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EP-4737562-A1 - METHOD FOR PRODUCING THREE-DIMENSIONAL CELL CULTURE, METHOD FOR CULTURING THREE-DIMENSIONAL CELL CULTURE, THREE-DIMENSIONAL CELL CULTURE, AND METHOD FOR EVALUATING TEST SUBSTANCE

EP4737562A1EP 4737562 A1EP4737562 A1EP 4737562A1EP-4737562-A1

Abstract

An object of the present invention is to provide a production method and a culture method of a three-dimensional cell culture containing human-derived neurons, human-derived astrocytes, and human-derived microglia capable of mimicking human brain functions; a three-dimensional cell culture containing human-derived neurons, human-derived astrocytes, and human-derived microglia capable of mimicking human brain functions; and an evaluation method of a test substance using the three-dimensional cell culture. According to the present invention, there is provided a production method of a three-dimensional cell culture, the production method including a step of adding, prior to co-culture of cells, human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel, and a step of co-culturing the astrocytes, the neurons, and the microglia in the culture vessel, in which a proportion of the astrocytes among all cells added to the culture vessel is 30% or more.

Inventors

  • KOBAYASHI HAYATO
  • ENDOH Setsu
  • KATO HIROSHI

Assignees

  • FUJIFILM Corporation

Dates

Publication Date
20260506
Application Date
20240628

Claims (19)

  1. A production method of a three-dimensional cell culture, the production method comprising: a step of adding, prior to co-culture of cells, human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel; and a step of co-culturing the astrocytes, the neurons, and the microglia in the culture vessel, wherein a proportion of the astrocytes among all cells added to the culture vessel is 30% or more.
  2. The production method according to claim 1, wherein the astrocytes, the neurons, and the microglia are simultaneously added to the culture vessel.
  3. The production method according to claim 1, wherein the step of adding, prior to co-culture of cells, human-derived astrocytes, human-derived neurons, and human-derived microglia to the culture vessel includes a step of suspending the human-derived astrocytes, the human-derived neurons, and the human-derived microglia in a culture medium, and a step of simultaneously adding the culture medium containing the astrocytes, the neurons, and the microglia obtained by the step to the culture vessel.
  4. The production method according to claim 1, wherein the astrocytes, the neurons, and the microglia are induced to differentiate from human-derived pluripotent stem cells.
  5. The production method according to claim 4, wherein the human-derived pluripotent stem cells are human iPS cells.
  6. The production method according to any one of claims 1 to 5, wherein a proportion of the microglia among all cells added to the culture vessel is 50% or less.
  7. The production method according to any one of claims 1 to 5, wherein a proportion of the neurons among all cells added to the culture vessel is 10% or more.
  8. The production method according to any one of claims 1 to 5, wherein the three-dimensional cell culture has a spheroid shape.
  9. A culture method of a three-dimensional cell culture, the culture method comprising: a step of adding, prior to co-culture of cells, human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel; and a step of co-culturing the astrocytes, the neurons, and the microglia in the culture vessel, wherein a proportion of the astrocytes among all cells added to the culture vessel is 30% or more.
  10. The culture method according to claim 9, wherein the astrocytes, the neurons, and the microglia are simultaneously added to the culture vessel.
  11. The culture method according to claim 9, wherein the step of adding, prior to co-culture of cells, human-derived astrocytes, human-derived neurons, and human-derived microglia to the culture vessel includes a step of suspending the human-derived astrocytes, the human-derived neurons, and the human-derived microglia in a culture medium, and a step of simultaneously adding the culture medium containing the astrocytes, the neurons, and the microglia obtained by the step to the culture vessel.
  12. The culture method according to claim 9, wherein the astrocytes, the neurons, and the microglia are induced to differentiate from human-derived pluripotent stem cells.
  13. The culture method according to claim 12, wherein the human-derived pluripotent stem cells are human iPS cells.
  14. The culture method according to any one of claims 9 to 13, wherein a proportion of the microglia among all cells added to the culture vessel is 50% or less.
  15. The culture method according to any one of claims 9 to 13, wherein a proportion of the neurons among all cells added to the culture vessel is 10% or more.
  16. The culture method according to any one of claims 9 to 13, wherein the three-dimensional cell culture has a spheroid shape.
  17. A three-dimensional cell culture comprising: human-derived astrocytes; human-derived neurons; and human-derived microglia, wherein the three-dimensional cell culture is obtained by the production method according to any one of claims 1 to 5.
  18. An evaluation method of a test substance, comprising: bringing the three-dimensional cell culture according to claim 17 into contact with a test substance.
  19. An evaluation method of a test substance, comprising: producing a three-dimensional cell culture by the production method according to any one of claims 1 to 5; and .bringing the three-dimensional cell culture into contact with a test substance.

Description

Technical Field The present invention relates to a production method of a three-dimensional cell culture and a culture method of a three-dimensional cell culture, using astrocytes, neurons, and microglia. Furthermore, the present invention relates to a three-dimensional cell culture containing astrocytes, neurons, and microglia. Moreover, the present invention relates to an evaluation method of a test substance using the three-dimensional cell culture. Background Art The central nervous system is composed of neurons and glial cells (astrocytes, microglia, and oligodendrocytes), and these cells exhibit normal brain functions by influencing each other (Non-Patent Document 1). For example, the neurons transmit information to other neurons by releasing glutamic acid into a synaptic cleft, and the astrocytes play a role in regulating the information transmission of the neurons by taking up glutamic acid (Non-Patent Document 2). In addition, it has been reported that microglia are activated by aging or inflammation to affect astrocytes, and the astrocytes further damage neurons (Non-Patent Documents 3 and 4). Therefore, a co-culture system capable of evaluating the intercellular interactions between neurons and glial cells is very useful for research on the central nervous system. In addition, in recent years, various three-dimensional culture systems have been reported to mimic the complicated structure of the human brain (Non-Patent Document 5). Furthermore, it is also known that there are species differences in neurons and glial cells (Non-Patent Documents 6 and 7), and it is very important to use human cells to reproduce the human brain. Patent Document 1 describes a three-dimensional co-culture containing astrocytes, neurons, and microglia derived from human iPS cells. Patent Document 2 describes a three-dimensional organoid containing neurons and glia derived from human iPS cells. Patent Document 3 describes a three-dimensional (spheroid) co-culture containing neurons and glia derived from human iPS cells. Prior Art Documents Patent Documents Patent Document 1: WO2023/039567APatent Document 2: WO2022/183015APatent Document 3: WO2022/178395A Non-Patent Documents Non-Patent Document 1: Francesca Cerbai et al., PLoS One, Vol. 7, Article number e45250, 2012Non-Patent Document 2: Jens V. Andersen et al., Neuropharmacology, Vol. 14, Article No. 108719, 2021Non-Patent Document 3: Shane A. Liddelow et al., Nature, Vol. 541, pp. 481-487, 2017Non-Patent Document 4: Laura E. Clarke et al., Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, pp. E1896-E1905, 2018Non-Patent Document 5: Eduarda G. Z. Centeno et al., Molecular Neurodegeneration, Vol. 13, Article No. 27, 2018Non-Patent Document 6: Anke M Tukker et al., Toxicological Sciences, Vol. 178, pp. 71-87, 2020Non-Patent Document 7: Leonid Tarassishin et al., Glia, Vol. 62, pp. 999-1013, 2014 Summary of Invention Object to be solved by the invention An object to be achieved by the present invention is to provide a production method of a three-dimensional cell culture containing human-derived neurons, human-derived astrocytes, and human-derived microglia capable of mimicking human brain functions, and a culture method of a three-dimensional cell culture containing human-derived neurons, human-derived astrocytes, and human-derived microglia capable of mimicking human brain functions. Another object to be achieved by the present invention is to provide a three-dimensional cell culture containing human-derived neurons, human-derived astrocytes, and human-derived microglia capable of mimicking human brain functions. Another object to be achieved by the present invention is to provide an evaluation method of a test substance using the above-described three-dimensional cell culture. Means for solving the object As a result of intensive studies to achieve the above object, the present inventors have found that a three-dimensional cell culture capable of mimicking human brain functions can be produced by adding human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel such that a proportion of the astrocytes among all cells added to the culture vessel is 30% or more prior to co-culture of the cells. The present invention has been completed based on these findings. That is, according to an aspect of the present invention, the following invention is provided. <1> A production method of a three-dimensional cell culture, the production method comprising a step of adding, prior to co-culture of cells, human-derived astrocytes, human-derived neurons, and human-derived microglia to a culture vessel, and a step of co-culturing the astrocytes, the neurons, and the microglia in the culture vessel, in which a proportion of the astrocytes among all cells added to the culture vessel is 30% or more.<2> The production method according to <1>, in which the astrocytes, the neurons, and the microglia are simultaneou