A scalable pipeline for designing reconfigurable organisms

Reconfigurable organisms were created using Xenopus embryos as donor tissue under methods approved by the Institutional Animal Care and Use Committee and Tufts University Department of Laboratory Animal Medicine under protocol number M2017-53.

Fertilized X. laevis eggs were reared in a 0.1×, pH 7.8, Marc’s Modified Ringers solution (MMR) using standard protocols and staged according to Nieuwkoop and Faber (32, 33). For shaping experiments, animal caps were manually cut at St. 9 using surgery forceps (Dumont, 11241–30 #4) and transferred to calcium- and magnesium-free medium for 5 min (50.3 mM NaCl, 0.7 mM KCl, 9.2 mM Na2HPO4, 0.9 mM KH2PO4, 2.4 mM NaHCO3, 1.0 mM edetic acid [EDTA], pH 7.3). The outer ectoderm layer was manually removed and discarded, while the inner layer was agitated until fully dissociated (cells are this stage are largely pluripotent, but differentiate into ectoderm without further intervention). Material from five animal caps was pooled and transferred to a welled dish containing 0.75× MMR. After 24 h at 14 °C, the spherical reaggregate was moved to a clean 1% agarose-coated dish containing 10 mL 0.75× MMR and 5 µl gentamicin (ThermoFisher Scientific, 15710072). Forty-eight hours after tissue reaggregation the resulting tissue (now fated to become specific epidermal cell lineages including ionocytes, small secretory cells, and goblet cells), was shaped using a combination of microsurgery forceps and a MC-2010 microcautery instrument with 13-μm wire electrodes (Protech International Inc., MC-2010, 13-Y1 wire tip cautery electrode). Tissue was reshaped as necessary for 3 h to create the desired anatomical outcome, after which it was moved to a clean 1% agarose-coated dish containing 10 mL 0.75× MMR and 5 µl gentamicin and raised at 14 °C.

For contractile movement experiments, cohorts of Xenopus embryos were microinjected with one of two synthetic mRNAs at the four-cell stage using standard protocols (32). mRNA for the fluorescent lineage tracer tdTomato (34) and the multiciliated cell inhibitor Notch ICD (20, 35) was synthesized using mMESSAGE transcription kits (ThermoFisher Scientific, AM1340). Injections were performed in 3% Ficoll solution using a pulled capillary to deliver 370 pg of mRNA for each transcript to all four cells. tdTomato microinjected embryos were reared for at 22 °C while Notch ICD injected embryos were reared at 14 °C. Twenty-four hours after injection, stage 10 Notch ICD injected embryos were moved to a 1% agarose-coated Petri dish containing 0.75× MMR, and animal caps were manually cut using surgery forceps as above. In addition, stage 23–24 tdTomato injected embryos were transferred to the same dish and the presumptive heart field was excised with the outer layer of ectoderm then removed and discarded. Presumptive heart tissue was then placed between two Notch ICD injected animal caps, and the three layers were allowed to heal for 1 h at 22 °C. Following healing, the tissue was moved to clean 1% agarose-coated dish containing 10 mL 0.75× MMR and 5 µl gentamicin and raised at 14 °C. For shaping, resultant tissue was sculpted as above using a combination of microsurgery forceps and a MC-2010 microcautery instrument.

All samples were imaged live in 0.75× MMR at 20 °C using a Nikon SMZ-1500 microscope equipped with both top and substage illumination. Still images were captured on a QImaging Retiga 2000R CCD camera and videos were captured using a Sony IMX234 at a sample rate of 30 frames per second. XY movement tracks were extracted for each run using Noldus Ethovision 14 software, and smoothed using a one-dimensional Gaussian filter (SI Appendix, section S9.1). The tdTomato lineage tracer was imaged using a standard tetramethylrhodamine isothiocyanate (TRITC) filter cube and fluorescent light source to verify cardiac muscle cell location, and GFPIII signal was imaged with a standard fluorescein isothiocyanate (FITC) filter cube to verify epidermal cell location (SI Appendix, section S9.2).

The source code necessary for reproducing the computational results reported in this paper can be found at GitHub (36).

This research was sponsored by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement Number HR0011-18-2-0022, the Lifelong Learning Machines program from DARPA/MTO. The content of the information does not necessarily reflect the position or the policy of the government, and no official endorsement should be inferred. Approved for public release; distribution is unlimited. This research was also supported by the Allen Discovery Center program through The Paul G. Allen Frontiers Group (12171), and M.L. gratefully acknowledges support from the National Science Foundation’s Emergent Behaviors of Integrated Cellular Systems Grant (Subaward CBET-0939511). This research was also supported by the National Science Foundation’s Emerging Frontiers in Research and Innovation (EFRI) Continuum, Compliant, and Configurable Soft Robotics Engineering (C3 SoRo) program (Subaward EFMA-1830870). We thank the Vermont Advanced Computing Core for the provided computational resources.

The source code necessary for reproducing the computational results reported in this paper can be found at GitHub (36).