Cell culture

Abstract

The disclosure relates to the fabrication of a three dimensional [3-D] cell culture membrane comprising one or more functionalized surfaces adapted to provide cell culture conditions suitable for the analysis of proliferation, differentiation or function of cells, typically eukaryotic or prokaryotic cells.

Claims

1. A cell culture vessel comprising a three dimensional cell culture substrate comprising: a perforated membrane with through holes, the perforated membrane having three membrane surfaces, namely an upper surface, a lower surface opposite to the upper surface, and a plurality of inner surfaces inside the through holes, the perforated membrane comprising a cured polymer adapted for cell culture having at least two modified cell culture surfaces among the three surfaces of the perforated membrane, wherein the first cell culture surface comprises at least one first cell culture agent for enhancing the proliferation and/or differentiation or function of the cells and the second cell culture surface does not comprise the first cell culture agent but a second different cell culture agent for enhancing the proliferation and/or differentiation or function of the cells, the first and the second cell culture surfaces being two surfaces out of the three membrane surfaces.

2. The cell culture vessel of claim 1, further comprising at least one cell, and cell culture medium.

3. The cell culture vessel according to claim 2, wherein said cell culture substrate is suspended and supported in said cell culture medium.

4. The cell culture vessel according to claim 3, wherein said cell culture substrate comprises one or more cell culture surfaces, wherein said cell culture surfaces do not contact a cell culture vessel surface.

5. The cell culture vessel according to claim 4, wherein said cell is genetically modified by transfection with an isolated nucleic acid or expression vector to recombinantly express a selected nucleic acid in said cell.

6. The cell culture vessel according to claim 1, wherein said cell is a mammalian cell or a prokaryotic cell.

7. The cell culture vessel according to claim 6, wherein said mammalian cell is an epidermal keratinocyte, a fibroblast cell, an epithelial cell, a neuronal glial cell, a neural cell, a hepatocyte, a hepatocyte stellate cell, a mesenchymal cell, a muscle cell, a kidney cell, a blood cell, a pancreatic β cell, cancer cell, or an endothelial cell.

8. The cell culture vessel according to claim 1, wherein said membrane comprises a plurality of perforations wherein said perforations are at least 5-1000 μm in diameter.

9. The cell culture vessel according to claim 8, wherein the perforations have an aspect ratio not greater than 2.

10. The cell culture vessel according to claim 1, wherein said curable polymer is UV curable.

11. The cell culture vessel according to claim 10, wherein said UV curable polymer is an acrylate based polymer.

12. The cell culture vessel according to claim 1, wherein said cell culture agent and/or said membrane is further modified by inclusion of a cross-linking agent that facilitates the cross-linking of the cell culture agent to said membrane to provide a modified cell culture surface.

13. The cell culture vessel according to claim 1, wherein said membrane has a refractive index of between about 1.30 to about 1.50.

14. The cell culture vessel according to claim 1, wherein said cell substrate comprises a network of interconnected cell culture microwells.

15. The cell culture vessel according to claim 14, wherein the network comprises a plurality of elongate cell culture microwells adapted to provide at least said first and second modified cell culture surfaces.

16. A method for the culture of cells, comprising: i) providing a cell culture vessel according to claim 1, comprising: a) cells; and b) cell culture medium sufficient to support the growth of said cells; and ii) providing cell culture conditions which promote the proliferation and/or differentiation and/or function of said cells.

17. The method according to claim 16, wherein said cell culture substrate is suspended and supported in said cell culture medium.

18. The method according to claim 17 wherein said cell culture substrate comprises one or more cell culture surfaces wherein said cell culture surface does not contact a cell culture vessel surface.

19. A method to screen for an agent that affects the proliferation, differentiation or function of a cell, comprising: i) providing a cell culture comprising at least one cell and a cell culture vessel according to claim 1; ii) adding at least one agent to be tested; and iii) monitoring the activity of the agent with respect to the proliferation, differentiation or function of said cells.

20. A method to test the liver toxicity of an agent, comprising: i) providing a cell culture comprising at least one hepatocyte cell and a cell culture vessel according to claim 1; ii) adding at least one agent to be tested; and iii) monitoring the activity of the agent with respect to the proliferation, differentiation or function of said hepatocyte cells as a measure of toxicity of the agent.

Description

(1) An embodiment of the invention will now be described by example only and with reference to the following figures:

(2) FIG. 1: Microwell fabrication and coating process (A) Schematic representation of the microwell fabrication process. A PDMS stencil is fabricated using classical soft lithography process (step 1) and placed on a flat substrate. The gaps are filled by capillarity (step 2) with a UV prepolymer mix (NOA 64). After UV curing, the stencil is removed and the NOA Membrane is incubated a solution of protein that will eventually coat the sides of the wells (step 3-4). The membrane is rinsed, dried and flipped onto a glass slide coated with the protein to be localized at the bottom of the wells (step 5-7). Pluronic acid was eventually used to passivate the top part of the wells (step 8-9). (B) Picture of microwell membrane in a 12 mm glass-bottom dish (scale bar=1 cm). (C) DIC image of hepatocytes in microwells (scale bar=100 μm). (D) Confocal images of the spatially structured coating of microwells. Pluronic acid (P, green, Alexa 488) and fibronectin (F, red, Alexa 561) were used. X/X indicates a side/bottom order of coating, where X is either F or P. (scale bar=5 μm)

(3) FIG. 2: A: Confocal imaging of circular wells (30 μm in diameter, 40 μm in height) with various differential coatings. Green: BSA-GFP+PEG, Red: Fibonectin-Alexa 568. B: Bright field image of the seeding density at 4× and 10× magnification. Hepatocytes were seeded at 0.5 million/ml and gently stirred according to the described protocol;

(4) FIG. 3: Cell survival assay. Fraction of microwells with all cells alive as a function of time. All conditions are represented with both polymers and three coating combination. In NOA wells the survival probability hardly depends on the coating combination and is clearly larger than for the MyPoly133;

(5) FIG. 4: Effects of the differential coating on the hepatocytes doublet morphology and shapes. Fixed samples with actin in green (phalloidin-Alexa 488), nucleus blue (DAPI) and MrP2 in red (antibody-Alexa 568). A: Hepatocytes doublet adhering on sides and bottom present a bile canaliculi with branched morphology (possibly due to the internal tension generated by the actin stress fibers). If only the bottom cell can adhere to the substrate the bile canaliculi rounds. B: If the cells attach only to the side the bile canaliculi is formed mainly vertically as demonstrated from the z projection on the right panel. This orientation is very useful to study accumulation of proteins from the cytoplasm;

(6) FIG. 5: 3D organization of cell-ECM adhesion influences the orientation of hepatocyte couplets and the BC morphology. 3D side views (right column, green, F-actin; blue, nucleus) and Z projection (middle column) of hepatocytes couplets culture for 2 days in the microwells with various coatings (left column) are demonstrated in (A-C). Couplets in F/F (A, n=39) and P/F (B, n=48) formed predominantly horizontal bile canaliculi. In F/P (C, n=44) the BC were mostly vertical. BC in F/F (A, XY) displayed in vivo-like tubular shape; in P/F (B, XY) they expanded into spherical shape. White dashed lines are virtual microwell boundaries for visual guidance. (scale bar=5 μm) (D) Quantitative analysis of BC orientation compared to microwell bottom. The parallel orientation corresponds to a null angle. (**, t-test, p<0.05);

(7) FIG. 6: Perturbations of cell-ECM adhesion or actomyosin contractility drive BC into spherical shape in F/F microwell. Treatment of (A) soluble RGD (250 μM for overnight), (B) blebbistatin +/− (100 μM for 4 hours), or (C) Y27632 (10 μM for 4 hours) converted tubular BC into spherical ones (blue, nucleus; green, F-actin; scale bar=5 μm). (D) Classification of BC morphologies according to the dimensionless morphological index and vertical aspect ratio. BC in control F/F wells (yellow circles, n=35) clearly display a higher degree of folding r with flatter tubular shape (low i) as compared to cells treated with soluble RGD (blue triangles, n=39), blebbistatin (green diamonds, n=44) or Y27632 (purple triangles, n=42). Drug treatments led BC morphology in F/F microwells to be undistinguishable from that in P/F coating (red squares, n=37);

(8) FIG. 7: Physical constraint combined with specific 3D ECM organization directs BC elongation via actomyosin contractility. Typical BC shapes stained with F-actin (i) and probability map of BC in microwells with different shapes (ii) on day 2. BC centers (black circles) and most distant apex of BC (white triangle) are overlaid. Circular F/F microwells (A, n=35) led to isotropically folded BC, whereas couplets cultured in F/F triangular microwells (B, n=29) formed BC with branches predominantly protruding towards the three vertices. BC elongated mainly along the long axis of F/P elongated microwells (C, n=46) containing ˜6 hepatocytes. Note the relative absence of BC protrusions along the short axis. (scale bar=5 μm);

(9) FIG. 8 Hepatic cord-like network of BC formed in intersecting elongated microwells with F/P coating. (A) Phase contrast image of hepatocytes inside intersecting elongated microwells (300 μm for each cross length) containing ˜60 hepatocytes on day 2. (B) Fluorescent image of CLF at the same view of (A). (C) Immunostaining image of bile canaliculi network formed in intersecting elongated microwells. F-actin (Green) and nucleus (Red) were stained. Note the relative absence of transverse BC. (D) Surface rendering image of (C). F-actin (Green) and pan-Cadherin (Red) were presented. (E) Zooming-in on one branch of (D). (scale bar=30 μm);

(10) FIG. 9 illustrates the viability of hepatocytes in the microwell device;

(11) FIG. 10 illustrates the functionality of bile canaliculi in the microwell device;

(12) FIG. 11 illustrates the staining of F-actin and ZO-1 in the microwell device;

(13) FIG. 12 illustrates the excretion of bile canaliculi in the microwell device; and

(14) FIG. 13 illustrates the morphologies of bile canniculi in a microwell device comprising different surface coatings.

MATERIALS AND METHODS

(15) Micro Well Fabrication

(16) An elastomeric stamp (Poly dimethysiloxane PDMS) is fabricated using standard soft lithography methods onto a silicon wafer coated with SU8. The typical size of the stamp features are between 20 and 40 μm both in height and width.

(17) 1 Cut out a piece of PDMS with pillar feature. Do not leave any flat surface around pillar features to allow efficient capillary filling at the next steps.

(18) 2 Put the PDMS piece onto a petri dish with pillars against the surface of petri dish. Use a stick to pick polymers and drop a little on one side of PDMS. Polymers would flow in between pillars by capillary effect.

(19) 3. Start UV lamp and allow the lamp to warm up for 15 min. After the polymer filled the spaces between pillars, put more polymers around the PDMS stamp.

(20) 4. Shine UV light onto the PDMS and polymers for 20 s if NOA is used or 5 min if Mypoly133 is used. Peel off the PDMS piece from the cured polymer sheet with knife. Microwells are formed in the polymer membrane.

(21) 5 Add a drop of a solution A containing the agent you wish to coat the inside of the wells with onto the polymer sheet; typically fibronectin at 1 μg/ml or 0.2% pluronic Acid™. Collagen, cadherins and carbohydrates and poly functional PEG can also be used.

(22) 6. Vacuum for 10 minutes to degas air trapped in the micro-wells and allow solution A into them.

(23) 7. Incubate micro-well with solution A for 1 h at room temperature.

(24) 8. Incubate a suitable container [e.g. glass, silica, plastics] with a solution B containing the agent that will coat the bottom surface of the wells for 1 h at room temperature. Typically the same agents used for the sides can be used here. Additionally clean glass can be used if supported lipid bilayers are to be further formed at the bottom of the micro-wells.
9. Rinse the micro-well and glass bottom dish with water for 3 times.
10. Suck up water and air dry the micro-well and glass bottom dish.
11. Cut off edges of polymer sheet. Peel the sheet off from petri dish and reversely lay it in glass bottom dish, press the polymer sheet gently with a flat PDMS.
12. Passivate the top of polymer sheet with 0.2% pluronic Acid™ for 30 min. 1% PEG/Methacrylate can also be used at that stage. Followed by a 1 min exposure under UV to ensure covalent bonding to the unreacted acrylate groups at the polymer surface.
13. Rinse polymer sheet with water for 3 times.
14. Keep polymer sheet in water and vacuum it for 10 minutes.
15. Rinse polymer sheet with water for 3 times. UV treat polymer sheet for more than 15 min for sterilization.
Cell Seeding 1. Exchange PBS in uWell with cell culture medium. Incubate it in 37 degree Celsius. 2. Seed 0.5 million hepatocytes for each dish. Distribute hepatocytes evenly by shaking the dish back and forth. Put the dish gently into incubator. 3. After 15 minutes of incubation, shake the dish again and put the dish gently into incubator. 4. Repeat step 3 twice. 5. Wash the dish with PBS twice and add cell culture medium.
Cell Culture

(25) Cells are cultured in 5% CO.sub.2 and 37 degree Celsius. Cell culture medium is changed on daily basis. Here a specific medium, William's E medium is used for cell culture. 0.1% BSA, 2 mM L-Glutamine, 100 nM dexamethasone, 100 unit/ml penicillin, 0.1 mg/ml streptomycin, 0.05 μg/ul linoleic acid and 0.3 μg/ml insulin are supplemented into William's E medium.

(26) Hepatocyte Isolation, Seeding and Culturing

(27) Hepatocytes were isolated from male Wistar rats by a two-step in situ collagenase perfusion method, as described in [33]. Animals were handled according to the IACUC protocol approved by the IACUC committee of the National University of Singapore. With a yield of >108 cells/rat, viability of the hepatocytes was tested to be >90% by Trypan Blue exclusion assay.

(28) Freshly isolated rat hepatocytes (0.5 million) were seeded onto microwells within 35 mm-glass bottom dish and cultured in 2 ml of William's E culture medium supplemented with 2 mM L-Glutamine, 1 mg/ml BSA, 0.3 μg/ml of insulin, 100 nM dexamethasone, 50 μg/ml linoleic acid, 100 units/ml penicillin, and 100 mg/ml streptomycin, all of which were purchased from Aldrich-Sigma in Singapore. Cells were incubated with 5% CO.sub.2 at 37° C. and 95% humidity. After 1 h incubation, culture medium containing the unattached cells was removed. The microwells were rinsed with PBS and replenished with fresh culture medium. After 1 h, another 0.5 million hepatocytes were added and incubated for 1 h. The unattached cells were removed by PBS rinsing and culture medium was replenished again. Culture medium was changed on a daily basis.

(29) Forming the Membrane and Index Matching

(30) First we form membranes with through holes according to the protocol as described. The membrane is fabricated either in NOA 73, a low viscosity UV curable resin from Norland Adhesive or in MyPoly 133 DC another UV curable resist from My polymer with a refractive index matched with that of water. NOA 73 is used preferentially for long term culture since it does not release solvent in the culture medium in the long run. MYPoly 133 is used for short term culture and high resolution imaging. When index matching with the medium is expected to be very good, MYPOLy 133 can be mixed with Mypoly134 in adequate proportions to obtain an index of 1.338 matched with DMEM media. The proportions of both polymers are adjusted accordingly with the media index or the wave length used. An alternative approach is to make the membrane in MYpoly 134 and complement the culture medium with Sucrose or Sorbitol until index matching of 1.34 is achieved.

(31) Viability Test and Bile Canaliculi Secretory Function Test

(32) Hepatocyte viability was assessed daily with propidium iodide (Sigma, 81845 FLUKA), a dye that only stains the dead cells. 5 μM PI in culture medium was incubated with hepatocytes in microwells for 30 mins at 37° C. After rinsed with PBS, the stained cells were kept in culture medium and observed under wide-field EVOS microscope (Life Technology). Viability ratio was calculated as the number of microwells with dead cells over the number of all microwells.

(33) Bile canaliculi [BC] secretion was assessed daily with CLF (BD, 451041), a dye that can only be secreted into functional bile canaliculi, where it becomes fluorescent. 5 μM CLF in culture medium was incubated with hepatocytes in microwells for 30 min in 37° C. After rinsing with PBS, the stained cells were kept in culture medium and observed under wide-field EVOS microscope. Functional bile canaliculi ratio was calculated as the number of microwells with functional bile canaliculi over the number of all microwells.

(34) Immunostaining and Image Acquisition

(35) For control, hepatocytes cultured for 48 hours in microwells were fixed in 4% para-formaldehyde (PFA) for 30 minutes. For ±Blebbistatin (Merck, 203390) or Y27632 (Sigma, Y0503) treatments, drugs were added 4 h before fixation, while soluble RGD (Sigma, G5646) was incubated with hepatocytes overnight. After fixation, the cells were rinsed by PBS and permeabilized for 30 min in TBST (0.2% Triton-X in TBS). The permeabilized cells were blocked with 1% BSA in TBST for 4 h and incubated overnight with pan-Cadherin antibody (sigma, C1821) and anti-ZO-1 antibody (life technology, 61-7300) at 4° C. as instructed in manuals. After rinsed with TBST, the cells were incubated in secondary antibodies (Life Technology, A10040 and A-31571) and phalloidin-alexa 488 (Life Technology, A12379) for 1 h in dark at room temperature. After rinsed with TBST again and incubated shortly with DAPI (Sigma, D9564), cells were mounted in mounting medium (DAKO, S3023). 3D stack of confocal microscopy images were acquired with 100×NA1.4 oil lens on a Nikon A1R Confocal Microscope.

(36) Image Analysis

(37) The selected slices of image stacks were reconstructed as maximum intensity projection or orthogonal section view. Manual segmentation was applied based on F-actin signal, because F-actin provides a better definition of BC boundaries, compared with ZO-1 (FIG. 11). Several features, such as parameter, thickness, horizontal spin size and area of bile canaliculi, were extracted subsequently.

(38) For bile canaliculi morphology analysis in triangle and elongated microwells, bile canaliculi were extracted and converted into binary images. All of these binary bile canaliculi were aligned according to the microwell orientation and overlaid together in sum. Resultant image was color-coded based on the probability of bile canaliculi appearance. The warmer the color is, the more frequent the bile canaliculi appear at the specific location.

(39) Statistical Analysis

(40) Data from at least 3 independent experiments were analysed, and values were represented as mean±standard error of means. The number of samples in each group was presented individually in the graphs. The Student t-test was used to analyse the statistical significance of the data. Values with a p-value less than 0.05 were considered statistically significant.

Example 1

(41) 3D Micro-Patterning of ECM Protein on a Chip

(42) To organize cell-ECM adhesion in 3D, we developed a new method to create microwells (FIG. 1) using standard soft lithography methods to fabricate a thin membrane (5-40 μm) with through holes [24]. A PDMS stamp was micro-moulded onto a textured wafer with microwells formed in SU8. The stamp was placed on a flat PDMS surface and the lumen was filled with NOA 64, a UV curable polymer. 30 s exposure (300 W Newport UV lamp operated at 20 mW/cm.sup.2 at 400 nm) is enough to form a solid film that can subsequently be used as a sticker [28]. The stamp was subsequently peeled. We then achieved three different coating configurations: F/F, F/P, P/F labelled according to Side/Bottom order where F stands for fibronectin and P for pluronic acid. The differential coating was achieved as listed below (FIG. 1) using a 10 μg/ml fibronectin and 0.2% pluronic acid solutions.

(43) i—Coating of the sides was achieved first. We incubated the membrane with the appropriate aqueous solution for 1 h. Degassing under vacuum was used to remove air bubbles trapped in the microwells. The membrane was subsequently rinsed and gently air-dried.

(44) ii—Coating of the bottom was achieved by incubating an acid-washed coverslip with desired solution for 1 h. The microwell membrane was flipped over and laminated onto the dried coverslip.

(45) iii—The top of the membrane was subsequently passivated for 1 h using pluronic acid. It ensures minimal adhesion of hepatocytes to the membrane top and maximal spontaneous localization of cells into the wells. The system was then sterilized under UV without appreciable loss of fibronectin (tested by spreading assay with MDCK cells, data not shown).

(46) Cell viability was assessed daily in each of the three coating configurations for 4 days using propidium iodide. The viability index was computed as the ratio of the number of microwells with no dead cells over the total number of microwells (FIG. 9). The survival rate was ˜70% after 4 days independent of the coating configuration.

(47) We then tested the maintenance of functional BC over 4 days (FIG. 10). A functionality index was computed as the ratio of the numbers of microwells displaying functional BC stained by choly-lysyl-fluorescein (CLF) over the total number of microwells. Typically 50% to 70% of the total number of wells displayed a functional BC. After 3 days, cells gradually detached from the substrates for any coating configuration. It is most likely due to enzymatic degradation of fibronectin coated on microwells. However, 75% of remaining couplets was functional. We performed all experiments on day 2 when BC have reached their equilibrium shapes (no difference compared to day 4) and when hepatocyte attachment is still unperturbed.

Example 2

(48) FIG. 2 illustrates the various combinations we used. Various other combinations where tested including BSA, cadherins and collagen. Though they are possible they are not used for hepatocyte cultures. The protein coating is mainly due to protein adsorption. Using a heterobifunctional PEG molecule Acrylate-PEG-NHS from NANOCS (nanocs.com), we are currently investigating the possibility to perform a direct chemical grafting. The acrylate moiety reacts with the polymer under UV curing and the NHS moiety is a standard functional group for protein functionalization.

Example 3

(49) Primary hepatocytes freshly obtained from rat liver according to [22], are seeded at a density of 0.5 million/mL onto the Petri dish. Protecting the top of the membrane with an antifouling treatment is a key component to help cells fall in the wells. Failure in the antifouling treatment will result in cells attaching to the membrane top and not falling in the microwells. The seeding is followed by a gentle shaking procedure described in protocol to ensure the maximum well filling capability. Following this protocol 90% of the wells are filled with at least one cell and 70% are filled with at least two. Better optimization of the doublet rate is under current investigation.

(50) The viability of the cells was tested daily since 24 h after cell seeding with propidium iodine solution (Sigma). In microwells made by MyPolymer, cells in more than 50% of filled wells are viable at 24 h after seeding even though the survival rate drops to about 20% at 48 h after seeding. However, for NOA, cells in more than 80% of filled wells are alive at 48 h after seeding no matter what coating configuration is used. The viability could sustain at more than 60% in microwells made by NOA polymer even at 96 h after seeding.

Example 4

(51) The hepatocytes can be fixed and stained with antibody within the wells. High resolution imaging within a single well without loss of optical resolution compared to normal culture conditions can then be performed. Live imaging with transfected protein is also possible. This offers the opportunity to monitor the evolution of a series of single doublets over time in a very easy way. As shown in FIG. 4 the morphology and orientation of the bile canaliculi can be tuned by changing the chemical coating of the wells. That property can be used to control the tension on the bile canaliculi and its correlation with drug testing. The formation of vertical canaliculi also allows high spatial and time resolution imaging of molecular transport from the cytoplasm to the bile duct. It is a first step towards the control of the formation of bile canaliculi spanning several cells. In addition the formation of vertical bile canaliculi makes it handier to collect bile secretion which is a key challenge for drug testing.

Example 5

(52) Actomyosin contractility couples 3D cell-ECM adhesion to BC morphologies between hepatocyte couplets.

(53) We investigated how the 3D adhesion of hepatocyte couplets to ECM influenced BC morphology. We chose 30 μm circular microwells to maximize the efficiency of couplet formation of hepatocytes, which have an average diameter of 25 μm. Cells were fixed on day 2, stained for F-actin and ZO-1, and imaged by confocal microscopy. Because F-actin signal provides a clearer definition of BC contour than ZO-1 (FIG. 11), we choose F-actin as a marker to demonstrate and quantify BC morphology in this work. The three ECM coating configurations induced three different BC morphologies (FIG. 5).

(54) When the side and bottom of microwells were ECM-coated (F/F configuration), the couplet became stacked vertically (FIG. 5A, 5D). The bottom cell adhered both to the bottom and side of the microwell, whereas the top cell adhered to the side only. The BC exhibited a flat (2-3 μm) isotropic tubular morphology localizing at the centre of the cell-cell junction. This is reminiscent of the BC morphology observed in vivo [23].

(55) When the microwell bottom was ECM-coated and the side was non-adhesive (P/F configuration), the couplet also remained stacked (FIG. 5B, 5D). Only the bottom cell of vertical couplet adhered to the bottom of microwell. The BC exhibited a large spherical morphology (5 to 15 μm) with few folds and villi, which is similar to the BC observed in freshly extracted couplets [26] or small organoids [27]. The BC with any morphology remained functional in microwells, which was confirmed by CLF test (FIG. 12).

(56) When cells adhered to the side of microwell only (F/P configuration), hepatocytes positioned side by side (FIG. 5C, 5D). The BC formed vertically, spanning most of the cell-cell contact area, exhibiting a flat morphology (2-3 μm thick). Our observations suggested that the spatial organization of cell ECM adhesion could control the mesoscale morphology of BC. To confirm the importance of cell-ECM adhesion in obtaining flat tubular BC, we treated the hepatocytes in F/F microwells on day 1 with soluble RGD peptides (250 μM, overnight) that antagonized the integrin-mediated cell-ECM adhesion while keeping the chemical signalling unperturbed. Indeed, as cells detached from the microwells, their BC rounded up to be morphologically equivalent to those observed in the P/F case (FIG. 6A).

(57) Cell-ECM interaction complex is intimately associated with actin cytoskeleton[28] and actomyosin contractility was shown to regulate BC dynamics [29]. Therefore, we tested whether actomyosin contractility was required for the differential control of BC morphology by the spatial organization of cell-ECM adhesion. We found that folded BC formed in F/F microwells rounded up upon inhibition of the myosin II activity through various pathways using blebbistatin (100 μM, 4 h) or Y27632 (10 μM, 4 h). The resulting morphologies were similar to those obtained in the P/F configuration (FIG. 6B, 6C).

(58) The BC morphologies in different conditions were quantitatively assessed by two dimensionless parameters: the degree of folding and vertical aspect ratio. The degree of folding r=P.sup.2/(4πA) was evaluated by measuring the ratio of the BC projected perimeter P to the projected area A. This morphological index r ranges from 1 (circle) upward (folded tubular structures). The vertical aspect ratio i was calculated as the ratio of the BC maximal height over maximal elongation in the XY plane. Higher vertical aspect ratio represents a thicker BC.

(59) When the degree of folding r was plotted versus the vertical aspect ratio i (FIG. 6D), the flat and tubular shape of BC in F/F microwells is reflected by a low vertical aspect ratio but a high degree of folding. In contrast, BC in P/F microwells, in F/F microwells with soluble RGD, in F/F microwells with blebbistatin, or in F/F microwells with Y27632 assumed a higher vertical aspect ratio and a lower degree of folding, characteristics of their inflated shape. The BC morphologies in F/F microwells group into a cluster clearly distinctive from the cases of the P/F configuration or the F/F configurations under drug treatments.

Example 6

(60) Physical Constraint Combined with Specific 3D ECM Organization Controls Bile Canaliculi Elongation

(61) Since actomyosin contractility participates to the distant regulation of BC morphology by cell-ECM adhesion, we hypothesized that the BC elongation would be affected by physical constraint, which is known to regulate actomyosin contractility [30]. Microwells with different shapes were utilized to impose physical constraint and modulate the actomyosin contractility. We seeded hepatocytes in F/F coated equilateral triangular microwells (45 μm side) since actomyosin contractility is developed in the vicinity of vertices by virtue of local actomyosin accumulation [31]. Two days after seeding, the hepatocytes remained stacked with their BC adopting a 3-lobed clover-leave shape pointing in the direction of the triangle vertices (FIG. 7B(i)). However, no BC formed in P/F triangle microwells since few couplets were observed (data not shown). Overlaying the binarized images of the BC after alignment of the microwell edges, we computed the probability of finding a BC at a given point of cell-cell contact inside triangle microwells (FIG. 7B(ii)). In addition, the distribution of the centres and the distribution of the most distant points of BC were also evaluated (FIG. 7B(ii)). It revealed that the average shape of BC was indeed a three-lobed structure with each branch extending towards a vertex of triangular microwell. As a control, we evaluated the probability map of BC in the round microwells in which the BC extension was isotropic (FIG. 7A(i)). The most possible BC shape in the circular microwells is a circle with its lobe ends distributed isotropically (FIG. 7A(ii)). Thus, these results supported that physical constraint could direct BC elongation via actomyosin contractility.

(62) Cell-cell interaction is equivalently likely to affect actomyosin contractility as cell-ECM interaction [32]. Cell-cell interaction is thus expected to impact the BC morphologies. To test this hypothesis, we seeded multiple hepatocytes into elongated microwells (F/P) to alter the cell-cell contact status. Compared to other coating configurations, the F/P elongated microwells best ensured that cells spontaneously accommodate into two facing rows with vertical interfaces (FIG. 13). Flat vertical BC with transverse branches are expected based on our observations on couplets in similar coating configurations (FIG. 5C). However, BC adopted long tubular structures extending in the direction of the major axis of the elongated microwells (FIG. 7C (i)) with only small transverse branches. The probability map of BC in these elongated microwells clustered in the centre along the major axis of the microwells (FIG. 7C (ii)).

(63) Taken together, our observations support the hypothesis that the actomyosin contractility, affected either by physical constraint, cell-ECM or cell-cell interactions, can determine the mesoscale BC morphology. We then developed a scaffolding biomechanical microenvironment with the combined effects of cell-cell/ECM interactions and physical constraint to control BC morphology. We mimicked hepatic cords using intersecting elongated microwells (30 μm wide and 300 μm long) with F/P coating configuration. We observed long functional bile canaliculi spanning the entire length of microwells (300 μm) (FIG. 8). This morphology was only observed in the F/P coating configuration with weak adhesion of hepatocytes on the bottom substrate, in which the actomyosin contractility likely distributed horizontally, thus connecting BC into elongated structures. The formed tubular network has similar dimension and morphology with a hepatic cord in vivo.

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