# Patent

## Title
Consumable cryopreserved cells transiently overexpressing gene(s) encoding drug transporter protein(s) and/or drug metabolizing enzyme(s)

## Abstract

The present invention discloses cryopreserved recombinant cells for screening drug candidates that transiently overexpress one or more drug transporter proteins and/or drug metabolizing enzymes. Advantageously, such cells provide a cost-efficient consumable product that streamlines the process of screening whether drug candidates are substrates or inhibitors of drug transporter proteins and/or drug metabolizing enzymes.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is filed under 35 U.S.C. §111 as a continuation application of U.S. application Ser. No. 14/972,012, filed on Dec. 16, 2015, which is a division of U.S. application Ser. No. 14/644,000, filed on Mar. 10, 2015, which is a continuation application of International Application No. PCT/US2013/059152, filed on Sep. 11, 2013, which designates the United States and claims priority to U.S. Provisional Patent Application No. 61/699,466, filed on Sep. 11, 2012, the entire contents of which are incorporated by reference herein.


FIELD OF THE INVENTION
The present invention relates to cryopreserved recombinant cells transiently overexpressing one or more genes encoding drug transporter protein and/or drug metabolizing enzyme such that activity of the encoded protein(s) is detectable in a population of said cells following thaw from cryopreservation.
BACKGROUND OF THE INVENTION
Drug development is a costly and time consuming endeavor whereby drug candidates must satisfy certain criteria established by government agencies such as the U.S. Food and Drug Administration and European Medicines Agency prior to receiving regulatory approval for marketing thereof. Importantly, assays are conducted to screen drug candidates to determine whether any are substrates or inhibitors of one or more drug transporter proteins and/or drug metabolizing enzymes as that can have a significant effect on the absorption, distribution, metabolism and elimination of such drugs, their toxicity and drug-drug interactions.
Although cell lines stably expressing a gene encoding a drug transporter proteins or a drug metabolizing enzyme may be used for such screening, significant time and resources are required to generate and maintain frozen stocks thereof. Plus, the level of recombinant protein expressed is typically variable (laboratory to laboratory) and may deteriorate and/or become more variable over time with passage of such cells. Alternatively, freshly plated cells either stably or transiently expressing a gene encoding a drug transporter protein or a drug metabolizing enzyme may be employed for such screening. However, freshly plated cells have a limited shelf life of a few days and are difficult to ship in a manner that maintains their viability. In addition, to generate stable cell lines, the foreign transfected gene is actually integrated into the host genome of the cells and carried along with it during cycles of cells division. The chromosomal integration in the host cells will lead to permanent modification of host genome, potentially leading to abnormal expression of other genes causing unexpected changes of host behavior and unreliable experimental results. Thus, there is a need for cells suitable for screening drug candidates that reduces the investment of time and resources associated with drug development and provide reliable results.
SUMMARY OF THE INVENTION
The present invention provides cryopreserved recombinant cells suitable for screening drug candidates to determine whether any are substrates or inhibitors of one or more drug transporter proteins and/or drug metabolizing enzymes that provide reliable results and ready convenience. Desirably, the level of activity in a population of the recombinant cells following cryopreservation is comparable to that of freshly transfected cells. Additionally, the cryopreserved recombinant cells are readily packaged in a vial and shipped with dry ice or dry shipper and conveniently stored at −135° C. in liquid nitrogen upon receipt with several years shelf life. Hence, such cells provide the end user a consumable “thaw and use” product which provides convenience in conducting experiments and reduces the investment of time and resources in creating and/or maintaining cell stocks for screening drug candidates.
In one aspect, the present invention provides cryopreserved recombinant cells including one or more transiently overexpressed genes encoding a protein selected from the group consisting of a drug transporter protein and a drug metabolizing enzyme, or a combination thereof wherein activity of the drug transporter protein or the drug metabolizing enzyme or combinations is detectable in a population of the cryopreserved recombinant cells following thaw from cryopreservation.
In another aspect, the present invention provides processes of preparing transiently transfected recombinant cells which transiently overexpresses one or more genes encoding a protein selected from a drug transporter protein and a drug metabolizing enzyme or a combination thereof including transiently transfecting cells with one or more genes encoding a drug transporter protein or a drug metabolizing enzyme and cryopreserving the transiently transfected recombinant cells within 48 hrs of transfection.
These and other features of the invention will be better understood through a study of the following detailed description.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a graph of the percentage of viable cells from cell stock, cells after electroporation (EP) and cells after thaw from cryopreservation for FreeStyleTM 293-F (FS293) cells and 293-F cells grown in suspension.
FIG. 2 are images of transfected cells 4 hrs (A), 24 hrs (B) and 48 hrs (C) after plating following thaw from cryopreservation.
FIG. 3 are images of 293-F cells transfected with pOATP1B1 expression plasmid plated at (A) 0.4×106 viable cells per well and (B) 0.2×106 viable cells per well in 24-well poly-D-lysine coated plates and cultured in plating media at 24 hrs post-plating (following thaw from cryopreservation).
FIG. 4 are images of 293-F cells transfected with MATE1, MATE2K OATP1B3, long isoform OAT1 (full length cDNA with 563 amino acids; referred to herein as “OAT1 long”), short isoform OAT1 (missing 13 amino acid at C-terminus 522-534, with 550 amino acids; referred to herein as “OAT1 short”), OAT3, and pCMV vector plated at 0.4×106 cells per well in 24-well poly-D-lysine coated plates at 24 hrs post-plating (following thaw from cryopreservation).
FIG. 5 are fluorescence images of adhered HEK293 cells transfected with 50 μg/ml, 100 μg/ml or 200 μg/ml green fluorescent protein (GFP) 24 hrs (A) and 48 hrs (B) following EP.
FIG. 6 is a graph of the percentage of viable cells following EP of adhered HEK293 cells using varying amounts of DNA.
FIG. 7A is a graph of estradiol-17β-glucuronide (E17βG) uptake activity following various incubation times in adhered HEK293 cells transfected with varying amounts of DNA (i.e., 0, 50 μg/ml, 100 μg/ml, 200 μg/ml or 400 μg/ml OATP2/OATP1B1) at 48 hrs post EP.
FIG. 7B is a graph of estradiol-17β-glucuronide (E17βG) uptake activity following various incubation times in adhered HEK293 cells transfected with varying amounts of DNA (i.e., 0, 50 μg/ml, 100 μg/ml, 200 μg/ml or 400 μg/ml OATP2/OATP1B1) at 96 hrs post EP.
FIG. 8 is a graph of signal to noise ratio of estradiol-17β-glucuronide (E17βG) uptake following various incubation times in adhered HEK293 cells transfected with varying amounts of DNA (i.e., 0, 50 μg/ml, 100 μg/ml, 200 μg/ml or 400 μg/ml OATP2/OATP1B1) at 48 hrs post EP.
FIG. 9 is a graph of estradiol-17β-glucuronide (E17βG) uptake activity in adhered HEK293 cells transfected with either OATP2/OATP1B1 using a small scale EP device (OC400), OATP2/OATP1B1 using a large scale EP device (CL2), or an empty vector control.
FIG. 10 is a graph of signal to noise ratio of estradiol-17β-glucuronide (E17βG) uptake following various incubation times in adhered HEK293 cells transfected with OATP1B1 gene using either “Control” (i.e., traditional lipid transfection reagent (lipofectamine 2000, available from Invitrogen)) or STX, MaxCyte scalable EP device.
FIG. 11 is a graph of signal to noise ratio of estradiol-17β-glucuronide (E17βG) uptake following various incubation times in adhered HEK293 cells transfected with OATP1B1 that are freshly plated or plated following thaw from cryopreservation.
FIG. 12 are images of HEK293 cells transfected with OATP1B1*1a (Gene Accession No. NM_006446.4), OATP1B1*1b (Gene Accession No. NM_006446.3), OATP1B3, pCMV vector, long isoform OAT1 (full length cDNA with 563 amino acids), OAT3, OCT1 or OCT2 using MaxCyte scalable EP device and scale-up process followed by cryopreservation, thawing, plating on Poly-D-Lysine plates and incubation for 24 hrs post-plating.
FIG. 13A is a graph depicting results of a time-dependent assay of p-Aminohippuric acid (PAH) (prototypical substrate for OAT1) uptake in HEK293 cells overexpressing OAT1 or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with PAH at a concentration of 3 μM.
FIG. 13B is a graph depicting results of a kinetic assay whereby uptake of PAH at a concentration in the range of 3 to 200 μM was measured in HEK293 cells overexpressing OAT1 following incubation for 5 min. Km and Vmax, calculated using Sigma-plot, are shown as insert in the graph.
FIG. 13C is a graph depicting results of an inhibition assay whereby HEK293 cells overexpressing OAT1 were incubated with PAH at a concentration of 15 μM and probenecid (OAT1 inhibitor) at a concentration in the range of 0-300 μM for 5 min. IC50, calculated using Sigma-plot, is shown as insert in the graph.
FIG. 14A is a graph depicting results of a time-dependent assay of Estrone-3-sulfate (E3S) (prototypical substrate for OAT3) uptake in HEK293 cells overexpressing OAT3 or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with E3S at a concentration of 1 μM.
FIG. 14B is a graph depicting results of a kinetic assay whereby uptake of E3S at a concentration in the range of 0.5 to 32 μM was measured in HEK293 cells overexpressing OAT3 following incubation for 1 min. Km and Vmax, calculated using Sigma-plot, are shown as insert in the graph.
FIG. 14C is a graph depicting results of an inhibition assay whereby HEK293 cells overexpressing OAT3 were incubated with E3S at a concentration of 4 μM and probenecid (OAT3 inhibitor) at a concentration in the range of 0-300 μM for 5 min. IC50, calculated using Sigma-plot, is shown as insert in the graph.
FIG. 15A is a graph depicting results of a time-dependent assay of TEA (prototypical substrate for OCT1) uptake in HEK293 cells overexpressing OCT1 or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with TEA at a concentration of 31 μM.
FIG. 15B is a graph depicting results of a time-dependent assay of metformin (prototypical substrate for OCT1) uptake in HEK293 cells overexpressing OCT1 or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with metformin at a concentration of 3.8 μM.
FIG. 15C is a graph depicting results of a concentration-dependent assay whereby uptake of TEA at a concentration of 1, 10 and 100 μM was measured in HEK293 cells overexpressing OCT1 or pCMV vector following incubation for 10 min.
FIG. 15D is a graph depicting results of a concentration-dependent assay whereby uptake of metformin at a concentration of 0.1, 1 and 10 μM was measured in HEK293 cells overexpressing OCT1 or pCMV vector following incubation for 10 min.
FIG. 15E is a graph depicting results of an inhibition assay whereby HEK293 cells overexpressing OCT1 were incubated with TEA and OCT1 inhibitor (quinidine, verapamil or decynium-22) at various concentrations in the range of 0.1-500 μM for 10 min.
FIG. 15F is a graph depicting results of an inhibition assay whereby HEK293 cells overexpressing OCT1 were incubated with metformin at a concentration of 3.8 μM and OCT1 inhibitor cimetidine at various concentrations in the range of 4 μM to 3 mM for 10 min.
FIG. 16A is a graph depicting results of a time-dependent assay of TEA (prototypical substrate for OCT2) uptake in HEK293 cells overexpressing OCT2 or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with TEA at a concentration of 31 μM.
FIG. 16B is a graph depicting results of a time-dependent assay of metformin (prototypical substrate for OCT2) uptake in HEK293 cells overexpressing OCT2 or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with metformin at a concentration of 3.8 μM.
FIG. 16C is a graph depicting results of a concentration-dependent assay whereby uptake of TEA at a concentration of 1, 10 and 100 μM was measured in HEK293 cells overexpressing OCT2 or pCMV vector following incubation for 10 min.
FIG. 16D is a graph depicting results of a concentration-dependent assay whereby uptake of metformin at a concentration of 0.1, 1 and 10 μM was measured in HEK293 cells overexpressing OCT2 or pCMV vector following incubation for 10 min.
FIG. 16E is a graph depicting results of an inhibition assay whereby HEK293 cells overexpressing OCT2 were incubated with metformin at a concentration of 3.8 μM and OCT2 inhibitor cimetidine at a concentration in the range of 4 μM to 3 mM for 10 min. IC50, calculated using Sigma-plot, is shown as insert in the graph.
FIG. 17A is a graph depicting results of a time-dependent assay of estradiol-17β-glucuronide (E17βG) uptake in HEK293 cells overexpressing OATP1B1*1a or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with E17βG at a concentration of 1 μM.
FIG. 17B is a graph depicting results of a time-dependent assay of estrone-3-sulfate (E3S) uptake in HEK293 cells overexpressing OATP1B1*1a or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with E3S at a concentration of 1 μM.
FIG. 17C is a graph depicting results of a time-dependent assay of rosuvastatin uptake in HEK293 cells overexpressing OATP1B1*1a or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with rosuvastatin at a concentration of 1 μM.
FIG. 17D is a graph depicting results of a concentration-dependent assay whereby uptake of E17βG at a concentration in the range of 0.25 to 40 μM was measured in HEK293 cells overexpressing OATP1B1*1a following incubation for 1 min. Km and Vmax, calculated using Sigma-plot, are shown as insert in the graph.
FIG. 17E is a graph depicting results of a concentration-dependent assay whereby uptake of rosuvastatin at a concentration in the range of 0.78 to 50 μM was measured in HEK293 cells overexpressing OATP1B1*1a following incubation for 5 min. Km and Vmax, calculated using Sigma-plot, are shown as insert in the graph.
FIG. 17F is a graph depicting results of an inhibition assay whereby uptake of E17βG at a concentration of 1 μM was measured in HEK293 cells overexpressing OATP1B1*1a following incubation with inhibitor cyclosporin A at a concentration in the range of 0.04 to 30 μM for 5 min. IC50, calculated using Sigma-plot, is shown as insert in the graph.
FIG. 18A is a graph depicting results of a time-dependent assay of E17βG uptake in HEK293 cells overexpressing OATP1B1*1b or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with E17βG at a concentration of 1 μM.
FIG. 18B is a graph depicting results of a time-dependent assay of E3S uptake in HEK293 cells overexpressing OATP1B1*1b or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with E3S at a concentration of 1 μM.
FIG. 18C is a graph depicting results of a time-dependent assay of rosuvastatin uptake in HEK293 cells overexpressing OATP1B1*1b or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with rosuvastatin at a concentration of 1 μM.
FIG. 18D is a graph depicting results of a concentration-dependent assay whereby uptake of E17βG at a concentration in the range of 0.25 to 40 μM was measured in HEK293 cells overexpressing OATP1B1*1b following incubation for 1 min. Km and Vmax, calculated using Sigma-plot, are shown as insert in the graph.
FIG. 18E is a graph depicting results of an inhibition assay whereby uptake of E17βG at a concentration of 1 μM was measured in HEK293 cells overexpressing OATP1B1*1b following incubation with inhibitor cyclosporin A at a concentration in the range of 0.04 to 30 μM for 5 min. IC50, calculated using Sigma-plot, is shown as insert in the graph.
FIG. 19A is a graph depicting results of a time-dependent assay of cholecystokinin (CCK-8) uptake in HEK293 cells overexpressing OATP1B3 or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with CCK-8 at a concentration of 1 μM.
FIG. 19B is a graph depicting results of a time-dependent assay of E17βG uptake in HEK293 cells overexpressing OATP1B3 or pCMV vector following various incubation times (i.e., 1, 2, 5, 10 and 15 min.) with E17βG at a concentration of 1 μM.
FIG. 19C is a graph depicting results of a concentration-dependent assay whereby uptake of CCK-8 at a concentration in the range of 0.5 to 20 μM was measured in HEK293 cells overexpressing OATP1B3 following incubation for 1 min. Km and Vmax, calculated using Sigma-plot, are shown as insert in the graph.
FIG. 19D is a graph depicting results of a concentration-dependent assay whereby uptake of rosuvastatin at a concentration in the range of 0.78 to 50 μM was measured in HEK293 cells overexpressing OATP1B3 following incubation for 5 min. Km and Vmax, calculated using Sigma-plot, are shown as insert in the graph.
FIG. 19E is a graph depicting results of an inhibition assay whereby uptake of CCK-8 at a concentration of 1 μM was measured in HEK293 cells overexpressing OATP1B3 following incubation with inhibitor cyclosporin A at a concentration in the range of 0.04 to 30 μM for 2 min. IC50, calculated using Sigma-plot, is shown as insert in the graph.



DETAILED DESCRIPTION OF THE INVENTION
As used herein the following terms shall have the definitions set forth below.
As used herein, the term “cell” includes both primary cells as well as established cell lines (e.g., human embryonic kidney HEK293 cells, Chinese hamster ovary CHO, Madin-Darby Canine Kidney Cells MDCK, Pig Kidney Epithelial Cells LLC-PK1, human epithelial colorectal adenocarcinoma cells Caco-2 and Chinese hamster lung fibroblast V79 cells).
As used herein, the term “drug transporter protein” refers to a membrane bound transport protein that includes, but is not limited to ATP binding cassette (ABC) transporters and solute carrier (SLC) transporters.
As used herein, the term “drug metabolizing enzyme” includes, but is not limited to, cytochromes such as cytochrome (CYP) P450; UDP-glucouronyl transferase and other non-CYP drug metabolizing enzymes such as alcohol dehydrogenase, monoamine oxidase and aldehyde oxidase.
As used herein, the term “detectable” means that the activity of a selected probe substrate in cells transfected with a drug transporter protein and/or drug metabolizing enzyme shall be higher than the activity of the same probe substrate in cells transfected with empty vector; desirably, the difference in activity will be at least 5-fold.
As used herein, the use of upper case letters in transporter nomenclature identifies the human protein/gene, i.e., MRP2/ABCC2, etc.; smaller case letters indicate the transporter derives from a preclinical (nonhuman mammalian) species, i.e., Mrp2/Abcc2, etc. Unless otherwise specified, a gene is derived from any species (e.g., human or other mammal).
As used herein, the terms “OATP1B1” “OATP2” and “SLCO1B1” are interchangeable and refer to a human protein/gene that corresponds to the nonhuman protein/gene Oatp2. Unless noted otherwise, reference to OATP1B1 is to OATP1B1*1b.
As used herein, the terms “OAT1” and “SLC22A6” are interchangeable and refer to an organic anion transporter 1. Unless noted otherwise, reference to OAT1 is to the full length cDNA encoding with 563 amino acids (also referred to herein as “OAT1 long”).
Exemplary ABC transporters include, but are not limited to those listed below in Table 1.








TABLE 1





GENE NAME
PROTEIN NAME







MDR1/P-gp/ABCB1
Multidrug Resistance Protein1


MRP1/ABCC1
Multidrug resistance protein 1


MRP2/ABCC2
Multidrug resistance-associated protein 2


MRP3/ABCC3
Multidrug resistance protein 3


MRP4/ABCC4
Multidrug resistance protein 4


MRP5/ABCC5
Multidrug resistance protein 5


MRP6/ABCC6
Multidrug resistance protein 6


MRP7/ABCC7
Multidrug resistance protein 7


MRP8/ABCC8
Multidrug resistance protein 8


BCRP/ABCG2
Breast Cancer Resistance Protein


BSEP/ABCB11
Bile Salt Export Pump









Exemplary SLC transporters include, but are not limited to those listed below in Table 2.








TABLE 2





GENE NAME
PROTEIN NAME







OSTα
Organic solute transporter α


OSTβ
Organic solute transporter β


OATP1B1*/SLCO 1B1/
Organic anion-transporting polypeptide 1B1


OATP2


OATP1B3/SLCO 1B3
Organic anion-transporting polypeptide 1B3


OAT1/SLC22A6
Organic anion transporter 1


OAT2/SLC22A7
Organic anion transporter 2


OAT3/SLC22A8
Organic anion transporter 3


OAT4/SLC22A11
Organic anion transporter 4


OCT1/SLC22A1
Organic cation transporter 1


OCT2/SLC22A2
Organic cation transporter 2


OCT3/SLC22A3
Organic cation transporter 3


OATP1A2/SLCO1A2
Organic anion-transporting polypeptide 1A2


OATP2B1/SLCO2B1
Organic anion-transporting polypeptide 2B1


PEPT1/SLC15A1
Peptide Transporter 1


PEPT2/SLC15A2
Peptide Transporter 2


OCTN1/SLC22A4
Organic cation/ergothioneine transporter


OCTN2/SLC22A5
Organic cation/carnitine transporter


MATE1/SLC47A1
Multidrug and toxin extrusion 1


MATE2K/SLC47A2
Multidrug and toxin extrusion 2K


URAT1/SLC22A12
Urate Transporter 1


ASBT/SLC10A2
Apical sodium/bile acid co-transporter


NTCP/SLC10A1
Sodium/taurocholate co-transporting peptide





*In this instance, OATP1B1 includes OATP1B1*1a and OATP1B1*1b.






Exemplary SLC transporters tested, but are not limited to those listed below in Table 3.









TABLE 3







GENE




ACCESSION


GENE NAME
FULL NAME
NUMBER







OATP1B1*1a/
Organic anion-transporting
NM_006446.4


SLCO1B1*1a
polypeptide 1B1 Wild Type



(388A)


OATP1B1*1b/
Organic anion-transporting
NM_006446.3


SLCO1B1*1b
polypeptide 1B1 SNP 388A > G


OATP1B3/SLCO1B3
Organic anion-transporting
NM_019844



polypeptide 1B3


OAT1/SLC22A6
Organic anion transporter 1
NM_004790


OAT3/SLC22A8
Organic anion transporter 3
NM_004254


OCT1/SLC22A1
Organic cation transporter 1
NM_003057


OCT2/SLC22A2
Organic cation transporter 2
NM_003058









Cells suitable for use in the present invention include mammalian cells, for example, as derived from human or non-human (e.g., mouse, rat, dog, monkey, hamster and pig, etc.). In certain embodiments, the cells are hepatocytes, or endothelial cells.
Gene delivery systems for introducing gene(s) into a population of cells are known to a skilled artisan. Virus-based gene delivery methods may be used but require special handling of the cells due to safety concerns. Although lipid-based transfection methods may be used, lipid-based transfection reagents are relatively costly and such methods are not amenable to large-scale manufacturing processes. Additionally, lipid-based transfection methods result in relatively low gene delivery efficiency and relatively delayed protein expression (generally 72 to 96 hours post transfection) (data not shown). Electroporation (EP) is preferable as it is amenable to large-scale manufacturing processes and avoids the safety issues of viral-based gene delivery methods. Further, EP results in relatively efficient gene delivery.
After gene delivery into a population of cells, gene(s) encoding a drug transporter protein and/or a drug metabolizing enzyme will be overexpressed such that activity of the protein(s) encoded therefrom are detectable following thaw from cryopreservation. Drug candidates can be tested to determine if any are substrates or inhibitors of the protein(s) encoded from the overexpressed gene(s) by incubation of the recombinant cells therewith. In particular, if a drug candidate is a substrate of a drug transporter protein and/or a drug metabolizing enzyme, the drug candidate will be affected. For instance, if the drug candidate is a substrate of a drug transporter protein, the drug candidate will be translocated in or out of the recombinant cell via the drug transporter protein. However, if the drug candidate is an inhibitor of the drug transporter protein, the drug candidate will inhibit translocation of a substrate of the drug transporter protein in or out of the recombinant cell.
Alternatively, assays can be conducted using whole cells or subcellular fractions thereof (microsome/cytosol).
Additionally, recombinant cells of the present invention may be further transfected with RNAi or siRNA of the transiently overexpressed gene(s) to knockdown/knockout the expression thereof as is desirable for certain assays. Primary cells (e.g., hepatocytes) can be transfected with RNAi or siRNA directed against any ABC transporters, SLC transporters or any other drug metabolizing enzymes to knockdown/knockout the expression of specific genes.
In one aspect (1), the disclosure provides a cryopreserved recombinant cell including one or more transiently overexpressed genes encoding a protein selected from the group consisting of a drug transporter protein and a drug metabolizing enzyme, or a combination thereof wherein activity of the drug transporter protein or the drug metabolizing enzyme or combinations is detectable in a population of the cryopreserved recombinant cell following thaw from cryopreservation.
In an aspect (2), the disclosure provides the invention of aspect (1), wherein said one or more genes encodes a drug metabolizing enzyme.
In an aspect (3), the disclosure provides the invention of aspect (2), wherein the drug metabolizing enzyme is selected from the group consisting of cytochrome P450, UDP-glucouronyl transferase, alcohol dehydrogenase, monoamine oxidase and aldehyde oxidase.
In an aspect (4), the disclosure provides the invention of aspect (1), which transiently overexpresses one or more genes encoding a protein selected from the group consisting of an ATP binding cassette transporter and a solute carrier transporter protein.
In an aspect (5), the disclosure provides the invention of aspect (4), wherein said one or more genes is selected from the group consisting of MDR1/Mdr1a/Mdr1b, MRP1/Mrp1, MRP2/Mrp2, MRP3/Mrp3, MRP4/Mrp4, MRP5/Mrp5, MRP6/Mrp6, MRP7/Mrp7, MRP 8/Mrp8, BCRP/Bcrp, BSEP/Bsep, OATP2/Oatp2, OATP1B3/Oatp1b3, OAT1/Oat1, OAT2/Oat2, OAT3/Oat3, OAT4/Oat4, OCT1/Oct1, OCT2/Oct2, OATP1/Oatp1, PEPT1/Pept1, PEPT2/Pept2, OCTN1/Octn1, OCTN2/Octn2, MATE1/Mate1, MATE2K/Mate2, URAT1/Urat1, ASBT/Asbt, and NTCP/Ntcp.
In an aspect (6), the disclosure provides the invention of aspect (4), wherein said one or more genes encodes a protein that is an ATP binding cassette transporter selected from the group consisting of MDR1/Mdr1a/Mdr1b, MRP1/Mrp1, MRP2/Mrp2, MRP3/Mrp3, MRP4/Mrp4, MRP5/Mrp5, MRP6/Mrp6, MRP7/Mrp7, MRP 8/Mrp8, BCRP/Bcrp, and BSEP/Bsep.
In an aspect (7), the disclosure provides the invention of aspect (4), wherein said one or more genes encodes a protein that is a solute carrier transporter selected from the group consisting of OATP2/Oatp2, OATP1B3/Oatp1b3, OAT1/Oat1, OAT2/Oat2, OAT3/Oat3, OAT4/Oat4, OCT1/Oct1, OCT2/Oct2, OCT3/Oct3, OATP1/Oatp1, PEPT1/Pept1, PEPT2/Pept2, OCTN1/Octn1, OCTN2/Octn2, MATE1/Mate1, MATE2K/Mate2, URAT1/Urat1, ASBT/Asbt, and NTCP/Ntcp.
In an aspect (8), the disclosure provides the invention of aspect (4), wherein said one or more genes is selected from OATP1B1*1a, OATP1B1*1b, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1 and MATE2K.
In an aspect (9), the disclosure provides the invention of aspect (1), wherein the one or more genes is derived individually from human or an animal species selected from mouse, rat, guinea pig, dog, and monkey.
In an aspect (10), the disclosure provides the invention of aspect (1), wherein said cell is derived from a mammal.
In an aspect (11), the disclosure provides the invention of aspect (10), wherein said cell is selected from the group consisting of HEK293, CHO, MDCK, LLC-PK1, Caco-2 and V79 cells.
In an aspect (12), the disclosure provides the invention of aspect (10), wherein the mammal is selected from the group consisting of human, monkey, dog, rat, mouse, porcine and hamster.
In an aspect (13), the disclosure provides the invention of aspect (1), wherein said cell comprises a hepatocyte.
In an aspect (14), the disclosure provides the invention of aspect (1), wherein said cell comprises an endothelial cell.
In an aspect (15), the disclosure provides the invention of aspect (1), wherein activity of the protein(s) is detectable in a population of said cell at least 24 hours post plating following thaw from cryopreservation.
In an aspect (16), the disclosure provides the invention of aspect (1), wherein activity of the protein(s) is detectable in a population of said cell at least 48 hours post plating following thaw from cryopreservation.
In an aspect (17), the disclosure provides the invention of aspect (1), wherein activity of the protein(s) is detectable in a population of said cell at least 72 hours post plating following thaw from cryopreservation.
In another aspect (18), the disclosure provides a process of preparing transiently transfected recombinant cells which transiently overexpresses one or more genes encoding a protein selected from a drug transporter protein and a drug metabolizing enzyme including transiently transfecting cells with one or more genes encoding a drug transporter protein or a drug metabolizing enzyme and cryopreserving the transiently transfected recombinant cells within 48 hrs of transfection.
In an aspect (19), the disclosure provides the invention of aspect (18), wherein the transient transfection step includes electroporation.
EXAMPLES
Cells were cultured under standard sterile practices for cell culture, and transiently transfected using EP. Following EP, cells were assayed for protein activity both before as well as after being frozen, thawed and plated. As detailed below, cells cultured in suspension and adherent cell cultures were both successfully transiently transfected and exhibited substantial activity of the recombinant protein following thaw from cryopreservation.
Cells Cultured in Suspension
In brief, on Day 1, FreeStyle 293 Cells and 293-F cells were each passaged into appropriate sized shaker flasks at a density of 0.7-1.0×106 cell/ml using supplemented CD293 medium (i.e., CD293 medium (available from Gibco, Cat. No. 11913-019, Life Technologies Corp., Carlsbad, Calif.) supplemented with 4 mM L-Glutamine (available from Gibco, Cat. No. 25030-081, Life Technologies Corp., Carlsbad, Calif.)) or supplemented ExcellTM 293 serum free media (available from Sigma, Cat. No. 14571C, Sigma-Aldrich, St. Louis, Mo.) supplemented with 6 mM L-Glutamine. Cell viability and cell number were determined using a Cellometer (available from Nexcelom Bioscience, Lawrence, Mass.).
On Day 2, EP of cells was executed. In short, following a determination of cell viability and cell density, cells were pelleted down by spinning at 100 g for 5 min, after which the media was aspirated and cells resuspended in 30 ml EP Buffer (available from MaxCyte, Cat. No. B201, MaxCyte Inc., Gaithersburg, Md.). The cell suspension was transferred to 50 ml Falcon tubes, pelleted down as described above, and resuspended in an appropriate amount of EP Buffer to reach 100×106 cells/ml which was used as the cell stock. DNAs to be used for EP were prepared in sterile water at a final concentration of 5 mg/ml. For each sample, 0.4 ml of cell stock and DNA was placed in a sterile 1.5 ml eppendorf tube resulting in a final concentration of 200 μg/ml (Table 4) or 300 μg/ml DNA (Table 10 and Table 11) and cell density of 40×106 cells per sample.













TABLE 4










CELL







STOCK




CELL

VOL.
[DNA]



SAMPLE #
TYPE
PLASMID(S)
(ml)
(ug/ml)





















A
1, 2
FS293
pOATP1B1
0.4
200


B
3, 4

pCMV6
0.4
200


C
 5

EP Buffer (16 μl)
0.4
—


D
6, 7
293-F
pOATP1B1
0.4
200


E
8, 9

pCMV6
0.4
200


F
10

EP Buffer (16 μl)
0.4
—









Samples were transferred into an OC-400 Processing Assembly (available from MaxCyte, Cat. No. OC-400R, MaxCyte Inc., Gaithersburg, Md.) which followed the manufacture instructions for EP of HEK cells. Following EP, the cells were carefully pipetted out and transferred into the bottom of a 125 ml shaker flask and incubated for 20 min at 37° C. with 8% CO2, after which pre-warmed 40 ml CD293 media was added into the shaker flask to reach cell density at 1×106 cells/ml. The cells were incubated for 30 min at 37° C. and 8% CO2. After 30 min recovery, cell viability and cell density were determined. A portion of cells (i.e., 20×106 cells) was used for plating and the rest was cryopreserved, or all of the cells were cryopreserved. It is contemplated that recombinant cells may be cryopreserved within 48 hrs of transfection and exhibit activity of protein(s) encoded from transfected gene(s) at a detectable level following thaw from cryopreservation.
For plating cells following EP, 20×106 cells were pelleted down by spinning at 100 g for 5 min and then resuspended in 20 ml pre-warmed plating media (DMEM with high glucose (available from Gibco, Cat. No. 11965092, Life Technologies Corp., Carlsbad, Calif.), supplemented with 0.1 mM non-essential amino acids (available from Gibco, Cat. No. 11140050, Life Technologies Corp., Carlsbad, Calif.), 10% FBS (available from SAFC Biosciences, Cat. No. 12016C, Sigma, St. Louis, Mo.)) (cell density of 1×106 cells/ml). Cells were placed in 24-well tissue culture plates poly-D-Lysine coated, Corning BiocoatTM (available from Corning Life Sciences, Tewksbury, Mass.) at a density of 0.2×106 cells/well and 0.4×106 cells/well and incubated at 37° C. with 8% CO2 so as to determine the impact of seeding density on uptake activity. Media was replaced 4 hours later and then every 24 hours until the day of assaying. On Days 4, cells were assayed for OATP1B1 activity as described below.
For cryopreservation, cells were pelleted then resuspended in freshly prepared ice-cold freezing media (9 parts supplemented CD293 medium and 1 part DMSO which was syringe filtered to sterilize) at a density of 10×106 cell/ml. Cryo vials were filled with 1 ml of this cell suspension, and placed on ice-cold Mr Frosty freezing container (available from Thermal Scientific), which was stored in −80° C. freezer overnight after which the vials were transferred into liquid nitrogen.
Cryopreserved cells were assayed for OATP1B1 activity as described below. In brief, on Day 1, cryopreserved cells were removed from liquid nitrogen to dry ice, and then thawed in a water bath at 37° C. for about 2 min. Cells were transferred into 10 ml of plating media as described above which is pre-warmed to a temperature of about 37° C. and the viability and cell density determined. Cells were pelleted down and resuspended in supplemented DMEM media at a cell density of 1×106 viable cells/ml. Cells were plated in the same manner described above for plating cells following EP (which had not been cryopreserved) and assayed for OATP1B1 activity at 24, 48 and 72 hrs following plating thereof.
Adherent Cell Cultures
In brief, HEK293 cells were cultured in 5 Layer Corning® CellStack® (available from Corning Inc. Life Sciences, Tewksbury, Mass.) using plating media containing DMEM (high glucose) available from Gibco Cat. No. 11965118, Life Technologies Corp., Carlsbad, Calif.; Penicillin-Streptomycin (10,000 units/ml) available from Gibco Cat. No. 15140-122, Life Technologies Corp., Carlsbad, Calif.; L-Glutamine (200 mM) available from Gibco Cat. No. 25030-081, Life Technologies Corp., Carlsbad, Calif.; Sodium Pyruvate, available from Gibco Cat. No. 11360, Life Technologies Corp., Carlsbad, Calif.; FBS available from Sigma-Aldrich Corp., St. Louis, Mo. in a ratio of 100:1:1:1:10. On Day 1, about 24 hrs before EP, HEK293 cells were trypsinized, cell viability and cell number determined after which cells were passaged to fresh multilayer chamber flasks at 30-40% confluency. Cells were incubated at 37° C. with 5% CO2.
On Day 2, EP of cells was executed. In short, cells were harvested, cell viability and cell number determined after which cells were pelleted down by spinning at 100 g for 5 min and the media aspirated. Cells were resuspended in EP buffer and pelleted down by spinning at 100 g for 5 min, then resuspended in an appropriate amount of EP Buffer to reach 50×106 cells/ml which was used as the cell stock. DNAs to be used for EP were prepared in sterile water at a final concentration of 5 mg/ml. For each sample used for OC-400 processing assembly, 0.4 ml of cell stock and DNA was placed in a sterile 1.5 ml eppendorf tube resulting in a final concentration of 50 μg/ml, 100 μg/ml, 200 μg/ml or 400 μg/ml DNA as indicated in FIGS. 5-9 and cell density of 40×106 cells per sample. For each sample used for CL-2 processing assembly, 10 ml of cell stock and DNA was placed in 50 ml sterile conical tube resulting in a final concentration of 100 μg/ml DNA.
Samples were transferred into an OC-400 or CL-2 processing assembly (available from MaxCyte, Cat. No. OC-400R and CL2-R, MaxCyte Inc., Gaithersburg, Md.) which followed the manufacture instructions for EP of HEK cells. Following EP, the cells were carefully pipetted out and transferred into 6-well tissue culture plates and incubated for 20 min at 37° C. with 5% CO2, after which cells were removed and placed in a 50 ml conical tube containing pre-warmed plating media. Cell viability and cell density were determined. A portion of cells (i.e., 20×106 cells) was used for plating and the rest was cryopreserved.
For plating cells following EP, cells were pelleted down by spinning at 100 g for 5 min and then resuspended in pre-warmed plating media (cell density of 1×106 cells/ml). Cells were placed in 24-well tissue culture plates (poly-D-Lysine coated, Corning BiocoatTM (available from Corning Life Sciences, Tewksbury, Mass.)) at a density of 0.4×106 cells/well and incubated at 37° C. with 5% CO2. Media was replaced 4 hours later and then every 24 hours until the day of assaying. On Days 4 and 6, cells were assayed for OATP1B1 activity.
For cryopreservation, cells were pelleted then resuspended in freshly prepared ice-cold freezing media (9 parts plating medium and 1 part DMSO which was syringe filtered to sterilize) at a density of 10×106 cell/ml. Cryo vials were filled with 1 ml of this cell suspension, and placed on ice-cold Mr Frosty freezing container (available from Thermal Scientific) stored in −80° C. freezer overnight after which the vials were stored in liquid nitrogen.
Cryopreserved cells were assayed for OATP1B1 activity. Notably, cells were plated in the same manner described above for plating cells following EP (which had not been cryopreserved) and assayed for OATP1B1 activity (as described below) at 48 hrs following plating thereof.
Assaying Transporter Activity
In brief, substrate solution was prepared for OATP1B1*1a and OATP1B1*1b using 2 μM estradiol-17β-glucuronide (99% of cold E17βG and 1% of [3H]-E17βG); for OATP1B3 using 2 μM CCK-8 (99% of cold CCK-8 and 1% of [3H]-CCK-8); for OAT1 short using 1 μM Para-aminohippurate (PAH) (90% of cold PAH and 10% of [3H]-PAH); for OAT1 long using 1 μM or 3 μM Para-aminohippurate (PAH) (90% of cold PAH and 10% of [3H]-PAH); for OAT3 using 1 μM or 2 μM Estrone-3-sulfate (99% of cold E3S and 1% of [3H]-E3S); for OCT1 and OCT2 using 30 μM Tetraethylammonium Bromide (100% [14C]-TEA); for MATE1 and MATE2K using 10 μM Metformin (100% [14C]-Metformin) or 10 μM Tetraethylammonium Bromide (100% [14C]-TEA); in Krebs-Henseleit Buffer pH 7.4 (available from Sigma, Cat. No. K3753, Sigma-Aldrich, St. Louis, Mo.) and incubated at 37° C. for at least 20 min. Culture media was aspirated from cells to be assayed and cells washed thrice with pre-warmed KHB Buffer. Cells were subsequently incubated with Uptake Buffer at 37° C. for 10 min. For MATE1 and MATE2K, cells were washed and pre-incubated with KHB buffer containing 20 mM NH4Cl for 10 min. Assays were initiated by adding 0.3 ml substrate solution into each well and incubated at 37° C. for 5 min with samples for OCT1 and OCT2 incubated for 10 min.
The reaction was quickly stopped after the incubation period by aspirating substrate solution from cells then washing cells thrice with cold Uptake Buffer. Cells were then incubated with lysing solution (0.1% SDS with 0.1% v/v 1M NaOH in Dulbecco's Phosphate-Buffered Saline (DPBS) buffer) for 15-20 minutes while being shaken. The substrate solution was triturated and 0.4 ml of the resultant cell lysis placed in 5 ml scintillation tube with 5 ml of scintillation liquid for analysis with scintillation counter.
As illustrated in FIG. 1, cell viability dropped 1-5% after EP relative to that of the cell stock. Additionally, after cryopreservation, cell viability dropped an additional 10-15% relative to that after EP. Nonetheless, even after EP and thaw from cryopreservation, cell viability is greater than 75%.
Cell morphology and uptake activity was examined following cryopreservation after 30 min recovery and 24 hrs recovery post transfection. Table 5 illustrated cell morphology and uptake activity with 24 hrs recovery was reduced compared to 30 min recovery.











TABLE 5







Cell





Recovery time prior
Confluency
Uptake Activity


SAMPLE
to cryopreservation
at 24 hrs
(pmole/mg/min)
S:N







OATP1B1
24 HOURS
40-50%
0.59
0.44


OATP1B1
30 MIN
70-75%
5.78
4.32


VECTOR
30 MIN
90-95%
1.34









Cell morphology and confluency of transfected cells thawed from cryopreservation were examined after various periods of time following plating at a density of 0.4×106 cells per well in 24-well poly-D-lysine coated Corning BiocoatTM plates. In particular, FIG. 2 illustrates OATP1B1 transiently transfected cells cultured at 4 hrs, 24 hrs and 72 hrs post plating. Additionally, cell confluency at 24 hrs, 48 hrs and 72 hrs post-plating of these cells is recorded in Table 6 below.












TABLE 6







CELLS
24 hrs
48 hrs
72 hrs









FS293 with pOATP1B1
80-90%
90-95% 
80-85%



FS293 with pCMV6 vector
70-80%
90-95% 
90%



293-F with pOATP1B1
90-95%
95-100%
80-85%



293-F with pCMV6 vector
90-95%
95-100%
80-85%










Desirably, after EP and cryopreservation, the cells form a monolayer on poly-D-lysine coated Corning BiocoatTM plates achieving 80-90% confluency at 24 hrs post-plating, 90%-100% confluency at 48 hrs post-plating.
FIG. 4 illustrates cells, transiently transfected with MATE1, MATE2K, OATP1B3, OAT1 long, OAT1 short, OAT3, and pCMV vector respectively, cultured at 24 hrs post plating after thawed from cryopreservation.
As illustrated in FIG. 5, the expression of Green Fluorescent Protein (GFP) in adhesion HEK293 cells was increased with increasing concentration of DNA. Additionally, GFP expression increased at the 48 hr timepoint relative to the 24 hr timepoint. In particular, GFP transfection efficiency by EP achieved 100% at 24 hrs with 200 μg/ml DNA and 100% fluorescent cell staining at 48 hrs with 100 μg/ml DNA. Hence, GFP protein expression level in transfected cells increased with increased DNA concentration and at 48 hrs relative to 24 hrs.
Uptake activity of suspension cultured 293 cells transfected with OATP1B1 (pOATP1B1) and control vector (pCMV) were assayed at various time points following EP. In brief, transfected cells were plated at a density of 0.4×106 cells/well in 24-well poly-D-lysine coated Corning BiocoatTM plates following EP or after thaw from cryopreservation. OATP1B1 uptake activity and uptake ratio were determined using probe substrate, estradiol-17β-glucuronide, in both fresh plated cells (“fresh”) and cryopreserved cells (“cryo”) at various timepoints post plating as detailed in Table 7 below.










TABLE 7







CELLS/CULTURE

UPTAKE ACTIVITY



MEDIA, FRESH OR
CELL PLATING
(pmol/mg/min)/confluence
UPTAKE











CRYO
TIME POINT (HR)
pOATP1B1
pCMV
RATIO














293-F in CD293, fresh
48
15.4 (85%)   
0.7 (90%)   
22.0


293-F in CD293, cryo
48
15.1 (95-100%)
0.9 (95-100%)
16.8


FS293 in Excell, cryo
24
36.4
1.9
19.2



48
10.0
0.7
14.3



72
6.6
1.0
6.6


293-F in CD293, cryo
24
27.4
1.5
18.3



48
15.1
0.9
16.8



72
9.9
1.0
9.9





Note:


The number appearing in parentheses is the cell confluency at assay time.






OATP1B1 uptake activity and uptake ratio in transfected cells following thaw from cryopreservation is consistent with those in freshly plated transfected cells. In both cells types 293-F and FS293, the highest uptake activity and uptake ratio is observed at 24 hrs post plating.
Morphology and cell confluency of transfected cells (FS293 or 293-F) were examined at 24 hrs, 48 hrs and 72 hrs post-plating in 24-well poly-D-lysine coated Corning BiocoatTM plates at plating density of either 0.4×106 cells/well or 0.2×106 cells/well after thaw from cryopreservation. Cell confluency at 24 hrs post-plating are summarized below in Table 8. Cell confluency at 48 hrs and 72 hrs are similar to those achieved at 24 hrs (data not shown). Additionally, FIG. 3 provides images of transfected cells plated at (A) 0.4×106 cells per well and (B) 0.2×106 cells per well 24 hrs post-plating following thaw from cryopreservation at a confluence of 90-95% and 60-70%, respectively.









TABLE 8





CELLS, CULTURE MEDIA,
0.4 × 106
0.2 × 106


TRANSFECTED DNA
CELLS/WELL
CELLS/WELL







FS293 with pOATP1B1 in Excell
80-90%
30-50%


FS293 with pCMV vector in Excell
70-80%
50%


293-F with pOATP1B1 in CD293
90-95%
60-70%


293-F with pCMV6 vector in CD293
90-95%
80%









For optimal assay performance, plating cells at a density of 0.4×106 is preferable to that of 0.2×106 as it achieves higher cell confluency and higher uptake activity.










TABLE 9









UPTAKE ACTIVITY




(pmol/mg/min)/confluence
UPTAKE










CELLS
pOATP1B1
pCMV6
RATIO













FS293 cells, 0.2 × 106 cells/well
10.5
3.0
3.5


FS293 cells, 0.4 × 106 cells/well
36.4
1.9
19.2


293-F cells, 0.2 × 106 cells/well
20.2
1.5
13.5


293-F cells, 0.4 × 106 cells/well
27.4
1.5
18.3









Following EP, cell viability was examined using trypan blue and hemocytometer or cellometer.
As illustrated in FIG. 6, when using adhesion HEK293 cells, cell viability post EP dropped with increasing amounts of DNA used in EP. Nonetheless, cell viability following transfection with pOATP1B1 was ranged from 89% to 77% and that following transfection with empty vector was 90%.
As illustrated in FIG. 7, when using adhesion HEK293 cells, OATP1B1 mediated uptake of Estradiol-17β-glucuronide in the fresh plated transient transfected adhesion HEK293 cells is time-dependent. Notably, uptake activity and uptake ratio increased with increasing amounts of DNA used in EP. However, OATP1B1 mediated uptake of Estradiol-17β-glucuronide reduced at the 96 hr timepoint relative to the 48 hr timepoint. Further, as illustrated in FIG. 8, the signal to noise ratio (i.e., uptake ratio) of estradiol-17β-glucuronide increased with the increase of amount of DNA and assay incubation time, in adhesion HEK293 cells transfected with OATP1B1 relative to empty vector at 48 hrs post EP.
As illustrated in FIG. 9, when using adhesion HEK293 cells, estradiol-17β-glucuronide uptake in OATP1B1 transiently expressed HEK293 cells using small scale EP device and large scale EP device is consistent for both uptake activity and signal to noise ratio (i.e., uptake ratio). 100 μg/ml DNA was used in the experiments.
As illustrated in FIG. 10, when using adhesion HEK293 cells, OATP1B1 uptake activity is compared between the cells transfected using traditional lipid transfection reagent (control: lipofectamine 2000, available from Invitrogen) and EP using STX, MaxCyte Inc., Gaithersburg, Md. Notably, cells transfected using EP resulted in a pronouncedly greater signal to noise ratio relative to those cells transfected with lipid transfection reagent.
As illustrated in FIG. 11, when using adhesion HEK293 cells, OATP1B1 uptake activity in both freshly plated EP transfected cells and cells following thaw from cryopreservation was detectable.
Uptake activity of suspension cultured 293 cells transfected with OATP1B1*1a, OATP1B1*1b, OATP1B3, OAT1 long, OAT1 short, OAT3, OCT1, OCT2, MATE1, MATE2K or control vector (pCMV) were assayed at 24 hrs post plating after thaw from cryopreservation. In brief, the transfected cells were plated at a density of 0.4×106 cells/well in 24-well poly-D-lysine coated Corning BiocoatTM plates following EP and after thaw from cryopreservation. SLC transporter uptake activity and uptake ratio were determined using probe substrates as indicated at 24 hrs post plating as detailed in Table 10 below.










TABLE 10









UPTAKE ACTIVITY




(pmol/mg/min)/











TRANS-

SLC

UPTAKE


PORTERS
SUBSTRATE
transporter
pCMV6
RATIO















OATP1B1*1a
2
μM E17bG
41.0
1.03
40


OATP1B1*1b
2
μM E17bG
32.6
0.88
37


OATP1B3
2
μM CCK-8
28.7
0.2
145


OATP1B3
2
μM CCK-8
77.0
0.79
98


OAT1 long
1
μM PAH
13.1
0.3
39


OAT1 short
1
μM PAH
9.7
0.3
29


OAT1 long
3
μM PAH
15.0
0.71
21


OAT3
1
μM E3S
44.7
1.2
38


OAT3
2
μM E3S
60.9
1.62
38


OCT1
30
μM TEA
127.6
5.63
23


OCT2
30
μM TEA
100.5
5.53
18


MATE1
10
μM Metformin
71.4
6.0
12



10
μM TEA
46.3
4.3
11


MATE2K
10
μM Metformin
33.5
5.2
6.5



10
μM TEA
46.6
6.1
7.6









As reflected in Table 10 above, the recombinant cells exhibited strong uptake activity towards their specific prototypical substrate each of which had an uptake ratio above 10. Notably, an uptake ratio above 5 indicates a successful process.
As reflected in Table 11, the post-thaw viability for recombinant cryopreserved cells was determined to be above 90%.










TABLE 11








Post-thaw



Cells
Viability









OATP1B1*1a
94.2%



OATP1B1*1b
96.1%



OATP1B3
95.5%



OAT1 long
93.5%



OAT3
93.8%



OCT1
95.1%



OCT2
96.1%










Each of these recombinant cells as well as a control vector (pCMV) was examined 24 hrs post-plating (after cryopreservation). Confluency for each of these cells 24 hrs post-plating was 85% or greater as reflected in Table 12 below.










TABLE 12







Transfected Cells
24-h confluency









OATP1B1*1a
90%



OATP1B1*1b
95%



OATP1B3
95%



OAT1 long
90%



OAT3
90%



Vector
95%



OCT1
95%



OCT2
85%










As illustrated in FIG. 12, each of the 8 cryopreserved recombinant cells formed a confluent monolayer following thawing, plating on Poly-D-Lysine plates and incubation for 24-hrs post-plating.
As illustrated in FIGS. 13-19 and Tables 13-14, the kinetic and inhibition profiles examined in cryopreserved recombinant cells expressing a transporter protein was consistent with reported values. Specifically, as illustrated in FIGS. 13A-13C, the kinetics of PAH uptake by recombinant cells expressing OAT1 and inhibition profile of probenecid thereof is consistent with reported values. As illustrated in FIGS. 14A-14C, the kinetics of E3S uptake by recombinant cells expressing OAT3 and inhibition profile of probenecid thereof is consistent with reported values. As illustrated in FIGS. 15A-15F, the kinetics of TEA and metformin uptake by recombinant cells expressing OCT1 as well as inhibition profile thereof is consistent with reported values. As illustrated in FIGS. 16A-16E, the kinetics of TEA and metformin uptake by recombinant cells expressing OCT2 as well as inhibition profile is consistent with reported values. As illustrated in FIGS. 17A-17F, the kinetics of E17βG, E3S and rosuvastatin uptake by recombinant cells expressing OATP1B1*1a as well as inhibition profile of E17βG uptake by cyclosporin A is consistent with reported values. As illustrated in FIGS. 18A-18E, the kinetics of E17βG, E3S and rosuvastatin uptake by recombinant cells expressing OATP1B1*1b as well as inhibition profile of E17βG uptake by cyclosporin A is consistent with reported values. As illustrated in FIGS. 19A-19E and Tables 13-14, the kinetics of CCK-8, E17βG and rosuvastatin uptake by recombinant cells expressing OATP1B3 as well as inhibition profile of CCK-8 uptake by cyclosporin A is consistent with reported values.








TABLE 13







SLC Transporter Cells
Literature Report














Km
Km
Test



Transporter
Substrate
(μM)
(μM)
System
Literature















OATP1B1*1a
E17βG
6.2
6.3
HEK293
P. Sharma, et al.






cells
Xenobiotica







40:24. 2010


OATP1B3
CCK-8
20.2
16.5
CHO
Poirier A, et al., J






Cells
Pharmacokinet







Pharmacodyn,







2009


OAT1
PAH
87.3
28
HEK293
Ueo H, et al.,






cells
Biochem







Pharmacol., 2005


OAT3
E3S
4.0
6.3
HEK293
Ueo H, et al.,






cells
Biochem







Pharmacol., 2005

















TABLE 14







Corning ® SLC TransportoCells TM
Literature Report
















IC50
IC50
Test



Transporter
Substrate
Inhibitor
(μM)
(μM)
System
Literature
















OATP1B1*1a
E17βG
Cyclosporin A
0.8
0.7
HEK293
MG Soars, et







Cells
al., Drug Metab








Dispos, 2012


OATP1B3
CCK-8
Cyclosporin A
0.7
0.6
HEK293
Bednarczyk D.







Cells
Anal Biochem.








2010


OAT1
PAH
Probenecid
7.2
6.5
CHO
Ho ES, et al., J








Am Soc








Nephrol., 2001


OAT3
E3S
Probenecid
8.8
9
S2
Takeda M, et








al., Eur J








Pharmacol.,








2001


OCT1
Metformin
Cimetidine
230
104
HEK293
Sumito I, et al.,







Cells
JPET, 2011


OCT2
Metformin
Cimetidine
195
124
HEK293
Sumito I, et al.,







Cells
JPET, 2011









It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.

## Claims

1. A recombinant cell comprising one or more transiently overexpressed genes encoding a drug transporter protein, wherein:
the recombinant cell is cryopreserved,
activity of the drug transporter protein is detectable in a population of the recombinant cells prior to cryopreservation,
activity of the drug transporter protein is detectable in a population of the recombinant cells following thaw from cryopreservation;
wherein the cryopreserved recombinant cell is transiently transfected with the one or more genes by a method comprising electroporation; and
wherein said one or more genes is OATP1B1*1a and wherein said cell is HEK293.

2. The recombinant cell of claim 1, wherein:
the detectable activity of the drug transporter protein prior to cryopreservation is the activity of the drug transporter protein towards a prototypical substrate for the drug transporter protein, and
the detectable activity in the population of the recombinant cells prior to cryopreservation is at an uptake ratio of from 5 to 25.

3. The recombinant cell of claim 1, wherein:
the detectable activity of the drug transporter protein following thaw from cryopreservation is the activity of the drug transporter protein towards a prototypical substrate for the drug transporter protein, and
the detectable activity in a population of the recombinant cells following thaw from cryopreservation at an uptake ratio of at least 5.

4. A process of preparing cryopreserved transiently transfected recombinant cells, the process comprising:
transiently transfecting cells with one or more genes encoding a drug transporter protein to provide the transiently transfected recombinant cells, and
cryopreserving the transiently transfected recombinant cells within 48 hours of transient transfection, wherein a population of the transiently transfected recombinant cells transiently overexpress the one or more genes encoding the drug transporter protein at a detectable level prior to cyropreserving the transiently transfected recombinant cells, wherein the transient transfection of the cells comprises electroporation, and wherein said one or more genes is OATP1B1*1a and wherein said cell is HEK293.

5. The process of claim 4, wherein the detectable level prior to cryopreserving is the activity of the drug transporter protein towards a specific prototypical substrate for the drug transporter protein, and wherein the detectable level prior to cryopreserving is an uptake ratio of at least 5.

6. The process of claim 4, wherein the detectable level prior to cryopreserving is the activity of the drug transporter protein towards a specific prototypical substrate for the drug transporter protein, and wherein the detectable level prior to cryopreserving is an uptake ratio of from 5 to 25.

7. The process of claim 4, wherein:
a population of the transiently transfected recombinant cells transiently overexpress the one or more genes encoding the drug transporter protein at the detectable level following thaw from cryopreservation,
the detectable level following thaw from cryopreservation is the activity of the drug transporter protein towards a specific prototypical substrate for the drug transporter protein, and
wherein the detectable level following thaw from cryopreservation is an uptake ratio of at least 5.

8. The process of claim 4, wherein the transiently transfected recombinant cells are cryopreserved at about 24 hours to about 48 hours post transient transfection.