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/pubmed?term=((Artificial+and+bioartificial+liver+devices%3A+present+and+future%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">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/pubmed?term=((Hepatocyte+transplantation%3A+Hepatocyte+transplantation%5BTitle%5D)+AND+%22American+Journal+of+Transplantation%22%5BJournal%5D)">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/pubmed?term=((Current+Status+of+Hepatocyte+Transplantation%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Current Status of Hepatocyte Transplantation.</a> Transplantation. 93 (4), 342-347 (2012).</li>
<li>Stéphenne, X., Najimi, M., Sokal, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatocyte+cryopreservation%3A+is+it+time+to+change+the+strategy%5BTitle%5D)+AND+%22World+Journal+of+Gastroenterology%22%5BJournal%5D)">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/pubmed?term=((Optimization+of+the+cryopreservation+and+thawing+protocol+for+human+hepatocytes+for+use+in+cell+transplantation%5BTitle%5D)+AND+%22Liver+Transplantation%22%5BJournal%5D)">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/pubmed?term=((Principles+of+cryopreservation%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Cryopreservation%3A+An+emerging+paradigm+change%5BTitle%5D)+AND+%22Organogenesis%22%5BJournal%5D)">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/pubmed?term=((Effect+of+common+cryoprotectants+on+critical+warming+rates+and+ice+formation+in+aqueous+solutions%5BTitle%5D)+AND+%22Cryobiology%22%5BJournal%5D)">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/pubmed?term=((Vitrification+as+an+approach+to+cryopreservation%5BTitle%5D)+AND+%22Cryobiology%22%5BJournal%5D)">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/pubmed?term=((Cell+encapsulating+droplet+vitrification%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((Bio-Inspired+Cryo-Ink+Preserves+Red+Blood+Cell+Phenotype+and+Function+During+Nanoliter+Vitrification%5BTitle%5D)+AND+%22Advanced+Materials%22%5BJournal%5D)">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/pubmed?term=((High+throughput+cryopreservation+of+cells+by+rapid+freezing+of+sub-%CE%BCl+drops+using+inkjet+printing--cryoprinting%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">High throughput cryopreservation of cells by rapid freezing of sub-μl 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/pubmed?term=((Blood+banking+in+living+droplets%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">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/pubmed?term=((High-Throughput+Non-Contact+Vitrification+of+Cell-Laden+Droplets+Based+on+Cell+Printing%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Bulk+Droplet+Vitrification%3A+An+Approach+to+Improve+Large-Scale+Hepatocyte+Cryopreservation+Outcome%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">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/pubmed?term=((Sterilization+of+liquid+nitrogen+with+ultraviolet+irradiation+for+safe+vitrification+of+human+oocytes+or+embryos%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">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/pubmed?term=((Cryoprotectant+Toxicity%3A+Facts%2C+Issues%2C+and+Questions%5BTitle%5D)+AND+%22Rejuvenation+Research%22%5BJournal%5D)">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&quot; 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>&lt;u&gt;&amp;nbsp;&lt;/u&gt;</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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5.6K 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

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Biochemistry

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Immunology and Infection

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 &#215; 106 cells per liver and cell viability of &#62;85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of &#62;35% in mouse hepatocytes.
To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.

This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Medicine

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Biology

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...