Directional freezing for the cryopreservation of adherent mammalian cells on a substrate

<div><p>Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices.</p></div

Transnational cryostage for directional freezing of cells adherent to a substrate.

<p>(A) Two independently controlled TECs controlled the temperatures on top of the 1.5 mm thick copper plate. “Cold” and “hot” bases of the thermal blocks separated by 2 mm slit. A motorized actuator enabled movement of the sample at a precise velocity. (B) Temperature field with a unidirectional gradient simulated with COMSOL Multiphysics. The temperature profile along the direction of the gradient displayed a steep linearity on top of the slit (inset). (C) Representative image sequence of a growing ice front in a 10% v/v DMSO solution. The front remained in the middle of the imaging frame while cells moved (displacement of a specific cell is indicated by a red arrow). The scale bar indicates 100 μm.</p

Viability of the HeLa and Caco-2 cells after directional freezing vs. non-directional slow freezing.

<p>Phase contrast images of the HeLa (left panel) and Caco-2 (right panel) cell cultures prior to freezing and 5 h after thawing. Cells frozen at 10% DMSO using a combination of directional freezing at a speed of 30 μm/sec and gradual cooling at 0.5°C/min (upper panel), or solely using gradual cooling at 0.5°C/min (lower panel). Scale bars indicate 100 μm for 10x and 50 μm for 40x magnification.</p

Quantification of cell viability.

<p><b>(</b>A) Adherent HeLa and Caco-2 cells labeled 5 h after thawing using the live/dead kit (green—<i>live</i>/ red- <i>dead</i> cells). Scale bars indicate 100 μm. (B) Quantification of live/dead labeling using flow cytometry performed on cells that were collected immediately after thawing compared the cell survival percentage obtained under combination of directional freezing and gradual cooling, non-directional gradual cooling (1°C/min) and direct freezing at a -80°C freezer.</p

The effect of gradual freezing rate from -20°C to -80°C on cell morphology in a 10% DMSO medium.

<p>Following directional freezing of the adhered IEC-18 epithelial cells and gradual freezing on the translational stage to -20°C, samples were subjected to gradual cooling to -80°C on the LN flow cooling stage at rates of 0.5°C/min or 1°C/min. As a control, a sample was transferred directly to the -80°C freezer after reaching -20°C. Phase contrast images (10x) were collected prior to freezing, after thawing, and after 5 h incubation post thawing in a humidified 5% CO₂ incubator at 37°C. Scale bars indicate 100 μm.</p

Effect of the directional freezing rate on cell morphology.

<p>IEC-18 epithelial cells cultured on a cover glass were subjected to directional cooling on the translational cryostage (10% DMSO). Sample movement velocities of 10 μm/sec (eq. 1.3°C/min), 30 μm/sec (eq. 3.8°C/min), and 90 μm/sec (eq. 11.3°C/min), were tested. Then the temperature was decreased gradually to -20°C, and the cells were transferred to -80°C. (A) Ice crystal morphology as a function of the ice front propagation. Left: during the process of freezing. Right: Frozen sample at -20°C. Scale bar indicates 400 μm. (B) Phase contrast images (10x) of IEC-18 cells prior to freezing, after thawing, and after 24 h incubation post thawing in a humidified 5% CO₂ incubator at 37°C,. Scale bar indicates 100 μm.</p

IEC-18 cell monolayer morphology after directional freezing at various concentrations of DMSO in the freezing medium.

<p>(A) Ice crystals during directional freezing (velocity = 30 μm/sec, eq. 3.8°C/min) of adhered IEC-18 cells in the presence of different concentrations of DMSO (0% - 10%). Scale bar indicates 100 μm. (B) Phase contrast images of IEC-18 cells frozen in deferent concentrations of DMSO (0% - 10%) collected after thawing and 5 h incubation in a humidified 5% CO₂ incubator at 37°C. Scale bars indicate 100 μm for 10x and 25 μm for 40x magnification.</p