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EP-4737584-A1 - METHOD FOR EVALUATING INTERCELLULAR INTERACTION OF NEUROINFLAMMATION

EP4737584A1EP 4737584 A1EP4737584 A1EP 4737584A1EP-4737584-A1

Abstract

An object of the present invention is to provide a method of evaluating intercellular interactions in neuroinflammation using a co-culture containing human-derived neural cells capable of mimicking human brain functions. According to the present invention, there is provided a method of evaluating intercellular interactions in neuroinflammation, the method including a step of producing a co-culture containing at least two cells selected from the group consisting of human-derived astrocytes, human-derived neurons, human-derived microglia, and human-derived oligodendrocytes, a step of applying an inflammatory stimulation to a first cell contained in the co-culture, a step of detecting at least one selected from the group consisting of an inflammatory response marker in the cells contained in the co-culture, neural activity of the cells, and cell morphology, and a step of evaluating, over time, a change in at least one selected from the group consisting of the inflammatory response marker, the neural activity, and the cell morphology in the first cell and a second cell different from the first cell contained in the co-culture.

Inventors

  • KOBAYASHI HAYATO
  • ENDOH Setsu
  • KATO HIROSHI

Assignees

  • FUJIFILM Corporation

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. A method of evaluating intercellular interactions in neuroinflammation, the method comprising: a step of producing a co-culture containing at least two cells selected from the group consisting of human-derived astrocytes, human-derived neurons, human-derived microglia, and human-derived oligodendrocytes; a step of applying an inflammatory stimulation to first cells contained in the co-culture; a step of detecting at least one selected from the group consisting of an inflammatory response marker in the cells contained in the co-culture, neural activity of the cells, and cell morphology; and a step of evaluating, over time, a change in at least one selected from the group consisting of the inflammatory response marker, the neural activity, and the cell morphology in the first cells and second cells different from the first cells and contained in the co-culture.
  2. The method according to claim 1, wherein the astrocytes, the neurons, the microglia, and the oligodendrocytes are induced to differentiate from human-derived pluripotent stem cells.
  3. The method according to claim 2, wherein the human-derived pluripotent stem cells are human iPS cells.
  4. The method according to any one of claims 1 to 3, wherein the first cells are human-derived microglia, human-derived astrocytes, or human-derived neurons.
  5. The method according to any one of claims 1 to 3, wherein, in a case where the first cells are human-derived astrocytes, the second cells are at least one selected from the group consisting of human-derived neurons and human-derived microglia.
  6. The method according to any one of claims 1 to 3, wherein, in a case where the first cells are human-derived neurons, the second cells are at least one selected from the group consisting of human-derived astrocytes and human-derived microglia.
  7. The method according to any one of claims 1 to 3, wherein, in a case where the first cells are human-derived microglia, the second cells are at least one selected from the group consisting of human-derived astrocytes and human-derived neurons.
  8. The method according to any one of claims 1 to 3, wherein the co-culture is a two-dimensional culture or a three-dimensional culture.
  9. The method according to any one of claims 1 to 3, wherein the inflammatory stimulation is a chemical stimulation.
  10. The method according to claim 9, wherein the chemical stimulation is a chemical stimulation with at least one substance selected from the group consisting of lipopolysaccharide, rotenone, amyloid-β protein, tau protein, α-synuclein, and TDP-43.
  11. The method according to any one of claims 1 to 3, wherein the inflammatory stimulation is a stimulation by adding cells different from the cells in the co-culture or adding a culture supernatant obtained by culturing the cells different from the cells in the co-culture.
  12. The method according to any one of claims 1 to 3, wherein the inflammatory stimulation is induced by aging of the first cells, a genetic mutation in the first cells, or gene introduction into the first cells in the co-culture.
  13. The method according to claim 12, wherein a protein produced by the genetic mutation in the first cells in the co-culture or the gene introduction into the first cells in the co-culture is at least one selected from the group consisting of amyloid-β protein, tau protein, α-synuclein, and TDP-43.
  14. The method according to any one of claims 1 to 3, wherein the inflammatory stimulation that stimulates the first cells is induced by a substance secreted from cells different from the first cells in the co-culture.
  15. The method according to any one of claims 1 to 3, wherein the inflammatory response marker is at least one selected from the group consisting of NF-κB, TNF-α, and STAT1.
  16. The method according to any one of claims 1 to 3, wherein the step of detecting the inflammatory response marker in the cells contained in the co-culture is a step of visualizing the inflammatory response marker protein by an immunostaining method.
  17. The method according to any one of claims 1 to 3, wherein the step of detecting the inflammatory response marker in the cells contained in the co-culture is a step of visualizing an mRNA expression level of the inflammatory response marker gene by a fluorescent in situ hybridization method.
  18. The method according to any one of claims 1 to 3, wherein the neural activity is at least one selected from the group consisting of intracellular calcium flux and action potential of the cells.
  19. The method according to any one of claims 1 to 3, wherein the step of detecting the neural activity in the cells contained in the co-culture is a step of visualizing intracellular calcium flux.
  20. The method according to claim 19, wherein the step of visualizing the intracellular calcium flux is calcium imaging.

Description

Technical Field The present invention relates to a method of evaluating intercellular interactions in neuroinflammation using a co-culture containing human-derived neural cells. Background Art The central nervous system is composed of neurons and glial cells (astrocytes, microglia, and oligodendrocytes), and these cells exert normal brain functions by influencing each other (Non-Patent Document 1). In addition to normal brain functions such as axonal conduction, synaptic transmission, and information processing (Non-Patent Document 2), it has been reported that intercellular interactions are also important in pathological conditions. For example, it has been reported that microglia are activated by aging or inflammation to affect astrocytes, and the astrocytes further damage neurons, thereby being deeply involved in the pathological mechanisms of neurodegenerative diseases (Non-Patent Documents 3 and 4). Therefore, a method capable of evaluating intercellular interactions between neurons and glial cells is very useful for evaluating the original functions of the human brain and for studying pathological mechanisms. With respect to intercellular interactions between neurons and glial cells, there is a report in which mutual influences have been evaluated by using co-culture systems of various combinations (Non-Patent Document 5). In this report, it is reported that the amount of cytokine in the culture medium after lipopolysaccharide (LPS) stimulation changes depending on various combinations of cells, including the three-cell-type co-culture of neurons, astrocytes, and microglia, and it can be inferred from this result that different cells influence each other. However, with this method, it is not possible to evaluate which cells are activated when and with which cells they interact. To study the effect of intercellular interactions on normal brain functions or the effect of intercellular interactions on pathological mechanisms, a method capable of evaluating which cells interact when and with which cells is useful. Patent Document 1 describes results that, in a case where the co-culture system of astrocytes, neurons, and microglia is subjected to LPS stimulation, the astrocytes release C3 in a microglia-dependent manner. Patent Document 2 describes the results of morphological observation by immunostaining and cytokine measurement in the culture medium in a case where the co-culture system of astrocytes, neurons, and microglia is subjected to LPS stimulation. Prior Art Documents Non-Patent Documents Non-Patent Document 1: Francesca Cerbai et al., PLoS One, Vol. 7, Article number e45250, 2012Non-Patent Document 2: R. Douglas Fields et al., Science, Vol. 298, pp. 556-562, 2002Non-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: Sudha R. Guttikonda et al., Nature Neuroscience, Vol. 24, pp. 343-354, 2021 Patent Documents Patent Document 1: JP2022-511385APatent Document 2: WO2023/039567A Summary of Invention Object to be solved by the invention An object to be achieved by the present invention is to provide a method of evaluating intercellular interactions in neuroinflammation using a co-culture containing human-derived neural cells, capable of mimicking human brain functions. Means for solving the object As a result of intensive studies to achieve the above object, the present inventors have found that intercellular interactions in neuroinflammation can be evaluated by applying an inflammatory stimulation to first cells contained in a co-culture containing at least two cells selected from the group consisting of astrocytes, neurons, microglia, and oligodendrocytes, and evaluating a change in an inflammatory response marker in the first cells and second cells over time. 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 method of evaluating intercellular interactions in neuroinflammation, the method comprising: a step of producing a co-culture containing at least two cells selected from the group consisting of human-derived astrocytes, human-derived neurons, human-derived microglia, and human-derived oligodendrocytes;a step of applying an inflammatory stimulation to first cells contained in the co-culture;a step of detecting at least one selected from the group consisting of an inflammatory response marker in the cells contained in the co-culture, neural activity of the cells, and cell morphology; anda step of evaluating, over time, a change in at least one selected from the group consisting of the inflammatory response marker, the neural activity, and the cell morphology in the first cells and second cells different from the first cells and contained in the co-culture.<2> The method acco