JP6733872B2 - How to collect hepatocytes - Google Patents

Description

[0003] The present invention relates generally to the collection and cryopreservation of hepatocytes from multiple donors.

[0004] Hepatocytes make up approximately 80% of the cells in the liver and are important for both activation and eventual detoxification of many pharmacological compounds, toxins or xenobiotics. Due to increasing demand for new drugs and availability, as well as for more stringent regulatory and safety studies prior to commercial approval, isolated primary hepatocytes can be used in the laboratory for drug metabolism, efficacy and efficacy. It is a valuable resource for studying toxicity.

[0005] In recent years, significant advances have been made in the isolation and cryopreservation of primary donor hepatocytes, allowing them to be thawed rapidly and used immediately in experiments. However, variations in sex, age, ethnicity, health status, genetic background, and other factors distort the test results, so that liver metabolism in hepatocytes isolated from one human liver (individual donor) may be altered. Studies have not accurately reflected liver function in the entire population. A more accurate measure of liver metabolism is to use a mixture or "pool" of individual donor cells to create a heterogeneous population of hepatocytes.

[0006] Several methods have been proposed for collecting hepatocytes. These protocols often use long procedures that expose the cells to both physical and chemical stresses that reduce the total number of viable cells. For example, the method of US Pat. No. 7,604,929 utilizes a density gradient centrifugation step prior to the second or final cryopreservation step. This places chemical and mechanical stress on the cells, resulting in cell loss (FIGS. 1A-B) or weakening cells to die during cryopreservation. Another method, disclosed in WO 2014/045202 A2, maintains hepatocytes in a cryopreservation solution through a process of pooling. Cryopreservation solutions contain toxic reagents known to cause cell death, such as dimethylsulfoxide (DMSO).

[0007] The proposed systems and methods employ techniques that reduce cell loss throughout the process, and thus aim to increase the total number of viable cells.

[0008] The present application aims to increase the number of viable cells resulting from the process of recruiting hepatocytes. One method for cryopreserving hepatocytes from multiple sources is:
A) Thawing hepatocytes from multiple sources;
B) collecting hepatocytes from multiple sources into a preservation solution;
C) Centrifuging the pooled hepatocytes to pellet both live and non-live hepatocytes;
D) removing the stock solution;
E) mixing pelleted viable and non-viable hepatocytes with a cryopreservative;
F) dispensing the pooled hepatocytes into a vial; and G) cryopreserving the hepatocytes in the vial.

[0009] The above method is selected from the group including human hepatocytes, porcine hepatocytes, monkey hepatocytes, dog hepatocytes, cat hepatocytes, bovine hepatocytes, horse hepatocytes, sheep hepatocytes and rodent hepatocytes. Various types of hepatocytes can be utilized, including those described.

[0010] Individual sources can be aggregated based on gender, race, age, metabolic status or health status. In some cases, the pool can be randomized based on the sample set that is present at the time.

[0011] The types of preservative solutions that can be used include University of Wisconsin solution, HypoThermosol-Base, or HypoThermosol-FRS (HypoThermosol-FRS), as well as the toxicity of cryopreservatives. Other similar preservative solutions that lower the blood pressure and/or provide hepatocytes with essential nutrients. For example, the preservation solution can include fetal calf serum.

[0012] The centrifugation step does not include a density gradient to reduce physical and chemical stress on the cells. This centrifugation step pellets both viable and non-viable hepatocytes.

[0013] The pellet may be combined with a cryopreservative prior to freezing. When combining cryopreservative and pelleted cells, pipetting up and down, vortexing, shaking the vial back and forth, tapping the vial or similar process can be used to Can be used to resuspend.

[0014] When dispensing pelleted hepatocytes via aliquots or other methods into vials, the aggregated cells can be distributed at densities in the range of 8-15 million cells/ml. .. In one embodiment, 133,300 cells/ml are used.

[0015] After the final thaw of the pooled hepatocytes, the user may perform a density gradient fractionation to separate viable and non-viable cells just prior to performing the experiment. In some cases, the density gradient centrifugation step is performed at 50-200 RCF. Density gradient fractionation may include density gradient centrifugation over polyvinylpyrrolidone-coated colloidal silica particles (Percoll).

[0016] The hepatocytes used in these processes may be plated or used in suspension.

It is a figure which shows the yield of the viable rat hepatocytes after continuous centrifugation in the presence or absence of a Percoll gradient. It is a figure which shows the outline of the method of collecting and cryopreserving hepatocytes from the hepatocytes from the individual donors which were cryopreserved beforehand. FIG. 3 is a diagram outlining a method for separating viable cells from non-viable cells from the final cryopreservation step in FIG. 2.

[0020] Described herein are improved systems and methods for preparing pools of cryopreserved cells (eg, hepatocytes) from hepatocytes obtained from previously cryopreserved individual donors. It is described in. This system and method reduces the physical and chemical stress on hepatocytes during the homing process, while reducing the collection of homing viable cells during the post-thaw centrifugation step performed by the end user. increase. Generally, the system and method involves thawing vials of individual donor hepatocytes and pooling them into a preservation solution. The pooled cells are then briefly centrifuged to pellet both viable and non-viable cells and then cryopreserved at high density in multiple vials. The end user may then perform density gradient centrifugation to separate viable cells from non-viable cells just prior to experimental use. Some of the advantages of the proposed method are that it reduces exposure to mechanical, chemical and other environmental factors that reduce the number of viable cells prior to experimenting with pooled hepatocytes.

[0021] In one embodiment, previously isolated and cryopreserved hepatocytes from individual donors are stored in liquid nitrogen vapor phase at a minimum of -150°C. The individual donor vials that are pooled together may be randomized or have specific metabolic activities (eg, ECOD, cytochrome P450, general phase I or phase II), age, race, gender, ethnicity, or other representation. It can be selected based on the type determinants. The number of thawed vials depends on the number of individuals included in the mixed population and the size of the pool produced. For example, the number of individual vials used for each pool of hepatocytes can be from 2 to 50 people. Each pool can range from 300 to 1000 vials. For example, a 10 donor pool with 300 vials would be expected to require approximately 30 vials from each donor.

[0022] Individual donor hepatocytes are thawed by soaking the vials in a water bath maintained at 37°C for approximately 2 minutes or until the ice axis is almost invisible, then at 4°C containing the stock solution. Immediately decant contents into container. The stock solution is used to dilute DMSO (and/or other reagents that are toxic to hepatocytes) found in cryopreservatives. In some instances, the preservation solution also provides hepatocytes with essential nutrients to improve viability during the homing process. The volume of stock solution depends on the size of the pool to be generated, but can be made up, for example, of a 1:1 ratio of stock solution to 1 mL of cryopreserved cells. The stock solution was a University of Wisconsin solution (10 mM potassium lactobionate, 25 mM KH2PO4, 5 mM MgSO4, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurinol, and 50 g/L hydroxyethyl starch) with or without fetal bovine serum (FBS). (Hyroxyethyl starch)), hypothermosol-base (HTS-Base), hypothermosol-FRS (HTS-FRS), or other such media. Hepatocytes from multiple vials are thawed, mixed in the same container, and maintained in storage solution at 4° C. while making a “populated” population of multiple donor cells. Depending on the number of thawed vials, thawed hepatocytes can be placed in the stock solution for 2-10 hours.

[0023] After thawing and collecting the hepatocytes in a plurality of vials, the cells are centrifuged in the range of 50 to 200 relative centrifugal force (RCF) for 8 to 10 minutes to remove both viable cells and non-viable cells. Pellet. The stock solution is removed from the pellet cells, eg by inhalation. The use of a density gradient-free centrifugation step at this point in the process reduces physical and chemical stress in hepatocytes prior to the second cryopreservation. 1A-B show the effect of density gradient (eg, Percoll) centrifugation on hepatocytes compared to centrifugation without density gradient. For each subsequent spin, the density gradient (Percoll) was used to reduce the total number of viable cells compared to hepatocytes centrifuged without Percoll. These results indicate that centrifugation with density gradient solution reduces the total number of viable cells.

[0024] After pelleting the cells and removing the stock solution, they are immediately resuspended in a cryoprotectant solution, which may consist of, for example, CryoStor(R) CS10 with 10% DMSO. Suspend. The pooled non-viable and viable hepatocytes are then distributed at 10-15 million cells/mL in 1-1.5 mL aliquots per vial. It should be noted that cell counting can be performed along any step of the process, eg, with or without trypan blue, acridine orange, or propidium iodide.

[0025] The mixing or resuspension process may consist of adding a cryopreservative to the pelleted hepatocytes. Pipette up and down mixed pellets and cryopreservatives, vortex, shake back and forth in vials, tap in vials, or other similar process to break up pellets and remove total cryopreservatives. Hepatocytes can be dispersed in the.

[0026] The vials containing the pooled hepatocytes are frozen using a rate controlled refrigerator and kept in liquid nitrogen at least -150°C for at least 3 days and up to 10 years prior to shipment. It Vials are shipped to end users on dry ice or liquid nitrogen in the vapor phase (eg Dewar) and stored in liquid nitrogen at a minimum of -150°C. Immediately prior to use, the end user can immerse the vial in a water bath maintained at 37° C. for approximately 2 minutes or thaw the hepatocytes pooled until the ice axis is barely visible.

[0027] The pooled non-viable and viable hepatocytes were applied to 20-30% colloidal silica coated with a polyvinylpyrrolidone (Percoll) gradient and centrifuged at 50-200 RCF for 8-10 minutes to remove viable cells. Separate from non-viable cells. Therefore, the maximum number of viable cells is isolated immediately before use without further exposure to cryopreservation or other freeze-thaw cycles. This process recovers from 5 to 8 million+ viable cells and is ready for use in experiments. Experiments include, but are not limited to, viability; metabolic activity; transporter activity; and foreign body uptake, metabolism, efficacy, and toxicity assays.

The procedure for preparation of pooled hepatocytes from individual cryopreserved donor hepatocytes is shown in Figure 2 and describes the following procedures:
1-A Vials of cryopreserved hepatocytes from 10 donors are thawed in a 37°C water bath for approximately 2 minutes until the ice axes are almost invisible. It should be noted that some pools can consist of between 500-900 vials per donor, or in this example 50-90 vials.

1-B Thawed hepatocyte suspension (1 mL) is pipetted into a 1 L beaker containing 500 mL of Hypothermosol-FRS stock solution and kept at 4° C. to generate hepatocyte pool.

1-C Collected hepatocytes are centrifuged at 100 G for 10 minutes from stock solution (FRS) to pellet both viable and non-viable cells.

The 1-D stock solution is removed by aspiration and the cells are gently resuspended (eg, rocked back and forth) in the cryoprotectant Cryostall® CS10 medium. Cells The cell density was determined using trypan blue exclusion, additional cryoprotectant solution optionally is added to achieve approximately cells 13.3 × 10 ⁶ cells / mL.

1-E 1.5 ml or approximately 20 million cells are aliquoted into individual vials.

1-F Collected vial of hepatocytes are cryopreserved in a rate-controlled freezer and stored in liquid nitrogen vapor phase at a minimum of -150°C.

The procedure for the separation of viable cells from the aggregated hepatocytes performed by the end user is shown in Figure 2 and described in the following procedure:
2-A Thaw the pooled hepatocyte vials in a 37°C water bath for approximately 2 minutes until the ice axes are almost invisible.

The 2-B hepatocyte suspension is carefully applied to a 20-30% Percoll density gradient.

The 2-C sample is centrifuged at 200 RCF for 10 minutes to separate viable and non-viable cells.

2-D A minimum of 5 million viable cells are collected and used immediately for experiments.

[0028] It should be noted that the yield of viable cells may vary with the dilution rate and the exposure time during the pooling process. For example, in larger batches, a 1:1 ratio of stock solution to thawed cryopreservative can be too costly or difficult (volume of junk container). Moreover, it takes more time to thaw a large number of vials. Therefore, cells are exposed to high concentrations of cryopreserved toxin for long periods of time. It may also be easier to do in smaller batches, where the cryopreservative is more diluted, has a shorter collection time, and generally results in a higher yield of viable hepatocytes. For example, Step 2-D can result in 5 million viable cells for a larger batch, but up to 8 million and more viable cells in a smaller batch. For smaller batches, there may be a 4:1 ratio of thawed cryopreservative to preservative solution.

[0029] The processes herein can be applied to hepatocytes used in suspension or plated.

[0030] Although the examples describe hepatocytes, the methods and processes can be applied to other cell types.

[0031] These embodiments and features illustrated and described herein are exemplary and not intended to limit the scope of the claims recited at the end of this application, It is not the claims themselves. Multiple combinations and equivalent components, ranges and steps are considered to be within the scope of this application.

[Description regarding copyright]
[0001] A portion of the disclosure of this patent application contains material which is subject to copyright protection, including drawings. The copyright owner has no objection to facsimile reproduction of either the patent document or the patent disclosure as it appears in the Patent and Trademark Office file or record, but otherwise all copyrights Reserve.

[Cross-reference and related applications]
[0002] This application claims the benefit of each of the following co-pending applications: US Patent Application No. 62/121,619, filed February 27, 2015, which is hereby incorporated by reference in its entirety. Incorporated in the book.

Claims (10)

A method for cryopreserving hepatocytes from multiple sources, comprising:
A) Thawing hepatocytes from multiple sources;
B) collecting hepatocytes from multiple sources into a preservation solution;
C) centrifuging the pooled hepatocytes to pellet both live and non-live hepatocytes , wherein centrifugation does not include a density gradient ;
D) removing the stock solution;
E) mixing the pelleted viable hepatocytes and the pelleted non-viable hepatocytes with a cryopreservative to form a pooled pelleted hepatocyte ;
F) Distributing the pooled pelleted hepatocytes into a vial; and G) Cryopreserving the pooled pelleted hepatocytes in the vial to form the pooled cryopreserved hepatocytes. A method comprising steps. The hepatocytes are selected from the group comprising human hepatocytes, pig hepatocytes, monkey hepatocytes, dog hepatocytes, cat hepatocytes, bovine hepatocytes, horse hepatocytes, sheep hepatocytes and rodent hepatocytes, The method of claim 1. 3. The method of claim 1 or 2 , wherein the plurality of sources is comprised of a random group or a preselected group based on gender, race, age, metabolic status or health status. 4. The method according to any one of claims 1 to 3, wherein the storage solution is composed of University of Wisconsin solution, hyperthermosol-based, or hyperthermosol-FRS. The method of claim 4, wherein the storage solution is further comprised of fetal bovine serum. The method according to any one of claims 1 to 5, wherein the distribution of the collected pelleted hepatocytes is performed at a density of more than 10 million cells/ml. 7. The method according to any one of claims 1 to 6 , further comprising thawing the pooled cryopreserved hepatocytes from step G) and applying the process of density gradient fractionation. The method of claim 7, wherein the step of density gradient centrifugation is performed between 50 and 200 relative centrifugal force . 9. The method of claim 7 or 8 , wherein the density gradient fractionation comprises density centrifugation with colloidal silica particles coated with polyvinylpyrrolidone. The step of mixing the pelleted hepatocytes with a cryopreservative may include one or more of pipetting up and down, vortexing, shaking the vial back and forth, or tapping the vial. The method according to any one of claims 1 to 9 , further comprising:

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