Device

Abstract

This disclosure provides a method for expanding organoids, comprising the steps of: i) dissociating organoids into single cells and encapsulating the cells in gel beads; ii) culturing the encapsulated cells under static conditions in an incubator; iii) transferring the encapsulated cells to a fluidized bed bioreactor of a cell expansion device using a pump that provides a flow velocity of approximately 0.1 mm / s to approximately 5 mm / s in the bioreactor to form organoids; and iv) recovering the gel beads from the fluidized bed bioreactor for organoid collection.

Description

[Technical Field]

[0001] The project leading to this application was funded by the European Union's Horizon 2020 Research & Innovation Programme under Marie Sklodowska-Curie Grant Agreement No. 665992.

[0002] (Field) This disclosure relates to a method for culturing organoids and a cell expansion apparatus. [Background technology]

[0003] (background) Before testing a novel drug candidate in vivo in animals or humans, an in vitro study is typically performed using either a primary cell culture or a cell line. However, results derived from this study can be unreliable because cell cultures do not mimic the in vivo system very well. This can lead to some good drugs being rejected at the in vitro stage, while some inadequate drugs may proceed to in vivo trials.

[0004] Organoids are three-dimensional structures of heterogeneous tissue that function like in vivo tissue. In other words, these three-dimensional tissue structures mimic organs better than conventional cell culture monolayers. Therefore, organoids offer an opportunity to create cellular models of diseases, which can be studied to better understand the causes of diseases and identify possible treatments.

[0005] Organoids are often derived from stem cells, which can differentiate into brain organoids, renal organoids, cardiovascular organoids, and other types of organoids. Organoid technology has also been used to create models of human rectal cancer progression. These organoids can be derived from normal intestinal cells mutated to transform into cancer cells, or they can be induced from tumor cells themselves. Organoids derived from tumors have been shown to be good reflections of the original tumor, offering improved in vitro drug testing opportunities. By providing improved predictive capabilities, the use of patient-derived organoids has the potential to revolutionize drug discovery, reducing the costs and time commonly associated with later clinical trial failures, resulting in greater patient benefits and reducing the use of animals.

[0006] While organoids are increasingly used in research, their high potential for industrial applications has yet to be fully realized. Several different aspects play a role in organoid formation, and the currently used manual static culture of organoids is not a suitable approach for translating this technology from basic research to streamlined manufacturing.

[0007] In reality, organoid culture is still in its nascent stages and is highly inefficient. 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). This is a labor-intensive and expensive process that yields small quantities and exhibits significant batch-to-batch variability. Furthermore, conventional cell culture in well plates, dishes, and flasks is notoriously wasteful in terms of culture medium components and disposable laboratory consumables. There is a significant need to optimize organoid generation into a streamlined, efficient, and reliable process in order to create organoid cultures suitable for mainstream adoption in the drug discovery process. [Overview of the Initiative] [Means for solving the problem]

[0008] (Summary) In static cell culture, mass transfer (i.e., exchange of nutrients and gases) is limited by a clear concentration gradient resulting from the accumulation of inhibitory waste products and nutrient depletion. Under static conditions, nutrient supply and waste removal occur solely by diffusion and are therefore limited to a distance of approximately 200 μm, which is roughly the distance over which oxygen movement is limited by diffusion in tissues. Alternatively, in dynamic culture using a bioreactor, cells exist in a more homogeneous environment due to mixing, providing improved mass transfer to the cells, which can simulate biological, physical, and mechanical environments and may facilitate the formation of complex structures.

[0009] Some dynamic systems include spinner flasks, rocking bioreactors, and wave bioreactors, but these do not adequately mimic the in vivo microenvironment within the body. A better alternative to organoid culture is a perfusion bioreactor, which can provide a downward, orthogonal flow environment or a microgravity environment. The latter includes rotating-wall bioreactors and fluidized-bed bioreactors, which provide greater mass transfer.

[0010] Bioreactors have been used to culture organoids in only a few cases (e.g., those summarized in Table 1). Some of these studies have suggested that the use of bioreactors not only substantially improves the generation of various 3D suspension organoids compared to conventional static organoid culture, but also increases the differentiation yield for organoids derived from pluripotent stem cells (PSCs) (Ovando-Roche et al., 2018). In fact, organoid generation from PSCs (embryonic PSCs or artificial PSCs) (e.g., brain organoids and retinal organoids) was increased by bioreactors. These bioreactors improved mass transport, allowing for longer culture times and larger organoid sizes, which are not possible under static conditions due to the concentration gradient and the lack of vascular structures in the organoids. For example, bioreactors were used to enhance nutrient absorption by brain organoids, allowing them to reach a diameter of approximately 4 mm and be cultured for nearly 10 months (Lancaster et al., 2013). Agitated tanks constitute the majority of bioreactors used, and they are not for large-scale production. Rather, they are primarily used for mass transfer purposes to improve organoid maintenance over long periods and to obtain larger organoid sizes (not organoid quality). This is difficult under static conditions due to the lack of vascular structure in the organoids, which leads to the formation of concentration gradients and, worse, necrotic cores. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0011] Accordingly, in a first aspect of the present disclosure, a method for expanding an organoid is provided, comprising the steps of: i) dissociating the organoid into single cells and encapsulating the cells in gel beads; ii) culturing the encapsulated cells under static conditions in an incubator; iii) transferring the encapsulated cells to a fluidized bed bioreactor of a cell expansion device using a pump that provides a flow velocity of approximately 0.1 mm / s to approximately 5 mm / s in the bioreactor to form an organoid; and iv) recovering the gel beads from the fluidized bed bioreactor for organoid collection.

[0012] In a second embodiment, the disclosure provides a cell expansion apparatus comprising: i) a fluidized bed bioreactor having a fluid inlet, a fluid outlet, a flow divider located proximal to the fluid inlet, and a minimum volume of 50 ml; and ii) a reservoir fluidized to a pump and a gas exchange unit, wherein the gas exchange unit is fluidized to either the fluid inlet or the fluid outlet of the fluidized bed bioreactor, and the fluid outlet of the fluidized bed bioreactor is fluidized to the reservoir.

[0013] In a further embodiment, the present disclosure provides a method for expanding an organoid, comprising the steps of: i) dissociating the organoid into single cells and encapsulating the cells in gel beads; ii) culturing the encapsulated cells under static conditions in an incubator; iii) transferring the encapsulated cells to a fluid-bed bioreactor using a pump that provides a flow rate of about 0.1 mm / s to about 5 mm / s in the bioreactor to form an organoid; and iv) recovering the gel beads from the fluid-bed bioreactor for organoid collection, wherein the gel comprises about 0.1% to about 1% arginate, about 0.001% to about 0.02% gellan gum, and about 2 mg / ml to about 10 mg / ml of extracellular matrix protein.

[0014] The inventors have determined that a fluidized bed bioreactor can be used as part of a cell expansion device to further scale up organoid culture, providing a more efficient and scalable expansion system. In a fluidized bed bioreactor, cell culture medium enters from the bottom of the bioreactor and exits from the top, suspending the encapsulated organoid particles, thereby allowing their entire surface to be in contact with the culture medium. As a result, the fluidized bed bioreactor is characterized by high-velocity transfer of matter and heat, low shear, and theoretically high cell density (and therefore smaller reactor volume for the same final cell number), all of which are beneficial for growing organoids encapsulated in a suitable matrix.

[0015] Fluidized bed bioreactors are generally advantageous for tissue engineering applications, but have been largely overlooked in favor of agitated tanks and flasks (which are simpler culture systems). Fluidized bed bioreactors are well-suited for particulate scaffolds, single cell populations, or spheroids (Lucena-Thomas et al., 2020) and can provide higher cell densities and a more defined culture environment than agitated tanks, as well as good mixing without the need for an impeller, and therefore lower shear levels than agitated tanks. Fluidized bed bioreactors have been widely used in wastewater treatment applications (Bello et al., 2017) and have been increasingly explored as artificial liver devices (Legallais et al., 2000, Lu et al., 2016, Naghib et al., 2016, David et al., 2004), but have yet to be introduced into the field of organoids.

[0016] The cell expansion device of the present disclosure is advantageous in that it can solve the problem of the need for a scalable bioreactor with good (non-rate-limiting) mass transfer conditions and high culture density. The advantages of this device include that less material (e.g., culture medium and other factors) is required per unit of organoids; more consistent time-averaged medium supply / waste removal per organoid; ease of collection from the bioreactor; easy scalability of the device; and that it can be more space-efficient than other known systems, particularly with respect to its footprint. Using a fluidized bed bioreactor can also be advantageous in that dead cells and / or any other debris present in the culture medium can be "counter centrifugally separated" (elutriated) from the bioreactor chamber as the culture medium passes through the bioreactor.

[0017] Another important advantage of the fluidized bed bioreactor is the low shear stress imposed on the cells, which is due to the absence of mechanical impellers (which can cause direct impact on the cells) and foam formation (which can cause cell damage due to microbubble rupture), which are common problems in stirred tank bioreactors. Furthermore, by operating the bioreactor under fluidization conditions in contrast to the packed state, a higher liquid-solid interfacial area can be achieved when the cells are suspended from each other by the liquid, and the resulting high degree of mixing significantly improves the transfer of mass and heat between the medium and the cells, supporting the kinetics of cell metabolism.

[0018] (Brief Description of the Drawings) The present disclosure will now be described in detail by way of example only with reference to the drawings.

Brief Description of the Drawings

[0019] [Figure 1] FIG. 1 shows a schematic diagram of a cell culture device (FBB pilot).

[0020] [Figure 2] Figure 2 shows representative images of organoids cultured in the cell culture system of this disclosure (A) and representative images of organoids cultured in CXP1 (B). These organoids exhibit similar morphologies.

[0021] [Figure 3] Figure 3 shows an analysis of organoid morphology, comparing organoids cultured in the cell culture system of this disclosure (FBB pilot) with organoids cultured in CXP1. Average diameter (A), organoid density relative to matrix volume (B), organoid density relative to Matrigel® volume (C), viability (D), and cell growth (E). No significant differences were observed. Error bars indicate standard deviation (SD). N=3.

[0022] [Figure 4] Figure 4 shows the results from drug response assays on organoids collected from the cell culture system of this disclosure (FBB pilot) and organoids collected from CXP1. These results indicate that the organoids behaved similarly. Error bars indicate the standard error (SEM) of the mean. N=1, as well as n=4 for CXP1 and n=12 for the CXP2 pilot (quadruple measurements from three vials). [Figure 5] Figure 5 shows a schematic diagram of the cell culture device (FBB50).

[0023] [Figure 6] Figure 6 shows representative images of organoids cultured in the cell culture system (FBB50) of this disclosure and representative images of organoids cultured in CXP1. These organoids exhibit similar morphology. Scale bar: 500 μm.

[0024] [Figure 7]Figure 7 shows representative images of organoids collected from the cell culture system (FBB50) of this disclosure, frozen, thawed, replated in a Matrigel® dome, and cultured for 7 days, and representative images of organoids collected from CXP1, frozen, thawed, replated in a Matrigel® dome, and cultured for 7 days. These organoid samples exhibit similar morphology. Scale bar: 500 μm.

[0025] [Figure 8] Figure 8 shows the MS3 count and CellTiter-Glo® (CTG) assay results from organoids collected from the cell culture system (FBB50) and CXP1 of this disclosure, frozen, thawed, replated in a Matrigel® dome, and cultured for 7 days. These organoid samples show similar growth (MS3 count) and metabolism / viability (CTG).

[0026] [Figure 9] Figure 9 shows the results from drug response assays on organoids collected from the cell culture system (FBB50) and CXP1 of this disclosure. The results indicate that the organoids behaved similarly. Error bars indicate the standard error (SEM) of the mean. N=1 and n=4.

[0027] [Figure 10] Figure 10 shows stiffness measurements from GAM (Matrigel® 4.25–5 mg / mL protein, arginate 0.25% w / v, gellan gum 0.005% w / v), Matrigel® alone (7.67 mg / mL protein), and 1% w / v arginate, using a uniaxial compression assay. These results indicate that GAM is a soft matrix with similar stiffness to Matrigel®. The stiffness of arginate was significantly higher than that of GAM and Matrigel®.

[0028] [Figure 11] Figure 11 shows schematic diagrams of cell culture devices (FBB15 and FBB25).

[0029] [Figure 12] Figure 12 shows representative images of colorectal cancer organoids and breast cancer organoids cultured in the cell culture system (FBB25) of this disclosure, as well as representative images of colorectal cancer organoids and breast cancer organoids cultured in a Matrigel® dome. These organoids exhibit similar morphologies. Scale bar: 200 μm. [Modes for carrying out the invention]

[0030] (explanation) In this specification, the cell growth apparatus refers to an apparatus in which each of the described components can be independently modified, substituted, or exchanged. This customizable apparatus offers particular advantages, especially with respect to scalability. Process conditions commonly considered during scalability are mixing, mass / heat transfer, and shear effects, all of which are known to be highly improved using the essential system and beneficial for organoid growth. Furthermore, the system offers flexible operation, where cell feeding and waste removal can be automated and controlled for more efficient process and reagent optimization based on the described sensor monitoring system, enabling higher densities to be achieved with a smaller footprint and reduced capital and operating costs.

[0031] This cell expansion device is particularly suitable for expanding organoids.

[0032] 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, 2014b). 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, with those cells performing 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 the intestinal crypt-villous structure in vivo, containing various cell types of the intestine (intestinal absorptive epithelial cells, goblet cells, Paneth cells, enteroendocrine cells, and stem cells) surrounding a cystic lumen (Sato et al., 2011).

[0033] In some embodiments, the cells described herein are eukaryotic cells, and in some embodiments, the cells are mammalian cells. Typically, cells are obtained from a multicellular organism (e.g., human) and then cultured into organoids according to the methods described herein before further processing. The cells may be cancer cells (e.g., cells obtained from a tumor biopsy). The organoids may be grown from pluripotent stem cells (embryonic pluripotent stem cells or induced pluripotent stem cells) or from tissue biopsies containing adult stem cells. The cells may be healthy or representative of a disease (e.g., cells obtained from healthy tissue or cells obtained from a diseased state (e.g., malignant tumor biopsy)). When grown in vitro under conditions supporting stem cell maintenance, the 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.

[0034] The term "expansion" refers to increasing the total number of cells in a culture by either increasing the total number of organoids in the culture, increasing the number of cells per organoid, or a combination of both. In contrast, "maintenance" of a culture (particularly a stem cell culture) refers to keeping its cells alive and viable (in the case of stem cells, keeping them in an undifferentiated "stem cell" state), without necessarily increasing the total number of cells present. Any "excess" cells generated during maintenance are usually discarded to maintain the overall number of unchanged cells.

[0035] Scaling up fluidized bed bioreactors has traditionally been done through hydraulic similarities based on dimensional parameters (e.g., the following simplified set proposed by Glicksman (Glicksman, 1984; Rudisuli et al., 2012)):

number

[0036] In the above equation, u0 is a predetermined air velocity; u mf g is the minimum fluidization velocity; g is the gravitational constant; d c and h c θ is the diameter and height of the bioreactor, respectively; ψ is the sphericity of the particles; and psd is the particle size distribution. These governing equations were initially created for gas-solid systems, but the same methodology was extended to liquid-solid systems due to similarities in design, geometry, hydraulic properties, and kinetic properties (Mendonca da Silva et al., 2020).

[0037] The fluidized bed bioreactor of this cell expansion device typically has a minimum volume of at least approximately 50 ml, at least approximately 250 ml, or at least approximately 1000 ml. The maximum volume of the fluidized bed bioreactor may be approximately 10 L, approximately 9 L, approximately 8 L, approximately 7 L, approximately 6 L, or approximately 5 L. In other words, the fluidized bed bioreactor may have a volume of approximately 50 ml to approximately 10 L, approximately 250 ml to approximately 10 L, or approximately 1000 ml to approximately 10 L.

[0038] Although not constrained by theory, the design of the fluidized bed bioreactor is r n The inventors have determined that the system can be scaled up while maintaining cell (e.g., organoid or spheroid) yield using the formula (wherein r = radius of the column and n = forces in the system, i.e., inertia, viscosity, shear, and gravity).

[0039] The fluid communication between the fluid outlet and reservoir of the fluidized bed bioreactor typically includes one or more sensors selected from oxygen sensors, pH sensors, pressure sensors, metabolite sensors, temperature sensors, or combinations thereof. Oxygen sensors and / or metabolite sensors allow for indirect monitoring of cell density over time by analyzing the cell culture medium with metabolic data from inline measurements (e.g., oxygen) and from offline measurements (e.g., glucose concentration, lactate concentration, glutamine concentration, ammonia concentration, glutamate concentration, or combinations thereof).

[0040] Data from one or more of these sensors can be continuously monitored and used to control the operating parameters of the fluidized bed bioreactor (e.g., flow rate, culture medium volume, gas exchange, or a combination thereof).

[0041] Preferably, the device includes at least one pressure sensor located between the fluid outlet of the fluidized bed bioreactor and the reservoir, and / or at least one pressure sensor located between the reservoir and the fluid inlet of the fluidized bed bioreactor. This allows the pressure of the fluid flowing into the fluidized bed bioreactor to be monitored, and / or the pressure of the fluid flowing out of the fluidized bed bioreactor to be monitored.

[0042] Therefore, pressure sensors can be used to monitor cell density, as the size of particles in the bioreactor (e.g., gel beads encapsulating cells or organoids) increases over time as the organoids grow. In particular, subtracting the pressure measured at or near the fluid outlet of the fluidized bed bioreactor from the pressure measured at or near the fluid inlet of the fluidized bed bioreactor provides a pressure drop across the fluidized bed bioreactor. Preferably, the cell expansion device is operated to provide a constant pressure drop across its fluidized bed bioreactor.

[0043] Metabolite sensors may include one or more lactate sensors. Lactate levels can provide a strong indicator of how well the organoid is growing, especially if the organoid is a cancerous organoid.

[0044] After passing through a fluidized bed bioreactor, the cell culture medium may be removed, recycled, or returned to the reservoir without recycling. Recycling of the cell culture medium typically involves passing the medium through a series of steps to remove waste molecules of various sizes. The recycled cell culture medium can then be returned to the reservoir. Alternatively, after passing through a fluidized bed bioreactor, the cell culture medium can be returned to the reservoir without recycling.

[0045] The pump maintains the fluidity of the encapsulated organoids in the fluidized bed bioreactor and ensures that the nutrient and oxygen demands of the cells being expanded in the apparatus can be met. Preferably, the pump is a positive displacement pump. Such a pump has been found to successfully maintain fluidity, successfully meet the nutrient and oxygen demands of the cells, and also successfully ensure that the cells are not washed out of the fluidized bed bioreactor by the flow of cell culture medium. However, the pump also allows the flow rate of the cell culture medium to be increased as desired, if desired, in order to counter-centrifuge the cells, or the gel beads containing the encapsulated cells or organoids, from the fluidized bed bioreactor.

[0046] The positive displacement pump may be a peristaltic pump. Such a pump typically includes a flexible tube mounted inside a pump casing. The rotor inside the casing has many “wipers” or “rollers,” which compress the flexible tubing as they rotate. The portion of the tube under compression is closed, forcing fluid to move through it, and as the tube opens to its natural state after passing through the rollers, more fluid is drawn into it. Typically, there are two or more rollers compressing the tube, trapping a large volume of fluid between them. This large volume of fluid is then moved through the tube to the pump outlet. The flexible tube contains the fluid in such a way that it does not come into direct contact with the pump or any of its components. This means that the fluid may be contained within sterile tubing, facilitating aseptic and sterilization processes.

[0047] The cell expansion apparatus may further include an air pump connected to a gas exchange unit and in fluid communication with the gas exchange unit. If necessary, the fluid communication between the air pump and the gas exchange unit may include a filter and / or an airflow meter.

[0048] The fluid communication between the gas exchange unit and the fluid inlet or outlet of the fluidized bed bioreactor may include one or more sensors selected from oxygen sensors, pH sensors, pressure sensors, metabolite sensors, temperature sensors, or combinations thereof, the sensors being as described above. As discussed above, the pressure sensor, oxygen sensor, and / or metabolite sensor allow for indirect monitoring of cell density.

[0049] The fluidized bed bioreactor typically includes a vertical column with a flow divider at its bottom. Fluid enters the column from the bottom and exits from the top. This upward flow of fluid suspends particles (e.g., gel beads containing cells or organoids), resulting in their entire surface being in contact with the fluid. The role of the flow divider is to support the bed and to provide the largest possible radially uniform fluid velocity distribution, thus minimizing flow irregularities (e.g., uneven flow and bulk circulation). Thus, the flow divider controls the flow of fluid (e.g., culture medium) into the fluidized bed bioreactor.

[0050] In some embodiments, the stream divider may be a porous substrate (e.g., a bed of solid particles or beads). The porosity of the substrate may be adjusted, for example, according to the size of the particles or beads, the size of the fluidized bed bioreactor, and / or the type of cells being cultured. The particles or beads are preferably formed from a biocompatible, sterilizable, inert material (e.g., plastic or glass). For example, the stream divider may contain solid particles or beads having a diameter of about 0.5 mm to about 5 mm, or about 2 mm to about 4 mm. In some embodiments, the stream divider is a bed of glass beads, the beads having a diameter of about 3 mm.

[0051] The flow divider may include one or more plates, each of which allows fluid to pass through. The fluid flow is typically controlled by the size, density, and pattern of the holes or voids in the plates. The number and diameter of the holes or voids in the plates should be balanced for good fluid distribution and pressure drop. If the pressure drop is too low, the fluid will not be distributed adequately; if it is too high, the system may leak or become dangerous. The plates are preferably formed from biocompatible and sterilizable inert materials (e.g., plastic or glass).

[0052] In some embodiments, the flow divider may include a flow divider plate and / or a flow divider substrate. The flow divider plate may be a plate containing holes or voids through which a fluid (e.g., culture medium) can pass. The size, density, and pattern of these holes or voids may be adjusted, for example, according to the size of the plate, the size of the fluidized bed bioreactor, and / or the type of cells being cultured. The holes or voids in the flow divider plate are preferably smaller than the particles cultured in the bioreactor (e.g., encapsulated organoids). This can prevent the particles from becoming trapped in the holes or voids and blocking the flow divider. For example, the flow divider plate may contain holes having a diameter of 3 mm or less, 2 mm or less, or 1 mm or less. Preferably, the flow divider plate contains holes having a diameter of about 1 mm.

[0053] The flow divider substrate is preferably a porous substrate (e.g., a sintered glass disk). The porosity of the substrate can be adjusted, for example, according to the size of the substrate, the size of the fluidized bed bioreactor, and / or the type of cells being cultured. For example, the flow divider substrate may contain pores having a diameter of about 20 μm to about 500 μm, preferably about 60 μm to about 300 μm, and more preferably about 100 μm to about 200 μm.

[0054] Preferably, the flow divider substrate is located between the fluid inlet and the flow divider plate of the fluidized bed bioreactor.

[0055] If necessary, the cell expansion system may include a flow divider structure (e.g., a ball ring) within the fluidized bed bioreactor to prevent or reduce aggregation between gel beads. This flow divider structure may be used in combination with or as a substitute for the flow divider plates and / or flow divider substrates described above.

[0056] The gas exchange unit may be particularly useful for oxygen enriching the cell culture medium passing through this cell expansion system. Suitable gas exchange units may include silicon membrane and / or hollow fiber gas exchange units.

[0057] Fluid communication within the expansion apparatus is typically provided by tubing through which the culture medium flows. This tubing may be flexible and formed from a biocompatible, sterilizable, and inert material (e.g., silicone). The tubing is preferably transparent, which allows for easy observation of any bubbles or flow instability. Other suitable materials include polypropylene-based thermoplastic elastomers and polypropylene-based thermoplastic elastomers (e.g., PharMed®). Such materials may be sufficiently suitable to withstand the severity of peristaltic pumping.

[0058] The minimum volume of this cell expansion device (including the cell culture medium reservoir, fluidized bed bioreactor, and tubing) is typically at least about 250 ml, or at least about 1 L, or at least about 5 L. In some embodiments, the maximum volume of this cell expansion device (including the cell culture medium reservoir, fluidized bed bioreactor, and tubing) may be about 50 L, or about 100 L, or about 150 L.

[0059] Preferably, the components of the apparatus described herein are sterile and / or aseptic in order to avoid contamination of the cells being expanded in the apparatus.

[0060] The cell expansion apparatus is preferably a closed system. If the cell expansion apparatus is a closed system, the entire apparatus is preferably sterile and / or aseptic. Indeed, the apparatus is advantageously easy to clean and reusable. The apparatus can also be easily sterilized between uses, for example, by autoclaving.

[0061] The disclosure also provides a method for expanding spheroids or organoids, the method comprising a method for expanding organoids, the method comprising: i) a step of dissociating organoids into a group of single cells and encapsulating those cells in gel beads; ii) a step of culturing the encapsulated cells in an incubator under static conditions; iii) a step of transferring the encapsulated cells to a fluidized bed bioreactor of a cell expansion device using a pump that provides a superficial flow velocity of about 0.1 mm / s to about 5 mm / s in the bioreactor to form organoids; and iv) a step of recovering the gel beads from the fluidized bed bioreactor for collection of those organoids.

[0062] Since the cells are in a fluid state and are not counter-centrifuged from the bioreactor chamber by the culture medium, the liquid needs to be supplied within its minimum fluidization rate and terminal velocity range. The pump can be operated to provide a superficial flow velocity of approximately 0.5 mm / s to approximately 4 mm / s in the bioreactor. Such a range has been found to be sufficient to maintain fluidity, meet the nutrient and oxygen demands of the cells, and also to ensure that the gel beads are not washed away from the fluidized bed bioreactor by the flow of cell culture medium. As discussed above, the flow rate can be adjusted as desired, if desired, to counter-centrifugate the gel beads.

[0063] The method may include (a) monitoring the pressure of the fluid flowing into the fluidized bed bioreactor, and / or (b) monitoring the pressure of the fluid flowing out of the fluidized bed bioreactor. If both (a) and (b) are monitored, the pressure drop can be calculated by subtracting the pressure in (a) from the pressure in (b). Preferably, the superficial flow velocity is adjusted to maintain a constant pressure drop.

[0064] The encapsulation of gel beads can be carried out, for example, using an electrospray system, as described in Park et al (Int J Pharmaceut. 427 (2012) 417-425) and Wright et al (J. Biomed. Mater. Res. 102 (2013) 3393-3400). Alternatively, an extrusion drop method may be used, in which cells are suspended in a gel solution and extruded into a gelling bath to form beads, which are then collected and transferred to a fluidized bed bioreactor.

[0065] The encapsulated cells from step (ii) are typically cultured under static conditions for at least about one day.

[0066] The encapsulated cells of step (iii) can be cultured to form organoids with a diameter of approximately 20 μm to approximately 700 μm. Organoids with a diameter greater than 700 μm have frequently been observed to develop necrotic cores, which means they cannot be used to accurately determine the effects of toxic compounds because they may have pre-existing necrotic cores and would produce atypical results. The encapsulated cells of step (iii) can be cultured to form organoids with a diameter of approximately 20 μm to approximately 500 μm, preferably approximately 20 μm to approximately 200 μm, and more preferably approximately 40 μm to approximately 90 μm.

[0067] The cells enclosed in step (iii) can be cultured for approximately 3 to 15 days, or approximately 4 to 7 days. Typically, the culture time can be determined by monitoring the size of the organoid, with a target diameter of approximately 40 μm to 85 μm.

[0068] In this specification, "approximately X days" of incubation time typically refers to X days ± 12 hours or X days ± 6 hours.

[0069] The cells can be encapsulated at a density of approximately 200 to 2500 cells per μL of gel. However, as those skilled in the art will recognize, the optimal seeding density varies within this range, depending on the cell line or organoid being expanded.

[0070] Preferably, the gel is 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 preferably isotonic and / or pH neutral, both of which are beneficial for cell health. A suitable aqueous buffer solution is phosphate-buffered saline.

[0071] Hydrogels typically have a Young's modulus of less than 10 kPa when measured by uniaxial unconfined oscillatory compression. For example, hydrogels may have a Young's modulus of approximately 5 kPa to 10 kPa when measured by uniaxial unconfined oscillatory compression.

[0072] The gel may contain about 0.2% to about 0.5% w / v of arginate or about 0.2% to about 0.4% w / v of arginate. In some embodiments, the gel may contain about 0.2% to about 0.3% w / v of arginate. The gel may contain about 0.25% w / v of arginate.

[0073] 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.

[0074] 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), 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.

[0075] The gel 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. In some embodiments, the gel may contain approximately 0.004% to approximately 0.006% w / v gellan gum. The gel may contain approximately 0.005% w / v gellan gum.

[0076] 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.

[0077] 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.

[0078] The gel may further contain a protein matrix, which provides extracellular matrix (ECM) proteins that can mimic the extracellular environment and act as a scaffold. The protein matrix typically contains ECM proteins (e.g., collagen I and / or collagen IV, as well as laminin). The protein matrix may further contain ECM proteins (e.g., entactin, perlecan, or gelatin, or a combination thereof). The protein matrix may be of artificial or biological origin.

[0079] The biologically derived protein matrices that may be used in the gels 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 many growth factors. Biologically derived protein matrices with reduced growth factors may also be used.

[0080] 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). Preferably, the biological protein matrix is ​​Matrigel®.

[0081] 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.

[0082] The gel may contain an ECM protein content derived from its protein matrix of approximately 2 mg / ml to approximately 10 mg / ml, or approximately 3 mg / ml to approximately 8 mg / ml. In some embodiments, the gel contains approximately 4 mg / ml to approximately 6 mg / ml of ECM protein or approximately 4.25 mg / ml to approximately 5 mg / ml of ECM protein. Thus, the gel reduces the amount of protein matrix required. Therefore, the protein matrix component of the gel is diluted by its other components (i.e., arginate and gellan gum). While sufficient protein matrix is ​​present to provide the biological cues required by cells or organoids, the problem associated with the variability of the protein matrix of biological origin is translated into dilution of this component. This dilution of the protein matrix component also allows for control of the final protein concentration in the composition.

[0083] The gel may further contain organoids or cells. In some embodiments, the gel contains organoids.

[0084] Therefore, the gel may contain about 0.2% w / v to about 0.5% w / v arginate, about 0.002% to about 0.01% gellan gum, about 3 mg / ml to about 8 mg / ml of ECM protein, and organoids or cells. This hydrogel mixture typically has a Young's modulus of about 5 kPa to about 10 kPa when measured by uniaxial unconfined oscillatory compression. In contrast, the inventors have determined that 1% (w / v) arginate alone (i.e., not used in combination with any other hydrogel) typically has a Young's modulus of about 36 kPa when measured by uniaxial unconfined oscillatory compression.

[0085] These gel beads may have a diameter of approximately 200 μM to 4000 μM. They may also have a diameter of approximately 1000 μm to 3000 μm. For example, these gel beads may have a diameter of approximately 2.1 mm, 2.2 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, or 2.9 mm.

[0086] The gel beads from step (iv) can be recovered by countercentrifugation. In other words, the superficial flow velocity in the bioreactor can be increased to flush the beads out of the fluidized bed bioreactor. Alternatively, the gel beads can be aseptically recovered from the fluidized bed bioreactor, for example, by removing the top section of the bioreactor or by inverting the bioreactor. If the flow of culture medium through the bioreactor is stopped, the gel beads will naturally settle to the bottom of the bioreactor, from which they can be easily recovered, for example, using an aspirator.

[0087] The gel beads can be dissolved to collect spheroids or organoids. Preferably, the gel beads are dissolved using a lysis buffer to break the gel and release the organoids 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 or gel blend contains Matrigel®, the lysis buffer preferably contains Cell Recovery Solution, which can remove Matrigel® while avoiding cellular dissociation of the organoids.

[0088] If necessary, the recovered organoids may be centrifuged to separate them from any remaining culture medium after the gel has been dissolved. Preferably, the centrifugation settings are adjusted to increase gravity and decrease braking speed to prevent resuspension of the cell pellet.

[0089] 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). [Examples]

[0090] (Examples) (Example 1) The pilot FBB expansion apparatus, as shown in Figure 1, includes an incubator 1, which includes a culture medium reservoir 5, a fluidized bed bioreactor 2 (which houses the encapsulated organoids and includes a flow divider 7), a peristaltic pump 4 for perfusion, a hollow fiber exchange module 3 and an air pump 6 for gas-liquid mass transfer, and an oxygen sensor / detector for measuring the oxygen difference across the fluidized bed bioreactor 2.

[0091] For the fluidized bed bioreactor, an Omnifit chromatography column (5 cm inner diameter) was repurposed for organoid culture using screw-type fixed ends (1 cm inner diameter inlet and outlet). The flow divider 7 consisted of a sintered glass filter disc (5 cm outer diameter × 4 mm, 100-160 μm pores) and a custom-made plastic flow divider (1 mm diameter holes). The volume of the bed in its unexpanded state was between 30% and 60% of the reactor volume.

[0092] The operating conditions for fluidizing the particles are, u mf=0.66mm / s and u t It was determined that the value should be between 4.22 mm / s. When operated at 0.94 mm / s, the fluidized bed bioreactor maintained a good oxygen concentration level at its outlet.

[0093] The process for culturing organoids using this cell culture system includes the following steps: 1. Organoids were dissociated into single-cell groups and encapsulated in hydrogel beads at a rate of 600 cells / μL. 2. The enclosed organoids were maintained in an incubator under static conditions for two days to enable cell retrieval after organoid dissociation. 3. The enclosed organoids were transferred to a fluidized bed bioreactor and operated at a flow rate of 0.94 mm / s for 3 days. 4. After achieving the desired size of organoids (40 μm to 85 μm for drug assays), the gel beads were recovered from the fluidized bed bioreactor by stopping the pump, removing the upper end of the fluidized bed bioreactor, and aseptically removing the gel beads. 5. Organoids were collected from the beads, characterized, and frozen in cryovials for future use.

[0094] The organoids collected from this cell culture system exhibited a similar morphology to those cultured in CPX1 (as described in WO2018 / 011558) (Figure 2).

[0095] Figure 3 shows that organoids collected from a fluidized bed bioreactor and organoids collected from CXP1 showed similar diameters, final organoid densities (relative matrix volume or hydrogel volume), viability, and increased cell percentages.

[0096] After being collected from a fluidized bed bioreactor, the organoids were replated in Matrigel®. These organoids showed comparable functional responses to three different drug groups (Figure 4).

[0097] Therefore, the cell expansion system of this disclosure provides a scalable bioreactor with good (rate-non-limiting) mass transfer conditions and high culture density. Other bioreactors used to date, which use gels (e.g., Matrigel®) to culture organoids, do not have the same level of high culture density or the ability to counter-centrifuge beads.

[0098] (Example 2) The FBB50 expansion apparatus includes an incubator, which contains a culture medium reservoir, a peristaltic pump for perfusion, a hollow fiber exchange module and air pump for gas-liquid substance transfer, and a fluidized bed bioreactor (50 mm inner diameter) (which includes a flow divider (3 mm glass beads) and houses the encapsulated organoids), all connected by tubing that allows communication between these components. The apparatus further includes temperature, oxygen, and pressure sensors / detectors to measure the oxygen, temperature, and pressure differences across the fluidized bed bioreactor, as shown in Figure 5. A lactate sensor / detector is also included between the fluid outflow from the fluidized bed bioreactor and the culture medium reservoir to monitor organoid growth and / or metabolism. Additional temperature and CO2 sensors are placed inside the incubator to monitor the overall culture conditions inside the apparatus. An air filter is provided facing the culture medium reservoir and between the air pump and the hollow fiber gas exchange unit. An airflow meter is placed between the air pump and the hollow fiber gas exchange unit. A pinch clamp is placed on the tubing between the hollow fiber gas exchange unit and the fluid inlet of the fluidized bed bioreactor.

[0099] The process for culturing organoids using this cell culture system includes the following steps: 1. Organoids were dissociated into single-cell groups and encapsulated in hydrogel beads at a rate of 600 cells / μL. 2. The enclosed organoids were maintained in an incubator under static conditions for two days to enable cell retrieval after organoid dissociation. 3. The enclosed organoids were transferred to a fluidized bed bioreactor and operated at a flow rate of 0.94 mm / s for 3 days. 4. After achieving the desired size of organoids (40 μm–85 μm for drug assays) (this took 3 days), the gel beads were recovered from the fluidized bed bioreactor by stopping the pump, removing the upper end of the fluidized bed bioreactor, and aseptically removing the gel beads. 5. Organoids were collected from the beads, characterized, and frozen in cryovials for future use.

[0100] The organoids collected from the FBB50 instrument showed a similar morphology to those cultured in CPX1 (as described in WO2018 / 011558) (Figure 6).

[0101] The FBB50 apparatus produced nearly 15 times more organoids compared to a comparative process performed using the CPX1, and reduced the amount of Matrigel® required by 40% (Table 1). [Table 1]

[0102] The FBB50 device further reduced the required culture medium volume by 33% compared to the CPX1 (Table 2). [Table 2]

[0103] Organoids generated using the FBB50 instrument and organoids generated using the CXP1 process were thawed and seeded in domes at a rate of 20 organoids / Matrigel® μl. After 7 days, the endpoint MS3 count (12 × 50 μl dome, 6-well plate) and CellTiterGlo assay (10 μl dome, 384-well plate) were performed. As shown in Figure 7, when organoid samples were collected from both the FBB50 and CXP1 instruments, frozen, thawed, and replated in Matrigel®, they showed similar morphology and growth. As shown in Figure 8, the MS3 count and CellTiterGlo assay results were similar for both organoid groups.

[0104] Organoids generated using the FBB50 instrument and organoids generated using the CXP1 process were thawed and seeded into domes at a rate of 20 organoids / μl of Matrigel®. After 7 days, an endpoint drug assay (10 μl dome, 384-well plate) was performed. As shown in Figure 9, when the organoids were collected from both the FBB50 and CXP1 instruments, frozen, thawed, and replated in Matrigel®, they showed similar responses to the three drug groups.

[0105] Therefore, this FFB cell expansion apparatus offers significant advantages in terms of increased scalability, reduced capital expenditure and operating costs, generation of a larger number of organoids, smaller equipment footprint, and more efficient process and reagent optimization.

[0106] (Example 3) Stiffness comparisons were performed using a uniaxial compression assay between GAM (Matrigel® 4.25 mg / mL protein, arginate 0.25% w / v, gellan gum 0.005% w / v), Matrigel® alone (7.67 mg / mL protein), and arginate alone (1% w / v).

[0107] More specifically, 10 mm diameter, 2 mm to 2.6 mm thick disks of Matrigel®, arginate, and GAM were prepared using the wells of a 48-well cell culture plate as molds. Similarly, gelation of Matrigel® occurred at 37°C, while gelation of arginate and GAM occurred at 37°C for 5 minutes with the gentle addition of a 135 mM calcium chloride solution, which was then removed by washing three times with culture medium.

[0108] After 24 hours in an incubator at 37°C, the gels were placed in culture medium, and the disks were gently removed from their wells, with excess liquid dried on tissue. The dimensions of the gel disks were measured using calipers before loading them between parallel plates in a dynamic mechanical analyzer (DMA1, commercially available from Mettler Toledo). The Young's modulus for each disk was determined by uniaxial unconfined oscillatory compression at 100 μm / sec for 5 minutes at 37°C.

[0109] The results are shown in Figure 10 and Table 3. [Table 3]

[0110] The values ​​for this 1% w / v arginate were consistent with literature using a similar uniaxial compression assay (38 kPa and 25 kPa for G-type and M-type arginate (1.2% w / v) from Ceccaldi (2012)).

[0111] While pure arginate is well-established as a biocompatible material for cell encapsulation and tissue transplantation, for organoid culture, arginate lacks the bioactive molecules (e.g., peptides or extracellular matrix proteins) necessary to support stem cell maintenance and replicate complementary biochemical, ultrastructural, and mechanical properties (e.g., cell adhesion, fate, and migration).

[0112] GAM has been shown to be a soft matrix with similar rigidity to Matrigel®, being decomposable, and simultaneously capable of producing robust beads that are stable over time. This is not possible with pure Matrigel®.

[0113] (Example 4) The FBB15 expansion apparatus, as shown in Figure 11, includes an incubator, which includes a culture medium reservoir, a fluidized bed bioreactor (which contains the encapsulated organoids and includes a flow divider), and a peristaltic pump for perfusion.

[0114] For the fluidized bed bioreactor, an Omnifit chromatography column (15 mm inner diameter) was repurposed for organoid culture using screw-type fixed ends (1 mm inner diameter inlet and outlet). The flow divider consisted of a polytetrafluoroethylene (PTFE) frit with 50 μm pores and a 10 μm nylon membrane at the top. The volume of the bed in its unexpanded state was between 30% and 60% of the reactor volume.

[0115] The operating conditions for fluidizing the particles are, u mf =0.66mm / s and u t It was determined to be between =4.22 mm / s. The following empty velocities (u) were investigated: 0.71 mm / s, 0.94 mm / s and 1.89 mm / s (umf <u<umf)。

[0116] The process for culturing organoids using this cell culture system includes the following steps: 1. Organoids were dissociated into single-cell populations and mounted in hydrogel beads at concentrations of 500 cells / μL, 600 cells / μL, or 1000 cells / μL. 2. The enclosed organoids were maintained in an incubator under static conditions for two days to enable cell retrieval after organoid dissociation. 3. The enclosed organoids were transferred to a fluidized bed bioreactor and operated for 3 days at flow rates of 0.71 mm / s, 0.94 mm / s, or 1.89 mm / s. 4. After achieving the desired size of organoids (40 μm to 85 μm for drug assays), the gel beads were recovered from the fluidized bed bioreactor by stopping the pump, removing the upper end of the fluidized bed bioreactor, and aseptically removing the gel beads. 5. Organoids were collected from the beads, characterized, and frozen in cryovials for future use.

[0117] As shown in Tables A and B below, all tested seeding densities and empty tower velocities produced organoids with a survival rate of >70%. [Table A]

[0118] [Table B]

[0119] (Example 5) The FBB25 expansion apparatus, as shown in Figure 11, includes an incubator, which contains a culture medium reservoir, a fluidized bed bioreactor (25 mm inner diameter) (which houses the encapsulated organoids and includes a flow divider (3 mm glass beads)), and a peristaltic pump for perfusion, all connected by tubing that allows communication between these components.

[0120] The volume of the floor in the non-expanded state was between 30% and 60% of the reactor volume. The operating conditions for fluidizing the particles were determined to be between u mf = 0.66 mm / s and u t = 4.22 mm / s. When operated at 0.94 mm / s, the fluidized bed bioreactor maintained a good oxygen concentration level at its outlet.

[0121] The process for culturing organoids using this cell culture system included the following steps: 1. Dissociated the rectal colon cancer and breast cancer organoid lines into single cell populations and encapsulated them in hydrogel beads at 600 cells / μL and 1000 cells / μL matrices respectively. 2. Maintained the encapsulated organoids under static conditions in an incubator for 2 days to enable cell recovery after organoid dissociation. 3. Transferred the encapsulated organoids to a fluidized bed bioreactor and operated it at a flow rate of 0.94 mm / s. 4. After 3 days (rectal colon cancer organoids) and 13 days (breast cancer organoids) to achieve organoids of the desired size (40 μm - 85 μm), the pumps were stopped, the upper end of the fluidized bed bioreactor was removed, and their gel beads were aseptically removed to recover their gel beads from the fluidized bed bioreactor. 5. The organoids were collected from their beads, characterized, and frozen in cryovials for future use.

[0122] The organoids collected from this cell culture system showed a similar morphology to the organoids cultured in Matrigel® domes for both organoid lines (Figure 12).

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Claims

1. A method for enlarging organoids, i) A step of dissociating the organoid into a single cell and encapsulating the cell in a gel bead; ii) A step of culturing the enclosed cells in an incubator under static conditions; iii) A step of transferring the encapsulated cells to a fluidized bed bioreactor of a cell expansion device using a pump that provides a flow velocity of approximately 0.1 mm / s to approximately 5 mm / s in the bioreactor, thereby forming organoids; and iv) The step of recovering the gel beads from the fluidized bed bioreactor for collection of the organoids. Methods that include...

2. The method according to claim 1, wherein the gel is a hydrogel.

3. The method according to claim 2, wherein the hydrogel has a Young's modulus of less than 10 kPa.

4. The method according to any one of claims 1 to 3, wherein the gel comprises about 0.1% to about 1% arginate, about 0.001% to about 0.02% gellan gum, and about 2 mg / ml to about 10 mg / ml of extracellular matrix protein.

5. The method according to any one of claims 1 to 4, wherein the pump is operated to provide an empty flow velocity of about 0.5 mm / s to about 4 mm / s.

6. (a) A step of monitoring the pressure of the fluid flowing into the fluidized bed bioreactor, (b) A step of monitoring the pressure of the fluid flowing out of the fluidized bed bioreactor. The method according to any one of claims 1 to 5, further comprising:

7. A step of calculating the pressure drop by subtracting the pressure in (a) from the pressure in (b), and (d) A step of adjusting the flow velocity of the empty tower to maintain a constant pressure drop. The method according to claim 6, further comprising:

8. The method according to any one of claims 1 to 7, wherein the gel beads have a diameter of approximately 200 μM to approximately 4000 μM.

9. The method according to any one of claims 1 to 8, wherein the encapsulated cells of step (ii) are cultured under static conditions for at least about one day.

10. The method according to any one of claims 1 to 9, wherein the encapsulated cells in step (iii) are cultured to form organoids measuring approximately 20 μm to approximately 700 μm.

11. The method according to any one of claims 1 to 10, wherein the encapsulated cells of step (iii) are cultured for about 3 to about 15 days.

12. The method according to any one of claims 1 to 11, wherein the cells are encapsulated in a quantity ranging from approximately 200 cells per μL of gel to approximately 2,500 cells per μL of gel.

13. A cell expansion device, i) A fluidized bed bioreactor having a fluid inlet, a fluid outlet, a flow divider located near the fluid inlet, and a minimum volume of 50 ml; ii) A reservoir connected to a pump and gas exchange unit; Includes, The gas exchange unit is in fluid communication with the fluid inlet or fluid outlet of the fluid bed bioreactor. A cell expansion device in which the fluid outlet of the fluidized bed bioreactor is in fluid communication with the reservoir.

14. The cell expansion apparatus according to claim 14, wherein the fluid communication between the fluid outlet of the fluidized bed bioreactor and the reservoir includes one or more sensors selected from an oxygen sensor, a pH sensor, a pressure sensor, a metabolite sensor, or a combination thereof.

15. The cell expansion apparatus according to claim 14, wherein the fluid communication between the fluid outlet and the reservoir of the fluidized bed bioreactor includes a pressure sensor.

16. The cell expansion apparatus according to any one of claims 13 to 15, wherein the pump is a positive displacement pump.

17. The cell expansion apparatus according to claim 16, wherein the positive displacement pump is a peristaltic pump.

18. The cell expansion apparatus according to any one of claims 13 to 17, further comprising an air pump connected to and in fluid communication with the gas exchange unit.

19. The cell expansion apparatus according to claim 18, wherein the fluid communication between the air pump and the gas exchange unit includes a filter and / or an airflow meter.

20. The cell expansion apparatus according to any one of claims 13 to 19, wherein the fluid communication between the gas exchange unit and the fluid inlet or fluid outlet of the fluid bed bioreactor includes one or more sensors selected from an oxygen sensor, a pH sensor, a pressure sensor, a metabolite sensor, or a combination thereof.

21. The cell expansion apparatus according to any one of claims 13 to 20, wherein the fluid communication between the reservoir and the fluid inlet of the fluidized bed bioreactor includes a pressure sensor.

22. The cell expansion device according to claim 14 or claim 20, wherein the metabolite sensor is a lactic acid sensor.

23. The cell expansion apparatus according to any one of claims 13 to 22, wherein the flow divider includes one or more plates.

24. The cell expansion apparatus according to any one of claims 13 to 23, wherein the gas exchange unit is a silicon membrane and / or hollow fiber gas exchange unit.

25. A method for enlarging organoids, i) A step of dissociating the organoid into a single cell and encapsulating the cell in a gel bead; ii) A step of culturing the enclosed cells in an incubator under static conditions; iii) A step of transferring the encapsulated cells to a fluidized bed bioreactor of a cell expansion device using a pump that provides a flow velocity of approximately 0.1 mm / s to approximately 5 mm / s in the bioreactor, thereby forming organoids; and iv) The step of recovering the gel beads from the fluidized bed bioreactor for collection of the organoids. A method comprising a gel containing about 0.1% to about 1% arginate, about 0.001% to about 0.02% gellan gum, and about 2 mg / ml to about 10 mg / ml extracellular matrix protein.

26. The method according to claim 25, wherein the gel comprises about 0.2% to about 0.5% w / v of arginate, about 0.002% to about 0.01% of gellan gum, and about 3 mg / ml to about 8 mg / ml of ECM protein.

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