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.

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Vitrification is typically handled by toxicity of cryoprotective agents. And re-inserted to small samples that can be cooled fast. This protocol enables vitrification of large cell quantities while using a low CPA concentration during pre-incubation.

Using rapid osmotic dehydration, the low inter-cellar CPA is concentrated in the fluid device directly ahead of vitrification. This eliminates the need of fully equilibrating toxic CPA concentrations. Bulk droplet vitrification may provide viable and metabolically active hepatocytes for using hepatocytes transplantation and bio-artificial liver devices which our currently hindered by inadequate outcomes after cryopreservation.

This method may potentially be adapted for vitrification of other cell types that need to be cryo-preserved in large quantities;such as blood products or stem-cells among others. To begin, make vitrification Solution 1 by adding 40 milligrams of BSA to 17.0 milliliters of University of Wisconsin Solution. Use a 0.22 micrometer filter to sterile filter the solution and store at four degrees Celsius.

Make vitrification Solution 2 as described in the protocol. And sterile filter it through a 0.22 micrometer filter. Load 3.5 milliliters of sterile-filtered V2 Solution in a three milliliter syringe and store at four degrees Celsius.

To prepare the bulk droplet vitrification apparatus, first, cut along one side of the mixing needles outer gray sleeve. Bend the sleeve open to remove it from the mixing needle. Then, cut the mixing needle to a length of 20 millimeters and insert its cutoff outlet into a 20 gauge injection needle.

Slide two 25 millimeter silicon tubing sections over the inlets of the mixing needle. And secure their position with glue. Sterilize this assembly by autoclaving.

Fill a sterile Dewar flask with liquid nitrogen. And sterilize the outside with isopropanol before placing it in a sterile Laminer flow-cell culture hood. Make sure to wear appropriate personal protective equipment as liquid nitrogen can cause serious burns.

To create the droplet collection device insert a funnel with the same outer diameter as the inner diameter of the Dewar, into a 50 milliliters conical tube. Using large forceps, slowly press the droplet collection device down in the liquid nitrogen in its final vertical position. Make sure that the conical tube rests in vertical position at the bottom of the Dewar.

And the liquid nitrogen level is one centimeter lower than the edge of the funnel. To mount a syringe pump at a vertical position on the wall of the cell culture hood, tie a string from the syringe pump's feet to screws protruding from the cell culture hood wall. Then, place the liquid nitrogen Dewar with the droplet collection device under the vertically mounted syringe pump.

After obtaining freshly isolated rat hepatocytes, count the stock concentration by tri-pan blue exclusion. And transfer 40 million viable hepatocytes into a 50 milliliter conical tube. Centrifuge the hepatocytes at 50 times G for five minutes without break.

Aspirate the supernatant. Re-suspend the hepatocytes in 3.4 milliliters of V1 Solution. And incubate on ice for three minutes.

Then, add 150 microliters of DMSO and 150 microliters of EG;mix gently and incubate on ice again for three minutes. Add 150 microliters of DMSO and 150 microliters of EG to the hepatocytes again and mix gently. Load 3.5 milliliters of this mixture in a three milliliter syringe.

Insert the syringe with pre-incubated hepatocytes and the syringe with V2 Solution onto the syringe pump adapter. Attach two Female Luer Lock hose-barb adapters to the syringes. And slide the silicon tubing of the mixing needle assembly over the barb fittings.

Place this entire assembly in the syringe pump. Three minutes after the last edition of DMSO and EG to the hepatocytes, start the pump at two milliliters per minute. After all hepatocytes have been added to the liquid nitrogen stop the pump.

Using a 20 gauge needle, puncture 10 small holes in the lid of a 50 milliliter conical tube. And wrap the outside of the lid with a flexible film, creating a valve that will enable the escape of evaporating left-over liquid nitrogen. To collect the vitrified hepatocyte droplets, first remove the funnel from the Dewar with liquid nitrogen.

Next, with long forceps, take out the conical tube that contains the droplets from the liquid nitrogen, and slowly pour out the excess liquid nitrogen from the conical tube. Close the conical tube with the punctured lid and then directly place the closed conical tube with vitrified hepatocyte droplets back in the Dewar with liquid nitrogen. Finally, transfer the conical tube from the liquid nitrogen into a liquid nitrogen cryotank.

After making the rewarming solution with sucrose and DMEM hepatocyte culture media, use a 0.22 micrometer filter to sterile filter it. Then warm it to 37 degrees Celsius. To rewarm the hepatocytes remove the conical tube with vitrified hepatocytes from the cryotank and transport it in a liquid nitrogen Dewar to the subculture hood.

Lightly loosen the cap and place the conical tube back in the liquid nitrogen. Working quickly, add all the vitrified hepatocyte droplets to an empty beaker, then quickly add 100 milliliters of 37 degree Celsius warm rewarming solution while stirring for 10 seconds. When the droplets are removed from liquid nitrogen intra-est freezing occurs within seconds if the droplets are not rapidly rewarmed.

Therefore, it is critical to directly add the rewarming media to the droplets while stirring. Divide the rewarming solution with hepatocytes into two 50 milliliter conical tubes. Centrifuge the tubes for two minutes at 100 times G at four degrees Celsius without break.

Aspirate the supernatants to leave a total volume of 12.5 milliliters using the tube's graduation mark as a reference. To dilute the rewarming solution to 50%add 12.5 milliliters of ice cold DMEM per conical tube. Re-suspend and incubate on ice for three minutes.

To dilute the rewarming solution to 25%add 25 milliliters of ice cold DMEM per conical tube. After centrifuging for five minutes at 50 times G at four degrees Celsius without break, aspirate the supernatant and re-suspend the hepatocytes in two milliliters of the desired culture medium. When measured by tri-pan blue exclusion testing, which determines membrane integrity, bulk droplet vitrification resulted in a direct post-preservation hepatocyte viability of 79%This was significantly higher than the 68%viability after classic optimized cryopreservation.

The yield of bulk droplet vitrification was 56%Which was 10%higher and more consistent than classic cryopreservation. Using presto-glue metabolization assay in long-term college and sandwich cultures, metabolic activity at the hepatocytes was measured to be significantly higher after bulk droplet vitrification then after classical cryopreservation. Albumin synthesis, as the most widely used parameter of synthetic function of hepatocytes, was greater by nearly two-fold after bulk droplet vitrification, then after classic cryopreservation.

Urea production was used to measure hepatocyte detoxification function in a one week culture. In bulk droplet vitrified hepatocytes, the urea production was significantly higher than that in classic cryopreserve hepatocytes. For consistent results after bulk droplet vitrification it is important to exactly follow timing during pre-incubation of CPAs and rewarming as detailed in this protocol.

After rewarming the vitrified hepatocyte droplets you will end up with a better sizing suspension which therefore can be used in all the same ways as if they were freshly isolated. As an improved preservation method, bulk droplet vitrification may increase the availability pri-ma for research and clinical applications.

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Journal (Bioengineering)

Journal (Bioengineering)

Bulk Droplet Vitrification for Primary Hepatocyte Preservation

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