Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

All methods described adhere to animal care guidelines of The University of North Carolina at Chapel Hill.

1. Preparation of micro-injection pipettes

1. Pull glass capillaries using a micropipette puller. For some injections, needle beveling is recommended as it provides an extremely sharp surface devoid of structural impurities. To do this, polish the end of a pulled needle on a beveling wheel at an angle of 45° for 15−20 min.
   NOTE: Exact settings for pulling will vary based on the puller being used. The final inner diameter of the bevel should be between 20−40 µm.
2. Coat the inner and external surfaces of the glass capillary with silicone. First dip the needles in the siliconizing agent to coat the external surface of the needle and then backload the siliconizing agent solution into each micropipette to coat the inner surface.
   NOTE: Coating of the glass capillaries should be done 24 h before the implantation experiment. Coating the glass capillaries with silicone provides a chemically inert surface to the glass. If the capillaries are left untreated, the cell suspension generated in later steps will adhere to the glass and plug the needle. Therefore, coating is necessary and vital to the success of the method.
   CAUTION: The siliconizing agent is a ready-made commercially available mixture of heptane and 1,7-dichloro-1,1,3,3,5,5,7,7-octamethyltetrasiloxane (Table of Materials). It is extremely flammable and acutely toxic. Always handle with proper PPE inside a fume hood.
   1. To backload the needle, load ~5−10 µL of the siliconizing agent into a micro-injection pipette tip (Table of Materials). Place the micro-injection tip in the wide end of the pulled glass capillary and position the tip as far down as possible (close to the glass needle tip). Eject the siliconizing agent while slowly removing the loading pipette in order to minimize air bubbles in the needle.
   2. Leave the siliconizing agent in the glass needle for 10 min, remove by aspirating with a new loading pipette and allow needles dry overnight in a fume hood.
3. The morning of the experiment, rinse the glass capillaries with deionized water following the procedure in step 1.2 and allow to dry for 3−4 h.

2. Preparation of solutions

1. Prepare 5 mL of trypsin neutralizing solution by supplementing 4.2 mL of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F12 (DMEM/F12) with 750 µL of Fetal Bovine Serum (FBS) and 50 µL of Penicillin/Streptomycin. Store at 37 °C until use.
2. Prepare 5 µM labeling dye solution by pipetting 5 µL of 1 mM stock labeling dye (in dimethyl sulfoxide [DMSO], Table of Materials) into 995 µL of Hank's balanced salt solution (HBSS). Vortex for 1 min and store at 37 °C until use.
3. Prepare fresh paraformaldehyde (PFA) by combining 10 mL of 32% PFA stock solution with 62 mL of molecular biology grade water and 8 mL of 10x Dulbecco's phosphate-buffered saline (DPBS). The final concentration is 4% PFA in 1x DPBS.

3. Preparation of host embryos

1. Incubate fertile chicken eggs in a horizontal orientation in a humidified incubator at 38 °C until Hamburger and Hamilton (HH) Stage 19¹⁰.
   NOTE: The stage chosen for manipulation is flexible and entirely dependent upon the aims of each individual experiment.
2. Score the "flat" end of the egg shell along the egg equator using angled forceps to make a small puncture >1 mm in diameter. Insert an 18 G needle with attached 10 mL syringe through the puncture and remove ~5 mL of albumin.
   NOTE: This anatomical location is external to the "air cell" inside the egg and keeps albumin from leaking once the puncture is made. This step is recommended as it "drops" the embryo away from the egg shell, preventing potential damage in the subsequent steps.
3. Apply transparent tape to the top of the egg shell. Score with angled forceps and cut a ~2.5 cm window using curved tenotomy scissors.
4. Inspect and stage the embryo based on criteria established by Hamburger and Hamilton¹⁰ and seal the puncture from step 3.2 with transparent tape.
   NOTE: Here, stage HH 19 embryos are used which have 37−40 somites extending into the tail bud. The puncture should not be sealed until after the window is opened along the top of the egg (step 3.3).
5. Inject ~200 µL of India Ink/HBSS mixture (1:5) beneath the embryo using a 32 G needle with attached 1 mL syringe.
   NOTE: India ink provides visual contrast between the embryo and the yolk beneath. Alternative dyes such as neutral red or commercially available cyan fluorescent protein (CFP) or blue fluorescent protein (BFP) fluorescence filter sets can be used to improve contrast.
6. Add 1 mL of HBSS dropwise onto the embryonic disc and seal windowed shells with paraffin film. Place eggs back in the humidified incubator until ready for injection.

4. Isolation of donor tissue

1. Incubate donor fertile chicken eggs in a humidified incubator at 38 °C until Stage HH 19 (or desired stage).
2. Remove the embryo from the egg and place in a 100 mm x 15 mm petri dish containing sterile HBSS at room temperature (RT).
3. Surgically microdissect atrial donor tissue from each embryo by first isolating the entire embryonic heart from the embryo and then by isolating the atria from the heart using forceps, tenotomy scissors, and microspatula under a stereo dissecting microscope. Pool in a sterile 1.5 mL microcentrifuge tube containing 1 mL of HBSS on ice.
4. Once all donor tissue has been collected, pellet the tissue by centrifugation at 1000 x g for 5 min at 4 °C in a fixed-angle microcentrifuge.

5. Trypsin digestion of donor tissue

1. Resuspend cell pellets in 1 mL of prewarmed 0.05% trypsin-EDTA and incubate at 37 °C for 15 min in a shaking heat block at 300 rpm. Alternatively, use a water bath with periodical agitation of the sample.
2. Pipette the digestion solution up and down to break up any remaining tissue, and pellet as in step 4.4.
3. Resuspend the pellet in 1 mL of the trypsin neutralizing solution and centrifuge as in step 4.4.
4. Resuspend the cells in 400 µL of red fluorescent labeling dye solution and incubate at 37 °C for 20 min in a heat block. Alternatively, use a water bath.
5. Once the labeling reaction is finished, pellet the cells as in step 4.4 and wash with 1 mL of HBSS (number of wash steps can be varied between 1 and 3).
6. Resuspend the labeled, pelleted cells at a concentration of ~50,000 cells/µL, which generally results in a 5−10 µL working volume depending on the total cell yield.
   NOTE: Cell concentrations below ~50,000 cells/µL can result in poor injection efficiency.

6. In-vivo injection

1. Backload the cell suspension into a silicone treated glass capillary pipette following the procedures in step 1.2. Mount the pipette into the pressure microinjector apparatus.
2. Remove host embryos from humidified incubator and place in an egg holder underneath the fluorescent stereo dissecting microscope.
   1. Open the vitelline membrane using sterile fine forceps and make a small incision (~0.5−1.0 mm in length) in the pericardium. Additional manipulation/dissection may be needed depending on target region for injection.
3. Position the microinjector such that the tip of the microinjection needle penetrates the target tissue.
   1. Pressure inject cells, and use the fluorescent label to determine that implanted cells are present in desired tissue. For typical injections, apply single pulses less than 0.5 s in duration ranging from 100-400 hectopascals in pressure.
      NOTE: Pulse length and absolute pressure will vary depending on the number of cells to be injected and can be modified to suit individual needs.
   2. Retract the microinjector apparatus and remove the egg from the holder after pressure injecting.
4. Add 1 mL of warm HBSS dropwise onto the embryo, seal eggs using transparent packing tape, and incubate in the humidified incubator at 38 °C for 24 h post implantation.

7. Isolation and analysis

1. Isolate host embryos in RT HBSS using forceps, tenotomy scissors, and microspatula similar to step 4.3, and fix in 4% PFA overnight at 4 °C with gentle rocking.
2. Wash embryos 3 x 5 min in HBSS at RT with gentle rocking, and store in HBSS at 4 °C for further downstream analysis (microscopy, immunohistochemistry, in-situ hybridization, etc.).