⇒ Controlled Rate Freezing of Multiwell and Microtiter Plates

⇒ Superior cell and tissue cryopreservation for LN₂-operated Freezers

The ILK Multiwell RACK combines the best from both worlds

Superior precision and temperature homogeneity as provided by electrically driven conduction freezers paired with the quick response characteristic and the superior cooling power of liquid nitrogen-operated convection freezers.
Further Information

Cost-efficient upgrade option for LN₂-operated freezers

Equipment and retrofitting of conventional, liquid nitrogen operated convection freezers with cell and tissue freezing capability in multi-well and microtiter plates.

• Proof of suitability for the successful cryopreservation of adherent cell cultures by use of 48-well culture plates.

• Test of the cryopreservation performance of cell-based in vitro-assays in the 96-well microtiter plate format.

• Information on the scientific background

• Information on design and function

A comparison of the temperature precision during freezing of 96 well microtiter plates with the ILK Multiwell RACK in a 15 years old convection freezer (SY-LAB IceCube 15M) and a novel computer-controlled conduction freezer with seeding technology (Asymptote VIA Freeze with IceStart) proves the outstanding cryopreservation accuracy of LN₂-operated freezers provided by the ILK Multiwell RACK.

Proof of the outstanding cryopreservation performance by use of the ILK Multiwell RACK:

• Clear difference of the temperature stability during the freezing run with both devices

• Six times lower variance of the sample temperatures compared to the nitrogen-free cutting-edge freezer

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Precise cryoprotocol application

The ILK Multiwell RACK enables:

• The application of cell and application specific freezing programs in convection freezers with a precision never reached before

• A constant supercooling of cell and tissue cultures as well as the defined induction of a synchronous ice formation by seeding.

• An elimination of the impact of the heat of crystallization released in the freezing process thank to the high cooling power of LN₂-based freezers

  • Prevention of a secondary warming of the samples (latent heat peak)

  • Immediate cooling of the samples and assurance of constant freezing rates

Best available temperature uniformity

The ILK Multiwell RACK obtains:

• A synchronization of the sample temperatures and freezing rates of all cavities of a multiwell or mictrotiter plate during the whole freezing cycle with the maximum accuracy

• Reduction of Temperature differences between identically processed samples by 95 % compared to the unaffected freezing in the convection freezer

Temperature profil of the cryopreservation of adherent fibroblasts in a 48-well cell culture plate moderated by the ILK Multiwell RACK:

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The cryopreservation performance of gel-embedded cells in 96-well microtiter plates is proved by the control of the cell numbers before and after the cryopreservation of the assay plate.

The cavities of the microtiter plate contain a cell carrier gel which contains human primary fibroblasts at five different cell densities between 120.000 and 960.000 cells per cavity (two rows per each cell density). The test evaluation was performed before and after cryopreservation with the ILK Multiwell RACK by a fluorescence photometric viability analysis.

The variance of the cell numbers of 12 identically seeded cavities is ±2.8 % before the cryopreservation. After cryopreservation it rises only slightly to ±4.7 %. The coefficient of determination for the calibration curve is 99% before the cryopreservation. After the cryopreservation it is still higher than 97 %.

The achieved testing accuracy exceeds the testing accuracy of the majority of cell-based in vitro-assays without a preceded cryopreservation.

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Differences between conductive and convective freezing

The freezing of biological samples, especially the cryopreservation of living cells and tissue requires the application of a defined sample specific freezing rate.

Two different types of computer controlled freezers are available to fulfill this task:

• Conduction freezers

Conduction freezers are electrical cooling devices which are operated by a refrigerating machine which directly cools a sample holder. The sample holder (metal block or contact plate) transfers the cold by direct contact to the sample package. Therefore, conduction freezers are able to cool a high sample count under very uniform temperature conditions.

Thus, conduction freezers are the best choice for the freezing of multiwell plates or small sample tubes (cryovials)

• Convection freezers

Convection freezers produce the required cold by the gassification of liquid nitrogen. The samples are placed in a freezing chamber and cooled by the sourrounding cooling gas flow. The simple design makes such freezers reasonable in price and service-friendly. The gasification of cryogenic nitrogen allows a high sample throughput and high cooling rates at the same time.

LN2-operated convection freezers are therefore the standard for the freezing of large-scale biological samples such as blood bags or bulk ware.

Disadvantages of LN₂-operated controlled rate freezers (convection freezers):

Dependency of the heat transfer on the flow conditions in the freezing chamber

• Local differences in the flow rate and flow direction cause inconsistent and non-reproducible sample temperatures and cooling rates

• Form and surface condition of the sample package affect the local flow behavior and heat transfer and, therefore lead to an inhomogeneous cooling of the samples

A successful cryopreservation of a high sample count is therefore only possible by the use of additional installations for the induction of a balanced heat transfer. Tests prove that the performance of standard installations, like racks or cassettes are sufficient for the freezing of blood products and stable cell suspensions but not for the cryopreservation of sensitive cell types, adherent cells, cell clusters or tissue.

For the successful cryopreservation of cells and tissue in multiwell plates we developed the ILK Multiwell RACK.

Cooling Performance with a convection freezer

The left picture shows the temperature distribution of a freestanding multiwell plate after 10 minutes of cooling in a LN₂-operated controlled rate freezer with a constant cooling rate of 2.5 K/min. After this short time span the temperature difference already gains a critical of 5 °C.

The ILK Multiwell RACK developed by the Centre of Cryo Competence in Life Sciences is able to reduce the temperature difference to a value of 1.5 K/min in the same time span. Even the uniformity of the temperature distribution within the multiwell plate is considerably better. Only this enables a successfully and reproducibly cryopreservation of living cells and tissue within the multiwell plate.

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The ILK Multiwell RACK is the key device to ensure a controlled heat transfer during the cell and tissue cryopreservation in multiwell plates or microtiter plates by the use of a computer controlled convection freezer.

The ILK Multiwell RACK consists of a framework which houses two thermally decoupled contact plates. The multiwell plate is fixed between those plates with a defined contact pressure. The special sample holder geometry enables the thermal transfer device to accommodate and cryopreserve any type of multiwell / microtiter plate (including special formats). The accessible cooling and heating performance is only limited by the technical specifications of the convection freezer used.

The sample specific thermal mass and surface of the contact plates allows an optimal heat transfer from the well plate to the cooling gas. An insulation belt (ISOBELT) prevents any direct thermal contact to enable a controlled heat transfer through the intended contact surfaces. The unique design of the heat exchange surface exhibits broad cooling channels and baffle-like fins by which the cooling gas is very efficiently lead to the contact plates and swirled there. Thus an unprecedented temperature homogeneity is achieved in the contact area of the samples combined with an ultimate heat transfer capability.

The ILK Multiwell RACK implements a seeding option for the mechanical triggering of the freezing process. With a vibration frequency of 300 – 350 Hz a synchronous ice formation can be induced. The synchronization of the freezing process leads to an identical cooling rate at any sample position.

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