NL2017834B1 - Hydrogel micro-patterning for embedding purposes - Google Patents

Abstract

The present invention is in the field of hydrogel micropatterning for embedding purposes. It relates to a mask-less method for isolating a single object of interest by patterning a pocket of a hydrogel, a pocket of hydrogel comprising said object of interest, and use of an isolated object of interest for study, such as cell study.

Description

FIELD OF THE INVENTION

The present invention is in the field of hydrogel micro-patterning for embedding purposes.

BACKGROUND OF THE INVENTION

The present invention is in the field of hydrogel micro-patterning for embedding purposes.

Hydrogels relate to a class of materials that can be widely applied in controlled drug release, soft matter actuators, cell encapsulation and in tissue engineering. They may be formed by self-assembly of low molecular weight precursors (e.g. monomers) or by cross-linking of polymeric chains. Hydrogel formation via cross-linking is often realized by photoinitiated radical polymerization or by catalyst initiated polymerization .

It would be highly valuable to perform imaging-based isolation of single cells from a population, for instance a heterogeneous population, such as in the field of biomedical research or forensic research, as it allows for selective follow-up studies e.g. molecular profiling or cell line development. In the prior art a majority of cell sorting methods is based on flow manipulation (flow cytometry) of fluorescently stained cells suspensions, e.g. fluorescence- activated cell sorting (FACS). However, when desired cells are e.g. attached to a certain substrate, which corresponds to the vast majority of cases in preclinical and clinical research, these techniques cannot be employed without significant manipulation and/or alteration of the samples. Therefore, efforts have been made to sort adherent cells and these have led to the development of several further techniques, e.g. Laser capture microdissection (LCM) or IsoRaftTM Array, and CellCelectorTM. These techniques generally require complex equipment, challenging manipulations and/or culture in specific supports. Developing a technology that enables online selection, isolation, and release of adherent cells after preliminary screening using common microscopy techniques would therefore be a major breakthrough.

Spatially controlled photo-patterning of hydrogels can be applied in cell studies, for example in the preparation of spatially controlled cell cultures, photo-patterning of cell adhesion molecules, and in cell isolation or release purposes. These approaches embed cells of interest by fabricating a hydrogel around them. Unfortunately, these methods require patterned surfaces and predefined photomasks, which drastically limits their applicability and is not flexible as it is limited by the necessity of designing and producing of a new photomask for every particular shape. An alternative approach is to covalently attach selected cells to a pre-treated surface by a photochemical reaction. Although this method was shown to be appropriate for single cell isolation, it requires using a custom-made digital micro mirror device (DMD) and was found to be prone to generating false positives.

Various gel-forming substances may be considered, such as Dextran. Dextran is considered biocompatible. Dextranbased hydrogels can be generated by the introduction of functional groups that are capable of cross-linking onto the saccharide based polymer backbone. For example, dextranmethacrylate (Dex-MA) was shown to form stable hydrogels via chemically induced crosslinking. These materials have been extensively studied in protein and drug release.

Some documents recite capturing living cells or components thereof.

For instance, US2012/270209 A recites that living cells can be selectively and reversibly bound to functionalized dissolvable material (e.g., cross-linked hydrogel compositions) and subsequently released from the composition as viable cells. In some examples, the cells are released by reducing the degree of cross-linking within a functionalized hydrogel composition and/or dissolving the functionalized hydrogel composition selectively bound to the cells. The functionalized hydrogel compositions can be adhered to silicon- and silicon-oxide containing surfaces, such as glass and aminated silicon. The hydrogel also functions as a support to the cells. The living cells can be isolated from biological samples, such as blood, by selectively binding certain cells from the sample to the functionalized hydrogel, removing unbound cells and later releasing viable bound cells from the functionalized hydrogel. The invention relies on a microfluidic device for cell-capturing. There is no patterning involved.

US2014/093911 A1 recites a method of predictive identification and separation of high-performing cells from a mixed population of cells includes distributing cells belonging to the mixed population to a plurality of open chambers; identifying open chambers containing desired cells and open chambers containing undesired cells; selectively sealing at least one open chamber containing undesired cells; and recovering the desired cells from the open chambers. Cells can be predictively assigned as desired or undesired based on an automated image analysis algorithm. The method relies on prefabricated cell-chambers. The chambers of the unwanted cells are polymerized.

W02010/042943 A1 recites techniques to produce and use non-spherical colloidal particles with independently tuned size, shape, flexibility, and chemical properties, typically using a photo-mask. A pre-polymer mixture for forming hydrogel particles includes a percentage of PEGDA selected to impart a target stiffness to the particles and includes, a percentage of acrylic acid selected to impart an independent target chemical function to the particles. The mixture also includes a percentage of photo-initiator to polymerize PEGDA upon exposure to a light source to impart an independently selected target size or shape or both to the particles.

W02015/010019 A1 recites photodegradable hydrogels and associated kits for selectively capturing and releasing cells. The hydrogels are typically used for coating of a surface, such as of a channel of a microfluidics. The hydrogels result from cross linking in the presence of a photo initiator, a macromer having a polymeric backbone structure, a photo labile moiety, and a first linking moiety, and a cell-binding moiety having a second linking moiety. These two components are cross-linked by a polymerization reaction of the linking moieties to form a photodegradable hydrogel incorporating the cell-binding moiety within the hydrogel. Such methods can be used to detect the presence and quantity of certain rare cell types in a biological fluid. The methods are considered to be micro-fluidic based.

WO 2008/088395 A1 recites an apparatus for particle sorting, particle patterning, and methods of using the same. The sorting or patterning is opto-fluidics based, in that particles are applied to individual chambers in the device, detection and/or analysis of the particles is carried out, such that a cell or population whose removal or conveyance is desired is defined, and the cell or population is removed or conveyed via application of an optical force and flow-mediated conveyance or removal of the part.

The present invention therefore relates to an improved method for isolating a single object of interest, which solve one or more of the above problems and drawbacks of the prior art, providing reliable results, without jeopardizing functionality and advantages.

SUMMARY OF THE INVENTION

The present invention relates to an improved method for isolating a single object of interest according to claim

1. Use is made in the present method of entities in a solution, typically an aqueous solution, that can form a hydrophilic hydrogel upon initiation by radiation. The entities are typically hydrophilic. They can polymerize, or cross-link, or both. For radiation any electro-magnetic source can be used; for sources having smaller wavelength, such as electrons, even smaller pockets as mentioned below could be formed. For most objects of interest, being in the micrometer scale or larger, a light source is typically considered. By providing focused initiation the hydrogel is formed locally, i.e. in the neighborhood of the object of interest; a spatial distribution of hydrogel is obtained. In an example a diameter of a focused beam is 3-100 pm, typically 5-50 pm, such as 10-15 pm. The focused initiation typically activates a so-called photoinitiator locally, though other initiators may be used as well, such as chemically activated initiators. A direct gelation of the aqueous solution is therewith obtainable. Preferably a means of relative movement of a beam through a sample

c.q. aqueous solution is used, such as an XY-stage, more pre5 ferably a controlled XY-stage. The XY-stage can control movement in an X and Y direction with an accuracy of ±1-5 pm or better, such as by using a step-motor. In addition a feedback loop may be used to improve the X-Y accuracy even further. The hydrogel forms a pocket around said object of interest, whereas the remainder of the solution can still be, and typically is, in a fluidic status. The hydrogel typically has a Youngs modulus of >1000 Pa, typically > 104 Pa. The object of interest as such is considered to be immobilized and can be manipulated further.

The present method thus provides a relatively fast method of locally providing a hydrogel, which method is flexible, scalable, has high resolution, is relatively simple applicable, and provides full separation of an object of interest .

The present method can be used for online imagingbased selective embedding, isolation, and subsequent release of micro-particles and live cells via spatial controlled hydrogel formation and subsequent enzymatic degradation. Interestingly, spatial controlled hydrogel fabrication was achieved by irradiation using a confocal laser-scanning microscope (CLSM). As imaging with CLSM may be performed via pointby-point illumination, which allows for irradiation of the sample exclusively at selected areas, it provides an opportunity to create an irradiation pattern and hereby spatially control hydrogel formation without applying a photomask. Advantages of the developed method include: (i) use of an CLSM; (ii) online image-based selection of areas chosen for hydrogel formation; (iii) fast hydrogel fabrication speeds (e.g. 3.71 ps/pm2); (iv) use of visible light (e.g. 405 nm) at moderate intensity; (v) application under physiological conditions;

(vi) biocompatible and readily biodegradable hydrogels, allowing for reversible embedding of micrometer objects e.g. cells. The present invention is also subject of a scientific paper entitled Microscope Controlled Hydrogel Formation for Cell Isolation, by S. Oldenhof et al. , submitted for publication, which paper and its contents are incorporated by reference. In said paper further details and supporting information are disclosed.

In a second aspect the present invention relates to a pocket of hydrogel obtainable by a method according to the invention, comprising a three dimensional polymeric network of hydrophilic and degradable polymers, and at least one spatially distributed degradable hydrogel pocket comprising a selected object of interest. The object of interest and the pocket are isolated from there environment and can therefore be freely and independently manipulated further, if required. Such distinguishes the present pocket over e.g. a hydrogel fully occupying a space such as in a container and having a number (more than one) of objects in the hydrogel, which pockets and objects are as a consequence not separated form one and another and can thus not be manipulated individually and independently .

In a third aspect the invention reflects that the pocket of hydrogel may typically be present on a surface, such as a glass surface. The surface may be functionalized, such as by providing for a given purpose suitable chemicals.

In a fourth aspect the present invention relates to a use of an immobilized cell obtained by a method according to the invention for cell-study, for study of a medical compound, for enzyme study, for genetic study, for study of controlled drug release, for study of soft matter actuators, for cell encapsulation, for tissue engineering, or for obtaining and/or isolating a viable cell. In certain situations, such as in experimental set-ups, some objects, typically biological cells, appear to deviate from an average and are therefore considered as objects of interest. These cells therefore need to be separated, such as for further study.

In a fifth aspect the present invention relates to a kit comprising an exemplary photo-initiator as well as a gel forming compound comprising polysaccharides, and optionally an enzyme capable of degrading said polysaccharides.

Thereby the present invention provides a solution to one or more of the above mentioned problems and drawbacks. Advantages of the present description are detailed throughout the description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in a first aspect to a method according to claim 1.

In an exemplary embodiment the present method further comprises the step of removing the solution, such as by rinsing the pocket of hydrogel. As the hydrogel is formed as a pocket a remainder of the initial solution is still there. For isolating the pocket the solution is typically removed, such as by rinsing, by washing, by decanting, etc.

In an exemplary embodiment the present method further comprises a step of isolating the at least one object of interest from its environment. As such the object of interest is considered to be freely manipulated.

In an exemplary embodiment of the present method the hydrogel in the pocket is enzymatically or chemically degraded therewith freeing the object of interest. Typical enzymes and chemicals may be used for this purpose, such as Glycosyl hydrolases, oxidases, and associated enhancing factors. In view of a biological cell a to the cell non-harmful degradation medium is preferred.

In an exemplary embodiment of the present method the object of interest is labelled, such as by a fluorescent compound, or a phosphorescent compound. As such the object can be viewed and studied under an optical microscope, such as in the initial solution, or later in a suitable environment. In addition by labelling certain objects can be distinguished from others, e.g. in terms of capacity to label, which may be considered indicative for further characteristics of the object. Compounds suitable for labelling may be selected from cyanines, rhodamines, radio-active tracers, MRI-contrast agents, nanoparticles, imaging molecules, a microbubble for ultrasound or opto-acoustic imaging, etc. It is noted that in an alternative, or in addition, objects may very well be selected based on characteristics, such as a morphology of a cell.

In an exemplary embodiment of the present method the entities are selected from polysaccharides and derivatives thereof, such as (meth)acrylate modified polysaccharides, such as dextran, and/or wherein the entities are provided in a concentration of 0.1-10 wt.%, preferably 0.2-7 wt.%, such as 0.58 wt. %. The modified polysaccharides typically have a nonterminal (meth)acrylate group. For the present invention all weight or volume percentages are relative to a total weight or volume, such as of the hydrogel solution, unless stated otherwise. It is found that especially polysaccharides provide good gel forming properties and do not interfere with e.g. requirements for maintaining and preserving a cell. A preferred example are (meth)acrylate modified polysaccharides; not only can the polysaccharides by modified and adapted easily, but also properties of the hydrogel are found to be tunable. Depending on the solution, hydrogel to be formed, object of interest, etc. an amount of polysaccharide can be varied.

In an exemplary embodiment of the present method the polysaccharides comprise 0.1-50 mole% (meth)-acrylate groups (relative to a number of monomers of the saccaharides), preferably 0.5-20 mole% (meth)-acrylate groups, such as 1-5 mole%, e.g. 1 acrylate group per 99 glycopyranose monomers attributes to 1 mole%. By varying an amount of acrylate groups it has been found that especially the characteristics of the hydrogel can be varied and tuned.

In an exemplary embodiment of the present method the entities are selected from compounds with a molecular weight of 100-1000 kDa, preferably 200-500 kDa, i.e. higher molecular weight compounds are preferred. In this case the molecular weight relates to a weight average molecular weight (Mw) which may be determined by light scattering.

In an exemplary embodiment of the present method a laser device is used for radiation initiation, such as a confocal laser scanning microscopy (CLSM). The laser provides a good focus, a good intensity, can be tuned to the polymerization reaction in terms of wavelength, etc. In order to combine a visual observation of the object of interest and local polymerization typically an (optical) microscope is provide and both features are preferably combined, such as in a CLSM.

In an exemplary embodiment of the present method a wavelength from 250-800 nm is used for polymerization, preferably 300-600 nm, more preferably 350-500 nm, such as 400-450 nm. The wavelength may also be tuned to a specific label. The wavelength is typically optimized in view of a photo-initiator used.

In an exemplary embodiment of the present method a photo-initiator is used, preferably an aromatic phosphinate, more preferably a bi-aromatic phosphinate, even more preferably a phenyl-benzoyl phosphinate, preferably comprising a mono-, di- or tri-alkyl benzoyl, wherein the alkyl is independently selected from C1-C6, such as methyl, ethyl, and propyl, preferably having a metallic counter ion, such as Li, and Mg, such as lithium-phenyl-2,4,6-trimethylbenzoyl-phosphinate. It has been found that certain photo-initiators do not perform well, whereas others, such as the present aromatic phosphinates, perform much better. Such is somewhat unexpected, as all photo-initiators tested are considered suitable for polymerization .

In an exemplary embodiment of the present method the photo-initiator is provided in a concentration of 0.1-2 wt.%, preferably 0.2-1 wt.%, such as 0.3-0.5 wt.%, that is relatively low concentrations are typically sufficient. The present photo-initiator is preferably compatible with other aspects, such as it dissolves well in the solution, does not interfere with the object of interest, is preferably used in low concentrations, provides good polymerization and hydrogel forming, etc .

In an exemplary embodiment of the present method the solution comprises 30-99.8 wt.% water, 0-50 wt.% alcohol, such as methanol, ethanol, and propanol, and 0-50 wt.% DMSO. The solution may be considered as aqueous, comprising optional further solvents. In addition also salts, buffers, acids and alkaline compounds may be added, typically in small amounts.

In an exemplary embodiment of the present method a radiation-initiation exposure time is 1-25 ps/pm2, preferably 2-15 ps/pm2, more preferably 5-10 ps/pm2. As such within relatively small time frames (ms), still relatively large hydrogel pockets can be formed, sufficient to encapsulate an object of interest, in a well-controlled and adaptable manner. As such the present method can be scaled-up easily.

In an exemplary embodiment of the present method a radiation source is used capable of producing a power of 1 mW10

W, preferably a 10 mW-2 W source, such as 30-100 mW. The amount of radiation may vary upon e.g. a chosen objective of the microscope, a nature of the hydrogel and/or polymers and of the photo-initiator, etc. Typically only a fraction of said power is found necessary for irradiation, such as 1-10%.

In an exemplary embodiment of the present method the pocket is immobilized on a surface, such as a glass surface, such as a functionalized glass surface.

In an exemplary embodiment of the present method the object of interest is a biological cell.

In an exemplary embodiment of the present method the hydrogel comprises a first layer of a first hydrogel, and at least one further layer such as of a second hydrogel. With the present method more than one layer of hydrogels may be formed, such as a stack of hydrogel layers. Such may be suitable for specific applications, where e.g. a growth medium is provided on top of a first hydrogel pocket comprising a biological cell.

In an exemplary embodiment of the present method at least one alignment marker is provided, preferably at least two alignment markers, preferably lithographic defined alignment markers. The alignment markers are preferably provided on the XY-stage. As such a very precise positioning of the stage, relative to e.g. an irradiation source and/or a microscope, can be provided, in an X-direction, in a Y-direction, in a combined x-y-direction, as well as in terms of rotation. Positioning with a precision of ±lpm is easily obtainable, which is considered more than sufficient such as in view of the typically less-well defined spatial polymerization.

In a second aspect the present invention relates to the isolated pocket of hydrogel according to claim 20.

In an exemplary embodiment of the present hydrogel pocket the object of interest in said pocket has a volume of 102-106 pm3, a length of 10-10.000 pm ±15 pm, preferably as 20-1.000 pm, more preferably as 25-100 pm, such as 30-50 pm, a width of 10-10.000 pm ±15 pm, more preferably as 25-100 pm, such as 30-50 pm, and a height of 1-500 pm, such as 2-100 pm, preferably 3-50 pm, such as 5-40 pm. As the pocket is typically somewhat larger than the object of interest, microscale up to mesoscale hydrogel pockets can be formed, though the present method is not considered to be directly limited to these sizes. Such sizes are very suitable for relative small objects of interest.

In a third aspect the present invention relates to a surface comprising a pocket of hydrogel according to the invention, such as a glass surface.

In a fourth aspect the present invention relates to a use of an immobilized cell obtained by a method according to the invention.

In a fifth aspect the present invention relates to a kit comprising an exemplary photo-initiator as well as a gel forming compound, and optionally a gel-degrading compound, such as an enzyme, such as dextranase. The photo-initiator and gel forming compound are further specified throughout the application, as well as advantageous effects thereof.

The one or more of the above examples and embodiments may be combined, falling within the scope of the invention.

FIGURES

Figure la-b: Hydrogel writing.

Figure 2 a-b: CLSM images.

Figure 3a-c, 4a-c, and 5a-c show selective embedding.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1. (a) Schematic representation of direct hydrogel writing experiment. First, a digital photomask is designed represented by the dashed lines. The selected areas are irradiated and hydrogels are fabricated, represented by the green squares. Subsequently, the obtained hydrogel objects can be isolated by rinsing the sample with water, (b) CLSM-image of six squared Dex-MA10 hydrogel objects fabricated with 10χ objective using exposure times of 3.71 ps/pm2. Scale bars 2 0 0 pm.

Figure 2. CLSM images of frame pattern containing 100 individual hydrogel objects (circle, triangle, square, and cross), scale bar 200 pm, and an enlarged section thereof.

Figure 3 (a) Illustration of selective embedding, isolation and release experiment of micro-particles. First, from a mixture of green and red objects, a digital mask is drawn (dashed lines) selectively surrounding the objects of interest e.g. red particles. After irradiation of designated areas, hydrogels are generated selectively embedding the red particles. After washing, the embedded objects are isolated and subsequently degradation of the hydrogel releases the particles of interest, (b) CLSM image of pre-hydrogel suspension containing a mixture of red and green fluorescent microparticles. A digital photomask, designated by the dashed lines is drawn around the red micro-particles (left). Hydrogels are obtained at the selected areas fully embedding the red microparticles, designated by the green dashed lines (right). In an alternative CLSM images of a coordinate-assisted embedding experiment may be obtained. The coordinates of selected microparticles are determined, such as in an automated fashion, and subsequently a digital mask is applied to all positions embedding selected micro-particles, (c) CLSM images of a hydrogel embedding a red micro-particle (t=0) being completely degraded by Dextranase in time hereby releasing the micro-particle (t=2-4) . After complete degradation a flow is induced (t=6) to show the red micro-particle is free-floating. Scale bars b, d: 100 pm c: 500 pm.

Figure 4. (a) Illustration of selective embedding, isolation and release experiment of a single cell from a mixture. First, from a mixture of cells (green and red), a digital mask is drawn (dashed lines) selectively surrounding the cell of interest. After irradiation of designated areas, a hydrogel is generated selectively embedding the cell of interest. After washing, the embedded cell is isolated and subsequent degradation of the hydrogel releases the cell, (b) CLSM image .

EXAMPLE

The invention is further detailed by the accompanying example, which is exemplary and explanatory of nature and are not limiting the scope of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.

Results

Hydrogelation system

In order to achieve controlled hydrogel formation followed by degradation, two criteria were considered. Firstly, in order to be able to achieve spatial control over hydrogel formation by localized irradiation, the formation of the hydrogel network was fast compared to the diffusion of the reactive reagents. Secondly, for selective embedding and subsequent release of objects, e.g. living cells, the obtained hydrogel may be biocompatible and biodegradable. Hydrogels prepared by cross-linking of polysaccharides have shown to meet these requirements. In particular, methacrylate modified dextran (Dex-MA) based hydrogels, which can be obtained by photoinitiated radical crosslinking are interesting because of their biocompatibility and biodegradability by Dextranases. Initial hydrogelation experiments with Dex-MA were performed on macroscopic scale to determine optimal conditions for hydrogel formation. High molecular weight dextran (500 kDa) was used in order to minimize diffusion effects. Dextran was functionalized with methacrylate groups to degrees of substitution (DS) ranging from 2.5 to 30%. As photo initiator, LAP (lithium-phenyl-2,4,6-trimethylbenzoyl-phosphinate) was chosen because of its high molecular extinction coefficient at 405 nm compared to typically applied 2-Hydroxy-4'-(2-hydroxyethoxy)2-methylpropiophenone (Irgacure® 2959). A high molecular extinction coefficient of the photo initiator at 405 nm was found relevant as this wavelength is applied for photoinitiation in subsequent CLSM hydrogel writing experiments described below. Macroscopic hydrogels were obtained by irradiating the pre-hydrogel solutions (5 wt. % Dex-MA and 0.5 wt. % LAP) with a high-pressure mercury vapor lamp (no filter, 130 W). The DS was found to affect the rate of hydrogelation and physical characteristics of the hydrogels obtained. Dextran modified with 2.5 % methacrylate groups (Dex-MA2.5) was found to require significantly longer exposure times (4-6 seconds) for hydrogel formation while DS > 10 (Dex-MA10/20/30) rapidly produce self-supporting hydrogels within one second of irradiation. Hydrogels obtained with Dex-MA20/30 were considered both significantly more hydrophobic than hydrogels obtained with Dex-MA2.5/10, and were found to shrink by expelling water (—25% by volume), and quickly turned opaque after irradiation. Applying Irgacure® 2959 as photo-initiator under the same experimental conditions was found to significantly increase the required irradiation for hydrogel formation by approximately six times. Next the inventors investigated the degradability of the Dex-MA hydrogels, the obtained hydrogels were exposed to a solution containing Dextranase. Hydrogels obtained from Dex-MA2.5/10 were found to be fully degraded within 10 minutes at 35°C. The rates of Dex-MA20 and Dex-MA30 hydrogel degradation were found to be significantly slower and full degradation only occurred after ~35 minutes at 35°C. From these experiments the optimal system for hydrogel formation and degradation was determined to be 5wt% of Dex-MA10 and 0.5 wt.% LAP.

Direct hydrogel writing

The areas exposed, i.e. where hydrogel is formed, is specified in a digital two-dimensional pattern, which are easily drawn in CLSM-software. For initial spatial controlled hydrogel formation experiments, a digital pattern consisting of six squared areas (each 200 x 200 pm) was applied to determine optimal experimental conditions, (see Figure 1A). The experiments were performed by placing solutions containing DexMA10 (5 wt.%), LAP (0.5 wt.%), and Dex-FITC-500 kDa (0.02 wt.%), between a glass- and a plastic-slide, thereby creating a thin layer. The glass slide was tethered with methacrylate groups in order to immobilize the hydrogels obtained to facilitate their isolation. The selected areas from the digital pattern were irradiated using different irradiation times, with the focal point in the middle of the sample. Afterwards the glass slide was rinsed with water and inspected for hydrogel objects with CLSM. Well-defined hydrogel objects were selectively obtained at the irradiated areas using optimized experimental parameters (optimized conditions: 10χ objective, exposure time: 3.71 ps/pm2, 40χ objective, exposure time: 4.36 ps/pm2, applying 100% and 10% of the maximum laser power of 30 mW respectively; see Figure IB). Applying a shorter exposure time led to the formation of weak and poorly defined hydrogels, while longer exposure led to the loss of spatial resolu15 tion as hydrogelation also occurred beyond the selected irradiated area. The dimensions of the hydrogel objects obtained with optimal experimental parameters were 216 ± 1.6 pm square, which is 8% bigger compared to selected 200 pm square, with a thickness of ~50 pm. This 8% enlargement is caused by swelling of the hydrogels when exposed to water, and likely also due to some diffusion of the photo-initiator/reactive polymer chains beyond the irradiated area. Applying Dex-MA2.5 required longer exposure times, which led to poor spatial control and the generation of weak hydrogels that were damaged upon washing with water. While direct hydrogel writing experiments with Dex-MA20 were successful applying the same conditions as with Dex-MA10, the hydrogels showed deformation and poor adhesion after washing with water.

Next the inventors investigated the resolution limits of the present method, which are defined as the minimal distance between two irradiated areas necessary to obtain two individual hydrogel objects. Resolutions were determined to be 35 pm and 15 pm for the 10χ and 40χ objectives respectively.

It was found that irradiation with a 10χ objective could result in formation of well-defined features down to approximately 50 pm, while irradiating through a 40χ objective significantly decreased the minimal feature size to 15 pm.

It was found that virtually any shape could be made. Also larger surface areas could be patterned with hydrogel objects such as by irradiation multiple frames in a tileirradiation mode. Hydrogel objects could be obtained in an automated fashion and were all well-defined and have high shape reproducibility.

Embedding, isolation and release of fluorescent microparticles

The ability of visualizing a sample combined with spatially controlled hydrogel formation is a practical approach for selective object isolation on a micrometer scale. This is achieved by applying a digital mask to positions containing an object of interest to selectively embed it into a hydrogel. After isolation of the fabricated hydrogels by a simple washing step, the objects of interest can be released by enzymatic degradation of the surrounding hydrogel (Fig.3A).

A four-steps approach was demonstrated by performing selective embedding experiments using a suspension of green and red fluorescent spherical micro-particles of ~25 pm in diameter. From a suspension the red micro-particles were selected and a digital mask was applied embedding particles 1, 5 and 3, see Figure 3B left. After embedding, the sample was rinsed with water and imaged by CLSM indeed showing particles 1, 3, 5 were successfully embedded in a gel object , that could remain bound to a glass surface, while all other particles were removed thereby isolating particles 1,3,5. After isolation of particles 1, 3, and 5 they were successfully released by the enzymatic degradation using by a Dextranase solution of their surrounding hydrogels. An enzymatic degradation of a hydrogel containing a red micro-particle is shown in Figure 3D. Addition of a diluted solution of Dextranase immediately lead to the degradation of the hydrogel as could be observed from the spreading of the Dex-FITC fluorescence. Within six minutes the hydrogel was fully degraded and the red micro-particle was completely released as could be observed from a flow induced below the micro-particle.

Initial embedding experiments were performed with a suspension of green fluorescent micro-particles, 25 pm in diameter. Applying a circular digital mask surrounding a microparticle, these could successfully be embedded. Embedding success rate were determined and could be dependent on the size of the applied digital photo-pattern. Applying a circular digital photo-pattern of 100-200 pm in diameter led to successful embedding of 113/113 of the selected micro-particles. Importantly, no false positives were detected during these experiments. When the circular diameter of the digital photomask was decreased to 60 pm the success rate of embedding dropped to 5-10% (1 out of 20-10) .

An interesting possibility of coordinate-assisted embedding and isolation of multiple objects of interest using this method is illustrated in Figure 3C. From an area visualized by CLSM containing 53 individual green fluorescent microparticles, 15/53 (as indicated) were selected and successfully embedded by applying a digital mask at their assigning coordinates. This shows the potential of this method for detection of an object, e.g. by fluorescents at a specific wavelength, followed by embedding fully automatically.

Cell embedding and release

A four-steps approach is in particularly interesting in the context of (single-) cell isolation (Figure 4A) . Thereto experiments were performed on a mixture of two adhered cells types, 3T3 and A549, labelled green and red respectively. This process can be automated by expressing for instance a certain detectable signal or containing a specific morphological feature. Peptide based hydrogels used for cell culturing, e.g. collagen, fibrin, can be enzymatically degraded by proteolysis, which may be damaging for cell surface receptors. On the contrary, enzymatic degradation of dextran relies on hydrolysis of a-(1,6)-alpha-glucosidic linkages and is therefore harmless to cells. It was found that the PBS buffer did not affect CLSM hydrogel writing.

Method

CLSM controlled hydrogel writing. A solution (29 pL) of Dex-MA10 (5 wt.%), LAP (0.5 wt.%) and dextran labelled with fluorescein (Dex-FITC, 0.017 wt.%) was placed upon a methacrylate tethered glass slide. The sample was covered with a plastic cover slide (24 χ 24 mm) to form a 50 pm thick layer.

A desired area of the sample was uni-directionally irradiated with 405 nm light focused in the middle of the sample. Experimental settings for 10χ magnification objective: frame 1024 χ 1024, pixel size: 0.83 χ 0.83 pm, pixel dwell: 1.27 ps, averaging: 2, laser-diode power at 100%, exposure time: 3.71 ps/pm2. Experimental settings for 40χ magnification objective: frame 1024 χ 1024, pixel size: 0.21 χ 0.21 pm, pixel dwell: 0.64 ps, averaging: 2, exposure time: 4.36 ps/pm2, laser-diode power at 10%. After irradiation the transparency sheet is carefully removed and the remaining solution washed away with water .

General

Commercially available materials were used as received unless stated otherwise. Dextran (500k) (Alfa Aesar), DMSO (Sigma Aldrich), DMAP (Sigma Aldrich), glyceryl-methacrylate (Sigma Aldrich), dimethyl phenyl phosphonite (Sigma Aldrich),

2,4,6-trimethylbenzoyl chloride (Sigma Aldrich), lithium bromide (Sigma Aldrich), 2-butanone (Sigma Aldrich), 3(Trichlorosilyl)propyl methacrylate (Sigma Aldrich), fluorescein isothiocyanate-dextran 500.000 conjugate, FITC : Glucose 1 : 100 (Sigma Aldrich), Dextranase from Chaetomium erraticum (Sigma Aldrich), Dulbecco's phosphate buffered saline (Sigma Aldrich), transparency sheet type retro copy S (Multicom) were cut and used as plastic cover slides. NMR spectra were recorded on a Bruker Avance-400 spectrometer (399.90 MHz for 1H, 100.56 MHz for 13C, and 161.92 MHz for 31P. Bulk hydrogelation experiments were performed using a Nikon intensilight C-HGFI equipped with a 1.5 meter optical fiber. Plasma cleaning was performed using a Harrick Plasma PDC-002 model plasma oven. Contact angles were determined using EasyDrop apparatus (KRUSS GmbH), Imaging of hydrogels were carried out on a Zeiss LSM 710 equipped with a Zeiss Axio Observer inverted microscope using a N-Achroplan 10x/0.25 M27 (420940-9900) and Objective

Fluar 40x/1.30 Oil M27 (420260-9900).

Further details on Dextran functionalization (DexMA), preparation of Lithium-phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), glass surface modification, macroscopic hydrogel preparation, macroscopic hydrogel degradation by dextranase, CLSM direct hydrogel writing procedure, experimental conditions using 10χ objective, experimental conditions using 40χ objective, resolution, illumination cone size and intensity profile, hydrogel shape analysis, micro-particle embedding, other than already mentioned above, can be found in the above referenced article.

For the purpose of searching prior art the following section is added, representing a translation of the last section in English:

1. Mask-less method for isolating a single object of interest, comprising providing the object of interest in a solution, selecting the object of interest, providing hydrophilic entities capable of radical polymerization in the solution, wherein the entities are monomeric, oligomeric, or polymeric entities, locally polymerizing the entities into a hydrogel by focused radiation-initiation with a single wavelength, preferably using an XY-stage, more preferably a controlled XYstage, thereby forming at least one pocket of the hydrogel comprising an object of interest, wherein the hydrogel is degradable, thereby immobilizing the object of interest.

2. Method according to claim 1, further comprising removing the solution, such as by rinsing the pocket of hydrogel.

3. Method according to claim 1 or 2, further comprising of isolating the at least one object of interest from its environment .

4. Method according to any of claims 1-3, wherein the hydrogel in the pocket is enzymatically or chemically degraded therewith freeing the object of interest.

5. Method according to any of claims 1-4, wherein the object of interest is labelled, such as by a fluorescent compound, or a phosphorescent compound.

6. Method according to any of claims 1-5, wherein the entities are selected from polysaccharides and derivatives thereof, such as (meth)acrylate modified polysaccharides, such as dextran, and/or wherein the entities are provided in a concentration of 0.1-10 wt.%, preferably 0.2-7 wt.%, such as 0.5-5 wt.%, wherein all weight percentages are relative to a total weight of the hydrogel solution.

7. Method according to claim 6, wherein the polysaccharides comprise 0.1-50 mole% (meth)-acrylate groups, preferably 0.5-20 mole% (meth)-acrylate groups per monomer of the polysaccharides .

8. Method according to any of claims 1-7, wherein the entities are selected from compounds with a molecular weight of 100-1000 kDa, preferably 200-500 kDa.

9. Method according to any of claims 1-8, wherein a laser is used for radiation initiation, such as a confocal laser scanning microscopy (CLSM).

10. Method according to any of claims 1-9, wherein a wavelength from 250-800 nm is used for polymerization, preferably 300-600 nm.

11. Method according to any of claims 1-10, wherein a photoinitiator is used, preferably an aromatic phosphinate, more preferably a bi-aromatic phosphinate, even more preferably a phenyl-benzoyl phosphinate, preferably comprising a mono-, di- or tri-alkyl benzoyl, wherein the alkyl is independently selected from C1-C6, such as methyl, ethyl, and propyl, preferably having a metallic counter ion, such as Li, and Mg, such as lithium-phenyl-2,4,6trimethylbenzoyl-phosphinate.

12. Method according to claim 11, wherein the photo-initiator is provided in a concentration of 0.1-2 wt.%, preferably 0.2-1 wt.%, such as 0.3-0.5 wt.%.

13. Method according to any of claims 1-12, wherein the solution comprises 30-99.8 wt.% water, 0-50 wt.% alcohol, such as methanol, ethanol, and propanol, and 0-50 wt.% DMSO.

14. Method according to any of claims 1-13, wherein a radiation-initiation exposure time is 1-25 ps/pm2, preferably 2-5 ps/pm2.

15. Method according to any of claims 1-14, wherein a 1 mW-5 W radiation source is used, preferably a 10 mW-2 W source.

16. Method according to any of claims 1-15, wherein the pocket is immobilized on a surface, such as a glass surface, such as a functionalized glass surface.

17. Method according to any of claims 1-16, wherein the object of interest is a biological cell.

18. Method according to any of claims 1-17, wherein the hydrogel pocket comprises a first layer of a first hydrogel, and at least one further layer such as of a second hydrogel .

19. Method according to any of claims 1-18, wherein at least one alignment marker is provided, preferably at least two alignment markers.

20. Isolated pocket of hydrogel obtainable by a method according to any of claims 1-19, comprising a three dimensional polymeric network of hydrophilic and degradable polymers, the hydrogel pocket comprising a selected object of interest .

21. Hydrogel pocket according to claim 20, wherein the object of interest has a volume of 102-106 pm3, a length of 1010.000 pm ±15 pm, a width of 10-10.000 pm ±15 pm, and a height of 1-500 pm.

22. Surface comprising a pocket of hydrogel according to any of claims 20-21, such as a glass surface.

23. Use of an immobilized cell obtained by a method according to any of claims 1-19 for cell-study, for study of a medical compound, for enzyme study, for genetic study, for study of controlled drug release, for study of soft matter actuators, for cell encapsulation, for tissue engineering, or for obtaining a viable cell.

24. A kit for capturing at least one object of interest in a fluid, comprising as a photo-initiator an aromatic phosphinate, more preferably a bi-aromatic phosphinate, even more preferably a phenyl-benzoyl phosphinate, preferably comprising a mono-, di- or tri-alkyl benzoyl, wherein the alkyl is independently selected from C1-C6, such as methyl, ethyl, and propyl, preferably having a metallic counter ion, such as Li, and Mg, such as lithium-phenyl-2,4,6-trimethylbenzoylphosphinate, and as hydrophilic entities capable of radical polymerization in a solution polysaccharides and derivatives thereof, such as (meth)acrylate modified polysaccharides, such as dextran, wherein the polysaccharides comprise 0.1-50 mole% (meth)-acrylate groups, preferably 0.5-20 mole% (meth)acrylate groups, per monomer of polysaccharide, and optionally a gel-degrading compound, such as an enzyme .

Claims (24)

CONCLUSIONS A maskless method for isolating a single object of interest, comprising providing the object of interest in a solution, selecting the object of interest, providing hydrophilic units capable of radical polymerization in the solution, the entities are monomeric, oligomeric or polymeric entities, locally polymerizing the entities into a single wavelength focused, focused, wavelength hydrogel, preferably with an XY platform, more preferably a controlled XY platform, thereby comprising at least one bag of the hydrogel the object of interest is formed, the hydrogel being degradable, thereby immobilizing the object of interest. The method of claim 1, further comprising removing the solution, for example, by rinsing the hydrogel bag. The method of claim 1 or 2, further comprising isolating the at least one object of interest from its environment. The method of any one of claims 1-3, wherein the hydrogel in the bag is degraded enzymatically or chemically, liberating the object of interest. The method of any one of claims 1-4, wherein the object of interest is labeled, such as by a fluorescent compound, or a phosphorescent compound. 6. A method according to any one of claims 1-5, wherein the units are selected from polysaccharides and derivatives thereof such as (meth) acrylate modified polysaccharides, such as dextran, and / or wherein the entities are provided in a concentration of 0.1-10. % by weight, preferably 0.2-7% by weight, for example 0.5-5% by weight, all weight percentages being based on a total weight of the hydrogel solution. The method of claim 6, wherein the polysaccharides comprise 1-50 mol% (meth) acrylate, preferably 2-30 mol% (meth) acrylate groups per monomer of the polysaccharides. A method according to any one of claims 1-7, wherein the units are selected from compounds with a molecular weight of 100-1000 kDa, preferably 200-500 kDa. The method of any one of claims 1-8, wherein a laser is used for radiation initiation, such as a confocal laser scanning microscope (CLSM). The method of any one of claims 1-9, wherein a wavelength of 250-800 nm is used for polymerization, preferably 300-600 nm. A method according to any one of claims 1-10, wherein a photo-initiator is used, preferably an aromatic phosphinate, preferably a bi-aromatic phosphinate, more preferably a phenyl-benzoyl phosphinate, preferably a mono-, di- or tri-alkylbenzoyl, wherein the alkyl is independently selected from C 1 -C 6 such as methyl, ethyl and propyl, preferably with a metallic counter ion such as Li and Mg such as lithium phenyl-2,4,6-trimethylbenzoyl phosphinate. A method according to claim 11, wherein the photoinitiator is provided in a concentration of 0.1-2% by weight, preferably 0.2-1% by weight, for example 0.3-0.5% by weight. The method of any one of claims 1 to 12, wherein the solution comprises 30-99.8% by weight of water, 0-50% by weight of alcohol, such as methanol, ethanol and propanol, and 0-50% by weight DMSO. The method of any one of claims 1-13, wherein a radiation initiation exposure time is 1-25 ps / pm ² , preferably 2-5 ps / pm ² . The method of any one of claims 1-14, wherein a 100 mW-5 W radiation source is used, preferably 200 mW-2 W source. The method of any one of claims 1-15, wherein the bag is immobilized on a surface, such as a glass surface, such as a functionalized glass surface. The method of any one of claims 1-16, wherein the object of interest is a biological cell. The method of any one of claims 1-17, wherein the hydrogel bag comprises a first layer of a first hydrogel, and at least one further layer, such as a second hydrogel. A method according to any of claims 1-18, wherein at least one target mark is applied, preferably at least two target marks. An isolated hydrogel bag obtainable by a method according to any of claims 1-19, comprising a three-dimensional polymer network of hydrophilic and degradable polymers, the hydrogel bag comprising a selected object of interest. The hydrogel bag of claim 20, wherein the object of interest has a volume of 10 ² -10 ⁶ µm ³ , a length of 10-10,000 µm ± 15 µm, and a width of 10-10,000 µm ± 15 µm, and a height from 1 to 500 pn. A surface provided with a hydrogel bag according to any of claims 20-21, for example a glass surface. Use of an immobilized cell obtained according to a method according to any of claims 1-19 for cell study, for study of a medical compound, for enzyme study, for genetic study, for study for regulated drug delivery, for study of soft matter actuators , for cell encapsulation, for tissue engineering, or for obtaining a viable cell. A kit for capturing at least one object of interest in a fluid, comprising as photoinitiator an aromatic phosphinate, preferably a bi-aromatic phosphinate, more preferably a phenyl-benzoyl phosphinate, preferably a mono-, di- or tri- alkylbenzoyl, wherein the alkyl is independently selected from C 1 -C 6, such as methyl, ethyl and propyl, preferably with a metallic counterion, such as Li and Mg, such as lithium-phenyl-2,4,6-trimethylbenzoyl phosphinate, and as hydrophilic units in capable of radical polymerization in a solution polysaccharides and derivatives thereof, such as (meth) acrylate modified polysaccharides, such as dex25 tran, wherein the polysaccharides contain 1-50 mol% (meth) acrylate, preferably 2-30 mol% (meth) acrylate groups per monomeric polysaccharide, and optionally a gel-degrading compound, such as an enzyme. W2 3 FIG. la After irradiation After rinsing Fig, lb