Funding from the US National Institutes of Health (R01DK096075, R01DK107875, and R01DK114506) and the US Department of Defense (DoD RTRP W81XWH-17-1-0680) is greatly acknowledged.

The primary hepatocyte isolations for this protocol were performed by the Cell Resource Core (CRC) at Massachusetts General Hospital, Boston, Massachusetts, USA. The animal protocol (#2011N000111) was approved by the Institutional Animal Care and Use Committee (IACUC) of Massachusetts General Hospital.
1. Bulk droplet vitrification
<ol>
	<li>Prepare the vitrification solutions.
	<ol>
		<li>Add 40 mg of bovine serum albumin (BSA) to 17.0 mL of University of Wisconsin solution (UW) to make vitrification solution 1 (V1). Sterile filter the V1 solution using a 0.22 &#181;m filter and store at 4 &#176;C. 
		NOTE: UW is a commonly used organ preservation solution. It contains 50 g/L hydroxyethyl starch, 35.83 g/L lactobionic acid, 3.4 g/L potassium phosphate monobasic, 1.23 g/L magnesium sulfate heptahydrate, 17.83 g/L raffinose pentahydrate, 1.34 g/L adenosine, 0.136 g/L allopurinol, 0.992 g/L total glutathione, 5.61 g/L potassium hydroxide, and sodium hydroxide to adjust the pH to 7.4.</li>
		<li>Combine 7.0 mL of UW, 6.5 mL of DMSO, 6.5 mL of ethylene glycol (EG), 40 mg of BSA, and 5.48 g of sucrose to make vitrification solution 2 (V2). Sterile filter the V2 solution using a 0.22 &#181;m filter. Load 3.5 mL of sterile filtered V2 solution in a 3 mL syringe and store at 4 &#176;C. 
		NOTE: To facilitate dissolving the CPAs, solutions are prepared in larger quantities than required.</li>
	</ol>
	</li>
	<li>Prepare the bulk droplet vitrification apparatus.
	<ol>
		<li>To prepare the mixing needle, first cut along one side of the mixing needle&#8217;s outer gray sleeve, and then bend the sleeve open to remove it from the mixing needle.</li>
		<li>Slide two 25 mm silicone tubing sections over the inlets of the mixing needle and secure their position with glue (Figure 1A).</li>
		<li>Cut the needle to a length of 20 mm (i.e., the length of the inner mixing helix) as shown in Figure 1A. 
		NOTE: The mixing needle length is proportional to the volume inside the needle and therefore can be altered to control the exposure time of the hepatocytes to the high CPA concentration solution.</li>
		<li>Insert the cutoff outlet of the mixing needle into a 20 G injection needle (Figure 1B). 
		NOTE: The injection needle size influences the droplet size.</li>
		<li>Sterilize the mixing needle assembly by autoclaving.</li>
		<li>Fill a sterile Dewar with liquid nitrogen. Sterilize the outside of the Dewar with isopropanol before placing the Dewar in a sterile laminar flow cell culture hood. 
		NOTE: Contamination is an important consideration during direct exposure of the droplets to liquid nitrogen. Although there was no contamination using non-sterilized liquid nitrogen in the current protocol, liquid nitrogen can be filtered or irradiated to assure sterility if needed16.</li>
		<li>Insert a funnel with the same outer diameter as the inner diameter of the liquid nitrogen Dewar into a 50 mL conical tube to create the droplet collection device (Figure 1C&#8722;E).</li>
		<li>Submerge the droplet collection device in the liquid nitrogen by slowly pressing it down in its final vertical position using large forceps. Ensure that the conical tube rests in a vertical position at the bottom of the Dewar (Figure 1D&#8722;E). 
		NOTE: The funnel should stay inserted in and resting on the conical tube. The liquid nitrogen level should be 1 cm below the inlet height of the funnel, as shown in Figure 1D.</li>
		<li>Mount a syringe pump in a vertical position on the wall of the cell culture hood by tying a string from the syringe pump&#8217;s feet to screws protruding from the cell culture hood wall.</li>
		<li>Place the liquid nitrogen Dewar with the droplet collection device under the vertically mounted syringe pump (Figure 1E).</li>
	</ol>
	</li>
	<li>Pre-incubate with CPAs.
	<ol>
		<li>After obtaining freshly isolated rat hepatocytes, count the stock concentration by Trypan blue exclusion and take 40 million viable hepatocytes. 
		NOTE: Gentle handling of hepatocytes in suspension is critical to prevent cell death.</li>
		<li>Centrifuge at 50 x g for 5 min without brake.</li>
		<li>Aspirate the supernatant and resuspend the hepatocytes in 3.4 mL of V1 solution.</li>
		<li>Incubate on ice for 3 min.</li>
		<li>Add 150 &#181;L of DMSO and 150 &#181;L of EG to the hepatocytes and mix gently.</li>
		<li>Incubate on ice for 3 min.</li>
		<li>Again, add 150 &#181;L of DMSO and 150 &#181;L of EG to the hepatocytes and mix gently.</li>
		<li>Load 3.5 mL in a 3 mL syringe.</li>
	</ol>
	</li>
	<li>Perform vitrification.
	<ol>
		<li>Insert the syringe with pre-incubated hepatocytes and the V2 solution syringes onto the syringe pump adapter.</li>
		<li>Attach two female Luer lock hose barb adaptors to the syringes and slide the silicone tubing of the mixing needle assembly over the barb fittings (Figure 1F).</li>
		<li>Place the entire assembly in the syringe pump (Figure 1E).</li>
		<li>Three min after the last addition of DMSO and EG to the hepatocytes, start the pump at 2 mL/min.</li>
		<li>Stop the pump after all hepatocytes have been added to the liquid nitrogen.</li>
	</ol>
	</li>
</ol>
2. Cryogenic storage
<ol>
	<li>Puncture 10 small holes in the lid of a 50 mL conical tube using a 20 G needle and wrap the outside of the lid with a flexible film, as shown in Figure 1B. This creates a valve that enables the escape of evaporating leftover liquid nitrogen.</li>
	<li>To collect the vitrified hepatocyte droplets, first remove the funnel from the liquid nitrogen Dewar. Next, use long forceps to take the conical tube that contains the droplets out of the liquid nitrogen and slowly pour out the excess liquid nitrogen from the conical tube.</li>
	<li>Close the conical tube with the punctured lid and directly place the closed conical tube with vitrified hepatocyte droplets back in the liquid nitrogen Dewar.</li>
	<li>Transfer the conical tube from the liquid nitrogen into a liquid nitrogen vapor cryotank.</li>
</ol>
3. Rewarming of the vitrified hepatocyte droplets
<ol>
	<li>Prepare the rewarming solution.
	<ol>
		<li>Dissolve 17.13 g of sucrose in 100 mL of DMEM hepatocyte culture media to make the rewarming solution. Sterile filter the rewarming solution using a 0.22 &#181;m filter and warm to 37 &#176;C.</li>
	</ol>
	</li>
	<li>Rewarm the hepatocytes.
	<ol>
		<li>Take the conical tube with vitrified hepatocytes from the cryotank and transport it in a liquid nitrogen Dewar to the cell culture hood.</li>
		<li>Lightly loosen the cap and put the conical tube back in the liquid nitrogen.</li>
		<li>Add the vitrified hepatocyte droplets to an empty beaker, instantaneously add the warm (37 &#176;C) rewarming solution, and immediately stir for 10 s. 
		NOTE: It is critical to work quickly to avoid freezing during rewarming. Depending on the study requirements, a few to all vitrified droplets can be rewarmed at once.</li>
	</ol>
	</li>
	<li>Unload the rewarming solution.
	<ol>
		<li>Divide the rewarming solution with hepatocytes over two 50 mL conical tubes.</li>
		<li>Centrifuge the two conical tubes for 2 min at 100 x g at 4 &#176;C without brake.</li>
		<li>Aspirate the supernatant to leave a total volume of 12.5 mL using the conical tube&#8217;s graduation mark as a reference and add 12.5 mL of ice cold DMEM per conical tube to dilute the rewarming solution to 50%.</li>
		<li>Resuspend the hepatocytes and incubate on ice for 3 min.</li>
		<li>Add 25 mL of ice cold DMEM per conical tube to dilute the rewarming solution to 25%.</li>
		<li>Centrifuge for 5 min at 50 x g at 4 &#176;C without brake.</li>
		<li>Aspirate the supernatant and resuspend the hepatocytes in 2 mL of the desired culture medium for use. 
		NOTE: Density gradient centrifugation can be used to remove dead hepatocytes from the cell suspension if desired.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Carpentier, B., Gautier, A., Legallais, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+and+bioartificial+liver+devices:+present+and+future.">Artificial and bioartificial liver devices: present and future.</a> Gut. 58 (12), 1690-1702 (2009).</li><li>Fox, I. J., Chowdhury, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+transplantation:+Hepatocyte+transplantation.">Hepatocyte transplantation: Hepatocyte transplantation.</a> American Journal of Transplantation. 4, 7-13 (2004).</li><li>Hughes, R. D., Mitry, R. R., Dhawan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Status+of+Hepatocyte+Transplantation.">Current Status of Hepatocyte Transplantation.</a> Transplantation. 93 (4), 342-347 (2012).</li><li>St&#233;phenne, X., Najimi, M., Sokal, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+cryopreservation:+is+it+time+to+change+the+strategy.">Hepatocyte cryopreservation: is it time to change the strategy.</a> World Journal of Gastroenterology. 16 (1), 1-14 (2010).</li><li>Terry, C., Dhawan, A., Mitry, R. R., Lehec, S. C., Hughes, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+cryopreservation+and+thawing+protocol+for+human+hepatocytes+for+use+in+cell+transplantation.">Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation.</a> Liver Transplantation. 16 (2), 229-237 (2010).</li><li>Pegg, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+cryopreservation.">Principles of cryopreservation.</a> Methods in Molecular Biology. 368, 39 (2007).</li><li>Baust, J. G., Gao, D., Baust, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation:+An+emerging+paradigm+change.">Cryopreservation: An emerging paradigm change.</a> Organogenesis. 5 (3), 90-96 (2009).</li><li>Hopkins, J. B., Badeau, R., Warkentin, M., Thorne, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+common+cryoprotectants+on+critical+warming+rates+and+ice+formation+in+aqueous+solutions.">Effect of common cryoprotectants on critical warming rates and ice formation in aqueous solutions.</a> Cryobiology. 65 (3), 169-178 (2012).</li><li>Fahy, G. M., MacFarlane, D. R., Angell, C. A., Meryman, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrification+as+an+approach+to+cryopreservation.">Vitrification as an approach to cryopreservation.</a> Cryobiology. 21 (4), 407-426 (1984).</li><li>Demirci, U., Montesano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+encapsulating+droplet+vitrification.">Cell encapsulating droplet vitrification.</a> Lab on a Chip. 7 (11), 1428 (2007).</li><li>El Assal, 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=Bio-Inspired+Cryo-Ink+Preserves+Red+Blood+Cell+Phenotype+and+Function+During+Nanoliter+Vitrification.">Bio-Inspired Cryo-Ink Preserves Red Blood Cell Phenotype and Function During Nanoliter Vitrification.</a> Advanced Materials. 26 (33), 5815-5822 (2014).</li><li>Dou, R., Saunders, R. E., Mohamet, L., Ward, C. M., Derby, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+cryopreservation+of+cells+by+rapid+freezing+of+sub-&#956;l+drops+using+inkjet+printing--cryoprinting.">High throughput cryopreservation of cells by rapid freezing of sub-&#956;l drops using inkjet printing--cryoprinting.</a> Lab on a Chip. 15 (17), 3503-3513 (2015).</li><li>Samot, J., 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=Blood+banking+in+living+droplets.">Blood banking in living droplets.</a> PloS One. 6 (3), 17530 (2011).</li><li>Shi, 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=High-Throughput+Non-Contact+Vitrification+of+Cell-Laden+Droplets+Based+on+Cell+Printing.">High-Throughput Non-Contact Vitrification of Cell-Laden Droplets Based on Cell Printing.</a> Scientific Reports. 5 (1), (2016).</li><li>de Vries, R. J., 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=Bulk+Droplet+Vitrification:+An+Approach+to+Improve+Large-Scale+Hepatocyte+Cryopreservation+Outcome.">Bulk Droplet Vitrification: An Approach to Improve Large-Scale Hepatocyte Cryopreservation Outcome.</a> Langmuir. , (2019).</li><li>Parmegiani, L., 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=Sterilization+of+liquid+nitrogen+with+ultraviolet+irradiation+for+safe+vitrification+of+human+oocytes+or+embryos.">Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos.</a> Fertility and Sterility. 94 (4), 1525-1528 (2010).</li><li>Best, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoprotectant+Toxicity:+Facts,+Issues,+and+Questions.">Cryoprotectant Toxicity: Facts, Issues, and Questions.</a> Rejuvenation Research. 18 (5), 422-436 (2015).</li></ol>

The authors declare competing financial interests. Drs. de Vries, Weng, Toner, Tessier, and Uygun have provisional patent applications relevant to this study. Dr. Uygun has a financial interest in Organ Solutions, a company focused on developing organ preservation technology. All researcher&#8217;s interests are managed by the MGH and Partners HealthCare in accordance with their conflict of interest policies.

Cryopreservation of hepatocytes by slow-freezing results in reduced viability and metabolic function. Vitrification offers a promising alternative for classic cryopreservation, as freezing injury is completely avoided9. However, pre-incubation with CPAs is required to lower the critical cooling rate8. Consequently, vitrification is hampered by CPA toxicity17 and limited to small sample volumes. In efforts to overcome these limitations the bulk drople...

Loss of cell viability and metabolic function after cryopreservation of hepatocytes is still a major issue that limits clinical applications1,2,3. The benchmark method of hepatocyte cryopreservation is slow-freezing, which is performed by pre-incubating the hepatocytes with CPA (dimethyl sulfoxide [DMSO], glucose, and albumin) and subsequent controlled rate freezing (at approximately 1 &#176;C/min) to cryogenic temperatures4,5. Despite many reported optimizations, CPA toxicity together with injurious osmotic imbalances during freezing and mechanical stress of ice formation remain fundamental drawbacks of slow-freezing6,7.
Vitrification offers an advantage over slow-freezing in that injury due to ice formation is completely avoided by a direct phase transition of water into the glass state6. However, to reach the glass transition temperature of pure water (-137 &#176;C), the water must be cooled at rates in the order of one million degrees Celsius per second (i.e., the critical cooling rate) to avoid ice formation above the glass transition temperature8. Addition of CPAs can lower the critical cooling rate and increase the glass transition temperature of aqueous solutions9. However, even with high CPA concentrations (e.g., 40% v/v DMSO or higher) fast cooling rates are nonetheless required for successful vitrification8,9.
The required cooling rates and high CPA concentrations result in two major drawbacks of vitrification. First, to enable fast cooling, the samples must have a low thermal mass. Second, to reach high CPA concentrations while avoiding osmotic injury, CPAs must be slowly introduced and fully equilibrated between the intra- and extracellular compartments6. This increases the exposure time of cells to toxic CPAs. Together, this makes vitrification a cumbersome process that is limited to a few small sized samples (microliters) at a time. Droplet vitrification has been proposed as a potential solution to these restrictions. By exposing miniscule cell-laden droplets (nanoliters) to liquid nitrogen the cooling rate is significantly increased, which consequently allows a considerable reduction in the CPA concentration10,11,12,13,14. Although multiple high frequency droplet-generating nozzles could potentially be used simultaneously, the extremely small droplet size limits the throughput to microliters per minute10, which precludes efficient vitrification of large cell volumes with magnitudes higher processing rates on the order of milliliters per minute.
Recently it was demonstrated that these inherent limitations of vitrification can be overcome by bulk droplet vitrification15. This novel method leverages rapid osmotic dehydration to concentrate an intracellular CPA concentration of 7.5% v/v ethylene glycol and DMSO immediately preceding vitrification, eliminating the need of full equilibration of toxic CPA concentrations. The cellular dehydration is performed in a fluidic device by a brief exposure of the hepatocytes to a high extracellular CPA concentration. Although this exposure causes rapid osmotic dehydration, it is too short for the high CPA concentration to diffuse into the cells. Immediately after dehydration, the cells are loaded in droplets that are directly vitrified in liquid nitrogen. This method eliminates the need of full intracellular uptake of high CPA concentrations while the high extracellular CPA concentration enables vitrification of large sized droplets, resulting in high throughput volumes with minimal CPA toxicity.
Droplet vitrification improves direct and long-term viability after preservation, as well as morphology and metabolic function of primary rat hepatocytes as compared to classical cryopreservation by slow-freezing15. This manuscript provides the methodological details for bulk droplet vitrification of primary rat hepatocytes.

<table>
	<tbody>
		<tr>
			<td>BD Disposable 3 mL Syringes with Luer-Lok Tips</td>
			<td>Fisher Scientific</td>
			<td>14-823-435</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>Sigma-Aldrich</td>
			<td>CLS1000-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Belzer UW Cold Storage Solution</td>
			<td>Bridge to Life</td>
			<td>BUW-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cole-Parmer Female Luer to 1/16&#34; low-profile semi-rigid tubing barb, PP</td>
			<td>Cole-Parmer</td>
			<td>EW-45508-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogentic stroage tank / Cryotank</td>
			<td>Chart Industries</td>
			<td>MVE 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, powder, high glucose, pyruvate</td>
			<td>Life Technologies</td>
			<td>12800-082</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Sigma-Aldrich</td>
			<td>107-21-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra long forceps</td>
			<td>Fisher Scientific</td>
			<td>10-316C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Higher-Speed Easy Reader Plastic Centrifuge Tubes - Flat top closure</td>
			<td>Fisher Scientific</td>
			<td>06-443-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Fishing line</td>
			<td>Stren</td>
			<td>SOFS4-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Airgas</td>
			<td>7727-37-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Platinum-Cured Silicone Tubing, L/S 14</td>
			<td>Cole-Parmer</td>
			<td>EW-96410-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix Tips, For Use With 3HPW1</td>
			<td>Grainger</td>
			<td>3WRL7</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Polypropylene Powder Funnel</td>
			<td>ThermoFisher</td>
			<td>4252-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle 20 ga</td>
			<td>Becton Dickinson (BD)</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M - Flexible film</td>
			<td>Sigma-Aldrich</td>
			<td>P7793-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor Blade</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Cole-Parmer</td>
			<td>EW-06369-18</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>Steriflip sterile filter</td>
			<td>Fisher Scientific</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose (Crystalline/Certified ACS)</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>New Era Pump Systems Inc.</td>
			<td>NE-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo-Flask Benchtop Liquid Nitrogen Container</td>
			<td>ThermoFisher</td>
			<td>2122</td>
			<td></td>
		</tr>
	</tbody>
</table>

bulk droplet vitrification for primary hepatocyte preservation

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. Vitrification requires extremely fast cooling rates to achieve transition of water into the glass phase while avoiding injurious ice formation. Although pre-incubation with cryoprotective agents (CPA) can reduce the critical cooling rate of biological samples, high concentrations are generally needed to enable ice-free cryopreservation by vitrification. As a result, vitrification is hampered by CPA toxicity and restricted to small samples that can be cooled fast. It was recently demonstrated that these inherent limitations can be overcome by bulk droplet vitrification. Using this novel method, cells are first pre-incubated with a low intracellular CPA concentration. Leveraging rapid osmotic dehydration, the intracellular CPA is concentrated directly ahead of vitrification, without the need to fully equilibrate toxic CPA concentrations. The cellular dehydration is performed in a fluidic device and integrated with continuous high throughput generation of large sized droplets that are vitrified in liquid nitrogen. This ice-free cryopreservation method with minimal CPA toxicity is suitable for large cell quantities and results in increased hepatocyte viability and metabolic function as compared to classical slow-freezing cryopreservation. This manuscript describes the methods for successful bulk droplet vitrification in detail.

Freshly isolated primary hepatocytes from five different rat livers were used for a direct comparison of bulk droplet vitrification to classic cryopreservation using the preeminent slow-freezing protocols reported in the literature4,5. In short, the hepatocytes were suspended in UW supplemented with BSA (2.2 mg/mL), glucose (333 mM), and DMSO (10% v/v) and frozen using a controlled rate freezer. After storage at -196 &#176;C, the samples were thawed in a warm wat...

Watch this Scientific Journal Video about Bulk Droplet Vitrification for Primary Hepatocyte Preservation at JoVE.com

Bulk Droplet Vitrification for Primary Hepatocyte Preservation

This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets.

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. ...

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5.5K Views.  Harvard Medical School, Massachusetts General Hospital. This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets.

Video: Bulk Droplet Vitrification for Primary Hepatocyte Preservation

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a blastocyst&#39;s volume changing during individual phases before and after vitrification. The method can be useful in searching for the most optimal timing of blastocyst exposure to different concentrations of cryoprotectants by observing blastocyst shrinkage and re-expansion in different pre- and post-vitrification phases. With this methodology, the blastocyst vitrification protocol can be optimized. For a better demonstration of the usefulness of this morphometric method, two different blastocyst preparation protocols for vitrification are compared; one with using an artificial blastocoel collapsing and one without this intervention before vitrification. Both blastocysts&#39; volume changes are followed by time-lapse microphotography and measured by photo-editing software tools. The measurements are taken every 20 seconds in previtrification phases and every 5 minutes in the post-warming period. The changes of the blastocyst dimensions per time unit are presented graphically in line diagrams. The results show a long equilibration previtrification phase in which the intact blastocyst first shrinks and then slowly refills the blastocoel, entering vitrification with a fluid-filled blastocoel. The artificially collapsed blastocyst remains in its shrunken stage through the entire equilibration phase. During the vitrification phase, it also does not change its volume. Since the blastocyst morphometry shows a constant volume of the artificially collapsed blastocysts during the previtrification step, it seems that this stage could be shorter. The described protocol provides many additional comparative parameters of blastocyst behavior during and after cryopreservation on the basis of the speed and intensity of the volume changes, the number of partial blastocoel contractions or total blastocyst collapses, and the time to a total blastocoel re-expansion or the time to hatching.

Here we present a time-lapse morphometric protocol to follow the intensity of blastocyst shrinkage and re-expansion during previtrification interventions and post-warming recovery. The application of the protocol is possible in in vitro fertilization laboratories equipped with time-lapse microscopes and is recommended in the development of an optimal blastocyst vitrification method.

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC&#160;subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...