FR3079524A1 - MICROPUTIC PLATES IN BIOCOMPATIBLE HYDROGEL - Google Patents

Abstract

The invention relates to a biocompatible hydrogel microwell plate adapted for the growth of cell aggregates, its method of preparation and its use for the growth of cell aggregates in wells, in particular for observing the response of cell aggregates grown in reaction. stimuli, such as exposure to toxic substances or drug candidates.

Description

BIOCOMPATIBLE HYDROGEL MICROWELL PLATES

The present invention relates to a plate of biocompatible hydrogel microwells suitable for the growth of cell aggregates, its preparation process and its use for the growth of cell aggregates in wells, in particular for observing the response of cell aggregates cultured in reaction. stimuli, such as exposure to toxic substances or drug candidates.

TECHNOLOGICAL BACKGROUND

It is now recognized that 2D cell culture represents little of the cellular environment in vivo and many systems are being developed to cultivate cells in 3D, with cell aggregates, particularly organized cell aggregates. One of the burgeoning models is the use of self-organizing cells into spheres (Multicellular Tumor Spheroids -MCTS).

There are standard 96-well plate systems with very low cell adhesion to form cell aggregates.

There are also plate systems on which the cell aggregates are cultured from a cell linked to the plate, such as the plates marketed under the brand ONCOSPHERES ™ by the company CYTOO (https://cytoo.com/). There are also PDMS microwell plates whose surface is coated with a biocompatible material such as polyethylene glycol (Karp et al., Lab Chip, 2007, 7, 786-794). However, these solutions have the drawback of offering a rigid wall to the cells, and therefore of strongly constraining the structure of the aggregates, which influences their reactions, in particular their ability to modify their volume and their organization as a function of a stimulus. .

There are also "micro-well" plates made of biocompatible materials such as agarose, allowing the formation of aggregates, such as the plates sold under the 3D Petri Dish® brand by the company Microtissues (www.microtissues .corn). Hydrogel micro-wells (agarose) have the advantage of allowing molecules of size <30 nm to diffuse freely. This ensures the supply of nutrients and molecules of interest. However, existing solutions do not allow observations by high resolution optical means, such as microscopy monitoring with high magnification objectives (x40, x63, x100), in particular due to the thickness of the hydrogel layer. Objectives compatible with this type of microwell can only go up to x20 magnification (Napolitano & al., BioTechniques, 2017, 43, No. 4).

The existing solutions and their preparation methods are notably recalled in the article "Three-dimensional culture Systems in cancer research: Focus on tumor spheroid model" (Nath & Devi, Pharmacology & Therapeutics, 2016, 163, 94-108).

The invention makes it possible to solve the drawbacks specific to each system, allowing culture in microwells with sufficiently flexible walls not to overly constrain the cell aggregates, in particular in response to stimuli to be observed, but also to allow the observation of these stimuli by high magnification optical means.

BRIEF DESCRIPTION OF THE INVENTION

The solution is provided by a microwell plate made of biocompatible hydrogel adapted to the growth of cellular aggregates which comprises a first layer of biocompatible hydrogel comprising the microwells placed on a support plate made of rigid material transparent to light radiation, the distance between the bottom microwells and the surface of the support plate ranging from 50 to 500 μm.

By cellular aggregates is meant according to the invention the self-assembly of one or more types of cells in 3 dimensions (also called neurospheres, spheroids, organoids)

According to a preferred embodiment of the invention, the biocompatible hydrogel is an agarose hydrogel.

The invention also relates to a method for preparing the microwell plate by molding a biocompatible gelling solution capable of forming a hydrogel at room temperature, on a mold comprising a substantially flat plate and fingers perpendicular to the plate which will form microwells of the hydrogel, to apply the support plate to the biocompatible gelling agent solution so as to spread said solution over a constant thickness in the mold, to allow the hydrogel to form and then to release the microwell plate obtained.

The invention also relates to the use of a microwell plate according to the invention for the cultivation of cellular aggregates in microwells, and in particular for the analysis of reactions of cellular aggregates cultivated in microwells in response to a stimulus , in particular by high magnification microscopy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a plate of microwells suitable for the growth of cellular aggregates, characterized in that it comprises a first layer of biocompatible hydrogel comprising the microwells placed on a support plate of rigid material characterized in that the distance between the bottom of the microwells and the surface of the support plate ranges from 50 to 500 μm, advantageously from 100 to 300 μm, preferably from 150 to 250 μm.

According to a preferred embodiment of the invention, the support plate is made of a rigid material transparent to light radiation, more particularly to allow use in optical microscopy at high magnification.

By "transparent to light radiation" is meant a material which is transparent to light radiation ranging from ultra violet to infrared, in particular in the visible light spectrum.

The support plate is advantageously a glass plate, or more particularly a glass slide, of a thickness suitable for observation by microscopy at high magnification, in particular a thickness of 100 to 170 μm.

Biocompatible hydrogels are well known to those skilled in the art, in particular agarose hydrogels (Napolitano & al., BioTechniques, 2017, 43, No. 4; Gong & al., 2015, PLOS ONE, 10 (6) ) or methacryloyl gelatin hydrogels (Loessner & al., 2016, Nature Protocol 11 (4): 727-746).

The biocompatible hydrogel according to the invention is stable at temperatures close to room temperature, ranging from 0 ° C to 30 ° C.

Preferably, the hydrogel is an agarose hydrogel, in particular a hydrogel essentially comprising agarose and water. Agarose and agarose hydrogels are well known to those skilled in the art, particularly in the field of biology. Hydrogels can be prepared with different stiffnesses depending on the agarose used and its concentration. The different types of agarose and the properties of the gels obtained are well known to those skilled in the art. Mention will in particular be made of the agaroses marketed under the names Agarose Standard, Agarose Low Gelling Temperature and Agarose Ultra-Low Gelling Temperature by the company Sigma-Aldrich (https://www.sigmaaldrich.com/) in qualities compatible with cell growth and their use to observe the effect of stimuli on cell growth.

According to a preferred embodiment of the invention, the biocompatible hydrogel comprises from 1 to 5% by mass of agarose relative to the total mass of the hydrogel, the remainder being essentially water.

The hydrogel may possibly include other constituents, insofar as they do not interfere with the biocompatibility of the hydrogel, nor with its capacity to allow the nutrients necessary for cell culture and small molecules, including the effect on the cellular aggregates will be observed, for example it is possible to add fluorescent nanoparticles of the desired size and concentration in the liquid agarose. These nanoparticles can advantageously be used as tracers to measure the forces exerted by cellular aggregates during their growth (Burnette, & al. (2014), The Journal of Cell Biology. 205 (1), 83-96; Delanoë-Ayari, & al. (2010) Physical Review Letters, 105 (24), 2-5; Style & al. (2014) Soft Matter, 10 (23), 4047-4055).

Advantageously, the biocompatible hydrogel layer adheres to the upper face of the support plate.

For this purpose, the upper face of the support plate in contact with the hydrogel comprises a material which promotes the adhesion of the biocompatible hydrogel layer. This material can be added to the upper face of the support plate by a surface treatment.

When the hydrogel is an agarose hydrogel, the upper face of the rigid support is advantageously treated by the addition of a layer of silane, in particular by grafting silane to the surface of the support plate.

According to a preferred embodiment of the invention, the support plate is a glass plate, the upper face of which is treated by the addition of a layer of silane. Those skilled in the art are well aware of the methods of modifying the surface of the support plate with silanes and the silanes capable of being used, in particular for glass supports with silanes, in particular by adopting the technologies used in the usual manner. to modify the surface of materials used in microfluidic systems (Séguin & al. (2010) Applied Surface Science, 256 (8), 2524-2531; Wu & al. (2011) Surface and Coatings Technology, 205 (10), 3182- 3189; Zhou & al. (2012) Electrophoresis, 33 (1), 89-104).

Among the silanes capable of being used, mention will be made in particular of mercapto-terminated silanes such as 3-mercaptopropyltrimethoxysilane and preferably amine-terminated silanes, which make it possible to have amine groups on the surface of the rigid support, thus facilitating the subsequent attachment of the gel. As amine-terminated silanes, mention will be made in particular of amino-propyl-triethoxysilane (APTS), 3-amino-propyl-trimethoxysilane, 3 amino- propyl-methyldiethoxysilane and methylamino-propyl-trimethoxysilane, preferably APTS.

According to a preferred embodiment of the invention, the support plate is a glass plate, particularly a glass slide, the upper face of which in contact with the hydrogel comprises a layer of silane, preferably APTS.

The thickness of the hydrogel layer will essentially depend on the depth desired for the wells. Advantageously, the hydrogel layer has a thickness of 200 to 1000 μm, preferably approximately 500 μm.

The microwells have dimensions suitable for the intended use for observing cellular aggregates. These are generally "standard" dimensions that are usually found in microwell plates used in biology laboratories, particularly adapted to the materials and tools, robots and devices used in these laboratories.

These standard dimensions will enable a person skilled in the art to use the plates according to the invention with the same tools, robots and laboratory devices as the usual microwell plates.

In particular, the microwells have a diameter of at least about 5 μm, or even at least 50 μm, generally from 100 to 500 μm, or even more.

The plates generally comprise 0.5 to 11 wells / mm ² which are advantageously distributed regularly over an orthonormal reference frame.

Advantageously, all the microwells of the same plate have the same dimensions.

These plates are advantageously sized to be used in standard multi-well plates (multi-wells of the Falcon or Corning brands widely used in biology for example) as well as plates dedicated to video microscopy (glass slides for example).

The invention also relates to a process for preparing a microwell plate according to the invention as defined above and in the examples, said process comprising the steps of preparing a mold inert with respect to the biocompatible hydrogel, said mold comprising a substantially flat plate and a plurality of fingers rising perpendicular to the plate, the plate corresponding to the upper face of the biocompatible hydrogel layer and each finger corresponding to a well of the biocompatible hydrogel layer, apply to the mold inert an appropriate quantity of a biocompatible gelling solution, apply to the biocompatible gelling solution a support plate in transparent rigid material so as to spread said solution on a constant thickness in the mold, let the gelling solution gel biocompatible hydrogel, and unmold the microwell plate obtained.

Advantageously, the mold is made of polydimethylsiloxane (PDMS).

This mold, negative of the microwell plate obtained, can be prepared by molding in a first mold incorporating the dimensional characteristics of the plate according to the invention that the skilled person seeks to obtain. This first mold can be advantageously prepared on a silicon wafer, by well-known photolithography techniques, used in the fields of electronics or microfluidics (Zhao & al. (1997), Journal of Materials Chemistry , 7 (7), 1069-1074).

In the process according to the invention, the step of preparing the mold comprises heating it to a temperature of 30 to 100 ° C., to receive the solution of biocompatible gelling agent, which is itself generally heated to remain in the form of a solution, gelling takes place at room temperature, or even, depending on the gelling agent used at a temperature close to 0 ° C.

A person skilled in the art will be able to determine the concentration of gelling agent used as a function of the gelling agent and of the rigidity sought for the hydrogel layer. He will also be able to determine the quantity of gelling solution to be applied to the inert mold as a function of the total volume of hydrogel, itself a function of the size of the microwells and their number.

The invention also relates to the use of a microwell plate according to the invention, defined above and in the examples, for the culture of cellular aggregates in the microwells and in particular for the analysis of the reactions of aggregates. cells grown in microwells in response to a stimulus.

The invention also relates to a method for observing the reactions of cellular aggregates in response to a stimulus, which comprises the steps of inoculating the microwells of a plate according to the invention with at least one cell, supplying the cells in the microwells the nutrients necessary for their growth and for the formation of cellular aggregates, subjecting the cellular aggregates formed to a stimulus and observing the reactions of the aggregates in response to this stimulus.

The observations can advantageously be made by microscopy at very high magnification (x40, x63, x100).

After submission to the stimuli, cell aggregates can be recovered for further analysis without affecting their structure and composition.

These different stimuli can be toxic products, drug candidates, reagents intended to identify the presence or absence of certain biological markers, etc.

The advantages of the microwell plates according to the invention are:

the possibility of manufacturing hundreds of aggregates, in particular of reproducible spheroids in a single pipetting, the possibility of controlling the final size of the aggregates, in particular spheroids, by adapting the diameter of the micro-wells, of having hydrogel allowing the contribution of nutrients and molecules of interest, elastic material with small deformations: it is possible to calculate the forces generated by the aggregates, in particular the spheroids, that all the aggregates, in particular spheroids, obtained are on the same optical plane, this which facilitates videomicroscopy, compatibility with high resolution microscopy, the possibility of incubating and washing aggregates, in particular spheroids, with different solutions without losing or mechanically damaging them, that it is easy to recover the aggregates, in particular spheroids, to perform molecular and genetic analyzes on the same samples illons (RT-PCR; western blots, histology, immunohistochemistry), transfer to cell biology laboratories / clinical analysis of biopsies is easily possible.

DESCRIPTION OF THE FIGURES

Figure 1 shows a sectional view of a microwell plate according to the invention.

Figure 2 shows an example of microwells arranged in a standard 24-well plate. (A) In this example, each well has 130 microwells 200 μm in diameter. (B) illustration of cell aggregates 200 µm in diameter (black spheres) obtained after 6 days of culture (taken under white light microscopy).

EXAMPLES

Step 1) Manufacture of the mold on silicon wafer (h = 250 pm, modular if necessary)

The manufacture of the mold uses conventional photo-lithography techniques. Briefly, a photosensitive resin (SU8 2100) is spread uniformly by centrifugation (500 rpm, 10 s then 1200 r / min, 30 s). This resin is then cured (75 ° C, 20 min, then 95 ° C, 1 h), then exposed to UVs (360 mJ / cm ² ). The resin is annealed in order to solidify the grip (75 ° C, 10 min then 95 ° C, 5 min). Finally, the pattern is developed (PGMEA solution, 18 min) in order to remove the resin not exposed to UVs. This first mold thus produced is then silanized overnight before being usable for molding the counter mold in PDMS (step 2). This mold can be reused for several years without deterioration.

Step 2) Manufacturing a PDMS counter mold

PolyDiMethylSiloxane (PDMS) is used as polymer to obtain a counter-mold which can be reused several times. The base and the crosslinker are mixed (10: 1 ratio by mass), then put under vacuum for 30 min in order to remove the bubbles. The PDMS is then spread on the silicon mold and annealed for 2 hours at 75 ° C.

After annealing, the PDMS is detached from the silicon mold. It constitutes our counter-mold on which we will mold the agarose (step 4).

Step 3) Functionalization of the glass slides to be able to adhere the micro-wells

The glass slides are functionalized with amino-propyltri (ethoxy) silane (APTS 1%, in a solution of acetic acid 5 mM, 20 min, room temperature with stirring). The coverslips are then washed with distilled water and annealed for 15 min at 100 ° C (to solidify the silane layer). The coverslips can be prepared in advance and stored at room temperature, protected from dust.

Step 4) Molding of agarose micro-wells

The micro-wells were molded from 2 types of agarose, to be chosen according to the desired stiffness (see table 1). The PDMS counter molds are placed on a hot plate (at 78 ° C or 50 ° C depending on the type of agarose used, see Table 1). The agarose is prepared by mixing the powder (% according to table 1) with distilled water. The agarose powder is dissolved in distilled water using an autoclave. The solution thus obtained is maintained in the liquid state using a dry bath maintained at the right temperature (Table 1). A volume of agarose (determined as a function of the size of the wells to be molded, cf. Table 2) is then deposited on the counter molds in hot PDMS. A lamella functionalized with APTS is then deposited on the agarose drop in order to spread it over a constant thickness. The whole (PDMS-agarose-coverslip mold) is then removed from the hot plate and left to gel according to the time and the temperature indicated in table 1.

Table 1. Parameters used for the manufacture of agarose microwells.

Rigidity agarose % Mold temperature Bath temperature Gelation temperature Gel time 3 kPa Ultra-Low 2 50 ° C 60 ° C 4 ° C 72h > 30 kPa standard 4.5 78 ° C 100 ° C ambient 10 minutes

Table 2. Volume of agarose to be deposited on the various PDMS molds for a 20 mm diameter mold

Well diameter (pm) 500 200 100 Mixed: 100 and 200 Agarose volume (pL) 100 120 120 120

Step 5) Seeding of cells in micro-wells

Once the agarose has gelified, the microwells are removed from the mold, placed in PBS and sterilized by UV treatment. These micro-wells can be kept for several days at 4 ° C without deterioration.

The micro-wells are then incubated with culture medium (2 h), before inoculating them with cells (1.2 × 10 ⁵ cells total, in 3 ml per well).

REFERENCES

- Burnette & al. (2014), The Journal of Cell Biology, 205 (1), 83-96

- Delanoë-Ayari & al. (2010) Physical Review Letters, 705 (24), 2-5.

- Karp et al., Lab Chip, 2007, 7, 786-794

- Gong & al., 2015, PLOSONE, 10 (6)

- Loessner & al., 2016, Nature Protocol 11 (4): 727-746

- Napolitano & al., BioTechniques, 2017, 43, No. 4

- Nath & Devi, Pharmacology & Therapeutics, 2016, 163, 94-108

- Séguin & al. (2010) Applied Surface Science, 256 (8), 2524-2531

- Style & al. (2014) 70 (23), 4047-4055

- Wu & al. (2011) Surface and Coatings Technology, 205 (10), 3182-3189

- Zhao & al. (1997), Journal of Materials Chemistry, 7 (7), 1069-1074

- Zhou & al. (2012) Electrophoresis, 33 (1), 89-104

Claims (15)

1. Plate of microwells adapted to the growth of cellular aggregates, characterized in that it comprises a first layer of biocompatible hydrogel comprising the microwells placed on a support plate of rigid material transparent to light radiation, the distance between the bottom of the microwells and the surface of the support plate ranging from 50 to 500 μm. 2. Microwell plate according to claim 1, characterized in that the biocompatible hydrogel is an agarose hydrogel. 3. Microwell plate according to one of claims 1 or 2, characterized in that the biocompatible hydrogel comprises from 1 to 5% by mass of agarose relative to the total mass of the hydrogel. 4. Microwell plate according to one of claims 1 to 3, characterized in that the biocompatible hydrogel layer adheres to the support plate. 5. Microwell plate according to claim 4, characterized in that the support plate is a glass plate whose upper face supporting the biocompatible hydrogel layer has been treated to allow adhesion between the plate and the hydrogel layer . 6. Microwell plate according to claim 5, characterized in that the face of the glass plate is treated by the application of a layer of silane. 7. Microwell plate according to one of claims 1 to 6, characterized in that the hydrogel layer has a thickness of 200 to 1000 µm. 8. Microwell plate according to one of claims 1 to 7, characterized in that the microwells have a diameter of at least 5 µm. 9. microwell plate according to claim 8, characterized in that the microwells have a diameter of 100 to 500 μm. 10. Microwell plate according to one of claims 1 to 8, characterized in that the microwells are distributed regularly on an orthonormal reference mark. 11. Method for preparing a microwell plate according to one of claims 1 to 10, characterized in that it comprises the steps of preparing a mold inert with respect to agarose, said mold comprising a plate substantially flat and a plurality of fingers rising perpendicularly to the plate, the plate corresponding to the upper face of the layer of biocompatible hydrogel and each finger corresponding to a well of the layer of biocompatible hydrogel, apply to the inert mold a appropriate amount of agarose solution, apply a support plate of rigid material to the agarose solution 5 transparent so as to spread said solution over a constant thickness in the mold, let the agarose solution gel in biocompatible hydrogel, and unmold the microwell plate obtained. 12. Method according to claim 11, characterized in that the mold is made of polydimethylsiloxane (PDMS). 13. Method according to one of claims 11 or 12, characterized in that the preparation of the mold comprises heating it to a temperature of 30 to 100 ° C. 14. Use of a microwell plate according to one of claims 1 to 10 15 for the cultivation of cell aggregates in microwells. 15. Use of a microwell plate according to one of claims 1 to 10 for the analysis of the reactions of cell aggregates cultivated in the microwells in response to a stimulus.