Composition

Abstract

This disclosure provides a method for encapsulating organoids, cells, or spheroids, the method comprising: (i) mixing an arginate solution and a gellan solution with a buffer solution to form a mixed solution; (ii) sterile filtering the mixed solution from (i) to form a sterile filtered solution; (iii) suspending organoids, cells, or spheroids in a protein matrix containing extracellular matrix proteins and mixing it 1:1 with the sterile filtered solution from (ii) to form a liquid suspension; (iv) exposing the liquid suspension from (iii) to calcium ions to induce gelation. A kit comprising arginate, gellan gum, and a protein matrix is ​​also provided; as well as a gel composition suitable for encapsulating organoids, cells, or spheroids, comprising about 0.1% w / v to about 1% w / v of arginate and about 0.001% w / v to about 0.02% w / v of gellan gum.

Description

Technical Field

[0001] The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 665992.

[0002] (Field) The present disclosure also provides compositions for encapsulating cells and three-dimensional cell cultures (e.g., organoids and / or spheroids), kits containing the components of such compositions, and methods of encapsulating cells, organoids or spheroids in such compositions.

Background Art

[0003] (Background) Three-dimensional cell cultures (e.g., organoids or spheroids) typically require an extracellular matrix support that mimics the extracellular environment and acts as a scaffold. Matrigel® is a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and is commonly used as an extracellular matrix. The major components of Matrigel® are structural proteins (e.g., laminin, collagen and entactin) which provide adherent peptide sequences that cultured cells would encounter in their natural environment. Growth factors such as TGFβ and EGF, which prevent the differentiation of many cell types and promote their growth, are also present. Growth factor-reduced Matrigel® is also available.

[0004] Traditionally, organoids are encapsulated in 10-50 μm adhesive droplets of Matrigel®, cultured under static conditions in well plates or dishes, and nutrient-supplied in semi-batch increments (Fatehullah et al., 2016). Such Matrigel®-based cell cultures have been shown to function well. However, Matrigel® is expensive, the process is labor-intensive, yields small quantities, and exhibits high batch-to-batch variability. This is less problematic when cells are cultured on a laboratory scale, but it means that it is not well-suited for large-scale production. Furthermore, due to Matrigel®'s animal-derived nature, its composition is complex and not well-defined, resulting in high batch-to-batch variability. Again, this is not suitable for large-scale production of organoids or spheroids, because a high level of consistency is required, especially when these organoids or spheroids are used for drug testing.

[0005] Synthetic alternatives to Matrigel® (e.g., QGel) have been developed. However, a drawback of these alternatives is the difficulty in completely adapting pre-established Matrigel® cultures to the new matrix, and their ability to manipulate these matrices into different shapes / forms is limited, especially for the rapid generation of large matrix volumes.

[0006] PEG-based synthetic matrices exhibit relatively lower organoid formation efficiency compared to Matrigel®. Synthetic scaffolds are often tailored to specific cell / organoid types, while Matrigel® is used universally. Various organoids, as well as the various cells within them, often require distinct physical and biochemical parameters to guide their cellular behavior; therefore, screening multiple synthetic scaffolds can be time-consuming, prohibitively expensive, and difficult. [Overview of the project] [Means for solving the problem]

[0007] (Summary) The inventors recognized the need for alternative scaffolds suitable for generating large quantities of three-dimensional cell cultures (e.g., organoids and spheroids) and for generating organoids and spheroids with consistent morphology and function. This would enable the widespread use of organoids and spheroids in drug testing and improve the comparability of test results.

[0008] This disclosure relates to gel compositions for encapsulating cells and three-dimensional cell cultures (e.g., organoids and / or spheroids). In particular, this disclosure provides gel compositions comprising about 0.1% w / v to about 1% w / v of arginate and about 0.001% w / v to about 0.02% w / v of gellan gum. A protein matrix can be combined with this arginate and gellan gum, which allows for balancing the amount of the protein matrix with the other components and controlling the final protein concentration. This is particularly advantageous when the protein matrix is ​​of biological origin (e.g., Matrigel®), due to the high levels of variation between different batches.

[0009] While not constrained by theory, the inventors believe that the composition may provide sufficient biological clues from its protein matrix while also utilizing the physical properties of its other components. Therefore, the composition provides a gel mixture capable of rapidly and easily forming self-supporting structures of any shape (e.g., particles or beads). These gel structures may facilitate the scaling up of any cell or organoid strain cultured in a protein matrix within more complex bioreactor systems, thereby enabling the large-scale production of organoids and spheroids with consistent morphology and function.

[0010] A kit containing the components of this gel composition, and a method for encapsulating cells, organoids, or spheroids in this gel composition are also provided.

[0011] (explanation) This disclosure provides gel compositions comprising about 0.1% w / v to about 1% w / v of arginate and about 0.001% w / v to about 0.02% w / v of gellan gum. The compositions described herein are particularly suitable for encapsulating cells and / or three-dimensional cell cultures. These cells (which may be in the form of three-dimensional cell cultures) are preferably eukaryotic cells, more preferably mammalian cells. Typically, cells are obtained from a multicellular organism (e.g., human) and then cultured into a three-dimensional cell culture (e.g., organoid or spheroid) before proceeding further. These cells may be healthy or represent a disease (e.g., cells obtained from healthy tissue or cells obtained from a diseased state (malignant tumor biopsy)). In some embodiments, the three-dimensional cell culture is an organoid.

[0012] The term organoid simply means something that resembles an organ. Organoids are typically defined by three characteristics: self-organization, multicellularity, and functionality (Lancaster and Knoblich, 2014). Thus, their cells align themselves in vitro to form a three-dimensional (3D) tissue that is characteristic of that organ in vivo, and the resulting structure consists of multiple cell types found in that particular organ, and those cells perform at least some of the functions they normally perform in that organ. For example, the prototype organoid, mouse intestinal organoid, grows as a simple epithelium organized into domains so that it resembles its in vivo intestinal crypt-villous structure surrounding a cystic lumen, containing various cell types of the intestine (intestinal absorptive epithelial cells, goblet cells, Paneth cells, enteroendocrine cells, and stem cells) (Sato et al., 2011).

[0013] Organoids can be grown from pluripotent stem cells (embryonic pluripotent stem cells or induced pluripotent stem cells) or from tissue biopsies containing adult stem cells. When grown in vitro under conditions that support stem cell maintenance, organoids resemble the organ from which they originate by replicating tissue-specific cell type and 3D structure, genetic function, and physiologically relevant function, while also being continuously expandable.

[0014] The term spheroid refers to a three-dimensional multicellular aggregate. Spheroids are typically formed from immortalized cell lines that have been previously grown under 2D culture conditions. Spheroids can be observed to have various 3D morphologies, but these do not necessarily represent the original source tissue type, and the cells containing these spheroids are typically homogeneous and not hierarchically organized.

[0015] Gellan gum is an extracellular polysaccharide secreted by the microorganism Sphingomonas elodea (ATCC 31461), formerly known as Pseudomonas elodea. It is available in two forms: high-acyl (typically containing 11–13% glyceryl groups and 4–5% acetyl groups, with the total amount of these acyl groups ranging from 15–18% (by weight)) and low-acyl (typically containing less than 1% glyceryl groups and less than 1% acetyl groups, with the total amount of these acyl groups being less than 2 wt%). The gellan gum used in the compositions described herein may be low-acylgellan gum. Low-acylgellan gum can be prepared, for example, by the method described in U.S. Patent No. 8,609,377.

[0016] Gellan gum forms a gel at low concentrations when a hot solution is cooled in the presence of gelation-promoting cations. The states of gellan gum described herein are appropriate to the state in which it is used. For example, when present in a gel composition (e.g., beads encapsulating organoids, cells, or spheroids), the gellan gum is in a gel state. Alternatively, if the gellan gum is handled before gelation, it is not in a gel state, but typically in a sol state.

[0017] Arginates are naturally occurring anionic polymers typically obtained from brown seaweed, known for their biocompatibility and ease of gelation. Arginates are known to comprise an entire family of linear copolymers containing blocks of (1,4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. These blocks consist of consecutive G residues, consecutive M residues, and alternating M and G residues. Arginates extracted from various sources vary in M ​​and G content, as well as the length of each block. Arginates used in the compositions described herein may have a low M / G ratio (e.g., G: approximately 65-70%; M: approximately 25-35%). G blocks have a higher calcium ion affinity than M blocks. Therefore, arginates with a higher M / G ratio can produce more permeable, flexible, and soft arginate gel matrices, while lower M / G ratios result in a stronger structure.

[0018] Arginates form gels in the presence of divalent and / or trivalent cations. The states of arginates described herein are appropriate to the state in which they are used. For example, when present in a gel composition (e.g., beads encapsulating organoids, cells, or spheroids), the arginate is in a gel state. Alternatively, if the arginate is handled before gelation, it is not in a gel state but typically in a sol state.

[0019] This gel composition may contain approximately 0.2% to approximately 0.5% w / v of arginate or approximately 0.2% to approximately 0.4% w / v of arginate, and may optionally contain approximately 0.2% to approximately 0.3% w / v of arginate. This gel composition may contain approximately 0.25% w / v of arginate.

[0020] This gel composition may contain approximately 0.002% w / v to approximately 0.01% w / v gellan gum or approximately 0.003% to approximately 0.009% w / v gellan gum, and optionally contain approximately 0.004% to approximately 0.006% w / v gellan gum. This gel composition may contain approximately 0.005% w / v gellan gum.

[0021] The gel composition may further comprise a protein matrix, which provides extracellular matrix (ECM) proteins that can mimic the extracellular environment and act as a scaffold. The protein matrix typically comprises ECM proteins (e.g., collagen I and / or collagen IV, as well as laminin). The protein matrix may further comprise ECM proteins (e.g., entactin, perlecan, or gelatin, or a combination thereof). The protein matrix may be of artificial or biological origin.

[0022] The biologically derived protein matrices that may be used in the compositions described herein are typically solubilized substrate preparations extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcomas. EHS mouse sarcomas are major sources of extracellular matrix (ECM) proteins (e.g., laminin, collagen IV, heparan sulfate proteoglycans, entactin / nidogen) and numerous growth factors. Biologically derived protein matrices with reduced growth factors may also be used.

[0023] Suitable biological protein matrices for use in this gel composition include protein matrices containing laminin, entactin, and collagen IV, and optionally containing heparin sulfate proteoglycan. Examples of commercially available matrices include Matrigel® (which contains laminin, entactin, and collagen IV), ECM Gel (which contains laminin, collagen IV, entactin, and heparin sulfate proteoglycan), Cultrex® (which contains laminin, entactin, collagen IV, and heparin sulfate proteoglycan), and / or Geltrex® (which contains laminin, entactin, collagen IV, and heparin sulfate proteoglycan). In some embodiments, the biological protein matrix is ​​Matrigel®.

[0024] Artificial protein matrices can be PEG-based hydrogels, which are typically bound to key peptide residues derived from ECM proteins (e.g., collagen and laminin). Polysaccharide products can also be used.

[0025] The protein matrix may form a gel in response to chemical and / or physical signals. For example, Matrigel® forms a gel when incubated at approximately 37°C. The states of the protein matrix described herein are appropriate for the state in which it is used. For example, if present in a gel composition (e.g., beads encapsulating organoids, cells, or spheroids), the protein matrix may be in a gel state. Alternatively, if the protein matrix is ​​handled before gelation, it may typically be in a sol state rather than a gel state.

[0026] This gel composition may contain an ECM protein content derived from its protein matrix that is from about 2 mg / ml to about 10 mg / ml, or from about 3 mg / ml to about 8 mg / ml. Optionally, the composition contains from about 4 mg / ml to about 6 mg / ml of ECM protein or from about 4.25 mg / ml to about 5 mg / ml of ECM protein. Thus, the gel composition described herein reduces the amount of protein matrix required. Thus, the protein matrix component of this gel composition is diluted by the other components thereof (i.e., alginate and gellan gum). There is sufficient protein matrix to provide the biological cues required by the cells, but the problems associated with the changing nature of the protein matrix of biological origin have been turned into dilution of this component. This dilution of the protein matrix component also allows the final protein concentration within the composition to be controlled.

[0027] This gel composition may further contain organoids, cells, or spheroids. Optionally, the gel composition further contains organoids or cells. In some embodiments, the gel composition contains organoids.

[0028] Thus, the gel composition disclosed herein may contain from about 0.2% w / v to about 0.5% w / v of alginate, from about 0.002% to about 0.01% of gellan gum, from about 3 mg / ml to about 8 mg / ml of ECM protein, and organoids, cells, or spheroids. Optionally, the gel composition disclosed herein may contain about 0.25% w / v of alginate, about 0.005% of gellan gum, from about 4 mg / ml to about 6 mg / ml of ECM protein, and organoids, cells, or spheroids.

[0029] The composition is a gel, preferably a hydrogel. As used herein, a hydrogel refers to a system in which hydrophilic polymer chains are dispersed in an aqueous solution (e.g., an aqueous buffer solution or water). Typically, the hydrogel is in a gel state (e.g., a semi-solid state that retains its shape). The aqueous buffer solution is optionally isotonic and / or pH neutral, both of which are beneficial for cell health. A suitable aqueous buffer solution is phosphate-buffered saline.

[0030] This gel composition typically has a Young's modulus of less than 10 kPa when measured by uniaxial unconfined oscillatory compression. For example, this gel composition may have a Young's modulus of approximately 5 kPa to approximately 10 kPa when measured by uniaxial unconfined oscillatory compression.

[0031] This disclosure also provides a kit comprising an arginate, a guerlain, and a protein matrix. Each of the arginate, guerlain, and / or protein matrix may be in the form of a solution. The arginate and guerlain may be combined. In some embodiments, the protein matrix is ​​provided separately.

[0032] This kit may contain a solution containing approximately 0.1% w / v to approximately 1% w / v of arginate and approximately 0.001% w / v to approximately 0.02% w / v of gellan gum.

[0033] The solution may contain approximately 0.2% to 0.5% w / v arginate or approximately 0.2% to 0.4% w / v arginate, and if necessary, the solution may contain approximately 0.2% to 0.3% w / v arginate. The solution may contain approximately 0.25% w / v arginate.

[0034] The solution may contain approximately 0.002% w / v to approximately 0.01% w / v gellan gum or approximately 0.003% to approximately 0.009% w / v gellan gum, and if necessary, the solution may contain approximately 0.004% to approximately 0.006% w / v gellan gum. The solution may contain approximately 0.005% w / v gellan gum.

[0035] The above-mentioned Guerlain components, arginate components, and protein matrix components are suitable for use in this kit.

[0036] This kit may further include instructions on how to use this kit to prepare gel compositions and how to encapsulate cells, organoids, or spheroids using the methods described below.

[0037] This disclosure further provides a method for encapsulating organoids, cells, or spheroids, the method being: (i) The step of mixing the arginate solution and the Guéran solution together with a buffer solution to form a mixed solution; (ii) A step of sterile filtering the mixed solution of (i) to form a sterile filtered solution; (iii) Suspending organoids, cells, or spheroids in a protein matrix and mixing them in a 1:1 ratio with the sterile filtered solution from (ii) to form a liquid suspension; (iv)(iii) The liquid suspension is exposed to calcium ions to induce gelation. Includes.

[0038] The inventors determined that suspending cells, organoids, or spheroids in a protein matrix before introducing them into a gellan gum / arginate mixture yielded the best results. In particular, a more uniform cell distribution was observed. This provided more consistent conditions and ultimately resulted in a final product with less variation. In contrast, they determined that directly mixing cells into the gellan gum / arginate mixture resulted in an insufficient distribution of organoids or spheroids within the gel particles or gel beads. While not constrained by theory, the inventors believe that calcium released from the cells or residual trace amounts of calcium present in the cell culture medium cause the arginate to initiate gelation around the cells, which leads to the formation of aggregates around the cells.

[0039] The arginate solution and the Guerlain solution may be mixed in step (i) in a volume ratio determined by the starting concentrations of the arginate solution and the Guerlain gum solution. For example, if both the arginate solution and the Guerlain solution are approximately 1% w / v, they may be mixed with the buffer solution in a volume ratio of approximately arginate 50: buffer 49: Guerlain 1.

[0040] Upon exposure to calcium ions (e.g., calcium chloride ions), the liquid suspension of step (iii) transforms into a gel state (i.e., a semi-solid state that retains its shape). Therefore, step (iv) may include extruding and / or dropping the liquid suspension of step (iii) into a solution containing calcium ions, thereby generating gel particles or gel beads (which include organoids, cells, or spheroids). The shape of the gel particles or gel beads is not particularly limited; however, spherical or oval shapes are commonly formed.

[0041] The solution containing the calcium ions is typically maintained at a temperature of approximately 37°C. The concentration of the calcium ions is, as needed, approximately 100 mM to approximately 200 mM, or approximately 120 mM to approximately 150 mM, or approximately 130 mM to approximately 140 mM. In some embodiments, the concentration of the calcium ions is approximately 135 mM.

[0042] The calcium ion-containing solution may further contain a surfactant to reduce surface tension, which facilitates the penetration of gel droplets into the liquid and thus prevents the surface tension from deforming the gel particles or gel beads. The surfactant may be a nonionic surfactant, preferably a polysorbate-type nonionic surfactant. Suitable surfactants include Tween® 20, Montanox 20, Polysorbate 20, PEG(20) sorbitan monolaurate, Alkest TW 20, and Scattics. In some embodiments, the surfactant is Tween® 20.

[0043] For example, when the liquid suspension of step (iii) is extruded from the needle, the flow rate and needle size may be controlled to control the size of the gel particles or gel beads. Suitable needles include 25G, 26G, 27G, 28G, 29G, or 30G needles, which may be used in a multi-injector manifold. If necessary, the needle is 27G. The flow rate may be approximately 1 mL / hour to approximately 20 mL / hour, or optionally approximately 5 mL / hour to approximately 15 mL / hour, or approximately 8 mL / hour to approximately 12 mL / hour. If necessary, the flow rate may be approximately 10 mL / hour.

[0044] The gel particles or gel beads may have a diameter of approximately 200 μM to approximately 4000 μM. The gel particles or gel beads may have a diameter of approximately 1000 μm to approximately 3000 μm. For example, the gel particles or gel beads may have a diameter of approximately 2.1 mm, or approximately 2.2 mm, or approximately 2.4 mm, or approximately 2.5 mm, or approximately 2.6 mm, or approximately 2.7 mm, or approximately 2.8 mm, or approximately 2.9 mm.

[0045] Exposure to a solution containing calcium ions is time-controlled as necessary to ensure that there are no causes of loss to the viability of cells, organoids, or spheroids. The gel particles or gel beads can be collected directly from the solution containing calcium ions, for example, by using a strainer to remove the gel particles or gel beads. Exposure may be limited to about 10 minutes or less, or about 8 minutes or less, or about 6 minutes or less as necessary. The minimum exposure time may be about 30 seconds or about 1 minute.

[0046] After being collected from a calcium ion-containing solution, the gel particles or gel beads can then be washed, for example, with a basic culture medium. The gel particles or gel beads can then be transferred to a static culture for up to 48 hours. During this time, the gel particles or gel beads equilibrate and typically shrink in size. For example, the gel particles or gel beads may have a diameter of approximately 2.5 mm to 2.9 mm when initially formed and equilibrate to a final diameter of approximately 2.3 mm to 2.6 mm.

[0047] The liquid suspension of (iii) may contain approximately 0.1% w / v to approximately 1% w / v of arginate, approximately 0.001% w / v to approximately 0.02% w / v of gellan gum, and approximately 2 mg / ml to approximately 10 mg / ml of extracellular matrix protein.

[0048] The liquid suspension of (iii) contains, as needed, approximately 0.2% w / v to approximately 0.5% w / v of arginate, approximately 0.005% to approximately 0.1% w / v of gellan gum, and approximately 3 mg / ml to approximately 8 mg / ml of extracellular matrix protein.

[0049] (i) The buffer solution may be isotonic and / or pH neutral. In other words, its pH is typically about 7. The buffer solution contains no calcium at all, because calcium ions induce gelation, which is undesirable until step (iv). A suitable buffer solution is phosphate-buffered saline (PBS).

[0050] The gel particles or gel beads can be lysed to collect cells, spheroids, or organoids. If necessary, the gel particles or gel beads are lysed using a lysis buffer to break the gel and release the cells, organoids, or spheroids without damage. The lysis buffer may contain one or more solvents and may be optimized based on the hydrogel or group of hydrogels used to form the gel beads. For example, if the gel contains a protein matrix of biological origin (e.g., Matrigel®), the lysis buffer may preferably contain a Cell Recovery Solution, which can remove the protein matrix while avoiding cellular dissociation of the spheroids or organoids.

[0051] If necessary, the recovered cells, organoids, or spheroids may be centrifuged to separate them from any remaining culture medium after the gel has been dissolved. The centrifugation settings may be adjusted to increase gravity and decrease braking speed to prevent resuspending of the cell pellet.

[0052] Cell culture media are well known in the art and are familiar to those skilled in the art. Typically, cell culture media contain amino acids, salts, glucose, and vitamins, and may also contain iron and phenol red. Culture media suitable for use in the cell expansion systems and methods described herein can be produced by modifying existing cell culture media. For example, such a cell culture medium may be Dulbecco's modified Eagle medium (DMEM) and may contain one or more additional components (e.g., nutrient mixtures (e.g., Ham F12), antibiotics / antifungal agents (e.g., penicillin / streptomycin), buffers (e.g., HEPES), glutamine, and n-acetylcysteine). Such a cell culture medium may further contain serum-free supplements (e.g., N2 supplements and / or B27 supplements).

[0053] (Brief explanation of the drawing) This disclosure will be described in detail below for illustrative purposes only, with reference to the drawings. [Brief explanation of the drawing]

[0054] [Figure 1] Figure 1 shows the results for various cell lines (MG63, C3A, Iso50) tested in a mixture of 2% v / v Matrigel® in 1% w / v arginate. A. Arginate / Matrigel® beads encapsulating MG63 cells (osteosarcoma). B. Arginate / Matrigel® beads encapsulating C3A cells (hepatocytes). C. Comparison between 100% Matrigel® cultures and arginate / Matrigel® cultures after 6 days of incubation.

[0055] [Figure 2] Figure 2 shows Iso50 organoids in the Guerlain / Matrigel® mixture or Matrigel® alone after three passages in each substance, and 5 days after the most recent passage. The scale bar represents 200 μM.

[0056] [Figure 3] Figure 3 shows Iso50 cells cultured in a 1:1 mixture of Matrigel® and 2% w / v arginate of the 5% oxidized form after four passages.

[0057] [Figure 4] Figure 4 shows beads produced from a 1:1 mixture of Matrigel® and 2% w / v arginate in its 5% oxidized form on day 0 (production day) and day 6. The scale bar represents 200 μM.

[0058] [Figure 5] Figure 5 shows an overview of the conditions under which successful bead formation was investigated under electrospray and non-electrospray conditions.

[0059] [Figure 6] Figure 6 shows the change in diameter of gel beads over time when the beads gel in a calcium chloride solution and then equilibrate in a culture medium.

[0060] [Figure 7] Figure 7 shows comparable cellular behavior in the Guérin / Arginate / Matrigel® (GAM) mixture compared to a standard Matrigel® culture. A: Representative images of organoids cultured in Matrigel® or the GAM mixture, showing comparable morphology. B: Cell viability collected from Matrigel® or GAM cultures. No significant difference was observed. C: Average diameter of organoids collected from Matrigel® or GAM cultures. No significant difference was observed. D: Number of organoids collected from Matrigel® or GAM cultures. No significant difference was observed.

[0061] [Figure 8]Figure 8 shows organoids collected from Matrigel® or GAM mixtures. These organoids were reseeded in Matrigel and subjected to drug response assays. These results demonstrate that organoids from various conditions behave similarly.

[0062] [Figure 9] Figure 9 shows that the Young's modulus of the GAM material mixture and Matrigel samples was approximately 6 kPa, and no statistical significance was observed in the difference between these two values. Error bars indicate the standard deviation, and n=4.

[0063] [Figure 10] Figure 10 shows images of organoids in beads when cell suspensions are mixed in Guerlain-arginate solution and then mixed with Matrigel® (A), and when cell suspensions are mixed in Matrigel® and then mixed with Guerlain-arginate solution (B). Scale bar: 500 μm. [Examples]

[0064] (Examples) (Comparative Example 1: Matrigel® in 1% w / v arginate) Various cell lines (MG63, C3A, Iso50) were tested in this mixture. C3A mouse hepatocytes showed some 3D structure formation (Figure 1B). MG63 osteosarcoma cells maintained viability in this mixture for 7 days (Figure 1A). However, the only organoid cell line tested showed very poor growth in this mixture compared to 100% Matrigel® cultures, and this formulation was not explored further (Figure 1C).

[0065] (Comparative Example 2: Matrigel® and gellan gum) ISO50 organoids were cultured (over three passages) in 0.25% w / v and 0.5% w / v gellan gum alone, as well as in 1:1, 2:1, and 3:1 gellan gum:Matrigel® mixtures. Only the 1:1 mixture of 0.25% gellan gum and Matrigel® proved successful over this period (Figure 2). However, this gellan was difficult to handle due to its high viscosity, and it was necessary to warm the gellan before use to reduce its viscosity for accurate pipetting, which contradicted the need to keep Matrigel® cold (approximately 4°C) during handling. The relatively high rigidity of the matrix containing higher concentrations of gellan likely limited cell growth / organoid formation.

[0066] We attempted bead formation with 0.25% w / v gellan gum, but this was not concentrated enough to form beads, and therefore this mixture was not explored further.

[0067] (Comparative Example 3: Matrigel® in a 1:1 ratio and 2% w / v oxidized arginate) Iso50 organoids were cultured three times in a mixture containing oxidized arginates at 2.5%, 5%, and 7.5% (to reduce the stiffness of the arginates). The best results were obtained using 5% oxidized arginates in static droplets / clumps of matrix (Figure 3).

[0068] While it was possible to form beads from this mixture, the resulting structures were irregular in shape and exhibited expansion / decomposition over the required incubation period (Figure 4).

[0069] (Example 1: Matrigel (registered trademark) / Arginate / Gellan gum) Since unmodified arginate is more stable than oxidized arginate, organoid growth was tested in low concentrations of arginate (to maintain low matrix rigidity). ISO 50 organoids grew successfully in a mixture of 0.5% w / v arginate and Matrigel® (mixed in a 1:1 ratio). It was also shown that bead formation is possible under standard "drop-in" conditions (Figure 5).

[0070] When electrospraying this mixture (to produce beads of a smaller diameter), this arginate / Matrigel® mixture produced irregular particles of non-uniform shape. Introducing gellan gum into this mixture increased the viscosity of the solution, resulting in beads of a more consistent shape. Adding a surfactant (Tween® 20) to this gelling bath was also important in producing round, regular beads during electrospraying (Figure 5).

[0071] Even when generating beads under these dropwise (i.e., non-electrospray) conditions, it was observed that the matrix containing Guerlain was more stable over time under agitated conditions.

[0072] To improve cell viability in this mixture, the encapsulation process was optimized by limiting the length of time the matrix was exposed to a calcium chloride / Tween® 20 gel bath.

[0073] (Example 2) This gel mixture, - Prepare a 1% w / v sodium alginate solution in PBS; - Prepare a 1% w / v gellan gum solution in distilled water; - Mix these two together with PBS (volume ratio: PBS:1% arginate:1% gellan gum = 49:50:1); - Sterilize and filter; and - When using, resuspend the cells in Matrigel® (8.5-10 mg / mL protein concentration) and mix with this arginate / Guélain solution in a 1:1 ratio. It was generated by [the specified method / system].

[0074] The final composition of this mixture was Matrigel® 0.425-0.5% w / v (4.25-5 mg / mL protein), arginate 0.25% w / v, and gellan gum 0.005% w / v.

[0075] This liquid mixture / cell suspension was kept on ice until use. Gelation occurred upon exposure to a sterile solution of 135 mM calcium chloride (with Tween® 20 added for bead production) for only 6 minutes.

[0076] More specifically, this GAM solution containing cells was extruded at 10 mL / h through a 27G needle using a syringe pump. Gelation was induced by dropping this solution into a 135 mM calcium chloride bath supplemented with 1 g / L Tween® 20 to reduce the surface tension of the bath and facilitate the penetration of gel droplets into the liquid. To process large volumes of GAM, the rate of bead generation was increased using a 5-channel multi-injector connector fitted with five 27G needles, and the GAM solution was extruded at 50 mL / h (i.e., 10 mL / h per needle). This gelling bath, held in a beaker, was heated to 37°C to aid in the gelation of Matrigel®, and continuous stirring at 100 rpm using a magnetic bar was performed to prevent bead adhesion, and intermittent stirring with a spatula was performed to gently remove the beads from the surface of the bath. After 6 minutes of bead collection, the beaker was replaced with a new beaker containing the same volume of gelling bath. The beads were removed from the gelation bath by filtration and rinsed in DMEM. These 6-minute intervals were repeated as needed for the required volume of GAM matrix. The beads were combined and transferred to a culture plate / dish containing basal medium supplemented with 10 μM ROCK inhibitor.

[0077] Organoids previously cultured in Matrigel® exhibit growth in this mixture consistent with their behavior in Matrigel®. They were enlarged by a modified version of the organoid enlargement process (WO2018 / 011558), which includes a first 1 or 2 day of culture under static conditions. During this static culture period, their beads contract as shown in Figure 6. Once they stabilize, they are transferred to a reactor for enlargement.

[0078] After expansion, the cells were collected by lysing the GAM mixture by physical disruption and chemical lysis using a solution of trisodium citrate, EDTA, sodium chloride, and a commercially available cell recovery solution. The successfully collected cells responded to a group of drugs in a solution equivalent to that of a Matrigel® culture alone. Figure 7 shows the cell behavior in the GAM mixture equivalent to that of a standard Matrigel® culture.

[0079] Once collected from the mixture culture, the organoids can be re-seed in Matrigel®, and the organoids exhibit comparable functional responses in the exemplary drug assay (Figure 8).

[0080] The Young's modulus of solid (gelled and hydrated) samples of this GAM material mixture and Matrigel® was evaluated by compression testing. Figure 9 shows that the Young's modulus of each material was approximately 6 kPa, and no statistical significance was observed in the difference between these two values.

[0081] Unlike Matrigel® alone (which gels as a result of temperature changes over a period of 10-20 minutes, making it difficult to form a support shape during the gelation period, and is also very soft, making it difficult to remove from a mold in its complete shape), this gel mixture enables the rapid and easy formation of self-supporting structures (e.g., particles or beads) of any shape from a solid gel matrix.

[0082] (Example 3: Mixing order of the mixture) Cells, organoids, or spheroids are suspended in a protein matrix containing extracellular matrix proteins (e.g., Matrigel®), and then mixed with a Guerlain-arginate solution (not the other way around) to avoid premature gelation of the Guerlain-arginate solution when exposed to cations derived from the cell suspension in DMEM / F-12 cell culture medium.

[0083] As shown in Figure 9, mixing the cell suspension with Matrigel® before combining it with the Guerlain arginate solution improved cell dispersion throughout the beads compared to cells that were first mixed with the Guerlain arginate solution and then combined with Matrigel® (Figure 9A), particularly cells from large batches (>50 mL of beads) (Figure 9B).

[0084] (References) FATEHULLAH, A., TAN, SH & BARKER, N. 2016. Organoids as an in vitro model of human development and disease. Nat Cell Biol, 18, 246-54. LANCASTER, MA & KNOBLICH, JA 2014. Organogenesis in a dish: modeling development and disease using organoid technologies. Science, 345, 1247125. SATO, T., STANGE, DE, FERRANTE, M., VRIES, RG, VAN ES, JH, VAN DEN BRINK, S., VAN HOUDT, WJ, PRONK, A., VAN GORP, J., SIERSEMA, PD & CLEVERS, H. 2011. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology, 141, 1762-72.

Claims

1. A method for encapsulating organoids, cells, or spheroids, (i) The step of mixing the arginate and Guerlain solutions together with a buffer solution to form a mixed solution; (ii) A step of sterile filtering the mixed solution of (i) to form a sterile filtered solution; (iii) Suspending the organoid, cell, or spheroid in a protein matrix containing extracellular matrix proteins, and mixing it in a 1:1 ratio with the sterile filtration solution of (ii) to form a liquid suspension; (iv) (iii) A step of exposing the liquid suspension to calcium ions to induce gelation. Methods that include...

2. The method according to claim 2, wherein step (iv) comprises extruding and / or dropping the liquid suspension of step (iii) into the calcium ion-containing solution.

3. The method according to claim 1 or 2, wherein the protein matrix is ​​an artificially derived protein matrix or a biologically derived protein matrix.

4. The method according to claim 3, wherein the biologically derived protein matrix comprises laminin and collagen IV.

5. The method according to claim 4, wherein the biologically derived protein matrix further comprises heparin sulfate proteoglycans.

6. The method according to claim 3, wherein the biologically derived protein matrix is ​​selected from one or more of Matrigel, ECM Gel, Cultrex, or Geltrex.

7. The method according to any one of claims 1 to 6, wherein the liquid suspension of (iii) comprises about 0.1% to about 1% w / v of arginate, about 0.001% to about 0.02% w / v of gellan gum, and about 2 mg / ml to about 10 mg / ml of extracellular matrix protein.

8. The method according to any one of claims 1 to 7, wherein the liquid suspension of (iii) comprises about 0.2% to about 0.5% w / v arginate, about 0.002% to about 0.01% w / v gellan gum, and about 3 mg / ml to about 8 mg / ml extracellular matrix protein.

9. The method according to any one of claims 1 to 8, wherein the buffer solution in (i) is phosphate-buffered saline (PBS).

10. (i) Arginate and (ii) Gellan gum and, (iii) Protein matrix and A kit that includes this.

11. The kit according to claim 10, wherein the arginate and gellan gum are combined in the form of a solution.

12. The kit according to claim 11, wherein the solution comprises about 0.1% to about 1% w / v of arginate and about 0.001% to about 0.02% w / v of gellan gum.

13. The kit according to claim 11 or claim 12, wherein the solution contains about 0.2% to about 0.5% w / v of arginate.

14. The kit according to any one of claims 11 to 13, wherein the solution contains gellan gum in an amount of about 0.002% to about 0.01% w / v.

15. The kit according to any one of claims 10 to 14, wherein the protein matrix is ​​an artificially derived protein matrix or a biologically derived protein matrix.

16. The kit according to claim 15, wherein the biologically derived protein matrix comprises laminin, enterin, and collagen IV.

17. The kit according to claim 16, wherein the biologically derived protein matrix further comprises heparin sulfate proteoglycans.

18. The kit according to claim 15, wherein the biologically derived protein matrix is ​​selected from one or more of Matrigel, ECM Gel, Cultrex, or Geltrex.

19. Arginate at approximately 0.1% to approximately 1% w / v, Gellan gum at approximately 0.001% to approximately 0.02% w / v A gel composition suitable for encapsulating organoids, cells, or spheroids, including the following.

20. The gel composition according to claim 19, wherein the composition comprises about 0.2% to about 0.5% w / v of arginate.

21. The gel composition according to claim 19 or 20, wherein the composition comprises about 0.002% to about 0.01% w / v of gellan gum.

22. The gel composition according to any one of claims 19 to 21, wherein the composition further comprises a protein matrix.

23. The gel composition according to any one of claims 19 to 22, wherein the protein matrix is ​​an artificially derived protein matrix or a biologically derived protein matrix.

24. The gel composition according to claim 23, wherein the biologically derived protein matrix comprises laminin, entactin, and collagen IV.

25. The gel composition according to claim 24, wherein the biologically derived protein matrix further comprises heparin sulfate proteoglycan.

26. The gel composition according to claim 23, wherein the biologically derived protein matrix is ​​selected from one or more of Matrigel, ECM Gel, Cultrex, or Geltrex.

27. The gel composition according to any one of claims 22 to 26, wherein the composition comprises about 2 mg / ml to about 10 mg / ml of extracellular matrix protein.

28. The gel composition according to any one of claims 22 to 27, wherein the composition comprises about 3 mg / ml to about 8 mg / ml of extracellular matrix protein.

29. The gel composition according to any one of claims 19 to 26, wherein the composition further comprises an organoid, a cell, or a spheroid.

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