Editor: Aeneas is sleepy
Just now, Fudan scientists successfully froze the human brain for 18 months, directly breaking the record in the field of cryogenic technology and appearing in the Cell sub-journal. Netizens have fried the pot: The three-body problem has come true?! Musk seems to be going to invest in this project. There are even more people who regret the frozen people who have been frozen in the liquid nitrogen tank for several years: Is it too early to freeze?
Just now, scientists have succeeded in resurrecting frozen human brains!
This technology is a major breakthrough in the field of cryogenic technology, paving the way for improved research methods for neurological diseases.
This month, the work of Dr. Shao Zhicheng's team at Fudan University was also officially published in the Cell sub-journal.
论文地址：https://www.cell.com/cell-reports-methods/pdfExtended/S2667-2375(24)00121-8
Previously, brain tissue could not survive the freezing and thawing process. This problem has created a huge obstacle to medical research.
Of course, this still doesn't stop the wealthy from spending huge sums of money in freezing their brains and even entire bodies in the hope of resurrecting them in the future.
Now, their dreams can come true!
Immunofluorescence staining imaging technique showing thawed brain organoids
In this regard, Professor João Pedro Magalhães of the University of Birmingham in the United Kingdom said that he was shocked.
You know, brain cells are very fragile and extremely sensitive to stress. It's a miracle that the team's approach has been able to prevent cells from dying and even retain their function.
Professor Magalhães boldly predicted that we can now fully imagine such a scenario -
Decades or centuries later, terminally ill patients can be cryopreserved, waiting for the day when there will be a cure.
Astronauts can be frozen and awakened to other galaxies.
It's a big gamble, and if you win, you could live forever
At this point, it seems that the three-body problem is about to come true, Yun Tianming's brain does not need to be intercepted by the three-body man, and we can resurrect him by ourselves.
Source: Zhihu "Wu Niu Panting Moon"
In this way, Musk will probably like this research very much, and cryonics technology may become a feasible investment.
Overall, the highlights of this study are-
The team has developed a new cryopreservation method (MEDY)
MEDY does not disrupt nerve cell structure or function
MEDY can be used to preserve a variety of brain organoids and human brain tissue
The interesting question arises - after the brain is thawed, will all the information/memories in the brain also be preserved intact?
So, do we have a soul or not?
Netizen: Please let me wake up inside the robot
As soon as this news came out, netizens were immediately shocked.
"The Frozen Era of humanity is coming, and we will cross the vast void ocean, where every star, every corner that has never been seen, will feel the touch of humanity."
"It's crazy, we can not only scan human brains, but also freeze them."
Previously, Google's ten-year neuroscience achievement, the Human Brain Atlas, was featured in Science. People were shocked by this 1 cubic millimeter nanoscale human cerebral cortex map
Some volunteers have already expressed their willingness to participate in human trials.
This said that he couldn't wait to freeze his brain and wake up in the robot's body.
Once you sleep through the singularity, you will wake up in a new era.
"When I'm old, freeze me and push me onto a colony ship, and let my brain operate the robot in a jar."
So, if someone had frozen Einstein's brain, we might have been able to resurrect it.
The most mentioned sci-fi in the comment area is the Three-Body Problem and Bobby Universe.
"Now we only need a probe traveling at 1% of the speed of light, and we can operate on our own power for millions of years, while avoiding space debris. The trimaran fleet has been gone for 200 years."
Of course, realistically, there needs to be a huge lead shield to protect the brain from cosmic radiation."
All in all, this research is incredible, and it can be said to break the barrier between science fiction and reality.
The discussion even rose to metaphysics.
Getting back to the point, those cryogenic people before, are they okay?
Currently, hundreds of people in the United States are frozen in -196°C liquid nitrogen tanks, waiting to be resurrected. The youngest frozen person is only 2 years old, and the cost is as high as 220,000 US dollars
Some knowledgeable netizens explained that in fact, these frozen people also used similar chemicals.
The biggest difference between freezing the whole whole and freezing only the brain is that after a person freezes, chemicals need to enter the brain's blood system as soon as possible. Freezing had to be done gradually to prevent the formation of ice crystals, and it had to be fast, so the task was urgent. Without active blood circulation, the brain quickly degenerates.
However, when only the brain is frozen, because it is a small sample, there are no such problems.
Technically, these people's bodies are not "frozen", but "vitrified". Once the body cools below freezing, the solution does not crystallize, but thickens and thickens. It is like a glass block that holds all the cells in place without any changes in the internal structure, so it does not cause any damage
In order to secure the future of the Frozen People, Alcor Corporation has set up a trust fund as an independent entity to manage and protect the funds of frozen patients in case the Frozen Companies are gone hundreds of years later
Freezing human brain tissue can now be resuscitated without damage
Just recently, the well-known scientific journal New Scientist did a special report on this research.
Using human embryonic stem cells, the Fudan University team cultivated brain organoids in three weeks, and these small clusters of self-organizing brain cells can develop into various types of brain cells.
The researchers then immersed the organoids in different compounds, including sugar and antifreeze, so that they could be frozen in liquid nitrogen for at least 24 hours.
After the samples were thawed, they monitored their growth and cell death for the next two weeks.
After experimenting with a variety of combinations of different compounds, the researchers found an ideal combination that would allow the tissue to thaw with the fewest cells that die and grow more.
This combination is a chemical mixture composed of methylcellulose, ethylene glycol, DMSO and Y27632, which was named "MEDY" by the researchers.
Some netizens found "Hua Dian" in the ingredients of MEDY - if you drink a shampoo with 3 of these ingredients, will you live forever?
Why does MEDY preserve fragile brain tissue cells? Researchers believe this is because MEDY interferes with a pathway that normally leads to brain cell death.
Subsequently, the team conducted a series of tests on MEDY, with brain organoids ranging from 28 days to more than 100 days.
These organs are frozen in MEDY for 48 hours before being frozen.
The team was pleasantly surprised to find that the thawed organoids were very similar in appearance, growth, and function to organoids of the same age that had never been frozen! This is true even for organoids that have been frozen in MEDY for 18 months.
They even set a record – after thawing, brain organoids can continue to grow and survive for up to 150 days!
The effectiveness of this combination has been demonstrated: the researchers removed 3 cubic millimeters of brain tissue from a 9-month-old girl with epilepsy, and the brain tissue remained active for at least two weeks after thawing.
How to preserve frozen brains
Here's how to freeze and thaw your brain.
First, brain organoids need to be cultured in medium containing 10 μM Y27632 for 1.5 h prior to cryopreservation.
Subsequently, we need to transfer it to a cryopreservation solution and let it sit for 1/6-5 hour at room temperature.
This resting time, depending on the diameter of the brain organoid, increases the room temperature pretreatment time by 20 minutes for every 1 mm increase in diameter.
The reason why pretreatment is carried out is to allow Y27632 to fully penetrate into the internal organ, thereby reducing vitrification and increasing osmolality.
Once pre-treatment, the organoids are ready to be stored in storage tubes at −80°C. After 24 hours, we need to transfer it to liquid nitrogen for long-term storage.
How do I thaw the freeze? Here's how it goes.
First, remove the organoids from liquid nitrogen and thaw them at 37°C as soon as possible, the faster the better.
Then, we need to carefully transfer the organoids to W4 medium containing 10 μM Y27632 for two days and then continue to culture at 37 °C.
After two days, the medium is changed every day.
From day 3 onwards, start using W4 medium without Y27632 for two more days.
Finally, the thawed organoids are wrapped in Matrigel and continue to culture.
With the above steps mastered, we can successfully freeze our brains.
Thesis results
Efficient preservation of cortical organoids
To address the challenge of long-term reliable storage of 3D brain organoids, the team developed a new cryopreservation method that includes precise control of the cryomedia composition and the freeze-thawing process (Figure 1A).
To further improve the efficiency of cryopreservation, the team tested different combinations of candidate reagents with the ROCK inhibitor Y27632 to form a new cryogenic medium (CM1–CM4).
综合来看，CM1（1% 甲基纤维素 + 10% 乙二醇 + 10% DMSO + 10μM Y27632）是脑类器官的最佳冷冻介质——即MEDY。
Figure 1. Establishment of MEDY cryopreservation technology
Maintain functional cellular structure
The structure of the ventricular region (VZ) and multiple cortical layers play an important role in maintaining the function of brain organoids, and it is important to preserve this functional structure after cryopreservation.
The team used MEDY to cryopreserved cortical organoids for 28 days and continued to culture for 3 weeks after thawing before immunostaining of cortical layer markers (Figures 2A and 2B).
On day 50, the VZ-like structures of Sox2+ and Pax6+ in thawed organoids were completely preserved (Figures 2C and 2D). MAP2+ and Tuj-1+ neurons with normal morphology and neurite outgrowth are evenly distributed near the outer layer of the VZ-like region (Figures 2C and 2D).
Not only that, but the team also examined organoids that had been revived after one and a half years of cryopreservation. Immunostaining showed that progenitor cells and neurons were well maintained, and most progenitor cells were still proliferating after thawing, which was similar to the normal group.
In summary, the functional cellular structure of cortical organoids was well preserved during the cryopreservation of MEDY.
Figure 2. Protection of MEDY on the functional structure of cortical organoids
Cell diversity and cell population
To explore whether cryopreservation of MEDY affects gene expression, the team performed whole RNA sequencing to examine the gene expression profiles of normal and MEDY cryopreserved organoids.
The results showed no significant difference in the expression of NPC markers (Figures 3A and 3B). Neuron-associated genes, including motor neurons, multilayered cortical neurons, and glial cells, have similar transcriptional profiles, suggesting that the cryopreservation process did not cause changes in gene expression (Figures 3A–3E).
To study the cell diversity and cell population of MEDY cryopreserved organoids, the team also performed single-cell RNA sequencing.
The results showed that both normal and thawed cortical organoids contained major neuronal cell populations, including multilayered cortical neurons, NPCs, and glial cells (Figures 3F and 3G). Among them, the number of NPCs did not decrease, suggesting that MEDY cryopreservation did not inhibit the neurodevelopmental processes of normal organoids (Figures 3H and 3I).
Figure 3. RNA sequencing and single-cell sequencing analysis of cell diversity in normal and MEDY cortical organoids
Functional activities can be maintained
To verify whether the thawed organoids still have functional neural activity, the team conducted calcium imaging experiments.
After approximately 20 sec of stimulation, strong calcium activity can be detected in MEDY cryopreserved organoids (Figures 4A–4C). These results suggest that glutamatergic synaptic connections are still maintained after cryopreservation.
To further confirm the neural network electrophysiological properties of the organoids, the team used a microelectrode array (MEA) to detect the synchronicity of neuronal activity on day 114 (Figure 4D).
Synchronous activity was detected in both normal and thawed organoids, indicating that the network burst was well preserved (Figures 4E–4G). The spike frequency of organoids was largely unaffected during cryopreservation (Figure 4H), and there was no difference in the total number of electrodes activated within 120 sec (Figure 4I). There was also no significant reduction in the number of network bursts, suggesting the complexity of neuronal functional connections in organoids (Figure 4J).
Overall, MEDY cryopreservation essentially preserves the functional connectivity of cryopreserved organoids.
Figure 4. Functional activity of normal and MEDY cortical organoids was detected by calcium imaging and MEA techniques
Multi-brain region-specific organoids
To further validate whether MEDY cryopreservation can be used to maintain various brain region-specific organoids, the team induced GABA organoids, SP organoids, and OVB organoids, respectively (Figure 5G).
The results showed that the axons of each organoid began to grow around day 6 and that almost no cellular debris was released from the organoids after cryopreservation of MEDY.
In addition, GAD67+ inhibitory neurons were well protected and NKX2.1+ inhibitory progenitor cells were at similar levels in the thawed GABA organoids.
In SP organoids, Hoxc9+ cells show the same structural features as human thoracic SP and have similar abundances in both groups (Figures 5I and 5K). NKX6.1+ ventral motor neuron progenitor cells and Pax6+ cells were also clustered with similar abundance (Figures 5H and 5J).
In OVB organoids, the Pax6+ and RX+ optic vesicle-like structures are also protected during cryopreservation, similar to normal.
Overall, the cell diversity and structure of various brain region-specific organoids were well preserved, suggesting that MEDY cryopreservation technology can be widely applied to different neural organoids.
Figure 5. MEDY cryopreservation can be used for the protection of long-term cultured cortical organoids and SP organoids
from the patient's brain tissue
To expand the potential clinical application of MEDY cryopreservation, the team prepared brain organoids based on induced pluripotent stem cells (iPSCs) from patients with epilepsy (Figure 6A).
On days 7 to 14 post-thaw, axonal growth is robust, exceeding 200 μm (Figure 6B). In addition, immunostaining showed no abnormalities in the cell populations of neural progenitor cells and neuronal cells (Figures 6C–6H). These results suggest that MEDY cryopreservation can be used to store neural organoids from patients.
It can be assumed that MEDY can also be used to cryopreserve fresh patient brain tissue with pathological features, which is essential for basic research to elucidate the pathogenesis of brain diseases (Fig. 6I).
The results showed that brain tissue approximately 3 mm in size survived after cryopreservation with MEDY, as a large number of viable cells could migrate out of the tissue on day 14 (Figure 6J). In addition, most neurons and astrocytes are well preserved (Figures 6K and 6L).
Overall, cerebral organoids from epilepsy patients and living brain tissues with pathological features can be preserved by MEDY.
Figure 6. MEDY cryopreservation is suitable for the preservation of cortical organoids and living brain tissue derived from children with epilepsy
Protective effect on nerves
In order to understand the neuroprotective mechanism of MEDY cryopreservation on brain organoids, the team performed whole RNA sequencing of cortical organoids cryopreserved with MEDY.
The results showed that the expression levels of MEDY on the four genes (APOL1, IL11, ULBP1, ULBP2) were similar compared to those not cryopreserved (Figure 7G), further suggesting that MEDY could prevent apoptosis in organoid cells by inhibiting the expression of these genes during cryoppreservation or thawing.
These results suggest that MEDY cryopreservation can maintain organoid survival and neurological function by inhibiting the endoplasmic reticulum-mediated apoptosis pathway (Figure 7H).
Figure 7. RNA sequencing reveals gene expression changes behind MEDY's cryopreservation technology
About the Author:
Zhicheng Shao graduated from Shanghai Jiao Tong University with a Ph.D. and has conducted postdoctoral research at the Institute of Neuroscience, Chinese Academy of Sciences, the University of Alabama, and Harvard University.
In February 2020, he joined the Institute of Translational Brain Science of Fudan University, doctoral supervisor, Shanghai Oriental Scholar Distinguished Professor, mainly engaged in somatic cell reprogramming, central nervous system regeneration, and pathogenesis of mental diseases.
The research group uses induced pluripotent stem cells of specific disease types, combined with 3D brain-like organoids and other technologies, to study the occurrence and development mechanism of mental diseases, find drug targets and establish drug screening platforms.
At the same time, organoid-on-a-chip was prepared by transdifferentiation technology combined with materials science to explore a new strategy for 3D-organoid transplantation in the treatment of central nervous system tissue regeneration.
目前，以第一作者和通讯作者在国内外重要学术刊物上发表多篇论文，其中包括Nature Biomedical Engineering、Nature Neuroscience、Biomaterials、Nature Communications、Cell Reports等。