Image-Guided Robotic Cell Microinjection: An Automated High-Throughput Technique for the Precise Delivery of Fluorescently-Labeled Polysaccharides Into Single Neuronal Cells in Organotypic Brain Slice Cultures

Overview

In this video, we demonstrate the robotic microinjection of fluorescently-labeled polysaccharides into pyramidal neurons in organotypic slices of mouse embryonic brain using an autoinjector. The autoinjector is a robotic platform that utilizes images acquired from a microscope for the precise delivery of biomolecules of interest into single cells.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Installation of Software

1. Follow instructions to install the software from https://github.com/bsbrl/Autoinjector.

2. Preparation of Reagents and Pipettes

1. Agarose: Prepare 3% agarose by separately dissolving 3 g of wide range agarose and 3 g low-melting point agarose in 100 mL of cell culture-grade PBS in two separate 200 mL glass bottles, respectively. Store at room temperature for up to 3 months.
2. Tyrode solution: Dissolve 1 g of sodium bicarbonate and Tyrode’s salt (use the content of the entire bottle) and 13 mL of 1 M HEPES in 1 L of distilled water. Adjust the pH to 7.4. Filter the solution through a 0.2 µm bottle-top filter.
3. Slice culture medium (SCM): Add 10 mL of rat serum, 1 mL of 2 mM glutamine, 1 mL penicillin-streptomycin (100x), 1 mL of N-2 supplement (100x), 2 mL of B27 supplement (50x) and 1 mL of HEPES (pH 7.3) buffer into 84 mL of Neurobasal medium. Aliquot 5 mL of SCM into 15 mL tubes. Store at -20 °C.
4. CO₂-Independent Microinjection Medium (CIMM): Prepare 5x DMEM modified low-glucose solution (without phenol red) by dissolving the powder in 200 mL of distilled water. Filter solution through a 0.2 µm bottle-top filter (for the DMEM powder, use the content of the entire bottle). To prepare 100 mL of CIMM, mix 20 mL of 5x DMEM modified solution, 1 mL of HEPES buffer, 1 mL of N2 supplement (100x), 2 mL of B27 supplement (50x), 1 mL of penicillin-streptomycin (100x), 1 mL of 2 mM glutamine and 74 mL of distilled water. Store the solution at 4 °C.
5. Reconstitution buffer: Prepare the reconstitution buffer by dissolving 262 mM NaHCO₃, 0.05 N NaOH, 200 mM HEPES in distilled water. Sterilize the solution by filtration through a bottle-top 0.22 µm filter system into a sterile glass bottle. Aliquot 500 µL of reconstitution buffer into airtight microcentrifuge tubes. Store at 4 °C.
6. Microinjection dye stock: Dissolve the fluorescently labeled Dextran in RNase free distilled water (final concentration 10 µg/µL). Prepare 5 µL aliquots and store at -20 °C until use.
7. Pull the microinjection pipettes from borosilicate glass capillaries (1.2 mm outer diameter, 0.94 mm inner diameter) using the micropipette puller. Protect the pipettes from dust. Do not store pipettes for more than 2–3 days. For this experiment, the pulling parameters were HEAT: ramp temperature +1 – 5; PULL: 100; VEL: 110; DEL: 100. HEAT and VEL are the parameters that affect the most shape and size of the pipette.
   NOTE: The optimal microinjection pipette has a long and flexible tip, to avoid cell damage during microinjection.

3. Tissue Slice Preparation

1. Melt the 3% wide range agarose using a microwave oven prior to the brain tissue dissection. Do not let the agarose solidify by keeping in a water bath at 37 °C prior to embedding. Ensure that the pipettes are protected from dust. Do not store pipettes for more than 2–3 days.
2. Thaw an aliquot of SCM and warm 10–12 mL CIMM and 20 mL of Tyrode’s solution to 37 °C using a water bath.
3. Mix the fluorescent tracer (Dextran-3000 or Dextran-10000-Alexa conjugated; final concentration 5–10 μg/μL) with the other chemical(s) to be injected. Centrifuge the microinjection solution at 16, 000 x g for 30 min at 4 °C. Collect the supernatant and transfer into a new tube. Keep the microinjection solution on ice until use.
4. Use the heads from E13.5–E16.5 mouse embryos to prepare organotypic tissue slices of the telencephalon. Remove the skin and open the skull using the forceps, moving along the midline. Dissect out the embryonic brain from the open skull and remove the meninges covering the brain tissue starting from the ventral side of the brain. Leave the dissected whole brain in Tyrode’s solution on a 37 °C heating block.
   NOTE: All dissection steps described in 3.4 must be performed in prewarmed Tyrode’s solution.
5. Pour the wide range melted agarose into a disposable embedding mold. When the agarose is cooled to 38–39 °C, carefully transfer the brains (a maximum of 4) into it using a Pasteur pipette. Always use cut tips for this step.
6. Stir the agarose around the tissue either using a spatula or a pair of Dumont #1 forceps without touching the tissue. Let the agarose solidify at room temperature. Once the agarose has solidified, trim the excess agarose surrounding the tissue.
7. Fill the buffer tray with PBS. Orient the brain with the rostro-caudal axis of the tissue perpendicular to the tray (use as landmark the olfactory bulbs, representing the rostral-most part of the brain). Cut 250 µm slices using a vibratome.
8. Fill a 3.5 cm Petri dish with 2 mL of prewarmed media. Using a plastic Pasteur pipette, transfer slices (10–15) to this dish. Once done, shift the Petri dish with the slices into the slice culture incubator. Maintain slices at 37 °C in a humidified atmosphere containing 40% O₂ / 5% CO₂ / 55% N₂ until use.

4. Microinjection

1. Turn on the computer, microscope, microscope camera, manipulators, pressure rig, and pressure sensor. Load the application by clicking the file “launchapp.py” in the main folder downloaded from GitHub and specify the device settings in the popup screen (see step 1.1 for install instructions).
2. Create an outward pressure to prevent unwanted clogging before submerging the pipette into the solution. To apply pressure to the pipette, slide the compensation pressure bar to 24–45% and click Set Values. Next, tune the pressure to a sufficient pressure by turning the mechanical pressure valve knob to 1–2 PSI (69–138 mbar) as indicated by the pressure sensor.
3. Transfer the slices to a 3.5 cm Petri dish containing 2 mL of pre-warmed CIMM. Place the slices to be microinjected in the centre of the Petri dish. Transfer the Petri dish to the preheated (37 °C) microinjection stage.
4. Load the microinjection pipette with 1.4–1.6 µL of microinjected solution (from step 3.3) using a long-tip plastic pipette. Insert the microinjection pipette onto the pipette holder.
5. Using the lowest magnification on the microscope, bring the slice into focus and guide the micropipette to this field of view (FOV) so that it is focused on the same plane as the slice target. Switch the output of the microscope to the camera to see the FOV in the application.
6. Click the magnification button in the top left of the interface to initiate device calibration. A window will prompt to select the magnification. Select the 10x magnification, or whatever magnification the lens is set to (e.g., 4x, 10x, 20x, 40x) and press Ok. The software assumes the internal objective lens is 10x (the most common objective lens magnification).
7. Refocus the pipette tip using the micrometric wheel of the microscope and click the pipette tip with the cursor. Next, press the step 1.1 button and press OK in the popup window. The pipette will move in the Y direction. Click the tip of the pipette and press the step 1.2 button. Lastly, enter 45 into the Pipette angle box and press the Set angle.
8. Enter desired parameters into the Automated microinjection controls panel. For microinjection into apical progenitors set the injection distance to 20–40 µm and depth to 10–15 µm. For microinjection into neurons set the injection distance 30–40 µm from the basal side, and depth to 10–30 µm depending upon what is being targeted. Always set speed to 100%. Click Set values.
   NOTE: The approach distance is the distance the pipette pulls out of the tissue before moving to the next injection distance, depth is the depth into tissue the microinjection goes, spacing is the distance along the line between sequential injections, speed is the speed of the pipette in µm/s.
9. Click the Draw edge button and drag the cursor along the desired trajectory in the popup window to define the trajectory of injection. For microinjecting progenitor stem cells, the ventral side of the telencephalon surface is targeted. Bring the pipette to the start of the line and click the tip of the pipette. Click Run trajectory to start microinjecting. Repeat this step for every plane of injection targeted (usually done for 3–4 planes with 40–75 injections per plane).

Materials

Name	Company	Catalog Number	Comments
Chemicals
Agarose, Low Melt	Carl Roth	Cat# 6351.2
Agarose, Wild Range	Sigma-Aldrich	Cat# A2790
Best-CA 221 Glue	Best Klebstoffe GmbH & Co.KG	Cat# CA221-10ml
B-27 Supplement	Thermo Fisher Scientific	Cat# 17504044
Distilled Water
DMEM-F12, CO2 independent (w/o Phenol red)	Sigma-Aldrich	Cat# D2906
DMEM-F12, CO2 independent (with Phenol red)	Sigma-Aldrich	Cat# D8900
HEPES-NAOH, pH 7.2, 1M (HEPES buffer)	Carl Roth	Cat# 9105.3
L-Glutamine, 200 mM	Thermo Fisher Sientific	Cat# 25030024
Mowiol 4-88	Sigma-Aldrich	Cat# 81381
N-2 Supplement	Thermo Fisher Scientific	Cat# 17502048
Neurobasal Medium	Thermo Fisher Scientific	Cat# 21103049
Nuclease-free water	Thermo Fisher Scientific	Cat# AM9937
O2 (40%), CO2 (5%), N2 (55%) Mix, 50 liters
PBS
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	Cat# 15140122
Rat serum	Charles River Laboratories
Sodium bicarbonate (NaHCO3)	Merck	Cat# 106323
Sodium hydroxide (NaOH)	Merck	Cat# 106482
Tyrode’s salt	Sigma	Cat# T2145-10x1L)
Equipment
Borosilicate glass capillaries, 1.2 mm outer diameter x 0.94 mm inner diameter	Sutter Instruments	Cat# BF-120-94-10
Bottle-top filter system, 500 mL	Corning	Cat# 430769
Computer PC
Custom pressure rig		Custom pressure rig
Electronic pressure regulator	Parker Hannifin	Cat# 990-005101-002
Falcon tubes, 15 mL	Corning	Cat# 430791
Falcon tubes, 50 mL	Corning	Cat# 430829
Flaming/ Brown micropipette puller	Sutter Instruments	Cat# P-97
Forceps, Dumont no. 3	Fine Science Tools	Cat# 11231-30
Forceps, Dumont no. 5	Fine Science Tools	Cat# 11255-20
Forceps, Dumont no. 55	Fine Science Tools	Cat# 11252-20
Heating block	Labtech International	Cat # Dri block Digi2
Inverted fluorescence microscope	Zeiss	Cat# Axiovert 200
Light source	Olympus	Cat# Highlight 3100
Manual pressure regulator	McMaster Carr	Cat# 0-60 PSI 41795K3
Microloader Tips	Eppendorf	Cat# 5242956.003
Microcontroller	Arduino	Cat# Arduino Due
Microscope camera Hamamatsu Orca Flash 4.0 V3
Motorized stage XY for microscope
Pasteur pipettes, plastic
Petri dish, 60 x 15 mm	Greiner	Cat# 628102
Petri dish, 35 x 10 mm	Nunc	Cat# 153066
Petri dish, 34 x 14 mm, including Microwell no. 1.5 cover glass	MatTek	Cat# P35G-1.5-14-C
Pipette holder	Warner Instruments	Cat# 64-2354 MP-s12u
Pipette and tips
Puller filament, 3.0-mm square box filament	Sutter Instrument	Cat# FB330B
Slice culture incubation box	MPI-CBG	Cat# custom made
Solenoid valve	Cat# LHDA053321H-A
Tabletop centrifuge	Heraeus	Cat# 5431622
Thermometer
Three-axis Manipulator	Sensapex Inc	Cat# tree-axis uMP
Vibratome	Leica	Cat# VT1000s
Whole-embryo-culture-system incubator	Ikemoto Company	Cat# RKI-10-0310
Waterbath
Software and Algorithms
Arduino	Arduino
Fiji	RRID: SCR_002285
Python	Python Software foundation	Python 2.7.12
ZEN		RRID: SCR_013672