This study was supported by the State Key Development Programs for Basic Research of China (2021YFC3100502).

The corals used for the present study were Seriatopora caliendrum, which are cultured in our lab. All the corals were kindly provided by the South China Sea Institute of Oceanology, University of Chinese Academy of Sciences.
1. Inspection and startup
NOTE: Each module must be tested for tightness and function individually before assembling the system. Deionized water should be used to test the module&#39;s tightness. Commercial details of all the module components are provided in the Table of Materials.
<ol>
	<li>Tightness test of the inter-module connection
	<ol>
		<li>Connect all the modules and the pumps (Figure 1), and ensure the water flows circularly over the system for at least 30 min.</li>
		<li>Check all the seams for possible leakage issues. If any leaking occurs in any of the bonding seams, apply bonding glue from the outside. If any leaking occurs in any of the connection seams, tighten the connection again, and check whether the sealing gasket needs to be changed.</li>
	</ol>
	</li>
	<li>Loading
	<ol>
		<li>After the tightness test, evacuate and dry the water inside.</li>
		<li>Load the appropriate contents. 
		NOTE: For example, the bacterial culture substrate is loaded into the microbial purification module, and the Chaetomorpha Algae is loaded into the algae purification module.</li>
	</ol>
	</li>
	<li>Assembling and testing the whole system
	<ol>
		<li>After loading, fix the modules onto the baseboard using the screws.</li>
		<li>Connect the inner circulation modules with the outer circulation ones (without the coral culture module).</li>
		<li>For the seawater perfusion, inject the seawater through the water inlet in the seawater store module. When the water level is 3 cm higher than the water inlets of the pumps, switch on the pumps, and continue the injection of the seawater until the inner circulation modules are full of the seawater, with room for air (3 cm high) in the seawater store module. 
		NOTE: The seawater is prepared using pure water and sea salt (see the Table of Materials).</li>
	</ol>
	</li>
	<li>System test
	<ol>
		<li>Turn on all the switches and set both the seawater pump voltages to 9 V. Set the water temperature to 25 &#176;C.</li>
		<li>Set the cycle timer to &#34;1 minute on and 1 minute off&#34;. Set all three channels of the three-channel timer to &#34;9:00 am on&#34; and &#34;5:00 pm off&#34;.</li>
		<li>Monitor the system for at least 24 h for any malfunction. If no problem is found, then the system is ready for the next step of the operation. 
		&#8203;NOTE: It is important to clear all the bubbles in all the modules except for the seawater store module. The whole system can be slightly lifted and shaken to move the bubbles from the module inlet to the outlet.</li>
	</ol>
	</li>
</ol>
2. Establishment of the microbial environment
NOTE: Establishing a coral-friendly microbial environment is necessary before coral transplantation. In order to culture microorganisms in the system, especially in the microbial purification module, the diluted probiotic solution should be added as the microbial source for the nitrification system.
<ol>
	<li>Adding the microbiome source
	<ol>
		<li>Add 1 mL of the commercial microbiome source solution (see the Table of Materials) into 500 mL of seawater with stirring.</li>
		<li>Inject 50 mL of the above diluted solution and 10 &#181;L of the commercial coral nutrition solution (see the Table of Materials) into the circulation system.</li>
	</ol>
	</li>
	<li>Microbiome culture
	<ol>
		<li>Switch on the inner circulation pump and an air pump to culture the microbiome for 21 days. The microbiome oxygen content requirements determine the on-time and off-time proportions of the air pump. 
		&#8203;NOTE: This step aims to culture the seawater purification microbiome and promote coral-beneficial microbiome growth in the system. In this process, the seawater starts to become muddy from the second day to the fourth day after the microbiome injection. After this microbiome culture process, the nutrient degradation capacity in the system should be established. It should be noted that to meet different experimental requirements, different microbiome sources can be used to establish the microbiome environment.</li>
	</ol>
	</li>
</ol>
3. Coral transplantation and growth
<ol>
	<li>Coral transplantation
	<ol>
		<li>Cut the raw coral branches with length scales of 3-5 cm, and then adhere these coral branches onto 3D-printed coral support bases (Supplementary Coding File 1).</li>
		<li>Put these coral branch samples back into the original seawater tank for at least 7 days for recovery.</li>
		<li>Fix the coral support base onto the rotation unit with glue. Assemble the coral culture module, and connect it to the outer circulation loop.</li>
	</ol>
	</li>
	<li>Imaging the coral growth 
	NOTE: The coral images over time need to be acquired to evaluate the coral growth. Using a demountable connection makes it convenient to remove the coral culture module from the whole system for imaging. To this end, a mini-photo studio with appropriate illuminating conditions is built. A camera with a macro lens (see the Table of Materials) is used to capture the coral surface morphologies in different periods. The coral rotation unit in the culture module can be operated outside the module using non-contact mode. By rotating the magnetic handle adjacent to the module, full-angle coral images can be captured.
	<ol>
		<li>Place the camera on the top of the studio, and capture the images from a vertical view.</li>
		<li>Place the coral culture module in the mini-photo studio with the coral positioned in the center and at the bottom.</li>
		<li>Capture the coral images by rotating the outside handle. 
		&#8203;NOTE: For coral survival reasons, the time period of imaging should be limited to 15 min.</li>
	</ol>
	</li>
</ol>
4. System routine maintenance
NOTE: The routine maintenance includes leakage inspection, malfunction inspection, additive addition, and seawater exchange.
<ol>
	<li>Leakage inspection
	<ol>
		<li>Inspect the baseboard for water stains or droplets. As the system&#39;s cover shell is transparent, visually inspecting the water leakage is easy and convenient. This inspection must be performed every day.</li>
	</ol>
	</li>
	<li>Malfunction inspection
	<ol>
		<li>Ensure that this step includes the inspection of the water temperature, pumps, light voltages, air pump status, and timer status, including visually checking and recording the set water temperature, real-time temperature, transformer output voltages, UV lamp settings, and timer working status. This inspection should be performed every day. 
		NOTE: Certain system malfunctions can be diagnosed based on abnormal sounds or unusual temperatures.</li>
	</ol>
	</li>
	<li>Additive addition 
	NOTE: Additive addition is the process of adding nutrients and other reagents into the system.
	<ol>
		<li>For example, extract 10 mL of the seawater using a syringe from the three-way valve between the activated charcoal module and seawater module.</li>
		<li>Dissolve the additives in the extracted seawater.</li>
		<li>Inject the solution back into the system through the three-way valve. In real cases, the types, amounts, and addition frequencies of the additives are decided by the system seawater quality, considering the experimental requirements.</li>
	</ol>
	</li>
	<li>Water exchange 
	NOTE: Routine water exchange can reduce toxic concentration and eutrophication in the culture system. If the experimental conditions allow, exchanging the seawater can be a routine operation.
	<ol>
		<li>Switch off the power for the whole system, and unplug the power cable for safety reasons.</li>
		<li>Remove the coral culture module.</li>
		<li>Connect the outside wastewater pipeline to the outlet in the seawater store module.</li>
		<li>Rotate the system, and place the system front-side down.</li>
		<li>Switch on the outlet. Let the inside seawater flow out of the system. 
		NOTE: Do not use any pump to draw the water out since the inside negative pressure may damage the system.</li>
		<li>Discharge an appropriate amount of seawater, and switch off the outlet. The amount of discharged seawater is decided by the physiological state of the corals.</li>
		<li>Reset the system, and inject the newly prepared seawater into the system through the water inlet.</li>
		<li>Install the coral culture module back into the system.</li>
		<li>Switch on the system power, and wait until the whole system goes back to normal.</li>
	</ol>
	</li>
</ol>
5. Module replacement
NOTE: If any module needs to be replaced due to malfunction or according to the experimental arrangement, it is important to change the module without suspending or negatively affecting the culture experiment.
<ol>
	<li>For the seawater store module, algae purification module, microbial purification module, or activated charcoal purification module, switch off the inner circulation pump, and loosen the fixing screws for the module.</li>
	<li>Disconnect the pipelines between the two joined modules, and disassemble the module to be replaced from the system. The final step is to assemble the new module into the system by connecting the pipelines and retightening the fixing screws. 
	NOTE: The replacement of the temperature control module is somehow different. First, all the wires need to be disconnected from the module. The fixing bolts are then unscrewed, and the pipelines are disconnected. Afterward, the heating panel is dismounted, and the module is disassembled from the system. The installation process for the temperature control module is the reverse process.</li>
</ol>
6. Shutting down the system and restoring the system to its initial state
NOTE: The system will eventually be shut down after the necessary coral culture experiment. The system needs to be restored to its original state.
<ol>
	<li>Shutting down the system
	<ol>
		<li>Switch off the system power, and unplug the power cable.</li>
		<li>Evacuate the seawater inside the system.</li>
		<li>Disassemble the modules in the following order: the coral culture module, the activated charcoal purification module, the seawater store module, the algae purification module, the microbial purification module, the UV lamp, the two circulation pumps, and the temperature control module.</li>
	</ol>
	</li>
	<li>System restoration
	<ol>
		<li>Clean all the modules with pure water and surface active agents (see the Table of Materials).</li>
		<li>Sterilize the modules with 3% hydrogen peroxide solution. 
		&#8203;NOTE: Do not use any organic solvent to wash the modules.</li>
		<li>Dry the modules at 40 &#176;C for 12 h. Ensure that all the water inside the system is evaporated.</li>
		<li>Clean all the pipelines and valves using the same surface active agents.</li>
	</ol>
	</li>
</ol>
7. Modification for the controlled microorganism environment
NOTE: Aside from the coral culture experiment, for certain special experiments, such as acquiring a controlled microorganism environment in the system, the microbiome species and abundance must be strictly controlled. The most innovative feature of our coral culture system is that the coral&#39;s physiological activity can be explored in a specific microbial environment in a relatively closed micro-ecosystem. Performing this function requires a different operating procedure.
<ol>
	<li>Pre-sterilization
	<ol>
		<li>Sterilize all the modules, pipelines, and valves using 3% hydrogen peroxide solution before assembling the system.</li>
		<li>Sterilize the bacterial culture substrate via autoclaving.</li>
		<li>Sterilize Chaetomorpha algae with 75% ethanol solution, and dry it using sterile paper.</li>
	</ol>
	</li>
	<li>System modification and sterilization
	<ol>
		<li>In assembling the system, add an air sterilizing filter (see the Table of Materials) between the air pump and the seawater store module.</li>
		<li>Add a water sterilizing filter between the inlet and the three-way valve. This step ensures the air and water injected into the system are sterilized.</li>
		<li>Introduce ozone into the system to eliminate the remaining microbiome.</li>
		<li>Wash off the remaining disinfectant agents with sterile seawater three times, and inject the sterile seawater into the system.</li>
		<li>For only the establishment of the microbial environment, inject the microbiome source solution through the water outlet. 
		NOTE: Do not use the water inlet to inject the microbiome source. Other reagents and seawater are still injected through the water inlet.</li>
	</ol>
	</li>
</ol>

A Combined Micro-Device System for Promoting and Monitoring Coral Growth

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The authors declare no competing financial interest.

This coral culture system is designed to simulate and provide a relatively natural or customized microenvironment for corals to be transplanted into and survive. Meanwhile, as a self-developed equipment, this system needs to be reliable, user-friendly, and safe. For example, in terms of temperature control, the seawater temperature should be controlled appropriately based on the daily environmental circumstances. The system was tested by culturing the coral for 1 month, confirming the system's reliability.
<p class="...

The deterioration of coral reef ecosystems has been occurring worldwide over the past 70 years. Considering all the major coral areas across Central America1, Southeast Asia2,3,4,5,6, Australia7,8, and East Africa9, the global coverage of coral reefs has halved since the 1950s10. This mass loss of coral reefs has resulted in ecological and economic problems. For example, by tracing the presence/absence and abundance of all kinds of coral-dependent fishes for 8 years, researchers concluded that the coral decline has directly caused a substantial decrease in fish biodiversity and abundance in Papua New Guinea11. This result proved that the coral decline can not only undermine coral reef-based biological systems but also reduce fishery incomes.
Over decades of field surveys, including direct monitoring, remote sensing, and data comparison, the scientific community has identified several factors causing the mass coral decline. One major reason for the mass coral decline is coral bleaching caused by high seawater temperatures12,13. By combining bleaching and meteorological records, scientists have concluded that coral bleaching is happening more frequently in El Ni&#241;o-Southern Oscillation phases14. Another reason for the coral decline is ocean acidification. Owing to the increased CO2 concentration in both the atmosphere and seawater, calcium carbonate dissolves faster than before, causing downscale net coral reef calcification15. Indeed, it has been concluded that when the CO2 concentration in the atmosphere reaches above 500 ppm, tens of millions of people will suffer, and the coral reefs will be at risk of significant deterioration and symbiodinium detachment16,17. There are other factors that can also affect coral survival, such as inshore pollutants causing or accelerating coral decline. Researchers in Hawaii measured the carbon, oxygen, and nitrogen isotopes in corals, along with the dissolved inorganic carbonate and the related nutrients (NH4+, PO43-, NO2&#8722;, and NO3&#8722;), and concluded that pollution from the land magnified the coastal acidification and bioerosion of corals18. Further to pollution, urbanization also endangers coral survival and causes relatively low architectural complexity in corals, as revealed by a study on the coral survival status in Singapore, Jakarta, Hong Kong, and Okinawa. Thus, the impact of anthropogenic stressors and the superimposed effects of climate change are leading to widespread reduced biodiversity on coral reefs and an associated decline in coral ecological function and resilience19.
It should also be noted that a large number of microorganisms participate in the physiological functions of corals, including nitrogen fixation, chitin decomposition, the synthesis of organic compounds, and immunity20, and these microorganisms should, thus, be included when considering coral reef deterioration. In natural environments, such as coral reefs, many factors cause hypoxic or anoxic conditions, including insufficient water circulation, algal exudate, and algal overgrowth. This phenomenon negatively affects the population distributions of coral and coral-related microorganisms. For example, Vietnamese scientists found that in Nha Trang, Phu Quoc, and Ujung Gelam, bacterial composition in the coral Acropora Formosa could be affected by dissolved oxygen at different locations21. Researchers in the United States explored hypoxic or anoxia conditions in corals and found that algal exudates can mediate microbial activity, leading to localized hypoxic conditions, which may cause coral mortality in the direct vicinity. They also found that corals could tolerate reduced oxygen concentrations but only above a given threshold determined by a combination of the exposure time and oxygen concentration22. Researchers in India found that when Noctiluca scintillans algae bloomed, the dissolved oxygen decreased to 2 mg/L. Below this concentration, about 70% of Acropora montiporacan died because of hypoxic conditions23.
All the abovementioned facts and factors suggest that environmental change leads to the deterioration of coral reefs. To culture and study reef corals under certain conditions, it is important to accurately and comprehensively build up a controllable microscopic environment for reef corals to inhabit. Normally, scientists focus on temperature, light, water flow, and nutrients. However, other features, such as the dissolved oxygen concentration, microorganism abundance, and microorganism diversity in the seawater, are commonly ignored. To this end, our group has explored the possibility of applying small equipment to culture coral polyps in a relatively controlled environment24,25. In this work, we designed and built up a modular micro-device system for coral culture. This modular micro-device system can provide a controllable micro-environment in terms of the temperature, light spectrum, dissolved oxygen concentration, nutrients, and microorganisms, etc., and has the capacity for expansion and upgrade.
Modules and functions of the device 
The micro-device system was inspired by the Berlin system26, but no live rocks are used in the current system. As shown in Figure 1, the current system comprises six main modules, two brushless motor pumps, one gas pump, one flow-through UV lamp, one power supply, certain electronic control components, and the related wires and screws. The six main modules include a seawater store module (with an air pump and temperature sensor), a temperature control module, an algae purification module, a microbial purification module, an activated charcoal purification module, and a coral culture module.
Device architecture 
As shown in Figure 2 and Figure 3, the overall micro-device system can horizontally be divided into two compartments with a temperature control module in between. For safety reasons, all the seawater-containing modules and parts are placed in the left compartment, named the culture compartment. The other electronic parts are placed in the right compartment, named the electronic compartment. Both compartments are sealed or packaged within shells. The temperature control module is fixed in a divider plate in between. The shell of the culture compartment includes a baseboard and three screw-fixing plates. This design ensures compartment tightness and facilitates the operation of the system. Additionally, the tightness favors accurate temperature control. The shell of the electronic compartment includes a baseboard, two screw-fixing plates, and one front control panel.
Water circulation 
An inner and outer seawater circulation loop connected to the seawater store module was pre-designed. The inner circulation loop successfully connects the seawater store module, temperature control module, flow-through UV lamp, algae purification module, and microbial purification module. This circulation loop aims to provide suitable physiochemical and physiological seawater conditions for the corals, and no frequent maintenance is needed. The algae purification module contains Chaetomorpha algae, which absorbs the extra nutrients (nitrate and phosphate) in the water. The microbial purification module contains the bacterial culture substrate, which cultivates the microbiome to transfer nitrite and ammonium into nitrate for water purification. All these modules need to be replaced only under critical circumstances.
The outer circulation loop successively connects the seawater store module, coral culture module, and activated charcoal module. This circulation loop aims to provide light, tightness, water current, and high seawater quality to the corals. The seawater can be refreshed through a water inlet and a water outlet. Additives are added through a three-way valve, and the seawater sample can also be extracted from this valve for inspection. Air can be pumped in through an air inlet and discharged from an air outlet.
Electronic design 
A 220 V AC power supply with a switch and a fuse is used for the whole system. The input power is divided into four branches. The first branch goes to a 12 V DC power supply, which directly powers the heating panel, cooling panel, and cooling fan. This branch also indirectly powers two pumps and two lighting panels through a four-channel DC transformer. The second branch goes to a PID temperature controller. The third branch goes to an air pump power supply. The last branch connects to a UV lamp power supply. A solid-state relay connects the PID temperature controller and the cooling panel in the temperature control module. A regular relay is used to connect the PID temperature controller and the heating panel. The four-channel DC transformer converts the voltage to that required.
There are two control panels on the right part of the system. There are four switches and one controller for the UV lamp on the top panel, including a main power switch, a UV lamp power switch, an air pump switch, and a temperature control switch. The main power switch controls the 12 V power supply of the system.
A PID temperature controller, a cycle timer, a four-channel DC transformer, and a three-channel timer are on the front panel. The PID temperature controller adjusts the water temperature by controlling the heating and cooling panels in the temperature control module. The temperature control module only works when the inner circulation pump is working and the water is flowing past the temperature control module. The cycle timer is connected to the air pump power line. Its purpose is to assign the working time period to the air pump. There is a three-channel timer deployed in the electronic compartment too. This timer controls the work time period for the air pump, coral light, and algae light.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12V DC power supply</td>
			<td>Delixi Electric Co., Ltd.</td>
			<td>CDKU-S150W</td>
			<td>12V12.5A</td>
		</tr>
		<tr>
			<td>3% hydrogen peroxide solution</td>
			<td>Shandong ANNJET High tech Disinfection Technology Co., Ltd</td>
			<td>NULL</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>75% ethanol solution</td>
			<td>Shandong ANNJET High tech Disinfection Technology Co., Ltd</td>
			<td>NULL</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Air pump</td>
			<td>Chongyoujia Supply Chain Management Co., Ltd.</td>
			<td>NHY-001</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Air sterilizing filter</td>
			<td>Beijing Capsid Filter Equipment Co., Ltd</td>
			<td>S593CSFTR-0.2H83SH83SN8-A</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>SONY</td>
			<td>&#913;7r4-ILCE-76M4A</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Coral nutrition solution</td>
			<td>Red Sea Aquatics Co., Ltd.</td>
			<td>22101</td>
			<td>Coral nutrition</td>
		</tr>
		<tr>
			<td>Coral pro salt (sea salt)</td>
			<td>Red Sea Aquatics Co., Ltd.</td>
			<td>R11231</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Cycle timer</td>
			<td>Leqing Shangjin Instrument Equipment Co., Ltd.</td>
			<td>CN102A</td>
			<td>220V version</td>
		</tr>
		<tr>
			<td>Double closed quick connector</td>
			<td>JOSOT Co., Ltd</td>
			<td>NL4-2103T</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Flow-through UV lamp</td>
			<td>Zhongshan Xinsheng Electronic technology Co., Ltd.</td>
			<td>211</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Four-channel transformer</td>
			<td>Dongguan Shanggushidai Electronic Technology Co., Ltd</td>
			<td>LM2596</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Macro lens</td>
			<td>SONY</td>
			<td>FE 90mm F2.8 Macro G OSS</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Microbiome source solution</td>
			<td>Guangzhou BIOZYM Microbial Technology Co., Ltd.</td>
			<td>303</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Mini-photo studio</td>
			<td>Shaoxing Shangyu Photography Equipment Factory</td>
			<td>CM-45</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>PID temperature controller</td>
			<td>Guangdong Dongqi&#160; Electric Co., Ltd.</td>
			<td>TE9-SC18W</td>
			<td>SSR version</td>
		</tr>
		<tr>
			<td>Pump (for water)</td>
			<td>Zhongxiang Pump Co., Ltd.</td>
			<td>ZX43D</td>
			<td>Seaswater version</td>
		</tr>
		<tr>
			<td>Pure water machine</td>
			<td>Kemflo&#160;(Nanjing) environmental technology Co, ltd</td>
			<td>kemflo A600</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Solid-state relay</td>
			<td>Delixi Electric Co., Ltd.</td>
			<td>DD25A</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Surface active agents</td>
			<td>Guangzhou Liby Group Co., Ltd.</td>
			<td>Libai detergent</td>
			<td>NULL</td>
		</tr>
		<tr>
			<td>Three-channel timer</td>
			<td>Leqing Changhong Intelligent Technology Co., Ltd.</td>
			<td>CHE325-3</td>
			<td>220V version</td>
		</tr>
		<tr>
			<td>Water sterilizing filter</td>
			<td>Beijing Capsid Filter Equipment Co., Ltd</td>
			<td>S593CSFTR-0.2H83SH83SN8-L</td>
			<td>NULL</td>
		</tr>
	</tbody>
</table>

an integrated micro-device system for coral growth and monitoring

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of the coral culture environment is highly in demand for coral conservation and study. Here, we developed a semi-closed coral culture micro-device system as a multi-functional platform, which can provide accurate and programmable temperature control, a sterile initial environment, long-term stable water quality, an adjustable dissolved oxygen concentration, and a customized light spectrum for corals. Owing to the modular design, the coral culture system can be upgraded or modified by installing desirable new modules or removing existing ones. Currently, under appropriate conditions and with proper system maintenance, the sample corals can survive for at least 30 days in a healthy state. Furthermore, due to the controllable and sterile initial environment, this coral culture system can support research into the symbiotic relationship between corals and associated microorganisms. Therefore, this micro-device system can be applied to monitor and investigate sea corals in a relatively quantitative manner.

Temperature control accuracy 
The system temperature is normally set to 23-28 &#176;C depending on the coral species. However, as one of the most important factors, temperature fluctuation can strongly affect coral survival. Hence, temperature control accuracy is a decisive factor for the coral culture system. A temperature sensor and an independent data collector with a temperature range from 9 &#176;C to 32 &#176;C can be used to test the temperature control accuracy in the coral culture module. W...

Watch this Scientific Journal Video about Advancing Coral Culture â€” Creating a Semi-Quantitatively Controlled Microenvironment System to Counter Current Limitations at JoVE.com

Author Spotlight: Advancing Coral Culture - Creating a Semi-Quantitatively Controlled Microenvironment System to Counter Current Limitations 

This protocol describes the development of a modular controllable micro-device system that can be applied for the long-term culturing and monitoring of sea corals.

An Integrated Micro-Device System for Coral Growth and Monitoring

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of ...

an-integrated-micro-device-system-for-coral-growth-and-monitoring

Research

JoVE Journal

Bioengineering

1.5K Views.  Southeast University. This protocol describes the development of a modular controllable micro-device system that can be applied for the long-term culturing and monitoring of sea corals.

Video: Author Spotlight: Advancing Coral Culture - Creating a Semi-Quantitatively Controlled Microenvironment System to Counter Current Limitations 

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

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 ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...