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Scientists are growing "mini brains" (also known as brain organoids) in culture dishes, hoping to simulate some brain functions through these clusters of brain nerve cells and deepen and change our understanding of brain neural development and diseases.

They strive to make them more like the human brain, and progress has been particularly rapid in recent years. They have discovered some surprising phenomena, such as neurons grown in vitro will spontaneously excite, which is one of the ways neurons grow and establish new connections in the human brain; active brain waves similar to those seen in the brains of premature babies have been observed in brain organoids, and this whole-brain coordinated electrical activity is one of the characteristics of conscious brain, etc.

So the question becomes urgent: Will these brain organoids eventually develop consciousness? Scientists are looking for the answer.

Written by | Xiaoye

In the 1980s, American philosopher Hilary Putnam proposed the famous "brain in a vat" thought experiment. Less than half a century later, biologists have been able to cultivate a real version of the "mini brain in a vat" in a laboratory dish - brain organoids.（brain organoids）。

Although this is a cluster of brain nerve cells that is only a few millimeters wide, it can already simulate some brain functions. And we soon face an important question: Can such brain organoids produce consciousness?

Brain organoid research is advancing rapidly

Organoids（organoid）Also called micro-organ（mini-organ）As the name suggests, it is a miniature model similar to a real organ. It is formed by self-organization through in vitro three-dimensional culture of pluripotent stem cells or adult cells. It is highly similar to the structure of human organs and can reproduce some of the functions of the imitated organs.

The origin of organoids can be traced back to 1907, when HV Wilson, a professor of zoology at the University of North Carolina, published a paper[1], revealing that sponge cells separated by mechanical separation can reaggregate and self-organize into a completely new sponge with the same normal life functions.

By the 1950s, other scientists had conducted similar experiments using other animal cells, demonstrating that vertebrate cells all have the ability to self-organize, laying the foundation for an important feature of organoid cultivation technology in the future: self-organization, which is like winding up cells. As long as a suitable culture environment is provided, the cells will perform their respective functions and self-organize to form organoids.[2]。

Stem cell technology is another key to the booming development of organoids. In the 1980s, the team of former Soviet scientist AJ Friedenstein conducted a series of cutting-edge experiments and discovered a type of osteoblast in the bone marrow.[3]bone marrow stromal stem cells[4], can generate a variety of bone tissues through in vivo experiments[5]In the 1990s, Arnold Caplan, a professor of biology at Case Western Reserve University in the United States, renamed it mesenchymal stem cells.（Mesenchymal Stem Cell, MSC）[6]MSC was finally accepted by the academic community.[7]It is a multipotent stem cell with the ability of self-renewal and multidirectional differentiation. It can be transformed into various cell types and has wide clinical application value.

Also in the 1980s, Professor James Thomson, a developmental biologist at the University of Wisconsin-Madison, also devoted himself to this field for a long time, exploring the potential of stem cells in primates. Until 1998, he used donated human embryos to construct the world's first human embryonic stem cell line.[8]In 2007, he collaborated with Shinya Yamanaka's team at Kyoto University in Japan and successfully induced human adult cells into pluripotent stem cells.（iPSC）[9]iPSC cells have the potential for unlimited proliferation in vitro. They can not only express stem cell markers in embryonic stem cells, but also have the potential to differentiate into cells or tissues of the three germ layers.[10]。

At this point, everything is ready. The rapid development of self-organization and stem cell fields has injected new vitality into organoid research. The first decade of the 21st century has ushered in a flourishing display of results: liver organoids[11], Intestinal Organoids[12]Organoids have been successfully cultivated in the fields of retina, prostate, lung, kidney, breast, and brain. Organoids have become a hot topic of research with their rapid development. In 2013, organoids were published by Science.（Science）Journal rated as one of the top ten technologies of the year[13]Another 10 years later, MIT Technology Review predicted in its 2023 “Top 10 Breakthrough Technologies in the World” forecast that engineered organ manufacturing technology will mature in the next 10-15 years as researchers explore how to design complex tissues from scratch and grow customized organs in factories.

Among the many organoids, brain organoids are a particularly important chapter. For hundreds of years, unraveling the mysteries of human brain development and neurological diseases has been a major challenge in the field of brain science and medicine. The academic community has made various efforts, not only to establish various in vivo and in vitro cell and animal models, but also to try to use two-dimensional methods to culture human brain neurons to analyze the mechanisms of related diseases. However, for animal models, due to species differences, laboratory model animal brain models cannot fully simulate the complexity of the human brain, and the experimental results may not be fully applicable to the human brain. The two-dimensional neurons grown in the culture dish are also far from the three-dimensional human brain in terms of spatial structure, cell type complexity, interactions, and microenvironment.[14]。

Brain organoids make up for the above shortcomings. In 2008, Japanese stem cell biologist Yoshiki Sasai（Yoshiki Sasai）The team found[15], neurospheres derived from spontaneous organization of stem cells can generate cortical-like structures, including cortical progenitor cells and functional neurons, which is the first primary brain organoid model. In 2013, Jürgen Knoblich of the Institute of Molecular Biotechnology of the Austrian Academy of Sciences and Madeline Lancaster, a developmental biologist at the University of Cambridge in the United Kingdom, published a paper in Nature.（Nature）Published papers[16], reported the first three-dimensional brain organoid derived from human pluripotent stem cells. The team used the biological gel matrigel to simulate the tissue surrounding the brain, and used a rotating bioreactor to help the absorption of nutrients and the diffusion of oxygen. In this continuous three-dimensional suspension culture, growth factors that promote neural development were added, and finally a further improved brain organoid culture was obtained, which contains multiple independent and interdependent brain region structures such as the forebrain, choroid plexus, hippocampus, and prefrontal lobe.

Subsequently, scientists around the world continued to explore various brain organoids with brain region specificity. They combined different small molecules and growth factors and successfully obtained brain organoids including the midbrain, thalamus, cerebellum, striatum, etc. Other scientists tried to assemble two or even more brain region organoids to form a "quasi-assembly".（assembloids）, further simulating the real-life human brain development, neuron migration and other processes. For example, a 2019 paper published in Cell Stem Cell（Cell Stem Cell）Papers in journals[17]The thalamic organoids were fused with the cortical organoids to simulate the bidirectional projection process between the thalamus and the cortex.[18]By assembling brain organoids with non-neural organoids such as muscle tissue and observing the control of nerves over other tissues, we obtained results similar to those in the real human body.

Simple diagram of the development of brain organoid technology, source: 10.1038/s41392-022-01024-9[19]

Differences from the real brain

In fact, brain organoids are only a few millimeters wide and are a cluster of cells similar to the brain. As a mini model cultivated in the laboratory, it has advantages that other brain research methods do not have. For example, when electrodes are connected to brain organoids, signal transmission between neurons can be triggered, spontaneously imitating the real brain.

So, are brain organoids miniature versions of the real brain? In reality, this is not the case. The current brain organoids do not completely match the real brain.

First of all, the most significant defect of brain organoids is that they stop growing after they grow to a few millimeters because there are no blood vessels to provide oxygen and nutrients. Unlike natural biological tissues, the growth of brain organoids depends on the nutrient solution that penetrates into the culture dish. Once they grow to a certain size, if there is not enough nutrition, the growth stops, and the cells in the center begin to die. Unfortunately, they die long before they grow like a real brain. Therefore, various teams have tried every possible way to grow blood vessels in brain organoids, culture vascularized organoids and fuse them with brain organoids, or artificially open channels in brain organoids to allow more nutrient solution to be infused into them to produce more mature synapses.[20]。

Second, unlike real brains, brain organoids lack sensory input from the surrounding environment, which is one of the keys to the development of brain circuits. Brain organoids have no eyes to see, no ears to hear, no nose to smell, and no mouth to taste. Isolated in a petri dish, brain organoids cannot autonomously encode experience and information without sensory input.[21]

A 2020 paper published in the journal Nature puts forward a relatively restrained view[22], indicating that the currently widely used brain organoid models are still unable to replicate the basic characteristics of real brain development and organization, let alone simulate the complex brain circuits required for complex brain diseases and normal cognition. The researchers found that one reason behind this is the "identity crisis" of organoid cells: brain organoid cells cannot normally differentiate into unique cell subtypes, and a "hodgepodge" of various genes can be found in cells of completely different types, throwing the developmental program into chaos. Another reason is that the laboratory culture method causes "great stress" to the cells: all brain organoid models express abnormally high levels of cellular stress response genes, which leads to abnormal cell behavior and the production of abnormal proteins, ultimately preventing the organoid cells from developing normally.[23, 24]。

The development of a real brain is like a symphony, with various instruments playing simultaneously and coordinating with each other under the coordination of a conductor to produce a beautiful and harmonious complex movement. Organoid scientists have just taken the first step to achieve such a level of complexity in brain organoids.

Can brain organoids develop consciousness?

Although brain organoids are still far from real brains, this does not prevent scientists from thinking ahead to a question: Will the "brain-like" in the petri dish eventually develop consciousness?

Based on the current research situation, most brain organoid scientists believe that brain organoids will not and cannot develop a form of consciousness.

Lancaster, the first person to grow brain organoids, believes that current brain organoids are still too primitive to generate consciousness, as they lack the anatomical structure necessary to create complex EEG patterns. Although brain organoids "may have neurons that communicate with each other without input and output, this does not necessarily mean anything like human consciousness."[25]In the view of Lancaster and most researchers, rejuvenating dead pig brains is more likely to produce consciousness than brain organoids.

In June this year, Kenneth Kosik, a neuroscientist at the University of California, Santa Barbara, wrote in Patterns（Patterns）A viewpoint article was published in the journal[26], suggesting that brain organoid research may eventually lead to the creation of consciousness in the laboratory, but this possibility does not exist based on current technology or even the technology of the near future.

First, as mentioned above, although the significant flaws of brain organoids indicate that they do not yet meet any operational definition of consciousness, scientists still have many obstacles to overcome to overcome these flaws. It is still too early to discuss whether organoids will generate consciousness.

Secondly, philosophers and scientists have been exploring the question of "what is consciousness" for thousands of years, and there are many different theories, but there is still no generally accepted definition. Modern science classifies consciousness as a scientific problem and explains it from the perspective of neural mechanisms, which can be divided into four categories of theories: high-level theory（HOT）, Global Neural Workspace Theory（GNWT）, Integrated Information Theory（IIT）As well as re-entry and pre-processing theories. These theories not only explore the issue of consciousness centered on the brain, but also emphasize the importance of the interaction between the subject's body and the environment, which affects the various abilities required for consciousness: representation, senses, perception, and so on. One of the most obvious features of brain organoids is that they are completely separated from the body, whether in movement or perception, and have no history of somatic experience. Although experiments have shown that the neural discharge activity of brain organoids is similar to the patterns related to brain encoding experience, there is still a problem: a framework that can encode experience but has no experience history.(Brain Organoids)Can consciousness be generated? Can consciousness exist without content?

As early as 2022, KosikNautilusLong article published in magazine[27]It is proposed that an important reason why brain organoids have no consciousness is that they do not possess the core property - abstract extraction ability. Consciousness requires an abstract process, which is based on the correlation between our impression of the sensory world and motor feedback. When we see a red apple on the table, the following process is triggered: the light reflected by the object activates the photoreceptors in the retina and transmits a signal to the brain; the signal contains rich information about the color, size and environment of the object. After years of life experience in the world, the discharge patterns corresponding to the two concepts of the words "red" and "apple" have been generated, and finally we "realize" that there is a red apple on the table. However, the neural discharge activity of brain organoids is not associated with anything in reality.

Of course, there are scientists who hold positive opinions. Anil Seth, a cognitive neuroscientist at the University of Sussex in the UK, said in a Nature podcast[28]He stated that he does not rule out the possibility of brain organoids developing consciousness. As the complexity of brain organoids and their similarity to the human brain continue to increase, even if their structure is not completely identical to the human brain, it is entirely possible that they can have conscious experiences.

Although most scientists hold a negative view, some interesting experiments suggest that the basic elements of consciousness may have gradually emerged.

In the lab of neuroscientist Alysson Muotri at the University of California, San Diego, there are hundreds of petri dishes filled with sesame-sized brain organoids. He uses a variety of unusual methods to manipulate brain organoids, and one of his experimental results has attracted widespread attention. In 2019, Moutri's team published a paper in Cell Stem Cell that showed that brain organoids can be used to grow and function.[29]The report created brain organoids that can produce coordinated activity waves, similar to the activity brain waves seen in the brains of premature babies. This whole-brain coordinated electrical activity is one of the characteristics of a conscious brain, so the team believes that the brain organoids basically simulate the early development of the human brain. However, there are also doubts about the results, mainly because brain waves similar to those of premature babies do not mean that brain organoids can be equated with infant brains. Moreover, infants' brain waves are different from those of adults, and often show very chaotic and irregular fluctuations.

A dish of brain organoids in Muotri's lab.Credit: David Poller/ZUMA Wire, via Alamy Live News

In the same year, Hideya Sakaguchi of Kyoto University （Hideya Sakaguchi）The team reports in the journal Stem Cell Reports[30], successfully visualized the network activity and connections between individual neurons in cortical spheroids. The team detected dynamic changes in calcium ion activity and discovered integrated activity between cells that were able to organize themselves into clusters and form networks with other nearby clusters. The manifestation of synchronized neural activity can serve as the basis for a variety of related brain functions, including memory. Another key finding of the study is that neurons grown in vitro will spontaneously excite, which is one of the ways neurons grow and establish new connections in the human brain.

Unavoidable ethical issues

The academic community has different opinions on the issue of consciousness, but scientists also recognize that it is much easier to create a conscious system than to define it. Therefore, brain organoid research also highlights a blind spot: scientists have no agreed-upon method to define and measure consciousness.

Even Muotri himself admitted that he did not know which definition to use to determine whether an organoid has reached a conscious state. Therefore, whether brain organoids produce consciousness has become a personal theoretical preference of researchers, which will affect their research methods and purposes.

So, plan ahead. Anil Set proposed that when there is no clear method to evaluate the conscious state of organoids, it is necessary to preemptively determine the ethical framework. Karen Rommelfanger, director of the Neuroethics Program at Emory University in the United States, also agreed that the differences between brain organoids and other body organoids research involve not only biological aspects, but also ethical aspects. Andrea Lavazza of the University of Pavia in Italy believes that in the future, organoids may show the ability to experience basic sensations such as pain, thereby demonstrating perception and even basic forms of consciousness. This requires us to consider whether brain organoids should be given moral status and what restrictions should be introduced to regulate research.[31]。

References

[1] https://onlinelibrary.wiley.com/doi/abs/10.1002/jez.1400090305

[2] https://www.360zhyx.com/home-research-index-rid-74706.shtml

[3] https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2184.1987.tb01309.x

[4] https://onlinelibrary.wiley.com/doi/10.1002/9780470513637.ch4

[5] https://linkinghub.elsevier.com/retrieve/pii/S1934590908001148

[6] https://onlinelibrary.wiley.com/doi/10.1002/jor.1100090504

[7] https://www.science.org/doi/10.1126/science.284.5411.143

[8] https://www.science.org/doi/10.1126/science.282.5391.1145

[9] https://www.science.org/doi/10.1126/science.1151526

[10] https://zh.wikipedia.org/wiki/%E8%AF%B1%E5%AF%BC%E6%80%A7%E5%A4%9A%E8%83%BD%E5%B9%B2%E7%BB%86%E8%83%9E

[11] https://www.liebertpub.com/doi/10.1089/ten.2006.12.1627

[12] https://www.nature.com/articles/nature07935

[13] https://www.science.org/content/article/sciences-top-10-breakthroughs-2013

[14] https://m.thepaper.cn/baijiahao_20603927

[15] https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(08)00455-4?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1934590908004554%3Fshowall%3Dtrue

[16] https://www.nature.com/articles/nature12517

[17] https://www.cell.com/cms/10.1016/j.stem.2018.12.015/attachment/3c78ab81-5238-4756-ace6-4b73fa2292d6/mmc1.pdf

[18] https://www.cell.com/cell/fulltext/S0092-8674(20)31534-8?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867420315348%3Fshowall%3Dtrue

[19] https://www.nature.com/articles/s41392-022-01024-9#citeas

[20] http://www.news.cn/health/2023-02/03/c_1211724659.htm

[21] https://www.livescience.com/health/neuroscience/we-can-t-answer-these-questions-neuroscientist-kenneth-kosik-on-whether-lab-grown-brains-will-achieve-consciousness

[22] https://www.nature.com/articles/s41586-020-1962-0

[23] https://www.ucsf.edu/news/2020/01/416526/not-brains-dish-cerebral-organoids-flunk-comparison-developing-nervous-system

[24] https://theconversation.com/brain-organoids-help-neuroscientists-understand-brain-development-but-arent-perfect-matches-for-real-brains-130178#:~:text=Organoid%20cells%20also%20don't,not%20reflected%20in%20the%20organoids.

[25] https://www.kepuchina.cn/more/202011/t20201117_2842435.shtml

[26] https://www.cell.com/patterns/fulltext/S2666-3899(24)00136-3#%20

[27] https://nautil.us/what-the-tiny-cluster-of-brain-cells-in-my-lab-are-telling-me-246650/

[28] https://www.nature.com/articles/d41586-020-03033-6#MO0

[29] https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(19)30337-6

[30] https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(19)30197-3

[31] https://link.springer.com/article/10.1007/s40592-020-00116-y

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