Method for generating hydrogel microparticles

Abstract

The invention relates to a microfluidic method for generating hydrogel microparticles (3a, 3b) for cultivating cells (9). A dispersion medium (44) flows through a second channel (24), and a medium (33) to be dispersed which comprises a hydrogel flows through a first channel (23). The dispersion medium (44) and the medium (33) to be dispersed meet at a connection point (25) of the first (23) and the second channel (24), leading to a pinch-off of spherical hydrogel microparticles (3a, 3b) of the phase (33) to be dispersed, said microparticles being further guided via a third channel (1). A hydrophilic culture medium (34) is used as the dispersion medium, and a hydrophilic hydrogel-cell suspension (33) is used as the medium to be dispersed. A polymerization process of the hydrophilic hydrogel-cell suspension (33) is carried out before the hydrophilic hydrogel-cell suspension meets the hydrophilic culture medium (44).

Description

[0001] Description

[0002] title

[0003] Process for producing hydrogel microparticles

[0004] The present invention relates to a microfluidic method for producing hydrogel microparticles for culturing cells according to the preamble of the independent claim.

[0005] State of the art

[0006] One subfield of microfluidics is so-called droplet-based microfluidics. In this technique, small droplets or discrete volumes are generated and used from immiscible liquid phases within the microfluidic system. For the microfluidic generation of the droplets, two liquid phases are used: the continuous phase (the medium in which the droplets are generated) and the dispersed phase (the medium of the droplets). Typically, oils, such as mineral oil or fluorinated oil, are used as the continuous phase. Aqueous solutions, such as hydrogels, are used as the dispersed phase.

[0007] For cell cultivation, cells can be absorbed into the droplets and cultivated within them. However, challenges in culturing include ensuring sufficient supply of nutrients and oxygen to the cells and maintaining a suitable pH. Furthermore, the continuous and dispersed phases must be biocompatible, and their effects on the cells and cultivation processes must be considered.

[0008] US 2019 / 0105279 A1 describes a device for producing hydrogel microparticles containing cells. A hydrophilic polymer precursor solution containing cells and a hydrophobic liquid, such as an oil, are brought together at a single point through two separate lines, forming hydrogel microparticles. Polymerization of the hydrogel microparticles occurs only after they have formed.

[0009] So-called lab-on-a-chip systems, or LoC systems for short, are microfluidic systems that integrate the functionalities of a macroscopic laboratory onto a plastic substrate for automated processing. Such systems enable biochemical processes to be largely or completely automated.

[0010] Lab-on-a-chip systems typically comprise two main components. The first is a test carrier, for example, in the form of a cartridge, which contains structures and mechanisms for manipulating a sample, particularly passive components such as channels, reaction chambers, or upstream reagents, or active components such as valves, pumps, or mixers. The second main component is a control unit for controlling the microfluidic processes in the cartridge.

[0011] Disclosure of the invention

[0012] According to the invention, a microfluidic method for producing hydrogel microparticles for culturing cells is provided with the characterizing features of the independent patent claim.

[0013] For example, tumor organoids, which accurately represent the in vivo conditions, are used to research tumor diseases. One method for producing tumor organoids is to remove individual cells or tissue fragments from a cancer patient's primary tumor and then cultivate them.

[0014] According to the invention, a microfluidic method for producing hydrogel microparticles for cell cultivation is provided, wherein a dispersion medium, also referred to as the continuous phase, flows through a second channel, and wherein a medium to be dispersed comprising a hydrogel, also referred to as the dispersed phase, flows through a first channel. At a junction of the first and second channels, the dispersion medium and the medium to be dispersed meet, resulting in the pinch-off of droplet- or spherical hydrogel microparticles of the phase to be dispersed, which are then carried further through a third channel.Here, a hydrophilic culture medium is used as the dispersion medium and a hydrophilic hydrogel cell suspension is used as the medium to be dispersed, whereby a polymerization process of the hydrophilic hydrogel cell suspension is initiated before it comes into contact with the hydrophilic culture medium.

[0015] In the process according to the invention, droplets still form during the microfluidic merging of the hydrophilic culture medium and the hydrophilic hydrogel-cell suspension, despite the hydrophilic culture medium instead of a hydrophobic liquid. The polymerization process of the hydrogel is initiated before contact with the culture medium, resulting in a significant difference in the rheological properties, particularly the elastic modulus and viscosity of the two phases. The already slightly polymerized hydrogel-cell suspension then encounters the aqueous culture medium, enabling the pinch-off of a droplet of the hydrogel-cell suspension.

[0016] Instead of a hydrophobic medium, such as an oil, a hydrophilic culture medium can be used as the dispersion medium or continuous phase. For example, the same culture medium is used that is also used in established laboratory-based cell and / or organoid cultivation processes. A particular advantage here is that an optimal culture medium for cell and / or organoid cultivation can be used. A hydrophobic medium, such as an oil, is not an ideal liquid in several respects: on the one hand, it leads to cytotoxicity and protein denaturation, and on the other hand, it does not serve to supply the cells with nutrients and oxygen like a culture medium.The advantage of using a hydrophilic culture medium as a dispersion medium is that it ensures and improves the necessary supply of nutrients and oxygen to the cells, as well as the establishment of a suitable pH value. Another particularly advantageous feature is that it prevents cytotoxic effects on the cells being cultured.

[0017] Furthermore, there is improved transferability and comparability between cell and / or organoid cultivation carried out manually in the laboratory and the microfluidic implementation of the use of a culture medium optimal for cell and / or organoid cultivation.

[0018] Dulbecco's Modified Eagle Medium (DMEM), for example, is used as the continuous phase. This is a standardized nutrient medium for cell culture that has been established for the cultivation of cells and / or organoids from various tissues or cells.

[0019] The cells to be cultured are suspended in hydrogel or a hydrogel / buffer solution, with a suitable cell concentration being adjustable. This allows, in particular, the desired number of cells to be set within the generated hydrogel microparticles. The size of the hydrogel microparticles is in the micrometer range and is primarily determined by the flow rates of the continuous and dispersed phases, the interfacial tension between the two phases, and the geometry of the microfluidic channels. It is selected, for example, so that one or more cells can be cultivated into organoids. The diameters of tumor organoids, for example, are between 100 and 700 pm.

[0020] Further advantageous embodiments of the microfluidic device emerge from the subclaims. In a first advantageous embodiment of the method according to the invention, the polymerization process of the hydrophilic hydrogel-cell suspension is thermally induced.

[0021] The thermal properties of the hydrogel are advantageously exploited here. The viscosity and elastic modulus of some hydrogels, such as Matrigel® (Corning), agarose, or gelatin, are known to be temperature-dependent. These two rheological properties and the polymerization process can thus be specifically controlled by varying the magnitude and duration of the temperature exposure.

[0022] It is advantageous if the thermal induction of the polymerization process of the hydrophilic hydrogel-cell suspension occurs before its introduction into the first channel by tempering the hydrophilic hydrogel-cell suspension. If Matrigel®, for example, is used as the hydrogel, the hydrophilic hydrogel-cell suspension is tempered to a temperature of 6–10°C. If a different hydrogel or mixtures of different hydrogels are used, the temperature is set to a specific temperature that initiates the polymerization of the respective hydrogel or mixture.

[0023] In addition, it is advantageous if the thermal polymerization process of the hydrophilic hydrogel cell suspension is supported during flow through the first channel by tempering the first channel.

[0024] If Matrigel® is used as a hydrogel, the first channel is heated to a temperature of 6 - 10°C, for example.

[0025] For this purpose, at least the first channel of the microfluidic device has a temperature control, for example by means of Peltier elements attached to it.

[0026] Alternatively, it is advantageous if the thermal polymerization process of the hydrophilic hydrogel cell suspension is initiated while flowing through the first channel. The hydrogel cell suspension is then introduced into the first channel of the microfluidic device in a liquid state in which the polymerization process has not yet begun. If Matrigel® (Corning) is used as the hydrogel, the Matrigel cell suspension is, for example, at 4°C when introduced into the first channel. The first channel is heated to initiate the thermal polymerization process of the hydrogel cell suspension. If Matrigel® is used as the hydrogel, the first channel is heated to a temperature of 6–10°C, for example.

[0027] For this purpose, at least the first channel of the microfluidic device is temperature-controlled, for example, by means of attached Peltier elements. In this way, the polymerization process of the hydrogel is thermally initiated in a targeted manner.

[0028] Temperature control of a Matrigel cell suspension and / or the first channel to 6-10°C is advantageous, as this initiates and supports the polymerization process of Matrigel® in a known or experimentally investigated manner. Furthermore, the viscosity and elastic modulus values ​​of Matrigel® are known for this temperature range, and data on the polymerization time are available.

[0029] Furthermore, it is advantageous if the hydrophilic culture medium is introduced into the second channel at a pre-tempered temperature. For this purpose, the hydrophilic culture medium is pre-tempered to 37°C, for example, if the dispersing phase comprises Matrigel®. Alternatively or additionally, the second channel is pre-tempered to a temperature of 37°C, for example, using Peltier elements.

[0030] The advantage here is that the formation of the spherical hydrogel microparticles is supported when the continuous phase and the phase to be dispersed meet, as well as the complete curing of the hydrogel microparticles during flow through the third channel of the microfluidic device.

[0031] In a further advantageous embodiment of the invention, the polymerization process of the hydrophilic hydrogel-cell suspension is induced by light. A photoactive hydrogel, for example, polyethylene glycol (PEG), is used. In this embodiment, at least the first channel is optically transparent to the respective light wavelengths.

[0032] The advantage here is that the location or area in which the polymerization process takes place is spatially precisely defined and only takes place where the incident light hits the hydrogel cell suspension.

[0033] In comparison, with thermal induction, adjacent areas are also heated, potentially influencing the polymerization process. Another advantage is that temperature changes do not affect the cells or cell culture, thus preventing potentially unwanted or undefined influences.

[0034] In a further advantageous embodiment of the invention, the polymerization process of the hydrophilic hydrogel cell suspension is induced by adding a reagent.

[0035] The reagent is added to the hydrophilic hydrogel-cell suspension before or during flow through the first channel. If the addition occurs within the microfluidic device during flow through the first channel, this can be done via a microfluidic mixer or another microfluidic channel.

[0036] The advantage here is that precisely defined molar and volume ratios can be set, allowing the polymerization process to proceed as desired. Furthermore, multiple reagents can be mixed or added individually one after the other. Compared to thermal induction of the polymerization process, this method eliminates the need for temperature control of adjacent areas and temperature-dependent undesirable effects on the cells and cell culture.

[0037] Of course, the aforementioned options for initiating the polymerization of the hydrogel-cell suspension can be combined in a suitable manner. This is particularly advantageous when using mixtures of several hydrogels or when using a hydrogel that offers more than one crosslinking activation option.

[0038] In a particularly advantageous embodiment of the invention, the hydrophilic hydrogel cell suspension has a modulus of elasticity of 0.1 - 20 Pa and a viscosity of 0.1 - 50 Pa-s upon contact with the culture medium.

[0039] In a further particularly advantageous embodiment, the complete polymerization of the hydrogel microparticles occurs during flow through the third channel by tempering the third channel, in particular to 37°C, and / or by light and / or by a reagent.

[0040] Setting the third channel to a temperature of 37°C is advantageous for cell cultivation. This is the standard cultivation temperature for 2D and 3D cell cultivation, especially in incubators, for example, in the laboratory.

[0041] In addition, Matrigel® and other thermally inducible hydrogels polymerize rapidly and completely at 37°C. To this end, at least the third channel of the microfluidic device has a temperature control, for example, by means of attached Peltier elements.

[0042] In hydrogels whose polymerization process is induced by light, the third channel is irradiated with light, for example, to achieve complete polymerization, so that the polymerization process proceeds without slowing down or stopping. For this purpose, the third channel is designed to be optically transparent to the respective light wavelengths.

[0043] For hydrogels whose polymerization process is reagent-induced, for example, a reagent is added to the third channel for complete polymerization. This addition occurs, for example, through a microfluidic mixer or another microfluidic channel. Of course, the aforementioned options for complete polymerization can be combined in a suitable manner.

[0044] In addition, it is further advantageously proposed that the hydrophilic hydrogel cell suspension comprises Matrigel® (Corning), agarose, gelatin, and / or PEG and / or alginate.

[0045] Matrigel® is the preferred hydrogel. It is standardly used for organoid cultivation and undergoes thermally induced polymerization or curing. At low temperatures of 4°C, Matrigel® is liquid and no polymerization occurs. The polymerization process begins at temperatures greater than or equal to 6°C and, in laboratory applications, typically occurs at room temperature or in a laboratory incubator at 37°C. Matrigel® is therefore proven and easy to handle.

[0046] Alternatively or additionally, agarose or gelatin, for example, can be used as a hydrogel, the polymerization of which is induced thermally. The advantage of using agarose is that it is not of animal origin, is inexpensive compared to other hydrogels, and is readily available.

[0047] Another alternative hydrogel is PEG, for example, whose polymerization is induced by light. The advantages of using PEG are its synthetic producibility and its flexibly adjustable mechanical properties.

[0048] Alternatively or additionally, alginate is used as a hydrogel, which hardens, for example, through the addition of calcium chloride.

[0049] A pump unit, such as a syringe, membrane, or peristaltic pump connected to the second microfluidic channel, is used to supply and advance the hydrophilic culture medium in the microfluidic device. This allows, in particular, the adjustment and variation of the flow rate of the hydrophilic culture medium. Likewise, a pump unit, such as a syringe, membrane, or peristaltic pump (not shown), connected to the first microfluidic channel, is used to supply and advance the hydrogel cell suspension. This allows the adjustment and variation of the flow rate.

[0050] Optionally, optical monitoring of the production of the spherical or droplet-shaped hydrogel microparticles, as well as the number of cells contained therein, can be additionally implemented. For this purpose, optical accessibility of the microfluidic device must be achieved by using a transparent material, at least partially, for its production. Relevant areas of the microfluidic device can be observed using a camera or microscope unit mounted above it.

[0051] In a further step, microfluidic cultivation of the cells within the generated, fully cured hydrogel microparticles can be carried out, particularly to form organoids. For this purpose, the hydrogel microparticles containing the cells are transported, for example, into a microfluidic device as described in patent application 102022214275.6, filed on December 22, 2022, and used, for example, for cell cultivation using the method described therein.

[0052] The method according to the invention can furthermore be carried out in a cartridge, in particular a microfluidic cartridge, as described for example in DE102016222072A1 or DE102016222075A1.

[0053] Short description of the drawing

[0054] Embodiments of the present invention are illustrated in the drawing and explained in more detail in the following description of the figures. It shows:

[0055] Fig. 1: the schematic representation of a cross section through a microfluidic device in which the method according to the invention for producing hydrogel microparticles for cultivating cells takes place, and Fig. 2: the schematic representation of a flow diagram of a

[0056] Embodiment of the method according to the invention.

[0057] Embodiments of the invention

[0058] Figure 1 shows a microfluidic device 10 having a first channel 23 and a second channel 24, which converge towards one another and meet at a junction 25 and then continue into a third channel 1 arranged orthogonally to the first channel 23 and second channel 24.

[0059] In the microfluidic device 10, for example, the method according to the invention for producing hydrogel microparticles 3a, 3b for cultivating cells 9 is carried out. Figure 1 shows an embodiment of the microfluidic method, wherein Matrigel® (Corning) is used as the hydrogel of the hydrogel-cell suspension 33, the polymerization process of which is thermally induced.

[0060] Via the second channel 24, the hydrophilic culture medium 44 (DMEM), for example, is introduced into the microfluidic device 10 as the dispersion medium 44 and conveyed in a second direction 15b. The hydrophilic culture medium 44 is pre-tempered to a temperature of 37°C, for example, and / or the second channel 24 is tempered to 37°C. Via the first channel 23, the hydrophilic Matrigel cell suspension 33 is introduced into the microfluidic device 10 as the medium 33 to be dispersed and conveyed in a first direction 15a. The hydrophilic Matrigel cell suspension 33 comprises suspended cells 9 with a set cell concentration and is introduced into the first channel 23 in the liquid state at 4°C. The first channel 23 is heated to a temperature of 6-10 °C so that the polymerization process of the Matrigel® is initiated thermally in a targeted manner.Thus, upon contact with the supplied culture medium 44, the Matrigel® exhibits a desired elastic modulus and viscosity, which differ significantly from the aqueous culture medium 44. These two rheological properties and the polymerization process are specifically controlled by the magnitude and duration of the temperature exposure. Since the Matrigel cell suspension 33 is already slightly cured upon contact at the junction 25 of the first 23 and second channel 24, a pinch-off of an already slightly polymerized Matrigel microparticle 3a occurs, ultimately breaking off and being transported further. In this way, slightly polymerized droplet- or spherical Matrigel microparticles 3a are dispersed in the culture medium 44 and transported further via the third channel 1 in a third direction 15c.

[0061] Further polymerization to fully polymerized hydrogel microparticles 3b occurs while flowing through the third channel 1 by tempering the third channel 1, for example, to a temperature of 37°C. A pump unit (not shown) connected to the first 23 and second microfluidic channels 24 is used to supply and advance the hydrophilic culture medium 44 and the Matrigel cell suspension 33 in the microfluidic device 10.

[0062] The cells 9 in the particularly fully cured Matrigel microparticles 3b are then cultured, for example, within the Matrigel microparticles 3b, in particular to form organoids.

[0063] Figure 2 shows a flow diagram of an embodiment of the microfluidic method. In a step 105, the hydrophilic hydrogel-cell suspension 33 is introduced as the medium to be dispersed into the first channel 23 of the microfluidic device 10 and conveyed further. In a step 106, the polymerization process of the hydrogel is specifically initiated. In a step 107, which occurs, for example, simultaneously with step 105, a hydrophilic culture medium 44 is introduced into the microfluidic device 10 via the second channel 24 as the dispersion medium. At the junction 25 of the first channel 23 and second channel 24, the partially polymerized hydrophilic hydrogel-cell suspension 33 and the hydrophilic culture medium 44 meet, whereby in step 108, hydrogel microparticles 3a are pinched off. These are transported further via the third channel 1 in a step 109 and completely polymerized.

Claims

1 . Microfluidic method for producing hydrogel microparticles (3a, 3b) for cultivating cells (9), wherein a dispersion medium (44) flows through a second channel (24), and wherein a medium (33) to be dispersed comprising a hydrogel flows through a first channel (23), and wherein the dispersion medium (44) and the medium (33) to be dispersed meet at a junction (25) of the first (23) and the second channel (24), resulting in the pinch-off of spherical hydrogel microparticles (3a, 3b) of the medium (33) to be dispersed, which are then conveyed via a third channel (1), characterized in that a hydrophilic culture medium (44) is used as the dispersion medium and that a hydrophilic hydrogel cell suspension (33) is used as the medium to be dispersed, wherein a polymerization process of the hydrophilic hydrogel cell suspension (33) before contact with the hydrophilic culture medium (44).

2. Microfluidic method according to claim 1, wherein the polymerization process of the hydrophilic hydrogel cell suspension (33) is thermally induced.

3. Microfluidic method according to claim 2, wherein the thermal induction of the polymerization process of the hydrophilic hydrogel cell suspension (33) before its introduction into the first channel (23) is carried out by tempering the hydrophilic hydrogel cell suspension (33), in particular to a temperature of 6 - 10°C.

4. Microfluidic method according to one of claims 2 or 3, wherein the thermal polymerization process of the hydrophilic hydrogel cell suspension (33) is initiated and / or supported during the flow through the first channel (23) by tempering the first channel (23), especially at a temperature of 6 - 10°C.

5. Microfluidic method according to one of the preceding claims, wherein the hydrophilic culture medium (44) is introduced into the second channel (24) in a pre-tempered state, in particular pre-tempered to a temperature of 37°C.

6. Microfluidic method according to one of the preceding claims, wherein the polymerization process of the hydrophilic hydrogel cell suspension (33) is induced by light, and wherein at least the first channel (23) is at least partially optically transparent.

7. Microfluidic method according to one of the preceding claims, wherein the polymerization process of the hydrophilic hydrogel cell suspension (33) is induced by adding a reagent which is added to the hydrophilic hydrogel cell suspension (33) before or during the flow through the first channel (23).

8. Microfluidic method according to one of the preceding claims, wherein the complete polymerization of the hydrogel microparticles (3a, 3b) during flow through the third channel (1) takes place by tempering the third channel (1), in particular to a temperature of 37°C and / or by light and / or by a reagent.

9. Microfluidic method according to one of the preceding claims, wherein the hydrophilic hydrogel cell suspension (33) has a modulus of elasticity of 0.1 - 20 Pa and a viscosity of 0.1 - 50 Pa-s upon contact with the hydrophilic culture medium (44).

10. Microfluidic method, wherein the hydrophilic hydrogel cell suspension (33) comprises Matrigel® (Corning), agarose, gelatin, polyethylene glycol and / or alginate.

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