Method and Apparatus for Large-Scale Spheroid Generation

Cross-reference to Related Applications

[001] The following application claims benefit of U.S. Provisional Application No. 62977874, filed 2/18/2020, which is hereby incorporated by reference in its entirety.

Background

[001] Over the past decade, there has been an intense effort to improve technologies for high- throughput screening (HTS) assays for various applications including, but not limited to, drug discovery targeting a wide variety of diseases. Of particular promise have been high content screening technologies that produce information-rich data sets from each individual screening experiment or well (e.g. flow cytometric analysis using the iQue Screener ^™ from IntelliCyt). Unfortunately, while useful for screening very large number of compounds, the value of any single-cell HTS assay to predict compound efficacy in vivo has not been established [1, 2]. This situation occurs in part due to the current HTS reliance on monolayer (2D) or single cell assays [3, 4]. Both have been well documented as poor models of cellular behavior in tissues or tumors, which is primarily due to their lack of cell-cell and cell-matrix interactions, as well as the lack of a heterogeneous chemical and cellular microenvironment such as that found in vivo [2, 5, 6]. This is a significant disadvantage for tissue-relevant drug screening, since cell cell signaling, cell-matrix interactions and microenvironmental effects are critical components of most disease states. Not only does the lack of these three-dimensional (3D) properties make screens on cells less predictive, single-cell systems cannot be used to screen directly for stimulation or inhibition of a specific cell-cell interaction. Just as importantly, the penetration of drugs into a tissue or tumor can profoundly affect drug efficacy in vivo. As a result, no current screening system can measure drug penetration in any high throughput manner.

[002] Increasing recognition of the need for 3D screening has led to efforts to develop engineered 3D HTS systems, primarily through two routes. Several microfluidic systems have been created for either mono- or co-culture of cells in different defined local environments [7- 9]. These systems suffer from some notable disadvantages for use in a truly high throughput format: they are complex, difficult and expensive to fabricate, and difficult to assay at a high rate. Introducing controlled amounts of different drugs into these microfluidic systems is also challenging, involving complex perfusion systems that also make true HTS impractical [10]. The other approach has been to create artificial 3D microenvironments by embedding cells within a variety of different matrix materials on top of a conventional culture dish or membrane [11-15]. While several of these systems are now commercially available, they also suffer from the disadvantages of being very low throughput and are difficult to assay due to non-uniform distribution of cells in a relatively large semi-solid matrix [16]. In addition, embedded cell systems have the further disadvantage of non-reproducible exposure to drugs, again due to the fact that cells or cell aggregates are randomly arrayed within a 3D matrix, which is a significant hindrance to drug penetration. Due to these issues, neither of the above approaches has been adopted for HTS.

[003] One 3D tissue/tumor model system that has been proposed to address most of these limitations is the multicellular spheroid [2, 17-21]. Spheroids are a simple system composed of spherical aggregates of cells grown in suspension or liquid-overlay culture (Fig. 1). After initial aggregation into small clusters, spheroids generally grow into compact, essentially spherical structures composed of cells and the extracellular matrix that they produce. Spheroids have been used for a wide variety of studies, including basic tumor biology, anticancer drug testing, cell-cell interactions, tissue and tumor modeling, normal cell culture, drug production, drug transport modeling, artificial organ development and stem cell biology [5, 19, 22-26]. Although most work using spheroids has been done with mono-cultures, a wide variety of tumor and normal cell spheroid co-cultures have been documented. As expected, histological analysis of spheroid co-cultures shows extensive cell-cell contacts and numerous examples of cell-cell interactions have been demonstrated [27-32]. Most work with spheroids has been done with fairly large aggregates (200 to >1000 pm diameter), since the emphasis to date has been on using this system as a model of the chemical and physiological microenvironment in spheroids. However, spheroids will generally assume a nearly spherical shape at sizes of 50-100 pm, containing only 20-150 cells (see Fig. 1). The spherical symmetry of this tissue model system is a distinct advantage: all of the chemical, physiological and cellular gradients within the spheroid are radially symmetric (Fig. 2). This symmetrical microenvironment is not only advantageous as a physical attribute of the model, but it also enables a wide variety of analysis techniques, from in situ imaging to selective dissociation of cell subpopulations from different regions [33-35].

[004] Unfortunately, current systems using spheroids for drug screening are not HT [36-39]. As pointed out in a number of publications [2, 40-43], one serious technical limitation to using spheroids for HTS is the lack of methods for producing large numbers of uniformly-sized spheroids. A second problem is the lack of methods for generating uniform spheroid co cultures (e.g. two or more cell types at a reproducible ratio within individual spheroids). Techniques do not currently exist for producing large numbers of uniformly-sized spheroids composed of a uniform distribution of different cell types. Common methods for producing large numbers of spheroids (e.g. agar overlay, aggregation in suspension) generate a very heterogeneous size distribution with a very non-uniform distribution of cell types. While the size distribution can be improved using post-aggregation sorting, most of these are laborious, inefficient and still result in a poor size distribution (e.g. >30% variation in volume and cell number, see Fig. 1) [44-46]. Post-aggregation sorting also does not address the non-uniform cell type distribution between spheroids. There are a variety of methods for generating a more uniform population of spheroids, including hanging-drop cultures (e.g. Sigma Millipore Perfecta3D® hanging drop plates), microfabricated culture surfaces (e.g. Corning® spheroid microplates) and microfluidic chambers [20, 47-51]. These systems can produce uniformly- sized populations and potentially uniform distributions of different cell types. However, they are complex, expensive, difficult to fabricate, hard to operate, only produce large spheroids and/or cannot produce large numbers (>10 ⁶ ) of spheroids for true HTS.

[005] Moreover, the above-described limitations also significantly inhibit application of the spheroid model system in several important research and clinical areas. One new clinical application for spheroids is in generating stem cells for transplantation. It has been demonstrated that culture as spheroids significantly improves the overall therapeutic potential, “sternness” and survival of multipotent stem cells after transplantation [41, 52, 53]. A second new clinical application is in the generation of multipotent stem cells using suspension cultures of spheroids. Stem cells exist in vivo in microenvironmental niches that are in many ways similar to the microenvironment within spheroids (e.g. hypoxia, nutrient limitations, specific cell-cell contacts, ECM interactions, see Fig. 2) [54-56]. Suspension cultures of spheroids containing pluripotent stem cells in the center surrounded by multipotent cells on the periphery could be stable for long term culture and continuously produce multipotent cells via cell shedding from the spheroid surface [57]. A final potential clinical application of spheroid co cultures is in their use to generate artificial organs for eventual transplant or for ex vivo operation. Several studies with both mono- and co-cultured spheroids have shown them to be superior for maintaining and preserving cells in artificial organ models [58-60]. On top of these direct clinical applications, a method for generating large numbers of uniform spheroid co cultures would remove a significant bottleneck to the further application of spheroids in many basic and clinical research areas.

Summary [006] The present disclosure provides methods and apparatus for easily and reproducibly producing large numbers of uniform spheroids including spheroid co-cultures.

Brief Description of the Drawings

[007] Fig. 1 depicts multicellular spheroids produced by a prior art agar overlay method and then sorted with screens: note size inhomogeneity (standard deviation/mean -12% in diameter, -36% in volume/cell number).

[008] Fig. 2 is a diagram of the internal gradients within a typical spheroid: cellular / physiological gradients are shown on the left and concentration gradients of chemicals / drugs on the right.

[009] Fig. 3 is a schematic illustration of one embodiment of a device for generating large numbers of uniform, cell-ECM droplets, by directing the droplet stream directly into a culture medium bath. These droplets are then used to generate uniform spheroids.

[010] Fig.4 is a schematic illustration of a second embodiment of a device for generating large numbers of uniform, cell-ECM droplets, by directing the droplet stream upwards into a gentle stream of heated air, causing the droplets to mix and then drop into the medium bath. These droplets are then used to generate uniform spheroids.

[Oil] Fig. 5 is a schematic illustration of droplet generation using a single nozzle and two cell types mixed 1 : 1 in the pre-gel. Mixed co-culture spheroids are generated after incubating the droplets.

[012] Fig. 6 is a schematic illustration of droplet generation using two nozzles with two cell types supplied in separate streams, generating droplets with the two cell types in different locations.

Detailed Description

[013] According to an embodiment the present disclosure provides methods and apparatus for easily and reproducibly producing large numbers of uniform spheroids including spheroid co-cultures. According to various embodiments, the present disclosure provides methods and apparatus that combine vibrational droplet generation with a temperature-transition zone through which the droplets pass.

[014] A first exemplary embodiment of an apparatus 1 for uniform spheroid generation is shown in Fig. 3. As shown, cells of interest are prepared in a single cell suspension in a solution of an extracellular matrix (ECM) extract. Suitable commercially available extracellular matrices include, but are not limited to, those sold under the trademarks Matrigel® (Coming, Inc., Coming N.Y.) or Cultrex® (RND Systems, Minneapolis, MN) or similar). According to an embodiment, the pre-gel solution may contain additional additives including, but not limited to, growth factors, metabolic factors, nutrients, immunomodulating agents, gene expression modulators or any form of chemical or drug that can alter the physiology of the cells in the droplet. For the current embodiment, the pre-gel solution 10 containing the cells and ECM is kept in a stirred vessel 12 and maintained at a sufficiently low temperature that the pre-gel solution will not undergo gelation. For example, the transition temperature of many commercially available ECMs is between 10 and 15 ° C and a suitable temperature for the vessel would thus be less than 5 ° C. According to specific embodiment, the vessel may be maintained at 2 ° C. Our embodiment envisions using gels that have a liquid temperature lower than the solidified temperature (e.g. they gel when the temperature is raised above the transition temperature), since there are many types of ECM gels that have this property. However, there are also gels that behave in the opposite manner: their liquid temperature is higher than the solidified temperature (e.g. they gel when the temperature is lowered below the transition temperature) and such gels would be suitable for use in our device merely by heating the pre gel solution and cooling the droplets. A variety of suitable methods for maintaining temperatures will be known to those of skill in the art and any such method may be employed. Vessel 12 is fluidly connected to nozzle 14, for example via temperature-controlled supply channel 16. Supply channel may be formed from tubing or any other suitable material. The pre-gel solution is then delivered to a nozzle 14 by pressurizing vessel 12, for example via pressure input 18 to push the solution through the temperature-controlled supply channel. Of course, it will be understood that other mechanisms for delivering the contents of vessel 12 to nozzle 14 other than pressure-driven force, including for example, mechanical means, may be similarly employed. In general, the temperature-controlled supply channel is cooled or refrigerated to maintain the pre-gel at a desired temperature (typically a temperature matching or similar to the temperature of vessel 12) until it exits the nozzle into air. Nozzle 14 is connected to or otherwise influenced by an acoustic transducer 20 which, when activated, vibrates the nozzle so as to generate uniform droplets 22 of the cell-containing pre-gel solution in air. While other methods of vibrating the nozzle, including mechanical vibrational means, may be employed, an advantage of using an acoustic transducer is that typical operating frequencies are 5 - 20 KHz, depending on the nozzle size, enabling in the production of thousands of droplets every second.

[015] The droplets then pass through a heated region 24 which brings the air temperature up to at least 37° C. As each droplet transitions this region, it undergoes gelation due to the temperature increase. The actual air temperature can be higher in order to cause faster gelation, with the caveat that the droplets do not reach a temperature above -40° C as this could be toxic. The gelled droplets then fall into a stirred vessel 26 containing tissue culture medium maintained at 37° C, which is the standard for mammalian cell culture and will maintain the droplets in a gelled form. For an embodiment in which the pre-gel solution is heated and the air is cooled to cause gelation, similar temperature ranges would apply (i.e. a maximum of 40° C for the pre-gel solution and 2-5° C for the gelation region and catch beaker. According to an exemplary embodiment, the heated region is comprised of a thermostat-regulated system for providing heated air and may be supplemented with infrared emitters or other methods (such as microwave irradiation) that heat the droplets directly.

[016] A second exemplary embodiment of an apparatus 2 for uniform spheroid generation is shown in Fig.4. This particular embodiment may be well-suited for formulations of pre-gel material and cells may take longer to gel than the time period afforded by the embodiment in the apparatus shown in Fig. 3. In the device shown in Fig. 4, the apparatus includes a cool zone 30 and a warm zone 32. Warm zone 30 includes vessel 36 which may be pressurized via pressure input 34 to enable delivery of the pre-gel 34 to nozzle 38 via channel 36. (As with the embodiment in Fig. 3, other non-pressure driven means may be employed.) Droplets 42 are generated in the separate cool zone and ejected from the nozzle into a separate warm zone. In this case, the nozzle is directed upwards at an angle such the droplet stream is directed to a point over a stirred medium bath 44. These droplets then “rain” down into the medium bath, after having spent a much longer time in the warm zone compared to the embodiment shown in Fig. 3, allowing sufficient gelation. The tip of the nozzle can be rotated as desired to better disperse the droplet stream over the medium bath. If desired, a slow stream of warm air 46 may be directed towards the droplet path, causing additional mixing improving droplet dispersion into the medium bath.

[017] A variety of suitable methods of stirring the contents of various vessels/baths will be known to those of skill in the art including, but not limited to, stirrers, agitators, shakers, etc. Similarly, a wide variety of methods for maintaining the temperatures of the various elements and regions will be known to those of skill in the art. As such, the apparatus of the present disclosure should be considered to include any such additional elements and needed or desired in order to stir the contents of the vessels/baths and maintain the desired temperatures throughout the apparatus.

[018] According to various embodiments, nozzles with different diameters can be used to create uniformly sized droplets of different mean diameters. By example, in the viscosity range of ECM pre-gel solutions nozzles having a diameter of 50 pm will produce droplets with a diameter of -100 pm. Accordingly, for a given pre-gel solution a population of droplets having a given average diameter can be produced simply by selecting a suitably sized nozzle and adjusting the vibration frequency. Accordingly, commercial embodiments of the devices described herein may include a variety of different nozzle sizes and/or mechanisms for changing or altering the nozzle or nozzle size in order to enable a user to select a desired nozzle/droplet/spheroid size.

[019] According to various embodiments, the embodiments in both Figs. 3 and 4 enable gelation of the droplets prior to impacting the medium surface, thereby preserving the original, very uniform, size distribution (<1% diameter variation) of the droplets. If a homogenous size distribution is desired, gelation prior to impact with a medium surface is important because pre-gel droplets that contact the surface of the medium will splatter and deform prior to gelation, resulting in an inhomogeneous droplet size distribution. Accordingly, it should be understood that a single apparatus may be configured to enable both the methods described and shown with respect to Figs. 3 and 4. Specifically, an apparatus may be designed such that the nozzle and/or a portion of the supply channel is movable such that it can be selectively positioned such that droplets formed at the tip of the nozzle are either dropped downward or sprayed upward at any number of angles (thus increasing or deceasing the amount of time afforded to the droplets to undergo gelation).

[020] Of course it will be understood that the amount of time afforded to the droplets to undergo gelation may also be affected by the speed at which the droplets leave the nozzle and the space between the nozzle and the vessel. (For example, the embodiment of Fig. 3 could include longer or shorter heating zones, as needed.)

[021] While not depicted in Figs. 3 or 4, it should be understood that the apparatus may include more than one reservoir and that such second (or more) reservoir(s) may include a second (or more) supply channel(s) which converges with the first supply channel prior to or at the point of delivery to nozzle 14. (Methods and apparatus that employ multiple nozzles are also contemplated and are discussed in greater detail, below.) Similarly, such additional reservoir(s) and supply channel(s) may contain additional temperature-controlling elements or may use the same temperature-controlling elements as those used in the first reservoir and supply channel. Moreover, such temperature-controlling elements may maintain the additional reservoir(s) and supply channel(s) at the same or a different temperature as the first reservoir and supply channel. [022] Spheroids are generated from the gelled, cell-containing droplets by continued culture of the recovered droplets for several days, for example in a spinner flask. In general, the cells within the droplets will attach to the ECM matrix and proliferate, reshaping the ECM as the cells fill out the gelled sphere. If cells are placed in the pre-gel at a high concentration, spheroids will be generated within a few days in culture. The number of cells in each droplet is very uniform due to the uniformity of the droplets generated by the acoustic method and the fact that the cells in the single-cell suspension are uniformly distributed throughout the pre-gel. The cells can be either a single cell type, or a mixture of two (or more) cell types mixed in the pre-gel.

[023] An example of a set-up for generation of a uniformly sized population of spheroids with a uniform distribution of different cell types in each spheroid is illustrated in Fig. 5. In this case, a co-culture 50 containing two (or more) cell types is introduced to a single nozzle 50. While Fig. 5 demonstrates two cell types in equal ratios, it will be understood that one could easily generate spheroids with a wide range of initial ratios of different cell types by merely adjusting the composition of the original cell suspension. Of course it will be understood that factors such as cell proliferation rate should be taken into account when determining the final distribution of cells within the spheroid, but such matters are believed to well within the knowledge of one of skill in the art and thus the present disclosure envisions mixtures of cells with different growth rates, or even mixtures of proliferating and non-proliferating cells. Accordingly, using the described methodology and apparatus, one could reproducibly generate a wide range of spheroid co-cultures by adjusting the number of cells per droplet, the droplet size, the cell type ratios and the incubation time. Importantly, it should be noted that the embodiment shown in Fig. 5 produces a spheroid with an even distribution of however many cell types are included in the co-culture throughout the spheroid.

[024] However, in some instances it may be desirable to produce a spheroid with concentric spherical layers of different cell types. Accordingly as shown in Fig. 6, the present application provides for a multi-nozzle system comprising nested nozzles. Fig. 6 depicts an exemplary two nozzle system wherein a smaller nozzle 60 is nested within a larger nozzle 62. The two different cell types are supplied to the nozzles from separate vessels (not shown), with one type delivered to the inner nozzle and the second to the outer nozzle. This creates an inner droplet 64 in the center of the outer droplet 66, thereby placing one cell type in the center and the second on the periphery. The relatively high viscosity of the pre-gel solutions ensures that there is little mixing of the two cell types in the short time between droplet formation and gelation. After gelation, the cell types are relatively fixed in their positions and will initially generate spheroids with the cells in different positions. Depending on the cell types used, the cells may mix together over time or they may remain separated. It should be understood that the present application envisions that either or both of the separate pre-gel solutions may contain more than one cell type, allowing construction of more complex co-cultures. It should also be understood that the present application envisions having more than 2 nested nozzles. Moreover, it should be understood that the present disclosure contemplates the use of multiple nozzles that are not nested, but rather positioned next to or near each other and/or the use of combinations of nested and not nested nozzles.

[025] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[026] All patents and publications referenced below and/or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

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