

Dual Solution pump connection

The sample carrier is placed into the chamber
The two lines are placed into the associated peristaltic pumps
The system is ready to go

Single Solution Fluid Path

DMSO
Pump 1
Pump 2
Sample carrier
Extraction line clamp
Waste collection
Pressure relief line
Filtered exhaust
Filtered inlet
Injection line
Extraction line

Single Solution Sterile Assembly

Sample carrier and fluids assembled using aseptic technique
Completed assembly can then be taken to the liquidus tracker unit

Stirrer

For single solution systems mechanical stirring is required; this is because of the large volumes
Sample carrier uses a magnetically coupled stirring system to ensure that the solution is thoroughly mixed
Stirs continuously through all three phases; injection, stand and extraction
Stirrer oscillates and speed can be adjusted by separate controller

The advantage of the Liquidus Tracking method is that ice is not allowed to form at all. This is achieved by progressively raising the concentration of cryoprotectant as cooling proceeds such that the conditions of the solution that the cells or tissue experience are similar to those that they would experience during conventional cryopreservation but without the formation of any ice.

Cooling and warming rates are flexible, making the method worth trying for some tissues that could not be cooled or warmed rapidly. Cartilage, ova, hepatocytes being possible candidates.

Available Models

Product Code	Description
PLLTPKryo360-1.7	Liquidus Tracker for Planer Kryo360-1.7 Chamber, Each
PLLTPKryo560-16	Liquidus Tracker for Planer Kryo560-16 Chamber, Each

Controller Specification:

Model	Liquidus Tracker - Controller
Dimensions	24 x 23.5 x 18.5 cm
Weight	2.8 kg
Display	2 x 16 LCD with backlight
Key pad	3 key switches
Number of stored profiles	10
Steps per Profile	32
Programmable Rates	0.01° - 99.9° C per minute (Preliminary)

Chamber Specifications

Model	PLKRYO360-1.7 Chamber	PLKRYO560-16 Chamber
Heating Rates	0.01° - 10° C per minute
Cooling Rates	0.01° - 50° C per minute
Temperature Range	+40° to -180° C
Weight	14.4 kg	23 kg
Capacity	1.7 L	16 L
Dimensions	20 x Ø15 cm	32 x 22.6 x 23 cm
Chamber Access	27.1 x 21.7 cm
Sample Carriers	Consult Planer PLC
Power Requirements	115 V, 50/60 Hz, 10 A	115 V, 50/60 Hz, 15 A
Warranty	1 Year

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