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Özet

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.

Abstract

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 °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.

Introduction

Loss of cell viability and metabolic function after cryopreservation of hepatocytes is still a major issue that limits clinical applications^1,2,3. 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 °C/min) to cryogenic temperatures^4,5. Despite many reported optimizations, CPA toxicity together with injurious osmotic imbalances during freezing and mechanical stress of ice formation remain fundamental drawbacks of slow-freezing^6,7.

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 °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 vitrification^8,9.

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 concentration^{10,11,12,13,14}. 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.

Protocol

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

1. Prepare the vitrification solutions.
   1. 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 µm filter and store at 4 °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.
   2. 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 µm filter. Load 3.5 mL of sterile filtered V2 solution in a 3 mL syringe and store at 4 °C.
      NOTE: To facilitate dissolving the CPAs, solutions are prepared in larger quantities than required.
2. Prepare the bulk droplet vitrification apparatus.
   1. To prepare the mixing needle, first cut along one side of the mixing needle’s outer gray sleeve, and then bend the sleeve open to remove it from the mixing needle.
   2. Slide two 25 mm silicone tubing sections over the inlets of the mixing needle and secure their position with glue (Figure 1A).
   3. 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.
   4. 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.
   5. Sterilize the mixing needle assembly by autoclaving.
   6. 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.
   7. 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−E).
   8. 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−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.
   9. Mount a syringe pump in a vertical position on the wall of the cell culture hood by tying a string from the syringe pump’s feet to screws protruding from the cell culture hood wall.
   10. Place the liquid nitrogen Dewar with the droplet collection device under the vertically mounted syringe pump (Figure 1E).
3. Pre-incubate with CPAs.
   1. 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.
   2. Centrifuge at 50 x g for 5 min without brake.
   3. Aspirate the supernatant and resuspend the hepatocytes in 3.4 mL of V1 solution.
   4. Incubate on ice for 3 min.
   5. Add 150 µL of DMSO and 150 µL of EG to the hepatocytes and mix gently.
   6. Incubate on ice for 3 min.
   7. Again, add 150 µL of DMSO and 150 µL of EG to the hepatocytes and mix gently.
   8. Load 3.5 mL in a 3 mL syringe.
4. Perform vitrification.
   1. Insert the syringe with pre-incubated hepatocytes and the V2 solution syringes onto the syringe pump adapter.
   2. 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).
   3. Place the entire assembly in the syringe pump (Figure 1E).
   4. Three min after the last addition of DMSO and EG to the hepatocytes, start the pump at 2 mL/min.
   5. Stop the pump after all hepatocytes have been added to the liquid nitrogen.

2. Cryogenic storage

1. 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.
2. 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.
3. 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.
4. Transfer the conical tube from the liquid nitrogen into a liquid nitrogen vapor cryotank.

3. Rewarming of the vitrified hepatocyte droplets

1. Prepare the rewarming solution.
   1. 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 µm filter and warm to 37 °C.
2. Rewarm the hepatocytes.
   1. Take the conical tube with vitrified hepatocytes from the cryotank and transport it in a liquid nitrogen Dewar to the cell culture hood.
   2. Lightly loosen the cap and put the conical tube back in the liquid nitrogen.
   3. Add the vitrified hepatocyte droplets to an empty beaker, instantaneously add the warm (37 °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.
3. Unload the rewarming solution.
   1. Divide the rewarming solution with hepatocytes over two 50 mL conical tubes.
   2. Centrifuge the two conical tubes for 2 min at 100 x g at 4 °C without brake.
   3. Aspirate the supernatant to leave a total volume of 12.5 mL using the conical tube’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%.
   4. Resuspend the hepatocytes and incubate on ice for 3 min.
   5. Add 25 mL of ice cold DMEM per conical tube to dilute the rewarming solution to 25%.
   6. Centrifuge for 5 min at 50 x g at 4 °C without brake.
   7. 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.

Representative Results

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 literature^4,5. 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 °C, the samples were thawed in a warm water bath. After all the ice melted, the DMSO was directly diluted while the glucose concentration was gradually lowered during multiple steps to reduce osmotic injury. The exact protocol can be found in detail elsewhere¹⁵. Preservation durations varied from 2 to 8 days and were matched between bulk droplet vitrification and freezing cryopreservation for each biological replicate to ensure equal comparison. Although shorter preservation times were tested for practical considerations, it should be noted that primary hepatocytes can be stored at -196 °C for years without loss of viability⁵.

Bulk droplet vitrification results in a direct post preservation hepatocyte viability of 79%, measured by Trypan blue exclusion testing, which determines membrane integrity (Figure 2A). This is significantly higher than the 68% viability after classic optimized cryopreservation. The yield of bulk droplet vitrification is 56% that is 10% higher, and importantly more consistent, as compared to classic cryopreservation (Figure 2B).

The metabolic activity of hepatocytes, measured using a Presto Blue metabolization essay in long-term collagen sandwich cultures, was significantly higher after bulk droplet vitrification than after classical cryopreservation. Albumin synthesis, which is the most widely used parameter of synthetic function of hepatocytes, was greater by nearly two-fold after bulk droplet vitrification as compared to classic cryopreservation (Figure 3A). Urea production is the most widely used parameter of hepatocyte detoxification function. After a one-week culture, the urea production of bulk droplet vitrified hepatocytes was significantly higher than that of classic cryopreserved hepatocytes (Figure 3B).

In summary, bulk droplet vitrification improves direct post-preservation viability and long-term metabolic function of primary rat hepatocytes in collagen sandwich cultures, as compared to cryopreservation by slow-freezing.

Figure 1: Bulk droplet vitrification experimental setup.
(A) Illustration of how to prepare the mixing needle and attach two silicone tubing sections to the inlets of the mixing needle. (B) Illustration of the insertion of the cut mixing needle in the injection needle to complete the mixing needle assembly. (C) Illustration of the funnel and 50 mL conical tube that constitute the droplet collection device. (D) Sketch showing the position of the conical tube, the funnel, and the liquid nitrogen level in the Dewar. (E) Representation of the complete experimental bulk droplet vitrification setup from bottom to top: the liquid nitrogen Dewar (gray); the droplet collection device that is placed inside the liquid nitrogen Dewar (clear); the mixing needle assembly (clear-yellow) attached to the syringes that are placed in the syringe pump (red). (F) Illustration of the syringes and mixing needle assembly. Two 3 mL syringes are clamped into the syringe pump adapter (blue); one syringe should be filled with pre-incubated hepatocytes in V1 solution and the other with the high CPA concentration solution (V2 solution). The mixing needle assembly is attached to the syringes by two female Luer lock hose barb adapters. (G) Illustration of how to create the conical tube lid for cryogenic storage which enables leftover evaporating liquid nitrogen to escape from the conical tube during storage of the vitrified hepatocyte droplets. Top: lid with punctured holes. Below: the punctured lid wrapped with flexible film. Scale bars: 1 cm. Please click here to view a larger version of this figure.

Figure 2: Hepatocyte viability and yield after preservation.
(A) Viability of fresh (gray), cryopreserved (blue), and bulk droplet vitrified (green) hepatocytes. (B) Preservation yield of cryopreserved and bulk droplet vitrified hepatocytes. Stars: p < 0.05 (Wilcoxon matched-pairs signed rank test). Whiskers: min to max. Please click here to view a larger version of this figure.

Figure 3: Metabolic activity in long-term collagen sandwich cultures.
(A) Albumin synthesis of fresh (gray), cryopreserved (blue), and bulk droplet vitrified (green) hepatocytes on culture days 3, 5, and 7. (B) Urea production of fresh, cryopreserved, and bulk droplet vitrified hepatocytes. Stars: p < 0.05 (paired one-way ANOVA followed by the Tukey correction for multiple testing). Error bars: standard deviation. Please click here to view a larger version of this figure.

Discussion

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 droplet vitrification method presented in this manuscript was developed to enable vitrification of large quantities of cells while using a low pre-incubated CPA concentration¹⁵. This bulk droplet vitrification leads to increased hepatocyte viability and metabolic function as compared to the most optimal slow-freezing cryopreservation method in the literature. Here, the comprehensive methods of bulk droplet vitrification and detailed insights in the practical aspects of the procedures are provided.

Multiple steps in the protocol are critical and require additional attention. Filling of the Dewar with non-sterilized liquid nitrogen potentially leads to semi-sterile conditions. Using the precautions explained in the protocol, no contamination was revealed during our long-term collagen sandwich cultures. However, additional sterilization measures can be taken if deemed necessary. Liquid nitrogen can be sterilized by radiation or filtering¹⁶ and the system may be completely closed with a lid on the liquid nitrogen Dewar with a chimney connected to the mixing needle. Alternatively, non-contact droplet vitrification methods may be explored, although this may limit larger volume throughput¹⁴.

Other essential parts of the protocol are the CPA pre-loading and unloading incubation durations. Longer durations could result in toxic CPA injury while shorter durations may cause osmotic injury. Also, it is important to ensure that the vitrified hepatocyte droplets always stay below the glass transition temperature because uncontrolled macro- and microscopic ice nucleation at higher temperatures lead to severe hepatocyte injury and cell death. From a practical perspective, the most critical step in bulk droplet vitrification is the rewarming of the vitrified hepatocyte droplets. When the vitrified droplets are removed from the liquid nitrogen they can freeze within seconds if not rapidly rewarmed by instantaneously adding the warm rewarming solution.

Future optimization of bulk droplet vitrification could further improve the preservation outcomes, such as the viability, yield, and metabolic function. Both permeable and non-permeable CPAs could be tested to optimize the osmotic dehydration ahead of vitrification. This could reduce the osmotic shock during dehydration and might also allow to decrease the pre-incubated CPA concentrations. Additionally, continuous fluidic, instead of batchwise, CPA pre-incubation would theoretically allow vitrification of unlimited cell quantities. Because only general laboratory equipment is needed for bulk droplet vitrification, it is a simple and cost-effective preservation method which could potentially be used for preservation of other cell types.

Materials

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