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1. (WO2019227063) METHODS, ASSAYS, AND KITS FOR EVALUATING THE PERFORMANCE OF ENGINEERED LIVER TISSUE CONSTRUCTS
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METHODS, ASSAYS, AND KITS FOR EVALUATING THE PERFORMANCE OF ENGINEERED LIVER TISSUE CONSTRUCTS FIELD OF THE INVENTION

[0001] This application relates to engineered liver tissue constructs, such as three- dimensional bioprinted liver tissue constructs, as well as methods, assays, and kits for evaluating the performance of the constructs.

BACKGROUND OF THE INVENTION

[0002] Conventional cell therapy and conventional tissue engineering approaches to treating liver diseases and injury are limited by low cell retention, poor engraftment, poor graft durability, and complications such as portal hypertension. U.S. Patent Nos.

9,222,932 and 9,442,105 provide examples of three-dimensional bioprinted liver tissue constructs developed by Organovo, Inc. (San Diego, California) for overcoming the complications and limitations of prior cell therapy and tissue engineering approaches, and are incorporated herein by reference. These bioprinted liver tissue constructs have the potential for broad clinical applicability ranging from treatment of inborn errors of metabolism to acute or chronic liver failure. For example, Organovo, Inc. demonstrated efficacy in a mouse model of alpha-1 antitrypsin deficiency (AATD), an inborn error of metabolism.

[0003] As engineered liver tissue constructs move forward towards human testing and therapies, there is a need for clinically translatable biomarkers for use in monitoring the performance of the constructs, including in vitro, pre-implantation, and post-implantation performances. While circulating exosomal RNAs (exRNAs) have been previously identified as potential biomarkers for liver regeneration after partial hepatectomy in mice (Yan et al., PLOS ONE 11(7):e0155888 (2016)), hepatocyte injury in alcoholic, drug- induced and inflammatory liver disease (Bala et al., Hepatology 56(5):1946-1957 (2012)), and hepatocellular carcinoma (HCC) (Kogure et al., Hepatology 54(4):1237- 1248 (2011)), there is an unmet need for biomarkers for evaluating the performance of engineered liver constructs, such as during in vitro culture (e.g., as pre-operative criteria for selecting suitable constructs for implantation) or following implantation into a patient (e.g., as post-operative criteria for determining efficacy of the implant).

BRIEF SUMMARY OF THE INVENTION

[0004] In one aspect, methods for evaluating post-implantation performance of an

implanted engineered liver tissue construct in a subject are provided, comprising:

obtaining a pre-implantation blood sample and a post-implantation blood sample from the subject; measuring expression of exosomal RNA (exRNA) comprising miR-122 and miR- 181a in the pre-implantation and post-implantation blood samples; and evaluating post- implantation performance of the construct, wherein an effective post-implantation performance comprises an increased expression of miR-122 and miR-181a in the post- implantation blood sample as compared to the pre-implantation blood sample.

[0005] In some embodiments, the exRNA further comprises miR-152 and an effective post-implantation performance further comprises increased expression of miR-152 in the post-implantation blood sample as compared to the pre-implantation blood sample.

[0006] In some embodiments, the method further comprises measuring the concentration of albumin in the pre-implantation and the post-implantation blood samples, and an effective post-implantation performance further comprises increased concentration of albumin in the post-implantation blood sample as compared to the pre-implantation blood sample.

[0007] In some embodiments, the expression of miR-122 and miR-181a in the post- implantation blood sample is dose responsive to the number of implanted constructs.

[0008] In some embodiments, the post-implantation blood sample comprises multiple samples obtained over a period of time. In some embodiments, an effective post- implantation performance comprises: an increase in the expression of miR-122 and miR- 181a in a post-implantation sample within about 7 days after implantation of the construct, followed by normalization in the expression of miR-122 and miR-181 in a post-implantation sample within about 28 days after implantation of the construct.

[0009] In some embodiments, the method further comprises implanting an additional engineered liver tissue construct based on the evaluation in order to obtain or further increase an effective post-implantation performance.

[0010] In some embodiments, the blood samples are serum or plasma samples.

[0011] In some embodiments, the construct comprises a three-dimensional bioprinted human liver tissue construct. In some embodiments, the three-dimensional bioprinted human liver tissue construct comprises an interior comprising parenchymal cells and a border comprising non-parenchymal cells, the parenchymal cells comprising human hepatocytes or human hepatocyte-like cells.

[0012] In some embodiments, the subject has a liver disorder. In some embodiments, the liver disorder is an inborn error of metabolism (IEM), and the IEM is alpha-1 antitrypsin deficiency (AATD).

[0013] In one aspect, methods for evaluating performance of an engineered liver tissue construct are provided, comprising: (a) preparing an in vitro sandwich culture comprising: incubating a layer of cells comprising mammalian hepatocytes in contact with an engineered liver tissue construct; (b) obtaining a sample of supernatant from the in vitro sandwich culture and a sample of supernatant from a control culture comprising the layer of cells without the construct or the construct without the layer of cells; (c) measuring the expression of an exRNA comprising miR-122 in the samples; and

(d) evaluating the performance of the construct, wherein an effective performance comprises an increased expression of miR-122 in the sample from the in vitro sandwich culture as compared to the sample from the control culture.

[0014] In some embodiments, the exRNA further comprises miR-21, miR-181a, miR- 152, or a combination thereof, and wherein the expression of miR-21, miR-181a, miR- 152, or a combination thereof is dose responsive to the number of constructs in the in vitro sandwich culture.

[0015] In some embodiments, the method further comprises measuring the concentration of albumin in the samples, wherein an effective performance comprises an increased concentration of albumin in the sample from the in vitro sandwich culture as compared to the sample from the control culture.

[0016] In some embodiments, the sample from the in vitro sandwich culture comprises multiple samples obtained at over a period of time. In some embodiments, an effective performance comprises an increase in the expression of miR-122 in the sample from the in vitro sandwich culture within about 3 days of incubation, followed by normalization of expression of miR-122 in the sample from the in vitro sandwich culture within about 7 days of incubation.

[0017] In some embodiments, the construct is a three-dimensional bioprinted human liver tissue construct. In some embodiments, the three-dimensional bioprinted liver tissue construct comprises an interior comprising parenchymal cells and a border comprising

non-parenchymal cells, the parenchymal cells comprising human hepatocytes or human hepatocyte-like cells.

[0018] In some embodiments, the hepatocytes in the layer of cells comprise diseased and/or healthy hepatocytes.

[0019] In some embodiments, the hepatocytes in the construct comprise diseased and/or healthy hepatocytes.

[0020] In some embodiments, the hepatocytes are from a subject with a liver disorder. In some embodiments, the liver disorder is an IEM, and the IEM is AATD.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figs.1A-1B illustrate the implantation of an exemplary three-dimensional

bioprinted therapeutic liver tissue (“BTLT”) construct into a mouse, with Fig.1A showing placement of the construct at the tip of the left liver lobe and Fig.1B showing the construct sutured to the liver.

[0022] Fig.2 illustrates the implantation of two exemplary BTLT constructs into a

mouse, with the individual sutured constructs indicated by number.

[0023] Figs.3A-3B illustrate the in vivo profiling of serum expression levels (fold

change, Log2FC) of exosomal ribonucleic acids (“exRNAs”) miR-122, miR-126, miR- 152, miR-181a, miR-192, miR-1, and miR-131a in a mouse at day 7 (Fig.3A) and day 28 (Fig.3B) post-implantation of an exemplary BTLT construct as compared to a sham- operated mouse (“placebo”).

[0024] Fig.3C depicts the concentration of human albumin (ng/ml) in the serum of the implanted mouse of Figs.3A-3B for up to 91 days post-implantation as compared to a sham-operated control mouse.

[0025] Figs.4A-4B illustrate the in vivo profiling of human albumin concentration

(ng/ml) (Fig.4A) and expression levels of exRNAs miR-152, miR-181a, miR-122, and miR-21 (Fig.4B) in the serum of mice implanted with one (1) or two (2) exemplary BTLT constructs. exRNA expression levels in Fig.4B are shown as fold change

(Log2FC) over 1 BTLT.

[0026] Figs.5A-5C illustrate multi-well plates with one (1) (Fig.5A), two (2) (Fig.5B), and three (3) (Fig.5C) exemplary BTLT constructs per well.

[0027] Figs.6A-6B illustrate the in vitro profiling of human albumin concentration

(ng/ml) (Fig.6A) and expression levels of exRNAs miR-152, miR-181a, miR-122, and

miR-21 (Fig.6B) in the culture media from individual wells of a multi-well plate containing (1), two (2), or three (3) exemplary BTLT constructs per well. exRNA expression levels in Fig.6B are shown as fold change (Log2FC) over 1 BTLT.

[0028] Figs.7A-7B illustrate the in vitro profiling of cell viability (%) in exemplary

BTLT constructs (Fig.7A), and expression levels of exRNAs miR-122, miR-152, and miR-21 (Fig.7B) in the culture media of exemplary BTLT constructs, following treatment with 0, 2.5, 5, 10, or 20% dimethyl sulfoxide (“DMSO”). Cell viability in Fig. 7A and exRNA expression levels (Log2FC) in Fig.7B are relative to the cell viability and exRNA expression levels, respectively, in a BTLT construct cultured in media containing 0% DMSO.

[0029] Fig.8 illustrates the in vitro profiling of cell viability (%) in an exemplary BTLT construct, a construct containing the nonparenchymal cell border of the BTLT construct but lacking a hepatocyte fill (“NPC” construct), and a construct containing the hepatocyte fill of the BTLT construct but lacking the nonparenchymal cell border (“Fill” construct), following treatment with 0, 2.5, 3.25, 5, 7.5, or 10% DMSO.

[0030] Figs.9A-9C illustrate the in vitro profiling of alanine transaminase (“ALT”)

activity (mU/mL) in the culture media of BTLT (Fig.9A), NPC (Fig.9B), and Fill (Fig. 9C) constructs following treatment with 0, 2.5, 3.25, 5, 7.5, or 10% DMSO for 2, 8, 24, or 72 hours.

[0031] Fig.10A-10C illustrate the in vitro profiling of lactate dehydrogenase (“LDH”) activity (mU/mL) in the culture media of BTLT (Fig.10A), NPC (Fig.10B), and Fill (Fig.10C) constructs following treatment with 0, 2.5, 3.25, 5, 7.5, or 10% DMSO for 2, 8, 24, or 72 hours.

[0032] Fig.11 illustrates a plot of LDH activity (mU/mL) from Figs.10A-C versus cell viability (%) from Fig.8 for BTLT, NPC, and Fill constructs.

[0033] Fig.12A-12B illustrate the in vitro profiling of expression levels for exRNAs miR-122, miR-152, and miR-181a in the culture media of BTLT, Fill, and NPC constructs following treatment with 0, 3.25, or 10% DMSO for 24 hours (Fig.12A) or 72 hours (Fig.12B). exRNA expression levels are shown as fold change (Log2FC) over constructs treated with 0% DMSO.

[0034] Fig.13A-13B illustrate the in vitro profiling of albumin concentration

(ng/mL/million cells) in the culture media of BTLT (Fig.13A) and Fill (Fig.13B)

constructs following treatment with 0, 2.5, 3.25, 5, 7.5, or 10% DMSO for 2, 8, 24, or 72 hours.

[0035] Fig.14 illustrates a plot of albumin concentration (ng/mL/million cells) from

Figs.12A-12B versus cell viability (%) from Fig.8 for BTLT and Fill constructs.

[0036] Fig.15A illustrates an in vitro sandwich culture containing a liver tissue construct on top of a layer of cells comprising mammalian hepatocytes.

[0037] Fig.15B illustrates the expression level of exRNA miR-22 in culture media after 72 hours and 1 week of culturing: a sandwich culture containing an exemplary BTLT construct on top of a layer of mouse hepatocytes, a layer of mouse hepatocytes, an exemplary BTLT construct, and a reverse sandwich control containing a layer of mouse hepatocytes on top of an exemplary BTLT construct. exRNA expression is shown in arbitrary units.

[0038] Fig.16 illustrates the in vitro profiling of cell viability (%) of exemplary BTLT constructs cultured under normal conditions (“negative control”) and after protocol deviations during production of the BTLT including excessive pipetting when preparing the hepatocyte fill (“excessive pipetting”), culturing for 4 hours at room temperature (“4 hr RT hold”), culturing for 4 hours without media (“4 hr no media hold”), and culturing in 10% DMSO (“positive control”). The cell viability is shown relative to cell viability of the negative control.

[0039] Figs.17-21 illustrate the in vitro profiling of expression levels of exRNAs miR- 122, miR-126, miR-21, and miR-181a in culture media at 4, 24, 48, and 72 hours after culturing the negative control (Fig.17), excessive pipetting (Fig.18), 4 hour room temperature hold (Fig.19), 4 hour no media hold (Fig.20), and positive control (Fig.21) constructs as described in Fig.16. exRNA expression levels are shown in arbitrary units.

[0040] Fig.22 illustrates the in vitro profiling of LDH activity (mU/mL) in culture media at 4, 24, 48, and 72 hours after culturing the negative control, 4 hour room temperature hold, excessive pipetting, 4 hour no media hold, and positive control constructs as described in Fig.16.

[0041] Fig.23 illustrates the in vitro profiling of the albumin concentration

(ng/mL/million cells) in culture media at 24, 48, and 72 hours after culturing the negative control, excessive pipetting, 4 hour room temperature hold, 4 hour no media hold, and positive control constructs as described in Fig.16.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present disclosure provides methods, assays, and kits for evaluating the

performance of engineered liver tissue constructs.

[0043] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. Similarly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

I. Certain Definitions

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

[0045] The singular forms“a,”“an,” and“the” as used herein include plural references unless the context clearly dictates otherwise. For example,“a” cell includes one cell, one or more cells, and a plurality of cells. The terms“a,”“an,”“the,”“one or more,” and“at least one,” for example, can be used interchangeably herein.

[0046] As used herein, the term“about” or“approximately” when used to modify an amount related to the invention, refers to variation in the numerical quantity that can occur, for example, through routine testing and handling; through inadvertent error in such testing and handling; through differences in the manufacture, source, or purity of ingredients employed in the invention; and the like. Whether or not modified by the term “about” or“approximately,” the claims include equivalents of the recited quantities. In some embodiments, the term“about” or“approximately” means plus or minus 10% of the noted amount.

[0047] The term“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include“A and B,”“A or B,”“A” (alone), and“B” (alone). Likewise, the term“and/or” as used in a phrase such as“A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0048] As used herein,“bioprinting,”“bioprinted,”“bio-printing,” or“bio-printed” means utilizing three-dimensional, precise deposition of cells (e.g., cell solutions, cell- containing gels, cell suspensions, cell concentrations, multicellular aggregates, multicellular bodies, etc.) via methodology that is compatible with an automated or semi- automated, computer-aided, three-dimensional prototyping device (e.g., a bio-printer). Suitable bioprinters include the Novogen Bioprinter® from Organovo, Inc. (San Diego, CA), and suitable bio-printers disclosed in U.S. Patent Nos.8,931,880, 9,227,339, 9,855,369, 9,499,779, and 9,315,043.

[0049] The terms“comprises,”“comprising,”“includes,”“including,”“having,” and their conjugates are interchangeable and mean“including but not limited to.” It is understood that wherever aspects are described herein with the language“comprising,” otherwise analogous aspects described in terms of“consisting of” and/or“consisting essentially of” are also provided.

[0050] The term“consisting of” means“including and limited to.”

[0051] The term“consisting essentially of” means the specified material of a

composition, or the specified steps of a method, and those additional materials or steps that do not materially affect the basic characteristics of the material or method.

[0052] As used herein, the term“effective” means sufficient to effect beneficial or

desired results and, as such, depends upon the context in which it is being applied.

[0053] As used herein, the term“substantially” refers to the qualitative condition of

exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0054] The terms“invention” and“disclosure” can be used interchangeably when

describing or used, for example, in the phrases“the present invention” or“the present disclosure.”

[0055] The term“as used herein” refers to any part of the application, including the

appended claims and Figures.

[0056] Throughout this application, various embodiments of this invention can be

presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible

limitation on the scope of the invention. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, unless the context clearly dictates otherwise. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range, such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 2, from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 3, from 2 to 4, from 2 to 5, from 2 to 6, from 3 to 4, from 3 to 5, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and subranges of less than whole number such as 1.1, 1.2, 1.3, 1.4, etc. This applies regardless of the breadth of the range. Furthermore, the expression levels, activities, or concentrations discussed herein can include ranges of any of the corresponding levels, activities, or concentrations based on values disclosed in the accompanying Figures and/or Examples, with any value in the Figures and/or Examples serving as an endpoint of a range. Such values can include the specific values disclosed in the Figures and/or Examples, or can be rounded to the closest whole number, or to the next lower or higher increment of 5 for values that are tenths or hundredths of a whole number (e.g., a value of 1.2 can be designated as 1.2, or rounded to 1, 1.0, or 1.5, and a value of 1.34 can be designated as 1.34, or rounded to 1, 1.0, 1.5, 1.00, 1.30, 1.35, or 1.50).

[0057] As used herein, the term“subject” or“patient,” means any subject, particularly a mammalian subject, who is undergoing diagnosis, prognosis, or therapy. In certain embodiments, the subject is a human subject or patient. The term“mammal” and derivatives thereof such as“mammalian” include, but are not limited to, humans;

primates such as apes, monkeys, orangutans, and chimpanzees; rodents such as mice, rats, hamsters and guinea pigs; and so on.

[0058] It is appreciated that certain features of the invention, which are, for clarity,

described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

II. Methods for Evaluating Post-Implantation Performance of an Engineered Liver Tissue Construct Using Biomarkers

[0059] In one aspect, the present invention is directed to a method for evaluating post- implantation performance of an engineered liver tissue construct in a subject comprising: obtaining a pre-implantation blood sample and a post-implantation blood sample from the subject; measuring expression of exosomal RNA (exRNA) comprising miR-122 and miR- 181a in the pre-implantation and post-implantation blood samples; and evaluating post- implantation performance of the construct, wherein an effective post-implantation performance comprises an increased expression of miR-122 and miR-181a in the post- implantation blood sample as compared to the pre-implantation blood sample. In some embodiments, the exRNA further comprises miR-152 and an effective post-implantation performance further comprises increased expression of miR-152 in the post-implantation blood sample as compared to the pre-implantation blood sample.

[0060] In some embodiments, the method further comprises implanting the engineered liver tissue construct into a subject. In some embodiments, implanting the engineered liver tissue construct comprises attaching the construct to the subject's liver. In some embodiments, implanting the engineered liver tissue construct into a subject comprises suturing the construct to the subject's liver and/or attaching the construct to the subject's liver with a bioadhesive. In some embodiments, implanting the engineered liver tissue construct comprises suturing and/or adhering the construct onto a surface of the subject's liver.

[0061] In some embodiments, the post-implantation performance comprises engraftment effectiveness (e.g., whether the construct is retained in the subject following

implantation), tissue health of the implanted construct, secreted proteins, metabolic activity, de novo vessel formation, alanine transaminase (ALT) activity, lactate dehydrogenase (LDH) activity, graft durability, complications of implantation (e.g., portal hypertension, inflammation, and/or host rejection), improvement in a liver function in a subject (e.g., as assessed by a liver function test), improvement in a symptom associated with a liver disease or disorder in a subject, or a combination thereof.

[0062] In some embodiments, the post-implantation performance comprises engraftment effectiveness. In some embodiments, effective engraftment comprises increased

expression of miR-122 and miR-181a in the post-implantation blood sample as compared to the pre-implantation blood sample. In some embodiments, effective engraftment further comprises increased expression of miR-152 in the post-implantation blood sample as compared to the pre-implantation blood sample. In some embodiments, the increased expression of miR-122 and miR-181a or the increased expression of miR-122, miR-181a, and miR-152 is a biomarker of early engraftment.

[0063] In some embodiments, the method further comprises assessing integration of the implanted engineered liver tissue construct with the subject's vasculature (e.g., integration of the construct with the vasculature of the subject's liver) and/or assessing vascular perfusion into the construct. For example, when there is effective vascular integration and perfusion with the host tissue of a patient, an implanted engineered liver tissue construct will likely have stable performance over a period of time. In some

embodiments, assessing integration of the implanted engineered liver tissue construct with the subject's vasculature and/or assessing vascular perfusion into the implanted engineered liver tissue construct comprises Doppler ultrasound imaging to verify blood flow into donor tissue.

[0064] In some embodiments, evaluating engraftment effectiveness further comprises assessing integration of the implanted engineered liver tissue construct with the subject's vasculature and/or assessing vascular perfusion into the construct. In some embodiments, effective engraftment further comprises integration of the implanted engineered liver tissue construct with the subject's vasculature and/or vascular perfusion into the implanted engineered liver tissue construct.

[0065] In some embodiments, the method further comprises measuring a liver function in the pre-implantation and post-implantation blood samples. In some embodiments, measuring a liver function comprises measuring the concentration of albumin, the concentration of ammonia, alkaline phosphatase (ALP) activity, gamma-glutamyl transferase (GGT) activity, prothrombin time (PT), activated partial thromboplastin time (aPTT), the concentration of bilirubin (direct and/or indirect), alanine transaminase (ALT) activity, aspartate transaminase (AST) activity, the total protein concentration, lactate dehydrogenase (LDH) activity, the concentration of alpha-feto protein (AFP), or a combination thereof.

[0066] In some embodiments, the method further comprises analyzing the samples with a liver test panel (LFT), wherein the LFT is for measuring a plurality of liver functions. [0067] In some embodiments, evaluating engraftment effectiveness further comprises measuring a liver function as described herein.

[0068] In some embodiments, evaluating engraftment effectiveness further comprises analyzing the samples with a LFT.

[0069] In some embodiments, the post-implantation performance comprises tissue health (e.g., viability and/or function), including health of specific cell types such as, but not limited to, hepatocyte health.

[0070] In some embodiments, effective tissue health comprises increased expression of miR-122 and miR-181a in the post-implantation blood sample as compared to the pre- implantation blood sample. In some embodiments, effective tissue health further comprises increased expression of miR-152, miR-21, or both in the post-implantation blood sample as compared to the pre-implantation blood sample. In some embodiments, the increased expression of miR-122 and miR-181a, the increased expression of miR-122, miR-181a, and miR-152, the increased expression of miR-122, miR-181a, miR-21, or the increased expression of miR-122, miR-181a, miR-152, and miR-21 is a biomarker of tissue health.

[0071] In some embodiments, evaluating tissue health further comprises measuring a liver function.

[0072] In some embodiments, evaluating tissue health further comprises analyzing the samples with a LFT.

[0073] In some embodiments, the method further comprises measuring the concentration of albumin in the pre-implantation and the post-implantation blood samples, and an effective post-implantation performance further comprises increased concentration of albumin in the post-implantation blood sample as compared to the pre-implantation blood sample.

[0074] In some embodiments, the exRNA further comprises a nonspecific exRNA, which also can be described as an exRNA that is nonspecific to liver (e.g., an exRNA that is not expressed in liver tissue or that is expressed in other tissues in addition to liver tissue).

[0075] In some embodiments, an effective performance (e.g., effective engraftment and/or effective tissue health) comprises an expression of a nonspecific exRNA that is about the same in the post-implantation blood sample as compared to the pre-implantation blood sample (i.e., where the expression of the nonspecific exRNA is stable (i.e., substantially unchanged) in the post-implantation blood sample as compared to the pre-

implantation blood sample). In some embodiments, the nonspecific exRNA comprises miR-1 and/or miR-131a.

[0076] In some embodiments, the exRNA further comprises miR-126 and/or miR-192.

[0077] In some embodiments, the implanted construct comprises one or more than one engineered liver tissue construct. In some embodiments, the implanted construct comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more engineered liver tissue constructs.

[0078] In some embodiments, the expression of miR-122 and miR-181a in the post- implantation blood sample is dose responsive to the number of implanted constructs. In some embodiments, the expression level of miR-122 and miR-181a can demonstrate effective engraftment and/or tissue health of multiple implanted constructs, such as whether all or less than all of the implants (including a specific number of the implants) demonstrate effective engraftment and/or tissue health.

[0079] In some embodiments, the engineered liver tissue construct comprises a three- dimensional bioprinted liver tissue construct. In some embodiments, the engineered liver tissue construct comprises a three-dimensional bioprinted human liver tissue construct. U.S. Patent Nos.9,222,932 and 9,442,105 provide examples of such three-dimensional bioprinted liver tissue constructs, and are incorporated herein by reference. In some embodiments, the three-dimensional bioprinted human liver tissue construct comprises an interior comprising parenchymal cells and a border comprising non-parenchymal cells. In some embodiments, the border surrounds the interior. In some embodiments, the parenchymal cells comprise human hepatocytes or human hepatocyte-like cells. In some embodiments, the non-parenchymal cells comprise endothelial cells and stellate cells. In some embodiments, the three-dimensional bioprinted human liver tissue construct comprises other cell types, cell ratios, and configurations, as disclosed in U.S. Patent Nos. 9,222,932 and 9,442,105. In some embodiments, the parenchymal cells are derived from one or more of the following sources: adult mammalian liver tissue; fetal mammalian liver tissue; embryonic stem cells (ESC); ESC-derived hepatocyte-like cells, induced pluripotent stem cells (IPSC); IPSC-derived hepatocyte-like cells; adult stem/progenitor cells derived from the liver; and adult stem/progenitor cells derived from a tissue other than liver. In some embodiments, the non-parenchymal cells comprise one or more of: vascular cells, endothelial cells, fibroblasts, mesenchymal cells, immune cells, Kupffer cells, stellate cells, biliary epithelial cells, biliary epithelial-like cells, sinusoidal endothelial cells, liver-derived stem/progenitor cells, and non-liver-derived

stem/progenitor cells. In some embodiments, the construct comprises one or more layers. In further embodiments, the construct comprises a plurality of layers, at least one layer compositionally or architecturally distinct from at least one other layer to create a laminar geometry.

[0080] The engineered liver tissue constructs of the methods can include constructs made from seeding cells or layers of cells onto a scaffold or constructs made from a

combination of seeding and bioprinting techniques.

[0081] The pre-implantation and/or post-implantation blood sample can be serum,

plasma, any component of blood in which biomarkers can be evaluated, or any combination thereof. In some embodiments, the blood samples are serum or plasma samples.

[0082] In some embodiments, the subject has a liver disorder, such as, but not limited to: non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH); inborn error of metabolism (IEM), such as, but not limited to, alpha-1 antitrypsin deficiency (AATD); hepatitis A, B, or C; cancer; fibrosis; cirrhosis; inflammation; autoimmune disease; genetic diseases; any other known liver disorder, disease, or condition; or a combination thereof. In some embodiments, the liver disorder is an IEM, and the IEM is AATD. In some embodiments, the subject is a human.

[0083] In some embodiments, the pre-implantation and/or post-implantation blood

sample comprises one or more than one blood samples. In some embodiments, the blood samples are obtained at a pre-determined frequency over a period of time. The pre- determined frequency can be any frequency, such as once approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; once approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days; approximately 1, 2, 3, or 4 weeks;

approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or at any frequency over any number of days, weeks, or months post-implantation. The period of time can also be any period of time post-implantation, such as approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days; approximately 1, 2, 3, or 4 weeks; approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months; or any number of days, weeks, months, or years post-implantation.

[0084] In some embodiments, the post-implantation blood sample comprises multiple samples obtained over a period of time.

[0085] In some embodiments, an effective post-implantation performance comprises an increase in the expression of miR-122 and miR-181a in a post-implantation blood sample as compared to a pre-implantation blood sample after implantation of the construct, followed by normalization in the expression of miR-122 and miR-181 in a post- implantation blood sample as compared to a pre-implantation blood sample. In some embodiments, an effective post-implantation performance comprises: an increase in the expression of miR-122 and miR-181a in a post-implantation sample within about 7 days (i.e., within about 1 week) after implantation of the construct, followed by normalization in the expression of miR-122 and miR-181 in a post-implantation sample within about 28 days (i.e., within about 4 weeks or about 1 month) after implantation of the construct.

[0086] In some embodiments, the method further comprises implanting an additional engineered liver tissue construct based on the evaluation in order to obtain or further increase an effective post-implantation performance. For example, if increased expression of miR-122, miR-181a, miR-152, miR-21, or a combination thereof is not observed in a post-implantation blood sample as compared to a pre-implantation blood sample, then additional implantations can occur until an increase is observed. Similarly, if evaluation of post-implantation performance does not indicate effective performance (e.g., effective engraftment and/or effective tissue health), then additional implantations can occur until an effective performance is achieved.

III. In Vitro Sandwich Cultures and Methods for Evaluating the Performance of

Engineered Liver Tissue Constructs

[0087] In another aspect, the present invention is directed to an in vitro sandwich culture comprising: a layer of cells comprising mammalian hepatocytes in contact with an engineered liver tissue construct.

[0088] The engineered liver tissue construct can be any engineered liver tissue construct as described herein. In some embodiments, the engineered liver tissue construct is a three-dimensional bioprinted liver tissue construct. In some embodiments, the engineered liver tissue construct is a three-dimensional bioprinted human liver tissue construct. In some embodiments, the three-dimensional bioprinted liver tissue construct comprises an interior comprising parenchymal cells and a border comprising non-parenchymal cells, the parenchymal cells comprising human hepatocytes or human hepatocyte-like cells

[0089] In some embodiments, the layer of cells is bioprinted or is a monolayer of cultured cells.

[0090] In some embodiments, the layers of cells are non-human mammalian cells (e.g., mouse or primate cells), and the construct is a human construct. In some embodiments, the layer of cells are mouse cells. In some embodiments, the layer of cells comprise mouse hepatocytes.

[0091] In some embodiments, the layer of cells are human cells and comprise human hepatocytes.

[0092] In some embodiments, the hepatocytes in the layer of cells comprise diseased and/or healthy hepatocytes.

[0093] In some embodiments, the hepatocytes in the construct comprise diseased and/or healthy hepatocytes.

[0094] In some embodiments, cells in the layer of cells, cells in the construct, and/or diseased hepatocytes are from a subject with a liver disorder as described herein. For example, if the liver disorder is NAFLD, then the cells exhibit characteristics of NAFLD, such as inflammation, lipid accumulation, and fibrosis. In some embodiments, the liver disorder is an inborn error of metabolism (IEM), and the IEM is alpha-1 antitrypsin deficiency (AATD). In some embodiments, the layer of cells comprising at least hepatocytes and/or the cells in the construct comprise healthy cells, diseased cells, or a combination of healthy and diseased cells.

[0095] In some embodiments, the construct is positioned on top of the layer of cells.

[0096] In some embodiments, the layer of cells is positioned on top of the construct.

[0097] In another aspect, the present invention is directed to a method for evaluating performance of an engineered liver tissue construct comprising: (a) preparing an in vitro sandwich culture comprising: incubating a layer of cells comprising mammalian hepatocytes in contact with an engineered liver tissue construct; (b) obtaining a sample of supernatant from the in vitro sandwich culture, and a sample of supernatant from a control culture comprising the layer of cells without the construct or the construct without the layer of cells; (c) measuring the expression of an exosomal RNA (exRNA) comprising miR-122 in the samples; and (d) evaluating the performance of the construct, wherein an effective performance comprises an increased expression of miR-122 in the sample from the in vitro sandwich culture as compared to the sample from the control

culture. The sandwich culture, layer of cells, and engineered liver tissue construct are as described herein.

[0098] In some embodiments, the exRNA further comprises miR-21, miR-181a, miR- 152, or a combination thereof, wherein the expression of miR-21, miR-181a, miR-152, or a combination thereof is dose responsive to the number of constructs in the in vitro sandwich culture.

[0099] In some embodiments, the sandwich culture comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more engineered liver tissue constructs. In some embodiments, the expression level of miR-21, miR-181a, miR-152, or a combination thereof can demonstrate effective performance of multiple constructs in the in vitro sandwich culture, such as whether all or less than all of the constructs (including a specific number of the constructs) demonstrate effective performance.

[0100] In some embodiments, the performance comprises tissue health of the construct, cell viability, secreted proteins, metabolic activity, de novo vessel formation, alanine transaminase (ALT) activity, lactate dehydrogenase (LDH) activity, improvement in a liver function of the layer of cells comprising hepatocytes, or a combination thereof.

[0101] In some embodiments, the method further comprises measuring cell viability of the layer of cells, including hepatocytes in the layer of cells, and/or measuring cell viability of the construct, including a portion thereof such as a nonparenchymal cell border of the construct as described herein or a fill of the construct as described herein, including hepatocytes in the fill.

[0102] In some embodiments, the method further comprises histological examination of the sandwich culture. In some embodiments, the histological examination comprises hematoxylin and eosin (H&E) staining.

[0103] In some embodiments, the method further comprises measuring a liver function in the samples. In some embodiments, measuring a liver function comprises measuring the concentration of albumin, the concentration of ammonia, ALP activity, GGT activity, PT, aPTT, the concentration of bilirubin (direct and/or indirect), ALT activity, (AST activity, the total protein concentration, lactate dehydrogenase (LDH) activity, the concentration of alpha-feto protein (AFP), or a combination thereof.

[0104] In some embodiments, the method further comprises analyzing the samples with a LFT, wherein the LFT is for measuring a plurality of liver functions.

[0105] In some embodiments, the performance comprises tissue health (e.g., viability and/or function) of the construct, including health of specific cell types such as, but not limited to, hepatocyte health. In some embodiments, effective tissue health comprises an increased expression of miR-122 in the sample from the in vitro sandwich culture as compared to the sample from the control culture.

[0106] In some embodiments, evaluating tissue health further comprises measuring a liver function as described herein.

[0107] In some embodiments, evaluating tissue health further comprises administering a LFT.

[0108] In some embodiments, the method further comprises measuring the concentration of albumin in the samples, wherein an effective performance (e.g., effective tissue health) comprises an increased concentration of albumin in the sample from the in vitro sandwich culture as compared to the sample from the control culture.

[0109] In some embodiments, the exRNA further comprises a nonspecific exRNA.

[0110] In some embodiments, an effective performance (e.g., effective tissue health) comprises an expression of a nonspecific exRNA that is about the same in the in vitro sandwich culture as compared to the sample from the control culture. In some

embodiments, the exRNA comprises miR-1 and/or miR-131a.

[0111] In some embodiments, the exRNA further comprises miR-126 and/or miR-192.

[0112] In some embodiments, the samples comprise one or more than one samples. In some embodiments, the samples are obtained at a pre-determined frequency over a period of time. The pre-determined frequency can be any frequency, such as once

approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; once approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days; approximately 1, 2, 3, or 4 weeks; approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or at any frequency over any number of days, weeks, or months of incubation. The period of time can also be any period of incubation, such as approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours; approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days; approximately 1, 2, 3, or 4 weeks; approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months; or any number of days, weeks, or months of incubation.

[0113] In some embodiments, the incubating is for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.

[0114] In some embodiments, the sample from the in vitro sandwich culture comprises multiple samples obtained at over a period of time.

[0115] In some embodiments, an effective performance comprises an increase in the expression of miR-122 in the sample from the in vitro sandwich culture within about 1, 2, or 3 days of incubation of the in vitro sandwich culture as compared to the control incubated for the same duration, followed by normalization of expression of miR-122 in the sample from the in vitro sandwich culture within about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days of incubation of the in vitro sandwich culture as compared to the control incubated for the same duration.

[0116] In some embodiments, the second layer of engineered liver tissue constructs is positioned above the first layer of cells of the host mammal, and the period of time for incubation is at least approximately four (4) hours.

[0117] In some embodiments, the performance is predictive of post-implantation

performance of the engineered liver tissue construct.

[0118] In some embodiments, an effective performance indicates that the engineered liver tissue construct is suitable for implantation into a subject.

[0119] In some embodiments, the in vitro sandwich culture or method of evaluating

performance of an engineered liver tissue construct comprising the in vitro sandwich culture can be used to design an engineered tissue construct, such as a bioprinted liver tissue construct. For example, the in vitro sandwich culture or associated method can be used to determine the best design elements in terms of cell lines, cell types, cell ratios, hydrogel, and geometry by evaluating the performance of constructs comprising variations in the elements.

IV. Diagnostic Assays and Testing Kits for Evaluating the Performance of Engineered Liver Tissue Constructs

[0120] In another aspect, the present invention is directed to a molecular diagnostic assay for evaluating post-implantation performance (e.g., engraftment effectiveness and/or tissue health) of an implanted engineered liver tissue construct in a subject. The engineered liver tissue construct can comprise a three-dimensional bioprinted human liver tissue construct or any other construct as described throughout this application. For

example, a three-dimensional bioprinted human liver tissue construct can comprise an interior comprising parenchymal cells and a border comprising non-parenchymal cells, the parenchymal cells comprising human hepatocytes or human hepatocyte-like cells.

[0121] In some embodiments, the molecular diagnostic assay is for evaluating post- implantation performance of an engineered liver tissue construct implanted in a subject, and the assay comprises one or more micro-RNA assays specific for detecting at least exRNAs miR-181a and miR-122 in one or more RNA samples derived from a bodily fluid of the subject. In some embodiments, the assay further comprises one or more micro-RNA assays specific for detecting miR-152 in one or more RNA samples derived from a bodily fluid of the subject. In some embodiments, the assay further comprises one or more micro-RNA assays specific for detecting miR-21 in one or more RNA samples derived from a bodily fluid of the subject. In some embodiments, the assay further comprises one or more micro-RNA assays specific for detecting miR-1 and/or miR-131a. In some embodiments, the assay further comprises one or more micro-RNA assays specific for detecting miR-126 and/or miR-192. In some embodiments, the bodily fluid comprises serum, plasma, blood (e.g., whole blood), urine, cerebrospinal fluid, or a combination thereof. In some embodiments, the subject has one or more liver disorders, as described throughout this application. In some embodiments, the subject is a human (e.g., a human patient).

[0122] In another aspect, the present invention is directed to a kit (e.g., a molecular

diagnostic testing kit) for evaluating post-implantation performance of an implanted engineered liver tissue construct in a subject, comprising a molecular diagnostic assay as described herein. In some embodiments, the kit comprises reagents necessary to conduct the assay, such as reagents for isolating exosomal RNA from a bodily fluid, or reagents for amplification of the RNA sample, including target exRNAs.

[0123] In some embodiments, the assays and kits comprise a real-time polymerase chain reaction, also known as quantitative polymerase chain reaction (qPCR). Typically, the first step is isolating the RNA sample from the bodily fluid. Optionally, if the RNA sample is low, then an amplification step can be conducted to increase the RNA sample. Next, a primer initiates reverse transcription on the RNA sample in order to create complementary DNA (cDNA). The cDNA is then used as the template for the qPCR reaction. During the qPCR reaction, sequence-specific probes will attach to the biomarker sequence of interest (for example, an exRNA as described herein, including, but not limited to, miR-181a, miR-122, miR-21, or miR-152). When the attached sequence-specific probes are cleaved, such probes will emit fluorescence signals that will be detected by the qPCR system. The emitted fluorescence signals can be correlated to determine the presence or absence of the target biomarkers, as well as the expression levels of the target biomarkers. Presently, qPCR systems are commercially available from Thermo-Fisher Scientific, Bio-Rad Laboratories, and Agilent Technologies.

Accordingly, the assays and kits can be used with commercially available qPCR systems and other molecular diagnostic systems capable of detecting microRNA biomarkers.

EXAMPLES

[0124] The following illustrative examples are representative of embodiments of the inventions, including the methods, described herein and are not meant to be limiting in any way.

Example 1

Biomarkers for Evaluating the Post-Implantation Performance of a Three-Dimensional Engineered Liver Tissue Construct

1. Implantation of Three-Dimensional Engineered Liver Tissue Constructs.

[0125] A three-dimensional, engineered liver tissue construct was implanted into a first  NOD/SCID mouse. The construct contained an interior of parenchymal cells comprising human hepatocytes and a border of nonparenchymal cells comprising human endothelial cells (ECs) and human hepatic stellate cells (HSCs). The Examples and Figures herein also refer to a contrast containing these characteristics as a bioprinted therapeutic liver tissue (i.e.,“BTLT”). Unless otherwise noted, liver tissue constructs used for implantation and in vitro experiments in this and the following Examples are BTLTs. As shown in Figure 1A, the BTLT was implanted on or near the apex of the left liver lobe of the mouse. Figure 1B shows the BTLT sutured to the liver of the mouse.

[0126] As shown in Figure 2, a second NOD/SCID mouse was implanted with two (2) BTLTs. Separately, a sham-operated placebo mouse as the control, which underwent similar liver suturing without implantation of a liver tissue construct.

2. Evaluating the Post-Implantation Performance of Three-Dimensional Engineered Liver Tissue Constructs.

[0127] To determine biomarkers for evaluating the post-implantation performance of a three-dimensional engineered liver tissue construct, serum samples were obtained at approximately day 7 and day 28 post-implantation from: (a) the first NOD/SCID mouse having one (1) implanted liver tissue construct, and (b) the placebo mouse (control). The obtained serum samples were profiled for circulating exosome RNAs (exRNAs) using standard protocols. Serum samples were also collected over approximately 91 days and analyzed for concentration of human albumin using standard protocols.

[0128] Figures 3A-3B depict the assessment of serum exRNA levels (Log2FC) from BTLT-implanted mice at approximately day 7 and day 28, respectively, with replicates from different mice. Significantly, as shown in Figures 3A-3B, exRNAs miR-122, miR- 152, and miR-181a each showed an acute response at post-implantation day 7 that normalized by day 28 in comparison to the placebo mouse, suggesting that these exRNAs are biomarkers of early engraftment. By contrast, no responses were observed with muscle specific exRNAs miR-1 and miR-131a, as depicted in Figures 3A-3B. Thus, specificity of the observed response with exRNAs miR-122, miR-152, and miR-181a was demonstrated with minimal movement of muscle-associated exRNAs miR-1 and miR- 131a. miR-126 is associated with endothelial cells, including those of the liver, but like miR-1 and mir-131a showed minimal movement. The lack of response for mir-126, being also an exRNA expressed in liver tissue, demonstrated specificity of the response of miR- 122, miR-152, and miR-181a..

[0129] Figure 3C shows that the concentration (ng/mL) of secreted human albumin, a measure of hepatocyte function specific to the human liver tissue construct in the implanted mouse, increased until approximately 21 days post-implantation and remained detectable throughout the 91 days that serum was collected, suggesting that albumin secretion is a biomarker for long-term functionality of engineered liver tissue constructs.

[0130] In an additional experiment, serum samples were obtained at approximately day 7 post-implantation from: (a) the first NOD/SCID mouse having one (1) implanted liver tissue construct, and (b) the second NOD/SCID mouse having two (2) implanted liver tissue constructs. The obtained serum samples were profiled for human albumin and circulating exosome RNAs (exRNAs).

[0131] Figure 4A shows the contrast in serum human albumin level (ng/mL) between the first and second NOD/SCID mice. Significantly, the second NOD/SCID mouse having two (2) implanted liver tissue constructs had a higher concentration of serum human albumin. Figure 4B shows the exRNA expression levels (fold change (Log2FC) from 1 liver tissue construct) for exRNA miR-21, miR-122, miR-152, and miR-181a.

Significantly, as shown in Figure 4B, exRNA miR-122 and miR-181a showed an acute response in terms of exRNA expression level, and thus, exRNA miR-122 and miR-181a were shown to be dose responsive with an increasing number of implanted liver tissue constructs.

[0132] These data support the use of exRNAs miR-122, miR-181a, and miR-152 as

biomarkers of effective post-implantation performance of implanted liver tissue constructs, including use in assessing engraftment, the amount of implanted tissue, and tissue health, either alone or in combination with assessment of post-implantation albumin concentration.

Example 2

In Vitro Tests of Biomarkers for Evaluating the Performance of a Three-Dimensional Engineered Liver Tissue Construct

[0133] In vitro tests were performed to further evaluate biomarkers of the performance of a three-dimensional engineered liver tissue construct, including pre-implantation and predicated post-implantation performance. BTLTs were maintained in appropriate culture medium in a multi-well plate as previously described. See, e.g., U.S. Patent Nos. 9,222,932 and 9,442,105. As shown in Figures 5A-C, each well in the multi-well plate contained either: (a) one (1) liver tissue construct (Figure 5A), (b) two (2) liver tissue constructs (Figure 5B), or (c) three (3) liver tissue constructs (Figure 5C). Supernatant concentrations of human albumin and exRNAs from each well were measured, as described further below. As used herein, "supernatant" with respect to in vitro cultures refers to the culture medium in which constructs or cells have been cultured.

[0134] Human albumin concentration (ng/mL) was measured at approximately day 5 and day 6, with results shown in Figure 6A. The human albumin concentration directly correlated with the number of liver tissue constructs in the respective wells.

[0135] Supernatant exRNAs miR-152, miR-181a, miR-122, and miR-21 were measured at approximately 48 hours for their expression levels (fold change (Log2FC) from 1 liver tissue construct). Significantly, as shown in Figure 6B, exRNAs miR-152, miR-181a, and mIR-21 were dose responsive with increasing number of liver tissue constructs, which further supports their utility as biomarkers for performance of the constructs, including in assessing tissue health, function, and dose-responsiveness of the liver tissue constructs. Surprisingly and unexpectedly, the most abundant, hepatocyte-specific exRNA miR-122 remained relatively constant in vitro with increasing number of liver tissue constructs, whereas exRNA miR-122 was dose responsive in vivo.

Example 3

Effects of Cell Viability on Biomarkers for Evaluating the Performance of a Three- Dimensional Engineered Liver Tissue Construct

[0136] Constructs were treated in vitro with dimethyl sulfoxide (“DMSO”) added to the culture medium to examine the effects of cell viability on biomarkers of construct performance.

1. Treatment with 0-20% DMSO for 24 hours

[0137] In one experiment, liver tissue constructs were treated with 0%, 2.5%, 5%, 10%, or 20% DMSO in culture medium for approximately 24 hours starting on day 3 after bioprinting. After approximately 24 hours of treatment, culture supernatant was analyzed for levels of albumin and exRNA expression to assess the effects of cellular toxicity induced by DMSO. Figure 7A shows the human albumin concentration (ng/mL) at approximately 24 hours after 0-20% DMSO treatment. Albumin concentrations were reduced in relation to increased cellular toxicity, showing that the cell viability and associated function of the liver tissue construct was inversely correlated with the percentage of DMSO treatment. Figure 7B shows the exRNA expression (fold change (Log2FC) from liver construct treated with normal media, i.e., 0% DMSO) for exRNAs miR-122, miR-152, and miR-21 after 0-20% DMSO treatment. Significantly, non- specific release of exRNAs into the supernatant was observed that was anti-correlative to cell viability, indicating that non-specific exRNA release provides a surrogate readout of liver tissue construct health in vitro. Additionally, because miR-122 is a hepatocyte- specific miR, the data indicate that monitoring release of miR-122 provides a surrogate readout of hepatocyte health in vitro, including in constructs comprising multiple cells types such as BTLTs.

2. Treatment with 0-10% DMSO for 72 hours

[0138] In another experiment, three different types of constructs were prepared: (1) a “BTLT” as described herein, (2) a“NPC” construct comprising a nonparenchymal cell border of a BTLT but lacking an internal fill of hepatocytes, and (3) a“Fill” construct comprising the hepatocyte fill of a BTLT but lacking a nonparenchymal cell border. Three days after bioprinting, each of the constructs was treated with culture medium containing 0%, 2.5%, 3.75%, 5%, 7.5%, or 10% DMSO for approximately 72 hours. No overt toxicity was observed based on visual analysis of the constructs during the 72 hours of treatment (data not shown).

(a) Cell Viability

[0139] Cell viability was assessed after approximately 72 hours of treatment with 0-10% DMSO using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega

Corporation, Madison, Wisconsin, USA) and manufacturer protocols. Figure 8 and

Table 1 show percent viability in samples for each construct after approximately 72 hours of treatment with 2.5-10% DMSO relative to average viability after approximately 72 hours of treatment with 0% DMSO (i.e., control). Viability was measured in triplicate for each percentage of DMSO for BTLTs and NPCs, and in duplicate for Fills.

[0140] As shown by Figure 8 and Table 1, a dose responsive effect on viability was observed with DMSO treatment, although NPCs and Fills were less sensitive to DMSO treatment than BTLTs.

(b) Alanine Aminotransferase Activity

[0141] Culture supernatant for each construct was obtained after approximately 2, 8, 24, and 72 hours of treatment with 0-10% DMSO and assessed for alanine aminotransferase (ALT) activity using standard protocols.

[0142] Figures 9A-9C and Tables 2-4 show ALT activity (mU/mL) in samples for each construct after approximately 2, 8, 24, and 72 hours of treatment with 0-10% DMSO. Activity was measured in duplicate for each construct at each timepoint and percentage of DMSO.

Table 2: Effect of DMSO Treatment on ALT Activity in BTLT Constructs


a = dimethyl sulfoxide; b = alanine aminotransferase; c = milliUnits/milliliter; d = bioprinted therapeutic liver tissue; e = average

Table 3: Effect of DMSO Treatment on ALT Activity in NPC Constructs


a = dimethyl sulfoxide; b = alanine aminotransferase; c = milliUnits/milliliter; d = nonparenchymal cell; e = average

Table 4: Effect of DMSO Treatment on ALT Activity in Fill Constructs


a = dimethyl sulfoxide; b = alanine aminotransferase; c = milliUnits/milliliter; d = average

[0143] As shown by Figures 9A-9C and Tables 2-4, DMSO-mediated ALT activity showed a variable downward trend for BTLTs and NPCs, and increased with Fills. The increased ALT activity at all timepoints for Fills showed an inverse correlation with cell viability at 72 hours in Fills. Compare Fill viability in Figure 8 with Fill ALT activity in Figure 9C, and cell viability with ALT activity at 72 hours shown in Tables 1 and 4, respectively.

(c) Lactate Dehydrogenase Activity

[0144] Culture supernatant for each construct was obtained after approximately 2, 8, 24, and 72 hours of treatment with 0-10% DMSO and assessed for lactate dehydrogenase (LDH) activity using standard protocols.

[0145] Figures 10A-10C and Tables 5-7 show LDH activity (mU/mL) in samples for each construct after approximately 2, 8, 24, and 72 hours of treatment with 0-10% DMSO. Activity was measured in duplicate for each construct at each timepoint and percentage of DMSO.

Table 5: Effect of DMSO Treatment on LDH Activity in BTLT Constructs



a = dimethyl sulfoxide; b = lactate dehydrogenase; c = milliUnits/milliliter; d = bioprinted therapeutic liver tissue; e = average ± standard deviation

Table 6: Effect of DMSO Treatment on LDH Activity in NPC Constructs


a = dimethyl sulfoxide; b = lactate dehydrogenase; c = milliUnits/milliliter; d = nonparenchymal cell; e = average ± standard deviation

Table 7: Effect of DMSO Treatment on LDH Activity in Fill Constructs


[0146] As shown by Figures 10A-10C in comparison with Figure 8, Tables 5-7 in

comparison with Table 1, and Figure 11, LDH activity level was inversely correlated with viability in DMSO-treated BTLTs and NPCs, but not Fills, at 72 hours. Figures 10A- 10C and Tables 5-7 show that LDH activity was primarily driven by NPCs, while the utility of LDH activity readouts for hepatocyte health was time-sensitive.

(d) exRNA Expression

[0147] Culture supernatant for each construct was obtained after approximately 24 and 72 hours of treatment with 0, 3.25, and 10% DMSO and assessed for exRNA (miR-122, miR-152, and miR-181a) expression. Measurements were made in duplicate except as otherwise shown in Figures 12A-12B.

[0148] As shown by Figure 12A (24 hour treatment) and Figure 12B (72 hour treatment) in comparison with Figure 8, exRNA levels (fold change (Log2FC) from liver construct treated with normal media, i.e., 0% DMSO) were inversely correlated with viability in DMSO-treated BTLTs. In particular, increased miR-122 expression was associated with decreased hepatocyte viability, demonstrating supernatant miR-122 levels as a non-lytic measure of hepatocyte specific viability.

(e) Albumin Levels

[0149] Culture supernatant for each BTLT and Fill construct was obtained after

approximately 2, 8, 24, and 72 hours of treatment with 0-10% DMSO and assessed for albumin concentration.

[0150] Figures 13A-13B and Tables 8-9 show albumin concentration (ng/mL/million cells) in samples for each BTLT and Fill construct after approximately 2, 8, 24, and 72 hours of treatment with 0-10% DMSO. Concentrations were measured in duplicate for each construct at each timepoint and percentage of DMSO.

Table 8: Effect of DMSO Treatment on Albumin Concentration in BTLT Constructs


a = dimethyl sulfoxide; b = nanograms/milliliter/million cells; c = bioprinted therapeutic liver tissue deviation; d = not determined

Table 9: Effect of DMSO Treatment on Albumin Concentration in Fill Constructs


a = dimethyl sulfoxide; b = nanograms/milliliter/million cells; c = not determined

[0151] As shown in comparing Figure 13A-13B and Tables 8-9, BTLTs had lower

baseline albumin with little additional accumulation from 2-8 hours, while Fills had higher baseline albumin with nearly 2-fold accumulation from 2-8 hours. Moreover, as shown by Figure 13A in comparison with Figure 8, Table 8 in comparison with Table 1, and Figure 14, secreted albumin concentration correlated with viability in DMSO-treated BTLTs.

Example 4

In Vitro Sandwich Culture Assay

[0152] In vitro sandwich cultures were developed to further assess the performance of engineered liver tissue constructs. Figure 15A shows the structure of the sandwich culture in which a liver tissue construct is placed on top of and in contact with a layer of cells comprising mammalian hepatocytes, which mimics the in vivo therapeutic implantation described in Example 1. Assays to assess performance of engineered liver constructs can be conducted using such sandwich cultures.

[0153] Figure 15B shows the results of an exemplary sandwich culture assay in which the supernatant levels of exRNA miR-122 were measured after approximately 72 hours and 1 week of incubation of: a sandwich culture containing a BTLT on top of and in contact with a layer of mouse hepatocytes, a layer of mouse hepatocytes, a BTLT, and a reverse sandwich control in which the layer of mouse hepatocytes was placed on top of and in contact with a BTLT. As shown in Figure 15B, supernatant levels of exRNA miR-122 had a response in vitro comparable to the in vivo response described in Example 1. This finding further indicates that exRNA miR-122 is a robust biomarker of the performance of liver tissue constructs. Furthermore, as discussed with respect to Figure 6B, miR-122 levels were not dose-responsive in vitro, suggesting that the miR-122 response observed in in vitro sandwich cultures is indicative of engraftment of the construct onto the layer of cells. This further indicates that miR-122 expression in the in vitro sandwich culture assay is predictive of post-implantation engraftment of the engineered liver tissue constructs.

Example 5

In Vitro Tests for Evaluating Protocol Deviations During Production

of a Three-Dimensional Engineered Liver Tissue Construct

[0154] Effects on biomarkers of tissue health and function were determined in relation to one of the following protocol deviations during production of BTLTs: (1) excessive pipetting when preparing the hepatocyte fill portion of the BTLT, (2) room temperature incubation of the BTLT for 4 hours, referred to herein and in the Figures as“4 hr RT hold,” and (3) incubation of the BTLT without culture medium for 4 hours, referred to herein and in the Figures as“4 hr no media hold.” BTLTs were printed and treated with one of the protocol deviations on Day 0 and then cultured normally for 72 hours. BTLTs cultured in 10% DMSO for 72 hours after printing served as positive controls for cellular toxicity. BTLTs cultured under normal conditions for 72 hours after printing served as negative controls. Overt toxicity was observed only in BTLTs treated with excessive pipetting, based on visual analysis of the constructs during the 72 hours of treatment (data not shown).

[0155] Effects of the protocol deviations on histology, cell viability, exRNA release, LDH activity, and albumin concentration were determined.

1. H&E staining

[0156] The histological effects of protocol deviations or DMSO treatment on hepatocyte levels and nuclear staining were assessed by H&E staining performed in cross-section and planar orientations using standard protocols in comparison to the negative control. H&E staining was performed at approximately 72 hours after treatment with the protocol deviation, or approximately 72 hours after bioprinting and incubation in normal culture medium or culture medium containing 10% DMSO for the negative and positive controls, respectively.

[0157] Excessive pipetting resulted in decreased hepatocyte level and decreased nuclear staining. A modest decrease in hepatocyte level and nuclear staining was observed in BTLTs treated with a 4 hour room temperature hold. A decreased hepatocyte level and decreased nuclear staining also was observed in BTLTs incubated without media for 4 hours and in BTLTs incubated in media containing 10% DMSO for 72 hours.

2. Cell Viability

[0158] Cell viability was assessed at approximately 72 hours after treatment with the protocol deviation, or approximately 72 hours after bioprinting and incubation in normal culture medium or culture medium containing 10% DMSO for the negative and positive controls, respectively, using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega Corporation, Madison, Wisconsin, USA) and manufacturer protocols. Figure 16 and Table 10 show percent viability relative to average viability of the negative control. Viability was measured in duplicate for each protocol deviation and control. Table 10: Effect of Protocol Deviations on Viability

a = average; b = room temperature

[0159] As shown by Figure 16 and Table 10, the DMSO-treated positive control and the 4 hour no media hold showed reduced cell viability.

3. exRNA Expression

[0160] Culture supernatant was obtained after approximately 4, 24, 48, and 72 hours following treatment of BTLTs with a protocol deviation or after bioprinting and incubation of BTLTs in normal culture media or culture media containing 10% DMSO for negative and positive controls, respectively. Supernatants were assessed for exRNA (miR-122, miR-126, miR-152, mir-21, and miR-181a) expression. Quadruplicate or triplicate measurements were made at each timepoint except where otherwise noted in Tables 11-15 and Figures 17-20.

[0161] As shown by Table 11 and Figure 17, supernatant exRNA levels (arbitrary units, normalized per hour) for untreated negative controls decline by approximately 100 fold over 72 hours.

[0162] For BTLTs treated with excessive pipetting, lowered miR-122 levels were

observed beginning at 48 hours after treatment as shown by Table 12 and Figure 18, suggesting that miR-122 levels can provide a specific measure of compromised hepatocyte health after bioprinting.

[0163] As shown by Table 13 and Figure 19 for BTLTs treated with a room temperature hold for 4 hours, a general acute lowered exRNA expression profile was observed at 4 hours for each of miR-122, miR-126, and miR-21, with expression normalized by 24 hours and unchanged compared to negative control through 72 hours. A similar profile was not observed for miR-181, most likely due to expression and detection limitations.

[0164] As shown by Table 14 and Figure 20 for BTLTs treated with a 4 hour no media hold, a general acute increased exRNA expression profile was observed at 24 hours, with expression normalized by 72 hours.

[0165] As shown by Table 15 and Figure 21 for positive controls treated with 10%

DMSO for 72 hours, a general sustained increased exRNA expression profile was observed from 24 to 72 hours.

4. LDH Activity

[0166] Culture supernatant was obtained after approximately 4, 24, 48, and 72 hours following treatment of BTLTs with a protocol deviation or after bioprinting and incubation of BTLTs in normal culture media or culture media containing 10% DMSO for negative and positive controls, respectively. LDH Activity (mU/mL) was measured in quadruplicate at each timepoint except as otherwise shown in Tables 16 and Figure 22.


T [0167] As shown by Table 16 and Figure 22, an acute increase in LDH activity was observed with media deprivation, similar to observed with exRNA release.

5. Albumin Concentration

[0168] Culture supernatant was obtained after approximately 24, 48, and 72 hours following treatment of BTLTs with a protocol deviation or after bioprinting and incubation of BTLTs in normal culture media or culture media containing 10% DMSO for negative and positive controls, respectively. Albumin concentration was measured in quadruplicate at each timepoint except as otherwise shown in Table 17 and Figure 23.


[0169] As shown by Table 17 and Figure 23, a decrease in albumin concentration was observed with excessive pipetting, which correlates with exRNA miR-122

downregulation observed with excessive pipetting. Other protocol deviations, including DMSO-treatment associated with the positive control, did not result in changes in albumin concentration.

[0170] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provide by way of example only. Numerous variations, changes and substitutions will occur to those skill in the art without departing from the invention. All patents, patent applications, and publications cited herein are fully incorporated by reference herein.