The authors would like to thank Dieter H&#228;ussler and Beat Gautschi for their assistance during the experimental set up. We are also grateful to Caroline Hyde for English proof reading.

1. Preparation of sucrose solutions
NOTE: The sucrose solutions are used as cheap, Newtonian model media with elevated viscosity and density for reduced turbulence conditions (see Table 1).
<ol>
	<li>Fill a Duran glass bottle with water and sucrose of different concentrations (20 - 60 %w/w).</li>
	<li>Mix the content with a magnetic stirrer until the sucrose has completely dissolved.
	<ol>
		<li>For sucrose concentrations in excess of 40 %w/w, add the sucrose intermittently and heat the glass bottle slightly (~ 50 &#176;C). Let the sucrose solution cool down to room temperature before use.</li>
	</ol>
	</li>
</ol>
2. Preparation of a measurement recipe and the data logging

<ol>
	<li>After starting the software, initiate the communication with the control unit by selecting the correct serial COM port from the dropdown menu and clicking the Connect button. 
	Note: The Connect button will change the colour to green and the LED below the dropdown menu will switch on, once the communication with the control unit is initiated.</li>
	<li>Set up the data file path inside the bioreactor control unit software in order to store the data on the operator PC.
	<ol>
		<li>Open the Settings tab page and hit the folder symbol next to the Data file location text field.</li>
		<li>In the file dialog window, browse to the desire folder, type a file name into the File name text field and click the OK button. 
		Note: The data log file path and name are displayed in the text box and the DAQ start button is enabled, once a valid file path is defined.</li>
	</ol>
	</li>
	<li>Set up a routine inside the recipe manager of the bioreactor control unit software in order to automate the measurement procedure.
	<ol>
		<li>Open the Recipe tab page and type the desired input values for the recipe phase elapsed time (min) and the corresponding impeller speed (rpm) into the text field boxes. The profile is automatically displayed in the chart. 
		Note: For example, the agitator speed is increased stepwise by 20&#160;rpm from 100&#160;rpm to 300&#160;rpm, and each value is maintained for 4&#160;minutes in order to guarantee a stable torque signal (see discussion below). The minimum and maximum speeds as well as the amount of the increase can be adjusted for different agitators and vessels. 
		Note: Select the speed range carefully with respect to the torque sensor resolution, the nominal torque and vortex formation. The latter often occurs in unbaffled bioreactors agitated at higher speeds and can cause damage to the torque meter.</li>
		<li>Click the Save button, browse to the desired file path and type a file name in the text field. Hit the OK button to save the file.</li>
	</ol>
	</li>
</ol>
3. Installation of the torque sensor
NOTE: The experimental setup is shown schematically in Figure 1.
<ol>
	<li>Install the torque transducer in a specially designed holder that incorporates the air bearing (see Figure 1) using screws to fix the sensor into place. The air bearing used in this study has a porous carbon bushing material with an inner diameter of 13 mm.
	<ol>
		<li>Mount the brushless servo agitator motor onto the top of the holder. Fix the torque transducer to the vertical holder mounting using four screws.</li>
		<li>Connect the motor shaft to the drive shaft of the torque transducer using a metal bellow coupling that can compensate small axial misalignments of the shafts and tighten the coupling using screws. Connect the agitator shaft to the measurement shaft of the torque transducer using another metal bellow coupling. 
		NOTE: In this study, specifically designed impeller shafts with a diameter of 13 mm (tolerance: - 0.0076 mm) and with lengths of between 270 mm and 520 mm were used for the different vessels investigated.</li>
	</ol>
	</li>
	<li>Mount the sensor holder onto the bioreactor head plate and install the impellers on the agitator shaft with the desired off-bottom clearance. Mount baffles and additional installations (e.g. sampling and harvest tubes, electrochemical sensors, etc.) inside the bioreactor if required.</li>
	<li>Install the desired bioreactor in the vessel holder if required (bioreactors #1, #3 to #10) or place the head plate onto the bioreactor tank (bioreactor #2) and tighten the head plate with screws.
	<ol>
		<li>For investigations of glass bioreactors, place the bioreactor glass vessel into the holder.</li>
		<li>For investigations of single-use bioreactors, disassemble the top mounted tubing ports and impeller shaft housing from the plastic head plates by using appropriate cutting tools. Place the plastic vessel into the holder.</li>
	</ol>
	</li>
	<li>Place a temperature sensor inside the bioreactor and connect it to the control unit. Connect the tubing for the pressurized air to the gas inlet port of the air bearing and apply a pressure of around 5.5 bar provided by a compressor. Connect the torque transducer to the A/D converter and power on the transmitter.</li>
</ol>
4. Configurations in the data acquisition software
<ol>
	<li>Open the software for the data acquisition of torque sensor signal and configure the measurement preferences.
	<ol>
		<li>Make sure that the first two channels in the DAQ channels window are initialized and active. In this study, the torque signal was set on channel 0 and the rotational speed signal was set on channel 1.
		<ol>
			<li>Click the Live update button to display the current measurement values.</li>
		</ol>
		</li>
		<li>Set the torque channel signal to zero if the absolute torque signal without rotation is larger than 0.1 mN&#183;m by using the right-mouse click on the channel item in the channel list and selecting the Zero balance option.</li>
		<li>Navigate to the DAQ job tab page and define a data acquisition rate of 2 Hz from the dropdown menu list. Use the options Immediately at job start and Duration from the dropdown lists to set the Start and Stop of the data acquisition, respectively.</li>
		<li>Define a time span for the Sample duration that is longer than the time required to finish the measurement (for example, use 1 h 0 m 30 s for a one hour recipe defined in the second step).</li>
		<li>Navigate to the Data storage settings page and select the option ASCII + channel info from the dropdown list to set the File format for the data save file. Set a file path on the PC hard drive for the measurement Output file.</li>
	</ol>
	</li>
</ol>
5. Perform the torque measurement
<ol>
	<li>Start the data acquisition for the torque signal in the control and data acquisition software for the torque meter by clicking the Start button on the DAQ job menu page.</li>
	<li>Start the data acquisition for the agitator speed and the temperature in the bioreactor control unit software by clicking the DAQ start button on the Settings tab page.</li>
	<li>Start the agitator control in the control unit software with a manual set-point or the pre-defined recipe scheme.
	<ol>
		<li>If a single measurement is conducted, use the control box entry on the Main tab page of the bioreactor control software. Type the desired set-point into the text box and click on the &#8216;Agitator control on&#8217; item.</li>
		<li>If multiple measurements with a recipe are conducted, navigate to the Phases tab page and click the Start button. 
		Note: The software will automatically disable all manual entry boxes for the duration of the recipe and a window opens automatically to confirm the end of the process.</li>
	</ol>
	</li>
	<li>In the data acquisition software, a window opens automatically after the pre-defined measurement duration. Save the data for each measurement on the operator PC, preferably on the hard drive, by clicking the Save data now button.</li>
	<li>Repeat the measurement for each desired agitator speed without and with liquid inside the bioreactor vessel.
	<ol>
		<li>Pour water (or the sucrose solution) through a funnel into the bioreactor. 
		Note: Make sure that the liquid completely covers the impellers since (partially) exposed impellers can result in undesired axial forces that could damage the torque sensor.</li>
	</ol>
	</li>
</ol>
6. Data evaluation
NOTE: The obtained torque values in the empty vessel (dead torque) correspond to the residual friction losses of the bearing and must be subtracted from the values determined in the liquid in order to obtain the effective torque values (see Eq. 1).
<ol>
	<li>Average the torque values for each agitator speed measured after a quasi-stable signal has been achieved (see discussion below). Ideally, calculate the mean value over a period of at least 2 min for each condition, corresponding to 240 data points at a measurement rate of 2 Hz.</li>
	<li>Use a Matlab code for the data processing by running the code from the software command line. 
	NOTE: The code is provided for download in the supplement section of this manuscript. This script imports the raw data file from the data recording, calculates the phase average rotational speed, Reynolds number (from Eq. 3 based on user inputs) and torque values for each of the phases, visualizes the outcomes, and stores the results in a second text file, which then can be used to further process the data.</li>
	<li>Subtract the torque values obtained in the empty vessel from those measured in the liquid to obtain the effective torque values.</li>
	<li>Calculate the power input and dimensionless power number from the time-averaged torque values according to Eq. 1 and Eq. 2.</li>
</ol>

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Eng. Res. Des. 79, 811-818 (2001).</li><li>Werner, S., Greulich, J., Geipel, K., Steingroewer, J., Bley, T., Eibl, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+propagation+of+Helianthus+annuus+suspension+cells+in+orbitally+shaken+bioreactors:+Improved+growth+rate+in+single-use+bag+bioreactors.">Mass propagation of Helianthus annuus suspension cells in orbitally shaken bioreactors: Improved growth rate in single-use bag bioreactors.</a> Eng. Life Sci. 14, 676-684 (2014).</li></ol>

The authors have declared no conflicts of interest.

Despite the importance of the (specific) power input for the engineering characterization and scaling-up/down of bioreactors, only a few publications on experimental investigations in benchtop scale bioreactors, particularly single-use systems in the one-digit liter volume range, can be found in the literature. One reason for this lack of data can be seen in the difficulties of accurate power input measurements in such small scales. In order to overcome some of these difficulties, the present study provides a detailed protocol for torque based power input measurements that are supported by an air bearing to minimize the friction losses in the bearing. The applicability of the method was demonstrated using three commercially available single-use bioreactors as well as multi-use bioreactors in scales between 1 L and 10 L working volume.
Based on our experience with the torque based measurements, the most critical factors to address are: 1) reducing the dead torque by minimizing the friction losses inside the bearings and seals, in particular in laboratory scale bioreactors, and 2) the selection of a suitable torque meter for the desired bioreactor size and agitation conditions. As has been shown earlier35, the dead torque can be dramatically reduced by the use of an air bearing. In the present study, a low cost air bushing made of porous carbon material was used. The residual torque in the empty vessels tested were typically below 0.5 mN&#183;m with agitation rates of up to 900 rpm, corresponding to impeller tip speeds of up to 3 m&#183;s-1. In contrast, the dead torque of the bioreactor #6 with the built-in mechanical shaft bearing was, for example, between 9.4 mN&#183;m and 20 mN&#183;m, and comparable values of around 3 mN&#183;m have been also reported for the bioreactor #732. This is about one order of magnitude higher than the values obtained in the proposed experimental setup.
Besides the air bearing, the torque meter used is the most critical component. A commercially available torque meter that is designed for measuring static and dynamic torque, rotation speed and angle of rotation was selected for this study. Considering the bioreactors of interest with maximum working volumes of 10 L and the corresponding agitators, a nominal torque of 0.2 N&#183;m was chosen. It was found that high reproducibility with relative standard deviation of replicates &#60; 5% and reliable measurements can be obtained for effective torques as low as 2 mN&#183;m, corresponding to only 1 % of the nominal torque. Hence, the measurement range of the sensor applied in the present study was significantly wider than results which have been published based on an inter-laboratory study of members of the German GVC-VDI working group on mixing41.
Nevertheless, the range of the agitator speed should be carefully selected with respect to the torque sensor resolution, the nominal torque and vortex formation. The latter often occurs in unbaffled bioreactors agitated at higher speeds and can cause damage to the torque meter. Both the minimum and maximum feasible agitator speeds can be limiting factors of the method described in this study. In addition to our previous work35, this study involved also the bioreactor #3, the smallest member in glass bioreactor family provided by the manufacturer, which is agitated by two-stage impellers with diameters of 42 mm. A comparable power characteristic to that in the geometrically similar bioreactor #4 was obtained with the presented experimental setup. This is notable since the torque scales with M <img alt="Proportional to" src="/files/ftp_upload/56078/56078pro.jpg" /> d5 for a given liquid density, impeller geometry (i.e. power number) and rotational speed (see Eq. 1 and Eq. 2). Consequently, an approximately 40% lower impeller torque results from a 10% smaller impeller diameter, for example. Nevertheless, higher rotational speeds in the 1 L scale than in the 2 L scale were required during operation to resolve the produced torque with the available torque meter. Due to the built-in baffles of the bioreactor #3, no vortex formation was observed, but this can become an issue with unbaffled vessels. It should be emphasized that the constant offset in the power numbers that was found between the two scales could result from measurement inaccuracies caused by the limited sensor resolution (in addition to geometrical differences). Further investigations are required to draw final conclusions on the minimum scale at which the proposed setup is still feasible.
Nevertheless, the same protocol was used for power input measurements in various glass vessels from different manufacturers with working volumes of between 1 L and 10 L in our laboratory. This highlights the transferability of the used method for the characterization of different bioreactor systems. The experimental effort could be reduced by automated measurements using the recipe management within the automation system provided by the control unit software and the automated data processing based on the universal Matlab language.
Further, it should be noted that, by using the sucrose containing, cheap Newtonian model media, a wide range of Reynolds numbers (100 &#60; Re &#60; 6&#183;104), depending on the agitator and scale, was covered. It should be also emphasized that the lower limit of the turbulence range is usually irrelevant for animal cell cultures with water-like media, even if very low impeller speeds are used. However, significant increases in the broth viscosity, which results in turbulence damping, and even non-Newtonian behavior have been described for fungi- and plant cell-based cultures. For example, apparent viscosities in plant cultures of up to 400-fold compared to water have been reported42, which leads to much lower Reynolds numbers.
Finally, using the bioreactor #7 as a first case study, it has been demonstrated that the proposed experimental setup can be used to study the effect of design modifications on the power input at laboratory scale. In combination with rapid prototyping techniques, this can be a powerful tool for impeller design studies, which will form parts of future work.

Power input is a key engineering parameter for the characterization and scaling-up of bioreactors because it relates to many unit operations, such as homogenization1,2,3, gas-liquid dispersion2,4,5, heat transfer6 and solid suspension7. Power input is associated also with shear stress, which can particularly affect growth and product formation in shear sensitive cell cultures8,9,10,11.
The most common techniques for the measurement of the power input in stirred bioreactors are based on electrical power draw12,13,14, calorimetry12,15 (i.e. stationary heat balance or dynamic heating through the agitation) or the torque upon the agitator. The latter can be experimentally determined by dynamometers, torque meters or strain gauges, which have been applied for a variety of agitators, including single or multi-stage Rushton turbines1,16,17,18,19,20,21,22,23,24,25, pitched blade impellers19,20,23,26,27, InterMig19,21 and Scaba impellers28,29. A detailed review is provided by Ascanio et al. (2004)30.
From the torque (T), the power input (P) can be estimated from Eq. 1, where N is the rotational speed of the agitator.
<img alt="Equation 1" src="/files/ftp_upload/56078/56078eq1.jpg" /> (1)
In order to account for losses occurring in the agitation (in bearings, seals and the motor itself), the effective torque (Teff) should be determined as the difference between the value measured in the empty vessel (TD) and in the liquid (TL). Finally, the dimensionless power number (P0, also known as Newton number), which is defined by Eq. 2 where &#961;L denotes the liquid density and d represents the impeller diameter, can be used to compare different agitators.
<img alt="Equation 2" src="/files/ftp_upload/56078/56078eq2.jpg" /> (2)
It is well-known that the power number is a function of the Reynolds number (i.e. the turbulence) and becomes constant under fully turbulent conditions. The impeller Reynolds number is defined by Eq. 3, where &#951;L is the liquid viscosity.
<img alt="Equation 3" src="/files/ftp_upload/56078/56078eq3.jpg" /> (3)
Nevertheless, power input measurements in small scale bioreactors are still challenging due to the relatively high friction losses inside mechanical bearings of the impeller shafts and the limited accuracy of most commercially available torque meters. Consequently, only a few reports about power input measurements in benchtop scale bioreactors have been published17,18,22,24,31,32. There is also a lack of data about the power input in single-use bioreactors, which are delivered by the manufacturers preassembled, sterilized and ready-to-use33,34. In contrast to their reusable counterparts, most single-use bioreactors are agitated by specially designed impellers, making comparisons difficult.
In order to close this gap, a reliable method for power input measurements with special focus on laboratory scale stirrers has been developed recently35. The torque values measured in the empty vessels, which were caused by the friction losses, were effectively reduced by the use of an air bearing. Consequently, a wide range of operational conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104) could be investigated and the power input of several multi-use and single-use bioreactors has been provided.
The present study provides a detailed measurement protocol of the previously developed method and describes how to set up, conduct and evaluate a torque-based power input measurement in laboratory scale bioreactors. Special focus is on commercially available single- and multi-use systems. An automated measurement procedure is used to reduce the experimental effort.

<table>
	<tbody>
		<tr>
			<td>T20WN torque meter</td>
			<td>HBM Hottinger Baldwin 
			Messtechnik GmbH</td>
			<td></td>
			<td>Nominal torque 0.2 Nm</td>
		</tr>
		<tr>
			<td>Spider-8</td>
			<td>HBM Hottinger Baldwin 
			Messtechnik GmbH</td>
			<td></td>
			<td>HBM Spider8 is no longer available for sale. QuantumX 
			DAQ system (especially the QuantumX modules MX840A and MX440A) are recommended.</td>
		</tr>
		<tr>
			<td>Catman easy software</td>
			<td>HBM Hottinger Baldwin 
			Messtechnik GmbH</td>
			<td></td>
			<td>Version 4.2.2</td>
		</tr>
		<tr>
			<td>Air bearing</td>
			<td>IBS precision engineering</td>
			<td></td>
			<td>13 mm air bushing</td>
		</tr>
		<tr>
			<td>Stainless steel impeller shaft</td>
			<td>Bioengineering AG</td>
			<td></td>
			<td>Shaft tolerance -0.0076 mm</td>
		</tr>
		<tr>
			<td>Brushless motor AKM2</td>
			<td>Kollmorgen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Metal bellow coupling</td>
			<td>Uiker AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Finesse RDPDmini control unit</td>
			<td>Finesse, a part of Thermo Fisher Scientific</td>
			<td></td>
			<td>No longer supported (the replacement product G3Lab universal controller can be used)</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Migros Schweiz AG</td>
			<td></td>
			<td>Food grade</td>
		</tr>
		<tr>
			<td>Matlab software</td>
			<td>Mathworks</td>
			<td></td>
			<td>Version R2017a</td>
		</tr>
		<tr>
			<td>Finesse &#956;TruBio PC software</td>
			<td>Finesse, a part of Thermo Fisher Scientific</td>
			<td></td>
			<td>Version 3.1 (no longer supported)</td>
		</tr>
		<tr>
			<td>SmartGlass 1L</td>
			<td>Finesse, a part of Thermo Fisher Scientific</td>
			<td></td>
			<td>referred to as Bioreactor 1L in Table 2</td>
		</tr>
		<tr>
			<td>SmartGlass 3L</td>
			<td>Finesse, a part of Thermo Fisher Scientific</td>
			<td></td>
			<td>referred to as Bioreactor 3L in Table 2</td>
		</tr>
		<tr>
			<td>SmartVessel 3L</td>
			<td>Finesse, a part of Thermo Fisher Scientific</td>
			<td></td>
			<td>referred to as Single-Use 3L Bioreactor in Table 2</td>
		</tr>
		<tr>
			<td>Mobius CellReady 3L</td>
			<td>Merck Millipore</td>
			<td></td>
			<td>referred to as Cell Ready Single-Use 3L Bioreactor in Table 2</td>
		</tr>
		<tr>
			<td>UniVessel SU 2L</td>
			<td>Sartorius Stedim Biotech</td>
			<td></td>
			<td>referred to as Single-Use 2L Bioreactor in Table 2</td>
		</tr>
	</tbody>
</table>

power input measurements in stirred bioreactors at laboratory scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

Watch this Scientific Journal Video about Power Input Measurements in Stirred Bioreactors at Laboratory Scale at JoVE.com

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

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17.6K 조회수.  Thermo Fisher Scientific. The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

비디오: Power Input Measurements in Stirred Bioreactors at Laboratory Scale

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

높은 처리량 MicroRNA의 프로파일 : 최적 멀티 플렉스 플루 동적 ArrayTM의 IFCs에 Nanoliter의 규모 qRT - PCR

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

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Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

실험실 규모의 합성 거미의 실크 생산

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

에 대한 중공의 생물 반응<em&gt; 생체</em&gt; -처럼 포유류 조직 문화

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

빠른 스캔 전자 스핀 공명 이미징 생리 학적으로 중요 매개 변수에 대한 새로운 길을 엽니 다<em&gt; 생체</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

작은 각도 중성자 회절 계 KWS-2에서 나노 미터와 마이크로 미터 크기에서 넓은 길이 규모 이상 소프트 물질과 생물학적 시스템을 공부

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

간단한 교반 용기를 사용하여 알긴산 염 비즈에서 포유류 세포 캡슐화

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

올레산의 Tripeptide 안정 Nanoemulsion

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

젤라틴 메타크릴 하이드로겔 기반 바이오잉크의 3D 바이오프린팅 프로토콜

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

중국 햄스터 난소 세포 재배에서 고처리량 자동화 마이크로 바이오리액터를 사용한 공정 최적화

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

오스트레일리아 싱크로트론의 직렬 결정학 측정을 위한 지질 주입 프로토콜

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...