WO2021168034A1 - Procédé et appareil pour la génération de sphéroïdes à grandes échelle - Google Patents

Procédé et appareil pour la génération de sphéroïdes à grandes échelle Download PDF

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Publication number
WO2021168034A1
WO2021168034A1 PCT/US2021/018453 US2021018453W WO2021168034A1 WO 2021168034 A1 WO2021168034 A1 WO 2021168034A1 US 2021018453 W US2021018453 W US 2021018453W WO 2021168034 A1 WO2021168034 A1 WO 2021168034A1
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WO
WIPO (PCT)
Prior art keywords
nozzle
droplets
cells
reservoir
temperature
Prior art date
Application number
PCT/US2021/018453
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English (en)
Inventor
James FREYER
Original Assignee
Benubio
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Publication date
Application filed by Benubio filed Critical Benubio
Priority to GB2217222.5A priority Critical patent/GB2610116A/en
Publication of WO2021168034A1 publication Critical patent/WO2021168034A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • B01L3/0268—Drop counters; Drop formers using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B01J2/02—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B01J2/02—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops
    • B01J2/06—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic by dividing the liquid material into drops, e.g. by spraying, and solidifying the drops in a liquid medium
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B01J2/18—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic using a vibrating apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1861—Means for temperature control using radiation
    • B01L2300/1866—Microwaves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1861—Means for temperature control using radiation
    • B01L2300/1872—Infrared light
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • HTS high- throughput screening
  • 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].
  • spheroids 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].
  • 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.
  • 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.
  • spheroids are 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].
  • 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.
  • the present disclosure provides methods and apparatus for easily and reproducibly producing large numbers of uniform spheroids including spheroid co-cultures.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 3 A first exemplary embodiment of an apparatus 1 for uniform spheroid generation is shown in Fig. 3.
  • cells of interest are prepared in a single cell suspension in a solution of an extracellular matrix (ECM) extract.
  • ECM extracellular matrix
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • pressurizing vessel 12 for example via pressure input 18 to push the solution through the temperature-controlled supply channel.
  • 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.
  • 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.
  • tissue culture medium maintained at 37° C, which is the standard for mammalian cell culture and will maintain the droplets in a gelled form.
  • similar temperature ranges would apply (i.e.
  • 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.
  • FIG. 4 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.
  • 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.
  • 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.
  • a slow stream of warm air 46 may be directed towards the droplet path, causing additional mixing improving droplet dispersion into the medium bath.
  • nozzles with different diameters can be used to create uniformly sized droplets of different mean diameters.
  • nozzles having a diameter of 50 pm will produce droplets with a diameter of -100 pm.
  • a population of droplets having a given average diameter can be produced simply by selecting a suitably sized nozzle and adjusting the vibration frequency.
  • 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.
  • 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.
  • 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).
  • 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.)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cells within the droplets will attach to the ECM matrix and proliferate, reshaping the ECM as the cells fill out the gelled sphere.
  • 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.
  • FIG. 5 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.
  • a co-culture 50 containing two (or more) cell types is introduced to a single nozzle 50.
  • 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.
  • 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.
  • the cell types are relatively fixed in their positions and will initially generate spheroids with the cells in different positions.
  • the cells may mix together over time or they may remain separated.
  • 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.
  • the present application envisions having more than 2 nested nozzles.
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne des procédés et un appareil permettant de produire facilement et de manière reproductible de grands nombres de sphéroïdes uniformes comprenant des co-cultures sphéroïdes par combinaison d'une génération de gouttelettes vibrationnelles avec une ou plusieurs zones de transition régulées en température.
PCT/US2021/018453 2020-02-18 2021-02-18 Procédé et appareil pour la génération de sphéroïdes à grandes échelle WO2021168034A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB2217222.5A GB2610116A (en) 2020-02-18 2021-02-18 Method and apparatus for large-scale spheroid generation

Applications Claiming Priority (2)

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US202062977874P 2020-02-18 2020-02-18
US62/977,874 2020-02-18

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1541739A (fr) * 1967-08-28 1968-10-11 Cie Pour L Etude Et La Realisa Pulvérisation par ultrasons de liquides ou de solides fusibles ou solubles
US20040121451A1 (en) * 2001-03-16 2004-06-24 Niko Moritz Treatment of sols, gels and mixtures thereof
US20100243753A1 (en) * 2007-06-22 2010-09-30 Arizona Board of Regents, a body corporate acting for and on behalf of Arizona State University Gas Dynamic Virtual Nozzle for Generation of Microscopic Droplet Streams
US20130274353A1 (en) * 2012-03-16 2013-10-17 The University Of Hong Kong System and method for generation of emulsions with low interfacial tension and measuring frequency vibrations in the system
US20190157060A1 (en) * 2017-11-22 2019-05-23 Labcyte, Inc. System and method for the acoustic loading of an analytical instrument using a continuous flow sampling probe

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1541739A (fr) * 1967-08-28 1968-10-11 Cie Pour L Etude Et La Realisa Pulvérisation par ultrasons de liquides ou de solides fusibles ou solubles
US20040121451A1 (en) * 2001-03-16 2004-06-24 Niko Moritz Treatment of sols, gels and mixtures thereof
US20100243753A1 (en) * 2007-06-22 2010-09-30 Arizona Board of Regents, a body corporate acting for and on behalf of Arizona State University Gas Dynamic Virtual Nozzle for Generation of Microscopic Droplet Streams
US20130274353A1 (en) * 2012-03-16 2013-10-17 The University Of Hong Kong System and method for generation of emulsions with low interfacial tension and measuring frequency vibrations in the system
US20190157060A1 (en) * 2017-11-22 2019-05-23 Labcyte, Inc. System and method for the acoustic loading of an analytical instrument using a continuous flow sampling probe

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GB202217222D0 (en) 2023-01-04

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