Patent Description:
Cancer remains the leading cause of death worldwide and among Canadians: about <NUM> in <NUM> Canadians will be diagnosed with cancer during their lifetime, with half of new cases being prostate, breast, lung and colorectal cancers (<NUM> in <NUM> will die and <NUM>% diagnosed with cancer will survive at least, <NUM> years after the diagnosis). For many cancer patients standard-of-care therapy involves surgical removal of the primary tumour. The success of this approach is often unsuccessful due to the growth of secondary tumours at distant sites. Therapies are required that are either directly toxic toward the proliferating metastatic cells, or that lock micrometastases in their dormant state. To eradicate metastasis, chemotherapy is administered after primary surgery, however there is a challenge to assess and predict the effectiveness of drugs for microtumors with dimensions in the range of tens to hundreds of micrometers that are grown in environments with different protective properties.

One of the major hurdles impeding the development of cancer therapeutics (and micrometastases, in particular) is the limitations of current model systems. Conventional models used for anticancer drug screening include in vitro studies conducted in a 2D format (in Petri dishes or well plates) and animal (zenograph) studies. Animal models are expensive, being both labor- and time-consuming. Moreover, animal models generally only provide endpoint analyses, in addition to issues of relevance for the human condition. Typically, immunocompromised murine models are used, however it is established that the immune system is crucial to the micrometastatic microenvironment. Those animal studies that do use syngeneic models (tumor tissues derived from the same genetic background as a given mouse strain) are also not fully representative of the human situation due to differences in cytokines and metabolism.

In vitro culture resolves some of the issues of animal studies, however most of conventional in vitro drug screening is conducted by using 2D culture systems. These systems lack important aspects which impact tumor behavior, such as 3D architecture to provide tissue depth for tumor intercalation, functional aspects, including fluid flow and control of oxygen content, and do not allow for time-extended culture. There is also a distinct absence of systems capable of recreating micrometastasis while concurrently providing for the evaluation of drug efficacy, toxicity and metabolism.

Cells cultured in 2D environments may be capable of forming multi-cell spheroids (MCSs), but these spheroids differ considerably in their properties and phenotype from those grown in physiological 3D environments, and may not serve as an efficient model for drug screening. Organotypical multicellular spheroids-3D aggregates of malignant cells that replicate many features of microtumors - serve as a bridge between 2D cell cultures and animal models to overcome some of the limitations listed above.

Cancer spheroids, namely, dense cancer cell clusters with typical sizes in the range from tens of micrometers to one millimetre, are extensively used as models of solid tumors for fundamental research in cell biology and for clinical applications, e.g., for studies of tumor response to chemo- and radiation therapies. They replicate many features of tumors, e.g., cell-cell adhesion, cell-matrix interactions and gradients in the concentration of nutrients and gases. Cancer spheroids are prepared in several different ways, that is, by partial or complete dissociation of tissues, by growth and proliferation of individual (isolated) cancer cells, or by aggregation of individual cancer cells in dense clusters.

However, existing MCS models currently used for drug screening have at least one of the following limitations:.

Another important consideration in the preparation and use of multicellular spheroids as tumorigenic models is the rate of their formation in vitro, which can vary from days to weeks, in order to reach the size of several hundred micrometers. Fast growth of multicellular spheroids in vitro is highly advantageous. Due to the rapid formation and growth, multicellular spheroids formed by aggregation of individual cells from their dense suspensions are particularly useful. Unfortunately, multicellular spheroids generated using this method, are generally formed in the absence of their extracellular matrix (ECM), which is one of the main components of their natural microenvironments, as it provides biophysical and biochemical cues to tumor growth. Although cancer cells in multicellular spheroids generated by cell aggregation can secret ECM components, the composition of these ECMs is complex, thereby making it is difficult to disentangle the effect of each ECM component on the growth and progression of cancer cells. On the other hand, multicellular spheroids formed by the growth of individual isolated cancer cells have been prepared in various ECMs, e.g., Matrigel, fibrin or collagen, however this process is slow and it lacks the physiological flow conditions.

Another important consideration in MCS generation and potential applications is the uniformity in their dimensions. Existing methods used for the generation of multicellular spheroids by cell aggregation, including rotary culture, cell aggregation on a non-adherent or a hanging drop method, yield multicellular spheroids with a broad size distribution. For example, a population of multicellular spheroids can have diameters varying from <NUM> to <NUM>. The high polydispersity of multicellular spheroids imposes uncertainty in diffusion of drugs to the spheroid center or the effect of hypoxia on MCS fate.

International patent application pub. No. <CIT> describes an array of microwells patterned in a microfluidic chip to allow easy access to a cell or group of cells under culture.

International patent application pub. No. <CIT> describes a method of manipulating micro drops including samples in a microfluidic system.

Article "<NPL>) describes an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations.

Article "<NPL>) describes platforms for evaluating drugs with a higher throughput and cell aggregates in more natural configurations.

International patent application pub. No. <CIT> describes a microfluidic platform for high throughput generation and analysis of clearly defined 3D cell spheroids with uniform geometry and versatility in terms of the spheroid sizes and cell composition.

Article "<NPL>) describes a multifunctional droplet microchip to address these issues and demonstrate the usefulness of this device for investigating post-embryonic development in individual C. elegans initiating at the larval L1 stage.

Article "<NPL>) describes various cell microencapsulation procedures based on a microfluidics, the optimal characteristics of microdevice intended for cell encapsulation, together with the used materials for production.

Korean patent application pub. No. <CIT> describes a scaffold for three-dimensional cell culture in which a hydrogel containing cells is confined in the form of a membrane using surface tension in an O-shaped loop.

Microfluidics (MFs) offers a platform for the generation of uniformly-sized multicellular spheroids in high efficiency. In particular, MF systems are advantageous as these devices enable the culture of multicellular spheroids under flow and deliver drugs to multicellular spheroids in flow, which enables close mimicking of natural dynamic conditions for tumor growth and progression, as well as chemotherapy. Microfluidics also offers the capability of multiplexing, that is, the exploration of a variety of different factors and their impact on the contained cells, e.g., MCS size and composition, or the effect of different drugs delivered in different dosages in a single series of experiments conducted on a single MF chip.

Thus, the present disclosure is drawn to a MF-MCS platform for drug screening, which can achieve the growth of large arrays of uniformly sized multicellular spheroids with various dimensions under continuous close-to-physiological flow conditions and the delivery of anticancer drugs to multicellular spheroids under dynamic conditions for throughput screening of drug efficacy for therapeutic treatment.

The advantages of the MF-MCS platform disclosed in the current application include, but are not limited to: the ability for the device to contain a large number of multicellular spheroids along each row (leading to statistically significant results), a large number of parallel MCS rows (enabling screening of a particular factor, e.g., drug dose, combinations of different drugs, or the role of MCS size on drug efficacy), a small number of malignant cells and amount of ECM needed for MCS growth (e.g., primary cells can be taken from a particular cancer patient) and the capability to parallelize several screening processes conducted under close-to-physiological flow conditions.

This particular MF-MCS platform holds great potential for cancer drug screening and in particular, as a predictive tool in the evaluation of the output of adjuvant chemotherapy for individual patients, thus enabling rapid decision making regarding the selection of the treatment strategy for a specific patient. Using this platform, screening of drugs can be achieved with higher accuracy, in a shorter time, and with fewer resources than using conventional drug screening procedures.

The present disclosure provides a method as set out in the appended set of claims.

A further understanding of the functional and advantageous aspects of the present disclosure can be realized by reference to the following detailed description and drawings.

Embodiments will now be described, by way of example only, with reference to the drawings. Embodiments of the method of the present invention are only illustrated in drawings 2A, 2B, 2C and <NUM> to <NUM>. Microfluidic devices illustrated in the drawings 1A, 1B, 1C and <NUM> do not form part of the invention. In the drawings:.

Various embodiments and aspects of the disclosure will be described with reference to details discussed below. The following description and drawings are illustrative of the disclosure and are not to be construed as limiting the disclosure. Numerous specific details are described to provide a thorough understanding of various embodiments of the present disclosure. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present disclosure.

As used herein, the terms "comprises" and "comprising" are to be construed as being inclusive and open ended, and not exclusive. Specifically, when used in the specification and claims, the terms "comprises" and "comprising" and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

As used herein, the terms "about" and "approximately" are meant to cover variations that may exist in the upper and lower limits of the ranges of values, such as variations in properties, parameters, and dimensions. In one non-limiting example, the terms "about" and "approximately" mean plus or minus <NUM> percent or less.

The present disclosure is drawn to a microfluidic (MF) device and method of formation of an array of multicellular aggregates onto the device. Multicellular cancer spheroids (MCSs) are three-dimensional cancer cell aggregates with dimensions from tens of micrometers to ~<NUM>, which replicate many features of solid tumors in vivo, including extracellular matrix (ECM) deposition between the cells, strong cell-cell junctions, and gradients in nutrient concentration (<NPL>). For the purpose of this disclosure, multicellular aggregate consists of an aggregate of cells being self-organized in a three-dimensional arrangement. Furthermore, the cells within the aggregate may have formed a specific common membrane. The multicellular aggregate may be an organoid obtained with stem cells or a combination of stem cells and other cells.

Alternatively, the multicellular aggregate may be a multicellular spheroid. The multicellular spheroid may be made of cells obtained from a cancer cell line or obtained from primary cells isolated from cancer patient tissue. Such cells may be obtained from separation and isolation of individual cells from tissue biopsy. Depending on the use of the current process or device disclosed herein, the multicellular aggregate may comprise one kind of cells or a plurality of different kinds of cells resulting in heterogeneous organoids or spheroids.

According to an embodiment, the device and method may be used for screening compounds for drug discovery, for understanding mode of action of the screened compounds and for the evaluation of pharmacodynamics and/or mechanistic biomarkers. According to an embodiment, the device and method may be particularly useful in screening/studying the effect of composition, structure and properties of the environment on MCS growth OR interactions between different cells, e.g., immune cells ad cancer cells and in screening/studying the role of the composition of the cell culture media. According to an embodiment, the device and method may be particularly useful in the study and/or screening of drugs for therapeutic treatment by delivering drugs such as anticancer drugs to multicellular spheroids or organoids, both of which may be acting as microtumors when obtained from high-density cell suspensions, under dynamic conditions.

According to an embodiment, the device and method may be useful in the study of cancer spreading and/or screening of cancer drugs on multicellular spheroids or organoids, both of which may be acting as metastatis microtumors when obtained from low-density cell suspensions, under dynamic conditions.

According to an embodiment, the present invention provides means to study different stages of cancerous tumors, e.g., early stage and later stages of cancerous tumors. Cancer spheroids obtained from low-density cell suspension may be used to study/mimic tumor growths formed from individual cells. Alternatively, cancer spheroids obtained with high-density cell suspension may be used to study/mimic later stage of tumor growth once the cells have organized themselves into a cancerous tumor.

According to an embodiment, the anticancer drugs may target various types of cancer. For example, the anticancer drugs may target breast cancer. Alternatively, the anticancer drugs may target pancreatic cancer.

One advantage of the present invention is the use of multicellular spheroids obtained from primary cells isolated from cancer patient tissue rendering possible a personalized screening of compounds. According to an embodiment, the device and method may provide the formation of a large arrays of uniformly-sized multicellular aggregates of various sizes under continuous close-to-physiological flow conditions in microfluidic devices. The multicellular aggregates may grow to become uniformly-sized multicellular cancer spheroids or organoids. According to an embodiment, the device and method may be useful in screening compounds by the delivery of such compounds to the multicellular aggregates under dynamic conditions.

According to an embodiment, the device and method disclosed herein may provide the formation of a large arrays of uniformly-sized multicellular aggregates, such as spheroids or organoids, which may be released from the device for retrieval of the spheroids or organoids from the microfluidic device. These spheroids or organoids may be released using different means known to the skilled person in the art (<NPL>;<NPL>; and <NPL>). They may be released by enzymatic or chemical-mediated hydrogel digestion/lysis, using mechanical hydrogel disruption, hydrogel photodegradation. Alternatively, the spheroids or organoids may be "washed away" by strongly increasing the flow rate in the microfluidic device. According to another embodiment, the spheroids or organoids may be released by inducing liquefaction of a temperature-responsive hydrogel at a reduced physiologically acceptable temperature.

According to an embodiment, as shown in <FIG>, a microfluidic device <NUM> has a plurality of parallel rows <NUM>. Each row <NUM> comprises a plurality of cylindrical microwells <NUM> in which a large arrays of multicellular aggregates may be grown in tissue-mimicking hydrogel scaffolds under close-to-physiological flow conditions in the microwells <NUM>. For each row <NUM>, a supplying channel <NUM> spans along the length of the row <NUM> and is in flow communication with the respective microwells <NUM>. The supplying channel <NUM> has an exposed opening at both of its ends <NUM> acting either as an entry opening or an exit opening for the circulation within a given row <NUM> of fluids such as wetting agents, solutions comprising aqueous suspensions of cells and an hydrogel precursor, and the cell culture medium for the supply of the nutrition medium and/or compound to be screened.

<FIG> shows an expanded view of three microwells <NUM> in one of the rows <NUM> from <FIG> along with the section of the supplying channel <NUM> that provides flow communication between the microwells <NUM> and the various reagent sources (not shown). As shown in <FIG>, the microwells <NUM> have a diameter <NUM> and a height <NUM> and the supplying channel <NUM> has a width <NUM> and a height <NUM>.

In an embodiment, the microwells <NUM> of the microfluidic device <NUM> may have a diameter <NUM> between the range of about <NUM> to about <NUM> and a height <NUM> of about between the range of about <NUM> to about <NUM>.

According to an embodiment the height <NUM> of the microwells is larger than the diameter <NUM> of the microwells <NUM>. Alternatively, the height <NUM> of the microwells <NUM> may be at least <NUM>% larger than the diameter <NUM> to avoid the overgrowth of the multicellular aggregates and the swelling of the hydrogels into the supplying channel <NUM>. Alternatively the height <NUM> of the microwells may be about <NUM>% larger than the diameter <NUM>. Since the dimensions of the device <NUM> may be tailored to the preferences of the user, as long as the ratio diameter/height for each microwell <NUM> is maintained, the diameter <NUM> of the microwells <NUM> may be varied over the microwells array in such ways that the resulting multicellular aggregates may have a distribution of diameters. Obtaining multicellular aggregates with a diversity of diameters allows the user to explore the drug efficiency for multicellular aggregates with varying dimensions.

According to an embodiment, to achieve the formation of the cell-laden precursor droplets and prevent overgrowth of the multicellular aggregates and the swelling of the hydrogels in the supplying channel <NUM>, the height <NUM> of the microwells <NUM> is larger than the height <NUM> of the supplying channel <NUM>. Alternatively, the ratio of the height <NUM> of the microwells <NUM> to the height <NUM> of the supplying channel <NUM> is at least about <NUM>. The ratio is dependent on the material used for the fabrication of the microfluidic device and the wetting agent used in the method and system of the present invention. Alternatively, the ratio of the height <NUM> of the microwells <NUM> to the height <NUM> of the supplying channel <NUM> is between about <NUM> to about <NUM>. This ratio was found to be good when the microfluidic device was made with polydimethylsiloxane (PDMS) and the used wetting agent comprised a fluorinated oil such as block copolymer perfluorinated polyether-b-(polypropylene glycol-polyethylene glycol-polypropylene glycol)-b-perfluorinated polyether. However, it will be appreciated that this ratio may change when different fabrication material and wetting agent combinations are used. In a preferred embodiment, the height <NUM> of microwells <NUM> is about <NUM>% larger than the diameter <NUM> of the microwells <NUM> and the ratio of the height <NUM> of the microwells <NUM> to the height <NUM> of the supplying channel <NUM> is between about <NUM> to about <NUM>. Furthermore, the excess swelling may be minimized by adjusting the concentration of hydrogel precursor in the aqueous solution.

Maintaining this ratio between dimensions <NUM> and <NUM> enables the formation of a relief volume between the top of the cell-laden droplet and the top of the microwell <NUM> that the droplet is contained in. This relief volume functions to limit the risk of hydrogel over-swelling or over-growth in multicellular aggregates by providing an extra volume for hydrogel swelling and growth of the cells to occur in the z-direction of the microwell <NUM>. The spillage of the cells into the supplying channel <NUM> is undesirable as it may lead to flow-driven loss of the cells from the multicellular spheroids.

According to one embodiment, as shown in <FIG>, the microfluidic device <NUM> may comprise four different layers. These layers include a first layer <NUM> comprising a rectangular silicone base <NUM> with a mounted glass slide <NUM> and a second layer <NUM> that is a silicone-based sheet <NUM> with a similar thickness to the first layer. The third layer <NUM> is likewise composed of silicone and contains an array <NUM> of microwells <NUM>, the array <NUM> is composed of one or more rows <NUM>, each row <NUM> having a plurality of microwells <NUM>. Each of the microwells <NUM> in the various rows <NUM> is in individual flow connection to a supplying channel <NUM> which spans the length of the row <NUM> and contains an exposed opening both of its ends of the supplying channel <NUM>. Lastly, a fourth layer <NUM> has a base <NUM> layer which is also composed of silicon and is attached to the bottom side of the device <NUM>. It is necessary for the MF-MSF platform to be robust, simple to fabricate and have a relatively low unit cost. It will be appreciated by those skilled in the art that polymeric materials other than silicone-based polymers may be used.

According to an embodiment, the four-layer device disclose herein may be partially fabricated in polydimethylsiloxane (PDMS) using soft lithography technique known in the art. Such technique may be found at http://www. com/microfluidic-tutorials/soft-lithography-reviews-and-tutorials/introduction-in-soft-lithography/introduction-about-soft-lithography-and-polymer-molding-for-microfluidic/. Soft lithography encompasses a collection of fabrication methods that are based on the use of a patterned layer of exposed PDMS. In one possible embodiment, the device's four silicon layers as disclosed above may be formed through the use of the soft-lithography deposition.

According to an embodiment, the device <NUM> may include at least one row or a plurality of parallel rows, with each row having at least one well. According to an embodiment, the device <NUM> may include anywhere from <NUM> to <NUM> parallel rows <NUM>, each row <NUM> may contain from <NUM> to <NUM> wells or alternatively from <NUM> to <NUM> wells, depending on the size of microwells <NUM>.

According to an embodiment, the device <NUM> may have overall dimensions of <NUM> x <NUM>. When having such dimensions, the device <NUM> may have <NUM>-<NUM> microwells depending on the microwell diameters <NUM>. The dimensions first layer <NUM> and second layer <NUM> are approximately <NUM> × <NUM> × <NUM>. These two layers are bonded together after fabrication. The third layer <NUM> having dimensions of <NUM> × <NUM> × <NUM> was bonded to the second layer <NUM> with the features of the supplying channels <NUM> and microwells <NUM> facing the second layer <NUM>. Finally, the fourth layer <NUM> is fabricated containing a reservoir filled with HBSS or additional saline solution to prevent evaporation of water from the multicellular aggregates and is then bonded to the third layer <NUM>. The device <NUM> is then heated in an oven for <NUM> hr at <NUM> to activate the hydrophobicity of the layers.

During the microfabrication of the microfluidic device layers, the silicon masters when prepared by 3D printing may adhere to PDMS molds, resulting in the distortion of the microwell shape and size. As both the size and shape of the microwells <NUM> are critical to the controlled formation of the multicellular aggregate, the fabrication method may include steps to address this issue. Specifically, in one non limiting example, the surface of the 3D printed masters may be treated with trichloro (<NUM>,<NUM>,<NUM>,<NUM>-perfluorooctyl) silane vapor. The treatment substrate for the 3D printed masters may be a variety of fluorinated/chlorinated aliphatic silanes which are suitable for achieving adequate fluid film lubrication. The surface of the masters is then, in this particular embodiment, controllably etched with H<NUM>SO<NUM>. The etching is needed in order to compensate for resolution limitations in the 3D printer which may result in the surface of microwells <NUM> being intrinsically rough.

In a non-limiting embodiment, the fabrication method of the microfluidic device layers and assembly into device <NUM> may utilize photolithography to fabricate microfluidic devices for MF-MCS platform, micromachining, etching or laser ablation.

In another embodiment, the fabrication method of the microfluidic device layers and assembly into device <NUM> may utilize 3D printing technologies. The utilization of 3D printing may make microfluidic devices more cost-efficient and versatile. Furthermore, the fabrication of the microfluidic device through 3D printing technology may enable growth of multicellular aggregates such as multicellular spheroids with dimensions larger than <NUM> and the growth of various sized multicellular spheroids in the same device, thus enabling drug screening for microtumors with different dimensions.

According to an embodiment, a method for producing of an array of multicellular aggregates onto the microfluidic device is provided. As previously mentioned, the multicellular aggregates consist of aggregates of cells being self-organized in a three-dimensional arrangement. The multicellular aggregates may be organoids obtained with stem cells or a combination of stem cells and other cells.

Alternatively, the multicellular aggregates may be multicellular spheroids. The multicellular spheroids may be made of cells obtained from a cancer cell line or cells obtained from primary cells isolated from cancer patient tissue. Such cells may be obtained from separation and isolation from tissue biopsy. Depending on the use of the current invention, the multicellular aggregates may comprise one kind of cells or a plurality of different kinds of cells resulting in heterogeneous organoids or spheroids.

According to an embodiment, the multicellular aggregates such as multicellular spheroids or organoids may be obtained from high-density cell suspensions or low-density cell suspensions.

According to an embodiment, multicellular aggregate such as multicellular spheroids or organoids may be obtained with varying dimensions in the hydrogels having different properties/characteristics in separate rows of the microfluidic device <NUM>.

According to an embodiment, multicellular aggregates such as organoids, multicellular spheroids or spheroids may be formed in a multi-step procedure, which includes the generation of uniformly-sized droplets from a dense suspension of cells in a solution of the hydrogel precursor in the microwells <NUM> of microfluidic device <NUM> and chemically or physically crosslinking of the hydrogel precursor, thus transforming droplets into cell-laden micrometer-size hydrogels and MCS formation and growth.

According to an embodiment, as shown in <FIG>, a method of rapidly producing uniformly-sized multicellular aggregates from a high-density cell suspension using the microfluidic device <NUM> of the present invention. The method may comprise the steps of:.

According to an embodiment, the solution <NUM> comprising the aqueous high-density suspension of cells and the hydrogel precursor <NUM> may have a cell concentration ranging from <NUM>×<NUM><NUM> to <NUM>×<NUM><NUM> cells/mL. For examples, the solution <NUM> may have a cell concentration of <NUM>×<NUM><NUM> cells/mL to obtain high-density spheroids.

According an embodiment, as shown in <FIG>, the method may further comprise the step of continuously flowing a second cell culture medium <NUM> into the supplying channel <NUM> of the at least one row <NUM> of the microfluidic device <NUM> for the during of the studies on the products in microwells <NUM>, wherein the cell culture medium <NUM> promotes cell growth of the suspension of cells <NUM> and the formation of the multicellular aggregates <NUM> within the hydrogels <NUM>.

According to an embodiment, when the cell culture medium <NUM> may be continuously flowing into the support channel <NUM> of the at least one row <NUM> of the microfluidic device <NUM>, the continuous flow may be maintained to promote the formation of multicellular aggregates such as multicellular spheroids or organoids within about <NUM> to about <NUM> days.

According to an embodiment, the first and second cell culture media are similar. Alternatively, they may be distinct.

According to another embodiment, multicellular aggregates may be formed using a multi-step procedure as described above but using a low-density cell suspension as shown in <FIG>.

According to an embodiment, the solution <NUM> comprising the aqueous low-density suspension of cells and the hydrogel precursor <NUM> may have a cell concentration ranging from <NUM> to <NUM> cells/µl. For examples, the solution <NUM> may have a cell concentration of <NUM> cells/µL to obtain low-density spheroids.

According to an embodiment, the multicellular aggregates such as multicellular spheroids and organoids may be obtained with the use of a solution comprising the aqueous suspension of cells having a plurality of different types of cells.

According to an embodiment, the multicellular aggregates such as multicellular spheroids or organoids may have diverse range of diameters suitable for a given application or use. Non-limiting diameters may range from at least <NUM>, and some are suitable in a range from about <NUM> to about <NUM> or from about <NUM> to about <NUM>, or higher than <NUM>. Alternatively, the multicellular aggregates may have a diameter ranging from about <NUM> to <NUM>.

According to an embodiment, the method may further comprise the step of releasing the multicellular aggregates <NUM> from the microwells <NUM> into the supplying channel <NUM> and moving the aggregates to the corresponding supplying exit for retrieval of the hydrogels <NUM> from the microfluidic device <NUM>. The multicellular aggregates <NUM> such as spheroids or organoids may be released using different means known to the skilled person in the art. They may be released by enzymatic or chemical-mediated hydrogel digestion/lysis, using mechanical hydrogel disruption, hydrogel photodegradation. Alternatively, the spheroids or organoids may be "washed away" by strongly increasing the flow rate in the microfluidic device. According to another embodiment, the spheroids or organoids may be released by inducing liquefaction of a temperature-responsive hydrogel at a reduced physiologically acceptable temperature.

According to an embodiment, the first agent 25a and second wetting agents 25b may be the same. The wetting agents 25a and 25b are a liquid that is immiscible in water. For example, the wetting agents 25a and 25b may be an organic liquid such as oil.

According to an embodiment, the wetting agent 25a and 25b comprise a fluorinated oil. According to another embodiment, the wetting agent 25a and 25b comprise a fluorinated oil and a surfactant. The surfactant may be, but is not limited to a block copolymer perfluorinated polyether-b-(polypropylene glycol-polyethylene glycol-polypropylene glycol)-b-perfluorinated polyether. Non-limiting examples of wetting agent are fluorinated oil is mixed with <NUM> wt%, <NUM> wt. % or <NUM> wt% block copolymer perfluorinated polyether-b-(polypropylene glycol-polyethylene glycol-polypropylene glycol)-b-perfluorinated polyether.

Because the aqueous solution <NUM> may wet the surface of microwells <NUM> and supplying channel <NUM> during the formation of cell-laden precursor droplets <NUM> and therefore interfere with the generation of uniformly-sized droplets <NUM> and by extent of this, with the formation of accurately sized multicellular spheroids or organoids <NUM> within the hydrogels <NUM>, wetting agent 25a is used to wet the walls of the microwells <NUM> and supplying channel <NUM>. When the aqueous solution <NUM> comprising cells <NUM> and a hydrogel precursor <NUM> enters the supplying channel <NUM> and microwells <NUM>, the aqueous phase consisting of the aqueous solution <NUM> is surrounded by the oil <NUM> in the microwells <NUM> adopts a more spherical shape than in the supplying channel <NUM> due to its reduced interfacial area.

A role of the wetting agent 25b is to form droplets in the wells <NUM>, which will form microgels upon gelation, and to wash the solution <NUM> from the supplying channel <NUM> while the solution <NUM> is contained within the microwells <NUM> as captured as droplets <NUM>. When introducing the wetting agent 25b into the supplying channel <NUM>, the mixture of precursor/cell becomes primed into droplets <NUM> and retained into the microwells <NUM> as the droplets <NUM> would have to deform in order to exit the microwells <NUM>.

According to an embodiment, the first step of introducing a first wetting agent 25a into the supplying channel <NUM> and corresponding microwells <NUM> may comprise the sub-step of first filling the supplying channel <NUM> and microwells <NUM> with a fluorinated oil and then replacing the fluorinated oil with a solution of fluorinated oil and surfactant such as a solution of fluorinated oil and <NUM> wt. % block copolymer perfluorinated polyether-b-(polypropylene glycol-polyethylene glycolpolypropyleneglycol)-b-perfluorinated polyether (PFPE-b-(PPG-PEG-PPG)-b-PFPE).

To achieve adequate functionality for a variety of screening applications, the multicellular spheroids or organoids may form in a variety of environments having different stiffness and permeability properties. The stiffness and permeability of the hydrogel scaffolds will mimic the characteristics of the environments in which the studies cells are usually found. As such, a skilled person would understand the need to select appropriate hydrogel precursors and the need to tune the formulation of solution of hydrogel precursors to obtain a hydrogel scaffold with suitable characteristics while mimicking the environment from which the cells forming the multicellular spheroids or organoids growth are derived.

According to an embodiment, the hydrogel scaffold may be obtained with the use of a hydrogel precursor of synthetic monomers or polymers, biopolymers or combination of polymers and/or biopolymers. Non-limiting examples of polymers or biopolymers for the formation of hydrogels are collagen, gelatin, fibrin, agarose, alginate, polyacrylamide, polyethylene glycol, hyaluronic acid, cellulose derivatives, polypeptides, and mixtures of these polymers and nanoparticles functionalized with biopolymers or synthetic polymers.

According to an embodiment, the hydrogel scaffold may be obtained via either chemical crosslinking, physical crosslinking or combination of chemical and physical crosslinking of the precursor as long as the hydrogel scaffold remains intact when subjected to continuous flows within the microfluidic device.

According to an embodiment, the flow within the microfluidic device may be set to flow rates up to <NUM>/hour without damaging the microfluidic device. According to another embodiment, flow rates between the range of <NUM> to <NUM>/hour may be used for cell culture and droplet generation.

The skilled person would know that any hydrogel precursor may be used as long as <NUM>) the precursor and resulting hydrogel scaffold is non-toxic and biocompatible to the cells, <NUM>) the desired gelation time, stiffness, permeability/diffusion, bioadhesion are obtained and <NUM>) the resulting hydrogel scaffold would be stable and withstand the shear forces when subjected to the continuous flows within the microfluidic device. The skilled person will also understand that the amount of precursor and degree of crosslinking may be varied to obtain hydrogel scaffolds with the desired characteristics, e.g., gelation time, stiffness, permeability/diffusion, as long as the cells may survive in the hydrogel scaffolds and formed multicellular spheroids or organoids.

According to an embodiment, the hydrogel precursor may be functionalized with growth factors and/or peptides fragments to promote cell growth, survival and transformation into a spheroid of organoid.

According to an embodiment, prior using the method for producing multicellular aggregates of the present disclosure, the skilled person may have use cell suspension in macroscopic gels as a first screening to identify which hydrogel precursor formulation would result in a hydrogel with suitable characteristics such as stiffness and permeability/diffusion. Once the screening has been done and the properties of the hydrogel scaffold have been tuned, the multicellular aggregates may be prepared using the microfluidic device.

According to an embodiment, depending on the selected precursor, the degree of crosslinking of the hydrogel scaffold and the total amount of precursor in the solution may be varied to enable the tuning of the stiffness of the hydrogel scaffold from tens of Pa to tens of kPa to provide the cells with an environment with suitable stiffness. According to an embodiment, the hydrogel scaffold may have a stiffness ranging between about <NUM> Pa to hundreds kPa or about <NUM> Pa about 100Kpa. According to another embodiment, the hydrogel scaffold may have a stiffness ranging between about <NUM> Pa to about 20kPa. The skilled person would understand that the mechanical properties of the hydrogel scaffolds should be tailored to the mechanical properties of the environments the hydrogel scaffolds are mimicking.

For example, for soft tissues and organs, the stiffness may range from <NUM> kPa to 1Mpa. The stiffness for brain tissues may range from <NUM> kPa to <NUM> kPa, the stiffness for skin, spleen or pancreas tissues may range from 1kPa to <NUM> kPA, the stiffness for gland and muscle tissues may range from <NUM> kPa to <NUM> kPa, the stiffness for tendon tissues may range from <NUM> kPa to <NUM> kPa, the stiffness for cartilage tissues may range from <NUM> kPa to <NUM> kPa, the stiffness for bone tissues may range from <NUM> MPA to <NUM> MPa and the stiffness for breast tissues may range from <NUM> kPA to <NUM> kPA (<NPL>;<NPL>;<NPL>; <NPL>; and <NPL>)The skilled person will understand that these ranges may vary depending on the publication source and also the method use to characterize the stiffness of the tissues.

Similarly, depending on the selected precursor, the gelation time, which is needed for the generation of precursor droplets and their gelation in the microwells <NUM> of the microfluidic device <NUM>, may be tuned by varying the degree of crosslinking of the hydrogel scaffold and the concentration of the hydrogel precursor. The gelation time may range between the order of about <NUM> minutes to hours depending on the nature of the hydrogel precursor, the concentration of the hydrogel precursor, the type of crosslinking and the degree of crosslinking of the hydrogel scaffold and the method of gelation being employed. For example, the time of gelation may range from about <NUM> minutes to about <NUM> hours and more preferably from about <NUM> to about <NUM> hours for enabling the generation of cell-laden droplets and support cell viability. Although longer gelation time may be possible, it will be appreciated that gelation times which are too long may be problematic and/or not suitable due to cell death.

According to an embodiment, depending on the selected precursor, the degree of crosslinking of the hydrogel scaffold and the total amount of precursor in the solution may be varied to enable the tuning of the permeability of the hydrogel scaffold. Proper permeability allows the transport of nutrients, chemicals or drugs from the media to the multicellular aggregates, e.g., spheroids or organoids. Furthermore, proper permeability promotes cell survival, cell growth, transformation into multicellular spheroids or organoids, survival of the multicellular spheroids or organoids and diffusion of the screened compounds to the multicellular spheroids or organoids. The permeability of the hydrogel scaffolds, depending on the selected precursor, the degree of crosslinking of the hydrogel scaffold and the total amount of precursor in the solution, may be at least <NUM>-<NUM>cm<NUM>. For example, the permeability may be between about <NUM>-<NUM> to about <NUM>-<NUM> cm<NUM>.

According to an embodiment, any chemically-crosslinked hydrogel scaffolds obtained via the covalent bonding between polymer chains may be used as long as the hydrogel scaffolds have suitable characteristics and allow the formation and survival of multicellular aggregates or spheroids. The crosslink formation may be carried out by the addition of small cross-linkers molecules, polymer-polymer conjugation, photosensitive agents or by enzyme catalyzed reaction.

According to another embodiment, a chemically crosslinked hydrogel scaffold may be derived from flexible biopolymers or polymers containing free amine groups and aldehyde-modified cellulose nanocrystals (a-CNCs). Because CNCs are rod-like, rigid nanoparticles with an average length and diameter of <NUM>-<NUM> and <NUM>-<NUM>, respectively, which assemble into a nanofibrillar network, the resulting a-CNC-derived hydrogel scaffolds may have a scaffolding structure that resembles the structure of the ECM proteins. CNCs may be surface-modified by oxidative cleavage at the C2-C3 bond in the presence of periodate, which yields dialdehyde groups at the respective carbon atoms. The aldehydes present on the a-CNCs react with the free amine groups present on the flexible biopolymers or polymers to form imine crosslinks resulting in a hydrogel scaffold that may be used in the synthesis of the hydrogel-based multicellular spheroids.

According to another embodiment, the chemically crosslinked hydrogel scaffold is obtained from the crosslinking of free amine groups present on gelatin and the aldehyde groups present on the a-CNCs as shown in <FIG>. Gelatin which is denaturated collagen. It contains Arg-Gly-Asp (RGD) sequences which are available to bind to integrin receptors on the cell surfaces, thereby advantageously making the hydrogel scaffold bioadhesive. The rationale behind the use of a-CNCs/gelatin hydrogels is that the a-CNCs may act as a filamentous building block (responsible for the nanofibrillar structure), while the gelatin may act bioadhesive soft component resulting in a hydrogel mimicking the ECM environment which may be used in the synthesis of the hydrogel-based multicellular spheroids.

An additional advantage of using a-CNCs/gelatin hydrogel is that both constituents are nontoxic and cytocompatible to a variety of cell types both in culture media and in hydrogel constructs.

Type A gelatin (<NUM> bloom), sodium periodate, butyl acrylate, <NUM>-vinyl anthracene, potassium persulfate, sodium dodecyl sulfate, and acetic acid were purchased from Sigma-Aldrich, Canada and used without further purification, unless otherwise specified. An aqueous <NUM> wt % suspension of CNCs was purchased from the University of Maine Process Development Center and dialyzed for <NUM> days against Milli-Q grade distilled deionized water (DI, <NUM> MΩ cm resistivity) before use.

Aldehyde-functionalized CNCs were prepared according to the protocol described by Prince at al. Briefly, the oxidation of the surface hydroxyl groups on CNCs to yield a-CNCs was performed by adding sodium periodate (NaIO4) to a <NUM> wt % suspension of CNCs at a NaIO4/CNC weight ratio of <NUM>:<NUM>. The pH was adjusted to <NUM> with acetic acid. The flask was covered with aluminum foil to prevent photodecomposition of NaIO4. The suspension was stirred at <NUM> for <NUM> and subsequently quenched by adding ethylene glycol. The suspension of a-CNCs was dialyzed against deionized water for <NUM> days, with replacement twice a day, and then concentrated by rotary evaporation.

The presence of aldehyde groups on the CNC surface was confirmed with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) using a Bruker Vertex <NUM> spectrometer with a <NUM> diameter diamond crystal. The aldehyde group content of the CNC surface was determined by first converting the aldehydes to carboxylic acid groups in <NUM> NaOH via the intramolecular Cannizzaro reaction and subsequently titrating with sulfuric acid to determine the consumption of hydroxide ions.

The TNBS (trinitrobenzenesulfonic acid) assay was used to quantify the number of primary amine groups in gelatin, as described by Prince et al. Briefly, gelatin was dissolved in <NUM> sodium carbonate buffer (pH = <NUM>) to a final concentration of <NUM> wt %. Trinitrobenzenesulfonic acid (TNBS) was added to the solution to a final concentration of <NUM> w/v %, and the solution was equilibrated for <NUM> at <NUM>. The absorbance of the solution was measured at λ = <NUM>. A calibration curve was prepared by measuring the absorbance of the solution at λ = <NUM> for standard solutions of beta alanine in <NUM> sodium carbonate buffer, containing <NUM> w/v % TNBS.

The molar ratio between the amine groups on gelatin and aldehyde groups on a-CNCs determines the number of complimentary cross-linkable groups. The molar ratio was determined by the following assay, as described by Prince et al. The concentration of aldehyde groups in the a-CNCs was characterized by first converting these groups to carboxylic acid groups in <NUM> NaOH using an intra-molecular Cannizzaro reaction, and subsequently titrating with sulfuric acid to determine the consumption of hydroxide ions. The aldehyde content was determined to be <NUM>µmol/gram CNCs. The concentration of primary amines in gelatin was determined by the TNBS assay and determined to be <NUM>µmol/g gelatin. Beta-alanine was used to prepare the calibration curve.

To achieve accurate control of the properties of the multicellular spheroids in gelatin/a-CNC hydrogels, a set of experimental runs utilizing macroscopic gelatin/a-CNC hydrogels and macroscopic gelatin/a-CNC hydrogels seeded with a suspension of breast cancer cells (MCF-<NUM>) were performed. For the macroscopic gelatin/a-CNC hydrogels seeded with a suspension of b MCF-<NUM> cells, the set of experimental runs utilized the suspension of MCF-<NUM> cells having a cell concentration of <NUM> × <NUM><NUM> cell/mL in a precursor solution of a-CNCs and gelatin.

The characterization and optimization of hydrogel formulations are performed using macroscopic hydrogels and by analyzing the hydrogels using scanning electron microscopy (SEM) imaging, rheometry, swelling, mechanical characterization, gelation time characterization, permeability experiments.

The Young's modulus of the hydrogels was determined in cyclic compression experiments using a Mach-<NUM> Mechanical tester (Biomomentum Inc. , QC) operating in parallel plate geometry. The hydrogel disks for mechanical testing were <NUM> in height and <NUM> in diameter. The disks were compressed by applying <NUM>% strain in the z-direction at a rate of <NUM>/s. The Young's modulus of the hydrogels was determined by fitting the linear portion of the resulting stress-strain curve. All hydrogels were equilibrated for <NUM> before the measurements.

<FIG> and <FIG> show the variation in the Young's modules of a-CNC gelatin hydrogels with varying concentration of a-CNCs and a gelatin concentration of <NUM> wt%. As the concentration of a-CNC increases the hydrogel stiffness also increases from <NUM>±<NUM>. 7kPa for <NUM> wt% of a-CNC to <NUM>±<NUM> kPa for <NUM> wt%.

<FIG> shows the effect of varying R on the hydrogel's Young's modulus at Ctotal of <NUM> and <NUM> wt %.

The hydrogel permeability was characterized by using the experimental setup as described by Prince et al. To determine Darcy permeability of the a-CNC/gelatin hydrogels, a hydrogel sample with the dimensions <NUM> × <NUM> × <NUM> (width × height ×length) was formed in a chamber fabricated in poly(dimethylsiloxane) (PDMS). Perfluoroalkoxyalkane tubing (IDEX Health & Science) was used to connect the ends of the chamber to inlet and outlet reservoirs containing HBSS solution. A pressure difference was applied across the hydrogel by varying the height of the inlet reservoir relative to that of the outlet reservoir. The HBSS solution was under the influence of the pressure drop. The value of the volumetric flow rate (Qp) of the HBSS solution perfused through the hydrogel sample was determined by measuring the change in the mass of the outlet reservoir over a particular time interval. The Darcy permeability was determined as: <MAT> where A is the hydrogel's cross-sectional area (<NUM><NUM>), L is the hydrogel length (= <NUM>), ΔP is the pressure drop across the hydrogel, and η is the viscosity of HBSS solution (taken as <NUM> cP, the viscosity of water at room temperature).

A hydrogel sample was prepared in a chamber with dimensions <NUM> × <NUM> × <NUM> (width x height x length), which was fabricated from poly(dimethyl siloxane) (PDMS). The two openings of the chamber were connected to an inlet and outlet reservoir, each containing <NUM> of HBSS. Perfluoroalkoxyalkane tubing (IDEX Health & Science) was used to connect the ends of the chamber to inlet and outlet reservoirs containing HBSS. A pressure difference was applied across the hydrogel by varying the height of the inlet reservoir relative to that of the outlet reservoir. To ensure that the pressure drop, ΔP, applied to the hydrogel does not lead to a substantial change in hydrogel structure, we ensured that a linear relationship exists between the ΔP and volumetric flow rate in the range <NUM>≤ΔP≤ <NUM> Pa for hydrogels, as expected for a porous network with a static structure.

<FIG> shows the variation in the Darcy permeability of the hydrogel used for cell culture. The total concentration of the hydrogel (Ca-CNC + Cgelatin) was <NUM> wt%. The permeability increases as the a-CNC-to-gelatin ratio ® increases. The change in permeability increased over <NUM> orders of magnitude by varying R from <NUM> to <NUM>.

<FIG> shows the variation in hydrogel permeability with varying R, at Ctotal of <NUM> and <NUM> wt %.

The gelation time for macroscopic a-CNC/ gelatin hydrogels was determined by the inversion test. A <NUM> vial containing <NUM>µL of the mixed suspension of a-CNCs and gelatin was inverted every <NUM> at room temperature. Gelation time was determined as the time when no flow was observed upon inversion. The variation in gelling time is achieved by varying the gelatin-to-a-CNC concentration ratio and additionally, by varying the total concentration of a-CNCs and gelatin. As shown in <FIG>, the gelation time vary depending on the ratio gelatin-to-a-CNC ratio and the total concentration of a-CNCs and gelatin. <FIG> shows that it is possible, with the exemplified formulations, to tune the gelation time between about <NUM>. to almost to <NUM>.

According to an embodiment, for the purpose of preparing multicellular spheroids in a-CNCs/gelatin hydrogels using a microfluidic device, it was found that the concentration of gelatin and the ratio of concentrations of a-CNCs and gelatin in the solution may be varied such that the time required for the formation of the hydrogel was tuned to be anywhere between about <NUM> minutes to hours but preferably between about <NUM> minutes to about <NUM> hours, and more preferably from about <NUM> to about <NUM> hours. Although longer gelation time may be possible, it will be appreciated that gelation times which are too long may be problematic and/or not suitable due to cell death.

Furthermore, when a-CNCs/gelatin hydrogels are used for the preparation of multicellular spheroids using a microfluidic device, the gelation time was controlled by changing the total concentration of a-CNCs and gelatin and the mass ratio of a-CNCs-to-gelatin to optimize the formation of droplets before a significant increase in viscosity (taking ~<NUM>).

Prior to the preparation of the macroscopic a-CNC/gelatin hydrogels seeded with human breast cancer MCF-<NUM> cells, the cells were cultured in <NUM> polystyrene tissue culture flasks. To each flask, <NUM> of Dulbecco's Modified Eagle Medium with <NUM>/L glucose, L-glutamine, and sodium pyruvate (DMEM-F12, GIBCO), supplemented with <NUM>% (v/ v) fetal bovine serum (FBS, Invitrogen), and <NUM>% (v/v) penicillin/ streptomycin were added. The flasks were incubated at <NUM> with a constant <NUM>% CO2 supply in the incubator. For cell passage, a Trypsin- EDTA solution (<NUM> wt %, GIBCO) was used to detach cells from the basement support. After detachment, <NUM> of fresh media was added and the suspension was centrifuged at <NUM> × g and <NUM> for <NUM>. The supernatant was removed, and the pellet was resuspended in <NUM> fresh media. <NUM>µL of the cell suspension was then transferred to fresh media in a new flask. Cells were passaged every <NUM> days.

Prior to the preparation of the macroscopic a-CNC/gelatin hydrogels seeded with human breast cancer MCF-<NUM> cells, an a-CNC suspension at a selected concentration and a gelatin solution at a selected concentration were prepared in Hank's balanced salt solution (HBSS) in order to obtain a a-CNC/gelatin hydrogel with a desired final wt % concentration of a-CNC and gelatin. Both solutions were sterilized by exposure to ultraviolet light (Sterilaire Lamp, <NUM>, <NUM>µW/cm2) for <NUM>. The gelatin solution and a- CNC suspension were then mixed with a cell suspension to give the final hydrogel composition. The cell density was <NUM> cells/well. Cells were cultured in a <NUM>-well plate and incubated at <NUM> at a constant <NUM>% CO2 supply for <NUM> before adding an additional <NUM>µL of fresh media to each well.

For example, prior to the preparation of the macroscopic a-CNC/gelatin hydrogels seeded with human breast cancer MCF-<NUM> cells, a <NUM> wt % a-CNC suspension and a <NUM> wt % gelatin solution were prepared in Hank's balanced salt solution (HBSS). Both solutions were sterilized by exposure to ultraviolet light (Sterilaire Lamp, <NUM>, <NUM>µW/cm2) for <NUM>. The gelatin solution and a-CNC suspension were then mixed with a cell suspension to give a final hydrogel composition of Ctotai = <NUM> wt %, Cgelatin = <NUM> wt % and Ca-CNC = <NUM> wt % (weight ratio of a-CNC to gelatin (R) is <NUM>). The cell density was <NUM> cells/well. Cells were cultured in a <NUM>-well plate and incubated at <NUM> at a constant <NUM>% CO2 supply for <NUM> before adding an additional <NUM>µL of fresh media to each well.

On day <NUM>, <NUM>, and <NUM> of cell culture, the cells were stained with calcein-AM (Invitrogen, Carlsbad; green fluorescence) and ethidium homodimer-<NUM> (Invitrogen, Carlsbad; red fluorescence) to identify live versus dead cells. To each well, <NUM>µL of the assay solution was added and incubated for <NUM> at <NUM>. The cells were then imaged by fluorescence microscopy (Nikon, Eclipse Ti).

<FIG> shows the viability and MCF growth from MCF-<NUM> cells in the hydrogels with a-CNC concentration of <NUM> wt% and gelatin concentration of <NUM> wt % after A) <NUM> day, B) <NUM> days and C) <NUM> days.

<FIG> shows cell viability assay over a period of <NUM> days using <NUM> wt%, <NUM> wt% and <NUM> wt% a-CNC and a gelatin concentration of <NUM> wt%. A fluorescent colorimetric dye Alamar blue was used to assess metabolic cell activity and cell viability. As seen in <FIG>, the cell viability is significantly greater at <NUM> days for hydrogel formulations of <NUM> wt% a-CNC and <NUM> wt% gelatin.

<FIG> shows the MCS obtained with MCF <NUM> cells from <NUM> day to <NUM> days with different concentration of a-CNCs and a gelatin concentration of <NUM> wt%. The variation in MCS diameter with time in hydrogels with varying concentration of a-CNCs , i.e., <NUM> wt%, <NUM> wt% and <NUM> wt% and a gelatin concentration of <NUM> wt%. For each a-CNC concentration, MCS showed significant growth for each successive time period (p<<NUM>). On day <NUM> and day <NUM>, MCS growth from MCF <NUM> cells in <NUM> wt% was statistically lower compared to <NUM> wt% and <NUM> wt% (p<<NUM>).

<FIG> shows the variation in MCF growth over a period of <NUM> days from MCF <NUM> cells in hydrogels with varying gelatin concentration, i.e., <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt% and <NUM>,<NUM> wt% and a-CNC concentration of <NUM> wt%. At gelatin concentration of <NUM>-<NUM> wt% significant MCS growth was observed for each successive time period (p<<NUM>). As seen in <FIG>, hydrogels with Cgelatin above <NUM> wt% were more favorable for MCS growth.

Immunostaining was used to evaluate the cell-to-cell interactions and cell nuclei organization. After <NUM> day culture, a-CNC/gelatin hydrogels with-multicellular spheroids formed at a cell concentration of <NUM>×<NUM> <NUM> cell/mL were fixed with <NUM>% paraformaldehyde diluted in HBSS. All the solutions used below were infused into the channels and microwells at a rate of <NUM>/h at room temperature. A solution of <NUM>% paraformaldehyde was perfused into the channels and wells for <NUM>. Subsequently, the paraformaldehyde solution was washed away by infusing a solution of <NUM> glycine in HBSS for <NUM>. Then, a solution of <NUM>% Triton X-<NUM> in HBSS was infused into the channels and wells to permeabilize the cells for <NUM>.

To remove excess Triton X-<NUM>, the Polymer-multicellular spheroids were washed by a washing solution (IF wash) consisting of <NUM> wt% NaN3, <NUM> wt% Bovine Serum Albumin, <NUM> vol% mL Triton X-<NUM>, <NUM> vol% mL Tween <NUM> in HBSS for <NUM>. After that, a block solution (<NUM>% goat serum in IF wash) was infused to the channels and wells for <NUM>. The block solution was replaced by a solution of the antibody (Alexa Fluor <NUM> E-Cadherin Rabbit monoclonal antibody, <NUM>:<NUM> dilutions in HBSS, Cell Signaling Technology), which was allowed to incubate overnight at <NUM>. To remove antibody excess, the Polymer-multicellular spheroids were perfused with the IF wash for <NUM> at room temperature. To stain cell nuclei, <NUM> ng/mL <NUM>',<NUM>-diamidino-<NUM>-phenylindole (DAPI, Life technologies) in HBSS was perfused into Polymer-multicellular spheroids for <NUM>. The structure of CSs was visualized and imaged by Nikon A1 confocal microscope.

Immunostaining of the a-CNC/gelatin-multicellular spheroids after <NUM> day culture was used to evaluate the cell-cell interactions and cell nuclei organization. <FIG> shows immunofluorescence staining of breast cancer MCS grown from MCF-<NUM> breast cancer cells formed in a-CNC/gelatin with a-CNC/ gelatin concentrations of <NUM> wt % a-CNC and <NUM> wt. % gelatin after <NUM>-day culture: <FIG>Immunostaining of the MCS by DAPI (Blue); <FIG> Immunostaining of the MCS by Alexa Fluor <NUM> E-Cadherin Rabbit monoclonal antibody (green); <FIG>Immunostaining of the MCS by Alexa Fluor <NUM> Phalloidin (Red); and <FIG>merged image composed of the fluorescence images in <FIG>. The results shown in <FIG> indicates that the multicellular aggregates transform from a cluster of cells into cancerous spheroids which may act as cancerous micro-tumors and are likely suitable to screen the effect of drugs on tumors.

Prior to the preparation of the macroscopic a-CNC/gelatin hydrogels seeded with patient derived pancreatic cells, the cells were cultured in <NUM>µL matrigel (<NUM> - <NUM>/mL) domes in a <NUM> well TC plate. To each well, <NUM> of Advanced Dulbecco's Modified Eagle Medium with <NUM>/L glucose, L-glutamine, and sodium pyruvate (Advanced DMEM-F12, GIBCO), supplemented with <NUM> GlutaMAX, <NUM> HEPES, <NUM> U/mL Antibiotic-Antimycotic, 1X B-<NUM> Supplement <NUM> N-Acetyl-L-cytseine, <NUM> gastrin (<NUM>-<NUM>), <NUM> ng/mL Recombinant Human EGF, <NUM> ng/mL Recombinant Human Noggin, <NUM> ng/mL Recombinant Human FGF-<NUM>, <NUM> A <NUM>-<NUM>, <NUM> Y-<NUM>, <NUM> Nicotinamide, <NUM>% v/v Wnt-3a conditioned media, and <NUM>% v/v Human R-Spondin1 conditioned media. The well plates were incubated at <NUM> with a constant <NUM>% CO2 supply in the incubator. For cell passage, <NUM> of a TryLE Express solution (Invitrogen) was used to detach cells and disintegrate the matrigel. After cell release, the suspension was added to <NUM> of fresh media and centrifuged at <NUM> × g and <NUM> for <NUM>. The supernatant was removed, and the pellet was resuspended in <NUM> fresh matrigel. <NUM>µL of the cell suspension was then transferred to a new well plate and <NUM> fresh media was added. Cells were passaged every <NUM> days.

Prior to the preparation of the macroscopic a-CNC/gelatin hydrogels seeded with patient derived pancreatic cells, a <NUM> wt % a-CNC suspension and a <NUM> wt % gelatin solution were prepared in Hank's balanced salt solution (HBSS). Both solutions were sterilized by exposure to ultraviolet light (Sterilaire Lamp, <NUM>, <NUM>µW/cm2) for <NUM>. The gelatin solution and a-CNC suspension were then mixed with a cell suspension to give a final hydrogel composition of Ctotal = <NUM> wt %, Cgelatin = <NUM> wt % and Ca CNC = <NUM> wt % (weight ratio of a-CNC to gelatin (R) is <NUM>). The cell density was <NUM> cells/well. Cells were cultured in a <NUM>-well plate and incubated at <NUM> at a constant <NUM>% CO2 supply for <NUM> before adding an additional <NUM>µL of fresh media to each well.

<FIG> shows the viability of patient-derived pancreatic cancer cells on Day <NUM> in the hydrogel with <NUM> wt% a-CNC and <NUM> wt. %, gelatin. The cells were stained by calcein AM (green) and Ethidium homodimer-<NUM> (red). Green color and lack of red color signify high cell viability in the hydrogel.

<FIG> shows the culture of primary pancreatic organoids in a-CNC/gelatin hydrogel with varying compositions compared to matrigel. The arrows show the presence of large cancer spheroids at days <NUM> and <NUM> for <NUM> wt% a-CNC and <NUM> wt% gelatin indicating that the hydrogels may be tuned for different cells.

Breast cancer cells were obtained from dissociated tissue prior to the preparation of the macroscopic a-CNC/gelatin hydrogels seeded with patient-derived breast cancer.

Prior to the preparation of the macroscopic a-CNC/gelatin hydrogels seeded with patient-derived breast cancer cells, the cells were cultured in <NUM>µL matrigel (<NUM> - <NUM>/mL) domes in a <NUM> well TC plate. To each well, <NUM> of Advanced Dulbecco's Modified Eagle Medium with <NUM>/L glucose, L-glutamine, and sodium pyruvate (Advanced DMEM-F12, GIBCO), supplemented with <NUM> GlutaMAX, <NUM> HEPES, <NUM> U/mL Antibiotic-Antimycotic, 1X B-<NUM> Supplement <NUM> N-Acetyl-L-cytseine, <NUM> gastrin (<NUM>-<NUM>), <NUM> ng/mL Recombinant Human EGF, <NUM> ng/mL Recombinant Human Noggin, <NUM> ng/mL Recombinant Human FGF-<NUM>, <NUM> A <NUM>-<NUM>, <NUM> Y-<NUM>, <NUM> Nicotinamide, <NUM>% v/v Wnt-3a conditioned media, and <NUM>% v/v Human R-Spondin1 conditioned media. The well plates were incubated at <NUM> with a constant <NUM>% CO2 supply in the incubator. For cell passage, <NUM> of a TryLE Express solution (Invitrogen) was used to detach cells and disintegrate the matrigel. After cell release, the suspension was added to <NUM> of fresh media and centrifuged at <NUM> × g and <NUM> for <NUM>. The supernatant was removed, and the pellet was resuspended in <NUM> fresh matrigel. <NUM>µL of the cell suspension was then transferred to a new well plate and <NUM> fresh media was added. Cells were passaged every <NUM> days.

Prior to the preparation of the macroscopic a-CNC/gelatin hydrogels seeded with patient-derived breast cells, a <NUM> wt % a-CNC suspension and a <NUM> wt % gelatin solution were prepared in Hank's balanced salt solution (HBSS). Both solutions were sterilized by exposure to ultraviolet light (Sterilaire Lamp, <NUM>, <NUM>µW/cm2) for <NUM>. The gelatin solution and a-CNC suspension were then mixed with a cell suspension to give a final hydrogel composition of Ctotal = <NUM> wt %, Cgelatin = <NUM> wt % and Ca CNC = <NUM> wt % (weight ratio of a-CNC to gelatin (R) is <NUM>). The cell density was <NUM> cells/well. Cells were cultured in a <NUM>-well plate and incubated at <NUM> at a constant <NUM>% CO2 supply for <NUM> before adding an additional <NUM>µL of fresh media to each well.

<FIG> shows the growth of multicellular cancer spheroids from breast tumor biopsy at A) day <NUM>, B) Day <NUM> and C) Day <NUM> (Images were taken from the same location in the hydrogel). The hydrogels contain <NUM> wt% a-CNC and <NUM> wt% gelatin. and D-F) different views of MCSs grown from breast cancer patient-derived cells in the a-CNC/gelatin hydrogel after day <NUM> (the concentration of a-CNCs and gelatin are <NUM> and <NUM> wt. %, respectively).

The results obtained with the breast cancer MCF <NUM> cells, the patient-derived pancreatic cancer cells and the patient-derived breast cancer cells indicate that the hydrogels may be tuned to have suitable characteristics to promote the formation of multicellular aggregates such as multicellular spheroids or organoids using the method, system and device of the present disclosure.

The microfluidic device <NUM> disclosed herein may be used in the formation of multicellular aggregates within a hydrogel scaffold (<FIG>, <FIG> and <FIG>). According to an embodiment, MCF-<NUM> breast cancer cell suspension was used to obtain cancer spheroids within an a-CNC/gelatin hydrogel droplets as shown in <FIG>, <FIG> and <FIG>.

In a first time, the surface of the microwells <NUM> and supplying channels <NUM> was first treated with fluorinated oil mixed with <NUM> wt. % block copolymer perfluorinated polyether-b-(polypropylene glycol-polyethylene glycol-polypropylene glycol)-b-perfluorinated polyether. Next, the supplying channel <NUM> and microwells <NUM> is filled with a suspension of MCF-<NUM> breast cancer cells in the precursor solution of gelatin and a-CNCs. The MCF-<NUM> breast cancer cells suspension has a concentration of <NUM> cells/µl for the low-density spheroids and <NUM>×<NUM><NUM> cells/µl for high-density spheroids and <NUM> wt. % gelatin and <NUM> wt. In the following step, the solution of cells/precursor in the supplying channel <NUM> is displaced with the fluorinated oil mixture while the solution of cells/ precursor is retained in the microwells <NUM>. This step leads to the formation of an array of cell-laden droplets <NUM>. After <NUM>-<NUM>, the precursor gelatin/a-CNC solution in the microwells <NUM> is transformed in a hydrogel <NUM> and the fluorinated oil mixture in the supplying channel <NUM> is replaced with the cell culture medium to induce cell growth and formation of multicellular spheroids.

The growth of the MCF-<NUM> breast multicellular spheroids is stimulated by placing the microfluidic device at <NUM> in an incubator. During incubation, the supplying channels <NUM> are supplied with a constant flow of the cell nutrition medium. The breast cancer cell viability and the progression of MCS formation are then tested through performing a variety of imaging steps, where the steps include the dyeing and fluoroscopic imaging of the cell cultures in the microwells, after various time exposure to the nutrition medium.

<FIG> shows the microfluidic preparation of MCF <NUM> laden a-CNC/gelatin microgels with a composition of <NUM> wt. % a-CNC, <NUM> wt. % gelatin and an initial cell concentration of <NUM> cells/µl for the hydrogel precursor solution.

<FIG> shows the viability of MCF <NUM> breast cancer cells on Day <NUM> in a-CNC/gelatin microgels with <NUM> wt% a-CNC and <NUM> wt. The left image corresponds to the brightfield image and the right image correspond to the fluorescence microscopy image within cell-laden hydrogels in the microfluidic device. The cells were stained by calcein AM (green) and Ethidium homodimer-<NUM> (red). Green color and lack of red color signify high cell viability in the microgels. According to an embodiment, favorable results regarding for low-density cancer spheroid generation may be obtained with hydrogel compositions: <NUM>-<NUM> wt. % gelatin, and <NUM>-<NUM> wt. % a-CNC and any cell density between <NUM> to <NUM> cells/µL. According to an embodiment, a preferred hydrogel composition is <NUM> wt. % gelatin and <NUM> wt.

According to an embodiment, favorable results regarding for high-density cancer spheroid generation may be obtained with hydrogel compositions: <NUM>-<NUM> wt. % gelatin and <NUM>-<NUM> wt. % a-CNC and any cell density between <NUM>-<NUM>×<NUM><NUM> cells/mL. According to an embodiment, a preferred hydrogel composition is <NUM> wt. % gelatin and <NUM> wt.

According to an embodiment, any physically-crosslinked hydrogel scaffolds obtained via polymer chain interactions such as hydrophobic, electrostatic, and hydrogen bonding between polymer chains may be used as long as the hydrogel scaffolds have suitable characteristics, allow the formation and survival of multicellular aggregates and remain intact when subjected to continuous flows within the microfluidic device.

According to an embodiment, the hydrogel precursor may be a physically gelling polymer such as agarose. This embodiment is advantageous as the thermosetting gelation by which agarose forms hydrogels results in a fully formed hydrogel scaffold which is both non-cytotoxic and biocompatible. Furthermore, the use of agarose is also advantageous for this particular application as it may be readily functionalized with growth factors or peptide fragments to make the gel structures bioactive.

According to an embodiment, when the precursor is agarose, the surface of the microwells and supplying channels may be treated with fluorinated oil (HFE <NUM>,<NUM>, Canada) mixed with <NUM> wt% block copolymer perfluorinated polyether-b-(polypropylene glycol-polyethylene glycol-polypropylene glycol)-b-perfluorinated polyether. In this particular embodiment of the cell encapsulation process a <NUM> wt% solution of PFPE-b-(PPG-PEGPPG)-b-PFPE in fluorinated oil is used. Next, the supplying channel and microwells may be filled with either an aqueous agarose solution or a cell suspension in an agarose solution therefore replacing the fluorinated oil mixture from the supplying channel and microwells. In the following step, the agarose solution or cell suspension in agarose solution in the supplying channel is replaced with a fluorination oil phase. Droplets of agarose solution are confined in the wells and cells are compartmentalized into the droplets. The temperature of the microfluidic device is then lowered to <NUM> to transform the droplets of agarose solution into gels. The resulting formations of such hydrogels are depicted in <FIG> which show a bright field image and fluorescence image of cell-free agarose gels. After gelation, the oil phase in the supplying channel is replaced with a cell culture medium.

<FIG> shows spheroids formed from a high-density of MCF-<NUM> cells laden in agarose microgels: <FIG>droplets of high-density MCF-<NUM> cell suspension (<NUM>%) in agarose solution at <NUM> [The droplets are suspended in fluorinated oil]. <FIG>agarose microgels laden with MCF-<NUM> cells (<NUM>%) in media after <NUM> day of cell culture.

According to an embodiment, the hydrogel scaffold may be a temperature-responsive hydrogel designed for <NUM>) mimicking the structure and mechanical properties of in vivo tumor environments and allowing the growths of the multi-cellular cancer spheroids or organoids and <NUM>) liquefying at a reduced, physiologically acceptable temperature for the subsequent release of the spheroids or organoids from the MF device.

According to an embodiment, the hydrogel precursor may be derived from cellulose nanocrystals (CNCs). For example, the hydrogel precursor may be an aqueous suspension of CNCs surface-functionalized with temperature-responsive polymer molecules. In non-limiting examples, such modified CNCs may be CNCs carrying surface-grafted molecules of the temperature-responsive polymer poly(N-isopropylacrylamide) (<NPL>; <NPL>) or CNCs functionalized with a copolymer of N-isopropylacrylamide and N,N'-dimethylaminoethyl methacrylate (<NPL>).

According to an embodiment, the device and method may be used for screening compounds for drug discovery, for understanding mode of action of the screened compounds and for the evaluation of pharmacodynamics and/or mechanistic biomarkers. According to an embodiment, the device and method may be particularly useful in the screening of drug efficacy for therapeutic treatment by delivering drugs such as anticancer drugs to multicellular spheroids or organoids under dynamic conditions. According to an embodiment, the anticancer drugs may target breast cancer or pancreatic cancer. One advantage of the present invention is the use of multicellular spheroids obtained from primary cells isolated from cancer patient tissue rendering possible a personalized screening of compounds.

According to an embodiment, a method for screening compounds using the microfluidic device <NUM> of the present disclosure. The method may comprise the steps of.

According to a non-limiting embodiment, the screened compound may be a drug, protein, hormone, antibody, nanoparticle or a toxin.

According to an embodiment, the screening method may be used with multicellular spheroids obtained from different cancer cells and the compound is an anticancer drug. Furthermore, the multicellular spheroids may be obtained from breast cancer and the anticancer drug targets breast cancer. Alternatively, the multicellular spheroids may be obtained from pancreatic cancer and the anticancer drug targets pancreatic cancer.

According to an embodiment, the MCCs may be obtained from primary cells isolated from cancer patient tissue for a personalized screening of compounds.

According to an embodiment, the multicellular spheroids may be obtained with the use of a solution comprising the aqueous suspension of cells having a plurality of different types of cells.

The method of the present disclosure provides the opportunity of screening a plurality of compounds on the microfluidic device. Furthermore, the present invention also provides the opportunity of screening a plurality of concentrations for each screened compound. According to an embodiment, solutions of individual drugs supplied in different doses or drug combinations may be supplied with a nutrition medium to the multicellular spheroids located in different rows <NUM>.

According to an embodiment, the screening may be performed on multicellular spheroids of different dimensions on the same microfluidic device.

According to an embodiment, the device and method may be used for screening compounds for drug discovery, for understanding mode of action of the screened compounds and for the evaluation of pharmacodynamics and/or mechanistic biomarkers.

According to an embodiment, the device and method may be particularly useful in the study and/or screening of drugs for therapeutic treatment by delivering drugs such as anticancer drugs to multicellular spheroids or organoids obtained either from high-density cell suspensions or low-density cell suspensions, under dynamic conditions.

In an additional non-limiting example, to demonstrate the use of the method and system of the present invention for studying and/or screening the effects of compounds on the spheroids or organoids, a fluorescent dye Fluorescein isothiocyanate-dextran (FITC-dextran) with hydrodynamic diameter of <NUM> was used to characterize the permeability of multi-cellular spheroids. Before the introduction of FITC-dextran solution, a 2D array of a-CNC/gelatin microgels with multi-cellular spheroids with a cell concentration of <NUM>×<NUM><NUM> cell/mL was formed in the microfluidic device and cultured under the flow rate of the medium at <NUM>/h for <NUM> days (<FIG>). Then, a solution of <NUM>/mL FITC-dextran in HBSS was infused into the a-CNC/gelatin microgels with multi-cellular spheroids at a flow rate of <NUM>/h. After a perfusion time, t, of <NUM>, the channels and contents of wells became fluorescent. After a perfusion time of <NUM>, the fluorescence intensity of the supplying microchannel and well contents did not change, anymore (<FIG>). After that, a dye-free cell culture medium was infused into the channels at a flow rate of <NUM>/h. <FIG> c shows the microgels after <NUM> or perfusion. After short perfusion time (f) of <NUM>, the fluorescent intensity in the supplying channels noticeably decreased. The fluorescence intensity of the a-CNC/gelatin microgels with multi-cellular spheroids also reduced. After <NUM> perfusion, the fluorescence intensity of the FITC-dextran in the a-CNC/gelatin microgels with multi-cellular spheroids very significantly diminished (<FIG>).

Claim 1:
A method for producing multicellular aggregates, in a microfluidic device comprising at least one row having at least one microwell, for each row, a supplying channel spanning along a length of the row and each microwell is in flow connection with the supplying channel, the method comprising the steps of:
introducing a first wetting agent comprising a liquid immiscible in water into the supplying channel and corresponding microwells of at least one row of the microfluidic device;
introducing a solution comprising an aqueous suspension of cells and a hydrogel precursor into the supplying channel and corresponding at least one microwell of the at least one row of the microfluidic device to replace the first wetting agent within the supplying channel and the at least one microwell with the solution;
introducing a second wetting agent comprising a liquid immiscible in water into the supplying channel of the at least one row of the microfluidic device to replace the solution within the supplying channel with the second wetting agent, wherein replacing the solution in the supplying channel with the second wetting agent induces the formation of a droplet containing the aqueous suspension of cells and the hydrogel precursor within each one of the at least one microwell of the at least one row of the microfluidic device, wherein the formation of the droplet is confined in the microwell;
inducing the gelation of the hydrogel precursor within the droplet to form a hydrogel seeded with the suspension of cells, wherein the gelation is induced after the droplet has formed and is being confined within the microwell; and
introducing a cell culture medium into the supplying channel of the at least one row of the microfluidic device to replace the second wetting agent in the supplying channel.