Name: electroporation-mediated delivery of cas9 ribonucleoproteins and mrna into freshly isolated primary mouse hepatocytes
Uploaded: 2022-06-02T13:10:00
Description: Watch this Scientific Journal Video about Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes at JoVE.com

RNC received funding from South Carolina Bioengineering Center of Regeneration and Formation of Tissues Pilot grant supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health, the American Association for the Study of Liver Diseases Foundation, and American Society of Gene &#38; Cell Therapy under grant numbers P30 GM131959, 2021000920, and 2022000099, respectively. The content is solely the responsibility of the authors and does not necessarily represent the official views of the American Society of Gene &#38; Cell Therapy or the American Association for the Study of Liver Diseases Foundation. The schematic for Figure 1 was created with BioRender.com.

The animal experiments were all performed in compliance with the Institutional Animal Care and Use Committee guidelines and approved protocols at Clemson University. Surgical procedures were performed in anesthetized wild-type C57BL/6J mice between 8 and 10 weeks old.
1. Animal surgery
<ol>
	<li>Preparation of solutions and instruments 
	NOTE: Perfusion solutions and anesthesia cocktail recipes are shown in the Table of Materials.
	<ol>
		<li>Prepare Pe...

<ol>
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The steps outlined in the protocol for hepatocyte isolation are challenging and require practice for proficiency. There are several key steps for the successful hepatocyte isolation from the liver. First, proper cannulation of the inferior vena cava is essential for complete liver perfusion. The absence of blanching in the liver after perfusion indicates displacement of the catheter (Table 1). The inferior vena cava (retrograde perfusion) was cannulated in the procedure because it is simpler and more acc...

IMDs of the liver are genetic disorders characterized by the deficiency of a crucial hepatic enzyme involved in metabolism that leads to the accumulation of toxic metabolites. Without treatment, IMDs of the liver result in organ failure or premature death1,2. The only curative option for patients with IMDs of the liver is orthotopic liver transplantation, which is limited due to the low availability of donor organs and complications from immunosuppressive therapy following the procedure3,4. According to recent data collected by the Organ Procurement and Transplantation Network, only 40%-46% of adult patients on the liver transplant waiting list receive an organ, while 12.3% of these patients die while on the waiting list5. Moreover, only 5% of all the rare liver diseases have an FDA-approved treatment6. It is clear that there is a critical need for novel treatments for IMDs of the liver. However, appropriate disease models are required to develop new therapeutic options.
Modeling human diseases using in vitro and in vivo systems remains an obstacle for developing effective therapies and studying the pathology of IMDs of the liver. Hepatocytes from patients with rare liver diseases are challenging to obtain7. Animal models are crucial for developing an understanding of disease pathology and for testing therapeutic strategies. However, one obstacle is generating models from embryos carrying lethal mutations. For example, attempts to create mouse models of Alagille Syndrome (ALGS) with embryos containing homozygous deletions of a 5 kb sequence near the 5&#8242;-end of the Jag1 gene resulted in the early death of the embryos8. In addition, generating mouse models by gene editing in embryonic stem cells can be time- and resource-intensive9. Lastly, mutations will appear outside the targeted tissue, leading to confounding variables that may impede study of the disease9. Somatic gene editing would allow for easier editing in liver tissue and bypasses the challenges associated with generating models using embryonic stem cells.
Electroporation enables the delivery of CRISPR-Cas9 directly into the nucleus by applying high-voltage currents to permeabilize the cell membrane and is compatible with many cell types, including those that are intransigent to transfection techniques, such as human embryonic stem cells, pluripotent stem cells, and neurons10,11,12. However, low viability is a potential drawback of electroporation; optimizing the procedure can yield high levels of delivery while limiting toxicity13. A recent study demonstrates the feasibility of electroporating CRISPR-Cas9 components into primary mouse and human hepatocytes as a highly efficient approach14. Ex vivo electroporation in hepatocytes has the potential to be applied to generate new mouse models for human IMDs of the liver.
This protocol provides a detailed step-by-step procedure for isolating mouse hepatocytes from the liver and subsequently electroporating CRISPR-Cas9 as RNP complexes, consisting of Cas9 protein and synthetic single-guide RNA (sgRNA), or Cas9 mRNA combined with sgRNA to obtain high levels of on-target gene editing. In addition, the protocol provides methods for quantifying gene editing efficiency, viability, and functionality following electroporation of CRISPR-Cas9 into freshly isolated mouse hepatocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 mL PCR 8-tube FLEX-FREE strip, attached clear flat caps, natural</td>
			<td>USA Scientific</td>
			<td>1402-4700</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Collagen Plates</td>
			<td>Advanced Biomatrix</td>
			<td>5073</td>
			<td></td>
		</tr>
		<tr>
			<td>accuSpin Micro 17R</td>
			<td>Fisher Scientific</td>
			<td>13-100-675</td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-one Fluorescent Microscope</td>
			<td>Keyence</td>
			<td>BZ-X810</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog Vortex Mixer</td>
			<td>VWR</td>
			<td>97043-562</td>
			<td></td>
		</tr>
		<tr>
			<td>ART Wide BORE filtered tips 1,000 &#181;L</td>
			<td>ThermoFisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Bio-Rad Laboratories</td>
			<td>TC20</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Wax Dissection Tray</td>
			<td>Braintree Scientific Inc.</td>
			<td>DTW1</td>
			<td>9&#34; x 6.5&#34; x 1/2&#34;+F21</td>
		</tr>
		<tr>
			<td>Cell scraper/lifter</td>
			<td>Argos Technologies</td>
			<td>UX-04396-53</td>
			<td>Non-pyrogenic, sterile</td>
		</tr>
		<tr>
			<td>Conical tubes (15 mL)</td>
			<td>Fisher Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes (50 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton applicators</td>
			<td>Fisher Scientific</td>
			<td>22-363-170</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Cooper Surgical</td>
			<td>62131</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes</td>
			<td>Falcon</td>
			<td>351029</td>
			<td>100mm,sterile</td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes</td>
			<td>VWR</td>
			<td>25373-100</td>
			<td>35mm, sterile</td>
		</tr>
		<tr>
			<td>Epoch Microplate Spectrophotometer</td>
			<td>BioTek Instruments</td>
			<td>250082</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell strainer (70 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Cooper Surgical</td>
			<td>61864</td>
			<td>Euro-Med Adson Tissue Forceps</td>
		</tr>
		<tr>
			<td>IV catheters&#160;</td>
			<td>BD</td>
			<td>382612</td>
			<td>24 G x 0.75 in</td>
		</tr>
		<tr>
			<td>IV infusion set</td>
			<td>Baxter</td>
			<td>2C6401</td>
			<td></td>
		</tr>
		<tr>
			<td>MyFuge 12 Mini Centrifuge</td>
			<td>Benchmark Scientific</td>
			<td>1220P38</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>Fisher Scientific</td>
			<td>05-561-20</td>
			<td>25 G</td>
		</tr>
		<tr>
			<td>Nucleofector 2b Device</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td>Program T-028 was used for electroporation in mouse hepatocytes</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Masterflex</td>
			<td>&#160;HV-77120-42</td>
			<td>10 to 60 rpm; 90 to 260 VAC</td>
		</tr>
		<tr>
			<td>Precision pump tubing</td>
			<td>Masterflex</td>
			<td>HV-96410-14</td>
			<td>25 ft, silicone</td>
		</tr>
		<tr>
			<td>Primaria Culture Plates</td>
			<td>Corning Life Sciences</td>
			<td>353846</td>
			<td>Nitrogen-containing tissue culture plates</td>
		</tr>
		<tr>
			<td>Serological Pipets (25 mL)</td>
			<td>Fisher Scientific</td>
			<td>12-567-604</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>BD</td>
			<td>329464</td>
			<td>1 mL, sterile</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad Laboratories</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>ALT</td>
			<td>27577</td>
			<td>Thermo Scientific Precision Microprocessor Controlled 280 Series, 2.5 L</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease V3</td>
			<td>IDT</td>
			<td>1081058</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Coulter AMPure XP, 5 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9959336</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap Cas9 mRNA</td>
			<td>Trilink Biotechnologies</td>
			<td>L-7606-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap&#160;EGFP mRNA</td>
			<td>Trilink Biotechnologies</td>
			<td>L-7201-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Matrix</td>
			<td>Corning Life Sciences</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher Scientific</td>
			<td>11885076</td>
			<td>Low glucose, pyruvate</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>VWR</td>
			<td>71001-652</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermoscientific</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatocyte Maintenance Medium (MM)</td>
			<td>Lonza</td>
			<td>MM250</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatocyte Plating Medium (PM)</td>
			<td>Lonza</td>
			<td>MP100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Albumin ELISA Kit</td>
			<td>Fisher Scientific</td>
			<td>NC0010653</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse/Rat Hepatocyte Nucleofector Kit</td>
			<td>Lonza</td>
			<td>VPL-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>OneTaq HotStart DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0481L</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10x pH 7.4&#160;</td>
			<td>Thermoscientific</td>
			<td>70011-044</td>
			<td>No calcium or magnesium chloride</td>
		</tr>
		<tr>
			<td>Percoll (PVP solution)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-500790A</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic acid</td>
			<td>Sigma-Aldrich</td>
			<td>P7875-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount Mounting Medium</td>
			<td>VWR</td>
			<td>100496-550</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickExtract DNA Extraction Solution</td>
			<td>Lucigen Corporation</td>
			<td>QE05090</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#8217;s fuchsin-sulfite reagent</td>
			<td>Sigma-Aldrich</td>
			<td>S5133</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td>Phenol red</td>
		</tr>
		<tr>
			<td>Vybrant MTT Cell Viability Assay</td>
			<td>ThermoFisher Scientific</td>
			<td>V13154</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Solution 1 (pH 7.4, filter sterilized)&#160;</td>
			<td></td>
			<td></td>
			<td>Stable at 4 &#176;C for 2 months</td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Fisher Scientific</td>
			<td>14155063</td>
			<td>Complete to 200 mL</td>
		</tr>
		<tr>
			<td>EGTA (0.5 M)</td>
			<td>Bioworld</td>
			<td>40520008-1</td>
			<td>200 &#181;L</td>
		</tr>
		<tr>
			<td>HEPES (pH 7.3, 1 M)</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ16924AE</td>
			<td>2 mL&#160;</td>
		</tr>
		<tr>
			<td>Perfusion Solution 2 (pH 7.4, filter sterilized)&#160;</td>
			<td></td>
			<td></td>
			<td>Stable at 4 &#176;C for 2 months</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H20 (1.8 mM)</td>
			<td>Sigma</td>
			<td>C7902-500G</td>
			<td>360 &#181;L of 1 M stock&#160;</td>
		</tr>
		<tr>
			<td>EBSS (no calcium, no magnesium, no phenol red)</td>
			<td>Fisher Scientific</td>
			<td>14-155-063</td>
			<td>Complete to 200 mL</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ16924AE</td>
			<td>2 mL&#160;</td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H20 (0.8 mM)</td>
			<td>Sigma</td>
			<td>30391-25G</td>
			<td>160 &#181;L of 1 M stock</td>
		</tr>
		<tr>
			<td>Perfusion Solution 3&#160;</td>
			<td></td>
			<td></td>
			<td>Prepared fresh prior to use</td>
		</tr>
		<tr>
			<td>Solution 2</td>
			<td></td>
			<td></td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Liberase</td>
			<td>Roche</td>
			<td>5401127001</td>
			<td>0.094 Wunsch units/mL</td>
		</tr>
		<tr>
			<td>Mouse Anesthetic Cocktail&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromzine</td>
			<td></td>
			<td></td>
			<td>0.25 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td></td>
			<td></td>
			<td>7.5 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td></td>
			<td></td>
			<td>1.5 mg/mL final concentration</td>
		</tr>
		<tr>
			<td>Software</td>
			<td>URL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benchling</td>
			<td>https://www.benchling.com/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TIDE: Tracking of Indels by Decomposition</td>
			<td>https://tide.nki.nl/</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Isolation of plateable primary hepatocytes from the liver 
The overall process of liver perfusion and hepatocyte isolation is illustrated in Figure 1. In this experiment, wild-type, 8-10-week-old C57BL6/6J mice were used. The procedure is expected to yield 20-50 &#215; 106 cells per mouse with a viability between 85% and 95%. If the viability is &#60;70%, percoll treatment should be followed to remove dead cells. Freshly isolated hepatocytes should be plated ...

Watch this Scientific Journal Video about Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes at JoVE.com

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

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 ...

The main advantage of our technique is that it is an easy, fast, and reproducible approach for editing genes in primary mouse hepatocytes as a way to generate in vitro and in vivo disease models. Using our technique, it may be possible to knock down a target gene in isolated hepatocytes and then transplant them back into the patient, to replace diseased cells with gene edited hepatocytes Inexperienced researchers might struggle with cannulation and keeping the catheter steady throughout the perfusion. The users are advised to carefully read and practice the procedure and the troubleshooting methods before getting started.Demonstrating the liver perfusion and hepatocyte isolation will be Tina Parker a clinical facility manager and Ilayda Ates, a graduate research assistant. The electroporation procedure will be demonstrated by Callie Stuart, a graduate research assistant from my laboratory. Under a surgical plane of anesthesia.Begin the liver perfusion by making a U-shaped lateral incision in the abdomen skin using scissors, and expand the hole through the sides. Continue opening the skin to the rib cage. Using scissors, cut through the peritoneum to expose the abdominal cavity up to the ribcage.Being careful not to nick any organs. Move the intestines to the right side using the back of forceps or the blunt surface of scissors. Identify the portal vein and the inferior vena cava.Start the pump to flush pre-warmed perfusion solution through the catheter. Stop the pump and remove the catheter. Then insert the tip of the needle connected to the catheter into the inferior vena cava at a 10 to 20 degree angle to the vein, remove the needle from the catheter.Then gently push the catheter into the vein in a movement parallel to the vein. Connect the tubing to the catheter without moving the catheter further into the vein. Start the pump with a flow rate of two milliliters per minute.Look for the liver immediately turning pale as a sign that the perfusion is successful. Cut the portal vein using scissors. Gradually increase the flow rate to five milliliters per minute.Once the perfusion solution three is flowing through carefully monitor the elasticity of the liver. To determine if the liver has softened, gently press the liver with a cotton swab or forceps and check whether indentations form on the liver. Be careful not to over perfuse to avoid loss of cell viability.Once the liver softens after 30 to 50 milliliters of perfusion solution three has flowed through, stop the pump, remove the catheter and the gallbladder. Careful not to spill its contents. Then carefully dissect the liver.Place the liver into a 100 millimeter Petri dish containing ice cold DME M with 10%FBS medium. Swirl the Petri dish gently to remove possible blood clots. Transfer the liver to a new Petri dish containing ice cold DME M and 10%FBS medium.To release cells from the liver capsule, place the Petri dish on ice then using two pairs of sterile forceps, gently tear apart the liver capsule on all lobes. Gently swirl the liver around in the medium using a cell lifter to release the hepatocytes. Continue this motion until the liver becomes very small and the suspension turns brown and opaque.Remove the liver tissue remains from the plate and using a 25 milliliter serological pipette set to low speed, carefully transfer the cell suspension to a 50 milliliter conical tube pre chilled on ice and fitted with a 100 micrometer cell strainer. Add fresh ice cold DMEM, and 10%FBS medium to the Petri dish to wash out and collect the remaining cells from the surface. Then, transfer to the 50 milliliter conical tube.Repeat this step until all the cells are collected. To wash and purify hepatocytes from non parenchymal cells, centrifuge the cells collected in the 50 milliliter conical tube at 50 RCF at four degrees Celsius for five minutes at low deceleration or without any breaks. Discard the super-naton containing debris and non-parenchymal cells.Then re-suspend the pellet in the leftover super-naton by gently swirling the tube. After pellet re-suspension add 30 milliliters of fresh ice cold DMEM and 10%FBS medium. Begin preparing the CRISPR CAS 9 substrates media, cells and the electroporation instrument by turning on the power button on the electroporation device.Then set the electroporation program by pressing the X button and then the down arrow button until the program T 0 28 appears on the screen. Prepared the destination wells for the electroporated hepatocytes by adding 1.5 milliliters of PM to six well collagen one coated plates. Incubate the plates in a 37 degree Celsius incubator until ready to use.In PCR split strip tubes, prepare the CAS 9 RMP and mRNA complexes as described in the text manuscript. Store the mRNA SG RNA mix on ice until electroporation. Separately, prepare a tube containing one microgram of E G F P mRNA to verify successful electroporation using fluorescence microscopy.Centrifuge the electroporation reaction at 100 RCF for two minutes at four degrees Celsius. Remove the supernaton from the cell pellet and add 100 microliters of nucleoffection solution per reaction along the side of the tube wall. Re suspend the hepatocytes in the electroporation solution by rocking the tube gently by hand.Once the hepatocytes are evenly dispersed in the electroporation solution, transfer 100 microliters of the hepatocyte suspension to the strip tubes containing the CAS nine complexes. Transfer the contents of the strip well to an electroporation vessel. Place the nucleo-vet vessel into the slot on the electroporation device.Electroporate the hepatocytes by pressing the X button. After electroporation, wait for a screen to pop up on the device that says:Okay. Press the X button again and remove the vessel.Incubate the electroporated vessel on ice for 15 minutes. Add 500 microliters a prew-armed PM to the electroporation vessel. Transfer 300 microliters of the electroporation reaction to each destination well on the pre-warmed plate.Incubate the plates overnight at 37 degrees Celsius. Add the membrane matrix to ice cold hepatocyte maintenance medium and mix by pipetting up and down 10 times. Remove the medium from the wells designated for gene editing analysis 24 hours after plating the cells.Pipette the overlay mixture slowly on top of the cells and place the plate back at 37 degrees Celsius. At 24 hours after plating, transfer the conditioned medium from the well designated for MTT and albumin assays and store in a micro-fuge tube until ready to perform the albumin assay. Proceed with analysis of on target CAS nine activity by extracting the genomic DNA and setting up the PCR as described in the text manuscript.Within 3 to 12 hours of plating the hepatocytes adhered to the plate and the cell morphology assumed a typical polygonal or hexagonal appearance within 24 hours The purity of the isolated cells was verified via glycogen staining using the periodic acid shift reagent at 24 hours after plating. The cytoplasm of the stained hepatocytes appeared magenta. The hepatocytes were imaged 24 hours after electroporation and on average, 89.8%of the hepatocytes were GFP positive.At three days after electroporation CAS nine induced in the HPD locus where analyzed and the results showed on target indels of 47.4%in hepatocytes electroporated with CRISPR Cas nine mRNA and 78.4%indels for the CAS nine RNP. The MTT assay showed 35.4 and 45.9%viability in hepatocytes treated with CRISPR Cas nine mRNA and R N P.Consistent with the MTT results the normalized albumin levels were 31.8%in hepatocytes treated with CAS nine RNA and 34.5%for CAS nine r p. The most important thing to remember is that proper cannulation affects all of the later steps and the success of the procedure.After this procedure, the hepatocytes can be transplanted and mice to investigate their engraftments. Moreover, the genomic DNA of plated hepatocytes can be extracted to analyze the efficiency and specificity of CAS nine editing.

electroporation-mediated-delivery-cas9-ribonucleoproteins-mrna-into

Research

JoVE Journal

Bioengineering

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

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 ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

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 ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Handling and Assessment of Human Primary Prostate Organoid Culture

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Compression of z-stacks creates a single in-focus image from which organoids are measured to quantify a variety of outputs, including circularity, roundness, and area.DNA, RNA, and protein can be collected from organoids recovered from the matrix gel. Cell populations of interest can be assessed by organoid dissociation and flow cytometry. Formalin-fixation-paraffin-embedding (FFPE) followed by sectioning is used for the histological assessment and antibody staining. Whole-mount immunofluorescent staining preserves organoid morphology and facilitates observation of protein localization in organoids in situ. Commercial assays that are traditionally used for 2D monolayer cells can be modified for 3D organoids. Used together, the techniques in this protocol provide a robust toolbox to quantify prostate organoid growth, morphologic characteristics, and expression of differentiation markers.

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process ...

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...