Main protocol

The following protocols in our lab are available for download.

  1. Microglia cell line cultivation
  2. Method of isolating microglial subtypes
  3. Method of isolating single cells from tissue slices with a laser micro-dissecting microscope equipped with specific cellular analysis technology and coordinating reproducing functions

1.Microglia cell line cultivation

There have been some reports on microglial cell lines. They almost express macrophage-like properties, and they also have oncogenes and genetic mutations, which seem to have the defect of making it difficult to control cellular proliferation. However, the microglial cell line that I have established has cells from the primary stage. They proliferate depending on growth factors, they lose their proliferation capability in the absence of such factors and they have a branched morphology (the morphology found in the normal brain). Due to this, these cells have the particular feature that it is possible reproduce in vivo microglial properties under cultivation. Also, since it has been observed that they exhibit highly similar properties to those of multiple microglial subgroups, which can be isolated in primary culture, we consider these properties to be useful in looking into the properties and roles of microglial subgroups in the brain.

We have set up multiple cell lines of mice strain microglia which have different properties, and for rats we have set up one cell line which proliferates well in rats. These cells express various particular properties, and therefore we obtained the following patents.
(The following patents were transferred to ACTGen Co., Ltd. in 2010, and Cosmo Bio started sales from 2011.)

Japanese patents

  1. Registration number: Patent number 3410738 (Japanese patent application H10-538383)
    Name of invention: A carrier for drugs from microglia
  2. Registration number: Patent number 4387108 (Japanese patent application 2003-679)
    Name of invention: A microglial cell line

US patent

  1. Registration number: 6,673,605 (Application number: 09/180,394)
    Name of invention: A microglial cell line and its use

European patent

  1. 1. Registration number: 0949331 (Application number: 98905808.6)
    Designated European countries: CH/LI, DE, FR, GB, IT
    Name of invention: A microglial cell line and its use

This protocol explains the method of cultivation of microglial cell lines which we established.

2.The method of isolation of microglial sub-types

Microglia have subtypes. So far, we have been successful in fractionating primary cells by different properties into at least two subtypes, namely Type I and Type II. For example, Type I and Type II have different strengths of expression of Mac1 (in rats, this applies to OX42), one of them expresses ERMP12, a bone marrow cell differentiation marker, and the other does not, they have different morphological responses to stimuli, they have different dependencies on proliferation factors, they have different cytokine production capabilities and phaogocytosis capabilities, and different surface antigens, and they express different proteins. This protocol explains the method of separating of mixed glial culture into Type I and Type microglia, and it is a part of a paper published in Protocols for Neural Cell Culture 4th eds. Ch 13.

3.Method of isolating single cells from tissue sections with a laser micro-dissecting microscope equipped with specific cellular analysis technology and coordinate reproduction functions

This protocol explains high-resolution tissue and cell imaging with a laser micro-dissecting microscope with specific cellular analysis technology and a coordinate reproducing function, and which is linked with our proprietary coordinate reproducing device, which is used with a high resolution imaging device developed at our laboratory. It also explains the identification of the target cell coordinates based on numerical data from such imaging analysis, and it explains the technology of collecting and separating cells using semi-automatic operation of a micro-dissecting microscope with a coordinate reproducing function using such coordinate data.