Insert for culturing tissue and method for culturing tissue using the insert

Abstract

The invention provides an insert (100) for culturing tissue, comprising: a base (110) comprising a groove (140) configured to provide guidance for the addition of cells to be cultured; a first strut (120) extending uprightly from the base and configured to support the first anchor; and a second strut (120) extending uprightly from the base (110) and configured to support a second anchor. The insert (100) is configured to enable cells to be cultured between the first and second struts (120) with an anchor for support to form tissue.

Description

Technical Field

[0001] The present invention relates to a cell culture insert, and particularly to a cell culture insert for use in a 3D cell culture method. Background Art

[0002] Culturing cells and tissues in the laboratory has many potential applications in drug development, medicine, and other fields. In drug development research, one of the most common mechanisms is the use of animal models. However, this has many drawbacks. Animals do not predict well the human response to drugs. Due to genetic and metabolic differences between laboratory animals such as mice and humans, the success rate is very low, and less than 5% of the therapeutic methods tested in animals can successfully enter human trials.

[0003] An alternative to animal models is the use of engineered human tissues. However, current 3D cell culture methods involve very expensive and complex bioreactors that many laboratories cannot use due to cost or the ability to operate and maintain them. Additionally, current 3D cell culture methods have a low throughput because they typically can only generate 2 or 3 tissue-engineered muscles at a time. This does not meet the needs of the pharmaceutical industry because they require high-throughput platforms to screen a large number of candidate drugs. Current high-throughput muscle culture methods involve culturing cells in a two-dimensional (2D) orientation (monolayer). Culturing muscle cells in 2D means that the structure of the tissue is not the same as that found in vivo. Therefore, using 2D culture means that human disease models are not accurate enough, or when used to treat a subject, the engineered tissue does not accurately match the tissue into which it is transplanted.

[0004] Outside of research, transplanted engineered tissues can be used to treat conditions such as volumetric muscle loss, esophageal defects, abdominal defects, or sports injuries. However, current 2D tissue culture methods are not well-suited for this. For example, for muscle tissue, 2D culture does not accurately replicate the structure of the subject's muscle or exhibit markers of functional maturity such as contractility. Additionally, current engineered muscle tissue culture techniques have limitations in the size of the tissue that can grow, with a maximum length of approximately 2 centimeters. Engineered muscle tissue culture techniques are also expensive and require high-end specialized equipment, which poses a limitation for many institutions.

[0005] Other uses of cell culture include the production of laboratory-based meat products. This is currently an area experiencing significant growth as laboratory-based meat products offer several advantages over using animals. For example, laboratory-based meat products do not involve the slaughter of livestock and can therefore be considered more ethical; additionally, the equipment used to produce meat products is more space-efficient, which makes laboratory-based meat products potentially applicable in space-constrained environments. Currently, laboratory-based meat production methods are very expensive, require a large amount of space, and face other challenges in becoming a viable food source.

[0006] The present invention aims to solve or ameliorate one or more of these problems. Summary of the Invention

[0007] According to a first aspect of the present invention, there is provided an insert for culturing tissue, comprising:

[0008] a base comprising grooves configured to provide guidance for adding cells to be cultured;

[0009] a first pillar extending upright from the base and configured to support a first anchor; and

[0010] a second pillar extending upright from the base and configured to support a second anchor,

[0011] wherein the insert is configured such that cells can be cultured between the first and second pillars using the anchors for support to form tissue.

[0012] The base comprises grooves configured to provide guidance for adding cells to be cultured. Cells can be added to the grooves themselves or inoculated on top of a hydrogel pre-injected into the grooves. The grooves can extend between the first and second pillars and pass through the center of the base. As previously mentioned, the grooves provide guidance for the user when adding cells to the insert. Advantageously, this enables the user to inoculate cells in the insert such that the cells grow between the two anchors and form tissue during the culturing process. The insert for initial inoculation and growth of cell culture can be referred to as a primary insert.

[0013] Advantageously, the tissue cultured using this insert has a structure highly similar to the equivalent tissue in a living organism. The tissue can be used as a research model or implanted into a subject to treat diseases or injuries. Further advantageously, the insert can be scalable and produced reliably, thereby reducing the inhibitive cost of research.

[0014] The position of the first and / or second anchor can be configured to be adjustable to stretch tissue. Advantageously, this can make tissue growth using the insert more closely mimic tissue growth in vivo.

[0015] During insert manufacture, the position of the struts on the base can vary, and the struts can be moved closer to or further from each other as needed. Additionally or alternatively, at least one strut can be adjustable. The adjustable strut can be moved closer to or further from another strut. Alternatively, the angle of the adjustable strut can be changed.

[0016] The groove can also include recesses. Advantageously, the recesses enable the user to co-culture additional cell types.

[0017] The position of the groove can be set to extend between the intersection points of the first and second struts with the base. The size of the groove can be designed such that it can accommodate cells suspended in the hydrogel. If there are more than two struts, the groove can extend between the intersection points of each strut with the base. In one embodiment, there can be up to 12 struts attached to the base. In this case, the groove can extend from the center out to each strut.

[0018] In one embodiment, the cells can be embedded in the hydrogel.

[0019] In one embodiment, the tissue is muscle tissue. In one embodiment, the muscle can be human muscle tissue. In one embodiment, the tissue can be livestock tissue, where the livestock can be a cow, pig, sheep, or chicken.

[0020] In one embodiment, the struts are made of a biocompatible material, optionally, the biocompatible material is a biocompatible resin. The advantage of the biocompatible material is that it does not affect cell culture except providing support for tissue growth. Examples of materials that can be used for the base and struts include but are not limited to organic materials such as resins, polycaprolactone, silicone, and inorganic materials such as silicon, silica, glass, metals, calcium phosphate, and mixtures of organic and inorganic materials.

[0021] In one embodiment, the struts include a biodegradable material. Biodegradable materials are advantageous because they decompose over time into non-toxic components that do not interfere with cell growth.

[0022] In one embodiment, the anchor includes a biocompatible nonwoven fabric. Similarly, the biocompatible nonwoven fabric is advantageous because it supports tissue during culture and allows the tissue to be stretched in a manner that mimics tissue in vivo.

[0023] In one embodiment, the anchor includes a biodegradable nonwoven fabric. A biodegradable anchor may be advantageous as it can be used to secure a tissue graft within a subject without interfering with normal physiological functions and then breaks down into non-toxic components. Examples of materials that can be used for the anchor include, but are not limited to, nonwoven or woven fabrics. These fabrics can be synthetic or made from natural materials such as cellulose and silk. These fabrics can have coatings or can be used alone without coatings. These fabrics can be stretchable or non-stretchable.

[0024] The anchor can be perforated with one or more holes. These holes can provide a way to attach the struts when the struts are passed through the holes to secure the anchor in place. Different holes can be used as attachment points at different stages of cell culture.

[0025] In one embodiment, the anchor is detachable from the strut. In one embodiment, the anchor can be detachable during cell culture to facilitate the transfer of the cultured tissue to a second set of struts. The second set of struts can be on the same insert or on a different insert.

[0026] Each anchor can include one or more holes. These holes can be used to attach the anchor to its respective strut.

[0027] The position of the anchor can be adjusted by using one or more holes in the anchor to stretch the engineered tissue. Alternatively, the engineered tissue can be stretched by transferring it to an insert with struts that are further apart or by sliding one of the struts to a more distant position. The position of the anchor can be adjusted manually, automatically, and semi-automatically.

[0028] In one embodiment, an insert can be stacked with other units of the insert. The base of the insert can include one or more recesses. These one or more recesses can be located on the bottom surface of the base. Each recess can be configured to receive a strut from another insert. The recesses can be arranged in a plane perpendicular to the grooves such that multiple inserts are stacked in a manner where they rotate 90 degrees between each layer (i.e., in the form of ABABAB…). Alternatively, the recesses can be arranged on the bottom surface of the base such that they correspond to the upright struts of the same insert. This allows the inserts to be stacked without rotation between each layer (i.e., in the form of AAAAA).

[0029] Other mechanisms for storing and / or stacking inserts can also be provided. For example, each of the multiple inserts can be located as a detachable "shelf" within a larger structure configured to accommodate multiple inserts. Advantageously, this allows a single insert to be removed without disturbing other adjacent inserts.

[0030] The insert may also include one or more spacers. Each of these one or more spacers may be disposed at the bottom of the strut. In embodiments providing two pairs of struts, one of the pairs of struts may be provided with a spacer. Advantageously, the provision of a spacer at the base of the strut can be used to elevate the supported anchor, such that the growing tissue grows in a suspended state. Advantageously, a cell culture growing in a suspended state facilitates the exchange of nutrients and gases during growth.

[0031] The spacer may include beads located at the bottom of the strut. The spacer may include a detachable collar configured to be placed on the strut. Advantageously, this may enable the end user to customize the insert by selecting which struts elevate the height of the associated anchor during use.

[0032] The insert may also include a third strut extending upright from the base and configured to support the anchor; and a fourth strut extending upright from the base and configured to support the anchor. The third and fourth struts may be disposed in a plane perpendicular to the groove. Optionally, at least one of the third and fourth struts may include a spacer at the bottom of the strut in question, such that the supported anchor is elevated above the base of the insert. The third and fourth struts may be referred to as a second pair of struts, such that the insert includes two pairs of struts with a rotational symmetry of 4. Considering the groove, the rotational symmetry of the entire insert may be 2, but the rotational symmetry of the struts may be 4.

[0033] Advantageously, providing the third and fourth struts in the above arrangement enables the user to replace the struts supporting the anchor, thereby changing the direction of the tension applied to the tissue growing using the insert. Optionally adding spacers provides a difference between the first and second struts and the third and fourth struts. By using the third and fourth struts to support the anchor, the tissue is suspended in the hydrogel rather than sitting in the groove of the insert.

[0034] Additionally or alternatively, the insert may include one or more additional struts for supporting one or more respective additional anchors, optionally or preferably, wherein the first, second, and one or more additional struts are equidistantly spaced around the base of the insert. Different from the embodiments described for the "two - pair" system, each strut may support a different anchor, rather than the user transferring the anchor from one strut.

[0035] In one embodiment, the first and second struts may be arranged to be spaced at least 10 mm, 20 mm, at least 22 mm, at least 25 mm, at least 28 mm, at least 30 mm, at least 32 mm, at least 35 mm, at least 38 mm, at least 40 mm.

[0036] Surprisingly, when culturing cells using the insert, tissue slices that are longer than ever before can be grown. Longer tissue slices have advantages in medical use because they are more versatile and can be used to treat a wider range of diseases.

[0037] In one embodiment, the struts can be arranged at different heights and angles in the same or different planes. Advantageously, this can change the position of the anchor and promote directional growth.

[0038] In one embodiment, the insert includes a force sensor, optionally or preferably, wherein the force sensor is connected to the first or second anchor. The force sensor can be used to measure contractility, thereby demonstrating normal muscle function. The force sensor can be manufactured simultaneously with the insert or separately and then connected. Contractility can be measured using physical sensors (such as piezoresistive sensors) or fiber optic sensors (such as optical fibers). The sensor interface circuit can be integrated as part of the insert or located outside the cell culture well plate.

[0039] In one embodiment, the insert is configured to provide electrical stimulation to the tissue grown using the insert. The electrical stimulation can be provided by the lid of the insert. The lid may include a pair of electrodes arranged along the surface, which are configured to contact the tissue grown in the insert. This pair of electrodes can be connected to an external power source.

[0040] According to a second aspect of the present invention, there is provided a system for culturing tissue, comprising:

[0041] An insert according to any embodiment of the first aspect; and

[0042] A plate including at least one well;

[0043] Wherein the at least one well houses the insert, and the engineered tissue is suspended in the insert.

[0044] The system may further include a lid, which is configured to at least cover the well storing the insert. The lid may include two or more electrodes, which are configured to provide electrical stimulation to the contents of the well. The electrodes can include metallic materials such as silver and copper, or conductive polymers such as PEDOT:PSS, or non-metallic materials such as carbon, graphene or graphite. The electrodes can be rigid, semi-flexible or flexible. The electrodes can be arranged in pairs. Alternatively, an odd number of electrodes can be provided. In an odd configuration, a common ground electrode can be shared with other electrodes, while in an even configuration, each pair of electrodes can have its own ground electrode. Each pair of electrodes can provide the same continuous stimulation. Alternatively, each pair of electrodes can provide different levels of stimulation at different intervals. The stimulation of each pair of electrodes can be provided simultaneously or sequentially.

[0045] The system described herein may also include a secondary insert, wherein the secondary insert includes: a body that includes an orifice; a first pair of struts that extend upright from the body and are configured to support a first pair of anchors; and a second pair of struts that extend upright from the body and are configured to support a second pair of anchors; wherein the secondary insert is configured to receive one or more pairs of anchors that are removed from the struts of the primary insert during tissue growth.

[0046] In one embodiment, the pairs of struts are parallel. Advantageously, parallel pairs of struts can be more space - saving than other alternative arrangements.

[0047] The secondary insert may include one pair of struts, two pairs of struts, three pairs of struts, four pairs of struts, five pairs of struts, or more than five pairs of struts.

[0048] The cells accommodated on the secondary insert may be from more than 1 primary insert, or from the same primary insert.

[0049] The orifice of the secondary insert may be a groove. The orifice of the secondary insert may include a plurality of grooves.

[0050] In one embodiment, the cells may be cultured on the primary insert for at least 48 hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1 month and then transferred to the secondary insert.

[0051] In another aspect of the present invention, an in vitro method of cell culture using the insert described herein is provided.

[0052] In one embodiment, the tissue is muscle tissue; optionally, wherein the tissue is mammalian tissue; optionally, wherein the tissue is human tissue; preferably, wherein the tissue is human muscle tissue. In one embodiment, the tissue may be avian tissue; preferably, wherein the tissue is from poultry.

[0053] The cells used in the method may be muscle cells including cardiomyocytes, or other cells such as smooth muscle cells, myoblasts, satellite cells, fibroblasts, muscle progenitor cells, induced pluripotent stem cells (iPSCs), and tendon cells.

[0054] Other cell types including adipocytes and / or motor neuron cells may also be cultured on the insert.

[0055] The cells may be cultured in a hydrogel. The hydrogel may include laminin, nestin, collagen, fibrin, Matrigel, and heparan sulfate proteoglycan.

[0056] Cells can be prepared for insert culture by amplifying and growing them using conventional methods before transferring them into the hydrogel and inoculating them onto the cell insert.

[0057] Cells can be inoculated into the grooves of the cell culture insert. Alternatively, cells can be inoculated into the grooves and on the base of the insert in a co-culture manner, or on top of the hydrogel itself. Once the hydrogel has solidified, cell culture medium can be added. The grooves can include recesses. The recesses can be configured for co-culture of adipocytes and / or motor neuron cells.

[0058] During the culture process, the cells will integrate with the anchors of the insert. The cells will form tissue with muscle fibers extending between the anchors.

[0059] In one embodiment, the insert includes up to 12 anchors, and the muscle tissue can form a patch.

[0060] In one embodiment, cells can be transferred from the first and second struts to a second pair of struts including third and fourth struts. The second pair of struts can extend upright from the base in a plane perpendicular to the plane defined by the first pair of struts as described in the first aspect. Advantageously, transferring the cells onto the second pair of struts enables uniaxial stretching and mechanical tensioning, which act as a maturation signal.

[0061] Cells can be cultured for at least 48 hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 1 month.

[0062] In another aspect of the present invention, the tissue cultured according to the method described herein is used for medicine.

[0063] In another aspect of the present invention, a treatment method is provided, including transplanting the tissue cultured using the insert described herein. The method can include implanting the anchors of the insert together with the tissue into a subject in need thereof.

[0064] In another aspect of the present invention, the tissue cultured as described herein is provided for the preparation of a medicament.

[0065] In one embodiment, the length of the tissue for medicine, treatment method, or medicament manufacture is at least 20 mm, at least 22 mm, at least 25 mm, at least 28 mm, at least 30 mm, at least 32 mm, at least 35 mm, at least 38 mm, at least 40 mm.

[0066] In one embodiment, the cultured tissue for medicine is used to treat muscle diseases and injuries caused by trauma. The muscle disease can be an abdominal wall or esophageal defect.

[0067] Tissues cultured using the methods described herein can be used in medicine. The tissues can be implanted into a subject in need thereof to treat a disease or injury. The medicine can be regenerative medicine.

[0068] The tissue can be transplanted using an anchor. The anchor can be used to fix the tissue in place.

[0069] The tissue can be used to treat muscle diseases or disorders, including: abdominal wall defects and esophageal defects, or volumetric muscle loss caused by trauma. Other uses can include treating: sarcopenia, Duchenne muscular dystrophy, Becker muscular dystrophy, Duchenne muscular dystrophy (DMD)-associated dilated cardiomyopathy, limb girdle muscular dystrophies (LGMD), LAMA2-related (merosin-deficient) congenital muscular dystrophy (Emery-Dreifuss muscular dystrophy), type VI collagen-related muscular dystrophy (Bethlem myopathy, Ullrich congenital muscular dystrophy), alpha-dystroglycanopathy (Walker-Warburg syndrome, muscle-eye-brain disease), laminopathy, distal muscular dystrophy, myofibrillar myopathy, nemaline myopathy, central core myopathy, central nuclear myopathy, congenital fiber type disproportion, multi- / mininucleated myopathy, cylindrical spirals myopathy, mitochondrial myopathy, glycogen storage disease, metabolic disease, inflammatory myopathy, motor neuron disease (such as amyotrophic lateral sclerosis and spinal muscular atrophy), spinal and bulbar muscular atrophy, Charcot-Marie-Tooth disease, and Kennedy's disease.

[0070] In one aspect of the invention, a cell insert is used for culturing meat products. The cells used for producing meat products can be livestock cells. The cells can be cells of cattle, sheep, poultry, or pigs.

[0071] The cell culture insert can be manufactured using additive manufacturing techniques (such as 3D printing) or subtractive techniques (such as milling). Alternatively, the insert can be manufactured using molding, forming, casting, machining, and joining.

[0072] One or more inserts can be used in series. For example, multiple inserts can be placed in a single system to produce multiple samples simultaneously. Additionally and / or alternatively, the insert can have more than one pair of struts and corresponding anchors. Advantageously, this allows for an expansion of muscle tissue production for research, medicine, or cultured meat products.

[0073] Anchor: In this context, the term "anchor" refers to a material that provides support for cells during the culturing process. The anchor can also be used to move the tissue after culturing for downstream experiments, as well as for culturing the tissue in suspension. The anchor can be made of a non-woven fabric. The material forming the anchor can be a mesh.

[0074] Base: In this context, the term "base" refers to the structure of the cell culture insert that provides support for the struts. Cells can also be seeded on the base. The base can be made of a biocompatible resin.

[0075] Cell: In this context, the term "cell" refers to those cells cultured on the cell culture insert. The cells can be mammalian cells, preferably human cells. The cells can be from livestock. The cells can be from cows, pigs, sheep, or chickens. The cells can be muscle cells. In one embodiment, the cells can be derived from an established cell line, such as C2C12 or human myoblasts. In one embodiment, the cells can be derived from a patient. In one embodiment, the cells are stem cells, optionally induced pluripotent stem cells. The cells can be skeletal muscle cells, cardiac muscle cells, smooth muscle cells, or tendon cells.

[0076] Biodegradable: In this context, "biodegradable" refers to a material that decomposes into non-toxic components over time.

[0077] Biocompatible material: As used herein, "biocompatible material" refers to a material that can be used to culture cells or implant in a subject without causing any damage or otherwise interfering with cell or tissue function. The material can be a resin. The material can be a non-woven mesh.

[0078] Muscle disease: The muscle diseases described herein can affect mammals, particularly humans. Muscle diseases can include abdominal wall defects, etc. Description of the Drawings

[0079] Embodiments of the present invention will now be described in more detail by way of example only and with reference to the accompanying drawings, in which:

[0080] Figure 1 A cell culture insert according to an embodiment of the present invention is shown;

[0081] Figure 2Shows a cell culture insert according to an alternative embodiment of the present invention;

[0082] Figure 3a Shows a plurality of cell culture inserts according to an embodiment of the present invention;

[0083] Figure 3b Shows a cell culture insert with in-situ muscle growth;

[0084] Figure 4 Shows a cell culture insert provided with a force sensor;

[0085] Figure 5 Figures 5a and 5b show evidence of engineered muscle growth using a cell culture insert of an embodiment of the present invention;

[0086] Figures 6a to 6c Shows three possible configurations of the struts on the insert. Detailed Description

[0087] Detailed Description of Exemplary Methods and Drawings

[0088] Figure 1 and Figure 2 Shows cell culture insert 100. The insert includes a base 110. The base 110 includes a biocompatible resin and provides a support structure for the other components of the insert 100. Two struts 120 extend upright from the base 110. In the illustrated example, the struts 120 are integrally molded with the base and provide connection points for the first and second anchors 130 ( Figure 2 ). The anchors 130 are made of a biocompatible nonwoven fabric and provide a scaffold for cells to use during tissue formation. The insert 100 also includes a groove 140. The groove 140 is formed in the base 110 and extends between the first and second struts 120. The groove is configured to provide a guide for the user to apply cells to the base 110. During use, cells are added to the groove 140 in the base 110 between the first and second struts 120.

[0089] Figure 1 Insert 100 is connected to a scaffold 150. The scaffold is used during the manufacture of the insert (e.g., as part of a 3D model print or mold), but is removed before being used for culturing cells.

[0090] Figure 3a and 3b Shows cell culture insert 100 during muscle growth. As compared with Figure 1 and Figure 2Like the inserts, each insert 100 includes a base 110, a pair of struts 120, an anchor 130, and a groove 140. Each insert 100 contains cells suspended in a hydrogel 320 and is located on a 6-well plate 310. Example compositions of the hydrogel 320 will be discussed below. Further along the muscle growth process, Figure 3b shows Figure 3a one of the inserts. Muscle tissue 430 is present between the two struts 120, using the hydrogel 320 as a growth scaffold.

[0091] Figure 4 Shows the use of a force sensor 500 to test the contractility of muscle tissue 430. The insert 100 has the same features as Figure 1 to 3. In the illustrated embodiment, the force sensor 500 is an "off the shelf" component. It is contemplated that versions of the insert 100 may include force sensor elements integrated into one or more components (e.g., the struts 120).

[0092] Figure 5 To confirm the growth of muscle tissue 430. Figure 5 Shows the muscle structure under a microscope, using two different stains 550 and 560. In 550, the nuclei of aligned myotubes are stained with DAPI. In 560, the cytoskeleton of aligned myotubes is stained with phalloidin.

[0093] Figure 6a Shows Figure 1 and Figure 2 a schematic diagram of the insert 100 in. In the insert 100 as described in Figure 6a , it can be seen that the struts 120 extend upright from the groove 140 provided within the base 110. The anchor 130 shown in Fig. 6 is not shown Figure 2 .

[0094] Figure 6b Shows an alternative insert 600. Similar to the insert 100 in Figure 6a , the insert 600 includes a base 110, and the struts 120 extend upright from the base 120 (specifically, from the groove 140). Different from the insert 100 in Figure 6a , the insert 600 includes a recess 610 within the groove 140. This recess 610 is configured to facilitate the co-culture of multiple cell types, including adipocytes and motor neurons. In this example, the recess 610 is formed at the center of the base 110 and includes two semi-circular indentations extending from the groove 140 into the base 110.

[0095] Figure 6c Shows an alternative insert 650. Similar to Figure 6aSimilar to the insert 100 in [reference], the insert 650 includes a base 110, and struts 120 extend upright from the base 120 (specifically, extend upright from the groove 640). Different from the insert 650 in Figure 6a [reference], multiple struts 120 are provided, so the configuration of the groove 640 is different from that of the groove 140 ( Figure 6a ). In the illustrated example in Figure 6c [reference], twelve struts 120 are provided, and they are equally spaced around the edge of the insert 650. Therefore, the groove 640 is similar to the shape of a twelve-pointed star, and a part of the groove 740 can be seen to extend between each pair of struts 120. For clarity, the anchor 140 associated with each strut 120 is not shown. The advantage of this more complex configuration is that the tissue can grow as "small pieces", which can be applicable to different tissue types (such as heart tissue).

[0096] Figure 7 An alternative insert 700 is shown. Similar to the insert 100 in Figure 6a [reference], the insert 600 includes a base 110, and struts 120 extend upright from the base 120 (specifically, extend upright from the groove 140). Different from the insert 100 in Figure 6a [reference], the insert 700 includes a second pair of struts 125. These struts are arranged in a plane perpendicular to the groove 140 such that the groove 140 bisects the path between the second pair of struts 125. Each of the second pair of struts 125 includes a spacer 170 provided at the base of each strut 125. The spacer 170 at the base of each strut 125 is used to raise the supported anchor (not shown for clarity). In the illustrated example, supporting the anchor with the second pair of struts 125 suspends the tissue in the hydrogel rather than sitting in the groove 140 of the insert 700. The user can transfer the anchor from the first pair of struts 120 to the second pair of struts 125 during the tissue culture process to achieve this change in growth conditions. The insert 700 also includes a pair of apertures 150 in its base 110. These apertures 150 facilitate microscopic observation of the tissue being cultured, especially when using the second pair of struts 125 for suspension. In the illustrated example, the apertures 150 are circular, but they can be of any shape. Similarly, a single aperture 150 can be provided. Multiple observation apertures 150 can be provided. The insert 700 also includes apertures 160. The apertures 160 are configured to allow the release of air bubbles that may be trapped under the insert 700. These air bubbles would prevent the insert 700 from floating. The provision of the apertures 160 reduces the risk of this happening.

[0097] Figure 8 An embodiment of a secondary insert 800 is shown. Similar to Figure 7The secondary insert 800 is similar to the insert 700 (which may be referred to as the primary insert 700), and includes a pair of struts 120 extending upright from the base 110. In the illustrated embodiment, the secondary insert 800 includes multiple pairs of struts 120. Different from the primary insert 700, the secondary insert 800 includes a central aperture 180 in the base 110, where the central aperture 180 is configured to accommodate cultured tissue, rather than a groove 140 for accommodating cells to be cultured. The struts 120 of the secondary insert 800 are configured to accommodate a pair of anchors during tissue growth. The multiple struts 120 allow multiple primary inserts 700 to be associated with each secondary insert 800. This improves the scalability of tissue culture. In the illustrated embodiment, a scaling ratio of 3:1 can be achieved.

[0098] Exemplary method - Production of tissue-engineered murine muscle using inserts and anchors

[0099] The murine immortalized myoblast cell line C2C12 (ATCC) was used to demonstrate that tissue-engineered muscle can be grown on the inserts.

[0100] Cell expansion:

[0101] The growth medium (gm) was prepared as follows: Medium (such as DMEM) with 20% fetal bovine serum and 1% penicillin-streptomycin added.

[0102] The differentiation medium (dm) was prepared as follows: DMEM + 1% penicillin-streptomycin and 2% horse serum.

[0103] The cells were seeded in gm at a density of approximately 5000 cells / cm 2 and incubated until they reached a confluence of ≤70%.

[0104] If plating from cryopreservation tubes, the gm was replaced the next day; otherwise, the gm was replaced every 2 days.

[0105] Subculture:

[0106] Once the cell confluence was ≤70%, the gm was removed, the cells were washed with DPBS(-Ca / -Mg), and then the cells were detached using a 0.25% (w / v) trypsin-0.53 mM EDTA solution.

[0107] The cells were incubated with trypsin at 37 °C for 5 minutes and examined under a microscope to confirm that the cells were suspended. An equal volume of pre-warmed complete gm was added to neutralize the trypsin (e.g., if 3 ml of trypsin was used, 3 ml of gm was added). The cell suspension was thoroughly mixed, for example, by gently pipetting up and down 10 times to avoid generating foam.

[0108] Cell counting:

[0109] Transfer 100 μl of cells into an Eppendorf tube, and add 400 μl of trypan blue to stain dead cells. After incubating at room temperature for several minutes, take out 10 μl and load it onto a hemocytometer (such as a Neubauer counting chamber). Then calculate the cell concentration.

[0110] Preparation of hydrogel

[0111] When preparing the hydrogel, all plasticware should be pre-frozen and the operations should be carried out on ice.

[0112] Use a type I collagen concentration of approximately 3.0 mg / ml as follows:

[0113]

[0114]

[0115] When the protein concentration is 8 mg / mL, Matrigel is used in undiluted form. Mix 65% v / v of type I rat tail collagen with 10% v / v of MEM, and then add 1 M NaOH dropwise to neutralize the solution until the color turns pink. Check the pH value to be approximately 7 using pH test paper. Add 20% v / v of Matrigel. Before use, store the hydrogel on ice for up to 2 hours.

[0116] Preparation of cell / hydrogel mixture:

[0117] Centrifuge the cell suspension at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the pellet in the solution of Matrigel and collagen.

[0118] Mix the cell suspension thoroughly, for example, by gently pipetting up and down 10 times to avoid generating foam. Keep the cell hydrogel mixture on ice before inoculation.

[0119] Prepare inserts with anchors:

[0120] Pierce the anchors with a syringe needle (2 holes in each anchor), and install one anchor on each of the first pair of struts of the insert. Sterilize the insert and the anchors. Under sterile conditions, place the insert in the well of a well plate.

[0121] Load the hydrogel and cells into the insert:

[0122] Mix the 200 μl of cell-hydrogel mixture thoroughly before pipetting it into the central groove of the culture insert. First, pipette a small drop of gel under each anchor. Then press and release the anchors multiple times. Finally, form a uniform cell strip by moving the pipette back and forth to fill the hydrogel into the top of the anchor and the groove.

[0123] Place the substrate with the culture plate in an incubator at 37 °C for 2 hours to polymerize the hydrogel.

[0124] After the gel has solidified, carefully add 3 ml of gm to each well.

[0125] Once the myoblasts reach ≥90% confluence (depending on the cell line), wash the cells with PBS and change the medium to dm.

[0126] If desired, another hole of the anchor can be used to stretch the tissue-engineered muscle and improve functional maturation. Alternatively or additionally, the tissue-engineered muscle can be transferred to struts 3 and 4 for suspension culture.

[0127] Myotube formation appears within 12 days.

[0128] The differentiation medium is changed every other day.

Claims

1. An insert for culturing tissue, comprising: A base including a groove configured to provide guidance for adding cells to be cultured; A first pillar extending upright from the base and configured to support a first anchor; And A second pillar extending upright from the base and configured to support a second anchor; Wherein the insert is configured such that cells can be cultured between the first and second pillars using the anchors for support to form tissue.

2. The insert according to claim 1, wherein the first and second pillars extend upright from the groove such that the groove extends from the bottom of the first pillar to the bottom of the second pillar.

3. The insert according to claim 1 or claim 2, wherein: The position of the first anchor and / or the second anchor can be adjusted to stretch the tissue.

4. The insert according to any one of claims 1-3, wherein the tissue is muscle tissue.

5. The insert according to any one of claims 1-4, wherein the first and second pillars are made of a biocompatible material, optionally, wherein the biocompatible material is a biocompatible resin.

6. The insert according to any one of claims 1-5, wherein the first and second pillars comprise a biodegradable material.

7. The insert according to any one of claims 1-6, wherein the first and second pillars are in a fixed position.

8. The insert according to any one of claims 1-7, wherein the position of at least one of the first pillar or the second pillar is adjustable.

9. The insert according to any one of claims 1-8, wherein the position of the first anchor and / or the second anchor can be adjusted by changing the position of the first pillar and / or the second pillar.

10. The insert according to any one of claims 1-9, wherein the position of the first anchor and / or the second anchor can be adjusted manually, automatically or semi-automatically.

11. The insert according to any one of claims 1-10, wherein the first and second pillars are arranged to be spaced apart by at least 10 mm, at least 20 mm, at least 22 mm, at least 25 mm, at least 28 mm, at least 30 mm, at least 32 mm, at least 35 mm, at least 38 mm, at least 40 mm.

12. The insert according to any one of claims 1-11, wherein the insert includes a force sensor, optionally or preferably, wherein the force sensor is configured to be connected to the first anchor or the second anchor.

13. The insert according to claim 12, wherein the sensor is a physical sensor, an optical sensor, a radio frequency sensor, an acoustic sensor or an optical fiber sensor.

14. The insert according to any one of claims 1-13, wherein the groove further includes a recess.

15. The insert according to any of the foregoing claims, further comprising: A third pillar extending upright from the base and configured to support an anchor; And A fourth pillar that extends upright from the base and is configured to support an anchor; wherein the third and fourth pillars are arranged in a plane perpendicular to the groove; and optionally or preferably, at least one of the third and fourth pillars includes a spacer at the base of the pillar such that the supported anchor is higher than the base of the insert.

16. The insert according to any one of the preceding claims, further comprising: One or more additional pillars configured to support one or more corresponding additional anchors, optionally or preferably, wherein the first pillar, the second pillar, and the one or more additional pillars are equidistantly spaced around the base of the insert.

17. A system for culturing tissue, comprising: An insert according to any one of claims 1-16; The first and second anchors; And A plate including at least one hole; Wherein the at least one hole receives the insert and a hydrogel is suspended in the insert.

18. The system according to claim 17, wherein the first and second anchors comprise a biocompatible nonwoven fabric; and optionally or preferably, wherein the first and second anchors comprise a biodegradable nonwoven fabric.

19. The system according to claim 17 or 18, wherein the first and second anchors include one or more holes for connecting the first and second anchors to the first and second pillars.

20. The system according to any one of claims 17 to 19, wherein the first anchor and / or the second anchor is detachable from the first pillar and / or the second pillar.

21. A method for culturing tissue in vitro, wherein the tissue is cultured using an insert according to any one of claims 1-16.

22. The method according to claim 21, wherein the tissue is muscle tissue; optionally, wherein the tissue is mammalian or avian tissue; optionally or preferably, wherein the tissue is human tissue; further optionally or preferably, wherein the tissue is human muscle tissue.

23. The method according to claim 22, wherein the insert comprises: A third pillar that extends upright from the base and is configured to support an anchor; And A fourth pillar that extends upright from the base and is configured to support an anchor, wherein the third and fourth pillars are arranged in a plane perpendicular to the groove; And Wherein the method comprises: a. Inoculating the cells into the groove between the first and second pillars and culturing; And b. During the growth of the tissue, transferring the anchor from the first and second pillars to the third and fourth pillars.

24. The method according to any one of the preceding claims, wherein the cells are livestock cells, optionally, wherein the cells are cells of cattle, sheep, poultry, or pigs.

25. Tissue cultured by the method according to any one of claims 21 to 24, for use in medicine.

26. Tissue cultivated by the method according to any one of claims 21 to 25, which is used as food.

27. The system according to any one of claims 17 - 20, further comprising: A secondary insert, wherein the secondary insert comprises: A body that includes an orifice; A first pair of struts that extend upright from the body and are configured to support a first pair of anchors; and A second pair of struts that extend upright from the body and are configured to support a second pair of anchors; Wherein the secondary insert is configured to receive one or more pairs of anchors removed from the struts of the primary insert during tissue growth.

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