Members of Takeuchi laboratory at The University of Tokyo including the authors, Prof. S. Takeuchi (the center of the front row), A. Shima (the third from the left in the back row), and S. Nagata (the third from the right in the back row).

Three-dimensional Co-culture of Blood-Brain Barrier-composing Cells in a Culture Insert with a Collagen Vitrigel Membrane

The Takeuchi lab specializes in biohybrid manufacturing, that combines cells with devices made by engineering techniques such as 3D printing and photolithography technology, as well as constructing micrometer- or millimeter-scale three-dimensional tissues. In this paper, we reported the three-dimensional co-culture method for blood-brain barrier (BBB)-composing cells in a culture insert with a collagen vitrigel membrane. BBB prevents toxic substances in the blood from invading into the brain. Therefore, testing BBB permeability is a key for drug development especially for drugs targeting the central nervous system. However, there is currently no reliable human BBB model. We co-cultured three types of BBB-composing human cells (brain endothelial cells (BECs), pericytes, and astrocytes) in layers across the collagen vitrigel membrane, mimicking the in vivo BBB-like layered structure. The unique point of this co-culture method is that the whole tissue including the collagen vitrigel membrane is detachable from the culture insert frame using acetone with the tissue fixed intact, and that the BBB tissue can be used for frozen sections to observe the tissue interior. Vertical sections showed the BECs lining up neatly at the bottom of the tissue. Also, this co-culture method can be applied for transendothelial electrical resistance (TEER) measurements, which is often performed to evaluate the barrier function of BBB model. Drug permeability assays by treating this model with some typical BBB permeable- or impermeable-reagents are planned to be implemented next. We are also exploring the possibility of using this BBB model as a tool for conducting basic research on BBB, for example, by introducing neurons.

Ai Shima, Shogo Nagata, Shoji Takeuchi.  Three-dimensional Co-culture of Blood-Brain Barrier-composing Cells in a Culture Insert with a Collagen Vitrigel Membrane.  In Vitro Cellular & Developmental Biology 56:500-504, 2020.

Pictured are: first row from left to right: Martina Garda (first author), Brett Hale, Naina Rao, Morgan Lowe. Second row from left to right: Megan Bright, Shayn Goodling, and Gregory C. Phillips (corresponding author) 

Soybean Androgenesis I: Identification of Pyramidal Stressors in Anther Cultures that Sustain Cell Divisions and Putative Embryo Fornation from Isolated Microspore Cultures

Androgenesis through anther culture or isolated microspore culture to produce doubled haploids has been challenging to develop among leguminous species.  Recent advances in model systems such as Brassica have identified pyramidal stressors that promote androgenesis which had not yet been evaluated in soybean.  Over a period of 6 years, we identified several stressor variables that appeared to promote soybean androgenesis through anther cultures, raising the frequency of putative gametic responses from approximately 2% up to 10%.  These variables included a 3-day cool-down of donor plants beginning to flower at low temperatures (8-10⁰C) followed by an overnight cold stress at 4⁰C.  Initial incubation conditions included a cool temperature (11⁰C) before increasing to 18⁰C and then 25⁰C.  A nitrogen starvation medium was beneficial during initial incubation.  These conditions were then tested for isolated microspore cultures with formation of putative embryos in >90% of the replicates.  Putative embryos obtained from both anther cultures and isolated microspore cultures arrested at the late globular to early heart stage.  Additional work is in progress to characterize early microspore embryogenesis.  If the developmental arrest of embryos can be overcome, this platform may be useful to develop doubled haploids for soybean.

Martina Garda, Brett Hale, Naina Rao, Morgan Lowe, Megan Bright, Shayn Goodling, Gregory Phillips. Soybean Androgenesis I: Identification of Pyramidal Stressors in Anther Cultures that Sustain Cell Divisions and Putative Embryo Fornation from Isolated Microspore Cultures.  In Vitro Cellular & Developmental Biology-Plant 56:415-429, 2020.