METHOD OF MANUFACTURING MICRODEVICES FOR LAB-ON-CHIP APPLICATIONS

Abstract

A method of manufacturing a microstructure comprises printing a positive mold structure, filling the positive mold structure with a second material to form an elastically deformable negative mold structure, filling the negative mold structure with a third material to form the microstructure, and releasing the microstructure from the negative mold structure. Advantageously, the negative mold structure can be stretched to facilitate the release of the microstructure. For example, the microstructure comprises a chamber with capped micropillars for the generation and/or analysis of muscle tissue.

Claims

1. A method of manufacturing a microstructure for generating or analyzing muscle tissue, the method comprising: using a 3D printing process to form a positive mold structure of a printable, first material, wherein the positive mold structure is a positive of the microstructure to be manufactured, wherein the microstructure comprises a set of two or more capped micropillars disposed in a microchamber for holding a liquid; filling the positive mold structure with a second material to form an elastically deformable negative mold structure, wherein the second material has a lower Young's modulus than the first material; filling the negative mold structure with a third material to form the microstructure; and releasing the microstructure from the negative mold structure, wherein the negative mold structure is stretched, during the releasing, to facilitate release of the microstructure from the negative mold structure.

2. The method according to claim 1, wherein the micropillars have a first diameter that is less than one millimeter, wherein a top of the micropillars is provided with a cap having a second diameter that is larger than the first diameter of the micropillar on which the cap is provided by at least fifty percent.

3. The method according to claim 1, wherein the micropillars comprise a widening section below the cap wherein a diameter of the widening section gradually decreases in a downward direction from the second diameter of the cap to the first diameter of the micropillar below.

4. The method according to claim 1, wherein the microchamber has a capacity for holding between five microliters and one hundred microliters of the liquid.

5. The method according to claim 1, wherein the second material forming the elastically deformable negative mold structure is more flexible than: the first material forming the 3D printed positive mold structure, and the third material forming the microstructure; and wherein the third material forming the microstructure is more flexible than the first material forming the 3D printed positive mold structure.

6. The method according to claim 1, wherein the second material forming the elastically deformable negative mold structure is reversibly stretchable by at least a factor of three without breaking.

7. The method according to claim 1, wherein the second material forming the elastically deformable negative mold structure comprises a silicone elastomer, and wherein the microstructure is formed of a biocompatible elastomer.

8. The method according to claim 1, wherein the 3D printing process comprises stereo-lithography, and wherein the first material comprises a liquid polymeric resin that is solidified by a laser spot or other light pattern.

9. A lab-on-chip comprising microstructures, wherein the microstructures comprise one or more microchambers, wherein a respective microchamber of the one or more microchambers has a capacity between five microliters and one hundred microliters for holding a liquid, wherein the respective microchamber comprises at least two micropillars, wherein a respective micropillar of the at least two micropillars has a first diameter less than a millimeter, wherein a top of the respective micropillar is provided with a cap having a second diameter that is larger than the first diameter of the micropillar below by at least fifty percent, wherein the respective micropillar comprises a widening section below the cap wherein a diameter of the widening section gradually decreases in a downward direction from the second diameter of the cap to the first diameter of the micropillar below, wherein the micropillars, including the cap and widening section there between are integrally formed of an elastomeric material.

10. (canceled)

11. A method for generating muscle tissue, the method comprising: providing a lab-on-chip comprising microstructures, wherein the microstructures comprise one or more microchambers, wherein each microchamber comprises at least two micropillars, wherein a respective micropillar of the at least two micropillars has a first diameter less than a millimeter, wherein a top of the respective micropillar is provided with a cap having a second diameter that is larger than the first diameter of the micropillar below by at least fifty percent, wherein the respective micropillar comprises a widening section below the cap wherein a diameter of the widening section gradually decreases in a downward direction from the second diameter of the cap to the first diameter of the micropillar below, wherein the micropillars, including the cap and widening section there between, are integrally formed of an elastomeric material; providing a respective microchamber of the one or more microchambers with myogenic progenitor cells, a hydrogel, and a culture medium, and culturing the myogenic progenitor cells to generate muscle tissue.

12. The method according to claim 11, wherein the hydrogel comprises: 0.2-4 mg/ml fibrinogen, extracellular matrix protein, and myogenic progenitor proliferation medium comprising antibiotics, fetal bovine serum (FBS) and fibroblast growth factor 2 (FGF2), in a concentration of 80-120 ng/ml; wherein the myogenic progenitor cells are added to the microchamber in a concentration of 10{circumflex over ( )}.sup.6-10{circumflex over ( )}.sup.9 cells/ml, and wherein the culture medium is: a proliferation medium comprising antibiotics, fetal bovine serum (FBS) and fibroblast growth factor 2 (FGF2) in a concentration of 80-120 ng/ml, comprising 6-aminocaproic acid in a concentration of 0.5-5 mg/ml, or aprotinin in a concentration of 60-100 μg/ml, or a differentiation medium comprising antibiotics, ITS-X in a concentration of 0.5-2.5% v/v, knock-out serum replacement in a concentration of 0.5-2.5% v/v, L-glutamine and 6-aminocaproic acid in a concentration of 0.5-5 mg/ml, or aprotinin in a concentration of 60-100 μg/ml, whereby the culture medium is initially the proliferation medium and after 1.5-3 days is replaced by the differentiation medium.

13. The method according to claim 11, wherein the generated muscle tissue is skeletal muscle bundles or muscle stem cells, and wherein the skeletal muscle bundles have a specific twitch force in vitro of more than 7 mN/mm.sup.2 and a specific tetanus force more than 33 mN/mm.sup.2.

14. The method according to claim 13 wherein the generated muscle tissue is used for in vitro screening of a test compound or drug.

15. The method according to claim 13 wherein the generated muscle tissue is used in therapy.

16. The method according to claim 15, wherein the therapy comprises at least one procedure taken from the group consisting of: regenerative therapy, and treatment of a muscle disorder.

17. The method according to claim 16, wherein the muscle disorder is a congenital muscle disease or congenital muscular dystrophy.

18. The method according to claim 17, wherein the muscle disorder is selected from the group consisting of: Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, limb-girdle dystrophy and facioscapulohumeral dystrophy, distal myopathies, myotonic syndromes, ion channel muscle diseases, malignant hyperthermias, metabolic myopathies, hereditary cardiomyopathies, congenital myasthenic syndromes, motor neuron diseases, hereditary ataxias, hereditary motor sensory neuropathies (HMSN), hereditary paraplegias, fibromyalgia, amyotrophic lateral sclerosis (ALS), myasthenia gravis, and Pompe disease.

19. The method according to claim 1, wherein the manufactured microstructure is used to generate muscle tissue by: providing the microchamber with myogenic progenitor cells, a hydrogel, and a culture medium; and culturing the myogenic progenitor cells to generate the muscle tissue.

20. The method according to claim 19, wherein the hydrogel comprises: 0.2-4 mg/ml fibrinogen, extracellular matrix protein, myogenic progenitor proliferation medium comprising antibiotics, fetal bovine serum (FBS) and fibroblast growth factor 2 (FGF2), in a concentration of 80-120 ng/ml, wherein the myogenic progenitor cells are added to the microchamber in a concentration of 10{circumflex over ( )}.sup.6-10{circumflex over ( )}.sup.9 cells/ml, and wherein the culture medium is: a proliferation medium comprising antibiotics, fetal bovine serum (FBS) and fibroblast growth factor 2 (FGF2) in a concentration of 80-120 ng/ml, comprising 6-aminocaproic acid in a concentration of 0.5-5 mg/ml, or aprotinin in a concentration of 60-100 μg/ml, or a differentiation medium, such as DMEM or DMEM high glucose, comprising antibiotics, ITS-X in a concentration of 0.5-2.5% v/v, knock-out serum replacement in a concentration of 0.5-2.5% v/v, L-glutamine and 6-aminocaproic acid in a concentration of 0.5-5 mg/ml, or aprotinin in a concentration of 60-100 μg/ml, whereby the culture medium is initially the proliferation medium and after 1.5-3 days is replaced by the differentiation medium.

21. The method according to claim 7, wherein the biocompatible elastomer comprises a polymeric organosilicon compound.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0043] These and other features, aspects, and advantages of the apparatus, systems and methods of the invention will become better understood from the following description, appended claims, and accompanying drawing wherein:

[0044] FIG. 1 illustrates fabrication methods of 3D culture chambers for tissue engineered skeletal muscles;

[0045] FIG. 2 illustrates generation of 3D culture chambers;

[0046] FIG. 3 illustrates Tissue Engineered Skeletal Muscles (TESMs) of different sizes generated with different 3D culture chambers;

[0047] FIG. 4 illustrates an overview on the different fabrication methods described herein;

[0048] FIG. 5 illustrates technical drawings showing the geometrical features and dimensions of the three different devices;

[0049] FIG. 6 illustrates a program interface for printing a master mold;

[0050] FIG. 7 provides an illustration of the “Direct Peeling method”;

[0051] FIG. 8 provides an illustration of the “ESCARGOT method”;

[0052] FIG. 9 provides an illustration of the “Ecoflex Replica method”;

[0053] FIG. 10 illustrates a 3D design;

[0054] FIG. 11 illustrates a comparison between 3D printed positive and PDMS replicas produced with the Ecoflex Replica method;

[0055] FIG. 12 illustrates bright field microscopy details of original master and PDMS replica made with the Ecoflex Replica method;

[0056] FIG. 13 illustrates Scanning Electron Microscopy pictures;

[0057] FIG. 14 illustrates bright field micrographs;

[0058] FIG. 15 illustrates spontaneous compaction of TESMs in different chambers;

[0059] FIG. 16 illustrates optimization generating muscle in a muscle-on-chip device;

[0060] FIG. 17 illustrates effect of fibrin concentration on contractile force of in vitro cultured 3D skeletal muscle.

[0061] FIG. 18 illustrates functional analysis of tissue engineered skeletal muscle cells (TESMs).

[0062] FIG. 19 illustrates Pax7-positive muscle stem cells formed by TEMs.

DESCRIPTION OF EMBODIMENTS

[0063] Terminology used for describing particular embodiments is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. The term “and/or” includes any and all combinations of one or more of the associated listed items. It will be understood that the terms “comprises” and/or “comprising” specify the presence of stated features but do not preclude the presence or addition of one or more other features. It will be further understood that when a particular step of a method is referred to as subsequent to another step, it can directly follow said other step or one or more intermediate steps may be carried out before carrying out the particular step, unless specified otherwise. Likewise, it will be understood that when a connection between structures or components is described, this connection may be established directly or through intermediate structures or components unless specified otherwise.

[0064] The invention is described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. In the drawings, the absolute and relative sizes of systems, components, layers, and regions may be exaggerated for clarity. Embodiments may be described with reference to schematic and/or cross-section illustrations of possibly idealized embodiments and intermediate structures of the invention. In the description and drawings, like numbers refer to like elements throughout. Relative terms as well as derivatives thereof should be construed to refer to the orientation as then described or as shown in the drawing under discussion. These relative terms are for convenience of description and do not require that the system be constructed or operated in a particular orientation unless stated otherwise.

[0065] FIG. 1 illustrates fabrication methods of 3D culture chambers for tissue engineered skeletal muscles. The present figure provides an overview of three different methods which are dubbed the “Direct Peeling method”, the “ESCARGOT method”, and the “Ecoflex Replica method”. It will be understood that where reference is made herein to the “Ecoflex Replica Method”, this method is preferably applied using Ecoflex or similar elastomeric materials, but of course can also be applied using other stretchable and moldable materials. (a) Direct Peeling method. Technical drawing of a single chamber and 3D CAD model of the negative mold (i), 3D printed acrylonitrile butadiene styrene (“ABS”) negative mold through Fused Deposition Modelling (“FDM”) printing (ii), detail of a polydimethylsiloxane (“PDMS”) positive replica (iii), detail of the pillar (iv). (b) ESCARGOT method. The same process of the Direct Peeling method from design to 3D printed mold is shared (i-ii), detail of PDMS replica (iii) and of a T-shaped pillar with cap, obtained after the dissolution of the ABS mold (iv). (c) Ecoflex Replica method. Technical drawing of a single chamber and 3D CAD model of the positive mold (i), 3D printed resin positive mold through Stereolithography (SLA) printing (top), negative molds in form of replicas made of Ecoflex (bottom), detail of a PDMS positive replica (iii) and a T-shaped pillar provided with conical cap (iv). (a-c) Examples of tissue engineered skeletal muscles within the three different 3D chambers (v). Scale bars: a-c, 5 mm (iii), 1 mm (iv, v). 3D models of each design were generated using CAD software. For the FDM printed devices, 3D models were converted and then uploaded to the 3D printer. For the SLA printed platforms, 3D models were sliced.

[0066] As illustration of the “Direct Peeling method” a first design is used including a 3D culture chamber of 50 μL in a “bone” shape with a total length of 11 mm, provided with two cylindrical pillars of 1 mm in diameter and 3.2 mm in height (FIG. 1a, i; FIG. 5a). This model was designed without overhanging feature of micrometric size, in order to be directly peeled from the 3D printed negative mold during the replica molding without incurring any damage to the final PDMS structure. A circular-based negative structure provided with a set of 23 chambers (FIG. 1a, ii), was printed using an FDM 3D printer (“Ultimaker 2+”) equipped with a 250 μm nozzle (FIG. 6). The circular shape of the base was chosen to fix the printed mold inside a 60 mm plastic Petri dish for convenient PDMS casting during the replica molding step (FIG. 7). For this method, e.g. polylactic acid (PLA) or acrylonitrile butadiene styrene (ABS) can be used as thermoplastic materials in 3D printing. ABS is preferred e.g. because PLA has a lower melting point compared to ABS and it can deform during the curing of the PDMS (>50° C.). This method can have advantages of being relatively fast and user-friendly. However, the method can also be limited by the difficulty of obtaining replicas with more complex geometries, e.g. due to the mechanical damage that the peeling from a rigid mold can cause. For example, the hard, non-deformable mold makes the retention of features such as long and thin or T-shaped pillars arduous. This obstacle has led the inventors to development of the next two methods.

[0067] As illustration of the “ESCARGOT method”, another design is used. Increasing the number of samples for experimental necessities often means reducing the volume of the sample itself (e.g. from a 12 wells plate to a 48 wells plate). However, reducing the diameter of a pillar can cause a contractile tissue to slip away from it due to the tension to which the bundle is subjected during culture. Hence, it is preferred to incorporate a protrusion in the pillar's design arises, leading to the more complex T-shaped pillar suitable for supporting small tissues. To this end, a negative model is used comprising a smaller, 8 mm long chamber of 30 μL with pillars of 750 μm in diameter and 3.2 mm in height in a T-shape (FIG. 1b i; FIG. 5b). To obtain a PDMS replica of such structure while still using FDM printing, we adapted another approach previously used in the fabrication of microfluidic systems in a monolithic PDMS [Saggiomo et al. “Simple 3D Printed Scaffold-Removal Method for the Fabrication of Intricate Microfluidic Devices”, Advanced Science 2, 1500125 (2015)]. This method, called Embedded Scaffold Removing Open Technology (ESCARGOT), replaces the peeling with the dissolution of the mold by exploiting the solubility of ABS in acetone. Briefly, an ABS FDM-printed negative mold (FIG. 1b, ii) was immersed in liquid PDMS, leaving at least one portion of the structure exposed. Once the PDMS was cured, the ABS scaffold was dissolved in an acetone bath overnight (FIG. 8). The resulting PDMS positive structure retains all the features of the original model (FIG. 1b, iii, iv), which would have been lost if peeled from the 3D printed mold. This method was developed to solve problems related to the need of smaller and more complex shapes of pillars, while still making use of the same 3D printing setup. However, for reproducible fabrication of pillars with a diameter of 750 μm, or less, a further improvement is desired.

[0068] As illustration of the “Ecoflex Replica method”, another design is used. The generation of 3D culture chambers with 15 μL volumetric capacity, containing T-shaped pillars with a diameter of 500 μm and a height of 3 mm (FIG. 5c) was made possible employing a new method, which combines stereolithography (SLA) printing and fully deformable elastic molds (FIG. 1c). SLA printers use a laser source to photo-crosslink a liquid polymeric resin, usually composed of acrylates, layer by layer. Thanks to the smaller laser spot, they are not only able to generate smaller features and thinner layers than FDM (down to 200 and 25 μm, respectively), but also to create complex geometries with overhanging structures. In some embodiments, a category of printers called masked SLA (mSLA) is used. For example, in these machines an array of 405 nm UV-LED can be used to replace the laser source.

[0069] Despite the improved resolution of SLA printers, it can still be difficult to directly peel the PDMS replicas from the 3D-printed molds without damaging the micrometric features. In a preferred embodiment, this is addressed by generating an intermediate “carrier” mold made of the highly elastomer, most preferably comprising or essentially consisting of polybutylene adipate terephthalate (PBAT). For example, Ecoflex 00-30 can be used which is a cheap, stretchable and durable silicone, mostly used in soft robotics. According to the datasheet Ecoflex™ 00-30 has Mixed Viscosity (ASTM D-2393) 3,000 cps; Specific Gravity (ASTM D-1475) 1.07 [g/cc], Specific Volume (ASTM D-1475) 26.0 [cu. in./lb.], Pot Life (ASTM D-2471) 45 min.; Cure Time 4 hours; Shore Hardness (ASTM D-2240) 00-30; Tensile Strength (ASTM D-412) 200 psi; 100% Modulus (ASTM D-412) 10 psi; Elongation at Break % (ASTM D-412) 900%; Die B Tear Strength (ASTM D-624) 38 pli; Shrinkage (ASTM D-2566)<0.001 in./in. So for example, the material can be stretched over 900 percent before breakage. In some embodiments, e.g. as shown, Ecoflex or similar material is used as negative mold to form PDMS replicas. In one embodiment, e.g. as shown, we used an SLA 3D printer, e.g. Formlab Form 2 SLA or a Photon Anycubic mSLA, to 3D print a positive structure comprising 37 culture chambers of 7×3×3 mm (L×H×W) equipped with 3 mm long T-shaped pillars of 500 μm diameter, provided with conical caps (FIG. 1c, i, ii; FIG. 2a; FIGS. 12, 13). The negative mold was then generated through Ecoflex Replica molding (FIG. 1c, ii, iii; FIG. 2b) and the final positive PDMS was easily peeled off after stretching the Ecoflex mold (FIG. 1, iii; FIG. 2c, d). In a preferred embodiment, both the 3D printed positive and the Ecoflex negative molds were subjected to a perfluorodecyltrichlorosilane (PFOTS) coating, which facilitates the unmolding process by making their respective surfaces inert (see FIG. 9). Being subjected to reiterated mechanical and thermal stresses, Ecoflex intermediate molds appear to acquire structural wear over time. Nevertheless, we found that a single Ecoflex mold with 37 chambers could be re-used at least 15 times without issues, yielding over 500 3D chambers with intact T-shaped pillars and showing only micro-cracking (FIG. 13).

[0070] FIG. 2 illustrates generation of 3D culture chambers through the Ecoflex Replica method. (a) Positive mold in acrylate resin produced with SLA 3D printing. (b) Single negative replica obtained by pouring liquid Ecoflex on the 3D printed positive from a, after curing of the elastomer. (c) Ecoflex Replica molding: PDMS was poured inside the Ecoflex negative mold. After curing the PDMS, the mold was stretched, allowing easy peeling of the PDMS positive replica. (d) Detail of the PDMS positive replica: the micrometric features of the chambers and the caps on the pillars are retained. Scale bar: d, 5 mm.

[0071] FIG. 3 illustrates Tissue Engineered Skeletal Muscles (TESMs) of different sizes generated with the three different 3D culture chambers. (a-c) 12 well plate containing TESMs within the 50 μL chambers obtained with the Direct Peeling method (a). Tissues were stained for the sarcomeric protein titin after 7 days of differentiation (b); the typical sarcomeric striation pattern of titin is visible at higher magnifications (c). (d-f) Smaller, 30 μL TESMs, obtained from the ESCARGOT fabricated chambers, inside a 24 well plate (d); engineered tissues display a similar morphology as in b with millimetres-long myotubes (e) with evident sarcomeric striation (f). (g-i) A 48 well plate including TESMs generated in the 15 μL chambers fabricated with the Ecoflex Replica method (g). Even in the smallest tissue, myogenic progenitors differentiated in long, multinucleated myotubes with an organized titin pattern (h, i). The dashed curved line at one extremity of the TESM indicates a loop-like structure originated from the tension to which the tissue is subjected (g, h). Scale bars: a, d, g, 1 mm; b, e, h, 200 μm; c, f, i, 50 μm.

[0072] Some aspects of the invention may relate to specific uses of the microscopic structures as described herein. As an example, we discuss generation of Tissue Engineered Skeletal Muscles (TESMs). We determined the generated 3D tissue chambers were able to promote the formation of TESMs and how downscaling affects formation of 3D tissues. TESMs were generated by mixing a suspension of human iPSC-derived myogenic progenitors, with a biocompatible hydrogel constituted of fibrin and Matrigel (see Methods) developed by adapting a previously published protocol [Hinds et al., “The role of extracellular matrix composition in structure and function of bioengineered skeletal muscle”, Biomaterials 32, 3575-3583 (2011)]. Corresponding volumes of 50 μL, 30 μL and 15 μL of cell-laden hydrogel were directly pipetted in the chambers obtained from the Direct Peeling method, ESCARGOT method and Ecoflex Replica method, which were fixed inside the wells of a 12 well plate, 24 well plate and 48 well plate, respectively (FIG. 3). As an example, from an initial cell-hydrogel volume of 500 μL it was possible to cast 10 units of 50 μL TESMs, 16 units of 30 μL TESMs, while from the same volume 33 TESMs could be casted in the devices obtained with the Ecoflex Replica method. In the first two days after casting, the cell-laden hydrogel underwent visible compaction (FIG. 15). After 7 days of differentiation, TESMs were fixed, immunostained and analysed by confocal imaging (see Methods). Engineered tissues in all the 3D culture systems showed a comparable organization, characterized by the presence of long, aligned and multinucleated myotubes that stained positive for the sarcomeric protein titin (FIG. 3b, e, h), whose typical striated pattern was immediately recognizable (FIG. 3c, f, i).

[0073] FIG. 4 illustrates an overview on the different fabrication methods described herein. (a) The Direct Peeling method is the fastest and easiest to the operator, involving a simple design and a reusable negative mold, thus maintaining a noticeable low cost. (b) With the same FDM setup, in the second method it is possible to generate thinner pillars provided with caps still in a single demolding step. In this case the 3D printed mold is dissolved in acetone, therefore not reusable, with a slight increase in production cost. (c) For higher throughput applications, the Ecoflex Replica method allows both an increment of resolution and production, thanks to the use of the SLA printer and the stretchable and reusable negative molds. (a-c). All the three methods can be operated in any standard biomedical laboratory without the need of specific biofabrication expertise. Ease of use indicates the number of steps and equipment required to obtain a certain device; complexity of features refers to the multiplicity of features that can be simultaneously incorporated in the final product; resolution indicates the capacity of the 3D printer to print small details; throughput indicates the number of devices that can be produced per unit of time and their suitability for standard multi-well systems; cost refers to the initial investment of equipment, while the cost of the fabrication materials are comparably low for each method.

[0074] The invention illustrates a fabrication pipeline which can be based, e.g., on off the shelf 3D printers, comprising three methods to generate PDMS-based tissue engineering culture devices. We have highlighted the adaptability of our approach by producing 3D culture chambers to support TESMs with different sizes and levels of complexity (FIG. 4). In principle, the structures realized with the Direct Peeling method can be produced with other fabrication techniques, such as milling or injection molding, thanks to their millimetric dimensions. However, 3D printing offers higher versatility, as the fabrication process can be fully tuned in-house from design to manufacturing to fit different needs and possible new necessities naturally arising during research. An example of this versatility is represented by the second method, developed using the same FDM printing setup but implementing the ESCARGOT strategy. To meet the need of thinner, T-shaped pillars provided with “caps” for a better retention of smaller engineered muscles, the ABS plastic molds were dissolved in acetone leaving the features intact.

[0075] In the Ecoflex Replica method, an SLA 3D printer was employed to further downscale the system with increased reproducibility for higher throughput applications. The devices produced with SLA 3D printing include the simultaneous presence of millimetric and micrometric features, a combination that is hardly possible with techniques such as photolithography, which is confined to the realization of structures within hundreds of micrometers. To replicate such structures in PDMS, a highly elastomer such as Ecoflex can used as a re-usable replica molding substrate for tissue engineering applications. To avoid accidental damage during demolding, other procedures may require supplementary steps solely to fix caps on each pillar or serial replica molding stages with high risk of failure. We inexpensively produced hundreds of faithful replicas with T-shaped pillars without additional fabrication steps thanks to the implementation of the stretchable molds. Considering the advantages, the low cost and the simplicity of use, the Ecoflex Replica strategy can be employed in a wide spectrum of applications: from the creation of supporting devices for other contractile or load bearing tissues such as heart and tendons, to the direct use as soft substrate for specific cell culture needs. The versatility, speed, low cost and ease of use of our methods can promote a larger diffusion of tissue engineering approaches in biomedical laboratories, and their implementation as tools for basic research, disease modeling and drug screening.

[0076] In the following we discuss specific devices materials, and methods which were used to produce some of the present results. Of course, it will be understood that the invention is not limited to these specifics. For example, where specific printing processes are described, also other similar processes can be used. For example, where reference is made to Ecoflex as the preferred material, also other elastomers can be used, e.g. allowing to be stretched before breaking by at least a factor two, three, five, ten, or more. For example, where reference is made to PDMS also other (viscoelastic) elastomers can be used.

[0077] In some embodiments, FDM printing is performed using a 3D printer, equipped with Acrylonitrile Butadiene Styrene (ABS) plastic. For example, the nozzle has 0.250 mm diameter, using a layer thickness of 0.060 mm and a controlled extruding temperature of 240° C. For the present results, SLA printing was performed both by means of a Form2 (Formlabs, USA) using grey resin v4 (Formlabs, USA) and a layer thickness of 0.050 mm and of an Anycubic Photon using Anycubic 405 UV clear resin (Anycubic, China) and a layer thickness of 0.050 mm. In one embodiment, after printing, the SLA products are left in isopropanol. In another or further embodiment, the SLA products are left in ethanol (EtOH). For example, the products are left in isopropanol and ethanol for twenty and ten minutes respectively. In some embodiments, the products are finally rinsed, e.g. with EtOH. In one embodiment, the prints are dried, e.g. with compressed air. In another or further embodiment, the prints are post-cured, e.g. using UV light and/or heat. For example, the prints are left in an 80 W UV chamber for 10 minutes. In some embodiments, the 3D printed molds are coated. For example, the molds are coated with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane (e.g. PFOTS, 97%, Merk). In one embodiment, the coating is applied using chemical vapour deposition (CVD), e.g. in a vacuum desiccator. For example, the 3D printed structure is first air plasma activated (e.g. for 30 seconds), then placed in a desiccator with a vial of PFOTS (e.g. 100 μL) and high vacuum is applied. In some embodiments, the desiccator is left under static vacuum for some time for CVD, e.g. for several hours. After PFOTS deposition, the 3D printed structure can be removed from the desiccator. In one embodiment, the structure is left in an oven, e.g. at 70° C. for 1 hour and finally washed with EtOH and isopropanol.

[0078] In some embodiments, Ecoflex 00-30 (e.g. Smooth-On Inc., PA) intermediate mold is generated by pouring the two pre-mixed liquid pre-polymers (1A:1B) on the 3D printed structure. This can be followed by a degassing step, e.g. inside a vacuum desiccator for 15 minutes. After degassing, platforms can be left curing, e.g. at room temperature (RT) for 8 hours. In one embodiment, the platforms can be peeled off the 3D printed original mold. In some embodiments, the Ecoflex mold is washed, e.g. with EtOH, and rinsed dry, e.g. with compressed air. In a preferred embodiment, before being utilized as negative molds for PDMS replica molding, Ecoflex molds can be subjected to a coating, e.g. PFOTS CVD coating using the same procedure described for the CVD of the 3D printed mold.

[0079] In some embodiments, Polydimethylsiloxane (PDMS, e.g. Dow Corning, MI) final chambers are produced by pouring the uncured elastomer. For example, the uncured elastomer is previously mixed in a 10:1 w/w ratio of pre-polymer and curing agent and degassed for 10 minutes, on the ABS or Ecoflex molds. In some embodiments, poured PDMS is again degassed for 20 minutes in order to remove any possible trapped air bubble and subsequently cured, e.g. at 75° C. in a ventilated oven for at least 3 hours. In other or further embodiments, cured PDMS replicas are peeled off the molds. Preferably, the replicas are rinsed, e.g. in isopropanol, to remove any impurities.

[0080] In some embodiments, ABS dissolution is achieved by immersing the 3D printed negative structure for a few hours, e.g. overnight, in an acetone bath, together with its PDMS positive still attached. Complete removal of ABS can be helped by washing the PDMS replica under running acetone flow for few seconds.

[0081] In some embodiments, in order to be utilized for 3D cell culture, each 3D chamber is cut from the whole PDMS block. In one embodiment, the chamber is fixed inside wells of e.g. 12, 24 or 48 well plates (e.g. CELLSTAR®, Greiner Bio-One, Germany) using PDMS (10:1) as glue and allowing it to solidify for 1 hour at 70° C. Preferably, the PDMS replicas from each fabrication method are sterilized, e.g. by rinsing in 70% ethanol for 15 minutes followed by 3×PBS washing and by UV treatment for 15 minutes, immediately before usage in cell culture.

[0082] In some embodiments, human iPSC-derived myogenic progenitors are generated following a previously published protocol [Saggiomo et al., “Simple 3D Printed Scaffold-Removal Method for the Fabrication of Intricate Microfluidic Devices”, Advanced Science 2, 1500125 (2015).] and cultured accordingly. For example, cells are expanded in monolayer culture in myogenic progenitor proliferation medium, consisting of DMEM high glucose (e.g. Gibco, Waltham Mass.) supplemented with 10% FBS (e.g. Hyclone, US), 1% penicillin-streptomycin-glutamine (P/S/G) (e.g. Gibco, Waltham, Mass.) and 100 ng/mL bFGF2 (e.g. Prepotech, Rocky Hill, N.J.); medium was refreshed, e.g. every 48 hours. Culture dishes of 100 mm (e.g. CELLSTAR®, Greiner Bio-One, Germany) were coated with ECM extract (1:200 diluted, e.g. Sigma-Aldrich, E6909) 40 minutes prior seeding. For passaging and harvesting, cells were detached in the incubator at 37° C. and 5% CO.sub.2 with TrypLE reagent (e.g. Gibco, Waltham, Mass.) diluted 1:1 with PBS (e.g. Gibco, Waltham, Mass.).

[0083] In some embodiments, hydrogel generated as extracellular matrix for the 3D culture is composed of bovine fibrinogen (e.g. Sigma-Aldrich) dissolved in DMEM high glucose (final concentration: 2 mg/mL), Matrigel growth factor reduced (20% v/v, e.g. Corning Life Sciences, Netherlands), thrombin from human plasma (e.g. Sigma-Aldrich) dissolved in 0.1% BSA in PBS (1% v/v, 0.5 U/mL final concentration), myogenic progenitor proliferation medium (69% v/v). Matrigel, previously stored at −20° C., was kept at 4° C. 2 hours before usage; fibrinogen, stored at −80° C., was kept at 4° C. 1 hour before usage. Myogenic progenitors used for the generation of the TESMs were harvested before passage 10. After detachment from culture dishes, cells were suspended in myogenic progenitor proliferation medium at a concentration of 16.Math.10.sup.6 cells/mL and the suspension was then mixed with fibrinogen and Matrigel. Lastly, thrombin was added to the mix immediately before pipetting the cell-hydrogel mix into the 3D chambers, which were coated with Pluronic F-127 (e.g. Sigma-Aldrich) for 1 hour at RT to prevent unwanted adhesion of the hydrogel. A volume of 50 μL, 30 μL and 15 μL was used for each TESM in the chambers obtained with the Direct Peeling, ESCARGOT and Ecoflex Replica methods, respectively. All the hydrogel components, as well as tubes and micropipette tips were kept on ice prior and during the duration of the experiments. The final solution was then left to polymerize for 30 minutes in the incubator at 37° C. and 5% CO.sub.2, before adding the myogenic progenitors proliferation medium supplemented with 6-aminocaproic acid (1.5 mg/mL, e.g. Sigma-Aldrich). After 48 hours, proliferation medium was switched to TESM differentiation medium, consisting of DMEM high glucose supplemented with 1% knock-out serum replacement, 1% ITS-X (all Gibco), 1% penicillin-G (e.g. Sigma-Aldrich), 1% Glutamax (e.g. Sigma-Aldrich), 6-aminocaproic acid (1.5 mg/mL, e.g. Sigma-Aldrich). Half volume of the TESM differentiation medium was replaced every 48 hours. TESMs were kept on agitation at 55 rpm (e.g. Celltron orbital shaker, Infors HT, Switzerland) at 37° C. and 5% CO.sub.2.

[0084] In some embodiments, Immunofluorescence Stainings are applied as follows. Samples were fixed in 4% paraformaldehyde (PFA, e.g. Sigma-Aldrich) for 1 hour at RT and then washed with PBS for three times. For whole-mount immunostaining, fixed samples were first subjected to a permeabilization/blocking step in a solution containing 0.5% Triton-X in PBS, 3% BSA in PBS, 0.1% Tween 20 in PBS on agitation, for 1 hour at RT. After washing with PBS, samples were incubated with primary antibody for titin, 9D 10-s (DSHB, IA), in 0.1% Triton-X in PBS, 0.1% BSA (e.g. Sigma-Aldrich) in PBS, 0.1% Tween 20 in PBS at 4° C., for 1 hour. Before incubation with the secondary antibody Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, Mass.), samples were washed in 0.1% Tween in PBS for 2 minutes and subsequently in PBS for 2 minutes. Secondary antibodies were diluted in the same solution used for the primary antibodies and samples were incubated for 1 hour at RT. Lastly, nuclear staining was performed through incubation with Hoechst 33342 (1:15000, e.g. Sigma-Aldrich) at RT for 15 minutes followed by washing in PBS. Samples were kept in PBS at 4° C. before imaging. In some embodiments, stained samples were imaged using a Leica TCS SP5 confocal microscope (Leica, Germany) equipped with LAS software (Leica, Germany), using 10× and 20× magnifications.

[0085] With reference to the “Direct Peeling” and “ESCARGOT” methods, the fabrication steps can be largely identical. However, different demolding steps can be used in order to obtain PDMS replicas from the different masters.

[0086] Fused Deposition Modeling (FDM) 3D printers are the most commonly used 3D printers. In short, a heated nozzle deposits a thermoplastic material layer by layer on a build plate. FDM printers are relatively simple to use and to maintain. Typically, FDM printers have a standard 400 μm nozzle. However, the most suitable option for structures that need more precision is the 250 μm nozzle. Although it is possible to print ABS with many printers, it is preferred to use a printer with an enclosure as the ABS can warp due to different temperatures experienced by the 3D printed material on the build plate.

[0087] Various programs can be used to design the 3D masters, depending on the difficulty of the program, operating system and personal preferences. Once the design is completed, the 3D design is typically saved as .STL file.

[0088] For 3D printing, the STL file is typically “sliced” in layers. This can be done with slicer programs. In the slicer program, many variables can be changed. For example, the nozzle size can be set to 250 μm, the layer height to 60 μm, 100% infill and print with the use of brim as build plate adhesion but without support (FIG. 6). Other variables such as extrusion temperature, bed temperature, speed and fan speed can also be changed according to the material to be printed. Once the slicer has finished, the file can be saved, e.g., as .gcode, and loaded on the 3D printer. Once the printer has finished, the 3D print can be easily detached from the build plate once it has cooled down to room temperature.

[0089] FIG. 5 illustrates technical drawings showing the geometrical features and dimensions of the three different devices. (a) 50 μL chamber produced with the Direct Peeling method. (b) 30 μL chamber obtained with the ESCARGOT method. (c) 15 μL chamber fabricated through the Ecoflex Replica method. The blue color highlights the pillar in each design.

[0090] FIG. 6 illustrates a program interface (“Cura”) and variables to be changed for printing the master.

[0091] In the Direct Peeling method, the 3D printed (negative) master can be fixed, e.g., to the bottom of a 6 cm plastic petri dish by means of common double sided tape or a thin layer of PDMS (10:1). This will facilitate the subsequent removing of the structure. PDMS is mixed in a 10:1 PDMS/curing agent ratio for few minutes and degassed under vacuum for 10 minutes. The PDMS mixture can be then poured on top of the 3D printed negative master and degassed again for 15 minutes to remove any air bubble trapped inside. The PDMS is then cured in an oven at 75° C. for 3 hours. Once the PDMS is cured, it can be peeled off from the negative master.

[0092] In the ESCARGOT method, instead of peeling off the PDMS from the negative mold, the structure comprised of the negative mold and the cured PDMS on top can be placed in an acetone bath overnight for dissolving the ABS. The acetone can be refreshed from time to time for speeding up the dissolution. Stirring and sonication can also speed up the dissolution process. After the mold dissolution, the remaining PDMS replica is quickly washed with running acetone and isopropanol, then air dried.

[0093] FIG. 7 provides an illustration of the Direct Peeling method. (a) The 3D CAD model is printed through FDM into an ABS negative mold. (b) The 3D printed mold is fixed inside a 6 cm plastic Petri dish with double sided tape; this will facilitate the subsequent pouring of uncured PDMS. (c) After curing of PDMS, the mold with PDMS still attached is removed from the dish. (d) The PDMS replica is carefully peeled off the negative mold; scale bar, 5 mm.

[0094] FIG. 8 provides an illustration of the ESCARGOT method. (a, b) The 3D CAD model is 3D printed into a negative mold and fixed inside a 6 cm plastic Petri dish, uncured PDMS is next poured in the dish and left to cure in the oven. (c, d) After curing of the elastomer and removal of the mold from the dish, the couple negative mold-PDMS replica is immersed in an acetone bath. (e) The negative mold will dissolve overnight. (f) The PDMS replica is finally washed with acetone; scale bar, 5 mm.

[0095] FIG. 9 provides an illustration of the Ecoflex Replica method. (a) The 3D CAD model is 3D printed through (m)SLA in a positive resin structure with a 1 cm thick base. (b) After a 30 seconds plasma activation step, the printed positive is coated with PFOTS overnight. (c) After treatment, a negative Ecoflex replica is obtained from the resin positive. The ecoflex replica is characterized by a 1 cm high wall which will facilitate pouring of PDMS inside the mold itself. (d) A second activation-coating step is performed on the Ecoflex negative. (e) Uncured PDMS is poured inside the mold, after curing it is easily peeled off by stretching the Ecoflex mold. (f) The final PDMS replica is obtained; scale bar, 5 mm.

[0096] SLA (stereolithography) printers, in contrast to FMD printers, use a liquid resin, typically made of (meth)acrylates mixtures, which is photo-crosslinked, e.g. using specific wavelengths. Some printers use a 405 nm laser to photopolymerize the liquid resin layer by layer. Another class of SLA printers are called LCD (liquid crystal display) or mSLA (mask stereolithography) 3D printers. These machines can use a 405 nm LED arrays as light source and an LCD to create a mask for the crosslinking of the liquid resin layer by layer.

[0097] In some embodiments, the positive master is designed as follows. In the Ecoflex replica method the printed structure preferably serves as a template to create the Ecoflex negative mold. For this reason, the master is printed as a positive, such as the final PDMS replica which will be its copy. The software used to create the positive 3D model is however independent of the type of structure or the 3D printer used, and various 3D modelling programs can be used.

[0098] In some embodiments, printing and slicing of the 3D master is performed as follows. To print the model, the 3D model can be sliced. Preferably, the master is printed in a horizontal orientation, lying flat directly on the build plate surface without the use of support structures. Printing parameters may vary between different resin materials. Preferably, a layer height of around 50 μm is used. Once printed, the object can be cleaned, e.g. by leaving in an isopropanol bath for 20 minutes and then in an ethanol bath for an additional 10 minutes, to remove the uncured resin. The object can then be washed and dried e.g. using a flow of air. The 3D printed object is then post-cured in a UV chamber, e.g. for 10 minutes.

[0099] FIG. 10 illustrates a 3D design. The design is printed flat directly on the build plate without the use of supporting material. The parameters in the box are the ones used for printing on the Anycubic Photon using the Anycubic clear resin.

[0100] In some embodiments, coating of the master is performed as follows. Uncured (meth)acrylates may react with Ecoflex and PDMS, forming covalent bonds between the two surfaces. To eliminate this problem, a chemical coating can be used in some embodiments. For example, the 3D printed object is activated using an air plasma oven, e.g. for 30 seconds. A PE-25 benchtop air plasma cleaner (e.g. Inseto Plasma Etch, Inc) was used at its maximum RF plasma power of 100 W with an air flow of ˜10 cc/min, which allowed for a vacuum pressure of 200-250 mTorr within the chamber during plasma treatment. After the plasma treatment, the 3D printed object is placed in a 22 cm desiccator together with an open vial containing 100 μL of (1H, 1H, 2H, 2H-perfluorooctyl)silane (PFOTS). The desiccator is evacuated (2×10-3 mbar) and left under static vacuum overnight. After that, the 3D objects are left in an oven at 70° C. for 1 hour and then washed with ethanol and isopropanol and dried with a flow of air.

[0101] In some embodiments, Ecoflex Replica and demolding is performed as follows. The 3D positive mold is glued on a petri dish using hot glue. Ecoflex 00-30 is mixed 1:1 part A/part B and stirred for 5 minutes. The mix is then poured on top of the 3D positive mold and left under vacuum for 15 minutes. The Ecoflex was then left curing at room temperature for 8 hours. Once set, the Ecoflex negative mold was gently peeled from the 3D printed positive mold, washed with ethanol and dried using compressed air. Ecoflex, bearing the same silicone components of the PDMS can react with it forming covalent bonds. For this reason, the same coating procedure used in (2.4) was used to coat the Ecoflex negative mold. To generate the PDMS replica from the Ecoflex negative elastic mold, uncured PDMS is poured inside the mold and, after curing at 75° C. for 3 hours, it is peeled off by stretching the Ecoflex. This procedure will allow the retention of the small and thin features of the PDMS replica. A visual comparison of the original 3D printed positive master and the PDMS positive replicas can be appreciated in FIGS. 11-14.

[0102] FIG. 11 illustrates a comparison between 3D printed positive and PDMS replicas produced with the Ecoflex Replica method. (a,b) Original positive resin structure 3D printed with a SLA printer. (c, d) Final PDMS replica: the original general structure is retained after the demolding process.

[0103] FIG. 12 illustrates bright field microscopy details of original master and PDMS replica made with the Ecoflex Replica method. (a) Original positive 3D printed single chamber. (b) PDMS replica of the same chamber. The micro-ridges of the original printed structure are retained in the PDMS replica, meaning that the intermediate Ecoflex negative mold can be used without losing resolution. (c) Detail of a pillar's cap. (d) PDMS replica of a pillar's cap. Even if a small defect is present, the general structure remains faithful to the original. (e) Side view of a 3D printed pillar (left) and its PDMS replica (right). Scale bars, a, b, e, 1 mm; c, d, 500 μm.

[0104] FIG. 13 illustrates Scanning Electron Microscopy pictures of an mSLA 3D printed pillar (a) and a PDMS replica (b). The SEM micrographs were acquired on a FEI Magellan 400 SEM. Scale bars, a, b, 500 μm.

[0105] FIG. 14 illustrates bright field micrographs. Structural integrity of Ecoflex Replica products after several cycles of replica molding. (a) Detail of a 3D printed original resin structure fabricated by means of an SLA printer. (b) After 3 cycles of replica molding using the same Ecoflex negative mold, the PDMS replica starts to display signs of micro-defects. (c-e) Re-using the same Ecoflex mold after 14 cycles of heating and stretching doesn't impair the structural integrity of the features in the PDMS replica, while the micro-defects show only a slight expansion. Scale bars, a-e, 200 μm.

[0106] We will now discuss advantageous uses of the microstructures obtainable by the methods of manufacturing as described herein. One particular use pertains to the generation of the TESMs in 3D chambers with microstructures made of PDMS.

[0107] FIG. 15 illustrates spontaneous compaction of the TESMs in the three different 3D chambers after 2 days in culture. (a) A TESM within its 50 μL 3D culture chamber right after the casting of the cell-laden hydrogel. (b) The same TESM after 2 days in culture under proliferation conditions: the fibrin-based hydrogel undergoes compaction around the pillars. (c, d) A 30 μL TESM inside a 3D chamber obtained from the ESCARGOT method before (c) and after (d) 2 days in culture. (e, f) 3D chambers fabricated with the Ecoflex Replica method provide equivalent results in terms of compaction of the cell-laden hydrogel. Scale bars, a-f, 1 mm.

[0108] FIG. 16 illustrates optimization generating muscle in a muscle-on-chip device. Panel A: example of method optimization for muscle generated in a 50 ul-sized muscle-on-chip device. Increasing the cell density and decreasing the fibrin concentration resulted in enhanced density of muscle fibers. Antibody stainings: Titin is a sarcomeric marker indicative of maturation; Dystrophin is a cell membrane marker that should be around the titin-positive fibers; Hoechst stains nuclei (please note that muscle fibers should be multinucleated). Panel B: as above, but now for muscle generated in a 15-20 ul-sized muscle-on-chip device. The figure shows enhanced quality of muscle tissue by reducing the size of the device and by increasing the cell density.

[0109] FIG. 17 illustrates effect of fibrin concentration on contractile force of in vitro cultured 3D skeletal muscle. On the X-axis, different concentrations of fibrin are shown (in mg/ml), on the Y axis the contractile force as % of the force at 4 mg/ml fibrin. The figure shows that the force at the commonly used fibrin concentration of 4 mg/ml results in the formation of muscle bundles with a force that is modest compared to muscle that was cultured at lower fibrin concentrations. The optimal fibrin concentration was 1 mg/ml, which resulted in a 5-fold increase in contractile force. The experiment was done in 2 parts, A and B.

[0110] FIG. 18 illustrates the specific twitch force and specific tetanus force obtained with the “Direct Peeling Method” and the Ecoflex Replica Method”. Control TESMs were either cultured in direct peeling (50 μl) or Ecoflex replica (15 μl) mold. After 7 days of differentiation TESMs were stimulated with a frequency of 1 Hz (twitch) or 20 Hz (tetanus). Displacement of the pillar was recorded and specific force was calculated.

[0111] FIG. 19 illustrates an example of TESM after 7 days of differentiation. Immunofluorescent stainings were performed using antibodies against the sarcomeric protein titin (green—here recolored as dark grey), and the muscle stem cell marker Pax7 (red—here recolored as lighter gray, some indicated by dashed circles). Arrows indicate examples of stained Pax7 and nucleic. Hoechst (blue—here recolored as white, some indicated by solid line circles) staining to visualize nuclei. Note that the Pax7-positive cells are located adjacent to the myofibers in agreement with their natural location in skeletal muscle tissue. This is the first time that formation of Pax7-positive muscle stem cells has been demonstrated within TESMs that were derived from human iPSCs obtained in a transgene-free manner. Upon culturing in 2D on petridishes of the myogenic progenitors used to generate the TESMs, Pax7 expression is rapidly lost, going from 100% after FACS sorting to 3% after culture in 2D), as published by us previously (see FIG. 5B in PMID: 29731431). The surprising result is the fact that upon culturing the same cells in a 3D environment in TESMs, they re-form large numbers of Pax7+ muscle stem cells. Therefore, the TESMs generated under the conditions employed in this application provide a novel tool for the in vitro generation of large numbers of transgene-free, human Pax7+ muscle stem cells that are anticipated to be useful for regenerative purposes.

[0112] As a specific example, 3D human Tissue Engineered Skeletal Muscles (TESMs) can be generated as follows.

[0113] Hydrogel generated as ECM for the 3D culture, composed of bovine fibrinogen (Sigma-Aldrich), is dissolved in DMEM high glucose (2 mg/mL final concentration), Matrigel growth factor reduced (20% v/v, Corning Life Sciences, Amsterdam, NL), thrombin from human plasma (Sigma-Aldrich) dissolved in 0.1% BSA in PBS (1% v/v, 0.5 U/mL final concentration), myogenic progenitor proliferation medium (69% v/v). Matrigel, previously stored at −20° C., was kept at 4° C. 2 hours before usage; fibrinogen, stored at −80° C., was kept at 4° C. 1 hour before usage. Myogenic Progenitors used for the generation of the TESMs were harvested before passage 10. After detachment from culture dishes, cells were suspended in myogenic progenitor proliferation medium at a concentration of 16.Math.10.sup.6 cells/mL and the suspension then mixed with fibrinogen and Matrigel. Lastly, thrombin was added to the mix immediately before pipetting the cell-hydrogel mix into 3D chambers. The 3D chambers were manufactured according to the methods of manufacturing a microdevice as described herein.

[0114] The chambers were previously coated with Pluronic F-127 (Sigma-Aldrich) for 1 hour at room temperature, to prevent unwanted adhesion of the hydrogel. Before the casting of the hydrogel inside the 3D chambers, the Pluronic F-127 is preferably removed after its incubation of a minimum of 1 hour at RT. This can be an important step: as minuscule traces of Pluronic still present as a result of an improper aspiration can affect the generation of a TESM. Tiny bubbles can form in the interface between the traces of Pluronic and the cell-laden hydrogel after casting. This can negatively influence the subsequent solidification and compaction of the hydrogel into the bundle-like shape of a TESM, leading to poor structural integrity or incomplete formation of the engineered tissue.

[0115] A volume of 15 μL was pipetted for each TESM in the chambers. All the hydrogel components, as well as tubes and micropipette's tips were kept on ice prior and during the duration of the experiments. The final solution was then left to polymerize for 30 minutes in the incubator at 37° C. and 5% CO2, before adding the TESM proliferation medium. This consisted in the myogenic progenitors proliferation medium supplemented with 6-aminocaproic acid (1.5 mg/mL, Sigma-Aldrich). After 48 hours, proliferation medium was switched to TESM differentiation medium, consisting of DMEM high glucose supplemented with 1% knock-out serum replacement, 1% ITS-X (all Gibco), 1% penicillin-G (Sigma-Aldrich), 1% Glutamax (Sigma-Aldrich), 6-aminocaproic acid (1.5 mg/mL, Sigma-Aldrich). Half volume of TESM differentiation medium was replaced every 48 hours. TESMs were kept on agitation at 55 rpm (Celltron orbital shaker, Infors HT, Switzerland) at 37° C. and 5% CO.sub.2 for all the duration of the culture time.

[0116] Twitch and tetanic contractions were induced by electrical stimulation with either a frequency of 1 Hz (twitch) or 20 Hz (tetanus) at 2.5 V/cm with 10% duty cycle using a function generator. During stimulation the pillar displacement was captured with a fast camera recording at 60 frames per second. The height of the TESM on the pillar was measured for each replica and included in the calculation. Contractile force was calculated using Young's Elastic Modulus (E) of PDMS, the geometrical properties and the position of the TESM on the pillar (R, a and L) and displacement (δ) of the PDMS pillar using the following Force in

[00002]$N=\frac{6E\pi R^4}{4a^2\left(3L-a\right)}\delta$

formula [Legant et al. “Microfabricated tissue gauges to measure and manipulate forces from 3D microtissues”, PNAS (25), 10097-10102 (2009)]. For the calculation of specific force paraffin based cross sections were generated from 2-3 TESMs and the specific force was calculated by dividing the force by cross sectional area. The results are shown in FIG. 17 and table 1, which also shows a comparison with the specific twitch force and specific tetanus force that have been measured in skeletal muscle bundles generated so far.

TABLE-US-00001 TABLE 1 Comparison of specific force in human skeletal muscle bundles Specific Time of force Specific force analysis after Study twitch tetanus Cell source differentiation Rao et al, 2018, DOI: ~0.8   ~3 mN/mm2 hiPSC; Pax7 2 weeks 10.1038/541467-017- mN/mm2   overexpression 02636-4   Madden et al, 2015, ~6  ~12 mN/mm2 Human primary 2 weeks DOI: mN/mm2   Myoblasts 10.7554/eLife.04885   Xu et al, 2019, DOI: N.R.  ~33 mN/mm2 hiPSC; Pax7 4 weeks 10.1002/adbi.   overexpression 201900005   Afshar et al, 2020, N.R.   ~5 mN/mm2 Human primary 2 weeks DOI:   Myoblasts 10.1038/s41598-020-   62837-8   Selvaraj et al, 2020, ~5   ~5 mN/mm2* hiPSC; 5 days DOI: mN/mm2* transgene-free 10.7554/eLife.47970 Mills et al, 2019, ~2 ~4.9 mN/mm2 Human primary 7 days DOI: mN/mm2 Myoblasts 10.1016/j.biomaterials. 2018.11.030 Direct Peeling 50 ul ~10  ~25 mN/mm2 hiPSC; 7 days (this study) mN/mm2   transgene-free Ecoflex replica 15 ul ~12  ~35 mN/mm2 hiPSC; 7 days (this study) mN/mm2 transgene-free N.R. Not Reported, *no specific force reported, calculation based on area of longitudinal staining.

[0117] For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments. While embodiments were shown for the manufacturing of specific microdevices using specific material and methods, also alternative ways may be envisaged by those skilled in the art having the benefit of the invention for achieving a similar function and result. For example, instead of 3D printing the first (positive) mold also other additive and/or subtractive manufacturing methods can be used. For example Jet fusion or selective laser sintering can be used. In principle, the positive first mold can be created by any process, such as injection molding, although this may be more difficult and less versatile. Alternatively, or in addition to stretching the elastically deformable negative mold structure it can also be envisaged to dissolve the negative mold structure. For example, the second material forming the negative mold structure can comprises or essentially consists of hydrogels, for example agarose, gelatin, or alginate. This may be less efficient because the negative mold can only be used once, but a new mold can be created from the positive, e.g. printed, first mold.

[0118] The various elements of the embodiments as discussed and shown offer certain advantages, such as the Generation of 3D human Tissue Engineered Skeletal Muscles. Of course, it is to be appreciated that any one of the above embodiments or processes may be combined with one or more other embodiments or processes to provide even further improvements in finding and matching designs and advantages. It is appreciated that this disclosure offers particular advantages to basic biological studies, 3D muscle-on-a-chip applications, drug screening, phase I-IV clinical trials, personalized medicine, and in general can be applied for any application wherein it is desired to easily manufacture and use microdevices or chips with reproducible sub-micron features. In interpreting the appended claims, it should be understood that the word “comprising” does not exclude the presence of other elements or acts than those listed in a given claim; the word “a” or “an” preceding an element does not exclude the presence of a plurality of such elements; any reference signs in the claims do not limit their scope; several “means” may be represented by the same or different item(s) or implemented structure or function.