Role of hepatocyte-derived extracellular matrix and coagulation factors in the fate recovery and maintenance of human hepatocytes

Abstract

Disclosed herein are human hepatocyte culture media and methods that better support the fate recovery and maintenance of cultured human hepatocytes. Cultivation of human hepatocytes is improved by supplementing hepatocyte clonal growth medium or hepatocyte maintenance medium supplemented with DMSO or DMSO2 with any one or combination of fibronectin, laminin, vitronectin, serpins, and coagulation factors.

Description

[Technical Field]

[0001] The present disclosure relates to cell culture media and uses thereof. [Background technology]

[0002] Chronic liver diseases (CLDs), such as viral hepatitis (HBV and HCV), alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), and many genetic liver disorders, can lead to end-stage liver disease (ESLD), including decompensated liver failure and liver cancer. Currently, ESLD is the 11th leading cause of death worldwide, and the only definitive medical intervention is liver transplantation (LT). However, LT is an invasive procedure, and the nationwide organ shortage significantly limits its usefulness. In addition, LT recipients must undergo lifelong immunosuppressive therapy, which is associated with significant morbidity, including opportunistic infections and malignant neoplasms.

[0003] Hepatocyte transplantation is expected to eventually replace LT. However, various obstacles remain, the most notable of which is the lack of a definitive technology that enables the complete maturation of iPSC-derived human hepatocytes, known as iHep. The maturity of currently available iHep is still far lower than that of primary human hepatocytes, at most being at the level of bipotent hepatic progenitor cells. Therefore, iHep cannot proliferate in human liver chimeric mice, which is in stark contrast to the characteristics of mature hepatocytes. Thus, significant challenges remain regarding the technology that promotes the definitive maturation of hepatic progenitor cells.

[0004] Additionally, it is important to note that the successful establishment of hepatocyte transplantation therapy requires further advances in our understanding of the molecular and cellular biology of the liver. One of the current fundamental limitations is the lack of definitive techniques that enable the maintenance of the cell fate of mature human hepatocytes in vitro.

[0005] The discoveries disclosed in the following sections are of significant value to the regenerative / stem cell biology field for the establishment of hepatocyte transplantation therapy. Furthermore, the disclosed inventions provide a significant benefit to basic research into liver biology and disorders. Summary of the Invention

[0006] The present invention provides the following. [1] A hepatocyte culture medium supplemented with at least one factor selected from the group consisting of fibronectin (FN), laminin (LAM), vitronectin (VTN), orosomucoid 1 (ORM1), serpin, collagen, haptoglobin (HP), transglutaminase 2 (TGM2), alpha-2 macroglobulin (A2M), coagulation factors including but not limited to fibrinogen (FG), tenascin-C (TNC), and combinations thereof. [2] The culture medium according to [1], which is a hepatocyte clonal growth medium (HCGM) or a hepatocyte maintenance medium (HMM). [3] The culture medium according to [1], wherein the laminin is laminin subunit gamma 1 (LAMC1), laminin subunit gamma 2 (LAMC2), or laminin subunit gamma 3 (LAMC3). [4] The culture medium according to [1], wherein the coagulation factor is fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB) or fibrinogen gamma chain (FGG). [5] The culture medium according to any one of [1] to [4], further supplemented with DMSO or DMSO2. [6] The culture medium according to any one of [2] to [5], wherein the HCGM comprises, consists of, or essentially consists of Dulbecco's modified Eagle's medium (DMEM), L-proline, insulin, dexamethasone, EGF, and L-ascorbic acid 2-phosphate (Asc-2P). [7] The culture medium according to [6], wherein the DMEM is DMEM-10, and the DMEM-10 comprises, consists of, or consists essentially of DMEM, HEPES, penicillin-streptomycin, and fetal bovine serum (FBS). [8] The culture medium of [6], wherein L-proline is 15 μg / mL in HCGM, insulin is 0.25 μg / mL in HCGM, dexamethasone is 50 nM in HCGM, EGF is 5 ng / mL in HCGM, Asc-2P is 0.1 mM in HCGM, DMSO or DMSO2 is 2% or 140.8 mM, respectively, in HCGM, HEPES is 20 mM in HCGM, penicillin is 100 IU / mL in HCGM, streptomycin is 100 μg / mL in HCGM, and FBS is 10% heat-inactivated FBS in HCGM. [9] The culture medium according to [6], wherein L-proline is 5 to 25 μg / mL in HCGM, insulin is 0.1 to 0.5 μg / mL in HCGM, dexamethasone is 10 to 100 nM in HCGM, EGF is 1 to 10 ng / mL in HCGM, Asc-2P is 0.01 to 1 mM in HCGM, DMSO or DMSO2 is 0.5% to 5% and 100 to 180 mM, respectively, in HCGM, HEPES is 10 to 50 mM in HCGM, penicillin is 10 to 300 IU / mL in HCGM, streptomycin is 10 to 300 μg / mL in HCGM, and FBS is 2 to 20% heat-inactivated FBS in HCGM.

[10] The culture medium according to any one of [2] to [5], wherein the HMM comprises, consists of, or essentially consists of Dulbecco's modified Eagle's medium (DMEM), L-proline, insulin, dexamethasone, and L-ascorbic acid 2-phosphate (Asc-2P).

[11] The culture medium according to

[10] , wherein the DMEM is DMEM-10, and the DMEM-10 comprises, consists of, or consists essentially of DMEM, HEPES, penicillin-streptomycin, and fetal bovine serum (FBS).

[12] The culture medium of

[10] , wherein L-proline is 15 μg / mL in HMM, insulin is 0.25 μg / mL in HMM, dexamethasone is 50 nM in HMM, Asc-2P is 0.1 mM in HMM, DMSO or DMSO2 is 2% or 140.8 mM, respectively, in HMM, HEPES is 20 mM in HMM, penicillin is 100 IU / mL in HMM, streptomycin is 100 μg / mL in HMM, and FBS is 10% heat-inactivated FBS in HMM.

[13] The culture medium according to

[10] , wherein L-proline is 5 to 25 μg / mL in HMM, insulin is 0.1 to 0.5 μg / mL in HMM, dexamethasone is 10 to 100 nM in HMM, Asc-2P is 0.01 to 1 mM in HMM, DMSO or DMSO2 is 0.5% to 5% or 100 to 180 mM in HMM, respectively, HEPES is 10 to 50 mM in HMM, penicillin is 10 to 300 IU / mL in HMM, streptomycin is 10 to 300 μg / mL in HMM, and FBS is 2 to 20% heat-inactivated FBS in HMM.

[14] The culture medium according to any one of [2] to

[13] , wherein FN is supplemented to 10,000 ng / mL in HCGM or HMM.

[15] The culture medium according to any one of [2] to

[13] , wherein laminin is supplemented to 1 to 10,000 pg / mL in HCGM or HMM.

[16] The culture medium according to any one of [2] to

[13] , wherein VTN is supplemented to 0.1 to 1000 ng / mL in HCGM or HMM.

[17] The culture medium according to any one of [2] to

[13] , wherein ORM1 is supplemented in HCGM or HMM to 1 to 10,000 μg / mL.

[18] The culture medium according to any one of [2] to

[13] , wherein serpins are supplemented to 0.1 to 1000 μg / mL in HCGM or HMM.

[19] The culture medium according to any one of [2] to

[13] , wherein collagen is supplemented to 1 to 10,000 ng / mL in HCGM or HMM.

[20] The culture medium according to any one of [2] to

[13] , wherein HP is supplemented to 0.01 to 100 mg / mL in HCGM or HMM.

[21] The culture medium according to any one of [2] to

[13] , wherein TGM2 is supplemented to 0.01 to 100 ng / mL in HCGM or HMM.

[22] The culture medium according to any one of [2] to

[13] , wherein A2M is supplemented to 0.1 to 1000 μg / mL in HCGM or HMM.

[23] The culture medium according to any one of [2] to

[13] , wherein the coagulation factor is supplemented to 0.1 to 1000 mg / mL in HCGM or HMM.

[24] The culture medium according to any one of [2] to

[13] , wherein TNC is supplemented to 0.01 to 100 ng / mL in HCGM or HMM.

[25] The culture medium according to any one of [1] to

[24] for use in growing terminally differentiated human hepatocytes, hepatocytes derived from chimeric animals having humanized livers, and / or cryopreserved and / or freshly isolated primary human hepatocytes.

[26] A culture medium according to any one of [1] to

[24] for use in differentiating hepatic bipotential progenitor cells (iHep) and / or chemically induced liver progenitors into terminally differentiated hepatocytes or mature hepatocytes, respectively.

[27] A culture medium according to any one of [1] to

[24] for use in co-culture with non-parenchymal cells and / or for use in suspending human hepatocytes for cryopreservation.

[28] A culture medium according to any one of [1] to

[24] for use in expanding and / or growing iHep and Chemically Induced Liver Progenitors (CLiPs) in the liver of an HLCM-host mouse.

[29] A method for growing terminally differentiated human hepatocytes, hepatocytes derived from a chimeric animal having a humanized liver, and / or cryopreserved and / or freshly isolated primary human hepatocytes, the method comprising culturing a predetermined amount of hepatocytes in a cell culture medium according to any one of [1] to

[24] .

[30] A method for differentiating hepatic bipotential progenitor cells (iHep) and / or chemically induced liver progenitors into terminally differentiated hepatocytes or mature hepatocytes, respectively, comprising culturing a predetermined amount of hepatocytes in a cell culture medium described in any one of [1] to

[24] .

[31] A method for co-culturing human hepatocytes with non-parenchymal cells and / or a method for suspending human hepatocytes for cryopreservation, the method comprising culturing a predetermined amount of hepatocytes in a cell culture medium according to any one of [1] to

[24] .

[32] A method for expanding and / or growing iHep and CLiP within the liver of an HLCM-host mouse, the method comprising culturing a predetermined amount of hepatocytes in a cell culture medium described in any one of [1] to

[24] . [Brief explanation of the drawings]

[0007] [Figure 1] 1 shows the detection of humoral factors derived from HLCM-HH in CM. CM was collected as described in Materials and Methods and examined by immunoblotting. All signals were detected at the expected molecular weights. [Figure 2]This figure shows the results of gene expression analysis of HLCM-HH cultured in dHCGM in the presence or absence of fibrinogen. HLCM-HH were seeded onto collagen-coated 24-well plates and cultured for 7 days in dHCGM or dHCGM supplemented with fibrinogen (50 μg / mL). The Y-axis title is shown in the upper left panel. Bars represent the mean ± SD. *: p<0.05, **: p<0.01, ***: p<0.001, #: p<0.0001. [Figure 3] Figure 1 shows the results of gene expression analysis of HLCM-HH cultured in dHCGM, CM, and CM containing coagulation cascade inhibitors. HLCM-HH were seeded onto collagen-coated 24-well plates and cultured for 7 days in dHCGM, CM, or CM containing F2i (dabigatran etexilate, 2 μg / mL) or F10i (rivoroxaban, 400 ng / mL). The Y-axis labels are shown in the upper left panel. Bars represent the mean ± SD. #: p<0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001 for FM, FM+F2i, and FM+F10i. [Figure 4]

[0033] Figure 1 shows the results of gene expression analysis of HLCM-HH cultured in a commercially available hepatocyte culture medium supplemented with or without fibrinogen. HLCM-HH were seeded onto collagen-coated 24-well plates and cultured for 7 days in IH or HCM in the presence or absence of fibrinogen (50 μg / mL). The Y-axis heading is shown in the upper left panel. Bars represent the mean ± SD. ****: p<0.0001. [Figure 5]This figure shows the results of gene expression analysis of HLCM-HH cultured in dHCGM containing CM components. HLCM-HH were seeded onto collagen-coated 24-well plates and cultured for 7 days in dHCGM supplemented with dHCGM, CM, or one or a combination of the following CM components: VTN: 50 ng / mL, FN: 100 ng / mL, TNC: 2 ng / mL, and HP: 0.03 U / mL. The Y-axis labels are shown in the upper left panel. Bars represent the mean ± SD. *: p<0.05, †: p<0.01, ‡: p<0.001, #: p<0.0001. [Figure 6] This figure shows the results of gene expression analysis of HLCM-HH cells transfected with A2M-specific siRNA. HLCM-HH cells were seeded onto siRNA-transfection plates coated with negative control or A2M-specific siRNA and cultured in dHCGM for 7 days. The Y-axis represents the expression level relative to that of freshly isolated HLCM-HH cells. Bars represent the mean ± SD. ***: p<0.001. DETAILED DESCRIPTION OF THE INVENTION

[0008] All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006);March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), will provide those of skill in the art with a general guide to many of the terms used in this application. Those of skill in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, several terms are defined below.

[0009] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to particular synthetic methods or to particular recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0010] As used herein, "and / or" means any one or more of the items in the list connected by "and / or." As an example, "x and / or y" means any element of the 3-element set {(x), (y), (x,y)}. As another example, "x, y and / or z" means any element of the 7-element set {(x), (y), (z), (x,y), (x,z), (y,z), (x,y,z)}. As used herein, the term "exemplary" means serving as a non-limiting example, case, or illustration. As used herein, the terms "eg" and "for example" introduce a list of one or more non-limiting examples, cases, or illustrations.

[0011] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if a value of "10" is disclosed, "about 10" is also disclosed. It is also understood that when a value is disclosed, "less than or less than" that value, "greater than or equal to" that value, and possible ranges between values ​​are also disclosed, as appropriately understood by one of ordinary skill in the art. For example, if a value of "10" is disclosed, "less than or less than 10" and "greater than or equal to 10" are also disclosed. Also, throughout the application, data are provided in several different formats, and it is understood that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point of "10" and a specific data point of 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, and between 10 and 15, are considered to be disclosed. It is also understood that each unit between two specified units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0012] The described components, steps, properties, objects, benefits, and advantages are merely exemplary. None of them, nor the discussion relating thereto, are intended to limit the scope of protection in any way. Many other embodiments are also contemplated. These include embodiments having fewer, additional, and / or different components, steps, properties, objects, benefits, and advantages. These also include embodiments in which the components and / or steps are arranged and / or ordered differently.

[0013] Unless otherwise indicated, all measurements, values, ratings, positions, dimensions, sizes and other specifications set forth in this specification, including the claims that follow, are approximate and not exact, and are intended to have a reasonable range consistent with the functions to which they relate and with those customary in the art to which they pertain.

[0014] "Comprising" is intended to mean that the composition, method, etc. includes the recited elements, but does not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean including the recited elements, but excluding other elements of any essential importance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, and the like. "Consisting of" is intended to mean excluding more than trace amounts of trace elements of other components, as well as substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0015] All articles, patents, patent applications and other publications cited in this disclosure are incorporated herein by reference.

[0016] The term "subject" refers to any individual who is the target of administration or treatment. The subject may be a vertebrate, such as a mammal. In one embodiment, the subject may be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or a veterinary patient. The term "patient" refers to a subject under the care of a clinician. Relationship terms, such as "first" and "second," may be used simply to distinguish one entity or action from another and do not necessarily require or imply any actual relationship or order between them.

[0017] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, several terms are defined below.

[0018] Applicants' invention can be used for the regenerative / stem cell biology field for the establishment of hepatocyte transplantation therapy. Furthermore, the present invention provides a substantial degree of benefit to basic research in liver biology and pathophysiology in general.

[0019] Applicants have discovered that there are several important hepatocyte-specific humoral factors contained in the culture medium of terminally differentiated human hepatocytes (HH) (hereafter referred to as human hepatocyte conditioned medium; CMHH). These factors can be used individually or in combination as nutritional supplements to DMSO- or DMSO2-containing hepatocyte clonal expansion medium or DMSO- or DMSO2-containing hepatocyte maintenance medium (Tables 1-3).

[0020] In various embodiments, as used herein, the combination of factors is a combination of two, or a combination of three, or a combination of four, etc. These humoral factors are fibronectin (FN), such as FN1; laminin, such as LAMC1, LAMC2 and LAMC3; vitronectin (VTN); orosomucoid 1 (ORM1) or alpha-1-acid glycoprotein (AGP1); serpin, such as SERPINA3; collagen, such as type 4 collagen, for example, collagen IV alpha 1 chain; haptoglobin (HP); transglutaminase 2 (TGM2); alpha-2 macroglobulin (A2M); tenascin C (TNC), and coagulation factors, such as fibrinogen (for example, FGA, FGB and FGG). The medium supplemented with humoral factors provides advantages for use in regenerative medicine and stem cell biology, for example, promotes the final cell maturation of iHep and maintains the cell fate of fully mature iHep. Additionally, the supplemented medium contributes to hepatocyte biology studies, such as maintaining the cell fate, function, and lifespan of in vitro cultured HHs, primary hepatocytes from other species (e.g., dog, mouse, rat, and monkey), hepatocytes derived from human-liver chimeric animals, and hepatic nonparenchymal cells (NPCs).

[0021] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors is hepatocyte clonal growth medium (dHCGM) containing DMSO, as shown in Tables 1 and 3. In some embodiments, the dHCGM comprises or consists essentially of a standard basal cell culture medium, L-proline, insulin, dexamethasone, EGF, and L-ascorbic acid 2-phosphate ester (Asc-2P). If particularly desired, the standard basal cell culture medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is fetal bovine serum (FBS) or human serum. In a preferred embodiment, the standard basal cell culture medium is Dulbecco's Modified Eagle's Medium (DMEM)-10.

[0022] Standard cell culture basal media are well known to those skilled in the art. Examples of standard cell culture basal media include, but are not limited to, DMEM, DMEM-10, minimum essential medium (MEM), RPMI-1640, Iscove's modified Dulbecco's medium (IMDM) or William's medium.

[0023] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors is hepatocyte maintenance medium (dHMM) supplemented with DMSO, as shown in Tables 2 and 3. In some embodiments, dHMM comprises or consists essentially of DMSO, standard cell culture basal medium, L-proline, insulin, dexamethasone, and L-ascorbic acid 2-phosphate ester (Asc-2P). If particularly desired, the standard cell culture basal medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is fetal bovine serum (FBS) or human serum. In a preferred embodiment, the standard cell culture basal medium is Dulbecco's Modified Eagle's Medium (DMEM)-10.

[0024] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors is not supplemented with DMSO, but is hepatocyte clonal growth medium (d2HCGM) supplemented with DMSO2, as shown in Tables 2 and 3. In some embodiments, the second HH culture medium is not supplemented with DMSO, but is hepatocyte clonal growth medium (tHCGM) supplemented with tetramethylene sulfoxide (TMSO).

[0025] In some embodiments, the medium, e.g., d2HCGM, supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, comprises or consists essentially of DMSO2, standard cell culture basal medium, L-proline, insulin, dexamethasone, EGF, and Asc-2P. If desired, the standard cell culture basal medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is fetal bovine serum (FBS) or human serum. In a preferred embodiment, the standard cell culture basal medium is Dulbecco's Modified Eagle's Medium (DMEM)-10.

[0026] In some embodiments, the medium, e.g., tHCGM, supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, comprises or consists essentially of TMSO, standard cell culture basal medium, L-proline, insulin, dexamethasone, EGF, and Asc-2P. If desired, the standard cell culture basal medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is fetal bovine serum (FBS) or human serum. In a preferred embodiment, the standard cell culture basal medium is Dulbecco's Modified Eagle's Medium (DMEM)-10.

[0027] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors is not supplemented with DMSO, but is hepatocyte maintenance medium (d2HMM) supplemented with DMSO2, as shown in Tables 2 and 3. In some embodiments, the second HH culture medium is not supplemented with DMSO, but is hepatocyte maintenance medium (tHMM) supplemented with tetramethylene sulfoxide (TMSO).

[0028] In some embodiments, a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, such as d2HMM, comprises or consists essentially of DMSO2, standard cell culture basal medium, L-proline, insulin, dexamethasone, and Asc-2P. If desired, the standard cell culture basal medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is fetal bovine serum (FBS) or human serum. In a preferred embodiment, the standard cell culture basal medium is Dulbecco's Modified Eagle's Medium (DMEM)-10.

[0029] In some embodiments, a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, e.g., tHMM, comprises or consists essentially of TMSO, standard cell culture basal medium, L-proline, insulin, dexamethasone, and Asc-2P. If particularly desired, the standard cell culture basal medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is fetal bovine serum (FBS) or human serum. In a preferred embodiment, the standard cell culture basal medium is Dulbecco's Modified Eagle's Medium (DMEM)-10.

[0030] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, such as dHCGM, d2HCGM, or tHCGM, is supplemented with at least 70 mM, 35 mM, and 35 mM of DMSO, DMSO2, or TMSO, respectively, in a standard basal cell culture medium, and the standard basal cell culture medium further comprises one or more of L-proline, insulin, dexamethasone, EGF, Asc-2P, HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0031] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, such as dHCGM, d2HCGM, or tHCGM, is supplemented with DMSO, DMSO2, or TMSO in an amount that reaches a concentration of about 281.6 mM, 140.8 mM, and 70.4 mM, respectively, in a standard basal cell culture medium, and the standard basal cell culture medium further comprises one or more of L-proline, insulin, dexamethasone, EGF, Asc-2P, HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0032] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, such as dHMM, d2HMM, or tHMM, is supplemented with at least 70 mM, 35 mM, and 35 mM of DMSO, DMSO2, or TMSO, respectively, in a standard basal cell culture medium, and the standard basal cell culture medium further comprises one or more of L-proline, insulin, dexamethasone, Asc-2P, HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0033] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, such as dHMM, d2HMM, or tHMM, is supplemented with DMSO, DMSO2, or TMSO in an amount that reaches a concentration of about 281.6 mM, 140.8 mM, and 70.4 mM, respectively, in a standard basal cell culture medium, and the standard basal cell culture medium further comprises one or more of L-proline, insulin, dexamethasone, Asc-2P, HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0034] In some embodiments, a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, such as dHCGM, d2HCGM, or tHCGM, is supplemented with a predetermined amount of DMSO, DMSO2, or TMSO to reach a concentration of about 2 (v / v)% in a standard basal cell culture medium, and the standard basal cell culture medium further comprises one or more of L-proline, insulin, dexamethasone, EGF, Asc-2P, HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0035] In some embodiments, a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, such as dHMM, d2HMM, or tHMM, is supplemented with a predetermined amount of DMSO, DMSO2, or TMSO to reach a concentration of about 2 (v / v)% in a standard basal cell culture medium, and the standard basal cell culture medium further comprises one or more of L-proline, insulin, dexamethasone, EGF, Asc-2P, HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0036] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors contains 281.6 mM or 2% (v / v) DMSO and a standard cell culture basal medium, the medium containing 15 μg / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin, and one or more of 10% heat-inactivated FBS or human serum. In a preferred embodiment, the standard cell culture basal medium is DMEM-10.

[0037] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors comprises 140.8 mM or 70.4 mM DMSO or TMSO, respectively, and a standard cell culture basal medium, the medium comprising 15 μg / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin, and one or more of 10% heat-inactivated FBS or human serum. In a preferred embodiment, the standard cell culture basal medium is DMEM-10.

[0038] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors contains 0.5%-5% (v / v) DMSO and a standard basal cell culture medium, the medium containing 5-25 μg / mL L-proline, 0.1-0.5 μg / mL insulin, 10-100 nM dexamethasone, 0-10 ng / mL EGF, 0.01-1 mM Asc-2P, 10-50 mM HEPES, 10-300 IU / mL penicillin, 10-300 μg / mL streptomycin, and one or more of 2-20% heat-inactivated FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0039] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors comprises 35-281.6 mM DMSO, DMSO2, or TMSO, and a standard basal cell culture medium, the medium comprising 5-25 μg / mL L-proline, 0.1-0.5 μg / mL insulin, 10-100 nM dexamethasone, 0-10 ng / mL EGF, 0.01-1 mM Asc-2P, 10-50 mM HEPES, 10-300 IU / mL penicillin, 10-300 μg / mL streptomycin, and one or more of 2-20% heat-inactivated FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0040] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains DMSO at a concentration of 150 mM to 350 mM, 200 mM to 300 mM, 220 mM to 290 mM, 240 mM to 285 mM, or 281.6 mM.

[0041] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains DMSO2 at a concentration of 100 mM to 200 mM, 120 mM to 180 mM, 140 mM to 160 mM, or 140.8 mM.

[0042] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains TMSO at a concentration of 40 mM to 100 mM, 50 mM to 90 mM, 60 mM to 80 mM, or 70.4 mM.

[0043] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains L-proline at a concentration of 5-25 μg / mL, 10-20 μg / mL, 12-18 μg / mL, or 15 μg / mL.

[0044] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains insulin at a concentration of 0.1-0.5 μg / mL, 0.15-0.45 μg / mL, 0.2-0.4 μg / mL, or 0.25 μg / mL.

[0045] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains dexamethasone at a concentration of 10-100 nM, 20-90 nM, 30-80 nM, 40-70 nM, or 50 nM.

[0046] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains EGF at a concentration of 0.0-10 ng / mL, 1-9 ng / mL, 2-9 ng / mL, 3-8 ng / mL, 4-7 ng / mL, 5 ng / mL, or 0 ng / mL.

[0047] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains Asc-2P at a concentration of 0.01-1 mM, 0.02-0.8 mM, 0.04-0.6 mM, 0.06-0.4 mM, 0.08-0.2 mM, or 0.1 mM.

[0048] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains HEPES at a concentration of 10-50 mM, 13-40 mM, 16-30 mM, 18-25 mM, or 20 mM.

[0049] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains penicillin at a concentration of 10-300 IU / mL, 30-250 IU / mL, 50-200 IU / mL, 70-150 IU / mL, or 100 IU / mL.

[0050] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains streptomycin at a concentration of 10-300 μg / mL, 30-250 μg / mL, 50-200 μg / mL, 70-150 μg / mL, or 100 μg / mL.

[0051] In some embodiments, the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors contains heat-inactivated FBS or human serum at a concentration of 2-20%, 4-18%, 6-16%, 8-14%, or 10%.

[0052] In some embodiments, the medium is supplemented with all of humoral factors, FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or clotting factors. In some embodiments, the medium is supplemented with some combination of humoral factors, FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or clotting factors. In some embodiments, the medium is supplemented with at least one of humoral factors, FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or clotting factors.

[0053] In some embodiments, FN is supplemented to the medium to a concentration of 1 to 10,000 ng / mL.

[0054] In some embodiments, laminin is supplemented to the medium to a concentration of 1-10,000 pg / mL.

[0055] In some embodiments, VTN is supplemented to the medium to a concentration of 0.1 to 1000 ng / mL.

[0056] In some embodiments, ORM1 is supplemented to the medium to a concentration of 0.1 to 1000 ng / mL.

[0057] In some embodiments, the serpin is supplemented to the medium to a concentration of 0.1 to 1000 μg / mL.

[0058] In some embodiments, collagen is supplemented to the medium to a concentration of 1-10000 ng / mL.

[0059] In some embodiments, HP is supplemented to the medium to a concentration of 0.01 to 100 mg / mL.

[0060] In some embodiments, TGM2 is supplemented to the medium to a concentration of 0.01 to 100 ng / mL.

[0061] In some embodiments, A2M (alpha-2 macroglobulin) is supplemented to the medium to a concentration of 0.1 to 1000 μg / mL.

[0062] In some embodiments, TNC (tenascin C) is supplemented to the medium to a concentration of 0.01 to 100 ng / mL.

[0063] In some embodiments, the clotting factors are supplemented to the medium to a concentration of 0.1 to 1000 mg / mL.

[0064] In some embodiments, the medium is supplemented with at least two of the following: fibronectin (FN), vitronectin (VTN), haptoglobin (HP), alpha-2 macroglobulin (A2M), fibrinogen, and tenascin-C (TNC). In some embodiments, the medium is supplemented with at least three of the following: fibronectin (FN), vitronectin (VTN), haptoglobin (HP), alpha-2 macroglobulin (A2M), fibrinogen, and tenascin-C (TNC). In some embodiments, the medium is supplemented with at least four of the following: fibronectin (FN), vitronectin (VTN), haptoglobin (HP), alpha-2 macroglobulin (A2M), fibrinogen, and tenascin-C (TNC). In some embodiments, the medium is supplemented with at least five of the following: fibronectin (FN), vitronectin (VTN), haptoglobin (HP), alpha-2 macroglobulin (A2M), fibrinogen, and tenascin-C (TNC). In some embodiments, the medium is supplemented with at least fibronectin (FN), vitronectin (VTN), haptoglobin (HP), alpha-2 macroglobulin (A2M), fibrinogen, and tenascin-C (TNC).

[0065] In some embodiments, the medium is supplemented with at least two of the following: fibrinogen, vitronectin, fibronectin, tenascin-C, and haptoglobin. In some embodiments, the medium is supplemented with at least three of the following: fibrinogen, vitronectin, fibronectin, tenascin-C, and haptoglobin. In some embodiments, the medium is supplemented with at least four of the following: fibrinogen, vitronectin, fibronectin, tenascin-C, and haptoglobin. In some embodiments, the medium is supplemented with at least five of the following: fibrinogen, vitronectin, fibronectin, tenascin-C, and haptoglobin. In some embodiments, the medium is supplemented with at least six of the following: fibrinogen, vitronectin, fibronectin, tenascin-C, and haptoglobin. In some embodiments, the medium is supplemented with at least two of fibrinogen, vitronectin, fibronectin, tenascin-C, and haptoglobin.

[0066] In some embodiments of the present invention, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors are used for rapid and robust recovery from cellular injury or stress associated with cell isolation procedures and freeze / thaw cycles.

[0067] In some embodiments of the present invention, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors are used to culture HH in vitro to exhibit a level of functionality comparable to that of human liver, thereby making them suitable for a variety of experimental applications.

[0068] In some embodiments of the present invention, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors is used to assay the performance of a candidate drug in promoting alcohol metabolism by HH, promoting xenobiotic metabolism by HH, or assaying the toxicity of a candidate drug to HH.

[0069] In some embodiments of the present invention, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors are used to assay the toxicity of drugs to human hepatocytes (HH) or the drug metabolism and pharmacokinetics (DMPK) of drugs.

[0070] In some embodiments of the assay method, the agent is an alcohol compound, including ethanol, methanol, ethylene glycol, isopropanol, or mixtures thereof.

[0071] In some embodiments of the assay method, the contacting and measuring are performed in a sealed device.

[0072] In some embodiments of the assay method, the level of toxicity is measured through quantifying the expression or function of xenobiotic metabolizing enzymes, such as, but not limited to, cytochrome P450 (CYP) enzymes, using amounts in the HH.

[0073] In some embodiments of the assay method, the level of toxicity is measured through quantifying the activation state of toxic metabolites of alcohol, such as acetaldehyde, or glutathione reduction, cell death pathways using the amount in HH.

[0074] In various embodiments, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors are used for hepatocyte biology research. A major limitation in hepatocyte biology research is the lack of a stable in vitro culture system for terminally differentiated HH. For example, cryopreserved and / or freshly isolated PHH are the primary tools utilized by most pharmaceutical industries for drug safety screening and drug metabolism and pharmacokinetic (DMPK) studies in the preclinical trial phase. The term "freshly isolated" refers to hepatocytes isolated less than 168 hours after isolation. For example, freshly isolated hepatocytes (e.g., PHH) include unfrozen hepatocytes isolated within 168 hours, 72 hours, 36 hours, 24 hours, 12 hours, or 6 hours. In a preferred embodiment of the present invention, freshly isolated hepatocytes are unfrozen hepatocytes isolated within 6 hours. The term "cryopreserved" refers to hepatocytes that have been frozen using, for example, liquid nitrogen, a programmed freezer, a deep freezer, or the like.

[0075] Thus, in various embodiments, hepatocytes are placed in contact with and cultured in medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors.

[0076] One aspect of the present invention provides culture media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors for use in growing hepatocytes derived from terminally differentiated PHH, HLCM-HH, another chimeric animal (e.g., rat, sheep, pig, monkey) with a humanized liver, primary hepatocytes from another species, e.g., dog, mouse, rat and monkey, and / or cryopreserved and / or freshly isolated hepatocytes derived from another chimeric animal (e.g., rat, sheep, pig, monkey) with a humanized liver, and primary hepatocytes from another species, e.g., dog, mouse, rat and monkey. Therefore, in various embodiments, terminally differentiated PHH and HLCM-HH are placed in contact with and cultured in a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors.

[0077] In some embodiments, the biological effect of medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors is to maintain the function of liver nonparenchymal cells (NPCs), such as hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), and liver macrophages (Kupffer cells). Thus, in various embodiments, liver nonparenchymal cells (NPCs), such as hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), and liver macrophages (Kupffer cells), are placed in contact with and cultured in medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors.

[0078] In some embodiments, medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors is used to prevent dedifferentiation of terminally differentiated in vitro cultured HH, HLCM-HH, hepatocytes derived from another chimeric animal with a humanized liver (e.g., rat, sheep, pig, monkey), PHH, and / or primary hepatocytes from another species, such as dog, mouse, rat, and monkey. Thus, in various embodiments, HH, HLCM-HH, hepatocytes derived from another chimeric animal with a humanized liver, PHH, and / or primary hepatocytes from another species are placed in contact with and cultured in medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors.

[0079] A further aspect of the present invention provides a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors for use in inhibiting the transdifferentiation of HH, HLCM-HH, hepatocytes derived from another chimeric animal having a humanized liver (e.g., rat, sheep, pig, monkey), PHH, and / or primary hepatocytes from another species, such as dog, mouse, rat, and monkey. Thus, in various embodiments, HH, HLCM-HH, hepatocytes derived from another chimeric animal having a humanized liver, PHH, and / or primary hepatocytes from another species are placed in contact with and cultured in a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors.

[0080] A further aspect of the present invention provides a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors for use in promoting the recovery of cryopreserved and / or freshly isolated HH, HLCM-HH, hepatocytes derived from another chimeric animal with a humanized liver (e.g., rat, sheep, pig, monkey), PHH, and / or primary hepatocytes from another species, such as dog, mouse, rat, and monkey. Thus, in various embodiments, cryopreserved and / or freshly isolated HH, HLCM-HH, hepatocytes derived from another chimeric animal with a humanized liver, PHH, and / or primary hepatocytes from another species are placed in contact with and cultured in a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors.

[0081] A further aspect of the present invention provides culture media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors for use in promoting recovery of cryopreserved and / or freshly isolated HH, HLCM-HH, hepatocytes derived from another chimeric animal (e.g., rat, sheep, pig, monkey) with a humanized liver, PHH, and / or primary hepatocytes from another species, e.g., dog, mouse, rat and monkey, from stress / injury associated with the cell procurement process. Thus, in various embodiments, cryopreserved and / or freshly isolated HH, HLCM-HH, hepatocytes derived from another chimeric animal with a humanized liver, PHH, and / or primary hepatocytes from another species are placed in contact with and cultured in a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors.

[0082] A further aspect of the present invention provides a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors for use in protecting hepatocytes derived from HH, HLCM-HH, another chimeric animal having a humanized liver (e.g., rat, sheep, pig, monkey), PHH, and / or primary hepatocytes from another species, such as dog, mouse, rat, and monkey, from the cellular stress associated with cryopreservation, which may further maintain cell recovery upon thawing. Thus, in various embodiments, HH, HLCM-HH, hepatocytes derived from another chimeric animal having a humanized liver, PHH, and / or primary hepatocytes from another species are placed in contact with and cultured in a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors.

[0083] A further aspect of the present invention provides a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or clotting factors for use in maintaining NPC function and morphology and function. In some embodiments, CMHH is provided for use in co-culture with NPCs and / or for use in suspending human hepatocytes for cryopreservation. Thus, in various embodiments, hepatocytes are placed in contact with a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or clotting factors and cryopreserved in the medium.

[0084] Another aspect of the present invention provides the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factor, for use in maintaining / improving the functionality and lifespan of the hepatocytes, PHH, and / or the primary hepatocytes from other species, such as dog, mouse, rat and monkey, from HH, HLCM-HH, other chimeric animals with humanized liver (for example, rat, sheep, pig, monkey) in the form of spheroid, organoid or 3D culture, and for the development of organ chip format.Therefore, in various embodiments, the spheroid, organoid or 3D culture of HH, HLCM-HH or hepatocytes is placed in contact with the medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factor, and is cultured in this medium.

[0085] A further aspect of the present invention provides a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors for use in improving the utility of hepatocytes derived from HH, HLCM-HH, other chimeric animals with humanized livers (e.g., rats, sheep, pigs, monkeys), PHH, and / or primary hepatocytes from other species, e.g., dogs, mice, rats, and monkeys, for the production of HLCM.

[0086] A further aspect of the present invention provides media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors for use in supporting the cell fate maintenance of hepatocytes derived from HH, HLCM-HH, another chimeric animal with a humanized liver (e.g., rat, sheep, pig, monkey), PHH, and / or primary hepatocytes from another species, e.g., dog, mouse, rat and monkey, thereby greatly enhancing the utility of these cells for various types of hepatocyte biology studies, e.g., DMPK, drug screening, infectious disease and metabolic disease. Thus, in various embodiments, hepatocytes derived from HH, HLCM-HH, another chimeric animal with a humanized liver, PHH, and / or primary hepatocytes from another species are placed in contact with and cultured in medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors in these studies.

[0087] In various embodiments, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors are used to maintain the cell fate of PHH and HLCM-HH, as well as HH derived from other chimeric animals with humanized livers, thereby greatly improving the utility of these cells for various types of liver cell biology studies, such as DMPK, drug screening, infectious disease, and metabolic disease.

[0088] In various embodiments, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors are used in regenerative medicine and stem cell biology. Media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors may be essential factors that enable the final stage of iHep maturation into terminally differentiated human hepatocytes. In some embodiments, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or clotting factors is used in the preparation of hepatocyte transplantation therapy, for example, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or clotting factors is used in the mass production of iPS cell-derived mature human hepatocytes, which can be a substitute for LT.

[0089] A further aspect of the present invention provides a culture medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or clotting factors for use in promoting further maturation of iHep (stem cell derived hepatic bipotent progenitor cells) into terminally differentiated hepatocytes.

[0090] A further aspect of the present invention provides culture media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or clotting factors for use in preventing the differentiation of iHep (stem cell derived hepatic bipotent progenitor cells) into cholangiocytes or other cell types.

[0091] A further aspect of the present invention provides a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors for use in promoting the redifferentiation of CLiPs (Chemically Induced Liver Progenitors) into mature hepatocytes.

[0092] A further aspect of the present invention provides a culture medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpin, collagen, HP, TGM2, A2M, TNC and / or coagulation factors for use in improving liver function of liver cancer cells (e.g., HepaRG cells, HepG2 and Huh7 cells).

[0093] In some embodiments, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors is used to improve liver function of HepaRG.

[0094] A further aspect of the present invention provides media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or clotting factors for use in enabling the expansion / growth of iHep and CLIp in the liver of an HLCM-host mouse. In some embodiments, the present invention provides media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or clotting factors for use in enabling the expansion / growth of primary hepatocytes from PHH and / or other species, such as dog, mouse, rat, and monkey, in the liver of an HLCM-host mouse.

[0095] A further aspect of the present invention provides culture medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors for use in promoting regression of chronic liver disease / liver fibrosis as a result of hepatocyte transplantation therapy.

[0096] A further aspect of the present invention provides a culture medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors for use in adjuvant therapy for liver cancer.

[0097] A further aspect of the present invention provides media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or clotting factors for use in supporting differentiation and cell fate maintenance of iHep, CLiP and HepaRG, which allows stable and long-term culture of these cells without compromising the characteristics of mature hepatocytes; therefore, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or clotting factors greatly enhances the utility of these cells for various types of hepatocyte biology studies, such as DMPK, drug screening, infectious diseases and metabolic diseases.

[0098] A further aspect of the present invention provides a medium supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors for use in differentiating CLiP (Chemically Induced Liver Progenitor) cells.

[0099] In some embodiments, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors is used to promote the redifferentiation of CLiPs into mature hepatocytes.

[0100] In various embodiments, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors are used to grow and maintain hepatocytes, which may include HH, PHH from other species, e.g., dog, mouse, rat, and monkey, hepatocytes derived from human liver chimeric mice (HLCM), or hepatocytes derived from another chimeric animal with a humanized liver (e.g., hepatocytes derived from rat, sheep, pig, or monkey).

[0101] In some embodiments, media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors is used for hepatocyte transplantation (e.g., iHep further differentiated with media supplemented with one or a combination of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC and / or coagulation factors) into patients suffering from any type of liver disease.

[0102] In certain embodiments, the HH comprises human liver chimeric mouse-derived human hepatocytes (HLCM-HH), primary human hepatocytes (PHH), hepatocytes derived from chimeric animals with humanized livers, such as rats, mice, sheep, pigs, or monkeys, or a combination thereof. In some embodiments, the HH is a PHH. In some embodiments, the HH is an HLCM-HH. In some embodiments, the HH is derived from a chimeric animal with humanized livers other than a mouse.

[0103] In certain embodiments, the HLCM-HH is obtained from the liver of a mouse that has been injected into the spleen with a PHH or a previously isolated HLCM-HH. In some embodiments, the HH is obtained from the liver of another animal (e.g., a rat, a mouse, a sheep, a pig, a monkey, etc.) that has been injected into the spleen with a PHH or a previously isolated HH from a human liver chimeric animal.

[0104] In some embodiments, the mouse has a human hepatocyte replacement index of at least 10% (as indicated by histological examination or blood concentrations of HH-derived factors (e.g., human albumin and human alpha-1 antitrypsin)) before the HLCM-HH is obtained, or the chimeric animal with a humanized liver has a human hepatocyte replacement index of at least 10% before the HH is isolated or obtained.

[0105] In a further embodiment, hepatocytes derived from HLCM-HH, PHH, another chimeric animal with a humanized liver, or a combination of any two or all three, are present in a concentration of 0.5x10 5 pieces / cm 2 ~5x10 5 pieces / cm 2 The cells are cultured at a cell density of .

[0106] In a further embodiment, the HH does not include HepG2 cells, Huh7 cells, or mouse hepatocytes. In another embodiment, the HH includes HLCM-HH, which may include, in addition to HH, some mouse hepatocytes and mouse non-parenchymal cells.

[0107] In a further embodiment, the HH comprises HLCM-HH, and the method further comprises obtaining HLCM-HH before the culturing step, and obtaining HLCM-HH comprises isolating hepatocytes derived from the liver of a mouse that has been injected into the spleen with PHH or previously isolated HLCM-HH, thereby obtaining HLCM-HH.

[0108] In a further embodiment, HH derived from mouse liver are isolated by collagenase perfusion of the mouse liver, the mouse having a human hepatocyte replacement index of at least 10% before the HH are isolated. [Example]

[0109] [Example 1] The role of hepatocyte-derived extracellular matrix and coagulation factors in the fate recovery and maintenance of human hepatocytes In exploring the mechanism by which CMHH exerts its fate restoration and protective effect on in vitro cultured HLCM-HH and PHH, the applicant's series of biochemical and genetic studies identified several important factors contained in CMHH: fibronectin (FN), such as FN1; laminin, such as LAMC1 and LAMC2; vitronectin (VTN); orosomucoid 1 (ORM1) or alpha-1-acid glycoprotein (AGP1); serpin, such as SERPINA3; collagen, such as collagen IV alpha 1 chain; haptoglobin (HP); transglutaminase 2 (TGM2); alpha-2 macroglobulin (A2M); tenascin C (TNC), and coagulation factors (e.g., FGA, FGB, and FGG). Based on this evaluation, supplementation of dHCGM or dHMM with these factors, either individually or in combination, particularly in the latter case, significantly supports the fate restoration and maintenance of in vitro cultured HLCM-HH and PHH.

[0110] Based on the fate-restoring and protective effects of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors on in vitro cultured HH, these factors have great potential as cell culture media supplements, either individually or in combination. The following are bullet points of utility:

[0111] Regenerative Medicine and Stem Cell Biology These studies demonstrate that FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors have potent fate-determining effects on in vitro cultured HH and, therefore, that these factors are required for the maturation of iHep into terminally differentiated human hepatocytes. This breakthrough discovery will enable the mass production of iPS cell-derived mature human hepatocytes, thereby bringing about a paradigm shift in the development of hepatocyte transplantation therapy as an alternative to LT. The following are critical characteristics of or for this category: 1. These factors serve as important components of the cell culture medium that promote further maturation of iHepatic Bipotential Progenitor Cells (iHePs) into terminally differentiated hepatocytes. 2. Cell culture media containing these factors prevents the transdifferentiation of iHepatic Bipotential Progenitor Cells (iHeP) into cholangiocytes. 3. Cell culture media containing these factors promote the redifferentiation of CLiPs (Chemically Induced Liver Progenitors) into mature hepatocytes. 4. Cell culture medium containing these factors improves liver function in HepaRG. 5. These factors serve as important components of the cell culture medium, allowing the expansion / proliferation of iHep and CLiP in the livers of HLCM-host mice. 6. Cell culture media containing these factors allows hepatocyte transplantation (e.g., iHep) into patients suffering from any type of liver disease. 7. Cell culture media containing these factors promotes regression of chronic liver disease / liver fibrosis. 8. These factors act as adjuvant therapy for liver cancer.

[0112] Hepatocyte biology research A major limitation in the study of hepatocyte biology is the lack of a stable in vitro culture system for terminally differentiated human hepatocytes. Supplementing human hepatocyte cell culture media with hepatocyte-specific humoral factors, including FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, offers numerous advantages over current standard human hepatocyte culture methods. Cryopreserved and / or isolated PHHs are the primary tools utilized by most pharmaceutical industries for drug safety screening and DMPK studies in the preclinical trial phase. Because supplementation with these humoral factors, either individually or in combination, allows for the complete fate recovery of cryopreserved and / or isolated PHHs and supports the maintenance of mature hepatocyte characteristics, this technological innovation has tremendous business potential. In addition, the biological effects of these humoral factors appear to support the function and fate of liver nonparenchymal cells (NPCs), such as hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), and liver macrophages (Kupffer cells). 1. These factors prevent dedifferentiation of terminally differentiated in vitro cultured human hepatocytes, thereby maintaining cell fate. 2. Cell culture media containing these factors inhibit the transdifferentiation of in vitro cultured human hepatocytes into cholangiocyte-like cells. 3. These factors promote the recovery of cryopreserved and / or freshly isolated human hepatocytes to establish stable in vitro cultures. 4. Cell culture media containing these factors protect PHH or HLCM-HH from cellular stress during cryopreservation, which may further support cell recovery upon thawing. 5. Cell culture media containing these factors supports the maintenance of NPC function and morphology. 6. These factors promote, maintain / enhance the functionality and longevity of HH or HLCM-HH in the format of spheroids, organoids or 3D cultures, and for the development of organ-on-a-chip formats. These factors promote and enhance the utility of PHH or HLCM-HH for the production of HLCM.

[0113] Unique and novel hepatocyte-specific humoral factor supplementation The biological effects of hepatocyte-specific humoral factors (hepatocyte fate recovery and maintenance) are robust, and therefore, their supplementation to hepatocyte culture medium greatly improves the utility of in vitro cultured hepatocytes, iHep, CLIP, and liver NPCs. Briefly, the production of one HLCM is achieved by injecting approximately 10 HLCM into the spleen of a host mouse (e.g., uPA-SCID strain). 5 PHH or HLCM-HH cells are required. This results in robust proliferation of PHH or HLCM-HH cells in the host mouse liver, where endogenous mouse hepatocytes with toxic transgene (uPA) overexpression are reduced as a result of competition with transplanted human hepatocytes. Eight weeks after cell transplantation, the host mouse liver is replaced by up to 95% of human hepatocytes. As a result, the liver of one HLCM mouse can contain 2-3 x 10 cells. 8 This expertise provides a stable supply of HLCM-HH cells, enabling the mass production of HLCM-HH. Additionally, HLCM-HH cells exhibit superior viability and plating ease compared to PHH cells (see review article PMID: 32074631 for details), preserving the authentic characteristics of terminally differentiated human hepatocytes during in vitro culture. This expertise led to the discovery that the culture medium for HLCM-HH cells (hereafter referred to as human hepatocyte conditioned medium: CMHH) contains hepatocyte-specific humoral factors necessary for the recovery and maintenance of human hepatocyte fate.

[0114] As described herein, applicants have identified key components mediating the physiological activity of CMHH, leading to the identification of hepatocyte-specific humoral factors FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors as critical nutritional supplements for human hepatocyte culture media. Several methodologies have been proposed to improve the maturation and function of in vitro cultured hepatocytes, such as Matrigel and a five-chemical combination (i.e., 5C) (PMID: 31023926). While there is some overlap between the biological effects of these methodologies and those of FN, laminin, VTN, ORM1, serpins, collagen, HP, TGM2, A2M, TNC, and / or coagulation factors, these factors, particularly in combination, have more robust and physiological effects than Matrigel or 5C-containing media. In addition, global transcriptome analysis reveals that the biological effects of hepatocyte-specific humoral factors (FN, laminin, VTN, ORM1, serpins, collagens, HP, TGM2, A2M, TNC, and / or coagulation factors) are highly distinct from the biological effects of Matrigel or 5C.

[0115] material and method: For human liver chimeric mice (HLCM), we first perform a quality assessment of the degree of human hepatocyte proliferation in the host mouse liver, i.e., the replacement index (RI). HLCM quality control can be performed through evaluation of serum / blood human albumin levels, which is widely accepted as a proxy for overall RI. We primarily utilize HLCM with blood albumin levels higher than 10 mg / ml, corresponding to an RI of over 70%. HLCM are then subjected to isolation of human hepatocytes (HH) via a collagenase perfusion-based method, followed by cell quality assessment (yield and viability). HLCM-derived human hepatocytes (HLCM-HH) are isolated at 2.1–4.2 x 10 cells. 5 pieces / cm 2Cells are seeded onto collagen-coated cell culture flasks / dishes at cell densities ranging from 0.1 to 1.5. Similarly, primary human hepatocytes (PHH) or HepaRG cells are seeded onto collagen-coated cell culture flasks / dishes. These cells are cultured in dHCGM, dHMM (see Appendix) or similar / equivalent media (e.g., but not limited to, dHCGM or dHMM containing DMSO) for up to 21 days. The culture medium is replaced every 24–48 h, and the medium incubated with the cells is collected and stored as human hepatocyte-conditioned medium (CMHH). The biological activity of CMHH in maintaining the cell fate of terminally differentiated hepatocytes is assessed by multi-readout assessment, including light microscopy for morphology, immunofluorescence microscopy for polarity, Western blotting for hepatocyte marker gene expression, and RT-qPCR. CMHH exhibits potent effects in supporting the recovery and maintenance of human hepatocyte fate, and therefore, key mediators of its biological effects are identified. First, CMHH was filtered through various types of size-exclusion columns to separate biomolecules by molecular weight and by their bioactivity, as assessed through the multi-readout approach described above for the role of protein molecules. Follow-up protein analysis revealed that CMHH is rich in both serum proteins and extracellular matrix. Next, the importance of each molecule was examined individually and in combination to further delineate the key determinants of CMHH bioactivity, leading to the identification of fibronectin, laminin, and fibrinogen as key components mediating the fate recovery and maintenance of human hepatocytes. Furthermore, analysis of HLCM decellularized liver tissue suggested that these molecules form a macromolecular complex that serves as a bioscaffold for human hepatocytes. Finally, in vitro studies targeting the mechanism of macromolecular complex formation, involving a series of specific inhibitors, determined that activation of the coagulation factor cascade plays a crucial role in the multimerization of fibronectin, laminin, and fibrinogen.

[0116] appendix

[0117] [Table 1]

[0118] [Table 2]

[0119] [Table 3]

[0120] [Example 2] material and method Isolation and culture of human hepatocytes from human liver chimeric mice (HLCM-HH) As described in our previous publication (Tateno 2015), human liver chimeric mice (HLCM) were generated through xenotransplantation of cryopreserved or freshly isolated primary human hepatocytes (lot: JFC, BioIVT, NY, USA) into severe combined immunodeficiency (SCID) mice carrying the albumin enhancer / promoter-driven transgene (cDNA), urokinase-type plasminogen activator (uPA) (i.e., cDNA-uPA Tg / SCID). HLCM with blood human albumin levels higher than 10 mg / mL were used for human hepatocyte isolation by two-step collagenase perfusion. Freshly isolated HLCM-derived human hepatocytes (HLCM-HH) were cultured on type I collagen-coated culture dishes (BioCoat, Corning, NY, USA) at a density of 1.05 x 10 cells. 5 pieces / cm 2The seeded HLCM-HHs were cultured in fresh dHCGM, INVITROGRO HI (HI, BioIVT), and hepatocyte culture medium (HCM, Lonza, MD, USA), or supplemented with the following factors: fibrinogen (catalog number F4883-500M, Sigma-Aldrich, Inc., MO, USA) or factor II inhibitor (F2i; dabigatran etexilate, catalog number SML2351-10mg, Sigma-Aldrich, Inc., MO, USA), factor X inhibitor (F10i; rivaroxaban, Sigma-Aldrich), vitronectin (VTN; catalog number 2349-VN, R&D systems, MN, USA), fibronectin (FN; catalog number 4305-FNB, R&D systems), and tenascin-C (TNC; catalog number 3358-TC, R&D systems). The cells were cultured for 7 days in these culture media supplemented with one or a combination of either HP (Haptoglobin; Catalog No. H3149-25KU, Sigma-Aldrich) or haptoglobin (HP; Catalog No. H3149-25KU, Sigma-Aldrich). To prepare HLCM-HH-derived conditioned medium, HLCM-HH cells were cultured in a collagen-coated T-75 flask (BioCoat, Corning) at a density of 2.1 × 10 cells. 5 pieces / cm 2 The culture supernatant was collected every 2 days from day 7 to day 21, pooled, and stored at −20° C. until use as conditioned medium.

[0121] Gene expression analysis Total RNA was extracted using the Zymo-Quick RNA Micro Kit (Zymo Research, CA, USA) according to the supplier's protocol. A total of 1 μg of RNA was used as a template for reverse transcription using Quanta cDNA Synthesis Supermix (Quantabio, MA, USA). Real-time quantitative PCR was performed using PowerUp SYBR Green Master Mix (ThermoFisher Scientific, MA, USA) and the QuantStudio 5 system (ThermoFisher Scientific) with the human-specific primer set summarized in Table 4. Relative gene expression levels were analyzed using the Delta-Delta Ct method.

[0122] [Table 4]

[0123] Immunoblotting analysis A total of 6 μL of fresh dHCGM and CM was mixed with Pro-Prep protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) supplemented with a protease inhibitor cocktail (MedChemExpress, NJ, USA). The mixture was subjected to SDS-PAGE and subsequently transferred to a PVDF membrane for protein detection. The antibodies and their dilution ratios are summarized in Table 5 (primary antibodies) and Table 6 (secondary antibodies).

[0124] [Table 5]

[0125] [Table 6]

[0126] Gene knockdown analysis For alpha2 macroglobulin (A2M) knockdown analysis, negative control and A2M-specific siRNA (Silencer select, catalog number: 4390824, assay ID: s819) were purchased from ThermoFisher Scientific. The siRNA was applied to a 96-well plate along with a transfection reagent and accelerator in CytoPathfinder (Tokyo, Japan). HLCM-HH cells were transfected onto the transfection plate at a density of 2.1 x 10 cells. 5 pieces / cm 2 and cultured in dHCGM for 9 days.

[0127] statistical analysis Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software, MA, USA). Significant differences were determined by Student's t-test. Multiple comparisons were performed using one-way or two-way ANOVA, followed by Dunnett's or Sidak's post-hoc test.

[0128] result Figure 1. Cultured HLCM-HH secreted various humoral factors into the culture supernatant. Immunoblotting analysis was performed to identify humoral factors secreted by HLCM-HH cultured with fresh dHCGM and CM. The results clearly showed that CM contained all investigated proteins, including FN, LAMC1, VTN, ORM, SERPINA3, COL4A1, HP, TGM2, A2M, and FGA.

[0129] Figure 2. Supplementation of dHCGM with fibrinogen enhances hepatic gene expression and prevents activation of cholangiocyte / hepatic progenitor cell marker expression. We supplemented fresh dHCGM with fibrinogen (50 μg / mL), one of the major components of HLCM-derived CM, and examined its effect on the expression of hepatic marker genes (CYP2C9, CYP2D6, OATP1B1) and cholangiocyte / liver progenitor cell marker genes (TGFB1, TGFB2, ANKRD1, KRT19). qPCR results clearly demonstrated that fibrinogen supplementation significantly improved the expression of hepatic marker genes. On the other hand, the expression of cholangiocyte / liver progenitor cell markers was significantly inhibited in HLCM-HH cultured in fibrinogen-containing dHCGM.

[0130] Figure 3. Inhibition of the coagulation cascade attenuated the effect of CM on hepatocyte phenotype. To investigate the importance of coagulation cascade activation for the beneficial effects of CM, HLCM-HH were cultured for 7 days with fresh dHCGM, CM, or CM containing F2i or F10i, and the expression profiles of hepatocyte and cholangiocyte / liver progenitor cell markers were analyzed. Compared with cells treated with FM, cells treated with CM showed both higher expression of hepatocyte markers and lower expression of cholangiocyte / liver progenitor cell markers. However, supplementation of CM with a potent coagulation inhibitor significantly reduced the positive effect of CM on maintaining the hepatocyte phenotype. These results suggest that activation of the coagulation cascade is one of the key pathways required for maximizing the effect of CM on maintaining hepatocyte function in cultured HLCM-HH.

[0131] Figure 4. Fibrinogen supplementation was able to improve the hepatocyte phenotype in HLCM-HH cultured in commercially available hepatocyte culture medium. To investigate whether fibrinogen can improve liver function in human hepatocytes cultured in commercially available hepatocyte culture media, HLCM-HH were cultured in two different hepatocyte culture media, Invitrogen HI (IH) and Hepatocyte Culture Medium (HCM), with or without supplemental fibrinogen. Expression of CYP2D6 and OATP1B1 was significantly activated by fibrinogen treatment. On the other hand, expression of cholangiocyte / hepatic progenitor cell markers, TGFB2 and ANKRD1, was significantly inhibited by fibrinogen treatment.

[0132] Figure 5. Effect of other CM components on maintaining hepatocyte gene expression profiles. Conditioned medium contains not only fibrinogen and coagulation factors, but also other hepatic matrisome components. We investigated whether these matrisome components also have a positive effect on hepatocyte gene expression profiles in cultured HH. HLCM-HH were cultured in fresh dHCGM (FM), CM, or FM supplemented with one or a combination of the following hepatic matrisome components (VTN, FN, TNC, HP) contained in CM. qPCR gene expression analysis showed that the tested hepatic matrisome components or their combinations at least partially mimicked the effects of CM on hepatocyte marker genes and cholangiocyte / liver progenitor cell markers.

[0133] Figure 6. Knockdown of alpha-2 macroglobulin (A2M) improved cholangiocyte marker expression in cultured HLCM-HH. To investigate the importance of A2M, we knocked down another conditioned medium component, A2M, by transfection with A2M-specific siRNA. At day 9 posttransfection, A2M expression was dramatically suppressed to less than 5% of that in HLCM-HH cells transfected with negative control siRNA. In addition, KRT19, a cholangiocyte / liver progenitor cell marker, was significantly upregulated in A2M-knockdown HLCM-HH cells. These results suggest that A2M, a CM component, plays an important role in maintaining the hepatocyte phenotype in cultured human hepatocytes.

Claims

1. Fibronectin (FN), laminin, vitronectin (VTN), orosomucoid 1 (ORM1), serpin, collagen, haptoglobin (HP), transglutaminase 2 (TGM2), alpha-2 macroglobulin (A2M), coagulation factors, tenascin C (TNC), and combinations thereof A hepatocyte culture medium supplemented with at least one factor selected from the group consisting of:

2. 2. The culture medium of claim 1, which is a hepatocyte clonal growth medium (HCGM) or a hepatocyte maintenance medium (HMM).

3. 2. The culture medium of claim 1, wherein the laminin is laminin subunit gamma 1 (LAMC1), laminin subunit gamma 2 (LAMC2), or laminin subunit gamma 3 (LAMC3).

4. 2. The culture medium of claim 1, wherein the coagulation factor is fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), or fibrinogen gamma chain (FGG).

5. The culture medium of any one of claims 1 to 4, further supplemented with DMSO or DMSO2.

6. 6. The culture medium of any one of claims 2 to 5, wherein the HCGM comprises, consists of, or consists essentially of Dulbecco's modified Eagle's medium (DMEM), L-proline, insulin, dexamethasone, EGF, and L-ascorbic acid 2-phosphate (Asc-2P).

7. 7. The culture medium of claim 6, wherein the DMEM is DMEM-10, and the DMEM-10 comprises, consists of, or consists essentially of DMEM, HEPES, penicillin-streptomycin, and fetal bovine serum (FBS).

8. 7. The culture medium of claim 6, wherein the L-proline is 15 μg / mL in the HCGM, the insulin is 0.25 μg / mL in the HCGM, the dexamethasone is 50 nM in the HCGM, the EGF is 5 ng / mL in the HCGM, the Asc-2P is 0.1 mM in the HCGM, the DMSO or DMSO2 is 2% or 140.8 mM, respectively, in the HCGM, the HEPES is 20 mM in the HCGM, the penicillin is 100 IU / mL in the HCGM, the streptomycin is 100 μg / mL in the HCGM, and the FBS is 10% heat-inactivated FBS in the HCGM.

9. The L-proline is 5 to 25 μg / mL in the HCGM, the insulin is 0.1 to 0.5 μg / mL in the HCGM, the dexamethasone is 10 to 100 nM in the HCGM, the EGF is 1 to 10 ng / mL in the HCGM, the Asc-2P is 0.01 to 1 mM in the HCGM, and the DMSO or the DMSO2 is 7. The culture medium of claim 6, wherein the concentrations of the HEPES and the penicillin are 0.5% to 5% and 100 to 180 mM, respectively, in the HCGM, the HEPES is 10 to 50 mM in the HCGM, the penicillin is 10 to 300 IU / mL in the HCGM, the streptomycin is 10 to 300 μg / mL in the HCGM, and the FBS is 2 to 20% heat-inactivated FBS in the HCGM.

10. 6. The culture medium of any one of claims 2 to 5, wherein the HMM comprises, consists of, or consists essentially of Dulbecco's modified Eagle's medium (DMEM), L-proline, insulin, dexamethasone, and L-ascorbic acid 2-phosphate (Asc-2P).

11. 11. The culture medium of claim 10, wherein the DMEM is DMEM-10, and the DMEM-10 comprises, consists of, or consists essentially of DMEM, HEPES, penicillin-streptomycin, and fetal bovine serum (FBS).

12. 11. The culture medium of claim 10, wherein the L-proline is 15 μg / mL in the HMM, the insulin is 0.25 μg / mL in the HMM, the dexamethasone is 50 nM in the HMM, the Asc-2P is 0.1 mM in the HMM, the DMSO or DMSO2 is 2% or 140.8 mM, respectively, in the HMM, the HEPES is 20 mM in the HMM, the penicillin is 100 IU / mL in the HMM, the streptomycin is 100 μg / mL in the HMM, and the FBS is 10% heat-inactivated FBS in the HMM.

13. 11. The culture medium of claim 10, wherein the L-proline is 5 to 25 μg / mL in the HMM, the insulin is 0.1 to 0.5 μg / mL in the HMM, the dexamethasone is 10 to 100 nM in the HMM, the Asc-2P is 0.01 to 1 mM in the HMM, the DMSO or DMSO2 is 0.5% to 5% or 100 to 180 mM in the HMM, respectively, the HEPES is 10 to 50 mM in the HMM, the penicillin is 10 to 300 IU / mL in the HMM, the streptomycin is 10 to 300 μg / mL in the HMM, and the FBS is 2 to 20% heat-inactivated FBS in the HMM.

14. The culture medium of any one of claims 2 to 13, wherein the FN is supplemented to 10,000 ng / mL in the HCGM or HMM.

15. The culture medium of any one of claims 2 to 13, wherein the laminin is supplemented to 1 to 10,000 pg / mL in the HCGM or HMM.

16. The culture medium of any one of claims 2 to 13, wherein the VTN is supplemented to 0.1 to 1000 ng / mL in the HCGM or HMM.

17. The culture medium of any one of claims 2 to 13, wherein the ORM1 is supplemented to 1 to 10,000 μg / mL in the HCGM or HMM.

18. The culture medium of any one of claims 2 to 13, wherein the serpin is supplemented to 0.1 to 1000 μg / mL in the HCGM or HMM.

19. The culture medium of any one of claims 2 to 13, wherein the collagen is supplemented to 1 to 10,000 ng / mL in the HCGM or HMM.

20. The culture medium of any one of claims 2 to 13, wherein the HP is supplemented to 0.01 to 100 mg / mL in the HCGM or HMM.

21. The culture medium according to any one of claims 2 to 13, wherein the TGM2 is supplemented to 0.01 to 100 ng / mL in the HCGM or HMM.

22. The culture medium of any one of claims 2 to 13, wherein the A2M is supplemented to 0.1 to 1000 μg / mL in the HCGM or HMM.

23. The culture medium of any one of claims 2 to 13, wherein the coagulation factors are supplemented to 0.1 to 1000 mg / mL in the HCGM or HMM.

24. The culture medium of any one of claims 2 to 13, wherein the TNC is supplemented to 0.01 to 100 ng / mL in the HCGM or HMM.

25. 25. The culture medium of any one of claims 1 to 24 for use in growing terminally differentiated human hepatocytes, hepatocytes derived from chimeric animals with humanized livers, and / or cryopreserved and / or freshly isolated primary human hepatocytes.

26. 25. The culture medium of any one of claims 1 to 24, for use in differentiating hepatic bipotent progenitor cells (iHep) and / or chemically induced liver progenitors into terminally differentiated hepatocytes or mature hepatocytes, respectively.

27. 25. The culture medium according to any one of claims 1 to 24, for use in co-culture with non-parenchymal cells and / or for use in suspending human hepatocytes for cryopreservation.

28. HLCM - A culture medium according to any one of claims 1 to 24 for use in the expansion and / or proliferation of iHep and Chemically Induced Liver Progenitors (CLiP) in the liver of a host mouse.

29. 25. A method for growing terminally differentiated human hepatocytes, hepatocytes derived from a chimeric animal with a humanized liver, and / or cryopreserved and / or freshly isolated primary human hepatocytes, the method comprising culturing a predetermined amount of hepatocytes in a cell culture medium according to any one of claims 1 to 24.

30. 24. A method for differentiating hepatic bipotent progenitor cells (iHep) and / or chemically induced liver progenitors into terminally differentiated hepatocytes or mature hepatocytes, respectively, comprising culturing a predetermined amount of hepatocytes in the cell culture medium according to any one of claims 1 to 23.

31. 24. A method for co-culturing human hepatocytes with non-parenchymal cells and / or suspending human hepatocytes for cryopreservation, comprising culturing a predetermined amount of hepatocytes in a cell culture medium according to any one of claims 1 to 23.

32. 24. A method for expanding and / or growing iHep and CLiP in the liver of a HLCM-host mouse, comprising culturing a predetermined amount of hepatocytes in a cell culture medium according to any one of claims 1 to 23.

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