Kit-of-parts and method for creating a three-dimensional structure for cell growth using a three-dimensional sacrificial template

Abstract

The present invention relates to a kit-of-parts for creating a three-dimensional structure for cell growth using a three-dimensional sacrificial template, wherein the kit- of-parts comprises a template coupling module configured to accommodate and hold the three-dimensional sacrificial template, and a fixating device configured to engage the template coupling module. The present invention further relates to the use of the kit-of-parts in a method for creating a three-dimensional structure for cell growth, a method for creating a three-dimensional structure for cell growth and to a three- dimensional structure for cell growth created by the method of the present invention.

Description

[0001] Title: Kit-of-parts and method for creating a three-dimensional structure for cell growth using a three-dimensional sacrificial template field

[0002] The present invention relates to a kit-of-parts for creating a three-dimensional structure for cell growth using a three-dimensional sacrificial template created from a polymer. The present invention further relates to the use of the kit-of-parts in a method for creating a three-dimensional structure for cell growth, a method for creating a three- dimensional structure for cell growth and to a three-dimensional structure for cell growth created by the method of the present invention.

[0003] It has been a major focus of tissue engineering to create increasingly more complex and representative 3D tissue constructs, either as more relevant in vitro platforms to study biological processes or to develop more competent replacement tissues. A critical challenge in achieving the goal has been the creation of lumen structures, a prevalent feature throughout the body including the intestine, blood vessels, bile duct, fallopian tubes, loops of Henle, etc. Whether they transport elements from one location to another or provide structural guidance for cell function, growth, and migration, the generation of a biologically relevant environment that incorporates appropriately sized lumen with meaningful organization has been an intense area of research in the translation from 2D cell biology to 3D tissue systems.

[0004] In view of this, there is research ongoing to create microchannels that would be capable of supporting and directing cell growth. Prior art options for creating microchannels include “gel-in-gel” constructions, wherein tracks of cells are deposited in an organized fashion within a host gel, producing general patterns that rely on cell organisation to form open channels. Alternatively, fugitive inks are similarly deposited within a host gel and are later removed, creating channel structures which lack the presence of cells and must be added later. Both techniques have limited resolution and feature size to approximately 100s of micrometres.

[0005] Other research is directed towards the use of sacrificial templates that form the desired structures embedded in a hydrogel. For instance, sugar is used, which has the drawbacks that it is soluble and must be coated, that it cannot easily be pre-seeded, that sugar is cytotoxic and that the template cannot be as small as desired.

[0006] One of the mechanisms for creating a sacrificial template is the use of polymers that have a thermally induced solubility. These polymers have a so-called Lower Critical Solubility Temperature (LCST). Above this temperature, the polymers are hydrophobic and retain their mechanical integrity allowing the structure to be embedded within a hydrogel. After cooling to a temperature below the LCST, the polymer will transition to its hydrophilic state, allowing it to become water soluble. One known polymer that is used is pNIPAM (Poly(N-isopropylacrylamide)). With this polymer, some desirable results are obtained, but it still has some drawbacks in that the temperature transition range is relatively narrow and difficult to control and that it is too hydrophilic to be pre-seeded with cells. It is also difficult to process and form well defined micron-sized template structures.

[0007] For example, Haigh et al. (Haigh, Jodie N., et al. "Hierarchically Structured Porous Poly (2-oxazoline) Hydrogels." Macromolecular rapid communications 37.1 (2016): 93-99) disclose a method using 3D printing of a sacrificial template composed of poly(8-caprolactone) inside a hydrogel, however a drawback of the method is that it does not allow seeding of living cells on the sacrificial template prior to embedding due to the application of acetone to dissolve the sacrificial template. Even further, the use of acetone results in a dehydration of the hydrogel, leading to changes in the hydrogel polymer network, e.g., densification.

[0008] International patent application published under number WO 2022 / 069618 A1 provides for a method for creating a three-dimensional structure for cell growth by creating a template from a polymer, applying a hydrogel support onto the template to embed the template in the hydrogel and removal of the template. The polymer used in this method is a PsecBuOx-stat-PAOx co-polymer (polyoxazoline) and was further disclosed in International patent application published under number WO 2022 / 069635 A1.

[0009] It was found that by using the method disclosed in WO 2022 / 069618 A1 the relatively benign trigger for template dissolution, i.e. lowering the temperature in an aqueous medium, allows the hydrogel to retain its original mechanical properties, and furthermore, biologicals (proteins, growth factors, cells) can be safely incorporated within the structure while retaining their viability throughout the dissolution process. Although the method disclosed in WO 2022 / 069618 A1 , also known as the FiberCyte technology developed by researchers at the MERLN Institute (Institute for Technology-Inspired Regenerative Medicine; Maastricht), is found to be very suitable for Organ-on-a-chip engineered tissues, to add further complexity, while maintaining a convenient method of manufacturing, further research is still ongoing. In order to add further complexity, one might would like to add additional 3D sacrificial template steps and / or loading the 3D sacrificial template with various types of cell material. To assembly of different parts of an engineered Organ-on-a-chip is a critical point during the fabrication of such fluidic structure systems as the structures are small and fragile. Especially in case more complexity is needed to be added to the fluidic structure system.

[0010] Detailed description of the invention

[0011] In order to facilitate the production of Organ-on-a-chip devices which are easy to use, have improved flexibility, and / or provides for an Organ-on-a-chip with improved complexity, in a first aspect, the present invention provides hereto a kit-of-parts for creating a three-dimensional structure for cell growth using a three-dimensional sacrificial template, wherein the kit-of-parts comprises: a fixating device, wherein the fixating device is made of a rigid material, such as a rigid metal material; and a template coupling module, wherein the template coupling module is made of a rigid material, such as a rigid polymeric material, and configured to accommodate and hold the three-dimensional sacrificial template, and wherein the template coupling module is provided with: an inner port configured to fluidly connect the template coupling module to the three-dimensional sacrificial template; and an internal channel, wherein the internal channel is fluidly connected on one end to said inner port of the template coupling module, wherein the template coupling module is either provided with: an outer port, said outer port is fluidly connected to another end of the internal channel, and wherein the outer port is configured to fluidly connect the template coupling module to a microfluidics apparatus; or a further inner port, said further inner port is fluidly connected to another end of the internal channel, and wherein the fixating device is either: fixed to the template coupling module and is, optionally, provided with coupling element for engaging at least one further template coupling module; or provided with coupling elements, said coupling elements are configured to engage the template coupling module.

[0012] By providing the kit-of-parts of the present invention, the creation of a three- dimensional structure for cell growth using a three-dimensional sacrificial template is now facilitated in a comprehensive and safe way, wherein the sacrificial template to be coupled with and held by the template coupling module can be safely transferred for further processing unit using the fixating device attached to or engaged with the template coupling module. By providing the kit-of-parts of the present invention, the template coupling module (and sacrificial template formed thereon) can be handled and transferred by handling and transferring the fixating device, such that the sacrificial template is not contaminated or that its structural integrity is not jeopardized.

[0013] The three-dimensional sacrificial template may be formed in a ‘loop’-like structure, wherein the template is held by the same template coupling module. In another embodiment of the present invention, the three-dimensional sacrificial template may be formed between two or more template coupling modules, e.g., in a ‘bridged’-like structure. In order to provide for the easy and safe handling of such a ‘bridged’-like structure, the present invention provides for a kit-of-parts for creating a three-dimensional structure for cell growth using a three-dimensional sacrificial template, wherein the kit-of-parts comprises: at least two template coupling modules, said at least two template coupling modules are configured to accommodate and hold the three-dimensional sacrificial template, wherein the at least two template coupling modules are each provided with an inner port configured to fluidly connect the respective template coupling module to the three-dimensional sacrificial template, an internal channel that is fluidly connected on one end to said inner port and wherein the internal channel is fluidly connected on another end to either another inner port configured to fluidly connect the template coupling module to the three-dimensional sacrificial template or an outer port configured to fluidly connect the template coupling module to a microfluidics apparatus; and a fixating device, said fixating device is configured to simultaneously releasably engage each of the at least two template coupling modules in order to fixate the at least two template coupling modules in space, orientation and distance from each other.

[0014] The kit-of-parts provided by the present invention overcomes the restraint of the lack of complexity and standardization of the solutions available up to date regarding the production of Organ-on-a-chip devices. As present solutions available on the market starts with a ‘simple’ Organ-on-a-chip design, adding further complexity to the initial design is limited. The present invention overcomes this limitation by providing the kit-of-parts of the present invention wherein a three-dimensional sacrificial template can be formed between and held by the template coupling modules and wherein the template coupling modules are subsequently fixed in space, orientation and distance from each other to allow further transferring, handling and processing of the three-dimensional sacrificial template after formation. As such, the kit-of-parts of the present invention thus facilitates the method of creating three- dimensional structures for cell growth which structures more closely resemble the complex three-dimensional environments found in the body. In fact, the kit-of-parts of the present invention may be used in creating complex organ-like structures.

[0015] In particular, in order to provide a kit-of-parts comprising two or more template coupling modules engageable with the fixating device, the present invention may provide for a kit-of-parts, wherein the kit-of-parts comprising the fixating device, and wherein the template coupling module further comprises at least one further template coupling module, wherein the at least one further template coupling module is made of a rigid material, such as a rigid polymeric material, and configured to accommodate and hold the three-dimensional sacrificial template, and wherein the at least one further template coupling module is provided with: an inner port configured to fluidly connect the further template coupling module to the three-dimensional sacrificial template; and an internal channel, wherein the internal channel is fluidly connected on one end to said inner port of the further template coupling module, wherein the at least one further template coupling module is either provided with: an outer port, said outer port is fluidly connected to another end of the internal channel, and wherein the outer port is configured to fluidly connect the further template coupling module to a microfluidics apparatus; or a further inner port, said further inner port is fluidly connected to another end of the internal channel, and wherein the fixating device is provided with coupling elements, said coupling elements are configured to simultaneously engage the template coupling module and the at least one further template coupling module in order to fixate the template coupling modules in space, orientation and distance from each other.

[0016] As used herein ‘the three-dimensional structure for cell growth’ refers to a three- dimensional structure that is able to grow cells ranging from single cell layers to complex cell structures, such as organoids. Other terms that might be used interchangeably for ‘cell growth’ may include, but are not limited to, ‘cell culture’, ‘culturing cells’, ‘cell expansion’, and the like.

[0017] It was recognized by the inventors that the method of creating a three- dimensional sacrificial template as provided by WO 2022 / 069618 A1 is a simple but yet versatile technology. The kit-of-parts of the present invention facilitates this process of creating three-dimensional sacrificial templates fixedly held between the template coupling modules of the present invention. By creating an assembly of fixated template coupling modules engaged in a releasable manner with the fixating device, the (still fragile) three-dimensional sacrificial template can be further processed, e.g., by further increasing the complexity of the three-dimensional sacrificial template and / or loading the three-dimensional sacrificial template with cells of interest. Given the kit-of-parts of the present invention, there is no need to add additional high-tech technology in adding further complexity to the three-dimensional sacrificial template and / or need of additional specific expertise in creating more complex three- dimensional structures for cell growth.

[0018] The kit-of-parts of the present invention further increases the flexibility of the user in creating three-dimensional sacrificial templates compared to the solutions available on the market nowadays. By providing a kit-of-parts that comprises multiple types of template coupling modules configured to hold three-dimensional sacrificial templates of different designs, the end-user is able to use different types of three- dimensional sacrificial templates. Preferably, each of the multiple types of template coupling modules is provided with an inner port configured to fluidly connect the template coupling module to the three-dimensional sacrificial template, an internal channel that is fluidly connected on one end to the inner port and wherein the internal channel is fluidly connected on another end to either another inner port configured to connect the template coupling module to the three-dimensional sacrificial template or an outer port configured to fluidly connect the template coupling module to a microfluidics apparatus. The template coupling modules may differ in dimensions, in number of inner ports and / or configuration of inner and outer ports. For example, the template coupling module may comprise an outer port for creating an inflow of fluid provided by the microfluidics apparatus into the three-dimensional structure created by the template and an outer port for creating an outlet for a microfluidic apparatus to collect and direct fluid discharged from the same three-dimensional structure, thereby providing a controlled fluid flow to sustain and direct cell growth. On the other hand, the template coupling module may comprise two inner ports fluidly connected via the same internal channel in order to create a fluid loop bringing the fluid discharged from one channel of the three-dimensional structure for cell growth into another channel of the three-dimensional structure for cell growth.

[0019] In an embodiment of the present invention, the template coupling modules may be provided with a first outer port and a first inner port fluidly connected to each other via a first internal channel, and wherein each of the template coupling modules is provided with a second outer port and a second inner port fluidly connected to each other via a second internal channel. Alternatively, the template coupling modules may be provided with multiple outer ports, multiple inner ports, and multiple internal channels, wherein each outer port is fluidly connected to a corresponding inner port via a corresponding internal channel. However, it has to be understood that the template coupling modules may have any configuration in order to provide the most efficient support for the three-dimensional sacrificial template and the subsequently formed three-dimensional structure for cell growth.

[0020] The fixating device of the kit-of-parts of the present invention may be an integral part of the template coupling module as defined above. By providing a fixating device being an integral part of the template coupling module, there is no need for the end user to touch the template coupling module as such, thereby preventing contamination of the template coupling module. In addition, such fixating device, being an integral part of the template coupling module, may be further provided with coupling elements for coupling (either fixatedly or releasably) to one or more other template coupling modules.

[0021] Alternatively, the fixating device of the kit-of-parts of the present invention is preferably provided with spaced apart coupling elements able to simultaneously releasably engage with corresponding coupling elements provided on two or more respective template coupling modules holding the three-dimensional sacrificial template. Depending on the design of the three-dimensional sacrificial template two or more template coupling modules may be involved in holding the three-dimensional sacrificial template. In case multiple template coupling modules are used, the fixating device is configured to simultaneously releasably engage multiple template coupling modules.

[0022] The fixating device of the kit-of-parts of the present invention may engage the template coupling modules by using magnets, clamps, knobs or the like. The use of magnets is preferred as a fixating device comprising magnets (and template coupling modules comprising the opposite poles of corresponding magnets) provides for the most easy, simplified and robust engaging of the template coupling modules with the fixating device.

[0023] Further, although the fixating device is made of a rigid material, such as a metal material, it is noted that the fixating device of the present invention may be configured such that the distance between different template coupling modules can be varied in distance and mutual orientation. For example, the fixating device of the present invention may be provided as a slidable fixating device, wherein the fixating device is able to switch between (at least) a retracted state and an expanded state. Instead of providing different dimensioned fixating devices, by providing a fixating device that is able to adjust the orientation of the template coupling modules, only one fixating device can be provided for forming multiple different dimensioned three-dimensional sacrificial templates.

[0024] Also, instead of providing a fixating device that forms an integral part of a template coupling module or providing a fixating device that is able to engage one or more template coupling modules using coupling elements such as magnets, it is noted that the coupling elements of the fixating device may be in the form of a recessed part as well. Such recessed part is able to support one or more template coupling modules, wherein the fixating device has the form of a supporting frame. Such frame may be used to transfer the one or more template coupling modules in a safe a releasable manner.

[0025] The kit-of-parts may further comprise hydrogel embedding unit. The hydrogel embedding unit comprises a frame provided with one or more, preferably at least two, template coupling module holders, wherein each template coupling module holder is dimensioned to releasably receive and hold a template coupling module. The hydrogel embedding unit is configured to embed the three-dimensional sacrificial template in the hydrogel. In a preferred embodiment, the frame of the hydrogel embedding unit may be further provided with a hydrogel chamber, wherein the hydrogel chamber is formed adjacent to the template coupling holder, preferably formed between at least two template coupling module holders. The hydrogel chamber is configured to receive a hydrogel solution. Once received by the hydrogel chamber, the hydrogel solution is stabilized and / or crosslinked to form a hydrogel support. Also the hydrogel chamber is dimensioned to embed the three-dimensional sacrificial template in the hydrogel support.

[0026] The hydrogel embedding unit allows for the formation of three-dimensional structure for cell growth embedded in the hydrogel support by embedding the three- dimensional sacrificial template in the hydrogel support and by the subsequent removal of the three-dimensional sacrificial template. Before embedding the three- dimensional sacrificial template in the hydrogel support, the three-dimensional sacrificial template may be further processed to add complexity to the three- dimensional sacrificial template and / or seeding the three-dimensional sacrificial template with cells.

[0027] In order to facilitate the further processing of the three-dimensional sacrificial template, the kit-of-parts may further comprise a further processing unit, such as a cell seeding unit, laser processing unit or the like. The further processing unit is provided with one or more, preferably at least two, template module holders, wherein each template module holder is dimensioned to releasably receive and hold a template module. The further processing unit is used for releasably holding at least one template coupling module accommodating a three-dimensional sacrificial template to apply a further processing step to the three-dimensional sacrificial template accommodated by the at least two template modules.

[0028] The further processing unit may include the cell seeding unit comprises a frame provided with at least two template coupling module holders, wherein each template coupling module holder is configured to hold a template coupling module. The cell seeding unit is configured to allow the three-dimensional sacrificial template to be preseeded with cells and / or precoated with proteins or other compounds, such that this preloaded template can then be transferred into a hydrogel and dissolved to form channel structures in the hydrogel that are already coated with cells, and the like, immediately after the channel is formed.

[0029] In a preferred embodiment, the frame of the cell seeding unit may be further provided with a seeding chamber, wherein the seeding chamber is formed between the at least two template coupling module holders. The seeding chamber is configured to receive cells or proteins in a suitable liquid suspension. Preferably, the seeding chamber is dimensioned to maximize and facilitate cell seeding onto the three- dimensional sacrificial template.

[0030] The further processing unit may include a laser processing unit. Such laser processing unit comprises a frame provided with at least two template coupling module holders, wherein each template coupling module holder is configured to hold a template coupling module. The laser processing unit is configured to allow the three- dimensional sacrificial template to be exposed to a laser treatment in order to further amend the design and / or dimensions of the three-dimensional sacrificial template.

[0031] In a second aspect of the present invention, the invention relates to the use of the kit-of-parts of the present invention in a method for creating a three-dimensional structure for cell growth.

[0032] In a third aspect of the present invention, the invention relates to a method for creating a three-dimensional structure for cell growth, the method comprising the steps of: a) providing the kit-of-parts of the present invention; b) creating a three-dimensional sacrificial template, wherein said three- dimensional sacrificial template is held by at least one template coupling module, preferably wherein the three-dimensional sacrificial template is formed between and held by the at least two template coupling modules; c) engaging the fixating device to the one or more template coupling modules in order to form an assembly of one or more fixated template coupling modules; and d) further processing the assembly by transferring the assembly to at least one further processing unit, such as a cell seeding unit.

[0033] By providing the kit-of-parts of the present invention, the kit-of-parts now provides for a method for creating a three-dimensional structure for cell growth enabling the step of transferring the assembly of one or more fixated template coupling modules, such as transferring the assembly of one or more fixated template coupling modules to a further processing, such as a cell seeding unit. Such further processing unit may be able to perform one or multiple further processing steps. In addition or alternatively, the further processing unit consists of two or more further processing modules, wherein each further processing module is able to perform a further processing step, and wherein the assembly of one or more fixated template coupling modules is transferrable between the two or more further processing modules. Examples of further processing steps may include sterilization procedures, surface modification of the template, further manufacture processing of the template, and the like.

[0034] Given the method of the present invention, it is noted that the further processing of the assembly of one or more fixated template coupling modules of step d) may comprise at least one of the steps of: performing at least one further processing step to the three-dimensional sacrificial template; transferring the assembly to a hydrogel embedding unit; releasing the fixating device from the one or more template coupling modules; and / or applying a hydrogel support onto the template to embed the template in the hydrogel.

[0035] The three-dimensional sacrificial template created in step b) is preferably created from a polymer, such as a PsecBuOx-stat-PAOx co-polymer (polyoxazoline) as described in International patent application published under number WO 2022 / 069618 A1.

[0036] It is further noted that the at least one further processing step may comprise on or more of the following further processing steps: seeding the three-dimensional sacrificial template with cells; surface modifying, such as by laser processing, the three-dimensional sacrificial template; and / or applying a sterilization procedure to the three-dimensional sacrificial template.

[0037] In a fourth aspect of the present invention, the invention relates to a three- dimensional structure for cell growth created by the method according to the present invention. Compared to the three-dimensional structures for cell growth available in the art, the three-dimensional structure for cell growth created by the method according to the present invention are improved in complexity.

[0038] Brief description of the figures

[0039] The following figures are illustrative of the present invention, and are not considered to be limiting the present invention, wherein:

[0040] Figure 1 shows an embodiment of the kit-of-parts of the present invention;

[0041] Figure 2 shows the assembly of fixated template coupling modules of the present invention;

[0042] Figure 3 shows the position of the three-dimensional sacrificial template formed between and held by the template coupling modules of the present invention;

[0043] Figure 4 shows the transfer and positioning of the three-dimensional sacrificial template in a further processing unit of the present invention;

[0044] Figure 5 shows the transfer and positioning of the three-dimensional sacrificial template in a hydrogel embedding unit;

[0045] Figure 6 shows a further embodiment of the assembly of fixated template coupling modules of the present invention;

[0046] Figure 7 shows another embodiment of the kit-of-parts of the present invention and the assembly of fixated template coupling modules of the present invention; and Figure 8 shows a ‘loop’-like three-dimensional sacrificial template accommodated by one template coupling module.

[0047] Detailed description of the figures

[0048] Figure 1 shows an embodiment of the kit-of-parts 10 of the present invention comprising template coupling modules 20, 30 and a fixating device 40. The template coupling modules 20, 30 are provided with inner ports 22, 32 and outer ports 24, 34. The inner and outer ports are fluidly connected to each other via internal channels 26, 36. The template coupling modules 20, 30 are further provided with coupling means 28, 38 (such as magnets) for cooperation with corresponding coupling means 42 (such as magnets) provided on the fixating device 40.

[0049] The fixating device 40 as shown in Figure 1 is able to fixate the template coupling modules 20, 30 in space, orientation and distance from each other. The fixating device 40 may further comprise handling means (not shown) for easy handling the fixating device 40.

[0050] Figure 2 shows the assembly of fixated template coupling modules 50 of the present invention. In Figure 2, the template coupling modules 20, 30 are fixed to the fixating device 40 via coupling of the coupling means 28, 38, 42.

[0051] Figure 3 shows the position of the three-dimensional sacrificial template 60 formed between and held by the template coupling modules 20, 30 of the present invention. The three-dimensional sacrificial template 60 is accommodated and held by the template coupling modules 20, 30 via inner ports 22, 32. In Figure 3, the three- dimensional sacrificial template 60 consists of two elongated bars 62. The left part of Figure 3 depicts the three-dimensional sacrificial template 60 accommodated between the template coupling modules 20, 30. The right part of Figure 3 depicts the three- dimensional sacrificial template in the assembly of fixated template coupling modules 50. In the assembly of fixated template coupling modules 50 the three-dimensional sacrificial template 60 can be transferred to further processing units (not shown in Figure 3) without deformation of the three-dimensional sacrificial template 60.

[0052] Figure 4 shows the transfer and positioning of the three-dimensional sacrificial template 60 in a further processing unit 70 of the present invention. The further processing unit 70 may be used for surface modifying the three-dimensional sacrificial template 60. The further processing unit 70 comprises two template coupling module holders 72, wherein each of the template coupling module holders 72 is able to hold a corresponding template coupling module 20, 30. Once the template coupling modules 20, 30 of the assembly of fixated template coupling modules 50 are positioned in the template coupling module holders 72, the fixating device 40 can be removed to allow further processing of the three-dimensional sacrificial template 60.

[0053] Figures 5A and 5B show the transfer and positioning of the three-dimensional sacrificial template 60 in a hydrogel embedding unit 80. The hydrogel embedding unit 80 comprises template coupling module holders 82 configured to receive and hold the template coupling modules 20, 30. In addition, the hydrogel embedding unit 80 comprises a hydrogel chamber 84 formed between the at least two template coupling module holders 82. The hydrogel chamber 84 is configured to receive a hydrogel support (not shown). The hydrogel chamber 84 is further dimensioned such that the hydrogel chamber 84 is able to embed the three-dimensional sacrificial template in the hydrogel support (Figure 5B).

[0054] Figure 6 shows a further embodiment of an assembly 50 the present invention using a fixating device 40 that is extendable. The left part of Figure 6 shows an assembly 50 wherein template coupling modules 20, 30 are engaged by the fixating device 40 and wherein the fixating device 40 is in its extended state. The right part of Figure 6 shows an assembly 50 wherein template coupling modules 20, 30 are engaged by the fixating device 40 and wherein the fixating device 40 is in its retracted state.

[0055] Figure 7 shows a kit-of-parts (left part of Figure 7) and an assembly 50 (right part of Figure 7) wherein the fixating device 40 is an integral part of template coupling module 20. The fixating device 40 is coupled to template coupling module 30 in order to provide the assembly 50 as shown in the right part of Figure 7.

[0056] Figure 8 shows a template coupling module 20, 30 accommodating a ‘loop’-like three-dimensional sacrificial template 60. Different to the ‘bridge’-like three- dimensional sacrificial template 60 as shown in, for example, Figure 4, the ‘loop’-like three-dimensional sacrificial template 60 only needs one template coupling module 20, 30.

Claims

CLAIMS1. Kit-of-parts (10) for creating a three-dimensional structure for cell growth using a three-dimensional sacrificial template (60), wherein the kit-of-parts (10) comprises: a fixating device (40), wherein the fixating device (40) is made of a rigid material, such as a rigid metal material; and a template coupling module (20), wherein the template coupling module is made of a rigid material, such as a rigid polymeric material, and configured to accommodate and hold the three-dimensional sacrificial template (60), and wherein the template coupling module (20) is provided with: an inner port (22) configured to fluidly connect the template coupling module (20) to the three-dimensional sacrificial template (60); and an internal channel (26), wherein the internal channel is fluidly connected on one end to said inner port (22) of the template coupling module (20), wherein the template coupling module (20) is either provided with: an outer port (24), said outer port (24) is fluidly connected to another end of the internal channel (26), and wherein the outer port (24) is configured to fluidly connect the template coupling module (20) to a microfluidics apparatus; or a further inner port (22), said further inner port (22) is fluidly connected to another end of the internal channel (26), and wherein the fixating device (40) is either: fixed to the template coupling module (20) and is, optionally, provided with coupling elements (42) for engaging at least one further template coupling module (20); or provided with coupling elements (42), said coupling elements (42) are configured to engage the template coupling module (20).

2. Kit-of-parts according to claim 1 , wherein the kit-of-parts further comprises at least one further template coupling module (30), wherein the at least one further template coupling module (30) is made of a rigid material, such as a rigid polymeric material, and configured to accommodate and hold the three-dimensional sacrificial template (60), and wherein the at least one further template coupling module (30) is provided with:an inner port (32) configured to fluidly connect the further template coupling module (30) to the three-dimensional sacrificial template (60); and an internal channel (36), wherein the internal channel is fluidly connected on one end to said inner port (32) of the further template coupling module (30), wherein the at least one further template coupling module (30) is either provided with: an outer port (34), said outer port (34) is fluidly connected to another end of the internal channel (36), and wherein the outer port (34) is configured to fluidly connect the further template coupling module (30) to a microfluidics apparatus; or a further inner port (32), said further inner port (32) is fluidly connected to another end of the internal channel (36), and wherein the fixating device (40) is provided with coupling elements (42), said coupling elements (42) are configured to simultaneously engage the template coupling module (20) and the at least one further template coupling module (30) in order to fixate the template coupling modules (20, 30) in space, orientation and distance from each other.

3. Kit-of-parts (10) according to claim 1 or 2, wherein the template coupling modules (20, 30) are selected from the group consisting of: first type template coupling modules (20, 30) provided with a first outer port (24, 34) and a first inner port (22, 32) fluidly connected to each other via a first internal channel (26, 36), and wherein said first type template coupling modules (20, 30) are further provided with a second outer port (24, 34) and a second inner port (22, 32) fluidly connected to each other via a second internal channel (26, 36); second type template coupling modules (20, 30) provided with a first inner port (22, 32) and a second inner port (22, 32) fluidly connected to each other via an internal channel (26, 36); and third type template coupling modules (20, 30) provided with multiple outer ports (24, 34), multiple inner ports (22, 32), and multiple internal channels (26, 36), wherein each outer port (24, 34) is fluidly connected to a corresponding inner port (22, 32) via a corresponding internal channel (26, 36).

4. Kit-of-parts (10) according to claim 2 or 3, wherein the fixating device (40) is provided with spaced apart coupling elements (42) configured to simultaneouslyreleasably engage with corresponding coupling elements (28, 38) provided on the respective template coupling modules (20, 30).

5. Kit-of-parts (10) according to any of the preceding claims, wherein the kit-of- parts (10) further comprises a hydrogel embedding unit (80), said hydrogel embedding unit (80) comprises a frame provided with at least one template coupling module holder (82), wherein the at least one template coupling module holder (82) is dimensioned to releasably receive and hold at least one template coupling module (20, 30).

6. Kit-of-parts (10) according to claim 5, wherein the frame is further provided with a hydrogel chamber (84), said hydrogel chamber (84) is formed adjacent to the at least one template coupling module holder (82), wherein the hydrogel chamber (84) is configured to receive a hydrogel support and wherein the hydrogel chamber (84) is dimensioned to embed the three-dimensional sacrificial template (60) in the hydrogel support.

7. Kit-of-parts (10) according to any of the preceding claims, wherein the kit-of- parts (10) further comprises a further processing unit (70) provided with at least one template coupling module holder (72), wherein the at least one template coupling module holder (72) is dimensioned to releasably receive and hold at least one template coupling module (20, 30).

8. Kit-of-parts (10) according to claim 7, wherein the further processing unit (70) is used for releasably holding the at least one template coupling module (20, 30) accommodating a three-dimensional sacrificial template (60)to apply a further processing step to the three-dimensional sacrificial template (60) accommodated by the at least one template coupling module (20, 30).

9. Use of the kit-of-parts (10) according to any of the preceding claims in a method for creating a three-dimensional structure for cell growth.

10. Method for creating a three-dimensional structure for cell growth, the method comprising the steps of: a) providing the kit-of-parts (10) according to any of claims 1-8; b) creating a three-dimensional sacrificial template (60), wherein said three-dimensional sacrificial template (60) is held by the at least one template coupling module (20, 30);c) engaging the fixating device (40) to the at least one template coupling module (20, 30) in order to form an assembly of a fixated template coupling module (50); and d) further processing the assembly of the fixated template coupling module (50) by transferring the assembly (50) to at least one further processing unit (70), such as a cell seeding unit.

11. Method according to claim 10, wherein the further processing of the assembly (50) of step d) comprises at least one of the steps of: performing at least one further processing step to the three-dimensional sacrificial template (60); transferring the assembly (50) to a hydrogel embedding unit (80); releasing the fixating device (40) from the at least one template coupling module (20, 30); and / or applying a hydrogel support onto the three-dimensional sacrificial template (60) to embed the three-dimensional sacrificial template (60) in the hydrogel.

12. Method according to claim 9 or 10, wherein the further processing unit (70): is able to perform multiple further processing steps; and / or consists of two or more further processing modules, wherein each further processing module is able to perform a further processing step, and wherein the assembly (50) is transferrable between the two or more further processing modules.

13. Method according to any of claims 10-12, wherein the three-dimensional sacrificial template (60) created in step b) is created from a polymer.

14. Method according to any of claims 10-13, wherein the at least one further processing step comprises one or more of the following further processing steps: seeding the three-dimensional sacrificial template (60) with cells; surface modifying, such as by laser processing, the three-dimensional sacrificial template (60); and / or applying a sterilization procedure to the three-dimensional sacrificial template (60).

15. Three-dimensional structure for cell growth created by the method according to any of claims 10-14.