3D Tissue Culture Materials and Processes for Producing Same

Abstract

A 3D tissue culture selected from the group consisting of hydrogel-based 3D tissue culture and cellular self-assembly 3D tissue culture as well as self-assembly 3D tissue culture. Additionally, disclosed is a method of preparing cells for 3D tissue culture, which method comprises the steps of plating the cells on a suitable surface, optionally, checking for their capability to adhere to said surface, discarding the cells which have not adhered to said surface, detaching the adhered cells and transferring them into a 3D tissue culture process.

Claims

1. A 3D tissue culture selected from the group consisting of hydrogel-based 3D tissue culture and cellular self-assembly 3D tissue culture, in which cells used in the 3D tissue culture process are prepared according to the method of: a) plating hepatocytes on a collagen coated surface of a cell-culture dish thereby forming a monolayer of hepatocytes; b) discarding the hepatocytes which have not adhered to the collagen coated cell-culture dish; and c) adding a cell detachment solution to detach the adhered hepatocytes from the collagen coated surface and transferring the hepatocytes into the hydrogel-based 3D tissue culture or the cellular self-assembly 3D tissue culture.

2. The 3D tissue culture of claim 1, wherein the time between the plating hepatocytes and the discarding the hepatocytes that have not adhered to the dish is between 30 minutes and 24 hours.

3. The 3D tissue culture of claim 2, wherein the hepatocytes self-assemble, further wherein the hepatocytes are not expanded any time prior to, during or after plating, and the hepatocytes are not passaged after plating.

4. The 3D tissue culture of claim 1, wherein the culture adopts the shape of a spheroid.

5. The 3D tissue culture of claim 1, wherein the hepatocytes are cryopreserved cells which are thawed before plating.

6. The 3D tissue culture according to claim 1, wherein the hepatocytes are non-frozen cells.

7. The 3D tissue culture of claim 1, wherein the cells are selected from the group consisting of primary cells, stems cells, tumor cells and immortalized cells.

8. The 3D tissue culture of claim 1, wherein the cells are not expanded prior to, during or after plating.

9. The 3D tissue culture of claim 1, wherein the cells are not passaged after plating.

10. The 3D tissue culture of claim 1, wherein the cells are mammalian cells.

11. The 3D tissue culture of claim 10, wherein the cells are selected from the group consisting of human cells, cynomolgus cells, pig cells, canine cells, and rat cells.

12. The 3D tissue culture of claim 1, wherein the tissue culture is utilized for at least one purpose selected from the group consisting of drug efficacy, toxicity screening, investigative toxicology, mechanistic toxicology, target identification, drug repositioning studies, pharmacokinetic assays, pharmacodynamic assays and regenerative medicine.

13. A cellular self-assembly 3D tissue culture created using a method comprising the steps of: a) plating hepatocytes on a suitable surface comprising a collagen coated surface of a cell culture dish, b) discarding those hepatocytes which have not adhered to the suitable surface, and c) adding a cell detachment solution to detach the adhered hepatocytes from the collagen coated surface and transferring the hepatocytes into the cellular self-assembly 3D tissue culture, wherein the hepatocytes are not expanded any time prior to, during or after plating, and the hepatocytes are not passaged after plating.

14. The 3D tissue culture of claim 13, wherein the culture adopts the shape of a spheroid.

15. The 3D tissue culture of claim 13, wherein the hepatocytes are cryopreserved cells which are thawed before plating.

16. The 3D tissue culture according to claim 13, wherein the hepatocytes are non-frozen cells.

17. The 3D tissue culture of claim 13, wherein the cells are selected from the group consisting of primary cells, stems cells, tumor cells and immortalized cells.

18. The 3D tissue culture according to claim 13, wherein the cells are mammalian cells.

19. The 3D tissue culture of claim 18, wherein the cells are selected from the group consisting of human cells, cynomolgus cells, pig cells, canine cells, and rat cells.

20. The 3D tissue culture according to claim 13, wherein the tissue culture is utilized for at least one purpose selected from the group consisting of drug efficacy, toxicity screening, investigative toxicology, mechanistic toxicology, target identification, drug repositioning studies, pharmacokinetic assays, pharmacodynamic assays and regenerative medicine.

Description

BRIEF DESCRIPTION OF FIGURES

[0092] FIG. 1 shows human liver microtissue formation without cell selection (cells seeded directly after thawing).

[0093] FIG. 2 shows human liver microtissue formation following vital cell selection.

[0094] FIGS. 3A and 3B show reproducibility of human liver microtissue formation following vital cell selection.

[0095] FIG. 4 shows microtissue formation of dog hepatocypes following vital cell selection.

[0096] FIGS. 5A and 5B show viability and functionality of 3D tissues.

[0097] FIG. 6A-6D show long-term toxicity and inflammation-mediated testing with human liver microtissues.

[0098] FIG. 7 shows structural integrity of human liver microtissues produced with and without pre-plating.

EXPERIMENTS AND FIGURES

[0099] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

Example 1: Optimized Method to Produce Human Liver Microtissues

[0100] 1. Thawing of Cryopreserved Hepatocytes [0101] Take chosen cell-vial from cryo-tank and transfer into water bath (37° C.); set timer on 2 minutes [0102] Pipette 40 ml of the Wash/Thawing medium into 50 ml tube [0103] Transfer the hepatocytes into the tube, wash with 1 ml medium [0104] Place 50 ml tube into centrifuge; start centrifugation for 5 min at 50 rcf (corresponds to 600 rpm) at RT. [0105] Remove supernatant [0106] Wash pellet with 20 ml wash buffer [0107] Place carefully 3 ml of Wash/Thawing medium into the 50 ml tube [0108] Re-suspend cell pellet with 2D cell culture medium [0109] Count hepatocytes with e.g. Trypan Blue

[0110] 2. Hepatocyte pre-plating [0111] Use a collagen coated cell-culture dish for pre-seeding [0112] Seed Hepatocytes in a 6 cm dish: 100000-250000 hepatocytes/cm.sup.2; 0.05-0.5 ml per cm.sup.2 [0113] Place at 37° C. in a CO.sub.2 containing incubator [0114] Optionally: Exchange medium after attachment with Serum-free 2D-culture medium [0115] Harvest hepatocytes after 12-96 hours (daily medium exchange if longer than 12 hours) [0116] 3×wash with pre-warmed phosphate buffered saline the non-adherent hepatocytes away [0117] Add cell detachment solution such as collagenase/accutase mixtures to culture dish to dislodge the hepatocytes from the well 50-500 ul/cm.sup.2 [0118] Incubate 5-30 min at 37° C. (visual observation until most cells are detached from the surface) [0119] Carefully transfer dispersed hepatocytes into a 50 ml tube prefilled with wash bufffer [0120] Centrifuge for 5 min with 50 rcf (corresponds to 600 rpm) at RT [0121] Aspirate supernatant, add 1-50 ml wash medium to the pellet and re-suspend pellet by gentle pivoting of the tube [0122] Repeat centrifugation step for 5 min with 50 rcf (corresponds to 600 rpm) at RT [0123] Add 1 ml re-aggregation medium to the pellet and dissolve hepatocytes by gentle pivoting of the tube [0124] Count hepatocytes

[0125] 3. Production of Microtissues

[0126] A. Preparation of GravityPLUS™ Plates (Insphero AG, CH) [0127] Fill Omnitray with 15 ml of 0.75x PBS/Amphothericin and add humidifier pad [0128] Place frame on Omnitray [0129] Put lid on the plate and label plate

[0130] B. Preparation of Cell Suspension [0131] Prepare cell suspension of defined cell number (i.e. 25′000 Heps/25′000 NPCs per ml) in falcon tubes [0132] Homogenize cell suspension by pivoting the tube

[0133] C. Seeding [0134] Use Viaflo electronic multichannel pipette (Integra Bioscience) [0135] Set Viaflo parameters: Repeat dispense: 40 ul, Speed 3, Repeat: lx (for 96-well Viaflo) [0136] Empty cell suspension in reservoir (don't pour more than 30 ml in the reservoir, to ensure proper distribution of cells) [0137] Remove lid from GravityPLUS plates [0138] Prior Aspiration of cell suspension, gently pivot reservoir for homogeneous distribution of cells [0139] Start Viaflo program: Repeat dispense [0140] Place Viaflo horizontally on the inserts, avoid tapping of the drop on the top by gentle pressure of the Viaflo on the inserts. [0141] Place lid back on plates

[0142] 4. Media Used

TABLE-US-00001 Wash 2D cell culture Re-aggregation Ingredient buffer medium medium Williams E Medium X X X (GE Healthcare) Insulin-Transferrin- X X X Selenium (Gibco) FBS (Fetal Calf 20% 10% 20% Serum) (Lonza) HGF (Hepatocyte 20 μg/ml Growth Factor) (Peprotech)

[0143] Further Ingredients in all media:

[0144] 2 mM Glutamine, Penecillin/Streptomycin, Amphothericin, 0.1 μM Dexamethasone

Example 2: Stability of 3D Tissue Culture

[0145] To test for the stability of 3D tissue culture obtained with the method according to the invention, 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. 5A).

Example 3: Use of 3D Tissue Culture Produced According to the Invention for Hepatoxicity Screening

[0146] 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 toxicological profile. Acetaminophen is the major cause of drug-induced liver injury (DILI) in humans. At therapeutic doses, a proportion of the drug undergoes bio-activation by 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).

[0147] Treatment of liver microtissues over 14 days with 3 re-dosing's resulted in a concentration-dependent increasing cell death with an IC50 value of 754.2 μM (FIG. 6A). Diclofenac is a non-steroidal 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 μM), long-term treated liver microtissues displayed an increased sensitivity toward this drug with an IC50 value of 178.6 μM (FIG. 6B). Most directly hepatotoxic compounds are detected during pre-clinical investigations. However, indirectly hepatotoxic compounds involving the immune system are not detected during pre-clinical phases, such as trovafloxacin. Recent animal experiments indicated that trovafloxacin is only hepatotoxic in combination with an inflammatory stimulus, such as lipopolysaccharide (LPS) or TNF-a. 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). The addition of LPS shifted the hepatotoxic threshold of trovafloxacin about threefold from 220 (without LPS) to 71 μM in the presence of LPS (FIG. 6D). Developed to overcome the limitations of conventional 2D culture, multi-cell type 3D 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.

FIGURES

[0148] FIG. 1 Incomplete microtissue formation if seeded directly after thawing

[0149] 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.1, 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.

[0150] FIG. 2 Vital cell selection allows for complete microtissue formation

[0151] 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 (GravityTRAP™) and imaged for microtissue appearance and-size. All 5 hepatocyte lots showed robust and uniform microtissue formation within 5 days of culture.

[0152] FIG. 3A-3B Reproducible formation of human liver microtissues with vital cell selection of primary human hepatocytes

[0153] FIG. 3A: Size variation of human liver microtissues produced after pre-plating of cryopreserved human hepatocytes. The diameter human liver microtissues of 24 production runs is shown, including the standard deviation (n=6). 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 um, with an relative size deviation of less than 10% between production runs.

[0154] FIG. 3B: Resulting assay variability. The intracellular ATP-content of 40 assays with human liver microtissues was quantified with CellTiter-Glo®-assay after 14 days. Measurements were performed in triplicates. The average RLU's from all 40 assays was set to 100%, the relative standard deviation of each measurement to the mean is depicted. Average relative standard deviation of all 40 measurements is 14.6%.

[0155] FIG. 4 Microtissue formation of dog hepatocytes is achieved only with vital cell selection

[0156] 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.

[0157] FIG. 5A-5B Viability and functionality of 3D tissues over 5 weeks in culture.

[0158] FIG. 5A: Intra-tissue ATP quantification. ATP content per microtissue is depicted (pmol ATP/MT) as an indicator of cell viability and vitality.

[0159] FIG. 5B: Quantification of secreted albumin by ELISA over time, normalized to the initial hepatocyte cell number and time.

[0160] FIG. 6A-6D Long-term toxicity and inflammation-mediated testing with human liver microtissues produced according to the invention

[0161] FIG. 6A: Dose—response of acetaminophen toxicity after 14 days treatment (3 re-dosing) resulted in an IC.sub.50 value of 754.2 μM

[0162] FIG. 6B: Dose—response curve of diclofenac supplemented for 14 days (3 re-dosing) resulted in an IC.sub.50 value of 178.6 μM.

[0163] FIG. 6C: Quantification of IL-6 secretion with ELISA measurement. Hepatosphere was induced for 48 h with 10 lg/ml LPS. Induction with LPS led to a tenfold increase in IL-6 secretion.

[0164] FIG. 6D: Dose—response of trovafloxacin induced toxicity in presence and absence of LPS. Presence of LPS decreased the IC.sub.50 threefold from 220 (−LPS) to 71 μM (+LPS)

[0165] FIG. 7 Structural integrity of human liver microtissues produced without pre-plating and with pre-plating

[0166] 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 24 h on a collagen-coated cell culture dish. 3-5 um thick sections were stained with Hematoxylin and eosin (H&E). Images were taken with a 10× objective.

[0167] The upper row of 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.

[0168] For donor #3 no microtissues were formed using the cryopreserved hepatocytes without the pre-plating step. For the other preparations, a high degree of necrotic areas apparent by dense eosinophilic staining and lack of nuclei within the tissues were visible in the groups without pre-plating (especially predominant for donor 2 and 4 which appear to have hardly any viable hepatocytes integrated). The pre-plating step significantly improves structural morphology of the liver microtissues.

REFERENCES

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