Mold and Method for Preparing a Hollow 3D Cell Tissue Structure

Abstract

The present invention relates to a mold for preparing a hollow 3D cell tissue structure such as an organoid, and uses thereof. Methods for preparing a hollow 3D cell tissue structure such as an organoid, in particular a human heart mimic, are also provided.

Claims

1. A mold for preparing a hollow 3D cell tissue structure such as an organoid, in particular a human heart mimic, comprising: an external body having an inner surface delimiting a cavity in the external body; an internal body having an outer surface, wherein the internal body is positioned in the cavity of the external body, wherein the inner surface of the external body and the outer surface of the internal body define a culture chamber there between for receiving cells therein, characterized in that the internal body is made in a predefined shape of a fugitive material to enable degradation of the internal body without significantly affecting an integrity of a formed cell structure and/or cell tissue.

2. A mold according to claim 1, characterized in that the internal body and the external body are made of a fugitive material.

3. A mold according to claim 2, characterized in that the internal body and the external body are formed from a different fugitive material.

4. A mold according to claim 3, characterized in that the fugitive material comprises gelatin, particularly at least 10% gelatin, and/or Pluronic F-127 and/or poly(vinyl alcohol) (PVA).

5. A mold according to any one of the preceding claims, characterized in that the fugitive material is adapted to degrade above or below a temperature threshold value and/or in the presence of an enzyme and/or in the presence of a reactive chemical compound and/or by sonication and/or in the presence of light with a wavelength above or below a threshold value.

6. A mold according to claim 5, characterized in that the fugitive material is adapted to degrade at a temperature between 10-50 degrees Celsius, more particularly between 25-40 degrees Celsius.

7. A mold according to any one of the preceding claims, characterized in that the fugitive material is substantially solid below 10 degrees Celsius and is adapted to dissolve and/or liquefy above 10 degrees Celsius, in particular to dissolve and/or liquefy at a temperature between 10-42 degrees Celsius.

8. A mold according to any one of claims 1-6, characterized in that the fugitive material is substantially solid above 15 degrees Celsius and is adapted to dissolve and/or liquefy below 15 degrees Celsius.

9. A mold according to any one of the preceding claims, characterized in that the internal body is made in an irregular shape, and/or any other desired shape, particularly a ventricle-like or atrium-like or heart-like shape.

10. A mold according to any one of the preceding claims, characterized in that the external body comprises at least one opening in fluid communication with the cavity.

11. A mold according to claim 10, characterized in that the external body comprises a first opening in fluid communication with the cavity for receiving there through a first conduit for guiding fluid to the cavity, and a second opening in fluid communication with the cavity for receiving there through a second conduit for guiding fluid away from the cavity.

12. A mold according to claim 11, characterized in that the external body comprises an inlet wall in which inlet wall both the first and second opening are provided.

13. Method for preparing a hollow 3D cell tissue structure such as an organoid, in particular a human heart mimic, comprising the steps, particularly consecutive steps, of: preparing an external body having an inner surface delimiting a cavity in the external body; preparing an internal body of fugitive material in a predefined shape and having an outer surface; placing the internal body inside the cavity of the external body, wherein the inner surface and outer surface define a culture chamber there between; providing cells, particularly stem cells, progenitor cells, cardiomyocytes and/or other cardiac cells into the culture chamber; subjecting the cells to incubating conditions for a period of time to promote the formation of a supported cell structure, and/or to incubating conditions to promote the formation of a cell tissue, in the culture chamber around the outer surface of the internal body; subjecting the internal body of fugitive material to a condition whereto the fugitive material reacts thereby degrading the internal body for removal from the cavity without significantly affecting an integrity of a formed supported cell structure or cell tissue in the culture chamber and maintaining a functioning of the supported cell structure or cell tissue.

14. Method according to claim 13, wherein the step of subjecting the internal body to a condition whereto the fugitive material reacts comprises liquefying and/or dissolving of the internal body by a change in temperature, particularly comprises dissolving the internal body by increasing the temperature from a start temperature below 10 degrees Celsius to a degradation temperature between 10 and 40 degrees Celsius.

15. Method according to claim 13 or claim 14, wherein the internal body is liquefied and/or dissolved by supplying a liquefying or dissolving agent to the internal body.

16. Method according to claim 15, wherein said liquefying or dissolving agent comprises an enzyme or chemical compound or composition.

17. Method according to claim 13 or claim 14, wherein the internal body is liquefied and/or dissolved by sonication and/or in the presence of light with a wavelength above or below a threshold value.

18. Method according to any one of claims 13-17, wherein the step of providing cells in the culture chamber comprises providing cells in the cavity of the external body followed by placing of the internal body inside the cavity of the external body to position the cells in the culture chamber.

19. Method according to any one of claims 13-18, wherein the preparing of the external body comprises preparing a first part of the external body comprising a first section of the cavity, preparing a separate second part of the external body comprising a second section of the cavity, and coupling of the first part and second part to each other such that the first section of the cavity and the second section of the cavity communicate with each other thereby forming the cavity.

20. Use of a mold according to any one of claims 1-12 for preparing a hollow 3D cell tissue structure, preferably an organoid with a cavity.

21. Use according to claim 20, wherein said organoid is a cardiac organoid, preferably a human cardiac organoid.

22. A bioreactor comprising: a mold according to any one of claims 1-12; and cells that are present in the culture chamber between the inner surface of the external body and the outer surface of the internal body of said mold.

23. A hollow 3D cell tissue structure obtainable by a method according to any one of claims 13-19.

Description

[0119] These and other objects and aspects of the present invention are hereinafter further elucidated by the appended drawing and the corresponding embodiment, which forms part of the present application. The drawing is not in any way meant to reflect a limitation of the scope of the invention, unless this is clearly and explicitly indicated. In the drawing:

[0120] FIG. 1A-1F show consecutive steps of the preparation of an embodiment of a mold and bioreactor according to the invention.

[0121] In this application similar or corresponding features are denoted by similar or corresponding reference signs. The description of the various embodiments is not limited to the example shown in the figures and the reference numbers used in the detailed description and the claims are not intended to limit the description of the embodiments, but are included to elucidate the embodiments by referring to the example shown in the figures.

[0122] In an embodiment as shown in FIG. 1, the invention concerns a mold comprising an internal body inside the cavity of an external body, which mold is formed as follows. In a first step an inner-mold (00) is 3D printed in a desired predetermined shape, herein in the shape of a human heart mimic with separate inlet channel and outlet channel, using a biocompatible resin. The inner-mold (00) is used to cast a negative (03) of the inner-mold (00) in polydimethylsiloxane as shown in FIG. 1A. The negative (03) is then cut in half (not shown), for instance by using a sharp blade to allow the inner-mold (00) to be removed from the inside of the negative (03). Next the two halves of the negative (03) are coupled together again to form the negative (03) with an empty cavity formed by the removed inner-mold (00). The two halves of negative (03) can be tightly clamped together in order to make the cavity at least substantially liquid-tight so as to avoid leakage of a liquid introduced into the cavity. A glass capillary (02) is inserted through each of the inlet and outlet spaces formed in the negative (03) as a result of the inner-mold (00) comprising respective inlet outlet channels. Each of the glass capillaries extends with an end into the human-heart shaped cavity of negative (03).

[0123] Gelatin (Sigma-Aldrich) is dissolved as 10% (w/v) in PBS or medium and autoclaved at 120 degrees Celsius. The gelatin solution is stored at 4 degrees Celsius. The stored gelatin solution is warmed to 37 degrees Celsius and is pipetted into the negative mold (03) through one of the glass capillaries (02) until the cavity of negative mold (03) is filled with gelatin. The negative mold (03) filled with gelatin is cooled to 4 degree Celsius in order to gelate the gelatin. The gelated gelatin forms a human-heart shaped inner body (07) of the mold according to the invention, as shown in FIG. 1B.

[0124] In a next step an outer-mold (01) is 3D printed using a biocompatible resin in a shape corresponding to the inner-mold (00) but larger in size. The outer-mold (01) is used to cast an outer negative (04) of outer-mold (01) in polydimethylsiloxane as shown in FIG. 1C. The outer negative (04) is then cut in half, for example using a sharp blade (not shown). This allows the outer-mold (01) to be removed from the inside of the outer negative (04). Optionally, the outer-mold (01) may be kept inside a part of the cut negative (04). However, a bottom part of the negative (04) is separated from a top part of the negative (04). The top part of the negative (04) together with the outer-mold (01), or optionally the outer-mold (01) alone, is then placed inside a cavity defined by support means, i.e. support container (05), with the cavity in the container (05) being sealed from an outside environment as shown in FIG. 1D due to tight fitting of the top part of negative (04) over container (05). Container (05) contains a pair of inlet/outlet cannulas that gives access to its interior independently of the inlet/outlets of the inner-body (07) and/or the tissue.

[0125] In order to prepare the external body, the stored gelatin solution previously described is warmed to 37 degrees Celsius and is introduced into the remaining free space in the cavity of the container (05) not occupied by the outer mold (01) through the inlet/outlet cannulas. The assembly of container (05), the top part of the negative (04), the outer-mold (01) and the introduced gelatin is cooled at 4 degrees Celsius in order to gelate the gelatin. After gelatin gelation the top part of the negative (04) and the outer-mold (01) are removed from the container (05), thereby leaving a gelatin external body (06) of the mold according to the invention behind inside the container (05).

[0126] The outer-mold (01) is next removed from the top part of the negative (04). An extra pair of inlet and outlet are made on negative (04) from an outside surface into a lumen surface.

[0127] Next, the negative (03) is opened and the solid inner-body (07) of gelatin is removed together with the inlet and outlet capillaries. The inner-body (07) is then partly placed in the space defined by the external body (06) in the container (05) and appropriately aligned therewith using the inlet and outlet capillaries. The external body (06) together with the inner-body (07) are thus positioned inside the container (05), as shown in FIG. 1E. The appropriate alignment is possible through the perfect match of the external body (06) and the inner body (07), creating a uniform gap between an outer surface of the inner-body (07) and an inner surface of the external body (06).

[0128] Beating cardiomyocytes are prepared as follows. Human embryonic stem cells (hESC) and/or human induced pluripotent stem cells (hiPSC) coming from in vitro cell cultures or from commercial available sources are cultured on Vitronectin Recombinant Human Protein (Life technologies) coated plastic plates in E8 medium (Life Technologies). The hESC and hiPSC are passaged using PBS (Life Technologies) containing EDTA 0.5 mM (Life Technologies) or TryplE (Gibco).

[0129] Differentiation into the cardiac lineage is induced in a monolayer as described previously (Elliott et al., 2011; (van den Berg, Elliott, Braam, Mummery, & Davis, 2016). Briefly, 25103/cm2 are seeded on plates coated with 75 g/mL (growth factor reduced) Matrigel (Corning) the day before differentiation (day 1). At day 0, cardiac mesoderm is induced by changing E8 to BPEL medium (Bovine Serum Albumin [BSA] Polyvinyl alcohol Essential Lipids; (Ng et al., 2008)), supplemented with a mixture of cytokines (20 ng/mL BMP4, R&D Systems; 20 ng/mL ACTIVIN A, Miltenyi Biotec; 1.5 M GSK3 inhibitor CHIR99021, Axon Medchem). After 3 days, cytokines are removed and a Wnt inhibitor (5 M, XAV939, Tocris Bioscience) is added for 3 days. BPEL medium is refreshed every 3-4 days.

[0130] To generate the cardiac tissue the beating cardiomyocytes are dissociated using TryplE 1 for 10 mins and resuspended in cell culture medium with 10% Serum (e.g. 1DMEM with 10% fetal calf serum).

[0131] The resuspended cells are mixed at a final density of 510.sup.6 to 2010.sup.6 cells/ml with 2 to 5 mg/ml bovine fibrinogen (stock solution: 200 mg/ml plus aprotinin 0.5 g/mg fibrinogen in NaCl 0.9%, Sigma F4753), 100 l/ml Matrigel (BD Bioscience 356235). To the final mix thrombin is added at 1:300 (100 U/ml, Sigma Aldrich T7513), resuspended well and the entire solution is pipetted into the gap between the inner body (07) and the external body (06) using the inlet existing on the negative (04). A thus formed bioreactor comprising the container (05) plus inner body and external body of the mold and the cell suspension, as shown in FIG. 1F, is kept at 4 degrees Celsius during the following procedure. The inlet is closed with a stopper and the bioreactor is placed in the fridge for 30 mins for polymerization of the fibrinogen.

[0132] After fibrinogen polymerization the bioreactor is transferred into a 37 Celsius, 5% CO.sub.2 humidified cell culture incubator in order to degrade the inner body (07) and the external body (06) by melting the gelatin. After the gelatin has melted a free standing tissue made of cells and fibrinogen remains inside the bioreactor. The inside of the tissue is perfused with medium at a flow rate of 100 ul per minute for 10 mins. Hence, the gelatin is removed by flushing the inner cavity of the tissue with medium. Furthermore, inlet and outlet of the tissue are closed and medium is perfused via the inlet/outlet cannulas present on the container (05) at a flow rate of 100 ul per hour. The tissue starts contracting 3 to 4 days after the making.

[0133] While the current application may describe features as part of the same embodiment or as parts of separate embodiments, the scope of the present invention also includes embodiments comprising any combination of all or some of the features described herein.

[0134] As used herein, the terms in particular, particularly and preferably are used interchangeably.

REFERENCES

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