This work was supported by NIH UF1NS115817 (G.P.). G.P. is partially supported by NIH grants R01NS072171 and R01NS098231. We would like to thank Patrick Zakrzewki and Mohammed Farhoud from Emit Imaging for the help and support in collecting and visualizing the data on the Xerra Imaging Platform. MSU has a research agreement with Bruker Biospin.

All the authors declare no conflicts of interest.

System Setup: 
The aquarium ecosystem has been developed in a way that both mechanical and biological methods of filtering and oxygenating the water are employed. The filtering elements of the system utilize sock filters, protein skimmers, and regular cleaning to maintain nitrogen and oxygen levels. More importantly, we also rely on marine microorganisms to consume the dangerous nitrogenous compounds and other biological waste as well as aerate the water through processes of photosynthesis. Addition...

New concepts in biomedical research and biomedical engineering are often inspired by identifying specific strategies that biological species possess to address environmental and physiological conditions and challenges. For example, understanding the fluorescence properties in fireflies has led to the development of new fluorescent sensors that can report cellular activity in other model organisms1; identifying ion channels activated by light in algae has led to the development of cellular and temporal specific light-based-neuromodulation2,3,4,5; discovering proteins in glass catfish that navigate according to the Earth&#39;s magnetic field has led to the development of magnetic-based-neuromodulation6,7,8,9,10,11; understanding the siphon reflex in Aplysia has been instrumental to understanding the cellular basis of behavior12,13,14.
Researchers continue to expand on the current bioengineering and phylogenetic toolbox by taking advantage of the unique strengths and novel perspectives on physiological functions that non-conventional lab species hold. Federal agencies are beginning to support these lines of studies by funding novel work performed on diverse species.
One genus of animals with unique anatomy and regeneration capabilities as well as the adaptive control of each of its arms, fascinating biologists and engineers, and captivating audiences from every part of the society is the Octopus17. Indeed, many aspects of the octopus&#39; physiology and behavior have been studied over the past decades15,16,17,18,19,20,21,22,23,24,25,26. However, recent developments in molecular and evolutionary biology, robotics, motion recording, imaging, machine learning, and electrophysiology accelerate discoveries related to octopus physiology and behavior and translate them to innovative bioengineering strategies27,28,29,30,31,32,33,34,35,36,37,38,39.
Here we describe how to set up and maintain octopus husbandry, which would be of interest and relevance to scientists and engineers from different backgrounds, scientific interests, and goals. Nevertheless, our results focus on the application of octopuses in neuroscience and neuroengineering research. The octopus has a highly developed nervous system with 45 million neurons in the central brain, 180 million neurons in the optic lobes, and additional 350 million neurons in the eight axial cords and peripheral ganglia; by comparison, a dog has a similar number of neurons and a cat only half of it40. Unlike the vertebrate nervous system, there are only 32K efferent and 140K afferent fibers connecting the millions of neurons in the octopus&#39; brain to the millions of neurons in each of their arm&#39;s axial cords40,41,42. These relatively few interconnecting fibers suggest that most of the details for the execution of the motor programs are performed in the axial cord itself, emphasizing the uniquely distributed neuronal control the octopuses possess. The octopus&#39;s arms have extraordinary fine motor control enabling them manipulation skills such as opening jar lids, even when they are inside the container. This highly developed prehensile motor capability is unique to the class of Cephalopods (octopus, cuttlefish and squid)43.
Indeed, through hundreds of millions of years of evolution, the octopus has developed a remarkable and sophisticated genome and physiological system43,44 that has inspired new development and progress across scientific and engineering fields. For example, a water-resistant adhesive patch based on the anatomical structure of the octopus&#39; suckers can stick to wet and dry surfaces45; a synthetic camouflaging material inspired by the&#160;octopus&#39; camouflage&#160;skin can transform a flat, 2D surface to a three-dimensional one with bumps and pits46. Miniature soft and autonomous robots (i.e., Octobots) that in the future could serve as surgical tools inside the body47; and an arm (i.e., OctoArm) attached to a tank-like robot48 have also been developed. Many species of octopuses are used in biomedical research e.g., Octopus vulgaris, Octopus sinensis, Octopus variabilis, and Octopus bimaculoides (O. bimaculoides); the&#160;O. vulgaris and O. bimaculoides being the most common34,49,50. The recent sequencing of different octopus genomes makes this genus of particular interest and opens new frontiers in octopus research34,43,51,52.
O. bimaculoides used in our set-up is a medium-sized species of octopus, first discovered in 1949, that can be found in shallow waters off the Northeast Pacific coast from central California to the South of Baja California peninsula17. It can be recognized by the false eyespots on its mantle below its eyes. Compared to&#160;Giant Pacific Octopus (Enteroctopus dofleini) and Common Octopus (O. vulgaris), the California Two-Spot Octopus (O. bimaculoides)&#160;is relatively small in size, starting out smaller than a few centimeters, growing fast as a juvenile. When raised within a laboratory, the adult mantle size can grow to an average size of 100 cm and weigh up to 800 g53,54. Octopuses have a rapid growth period within their first 200 days; by then, they are considered adults and continue to grow throughout the rest of their life55,56,57. Octopuses can be cannibalistic, especially when&#160;both sexes are housed together within a tank; therefore, they need to be housed individually in separate tanks58.

<table><tbody><tr><td>1-3/4 in. Drill Bit</td><td>Home Depot</td><td>204074205</td><td>Glass cutting tool&lt;br/&gt; Part number:1</td></tr><tr><td>1&quot; flow sensors</td><td>Neptune Systems</td><td>Local Dealer</td><td>Pipe with sensor to measure water flow&lt;br/&gt; Part number:2</td></tr><tr><td>1&quot; Slip Bulkhead Strainer</td><td>Bulk Reef Supply</td><td>207113</td><td>Strainer for water leaving tank&lt;br/&gt; Part number:3</td></tr><tr><td>10 gallon tank</td><td>Preuss Pets</td><td>Local Dealer</td><td>Fiddler crab holding tank&lt;br/&gt; Part number:4</td></tr><tr><td>4 inch X 12 inch 200 Micron Nylon Monofiliment Mesh Filter Sock w/ Plastic Ring</td><td>AQUAMAXX</td><td>UJ41171</td><td>Filter for large organic matter in sump&lt;br/&gt; Part number:5</td></tr><tr><td>40 gallon aquarium</td><td>Preuss Pets</td><td>Local Dealer</td><td>4 Food aquarium tanks&lt;br/&gt; Part number:6</td></tr><tr><td>60g poly tanks - rectangle</td><td>Preuss Pets</td><td>Local Dealer</td><td>2 Water Storage (salt and freshwater)&lt;br/&gt; Part number:7</td></tr><tr><td>Active Aqua 1/10th HP Hydroponic or Aquarium Chiller 2018 Model</td><td>WayWe</td><td>719574198463</td><td>For cooling water continuously&lt;br/&gt; Part number:8</td></tr><tr><td>ALAZCO 2 Soft-Grip Handle Heavy-Duty Tile Grout Brush</td><td>ALAZCO</td><td>B06W2FT5V5</td><td>Tank Cleaning&lt;br/&gt; Part number:9</td></tr><tr><td>Ammonia Testing Kit</td><td>Aquarium Pharmaceuticals</td><td>33D</td><td>For water testing&lt;br/&gt; Part number:10</td></tr><tr><td>Apex system WiFi</td><td>Neptune Systems</td><td>Local Dealer</td><td>System connection for off site monitoring&lt;br/&gt; Part number:11</td></tr><tr><td>API Aquarium Test Kit</td><td>Amazon</td><td>B001EUE808</td><td>For water testing&lt;br/&gt; Part number:12</td></tr><tr><td>API Copper Test Kit</td><td>Amazon</td><td>B0006JDWH8</td><td>For water testing&lt;br/&gt; Part number:13</td></tr><tr><td>Aqua Ultraviolet Classic UV 25 Watt Series Units</td><td>Aqua Ultraviolet</td><td>A00028</td><td>For removing bacteria leaving sump system&lt;br/&gt; Part number:14</td></tr><tr><td>AquaClear 50 Foam Filter Inserts, 3 pack</td><td>Aquaclear</td><td>A1394</td><td>Food Tank Carbon Filter Inserts&lt;br/&gt; Part number:15</td></tr><tr><td>Aqueon QuietFlow LED PRO Aquarium Power Filter 30</td><td>Aqueon</td><td>100106082</td><td>Food tank filtering units&lt;br/&gt; Part number:16</td></tr><tr><td>Auto Top Off Kit (ATK) (Each includes 1 FMM module, 2 optical sensors and 1 float)</td><td>Neptune Systems</td><td>Local Dealer</td><td>For freshwater tank&lt;br/&gt; Part number:17</td></tr><tr><td>Automatic top off from RODI (LLC)</td><td>Neptune Systems</td><td>Local Dealer</td><td>From water storage to octopus tanks&lt;br/&gt; Part number:18</td></tr><tr><td>Banded Trochus Snail</td><td>LiveAquaria</td><td>CN-112080</td><td>For algae bin&lt;br/&gt; Part number:19</td></tr><tr><td>Chaetomorpha Algae, Aquacultured</td><td>LiveAquaria</td><td>BVJ-76354</td><td>For algae bin&lt;br/&gt; Part number:20</td></tr><tr><td>Clams - Live, Hard Shell, Cherrystone, Wild, USA Dozen</td><td>Fulton Fish Market</td><td>N/A</td><td>Live food&lt;br/&gt; Part number:21</td></tr><tr><td>Classic Sea Salt Mix - Tropic Marin</td><td>Bulk Reef Supply</td><td>211813</td><td>Salt for tank water&lt;br/&gt; Part number:22</td></tr><tr><td>Clear Masterkleer Soft PVC Plastic Tubing, for Air and Water, 3/4&quot; ID,&amp;nbsp;1&quot; OD</td><td>McMaster</td><td>5233K71</td><td>Cleaning tool&lt;br/&gt; Part number:23</td></tr><tr><td>Continuum Aquablade-P Acrylic Safe Algae Scraper W/ Plastic Blade - 15 Inch</td><td>Marine Depot</td><td>4C31001</td><td>Cleaning tool&lt;br/&gt; Part number:24</td></tr><tr><td>Copper Testing Kit</td><td>Aquarium Pharmaceuticals</td><td>65L</td><td>For water testing&lt;br/&gt; Part number:25</td></tr><tr><td>Curve Refugium CREE LED Aquarium Light</td><td>Eshopps</td><td>6500K</td><td>Algae bin light&lt;br/&gt; Part number:26</td></tr><tr><td>Eheim 1262 return pumps</td><td>EHEIM</td><td>1250219</td><td>Pump for storage tanks&lt;br/&gt; Part number:27</td></tr><tr><td>Eshopps R-100 Refugium Sump GEN 3</td><td>Eshopps</td><td>15000</td><td>Sump system&lt;br/&gt; Part number:28</td></tr><tr><td>Ethyl Alcohol, 200 Proof</td><td>Sigma-Aldrich</td><td>64-17-5</td><td>Anesthesia&lt;br/&gt; Part number:29</td></tr><tr><td>Extech DO600 ExStik II Dissolved Oxygen Meter</td><td>Extech</td><td>DO600</td><td>Oxygen measurement&lt;br/&gt; Part number:30</td></tr><tr><td>Fiddler Crabs; live; dozen</td><td>NORTHEAST BRINE SHRIMP</td><td>N/A</td><td>Live food&lt;br/&gt; Part number:31</td></tr><tr><td>Filter Cartridges</td><td>Aqueon</td><td>100106087</td><td>Food tank filters&lt;br/&gt; Part number:32</td></tr><tr><td>Florida Crushed Coral Dry Sand - CaribSea</td><td>Bulk Reef Supply</td><td>212959</td><td>Sediment for bottom of tank&lt;br/&gt; Part number:33</td></tr><tr><td>FMM module</td><td>Neptune Systems</td><td>Local Dealer</td><td>Controller for apex system&lt;br/&gt; Part number:34</td></tr><tr><td>Fritz-Zyme TurboStart 900 - Fritz</td><td>Bulk Reef Supply</td><td>213036</td><td>Bacteria start&lt;br/&gt; Part number:35</td></tr><tr><td>Hand Operated Drum Pump, Siphon, Basic Pump with Spout, For Container Type Bucket, Pail</td><td>Grainger</td><td>38Y789</td><td>Water Hand Pump&lt;br/&gt; Part number:36</td></tr><tr><td>High pH Testing Kit</td><td>Aquarium Pharmaceuticals</td><td>27</td><td>For water testing&lt;br/&gt; Part number:37</td></tr><tr><td>Imagitarium Fine Mesh Net for Shrimp</td><td>Petco</td><td>2580993</td><td>Shrimp and fish transfer net&lt;br/&gt; Part number:38</td></tr><tr><td>Leak Detection Kit (LDK) - Includes FMM module plus 2 ALD sensors</td><td>Neptune Systems</td><td>Local Dealer</td><td>Placed on floor to detect water&lt;br/&gt; Part number:39</td></tr><tr><td>Lee`S Algae Scrubber Pad Jumbo - Glass</td><td>Marine Depot</td><td>LE12007</td><td>Cleaning tool&lt;br/&gt; Part number:40</td></tr><tr><td>Live rocks</td><td>Preuss Pets</td><td>Local Dealer</td><td>Habitat for octopus&lt;br/&gt; Part number:41</td></tr><tr><td>Long Bottle Cleaning Brush 17&quot; Extra Long</td><td>Haomaomao</td><td>B07FS7J7PN</td><td>Tank Cleaning&lt;br/&gt; Part number:42</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M1028-100ML</td><td>Euthanasia&lt;br/&gt; Part number:43</td></tr><tr><td>Magnetic Probe Rack</td><td>Neptune Systems</td><td>Local Dealer</td><td>For holding apex sensor probes&lt;br/&gt; Part number:44</td></tr><tr><td>Marine Ghost Shrimp</td><td>NORTHEAST BRINE SHRIMP</td><td>N/A</td><td>Live food&lt;br/&gt; Part number:45</td></tr><tr><td>Marineland C-Series Canister Carbon Bags Filter Media, 2 count</td><td>Chewy</td><td>98331</td><td>For elevated copper levels&lt;br/&gt; Part number:46</td></tr><tr><td>Nitra-Zorb Bag</td><td>Aquarium Pharmaceuticals</td><td>AP2213</td><td>Absorbs nitrogen compounds&lt;br/&gt; Part number:47</td></tr><tr><td>Nitrate Testing Kit</td><td>Aquarium Pharmaceuticals</td><td>LR1800</td><td>For water testing&lt;br/&gt; Part number:48</td></tr><tr><td>Nitrite Testing Kit</td><td>Aquarium Pharmaceuticals</td><td>26</td><td>For water testing&lt;br/&gt; Part number:49</td></tr><tr><td>Pawfly 2 Inch Air Stones Cylinder 6 PCS Bubble Diffuser Airstones for Aquarium Fish Tank Pump Blue</td><td>Amazon</td><td>B076S56XWX</td><td>Aerate water&lt;br/&gt; Part number:50</td></tr><tr><td>Penn Plax Airline Tubing for Aquariums &amp;ndash;Clear and Flexible Resists Kinking, 8 Feet Standard</td><td>Amazon</td><td>B0002563MM</td><td>Tubing for connecting air pump to air stone&lt;br/&gt; Part number:51</td></tr><tr><td>Plumbing with unions/valves plus 3/4&quot; flex hose</td><td>Preuss Pets</td><td>Local Dealer</td><td>Water transport&lt;br/&gt; Part number:52</td></tr><tr><td>PM1 module</td><td>Neptune Systems</td><td>Local Dealer</td><td>Power control module for apex&lt;br/&gt; Part number:53</td></tr><tr><td>Protein skimmer</td><td>Reef Octopus</td><td>AC20284</td><td>Removes biowaste from system&lt;br/&gt; Part number:54</td></tr><tr><td>PVC Apex Mounting board, grommets, wire mounts</td><td>Neptune Systems</td><td>Local Dealer</td><td>Helps ensure organization for wires and tubing within system&lt;br/&gt; Part number:55</td></tr><tr><td>PVC Regular Cement and 4-Ounce NSF Purple Primer</td><td>Amazon</td><td>Oatey - 30246</td><td>For connecting PVC pipes&lt;br/&gt; Part number:56</td></tr><tr><td>RODI unit</td><td>Neptune Systems</td><td>Local Dealer</td><td>RO Water&lt;br/&gt; Part number:57</td></tr><tr><td>Salinity Probes</td><td>HANNA probes</td><td>HI98319</td><td>Measures salinity of water&lt;br/&gt; Part number:58</td></tr><tr><td>Seachem Pristine Aquarium Treatment</td><td>Seachem</td><td>1438</td><td>Provides bacteria that break down excess food, waste and detritus&lt;br/&gt; Part number:59</td></tr><tr><td>Seachem Stability Fish Tank Stabilizer</td><td>Seachem</td><td>116012607</td><td>Seachem Stability will rapidly and safely establish the aquarium biofilter in freshwater and marine systems&lt;br/&gt; Part number:60</td></tr><tr><td>Set of lexan tops</td><td>Preuss Pets</td><td>Local Dealer</td><td>Aquarium tank lids&lt;br/&gt; Part number:61</td></tr><tr><td>Set of Various extended length aquabus cables</td><td>Neptune Systems</td><td>Local Dealer</td><td>Cables for Apex system&lt;br/&gt; Part number:62</td></tr><tr><td>SLSON Aquarium Algae Scraper Double Sided Sponge Brush Cleaner Long Handle Fish Tank Scrubber for Glass Aquariums</td><td>Amazon</td><td>B07DC2TZCJ</td><td>Cleaning tool&lt;br/&gt; Part number:63</td></tr><tr><td>Standard-Wall PVC Pipe Fitting for Water, 45 Degree Elbow Adapter, 3/4 Socket Female x 3/4 Socket Male</td><td>McMaster</td><td>4880K189</td><td>PVC pipe&lt;br/&gt; Part number:64</td></tr><tr><td>Standard-Wall&amp;nbsp;PVC Pipe Fitting for Water, 90 Degree&amp;nbsp;Elbow&amp;nbsp;Adapter,&amp;nbsp;1 Socket Female x 1 Socket Male</td><td>McMaster</td><td>4880K773</td><td>PVC pipe&lt;br/&gt; Part number:65</td></tr><tr><td>Standard-Wall&amp;nbsp;PVC Pipe Fitting for Water, Adapter,&amp;nbsp;1&amp;nbsp;Socket-Connect&amp;nbsp;Female x 1 Barbed Male</td><td>McMaster</td><td>4880K415</td><td>PVC pipe&lt;br/&gt; Part number:66</td></tr><tr><td>Standard-Wall&amp;nbsp;PVC Pipe Fitting for Water, Straight&amp;nbsp;Reducer,&amp;nbsp;2 Socket Female x&amp;nbsp;3/4&amp;nbsp;Socket Female</td><td>McMaster</td><td>4880K008</td><td>PVC pipe&lt;br/&gt; Part number:67</td></tr><tr><td>Standard-Wall&amp;nbsp;PVC Pipe Fitting for Water, Tee Connector,&amp;nbsp;White,&amp;nbsp;1 Size&amp;nbsp;Socket-Connect&amp;nbsp;Female</td><td>McMaster</td><td>4880K43</td><td>PVC pipe&lt;br/&gt; Part number:68</td></tr><tr><td>Standard-Wall&amp;nbsp;Unthreaded Rigid PVC Pipe for Water, 1 Pipe Size, 10 Feet Long</td><td>McMaster</td><td>48925K13</td><td>PVC pipe&lt;br/&gt; Part number:69</td></tr><tr><td>Standard-Wall Unthreaded Rigid PVC Pipe for Water, 3/4 Pipe Size, 5 Feet Long</td><td>McMaster</td><td>48925K92</td><td>PVC pipe&lt;br/&gt; Part number:70</td></tr><tr><td>Structural FRP Fiberglass Sheet, 48&quot; Wide x 96&quot; Long, 1/2&quot; Thick</td><td>McMaster</td><td>8537K15</td><td>Table top material&lt;br/&gt; Part number:71</td></tr><tr><td>Structural FRP Fiberglass Square Tube, 10 Feet Long, 2&quot; Wide x 2&quot; High Outside, 1/8&quot; Wall Thickness</td><td>McMaster</td><td>8548K33</td><td>Structural table material&lt;br/&gt; Part number:72</td></tr><tr><td>Tank Sediment</td><td>TopDawg Pet Supply</td><td>8479001207</td><td>Sediment for bottom of fiddler crab tank&lt;br/&gt; Part number:73</td></tr><tr><td>Temperature probe</td><td>Neptune Systems</td><td>Local Dealer</td><td>Temperature probe for tanks&lt;br/&gt; Part number:74</td></tr><tr><td>Tetra TetraMarine Large Saltwater Flakes for all Marine Fish</td><td>Amazon</td><td>B00025K0US</td><td>Fish, shrimp, and crab food&lt;br/&gt; Part number:75</td></tr><tr><td>Tetra Whisper Aquarium Air Pump for 10 gallon Aquariums</td><td>Petco</td><td>2335234</td><td>Air pump for smaller tanks&lt;br/&gt; Part number:76</td></tr><tr><td>Thick-Wall Through-Wall Pipe Fitting, for Water, PVC Connector, 1 Socket-Connect Female</td><td>McMaster</td><td>36895K843</td><td>PVC pipe&lt;br/&gt; Part number:77</td></tr><tr><td>Vectra s2 pump</td><td>Bulk Reef Supply</td><td>212141</td><td>Aquarium Pump&lt;br/&gt; Part number:78</td></tr><tr><td>Water Pump</td><td>TACKLIFE</td><td>GHWP1A</td><td>Pump for cleaning tanks&lt;br/&gt; Part number:79</td></tr><tr><td>Wyze Cam v2 1080p HD Indoor WiFi Smart Home Camera with Night Vision</td><td>Amazon</td><td>B076H3SRXG</td><td>DeepLabCut Recording&lt;br/&gt; Part number:80</td></tr></tbody></table>

establishing an octopus ecosystem for biomedical and bioengineering research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

All the animals in our studies were obtained from the wild, and thus their exact age could not be determined and their stay in the lab was variable. Octopus condition was observed daily. We did not see parasites, bacteria, skin damage, or abnormal behavior. The average weight of animals was 170.38 +/- 77.25 g. Each animal inhabited their own 40-gallon tank. The mean &#177; standard deviation for the parameters recorded for a tank over a week were: pH 8.4 &#177; 0.0, salinity 34.06 &#177; 0.61 ppt, temperature 18.7 &#177;...

Watch this Scientific Journal Video about Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research at JoVE.com

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...

establishing-an-octopus-ecosystem-for-biomedical-bioengineering

Research

JoVE Journal

Bioengineering

2.7K Views.  Michigan State University. Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

Video: Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

BioMEMS: Forging New Collaborations Between Biologists and Engineers

This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons.  This device is compatible with live-cell optical microscopy (DIC and phase contrast), as well as confocal and two photon microscopy approaches.  This method uses precision-molded polymer parts to create miniature multi-compartment cell culture with fluidic isolation. The compartments are made of tiny channels with dimensions that are large enough to culture neurons in well-controlled fluidic microenvironments.  Neurons can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration.

This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons.  This device is compatible with ...

Biology

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact genome. For these reasons, it is considered a useful model organism for marine ecological studies, evolutionary developmental biology, and genomics. As research often requires a steady supply of animal resources, it is useful to establish a reliable, low-maintenance culture system. Here we describe a step-by-step method for establishing an O. dioica culture. We describe how to select potential sampling sites, collection methods, target animal identification, and the set-up of the culturing system. We provide troubleshooting advice based on our own experiences. We also highlight critical factors that help sustain a robust culture system. Although the culture protocol provided here is optimized for O. dioica, we hope our sampling technique and culture setup will inspire new ideas for maintaining other fragile pelagic invertebrates.

Streamlined Sampling and Cultivation of the Pelagic Cosmopolitan Larvacean, Oikopleura dioica

Oikopleura dioica is a tunicate model organism in various fields of biology. We describe sampling methods, species identification, culturing setup, and culturing protocols for the animals and algal feed. We highlight key factors that helped strengthen the culture system and discuss the possible problems and resolutions.

Oikopleura dioica is a planktonic chordate with exceptional filter-feeding ability, rapid generation time, conserved early development, and a compact ...

Environment

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions that include high light levels, rapid water flow, and low nutrient levels. Poor water quality and other anthropogenic stressors have caused coral mortality in recent decades, leading to trophic downgrading and the loss of biological complexity on many reefs. Solutions to reverse the causes of trophic downgrading remain elusive, in part because efforts to restore reefs are often attempted in the same diminished conditions that caused coral mortality in the first place. 
Coral Arks, positively buoyant, midwater structures, are designed to provide improved water quality conditions and supportive cryptic biodiversity for translocated and naturally recruited corals to assemble healthy reef mesocosms for use as long-term research platforms. Autonomous Reef Monitoring Structures (ARMS), passive settlement devices, are used to translocate the cryptic reef biodiversity to the Coral Arks, thereby providing a "boost" to natural recruitment and contributing ecological support to the coral health. We modeled and experimentally tested two designs of Arks to evaluate the drag characteristics of the structures and assess their long-term stability in the midwater based on their response to hydrodynamic forces. 
We then installed two designs of Arks structures at two Caribbean reef sites and measured several water quality metrics associated with the Arks environment over time. At deployment and 6 months after, the Coral Arks displayed enhanced metrics of reef function, including higher flow, light, and dissolved oxygen, higher survival of translocated corals, and reduced sedimentation and microbialization relative to nearby seafloor sites at the same depth. This method provides researchers with an adaptable, long-term platform for building reef communities where local water quality conditions can be adjusted by altering deployment parameters such as the depth and site.

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Moored midwater geodesic structures called Coral Arks provide a modular, scalable, and vertically adjustable research platform that can be used to build, monitor, and perturb coral reef communities in previously inoperative areas, including offshore.

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Immunology and Infection

A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, known as the Aristotle's lantern, is a compelling source of bioinspiration with an intricate network of musculature and calcareous teeth that can scrape, cut, chew food and bore holes into rocky substrates. We describe the bioinspiration process as including animal observation, specimen characterization, device fabrication and mechanism bioexploration. The last step of bioexploration allows for a deeper understanding of the initial biology. The design architecture of the Aristotle's lantern is analyzed with micro-computed tomography and individual teeth are examined with scanning electron microscopy to identify the microstructure. Bioinspired designs are fabricated with a 3D printer, assembled and tested to determine the most efficient lantern opening and closing mechanism. Teeth from the bioinspired lantern design are bioexplored via finite element analysis to explain from a mechanical perspective why keeled tooth structures evolved in the modern sea urchins we observed. This circular approach allows for new conclusions to be drawn from biology and nature.

A protocol for bioinspired design is described for a sampling device based on the jaws of a sea urchin. The bioinspiration process includes observing the sea urchins, characterizing the mouthpiece, 3D printing of the teeth and their assembly, and bioexploring the tooth structure.

Bioinspired design is an emerging field that takes inspiration from nature to develop high-performance materials and devices. The sea urchin mouthpiece, ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

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A Protocol for Bioinspired Design: A Ground Sampler Based on Sea Urchin Jaws

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Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues