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• • G—PHYSICS
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  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5067—Liver cells
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material

Definitions

• the present invention relates to the field of 3D tissue cultures.
• a 3D (three dimensional) tissue culture is an artificially-created environment in which biological cells are permitted to grow or interact with its surrounding environment in all three dimensions.
• 3D cultures are an improvement over 2D (two dimensional) cultures (also called “monolayers”) for many reasons.
• 2D two dimensional cultures
• cells exist in 3D micro environments with intricate cell-cell and cell-matrix interactions and complex transport dynamics for nutrients and cells.
• Standard 2D, or monolayer, cell cultures are inadequate representations of this environment.
• the main applications for 3D tissue culture lie in drug efficacy and/or toxicity screenings, investigative/mechanistic toxicology, target disco very/identification, drug repositioning studies, and pharmacokinetics and pharmacodynamics assays, although other applications are also available, like for regenerative medicine.
• 3D constructs more closely resemble in vivo tissue in terms of cellular communication, the formation of biochemical and physico-chemical gradients and the development of extracellular matrices.
• 3D constructs are thus improved models for cell migration, differentiation, survival, and growth.
• 3D tissue cultures provide more accurate depiction of cell polarization, since in 2D, the cells can only be partially polarized. The latter is particularly important for hepatocytes, which are one of the preferred cell types in 3D tissue cultures used for drug efficacy and toxicity screenings.
• hepatocytes are one of the preferred cell types in 3D tissue cultures used for drug efficacy and toxicity screenings.
• cells grown in 3D exhibit different gene expression than those grown in 2D (Kelm et al 2010, Ridky et al 2010, Cody et. al. 2008).
• a real 3D environment is often necessary for cells in vitro to form important physiological structures and functions.
• the third dimension of cell growth provides more contact space for mechanical inputs and for cell adhesion, which is necessary for integrin ligation, cell contraction and even intracellular signaling.
• Normal solute diffusion and binding to effector proteins is also reliant on the 3D cellular matrix, so it is critical for the establishment of tissue scale solute concentration gradients.
• the state of the art provides cell viability assays which yields information on the viability of cells used in a culture.
• a method of preparing cells for 3D tissue culture comprises the steps of a) plating the cells on a suitable surface
• the said process is also called "vital cell selection".
• cell viability live or dead
• cell viability assays which determine the ability of cells to maintain or recover their viability.
• suitable viability assays which determine the ability of cells to maintain or recover their viability.
• the trypan blue test which is a membrane leakage assay, tests whether cells are permeable to trypan blue. If yes, they are considered non-viable.
• Cell viability is for example calculated as the number of viable cells divided by the total number of cells within the grids of a hemacytometer, onto which a subsample of the cells that are meant to be transferred into a 3D tissue culture process is placed.
• the respective test provides a overall signal on cell viability, which, although it has been obtained by assessment of individual cells, does not provide any information on the viability of individual cells which are meant to be transferred to the, 3D tissue culture process, mainly because the cells which are actually subjected to said test are discarded later on, and are not being further used for the 3D tissue culture, because they have been treated with the testing agent.
• the viability assay is in most cases performed with a sample of cells which is meant to be representative for the cells that are used for the 3D tissue culture process.
• viability tests do not allow the separation of dead or unsuitable cells from the total cell pool which is meant to be used for the 3-D tissue culture process. They only provide information about the overall viability of the cells which are meant to be used.
• a 3D tissue is characterized by (i) a high density of cells, (ii) the development of an extracellular matrix, and (iii) the establishment of cell-cell contacts, the presence of nonviable cells and cells with a low vitality can seriously affect the function of a 3D tissue, with the ultimate effect that the 3D tissue becomes dysfunctional in case the amount of nonviable cells in the tissue is too high.
• cells which are not capable of integrating into a 3D tissue may still pass the viability test, for example because their membrane is still intact and thus impermeable to the test reagent, e.g. trypan blue.
• cells may be sorted through FACS prior to using them, in order to separate viable from non-viable cells, e.g., on the basis of a dual fluorescent CalceinAM/PI approach.
• FACS assay delivers a cell-specific information which helps to sort out viable from non-viable cells
• the group classified as "viable” encompasses cells would not be fully functional in a 3D tissue.
• the latter group forms a subgroup of all viable cells, which can not be sorted out by the above techniques.
• the method according to the invention solves this problem by providing a tool which allows an efficient selection of those cells which are viable enough that they can be used for forming a 3D tissue, while it further allows to discard those cells which lack such viability.
• the standard applied by the method according to the invention relies on the plateability of the cells, i.e., on their capability to adhere to a cell culture surface, which again is an excellent measure for their viability, and for their suitability to integrate into a 3D network of cells as it occurs in 3D tissue culture.
• the plateability depends on the cell's capability to form an extracellular matrix, which in turn is a feature that is vital for their integration into a 3D network of cells.
• Plating cells is a standard approach that is mainly used in 2D cell culture, not in 3D cell culture.
• 3D cell culture such step is apparently useless, because it adds further effort to a cell culturing method, which as such is subject of many uncertainties anyway, and it subjects the cells to additional stress, with the risk that cells get lost, or lose viability, which otherwise could still be used in 3D cell culture.
• hepatocytes which were obtained from a human donor liver by means of collagenase digestion, as disclosed in Pichard et al. (2006). This process yielded up to 300 vials with up to 8 x 10 6 hepatocytes each.
• the plating step that is implemented according to the invention prior to the 3D tissue fabrication can causes a loss of up to 60 % of the cells, i.e., 4.8 out of 8 million cells get lost because they do not adhere sufficiently to the plate surface, and 3.2 million hepatocytes are retained, which can then be used for the 3D tissue fabrication, providing up to 3000 microtissues of superior quality.
• the plating process thus results in a loss of cells, but helps to improve the quality of the 3D tissues, which otherwise would have been wasted due to the negative interference of cells with low vitality.
• the step of plating the cells and discarding those cells which have not adhered is not a viability assay according to the state-of-the-art. In strictu sensu, it is not an assay at all, because, in its broadest sense, it simply involves a plating step and a subsequent washing step to remove the cells that have not adhered.
• the plating step as such may last anything between 30 mins und 98 firs. Further details will be discussed elsewhere herein.
• the method of the invention does not encompass the application of a viability assay throughout steps a) - d).
• viability assay provides an overall signal on cell viability only, (ii) does not provide any information on the viability of individual cells, (iii) does not allow the separation of unsuitable cells and (iv) does not help to discard those cells which, due to lack of viability or vitality, would deteriorate the 3D tissue culture process.
• the 3D tissue culture process is at least one selected from the group consisting of
• Preferred examples for cellular self-assembly are hanging drop cultures, liquid overlay cultures or cultures on micro-patterned surfaces.
• the hanging-drop technique was initially developed for cultivating stem ceil embryo id bodies.
• ceil suspension drops are suspended at the lower side of a suitable plate.
• so-called hanging drop plates are used for this purpose which resemble microtiter plates, and come in similar formats, but have holes through which the ceil suspension liquid can be delivered.
• Such type of system is available from InSphero AG, Switzerland, under the brand name "GravityPLUSTM 3 D Culture and Assay Platform.”
• the hanging drops can assume v olumes of between 2 and 100 ⁇ , and accommodate between 100 and 50000 cells which then are meant to form the D tissue. In each drop the cells are concentrates at the bottom of the drop simply by gravity, which then form single spheroids at the air- liquid interface. Background and protocols and are disclosed in Kelm et al, 2003, and Kelm & Fussenegger 2004.
• the hanging-drop technique allows ceils to form a tissue which in other vessels would tend to adhere to a so lid- liquid interface, like the flat plane of culture dishes.
• a hanging drop is devoid of such interfaces, thus allowing cells to form a 3D construct that resembles, in its structure, a true tissue.
• the liquid overlay culture technique is another technique to form 3D tissue spheroids.
• a multititer plate is made non adherent, e.g., by coating with agarose or by photodynamic chemical patterning. Cells are then seeded in the wells at a density of 100 - 10000 cells per well and are then incubated in culture medium. Protocols are disclosed, e.g., in Yuhas et al (1977) or Metzger et al (2011).
• 3D bio-printing is a process in which cells are plotted on a matrix where they form a tissue.
• 3D bio-printing may involve techniques like photolithography, magnetic bioprinting, stereo lithography, and cell extrusion.
• Scaffold-based 3D tissue culture uses a scaffold on which cells are seeded.
• the scaffold may, or may not, be biodegradeable (e.g., chitosan, polylactic acid, polystyrene, etc).
• biodegradeable e.g., chitosan, polylactic acid, polystyrene, etc.
• Hydrogel-based 3D tissue culture (also called “scaffoldless 3D tissue”) is an approach were cells are grown in a hydrogel matrix and form a 3D tissue. This technique is for example disclosed in Geckil et al. (2010).
• One key feature of the present invention is that nonviable cells are sorted out prior to the culture process. Therefore, the quality of the resulting tissue increases significantly, because dysfunctional cells which could disturb the tissue- forming process have been removed.
• This advantage does not only apply to the hanging drop technique, for which examples are shown herein, but also for scaffold-based 3D tissue culture, hydrogel-based 3D tissue culture, 3D bioprinting, and other cellular self-assembly 3D tissue culture methods, like liquid overlay.
• the cells are crypopreserved cells which are thawed before plating.
• cryopreserved Most of the cell types discussed herein may be cryopreserved, and can still be used after thawing thereof.
• the potential of cryopreservation is important to have sufficient cell mass on stock for on demand production capacities of 3D tissues of a given kind.
• the cells are non-frozen cells. This approach allows on-the-fly production of 3D tissues if desired so, but requires the availability of unfrozen, viable cells, e.g., obtained directly from a donor organ.
• the cells used are selected from
• Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumors, most primary cell cultures have limited lifespan. After a certain number of population doublings (called the Hayflick limit), cells undergo the process of senescence and stop dividing, while generally retaining viability.
• Stem cells are undifferentiated, or partly differentiated, biological cells that can differentiate into specialized cells and can divide (through mitosis) to produce more stem cells. They are found in multicellular organisms. In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues.
• hPSC human pluripotent stem cells
• An immortalized cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells can therefore be grown for prolonged periods in vitro. The mutations required for immortality can occur naturally or be intentionally induced for experimental purposes. Immortal cell lines are a very important tool for research into the biochemistry and cell biology of multicellular organisms.
• HepaRG cells which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line that retains many characteristics of primary human hepatocytes.
• the cells are not expanded prior to, during or after plating, and/or the cells are not passaged after plating.
• Plating of cells plays a role only in one particular subdivision of 3D cell culture, namely in those types of cell culture where cells are expanded prior to transferring them into a 3D tissue culture process. As discussed above this applies only to a very limited number of primary cells, as well as to stem cells, tumor cells and immortalized cells.
• expansion requires serial passaging of the cells, which is performed mainly in monolayer cultures such as T-flasks or Roller bottles later in the process.
• One major drawback of cell expansion is potential loss of the native cell phenotype (de-differentiation), which is followed by a loss of native functionality, and genetic/chromosomal alterations.
• the cells are mammalian cells, preferably selected from the group consisting of:
• Hepatocytes are cells of the main tissue of the liver, which make up 70-85% of the liver's cytoplasmic mass.
• Primary cultures of human hepatocytes are an in vitro model widely used to investigate numerous aspects of liver physiology and pathology, as well as for screening purposes, e.g., toxicity assays and the like.
• the technique used to isolate human hepatocytes is based on a two-step collagenase perfusion of a donated liver.
• hepatocytes The functionality of hepatocytes is highly dependent on their capability of forming a polar phenotype. This does not only affect their usability for screening purposes, but also their life span, which under sub optimal conditions does rarely exceed 5 days.
• Such polar phenotype is only established in 3D culture, which mimics the cell's natural environment, but not in 2D culture. 3D tissues made from hepatocytes are thus often nicknamed "organ on a chip or "organ in a well".
• a 3D tissue culture process is provided, said process being selected from the group consisting of
• a 3D tissue culture is provided that has been obtained with the above process and/or from cells that have been prepared according to the method of the invention.
• said which 3D tissue culture adopts the shape of a spheroid, which assumes preferably, a volume of between > 2 and ⁇ 500 id, and accommodates, preferably, between > 100 and ⁇ 50000 cells which then are meant to form the D tissue.
• a 3D tissue according to the invention for at least one purpose selected from the group consisting of
• Example 1 Optimized method to produce human liver microtissues 1. Thawing of cryopreserved Hepatocytes
• liver microtissues were monitored over time. It turned out that they remained stable over 5 weeks in culture as shown by a constant ATP content (Fig. 5a). This extended life span compared to 2D cultures of hepatocytes is most likely due to extensive cell-cell contacts, which are essential for maintaining the differentiated status of hepatocytes. Besides the stable viability, functionality of liver microtissues is preserved over 5 weeks, as indicated by persistent albumin secretion (Fig. 5b).
• Example 3 Use of 3D tissue culture produced according to the invention for hepatoxicity screening
• the prolonged lifetime and functionality of the 3D tissue cultures produced according to the invention allows for long-term studies with repeated dosing to evaluate chronic hepatotoxic effects.
• the hepatotoxic compounds acetaminophen and diclofenac were tested with respect to their long-term toxico logical profile.
• Acetaminophen is the major cause of drug-induced liver injury (DILI) in humans.
• DILI drug-induced liver injury
• CYP2E1, CYP1A2 and CYP3A4 The reactive intermediate depletes intracellular glutathione pools leading to hepatocyte cell death. So far, 2D cultures of hepatocytes have not been able to convincingly recapitulate acetaminophen-induced toxicity in vitro (Fey and Wrzesinski 2012).
• Diclofenac is a nonsteroidal anti-inflammatory drug that has a strong association with hepatotoxicity. The mechanism is thought to involve phase I enzyme activity (multiple P450-catalyzed oxidations), phase II enzyme activity (glucoronylation) and mechanism-based inhibition. In comparison with 2D cultures of human hepatocytes (calculated IC50 value of 331 ⁇ ), long- term treated liver microtissues displayed an increased sensitivity toward this drug with an IC50 value of 178.6 ⁇ (Fig. 6b).
• trovafloxacin is only hepatotoxic in combination with an inflammatory stimulus, such as lipopolysaccharide (LPS) or TNF-a.
• LPS lipopolysaccharide
• TNF-a a inflammatory stimulus
• the mechanism is thought to involve enhanced cytokine secretion and accumulation in the liver, causing caspase activation and subsequent liver injury.
• Induction of the inflammatory response in liver microtissues by LPS resulted in elevated levels of IL-6 secretion, verifying the responsiveness of incorporated macrophages in the liver microtissues (Fig. 6c).
• liver microtissues resemble liver-like cell composition and an extended stability in culture.
• the long-term viability and functionality of liver microtissues allows for routine compound testing as well as chronic and inflammation-mediated toxicity.
• the 96-well format allows for microtissue mass production enabling the implementation of an organotypic liver model at an early time point in drug development.
• Fig. 1 Incomplete microtissue formation if seeded directly after thawing Human liver microtissue formation was initiated with cryopreserved, plateable primary human hepatocytes, which were seeded directly after thawing in hanging drop plates. Two different medium compositions were tested (Medium A + B). Three representative hanging drops (Nr. l, 2, 3) were imaged directly after seeding and after 1 and 4 days in hanging drops. The hepatocytes accumulated at the meniscus of the in hanging drop and formed loose aggregates until day 4 in culture. The same hepatocyte lot displayed attachment to 2D collagen-I coated culture dishes after thawing, showing the suitability of this hepatocyte for 2D culture.
• Human liver microtissue formation was initiated with 5 independent lots (donor 1-5) of cryopreserved, plateable primary human hepatocytes, which were seeded directly after thawing on collagen-I coated 2D culture dishes for 24 hours. After vital cell selection, the cells were detached from the culture dish and seeded in hanging drop plates. The hepatocytes accumulated at the meniscus of the in hanging drop and formed tight microtissues (also called "hepatospheres") within 5 days of culture. The microtissues were transferred to a receiver plate (GravityTRAPTM) and imaged for microtissue appearance and -size. All 5 hepatocyte lots showed robust and uniform microtissue formation within 5 days of culture.
• Fig. 3 Reproducible formation of human liver microtissues with vital cell selection of primary human hepatocytes
• A Size variation of human liver microtissues produced after pre-plating of cryopreserved human hepatocytes.
• the diameter of human liver microtissues of 24 production runs with the same hepatocyte lot was determined.
• the average diameter and standard deviation of the size measurement of 6 microtissues per run is shown.
• the microtissues showed less than 5% size variation within each production run.
• the average microtissue size of all 24 productions was 280 ⁇ , with an relative size deviation of less than 10% between production runs.
• Dog liver microtissue formation was initiated with cryopreserved, plateable primary dog hepatocytes, which were seeded either directly in the hanging drops (B) or pre-plated on collagen-I coated 2D culture dishes for 24 hours (C, D). Dog liver microtissue formation was not observed after direct seeding of cryopreserved hepatocytes until 4 days in culture. Vital- cell selected dog hepatocytes formed dense microtissues within 4 days in hanging drops (C). (D) Image of a dog liver microtissue transferred from the hanging-drop to the receiver plate.
• Fig. 5 Viability and functionality of 3D tissues over 5 weeks in culture.
• Presence of LPS decreased the IC50 threefold from 220 (-LPS) to 71 fM (+ LPS)
• Fig. 7 Structural integrity of human liver microtissues produced without pre-plating and with pre-plating
• Histological preprations were made from formalin fixed and paraffin embedded human liver microtissues produced either directly after thawing of the cryopreserved hepatocytes or after pre-plating of the hepatocytes for 24h on a collagen-coated cell culture dish. 3-5um thick sections were stained with Hematoxylin and eosin (H&E). Images were taken with a lOx objective.
• Fig. 7 shows microtissues which have been created without prior pre- plating, while the low reo shows microtissues which have been created with prior pre-plating.
• Kelm et al Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol. Bioeng. 2003, 83, 173-180.

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