This work was supported by the Australian Research Council Centre of Excellence in Advanced Molecular Imaging (CE140100011) (http://www.imagingcoe.org/). This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector.

1. Preparation of crystals in a high viscous media using glass syringes
<ol>
	<li>Centrifuge the crystal solution gently (~1,000 x g, ~10 min at 22 &#176;C) to form a soft crystal pellet and remove the excess buffer. This will result in a high concentration of crystals in the pellet which can be used for data collection. 
	NOTE: To prevent dilution of the viscous media increase the crystal concentration at this step. Optimize the ratio of viscous media to crystal volume for each sample to obtain a high concentration of crystals whilst maintaining a high viscosity for the media. Centrifuge the crystal solution to form a pellet and remove excess buffer to increase the crystal concentration in the solution, as described in Darmanin et. al.29.</li>
	<li>Set up two 100 &#181;L syringes with a coupler.</li>
	<li>Fasten the coupler to the end of the first syringe and add 28 &#181;L of crystal solution to the top of the syringe. Slowly, insert the plunger into the top of the syringe and gently push the solution down to the end of the coupler tip, removing any air bubbles that form. Do this by following one of the two methods discussed below. 
	NOTE: The volume of crystals to be added to the high viscous media can be varied if it does not significantly change the viscosity of the overall sample.
	<ol>
		<li>First method: Use rapid pressure changes to burst the air bubbles.
		<ol>
			<li>Carefully place one gloved finger on the top of the blunted coupler needle point and (very gently) apply pressure to create a seal. Do not apply too much pressure as this may result in a needle stick injury.</li>
			<li>Now pull the plunger back to draw the solution away from the needle point, this will generate a buildup of pressure in the syringe.</li>
			<li>Quickly release the pressure at the top of the syringe by removing the gloved finger from the blunted needle tip. Be careful, if the sample is too close to the tip when the finger is removed it may spray out resulting in loss of sample. The rapid change in pressure within the syringe will burst the air bubbles. Repeat until all bubbles have been removed.</li>
		</ol>
		</li>
		<li>Second method: Using the &#8216;human centrifuge&#8217; to remove air bubbles.
		<ol>
			<li>Place the syringe in one hand with the needle facing upwards and the plunger wedged between two fingers, so that it cannot move.</li>
			<li>Quickly rotate the arm holding the syringe in one direction 2&#8211;3 times, the resulting centrifugal force on the sample will force any air bubbles out of the syringe. Be careful, if done too slowly sample can be lost.</li>
			<li>Inspect the syringe for air bubbles, if bubbles remain, repeat steps 1.3.2.1 &#8211; 1.3.2.2 until all the air bubbles are removed.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Using a fine spatula, add ~42 &#181;L of high vacuum silicone grease directly to the top of the second syringe. Push the plunger all the way down to the end, removing all the air bubbles and ensuring that there is no air gap in the end of the syringe. 
	NOTE: The precise composition of the silicone grease is not critical as it is acting as an inert carrier. A viscosity value of &#62;10 Pa.s or ratio of approximately 60:40 silicone grease to crystal solution is optimal for injection, however, it is possible this may vary depending on the exact characteristics of the sample. Adjust the volume of crystal solution added to the silicone grease to optimize the crystal concentration in the mixture, as outlined in the discussion. High-viscous media other than silicone grease can be used as long as they are compatible with the sample30,31,32,33.</li>
	<li>Ensure there are no air bubbles in either of the two syringes and attach the syringes together using the coupler. Hold the syringes vertically by griping just the end of the syringe as warming up the syringe due to the heat generated from fingers can affect the sample.</li>
	<li>Mix the sample together by gently depressing the plunger on the crystal solution side so that it mixes into the silicone grease and then push the plunger on the silicone grease side, so that it pushes the sample back towards the crystal solution side. Gently, repeat this process 50 to 100 times so that the sample is mixed thoroughly and looks homogeneous. 
	NOTE: If the crystals are grown directly in highly viscous media (e.g., lipidic cubic phase, LCP), steps 1.1&#8211;1.6 are not required. Protocols for growing crystals within the syringes can be found in Liu et.al.34,35. Follow this protocol up to sample consolidation, step 10, where all the sample is now contained in one syringe and the crystallization buffer is removed. The addition of 7.9MAG is not required for this protocol. Instead, remove all excess liquid from the syringe by gently pushing the plunger down in the sample until no more liquid remains in the syringe.</li>
	<li>Visualize the crystals under an optical microscope either through the syringe or, for best results, extract a small amount (~1 &#181;L) on to a glass slide and place a cover slip on the top.</li>
	<li>Check crystal concentration.
	<ol>
		<li>Determine the crystal concentration in the silicone grease by counting the number of crystals in a specific area using the optical microscope images. For best results, a high density of crystals uniformly dispensed throughout the media is ideal (&#62;106 crystals/mL) in order to obtain a high crystal-hit rate.</li>
		<li>Adjust the crystal concentration in the syringe by changing the ratio of crystals to viscous media in steps 1.3 and 1.4.
		<ol>
			<li>To decrease the crystal concentration, dilute the crystals in the syringe by increasing the amount of silicone grease/HV media at step 1.4 or by diluting the crystal pellet in solution with the crystallization buffer prior to setting up the syringes in step 1.1.</li>
			<li>To increase the crystal concentration, decrease the amount of silicone grease in step 1.4 but be mindful not to reduce the viscosity below 10 Pa.s. 
			NOTE: The crystal concentration reported here was optimized for this specific sample and crystal size. However, the ideal crystal concentration will depend on the crystal size, beam size, the needle Internal Diameter (I.D.), and the incident X-ray flux. This can be determined from the hit rate with an optimal hit rate of ~30% considered as &#39;good&#39;. In practice, the crystal concentration must be optimized for different samples during beam time to obtain the desired hit rate. Start with a high concentration of crystals, 109 crystals/mL. The procedure for adjusting the crystal concentration is given in Liu et.al.35.</li>
		</ol>
		</li>
		<li>The protocol can be paused here until the injector is ready for data collection.
		<ol>
			<li>If the crystals are grown in LCP, then seal&#160;them and let them remain in syringes for up to a few days at room temperature.</li>
			<li>If the crystals have been transferred to a different high viscous media (e.g., silicone grease), then&#160;a stability test of the crystals over time should be carried out prior to the measurement. This will determine how long the crystals are stable in the inert media (ideally &#62; 8 h) and the time limit for data collection. Here, lysozyme crystals were stable for days in silicone grease and did not show signs of dissolving when inspected using an optical microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Move all the sample into one syringe prior to disconnecting the coupler. Disconnect the coupler from the syringe and attach the injection needle. Screw the needle firmly into the base of the syringe and ensure that it is fastened tightly to prevent any leakages. A 108 &#181;m I.D. needle (needle length 13 mm, point style 3) was used for this experiment. Depending on the crystal size and the beam size attach either a 51 &#181;m or 108 &#181;m I.D. needle. 
	NOTE: Pre-testing of the sample stream prior to beam time is required to be able to select the correct injector needle size. Depending on the sample characteristics (i.e., viscosity, charging, buffer composition) and the needle I.D., the samples will flow differently. Therefore, it is recommended to test a variety of needle sizes to produce the most stable stream with the smallest possible I.D. needle to maximize the signal-to-noise ratio during data collection.</li>
	<li>If the injector is already mounted and aligned on the beam line move to section 3 and mount the sample syringe directly onto the injector. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Injector mounting and control
<ol>
	<li>Mount the injector onto the beamline. Remove the cryogenic nozzle on the MX2 beamline and replaced it with the injector. Loosen the clamping screw that holds the cryogenic nozzle and attach it to the bracket located next to the motorized stage.</li>
	<li>Lift the injector up from its trolley by holding the black handle and place it on the motorized stage.
	<ol>
		<li>To fasten the injector to the motorized stage; hand tighten the black clamping screw knob.</li>
	</ol>
	</li>
	<li>Mount an empty syringe onto the injector
	<ol>
		<li>In the injector computer control interface (i.e., EPOS position control software), select Homing Mode under Tools in the software menu. Then select Negative Limit Switch, and Start Homing, let the software run until the screw is retracted sufficiently for the syringe to fit under the screw. If left to run unsupervised the limit switch stops the screw automatically.</li>
		<li>Mount an empty (&#8216;dummy&#8217;) syringe onto the injector by inserting the needle through the slit in the syringe holder. Line up the syringe against the bracket.</li>
		<li>Fasten the syringe with two O-rings.
		<ol>
			<li>Wrap the first O-ring across the mid-section of the syringe attaching it to the hooks on either side of the syringe.</li>
			<li>Loop the second O-ring around the hooks on the upper section of the syringe and then put one part of the O-ring on the top of the glass syringe as shown in the demonstration and&#160;Figure 1B.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Align the injector to X-ray beam
	<ol>
		<li>Move the motorized stage with the injector towards the X-ray interaction point via the beamline control software. This&#160;can be visualized using the&#160;inline camera. The size and position of the beam is denoted by a red cross on the screen and the motorized stage can be moved to align the needle with the X-ray interaction region.</li>
		<li>Adjust the x and y position of the stage to align the needle with the red cross.</li>
		<li>Adjust the z position until the needle tip comes into focus.</li>
		<li>Verify alignment of the needle tip and X-ray beam visually by checking that the syringe tip meets the crosshairs which are visible in the optical microscope image generated by the beamline camera.</li>
		<li>Once the needle tip is aligned move the tip above the cross hairs ~100 &#181;m. This eliminates&#160;x-ray scatter from the needle tip. 
		NOTE: Mounting and aligning the injector on the MX2 beamline at the synchrotron must be carried out by the beamline scientists and can be completed in less than 30 min.</li>
	</ol>
	</li>
</ol>
3. Mounting the sample syringe
<ol>
	<li>Replace the empty syringe with the sample syringe by following step 2.3.</li>
	<li>Place the injector cap on the sample syringe plunger head.</li>
	<li>Move the drive screw to the top of the cap.</li>
	<li>In the injector control software select Velocity Mode.</li>
	<li>Input 3000 rpm (~115 nL/s) as the Setting Value and press Apply Setting Value.</li>
	<li>When the drive screw touches the cap on the syringe plunger head, stop the motor.
	<ol>
		<li>Set the rpm value to zero in the injector control software by changing the Setting Value to &#8216;0&#8217; as the screw approaches the cap.</li>
		<li>Once contact is made, activate the preset value by pressing Apply Setting Value to stop the screw instantly.</li>
	</ol>
	</li>
	<li>Gently wiggle the cap to make sure that it is firmly held in place. 
	NOTE: Whenever a new set value is entered in the Setting Value box in the control software it is only activated after pressing Apply Setting Value.</li>
</ol>
4. Running the injector
<ol>
	<li>After the cap drive screw has made firm contact with the top of the plunger change the rpm Setting Value to 100. This is equivalent to ~ 4 nL/s.</li>
	<li>Visually inspect the needle tip when the sample first comes out or look at the beamline camera image of the needle to observe when the sample starts to extrude from the needle tip.</li>
	<li>Perform a search of the hutch, close the hutch door, and turn on the X-ray beam. From this point the injector must be operated remotely from outside of the hutch.</li>
	<li>Tune the rpm until a stable stream is generated.
	<ol>
		<li>Decrease the rpm Setting Value to 90.</li>
		<li>Repeat step 4.4.1, decreasing the rpm Setting Value in increments of 10, to slow down the stream but still maintain a stable stream. For silicone grease a value of 30 rpm was used. 
		NOTE: The typical rpm range varies between 30 &#8211; 100 rpm (1 &#8211; 4 nL/s) depending on the sample characteristics and X-ray exposure time. In general, the rpm should be tuned to produce the slowest flow rate (to minimize sample consumption) whilst maintaining a stable stream.</li>
	</ol>
	</li>
	<li>Managing a sample stream that does not flow well.
	<ol>
		<li>Keep increasing the rpm Setting Value in increments of 10 until the stream gets straighter. If the stream does not stabilize, even after increasing the rpm Setting Value to the maximum value of 100 rpm try to improve the stability by implementing one of the two methods described below.
		<ol>
			<li>First method: Insert a polystyrene sample catcher underneath the sample stream to help guide the sample. This method works well for highly charged sample streams.</li>
			<li>Second method: Insert a venturi suction funnel to the sample catcher. To supply air to the funnel, connect it to the air outlet tube located in the hutch. This method can aid in directing and stabilizing the sample flow independent of the charge of the stream.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Once a constant and steady stream is achieved, start data collection, and optimize the detector distance as per a normal X-ray crystallography experiment. 
	NOTE: The rpm is converted to flow rate using the supplied conversion table in the Supplementary Information, &#8216;Lipidico Device Calculator&#8217;. Enter the rpm value, needle diameters, and syringe volumes to calculate the flow rate using this calculator.</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+inert+crystal+delivery+medium+for+serial+femtosecond+crystallography.">A novel inert crystal delivery medium for serial femtosecond crystallography.</a> International Union of Crystallography. 2, 421-430 (2015).</li><li>Sugahara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grease+matrix+as+a+versatile+carrier+of+proteins+for+serial+crystallography.">Grease matrix as a versatile carrier of proteins for serial crystallography.</a> Nature Methods. 12 (1), 61-63 (2015).</li><li>Sugahara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil-free+hyaluronic+acid+matrix+for+serial+femtosecond+crystallography.">Oil-free hyaluronic acid matrix for serial femtosecond crystallography.</a> Scientific Reports. 6, 24484 (2016).</li><li>Fromme, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+femtosecond+crystallography+of+soluble+proteins+in+lipidic+cubic+phase.">Serial femtosecond crystallography of soluble proteins in lipidic cubic phase.</a> International Union of Crystallography. 2, 545-551 (2015).</li><li>Ishchenko, A., Cherezov, V., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Delivery+of+Protein+Microcrystals+in+Lipidic+Cubic+Phase+for+Serial+Femtosecond+Crystallography.">Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.</a> Journal of Visualized Experiments. (115), e54463 (2016).</li><li>Liu, W., Ishchenko, A., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+microcrystals+in+lipidic+cubic+phase+for+serial+femtosecond+crystallography.">Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography.</a> Nature Protocols. 9 (9), 2123-2134 (2014).</li><li>Hadian-Jazi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peak-finding+algorithm+based+on+robust+statistical+analysis+in+serial+crystallography.">A peak-finding algorithm based on robust statistical analysis in serial crystallography.</a> Journal of Applied Crystallography. 50, 1705-1715 (2017).</li><li>Kong, F. W., Yuan, L., Zheng, Y. F., Chen, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Liquid+Handling+for+Life+Science:+A+critical+review+of+the+current+state+of+the+art.">Automatic Liquid Handling for Life Science: A critical review of the current state of the art.</a> Journal of Laboratory Automation. 17 (3), 169-185 (2012).</li></ol>

MK works for Kusel designs. Kusel Design, a custom laboratory device developer, was engaged by Dr Peter Bentsen of La Trobe University and Dr Tom Caradoc-Davies of ANSTO and to develop a low-cost device to enable high viscous studies in the MX2 beam line at the Australian Synchrotron. The device was developed in close consultation with Dr. Caradoc-Davies. The authors have no competing financial interests.

An alternative HVI has been developed, ideal for carrying out SX experiments at synchrotron sources. It has two key advantages over existing HVIs. First, it is easy to install on the beamline allowing rapid switching between conventional crystallography and SX, just ~30 minutes is required for installation&#160;and alignment on MX2. Second, the sample syringes used to grow crystals can be directly used as the reservoirs for injection, limiting wastage during sample transfer. The protocol for changing samples has been des...

Serial crystallography (SX) is a technique that was developed initially in the context of X-ray Free Electron Lasers (XFELs)1,2,3,4. Though fixed target approaches can be used for SX5,6,7, typically, injector systems are employed to deliver crystals in a continuous stream to the X-ray beam. Because it combines data from a large number of crystals, SX avoids the need for any crystal alignment during the experiment, and enables data to be collected at room temperature8,9. With the aid of a suitable injector, the crystals are streamed one-by-one into the X-ray interaction area and the resulting diffraction data is collected on an area detector9,10. To date, SX has been successful in solving a number of protein structures1,11,12,13 including crystals too small to measure using conventional crystallography. It has also provided new insights into time-resolved molecular dynamics by exploiting the femtosecond pulse duration of the XFEL. By initiating pump-probe reactions with optical laser sources, in-depth studies have been carried out on photosystem II14,15, photoactive yellow protein16,17, cytochrome C oxidase18, as well as bacteriorhodopsin19,20,21. These studies have probed the electron transfer dynamics that occur following light activation demonstrating the significant potential of serial crystallography for understanding time-resolved biological processes.
Development of serial crystallography is also becoming increasingly prevalent at synchrotron sources9,12,20,22,23,24. Synchrotron based SX allows for large numbers of individual crystals to be measured efficiently at room temperature using an appropriate injector system. This approach is suitable for smaller crystals hence, in addition to requiring a fast frame-rate detector to collect the data, a micro-focused beam is also required. Compared to conventional crystallography, SX does not involve the mounting and alignment of individual crystals in the X-ray beam. Because data from a large number of individual crystals is merged, the radiation dose received by each crystal can be substantially reduced compared to conventional crystallography. Synchrotron SX can also be applied to the study of time-resolved reactions, even down to the millisecond regime, provided a detector with a sufficiently high frame rate is available (e.g., 100 Hz or more). Several serial crystallography experiments have been carried out at the synchrotron using injectors that were initially developed at XFEL sources20,22,23. The two most common types of injector are the Gas Dynamic Virtual Nozzle (GDVN)25 and High Viscous Injector (HVI)9,24,26,27,28. The GDVN is ideal for injecting low viscosity, liquid samples, but requires high flow rates to achieve stable streams, which in turn leads to high sample consumption rates. By contrast, HVI&#8217;s are suitable for high viscosity samples which allows generation of a stable stream at much lower flow rates, leading to much lower sample consumption. The HVI injector, therefore, favors delivery of samples where a viscous carrier is preferable (e.g., lipid-based for membrane proteins) and/or large quantities of sample are not available. SX injectors are generally challenging to use and require extensive training to operate. They also involve lengthy sample transfer protocols, as the sample needs to be loaded into a specialized reservoir, this generally has a high risk associated with it of sample being lost either in the &#8216;dead volume&#8217; or via leakages in the connections. Therefore, it is desirable to optimize the injector design to mitigate any losses prior to the sample reaching the X-ray beam.
Recently, the first SX results were published using Lipidico23&#160;with a lysozyme target,&#160;using an Eiger 16M detector. This injector design limits sample wastage by minimizing the number of steps involved in going from initial crystallization to the transfer of crystals into the injector followed by the delivery of sample to the X-ray beam. This manuscript describes and demonstrates the sample transfer procedure starting from sample preparation, moving on to the injection process, and finally data collection, using the same crystallization vessel. The operation of the injector is also described.

<table>
	<tbody>
		<tr>
			<td>Hen eggwhite lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td>Used to grow crystals for testing the injector and the crystals are transferred into silicon grease. https://www.sigmaaldrich.com/</td>
		</tr>
		<tr>
			<td>High vacuum silicon grease</td>
			<td>Dow Corning</td>
			<td>Z273554-1EA</td>
			<td>Used for testing of injector. https://www.sigmaaldrich.com/</td>
		</tr>
		<tr>
			<td>Injector needle (108 &#181;m ID)</td>
			<td>Hamilton</td>
			<td>part No: 7803-05</td>
			<td>www.hamiltoncompany.com</td>
		</tr>
		<tr>
			<td>Glass gas-tight syringes, 100 &#181;l</td>
			<td>Hamilton</td>
			<td>part no: 7656-01</td>
			<td>Syringes used for sample injection. www.hamiltoncompany.com</td>
		</tr>
		<tr>
			<td>LCP syringe coupler</td>
			<td>Formulatrix</td>
			<td>209526</td>
			<td>Syringe coupler to mix the samples</td>
		</tr>
		<tr>
			<td>Lipidico injector</td>
			<td>La Trobe Univerity/ANSTO</td>
			<td></td>
			<td>This is a specific piece of equipment that can be accessed through La Trobe University / ANSTO Australian Synchrotron Facility</td>
		</tr>
	</tbody>
</table>

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.

Lipidico is an HVI built as an alternative delivery system for use on MX2 (Figure 1). It is ideally suited for SX where crystals are either grown in lipidic cubic phase or transferred to a high viscous inert media.
To demonstrate the injector application silicone grease mixed with lysozyme crystals was used to collect SX data at the MX2 beamline at the Australian synchrotron. To mount the injector on the MX2 beamline the cryogenic nozzle is removed and replaced by...

Watch this Scientific Journal Video about Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron at JoVE.com

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

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.

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

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3.2K Views.  La Trobe University, Melbourne, Australia, 3086. 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.

Video: Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

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.

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 ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and ...

Ceramic Omnidirectional Bioprinting in Cell-Laden Suspensions for the Generation of Bone Analogs

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized matrix. This organization is challenging to replicate due to the heterogeneous environment of bone. Ceramic omnidirectional bioprinting in cell-suspensions (COBICS) is a microgel-based bioprinting technique that uniquely replicates the mineral and cellular structure of bone. COBICS prints complex, biologically relevant constructs without the need for sacrificial support materials or harsh postprocessing steps (e.g., radiation and high-temperature sintering), which are two of the biggest challenges in the additive manufacturing of bone mimetic constructs. This technique is enabled via the freeform extrusion of a novel calcium phosphate-based ink within a gelatin-based microgel suspension. The yield-stress properties of the suspension allow deposition and support the printed bone structure. UV crosslinking and nanoprecipitation then "lock" it in place. The ability to print nanostructured bone-mimetic ceramics within cell-laden biomaterials provides spatiotemporal control over macro- and micro-architecture and facilitates the real-time fabrication of complex bone constructs in clinical settings.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Structurally, bone tissue is an inorganic-organic composite containing metabolically active cells embedded within a hierarchical, highly mineralized ...

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a chemotherapeutic might be functionalized with an antibody to enhance binding to cancer cells (&#34;targeting&#34;) or functionalized with polyethylene glycol to help evade immune cell recognition (&#34;stealth&#34;). Even at a cellular level, optimizing the binding and uptake of a drug carrier is a complex biological design problem. Thus, it is valuable to separate how strongly a new carrier interacts with a cell from the functional efficacy of a carrier&#39;s cargo once delivered to that cell.
To continue the chemotherapeutic example, &#34;how well it binds to a cancer cell&#34; is a separate problem from &#34;how well it kills a cancer cell&#34;. Quantitative in vitro assays for the latter are well established and usually rely on measuring viability. However, most published research on cell-carrier interactions is qualitative or semiquantitative. Generally, these measurements rely on fluorescent labeling of the carrier and, consequently, report interactions with cells in relative or arbitrary units. However, this work can be standardized and be made absolutely quantitative with a small number of characterization experiments. Such absolute quantification is valuable, as it facilitates rational, inter- and intra-class comparisons of various drug delivery systems-nanoparticles, microparticles, viruses, antibody-drug conjugates, engineered therapeutic cells, or extracellular vesicles.
Furthermore, quantification is a prerequisite for subsequent meta-analyses or in silico modeling approaches. In this article, video guides, as well as a decision tree for how to achieve in vitro quantification for carrier drug delivery systems, are presented, which take into account differences in carrier size and labeling modality. Additionally, further considerations for the quantitative assessment of advanced drug delivery systems are discussed. This is intended to serve as a valuable resource to improve rational evaluation and design for the next generation of medicine.

Experimental Quantification of Interactions Between Drug Delivery Systems and Cells In Vitro: A Guide for Preclinical Nanomedicine Evaluation

A workflow is demonstrated for the absolute quantification of drug carrier-cell interactions using flow cytometry to allow better rational evaluation of novel drug delivery systems. This workflow is applicable to drug carriers of any type.

A major component of designing drug delivery systems concerns how to amplify or attenuate interactions with specific cell types. For instance, a ...

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Author Spotlight: A Novel Cell Injection Method with Minimum Invasion  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...