Total Recall: Proving that Complex Mammalian Memory can be Cryopreserved

We propose allocating $259,000 USD to fund the first year of Total Recall, a research project at 21st Century Medicine (21CM), with support from Dr. Brett Kagan and Cortical Labs. The project will test whether mammalian neural systems retain previously learned responses after vitrification and rewarming.

Two complementary approaches will be pursued: recovering trained behavior from cryopreserved neural cultures or hippocampal slices, and assessing whether training-associated molecular changes survive the same process. Together, these approaches target a central question for cryopreservation: can the information underlying memory be preserved?

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Functional Recall‍

Total Recall will combine 21CM's vitrification methods with Cortical Labs' CL-1 neural training platform. Cells or hippocampal slices will be trained on a removable multielectrode array (MEA), which stimulates tissue and records its responses. Vitrifying the specimen directly on the same MEA is intended to preserve electrode contacts so that the trained response can be evaluated after rewarming.

Training will begin with a practical, simpler task, potentially character recognition, with more complex tasks such as Pong pursued as the platform matures. The task remains to be selected experimentally. To address the short duration of earlier neural-culture learning effects, the team will investigate repeated training and hippocampal-slice cultures, aiming for responses that persist long enough to undergo cryopreservation.

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Molecular preservation

‍Imaging mass spectrometry will compare trained and untrained samples before and after vitrification to assess the preservation of molecular changes associated with training. This provides a second route to evidence if functional recall cannot be demonstrated. The budget includes 20 samples: five in each of four groups, trained and untrained, with and without vitrification.

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Team and Research Setting

Dr. Regina R. Monaco will serve as principal investigator (PI), carrying out the work at 21CM with a dedicated technician or postdoctoral researcher. Dr. Gregory M. Fahy will supervise the project. Dr. Monaco brings experience in cell culture, molecular biology and computational modeling; Dr. Fahy contributes expertise in vitrification and brain cryobiology.

Dr. Brett Kagan's team at Cortical Labs will support neural training and evaluation, including remote operation of the CL-1 when needed. The proposal also describes collaboration with Nick Llewelyn at Alcor on hippocampal-slice culture, as well as assistance from Drs. Ralf Spindler, Brian Wowk and Alexandra Stolzing at 21CM, and Professor Gregory Brewer at UC Irvine.

21CM will provide existing tissue-culture, electrophysiology, microscopy and solution-preparation facilities. The team reports preliminary on-site CL-1 testing, hippocampal-slice stimulation and recording, and an MEA cooling and rewarming test without observed cracking. These support the experimental platform; they are not demonstrations of preserved memory.

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Timeline

The research is planned over four quarters beginning upon receipt of funding.

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Quarter 1: Establish the platform

Relocate the PI, hire and train an assistant, acquire the CL-1, MEAs and supplies, establish cultures, adapt cryoprotection equipment, and master at least one training paradigm. Establish baseline light and electron microscopy for trained and untrained cultures.

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Quarter 2: Consolidate training and optimize preservation

Establish trained responses lasting at least five hours, and optimize cryoprotectant loading and unloading for cells and slices while maintaining acceptable osmotic changes. Preservation protocols will first be optimized in untrained samples, using at least two days of post-treatment viability and structural integrity as evaluation criteria.

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Quarter 3: Test retention after cryopreservation

Train, vitrify and rewarm neural systems, then measure the effects of cryopreservation and storage on training retention. The team aims to progress toward more complex tasks, potentially including Pong.

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Quarter 4: Complete experiments and write up results

Finish year-one experiments and write up the results. Positive findings would support a separate proposal to extend the approach toward intact rabbit brains.

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Budget

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In-kind support

Dr. Monaco's home institution will cover her salary and air travel. Dr. Fahy's time, engineering support and solution-preparation support will be donated. 21CM will provide a workstation, software and desk space, and waive overhead estimated at $65,000. These contributions are not additional amounts requested from CryoDAO.

CryoDAO will retain ownership of the CL-1 system purchased through this funding. The system will be made available for the proposed research and subsequently transferred to CryoDAO’s upcoming laboratory to support future cryopreservation research.

Recovering viable tissue, electrical activity, or the ability to induce new long-term potentiation (LTP), a laboratory model of learning, does not establish that a response learned before cryopreservation has been retained. The 21CM proposal builds on reported recovery of hippocampal-slice function but targets a different endpoint: retention of prior training, rather than the capacity to learn again afterward.

View the full research proposal: https://drive.google.com/file/d/1nX1WKp8nSfBYF729ZMxee0cXt5nktknW/view?usp=sharing