Source: Lenovo Venture Capital
Just now, scientists from Fudan successfully revived the human brain that had been frozen for 18 months, directly breaking the record in the field of cryogenics and appearing in Cell sub journal. Netizens are frying pan one after another: Did the three bodies come true?! It seems that Musk will invest in this project. Some people feel sorry for the cryogenics who have been frozen in liquid nitrogen tanks for several years: Is it too early?
Just now, scientists have successfully revived the frozen human brain!
This technology is a major breakthrough in the field of hypothermia technology, paving the way for improving the research methods of nervous system diseases.
This month, Dr. Shao Zhicheng of Fudan University This work has also been officially published in Cell sub journal.
Address: https://www.cell.com/cell-reports-methods/pdfExtended/S2667-2375 (24)00121-8
Previously, brain tissue could not survive freezing and thawing. This problem has caused huge obstacles to medical research.
Of course, this still can't stop the rich from making huge investments to freeze their brains and even their whole bodies in order to revive in the future.
Now, their dreams can come true!
Immunofluorescence staining imaging technology shows brain like organs that understand freezing In this regard, Professor Jo ã o Pedro Magalh ã es of Birmingham University, UK, said that he was shocked.
You should know that brain cells are very fragile and sensitive to pressure. However, the method adopted by the team can successfully prevent cells from dying, or even let them retain their functions, which has to be said to be a miracle.
Professor Magalh ã es boldly predicted that we can now imagine such a scenario——
Decades or centuries later, terminally ill patients can be frozen, waiting for the day when there is a cure.
Astronauts can be frozen and sent to other galaxies when they wake up.
This is a big bet. If you win, you may be immortal So far, it seems that the three bodies will come true. Yun Tianming's brain does not need to be intercepted by the three bodies, but can be revived by ourselves.
Source: Zhihu "Wu Niu gasps for the moon" It seems that Musk will probably like this research very much. Human body freezing technology may become a feasible investment.
In general, the highlight of this study is——
The team developed a new method of cryopreservation (MEDY)
MEDY will not damage the structure or function of nerve cells
MEDY can be used to preserve various brain organs and human brain tissues
An interesting question arises - will all the information/memory in the brain be preserved intact after the brain is unfrozen?
So, do we have souls?
01
Netizen: Please wake me up inside the robot
The netizens were shocked at the news.
"The frozen age of mankind is coming, and we will cross the vast empty sea, where every star, every corner that has never been seen, will feel the touch of mankind."
"It's crazy. We can not only scan human brains, but also freeze them."
Previously, Google's ten year neuroscience achievement, the atlas of human brain, was listed in Science. People were shocked by this 1 cubic millimeter nanoscale map of human brain cortex Some volunteers have expressed their willingness to participate in human experiments.
This person said that he could not wait to freeze his brain and wake up in the robot body.
Once you sleep through Singularity, you will wake up in a new era.
"When I get old, freeze me and push me to a colonial ship, so that my brain can operate the robot in a jar."
So if someone had frozen Einstein's brain, we might have revived it.
The science fiction most mentioned in the comment area is the three body and the Bobby universe.
"Now we only need a probe traveling at 1% of the speed of light to run for millions of years on its own power, while avoiding space debris. The trimaran fleet has gone 200 years."
Of course, from a practical point of view, a huge lead shield is needed to protect the brain from cosmic radiation. "
In a word, this research is really incredible, which can be regarded as breaking the barrier between science fiction and reality.
Debate The theory even rose to metaphysics.
To get down to business, how are those cryogenics before them?
At present, hundreds of people in the United States have been frozen in liquid nitrogen tanks at - 196 ℃, waiting for revival. The youngest freezer is only 2 years old and costs up to 220000 dollars Some knowledgeable netizens explained that these cryogenics also used similar chemicals.
The biggest difference between freezing a person and only freezing the brain is that after a person is frozen, it is necessary to let chemicals enter the brain's blood system as soon as possible. The freezing must be carried out gradually to prevent the formation of ice crystals. The speed must be very fast, so the task is urgent. If there is no active blood circulation, the brain will soon degenerate.
When only the brain is frozen, these problems do not exist because it is a small sample.
Technically, these people's bodies are not "frozen", but "vitrified". Once the body cools below zero, the solution will not crystallize, but will become thicker and thicker. It is like a glass block, which fixes all cells in place without any internal structural changes, so it will not cause any damage
In order to ensure the future of cryogenics, Alcor set up a trust fund as an independent entity to manage and protect the funds of cryogenics, in case that the cryogenics company will disappear hundreds of years later
02
Frozen human brain tissue can now be resuscitated without damage
Recently, New Scientist, a famous scientific magazine, also made a special report on this research.
The team of Fudan University used human embryonic stem cells to cultivate brain like organs in three weeks. These small clusters of self-organized brain cells can develop into various types of brain cells.
The researchers then immersed these organs in different compounds, including sugar and antifreeze, so that they could be frozen in liquid nitrogen for at least 24 hours.
After thawing the samples, they monitored their growth and cell death in the next two weeks.
After experimenting with the combination of various compounds, the researchers found an ideal combination that can make the number of dead cells in the tissue after thawing minimum and grow more.
This combination is a chemical mixture composed of methyl cellulose, ethylene glycol, DMSO and Y27632, which was named "MEDY" by the researchers.
Some netizens found "Huadian" in the ingredients of MEDY - if you drink shampoo with three ingredients, will you live forever?
Why can MEDY preserve fragile brain cells? The researchers believe that this is because MEDY interferes with a path that usually leads to brain cell death.
Subsequently, the team conducted a series of tests on MEDY, including brain organs from 28 days to more than 100 days.
These organs will be frozen in MEDY for 48 hours before freezing, and then thawed.
The team was surprised to find that the unfrozen organs were very similar to those of the same age in appearance, growth and function! This is true even for organ like organs frozen in MEDY for 18 months.
They even set a record - after thawing, brain like organs can continue to grow and survive for 150 days!
The effectiveness of this combination has been proved: the researchers took 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 brain
Here are the specific steps for freezing and thawing the brain.
First, before cryopreservation, brain like organs need to be cultured in the medium containing 10 μ M Y27632 for 1.5 hours.
Then, we need to transfer it to the cryopreservation solution and let it stand at room temperature for 1/6-5 hours.
This standing time depends on the diameter of brain like organs. Every increase in its diameter of 1mm will increase the room temperature pretreatment time by 20 minutes.
The reason for pre-treatment is to allow Y27632 to fully penetrate into the organ, so as to reduce vitrification and increase osmotic pressure.
After pre-treatment, the like organs can be put into storage tubes and stored at − 80 ° C. After 24 hours, we need to transfer it to liquid nitrogen for long-term preservation.
How to unfreeze after freezing? This is the process.
First of all, take out organs like compounds from liquid nitrogen and thaw them at 37 ° C as soon as possible, the faster the better.
Then, we need to carefully transfer the organoid to W4 medium containing 10 μ M Y27632 for culture for two days, and then continue to culture at 37 ° C.
After two days, the medium should be changed once a day.
From the third day, the W4 medium without Y27632 will be used for another two days.
Finally, the unfrozen organs were wrapped with Matrigel and continued to be cultured.
After mastering the above steps, we can successfully freeze our brains.
03
Thesis results
high Effectively preserve cortical organs
In order to solve the challenge of long-term reliable storage of 3D brain organs, the team developed a new method of cryopreservation, including precise control of the composition of the freezing medium and the freezing and thawing process (Figure 1A).
In order to further improve the efficiency of cryopreservation, the team tested different combinations of candidate reagents and ROCK inhibitor Y27632, forming a new cryopreservation medium (CM1 – CM4).
In a word, CM1 (1% methylcellulose+10% ethylene glycol+10% DMSO+10 μ M Y27632) is the best freezing medium for brain organs, namely MEDY.
Figure 1 Establishment of MEDY cryopreservation technology Maintain functional cellular structure
The structure of the ventricular zone (VZ) and multiple cortical layers play an important role in maintaining the function of brain like organs. Whether this functional structure can be preserved after cryopreservation is crucial.
The team used MEDY to freeze and preserve cortical organs for 28 days, and then continued to culture for 3 weeks after thawing, and then carried out immunostaining of cortical markers (Fig. 2A and 2B).
On the 50th day, the VZ like structure of Sox2+and Pax6+in the unfrozen organ like structure was completely preserved (Fig. 2C and 2D). MAP2+and Tuj-1+neurons with normal morphology and neurite growth are evenly distributed near the outer layer of VZ like region (Fig. 2C and 2D).
Not only that, the team also checked the organs that recovered after 1 and a half years of cryopreservation. Immunostaining showed that the progenitor cells and neurons were well maintained, and most progenitor cells were still proliferating after thawing, which was similar to the normal group.
In conclusion, the functional cellular structure of cortical organs was well preserved during MEDY cryopreservation.
Figure 2 MEDY protects the functional structure of cortical organs Cell diversity and cell population
In order to explore whether MEDY cryopreservation will affect gene expression, the team conducted overall RNA sequencing and checked the gene expression profiles of normal and MEDY cryopreserved organs.
The results showed that there was no significant difference in the expression of NPC markers (Fig. 3A and 3B). The genes related to neurons, including motor neurons, multilayer cortical neurons and glial cells, have similar transcriptional profiles, indicating that the cryopreservation process did not cause changes in gene expression (Fig. 3A – 3E).
In order to study the cell diversity and cell population of MEDY cryopreserved organs, the team also conducted single cell RNA sequencing.
The results showed that both normal and unfrozen cortical organs contained major neuronal populations, including multilayered cortical neurons, NPC and glial cells (Fig. 3F and 3G). Among them, the number of NPC has not decreased, which indicates that MEDY cryopreservation does not inhibit the neurodevelopment of normal organs (Fig. 3H and 3I).
Figure 3 RNA sequencing and single cell sequencing analysis of cell diversity in normal and MEDY cortical organs Functional activities can be maintained
In order to verify whether the unfrozen organs still have functional neural activity, the team conducted calcium imaging experiments.
After about 20 seconds of stimulation, strong calcium activity can be detected in MEDY cryopreserved organs (Fig. 4A – 4C). These results suggest that glutamate synaptic connections can still be maintained after cryopreservation.
In order to further confirm the electrophysiological characteristics of the neural network of the organoid, the team used the microelectrode array (MEA) to detect the synchronization of the primitive activities of the Shenjing on the 114th day (Figure 4D).
Synchronous activity can be detected in normal and unfrozen organ like organs, indicating that network bursts are well preserved (Figure 4E – 4G). The peak frequency of organoid was basically unaffected during cryopreservation (Figure 4H), and there was no difference in the total number of activated electrodes within 120 seconds (Figure 4I). The number of network bursts also did not decrease significantly, indicating the complexity of functional connections of neurons in organs like this (Figure 4J).
In general, MEDY cryopreservation basically protects the functional connection of cryopreserved organs.
Figure 4 Detection of functional activity of normal and MEDY cortical organs by calcium imaging and MEA technology Multi brain specific organ like
To further verify whether MEDY cryopreservation can be used to maintain various brain region specific organs, the team induced GABA organs, SP organs and OVB organs (Figure 5G).
The results showed that the axons of each organoid began to grow around the 6th day, and almost no cell fragments were released from the organoid after MEDY cryopreservation.
In addition, GAD67+inhibitory neurons in unfrozen GABA like organs were well protected, and NKX2.1+inhibitory progenitor cells were also at a similar level.
In SP organs, Hoxc9+cells show the same structural characteristics as human thoracic vertebrae SP, and have similar abundance in the two groups (Fig. 5I and 5K). NKX6.1+ventral motor nerve progenitor cells and Pax6+cells were also clustered with similar abundance (Fig. 5H and 5J).
In OVB organs, Pax6+and RX+optic vesicle like structures were also protected during cryopreservation, similar to normal.
In general, the cell diversity and structure of various brain region specific organs have been well preserved, which indicates that MEDY cryopreservation technology can be widely applied to different neural organs.
Figure 5 MEDY cryopreservation can be used for the protection of long-term cultured cortical organs and SP organs Brain tissue from patient
In order to expand the potential clinical application of MEDY cryopreservation technology, the team prepared brain like organs based on induced pluripotent stem cells (iPSCs) of epilepsy patients (Figure 6A).
From the 7th to the 14th day after thawing, axons grew strongly, exceeding 200 μ m (Fig. 6B). In addition, immunostaining showed that there was no abnormality in the cell population of neural progenitor cells and neuron cells (Fig. 6C – 6H). These results indicate that MEDY cryopreservation can be used to store neural organs from patients.
It can be inferred that MEDY can also be used to freeze the fresh brain tissue of patients with pathological characteristics, which is crucial for basic research to clarify the pathogenesis of brain diseases (Figure 6I).
The results showed that the brain tissue with the size of about 3mm survived after MEDY cryopreservation, because a large number of living cells could migrate out of the tissue on the 14th day (Fig. 6J). In addition, most neurons and astrocytes were well preserved (Fig. 6K and 6L).
In general, MEDY can preserve brain like organs and living brain tissues with pathological characteristics from epilepsy patients.
Figure 6 MEDY cryopreservation is applicable to the preservation of cortical organs and living brain tissues from epileptic children Neuroprotective effect
In order to understand the neuroprotective mechanism of MEDY cryopreservation on brain like organs, the team conducted overall RNA sequencing on cortex like organs after MEDY cryopreservation.
The results show that MEDY has similar expression levels on four genes (APOL1, IL11, ULBP1, ULBP2) compared with non cryopreserved organ like cells (Fig. 7G), which further indicates that MEDY can prevent apoptosis of organ like cells by inhibiting the expression of these genes during cryopreservation or thawing.
These results indicate that MEDY cryopreservation can maintain organ like survival and neural function by inhibiting the endoplasmic reticulum mediated apoptosis pathway (Figure 7H).
Figure 7 RNA sequencing reveals gene expression changes behind MEDY cryopreservation technology 04
Author Introduction
Shao Zhicheng, graduated from Shanghai Jiaotong University with a doctor's degree, has carried out postdoctoral research in the Institute of Neuroscience, Chinese Academy of Sciences, the University of Alabama and Harvard University.
In February 2020, he joined the Institute of Brain Science Transformation of Fudan University as a doctoral supervisor and a distinguished professor of Shanghai Oriental Scholars, mainly engaged in somatic reprogramming, central nervous system regeneration, and the pathogenesis of mental diseases.
The research group uses induced pluripotent stem cells of specific disease types, combined with 3D brain like organs and other technologies, to study the mechanism of the occurrence and development of mental diseases, find drug targets and establish drug screening platforms.
At the same time, we used transdifferentiation technology and materials science to prepare organ like chips to explore a new strategy of 3D organoid transplantation for central nervous system tissue regeneration.
At present, he has published many papers in important academic journals at home and abroad as the first author and corresponding author, including Nature Biomedical Engineering, Nature Neuroscience, Biomaterials, Nature Communications, Cell Reports, etc.
reference resources Data:
https://www.newscientist.com/article/2431153-frozen-human-brain-tissue-can-now-be-revived-without-damage/
https://www.cell.com/cell-reports-methods/fulltext/S2667-2375 (24)00121-8
Source: New Zhiyuan
