Pierre, Elena, and their team returned to the main chamber with renewed energy. The earlier simulation results were promising: the latest cryoprotectant mixture showed a dramatic reduction in tissue damage, with cooling and rewarming curves within stable parameters. Now, it was time to test the theory on a real biological sample.
Elena and her team prepared the setup with meticulous precision. The test chamber was calibrated, and Pierre stood beside her, assisting with the procedural checklist. They started with a rabbit brain, acquired minutes after euthanasia to minimize pre-preservation degradation. Following their theoretical and simulation-based protocol, the results on the rabbit brain were encouraging. Next, they moved to a human brain sample, obtained through consent from a patient on the mainland. Due to transportation, the sample had sustained slight damage, but it was still viable. They followed the same protocol, carefully perfusing the new cryoprotectant formula into the specimen. The chamber began to cool slowly, dropping from room temperature to 77 Kelvin and eventually to 4 Kelvin, just above absolute zero.
Pierre watched the sensors with intense focus. "No signs of ice crystallization so far," he noted.
"Cryoprotectant distribution is homogeneous," Elena confirmed, tapping the 3D imaging panel. "Osmotic balance looks good. Membranes are holding," her team reported, reading the data.
After reaching the target temperature, they let the sample stabilize for an hour before initiating the rewarming phase. The entire lab grew quiet, sensing the experiment's critical nature. The automated rewarming protocol activated, gradually raising the temperature in precise increments. Elena and Pierre monitored every spike and plateau on the neural activity graphs and temperature gradients.
Halfway through the rewarming process, something changed.
"Wait," Pierre said, pointing to a slight irregularity in the tissue conductivity readout.
"I see it too," Elena replied, pulling up the high-resolution scan overlay. "There's a localized but significant drop in cell density—a necrotic bloom."
Within seconds, more blue and gray zones appeared on the display.
"The protective agents aren't holding through the transition zone," Elena muttered. "There's microfracturing across the midbrain—damn it!"
Pierre clenched his fists. "That was the most stable cooling curve we've achieved. We followed the rewarming gradient to the letter."
"It's not the curve," Elena said sharply. "It's the perfusion depth. The brain's center is slightly behind in temperature—see? A thermal lag of just half a degree. That's enough to compromise the inner core."
Moments later, nearly half the simulation panel turned gray.
"Cellular collapse confirmed," Elena said quietly.
Pierre leaned back against the wall, frustrated. "Another failed test."
Elena sighed, visibly struggling to maintain her composure. "This should have worked. The equations held, and the test model was solid. Maybe the mixture needs rebalancing, or perhaps we still don't fully understand how molecular damage propagates during thermal stress."
Pierre turned away from the monitor. "No, we're close. This failure shows us the threshold. We dial it back, try another concentration profile, and maybe add localized thermal buffers in the core."
The team regrouped quickly, discussing over the whiteboard and recalculating the cryoprotectant formulation. Their theory was straightforward: the prior sample failed due to incomplete perfusion, particularly toward the brain's core. The viscosity of the trehalose-heavy mixture was likely the culprit. Although trehalose offered excellent vitrification and membrane stabilization, its high molecular weight as a disaccharide limited its ability to diffuse evenly through dense neural tissue.
"We'll reduce the trehalose ratio by 25%," Pierre suggested. "That should lower viscosity and improve penetration."
"But at the risk of losing vitrification quality," Elena warned. "If the membranes destabilize, we'll face intracellular freezing."
"It's a trade-off," Pierre said. "But we need to see if we can at least solve the perfusion issue."
They prepared another brain slice sample, perfused it with the revised solution, and began the cooling process. So far, the metrics looked promising—perfusion had vastly improved. The cryoprotectant reached the core tissues efficiently, and the brain's structural integrity held well.
"Perfusion depth is good," Elena said, eyes on the 3D overlay. "Temperature lag is under 0.2 degrees across the core. That's our best uniformity yet."
Encouraged, they proceeded to rewarming. But only minutes into the process, early warning signals flared on the screen. Some regions lit up in red.
"What is that?" Pierre asked.
Elena's expression grew serious. "Cellular toxicity markers. The inorganic cryoprotectant diffused and compromised membrane integrity."
A new layer of blue and gray spread quickly across the image. The brain's structural preservation had improved, but many cells were biologically dead.
Elena looked at him. "We're not going to solve this today."
"No," Pierre admitted. "But we will solve it."
They stood in silence for a moment, watching the now-gray representation of what had been, moments ago, a living brain. The cold hum of the cryo-chamber filled the room like an echo of defeat.
Elena finally shut down the console. "Let's document everything. Then we'll try again." They spent the rest of the day documenting and writing up the day's progress. Pierre had already headed back to his office on the fourth floor. Elena remained in the lab with her team, summarizing the disappointing results. "The rewarming process still needs more work, or every step may need revision," she murmured.
After finishing her report for the day, Elena realized her team had already left. Perhaps she should head home too. Then she remembered that Pierre might still be in his office. Since she needed to stop by her lab on the fourth floor to check for letters and other paperwork, she decided to visit Pierre's office as well. She packed up her things, locked the lab, and headed to the fourth floor.
Having returned from Elena's lab after another failure, Pierre couldn't hide his frustration. His watch blinked red more frequently, displaying: Stress 75%. "What am I missing?" he muttered to himself, recalling past mistakes.
He turned to his desktop and opened a terminal. Accessing Elena's lab data, he reviewed the results again. The data suggested that the trehalose content was significant, though it reduced diffusivity.
They couldn't decrease the trehalose below this threshold without significantly increasing toxicity. The rewarming protocol had been perfect until a local inhomogeneity caused a damaged cell to trigger widespread collapse.
"It was only a few small regions, but they can cause severe damage to the entire brain," Pierre murmured.
Pierre updated his simulation software with the latest data. The new simulation now aligned with the current experimental results. He examined it closely.