Proline-conditioning and chemically-programmed ice nucleation protects spheroids during cryopreservation

Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants.


Fabrication of spheroids
3D Petri Dishes® 1 g of pure agarose powder (Sigma) was measured and placed in a dry 100 mL autoclave-safe glass bottle for autoclave 30 minutes. Swirling the bottle after 50 mL of sterile saline [0.9% (w/v) NaCl] was added to the bottle containing agarose power in a biosafety cabinet. The solution was then heated using a microwave oven to boil and completely dissolve the power.
Pipetting 500 µL of agarose solution in 3D Petri Dishes® for 10 min for gelation. The agarose micro-moulds were subsequently removed from the 3D Petri Dishes before transferring them in a 12-well plate and equilibrating with cell culture medium for 30 minutes in an incubator.
Extracting all culture medium before seeding cells in a micro-mould and 190 µL of A549 cells S4 suspension was placed in each chamber containing 648,000 cells (~8000 cells/spheroid). Plates left to set for at least 10 min and then adding 2.5 mL medium to each well outside of the mould.
Spheroids were cultured for up to 7 days. A549 cells were grown in F-12 K medium and placed in an incubator and renew medium every third day. HepG2 cells were cultivated using MEM supplemented with 10% (v/v) FBS, 1% antibiotic-antimycotic solution 100x, 1% MEM Non-Essential Amino acid solution 100X in the same formation method as A549 spheroids. An inverted microscope (Olympus, CX41, Southend-on-Sea, UK) were applied to monitor spheroid formation and spheroids images analysis used ImageJ software v1.52.

Slow freezing spheroids
a. Three ways for cryopreservation of spheroids: A) Spheroids were frozen directly in agar micro-moulds with 1 mL cryoprotectant solution in 12-well plates in a controlled-rate freezer (VIA freezer TM ), -1 °C/ min until it reached -80 ℃, then plates were transferred to -80 ℃ freezer for 24 hours. B) Spheroids were frozen directly in agar micro-moulds, however, we optimised the method with 200 µl cryoprotectant solution, 12-well plates were placed on a CoolCell® MP plate (BioCision, LLC, Larkspur, CA) and transferred to a -80 ℃ freezer for 24 hours. C) To further improve the recovery, spheroids were transferred to U-bottom 96-well plates, one spheroid with 50 µl cryoprotectant solution each well and then plates were placed on CoolCell® MP plates and were stored in a -80 ℃ freezer directly. b. Thawing methods S5 A) B) After 24 h storage at -80 ℃, the 12-well plates were thawed by adding 1 mL pre-warmed complete cell culture medium, plates were placed in the incubator for 5 min to fully thaw and replaced with 1 mL complete cell culture media for the other 24 hours incubation. C) Complete cell culture medium warmed to 37 ℃ was added into U-96 well plates, 100 µl/well. Plates were placed in a humidified atmosphere for 5 min to thaw and the cell culture media replaced with 100 µl complete cell culture media before moving back to the incubator for 24 h culture.
The spheroids were transferred individually to a white 96 well plate for the luminescence assay to determine the recovery and the viability before and after thawing. Unless stated otherwise, the spheroids were 8000 cells for all tests. Images of before/after freezing were captured by an Olympus CX41 microscope.

Evaluation of cell viability morphology
Before freezing and post-thaw, we used a CellTiter-Glo® 3D Cell Viability Assay (Promega, G9682, USA), which is a homogeneous method to determine cell viability in 3D cell culture based on quantitation of the ATP present. ATP is the marker for the present of metabolically active cells. The kit is a involves with more robust lytic capacity and is designed for microtissues sample. We picked one spheroid with 100 µL media to each well of a 96-well white plate, added 20 µl/well, mixed by a BioTek Synergy HT microplate reader shaking for 5 min to lyse cells and stabilize ATP. The plate was placed in dark for 25 min at room temperature. Subsequently, a luminescence BioTek Synergy HT microplate reader was used to analyse the viable cells.
The LIVE/DEAD® Viability/Cytotoxicity Assay Kit was used to observe apoptotic cell death and morphology of spheroids. It provides a two-colour fluorescence cell viability assay that is based on the simultaneous determination of live and dead cells with two probes that S6 measure recognized parameters of cell viability-intracellular esterase activity and plasma membrane integrity. For this assay, remove the media from each well, the spheroids were kept in the mould and washed with DPBS for two times. An approximately 2 µM of calcein AM (5 µL) and 4 µM of ethidium homodimer-1 (20 L) were added to 10 mL of sterile D-PBS in a 15 mL tube with 33 μM Hoechst 33342 (Life Technologies, Carlsbad, CA), vertexing to ensure thorough mixing. Then, 200 µL solution was added in each mould and incubated at room temperature for 1 h, spheroids were then transferred to confocal dishes and imaged using a FV3000 confocal laser-scanning microscope (Olympus, Tokyo, Japan). The polyanionic dye calcein AM retained within live cells, showing an intense uniform green florescence in live cells at ex/em ~495 nm/ ~515 nm. EthD-1 entered cells with damaged membranes, binding to nucleic acids and producing a bright red fluorescence in dead cells at ex/em ~495 nm/ ~635 nm.

Cytoskeleton
Samples (8000 cells/spheroid) were fixed with 4% paraformaldehyde (PFA) in PBS for 20 minutes at room temperature. Permeabilize the spheroids with 0.1% Triton X-100 in PBS for 15 minutes, washing twice with DPBS and then spheroids were incubated with primary antibody for cytoskeleton dye (Incitrogen ActinGreen TM ,488 ReadyProbe reagent overnight at 4 degrees in the fridge. The samples were washed once with DPBS before imaging with a FV3000 confocal laser-scanning microscope (Olympus, Tokyo, Japan), ex/em ~ 499/520 nm. F-actin mean fluorescent intensity of the images were analysed by using ImageJ v1.52. The spheroids cultured for 6 days as a control and spheroids cryopreserved with 10D or Pro-10D+IN group were analysed. The percentage of fluorescence intensity of F-actin is equal to the value of each group divided by the mean of the control group.

S7
ROS detection reagent (Invitrogen, D399) was applied for spheroids imaging. Samples of before and after-freezing were washed with a DPBS buffer before incubating with 200 µl dichlorodihydrofluorescein diacetate (DCFDA) solution (20 µl of 7.5 mM diluted stock in PBS)/ well in U-96 well plates for 30 minutes. The spheroids were transferred to confocal dishes and captured using the FV3000 confocal laser-scanning microscope (Olympus, Tokyo, Japan) with fluorescence excitation and emission in ~495/515 nm. ImageJ v1.52 was used to analyse the mean fluorescent intensity of ROS. Three images of each group were measured, the value of mean fluorescence was calculated using mean fluorescence of the spheroids minus that of background.

Ice Nucleation Assay
Ice nucleation temperatures of 5 µl droplets were measured using a custom-built droplet freezing assay. The pollen washing water (PWW) solution for nucleation measurements was prepared by adding 0.04 g of Carpinus betulus pollen (Pharmallerga®) to 2 ml of a 10% DMSO solution. The pollen suspension was refrigerated overnight before filtering through a 0.2 μm syringe filter into a clean glass vial. For the ice nucleation measurements, twenty 5 µl droplets of the filtered PWW were pipetted onto a 22 mm diameter Hampton Research HR3-231 siliconized glass slide using a Sartorius Picus® electronic micropipette. This slide was placed onto the cold stage, which was used to reduce droplet temperature at a rate of 2 °C/min. Droplet freezing was monitored using a digital camera, allowing the fraction of droplets frozen as a function of temperature to be determined.
Statistical Analysis: Origin 2022b, ImageJ v1.52 and GraphPad Prism 9 software were used to analyse the data. To determine significance between the means of two groups, an unpaired two-sided T-test was conducted using GraphPad Prism 9 Software.    HepG2 spheroids, the data was plotted relative to the non-frozen control group. The scale bar is 100 µm (A). * P<0.05, ** P<0.01.