Magnetic Alignment of Microelements Containing Cultured Neuronal Networks for High-Throughput Screening

Abstract

High-throughput screening (HTS) on neurons presents unique difficulties because they are postmitotic, limited in supply, and challenging to harvest from animals or generate from stem cells. These limitations have hindered neurological drug discovery, leaving an unmet need to develop cost-effective technology for HTS using neurons. Traditional screening methods use up to 20,000 neurons per well in 384-well plates. To increase throughput, we use "microraft" arrays, consisting of 1600 square, releasable, paramagnetic, polystyrene microelements (microrafts), each providing a culture surface for 500-700 neurons. These microrafts can be detached from the array and transferred to 384-well plates for HTS; however, they must be centered within wells for automated imaging. Here, we developed a magnet array plate, compatible with HTS fluid-handling systems, to center microrafts within wells. We used finite element analysis to select an effective size of the magnets and confirmed that adjacent magnetic fields do not interfere. We then experimentally tested the plate's centering ability and found a centering efficiency of 100%, compared with 4.35% using a flat magnet. We concluded that microrafts could be centered after settling randomly within the well, overcoming friction, and confirmed these results by centering microrafts containing hippocampal neurons cultured for 8 days.

Keywords: finite element analysis; high-throughput screening; magnetic centering; microfabrication; microraft arrays; primary neurons.

Figure 1

Microraft arrays and three-dimensional modeling and drawings of the magnet array plate. (A) Photograph of microraft array with schematic drawing showing dimensions; (B) MAP2 immuno stained neurons cultured on a single microraft; (C–D) computer-aided design (CAD) drawings of magnet array plate, plan and section views; (E) photograph of magnet array plate; and (F) CAD renderings of the magnet array plate in isometric view.

Figure 2

Results of the magnetic field analysis of a microraft at various heights within a microtiter plate well. The magnetic field peaks in the center and edge in the axial and radial directions of the microwell, respectively. Adjacent magnetic fields do not interfere. (A) Analysis positions of microrafts (in μm) in the axial (Z) direction. Red rectangles represent microrafts. (B) Magnetic field in the axial direction. (C) Magnetic field in the radial direction. (D) Magnetic field decay curves. (E) Dual magnet field in the axial direction. (F) Dual magnet field in the radial direction. For B and C, the red dashed line represents the outside edge of the cylinder magnet. For E and F, the red dashed line represents the center axis of the cylinder magnet.

Figure 3

Magnetic force analysis of microrafts at different axial (Z) and radial positions. (A) Analysis positions of microrafts (in μm) in the axial direction in the center, quarter, and edge of the well, respectively. Rectangles represent microrafts. (B) Magnetic force on microraft in the radial direction. (C) Magnetic force on microraft in the axial (Z) direction.

Figure 4

The magnet array plate effectively centers the microrafts. (A) Percent centering metric in which 100% represents the center of the microwell and 0% represents the microrafts abutted against the microwell. d represents the distance of the microraft from the center of the microwell, and L is the distance between 0% and 100% centering. (B) Centering efficiency in which the red dashed line represents a ≥75% centered value. (C) Representative images of Test 1 centering. (D) Test 1 mean percent centering. (E) Test 1 centering efficiency. (F) Percentage of microrafts flipped on edge. (G) Representative images of Test 2 centering. (H) Test 2 mean percent centering. (I) Overall technique efficiency. For all n = 30, **** = p < 0.0001; scale bar = 500 μm.

Figure 5

Cell viability was not affected by the transfer and centering process. (A) Neurons cultured for 8 days on released and transferred microrafts are immunolabeled for MAP2 (red), a neuron specific marker; scale bar = 250 μm. (B) Merged image of MAP2 immunolabeling, Hoechst (cyan) to label all nuclei, SYTOX (green) to label dead cells, and differential interference contrast. (C) Percent viability results (n = 12 wells). Stain located off of microraft is dead cell debris.