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US-12618033-B2 - Multi-well plate and method for preparing same

US12618033B2US 12618033 B2US12618033 B2US 12618033B2US-12618033-B2

Abstract

The invention relates to a multi-well plate comprising a support, the upper surface of which is at least partially covered with a continuous layer of a hydrogel in contact with the lower surface of a bottomless multi-well plate, the support, the continuous layer, and the bottomless multi-well plate being adhered by means of an adhesive which extends from at least certain portions of the lower surface of the bottomless multi-well plate up to certain portions of the upper surface of the support by passing through the continuous layer of hydrogel, each well of the bottomless multi-well plate being entirely surrounded by the at least certain portions of the lower surface. The application also relates to a method for preparing the multi-well plate and the use thereof for in vitro cell culture.

Inventors

  • Alice NICOLAS
  • CAMILLE MIGDAL

Assignees

  • CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
  • Commissariat à l'énergie atomique et aux énergies alternatives
  • UNIVERSITE GRENOBLE ALPES

Dates

Publication Date
20260505
Application Date
20200709
Priority Date
20190712

Claims (16)

  1. 1 . A multi-well plate comprising a support, an upper surface of said support being at least partially covered with a continuous layer of a hydrogel which has a stiffness of from 0.05 kPa to 100 kPa, as measured by atomic force microscopy, said continuous layer of the hydrogel being in contact with a lower surface of a bottomless multi-well plate, wherein said support, said continuous layer of the hydrogel, and the said bottomless multi-well plate are adhered by an adhesive which extends from at least certain portions of the lower surface of the bottomless multi-well plate up to certain portions of the upper surface of said support by passing through the continuous layer of hydrogel, said continuous layer of the hydrogel being partitioned into compartments by means of the adhesive, a bottom of each well of the multi-well plate being entirely surrounded by said at least certain portions of the lower surface of the bottomless multi-well plate.
  2. 2 . The multi-well plate according to claim 1 , wherein the hydrogel comprises a polymer matrix comprising a polymer selected from the group consisting of: polyacrylamides; polyethylene glycols, polypropylene glycols and ethylene glycol or propylene glycol copolymers, these latter optionally comprising units resulting from the polymerisation of (meth)acrylate compounds; polysaccharides, optionally comprising repeating units resulting from the polymerisation of (meth)acrylate compounds; (co)polymers resulting from the polymerisation of diacrylate and/or (meth)acrylate compounds; polyvinyl alcohols comprising repeating units resulting from the polymerisation of (meth)acrylate compounds; dextrans comprising repeating units resulting from the polymerisation of (meth)acrylate compounds; polypropylene fumarates and poly(propylene fumarate-co-ethylene glycol); and combinations thereof.
  3. 3 . The multi-well plate according to claim 2 , wherein the polymer matrix comprises a polyacrylamide.
  4. 4 . The multi-well plate according to claim 1 , wherein within the same given well, the variability in the stiffness of the hydrogel at the micrometer scale is less than 10%.
  5. 5 . The multi-well plate according to claim 4 , wherein within the same given well, the variability in the stiffness of the hydrogel at the micrometer scale is less than 5%.
  6. 6 . The multi-well plate according to claim 1 , wherein, within the same given well, the hydrogel comprises at least two contiguous zones of distinct stiffness exhibiting a stiffness gradient greater than or equal to 0.05 kPa/μm.
  7. 7 . The multi-well plate according to claim 1 , wherein the surface of the hydrogel of at least one of the wells is functionalised with a polysaccharide and/or a protein and/or a peptide.
  8. 8 . The multi-well plate according to claim 7 , wherein the surface of the hydrogel of at least one of the wells is functionalised with a protein and/or a peptide able to induce cell adhesion via integrins.
  9. 9 . The multi-well plate according to claim 8 , wherein the surface of the hydrogel of at least one of the wells is functionalised with fibronectin, fibrinogen, collagen, laminin, vitronectin or an RGD peptide.
  10. 10 . The multi-well plate according to claim 7 , wherein the surface of the hydrogel of each well is functionalised with a polysaccharide and/or a protein and/or a peptide.
  11. 11 . A cell culture method in which the multi-well plate according to claim 1 is seeded with cells, and the cells are then cultured.
  12. 12 . The cell culture method according to claim 11 , wherein the cells are stem cells and wherein the stem cells are cultured and differentiated.
  13. 13 . A plate preparation method for preparing the multi-well plate according to claim 1 , said method comprising: a) providing a substrate comprising said continuous layer of the hydrogel which has a stiffness of from 0.05 kPa to 100 kPa, as measured by atomic force microscopy, said continuous layer at least partially covering the upper surface of the support; b) applying the adhesive at 20° C., the adhesive being in liquid form at 20° C., over said at least certain portions of the lower surface of a bottomless multi-well plate, the bottom of each well of the multi-well plate being entirely surrounded by the at least certain portions of the lower surface of the bottomless multi-well plate; and c) assembling the substrate and the bottomless multi-well plate by bringing into contact the surface of the hydrogel of the substrate with the lower surface of the bottomless multi-well plate that is at least partially covered with the adhesive, whereby the adhesive at least partially penetrates within the continuous layer of the hydrogel so that said continuous layer of the hydrogel is partitioned into compartments by means of the adhesive.
  14. 14 . The method according to claim 13 , wherein during c), the liquid adhesive penetrates totally within the continuous layer of hydrogel and passes through it until it reaches certain portions of the upper surface of the support.
  15. 15 . The method according to claim 13 , wherein: between a) and c), said method further comprises an element a1) wherein a polysaccharide and/or a protein and/or a peptide is applied onto at least a portion of the surface of the hydrogel of the substrate; or after c), said method further comprises an element d) wherein a polysaccharide and/or a protein and/or a peptide is applied onto the surface of the hydrogel of at least one well.
  16. 16 . A screening method for screening of therapeutic molecules, which includes: deposition in the wells of the multi-well plate according to claim 1 of a ligand; bringing into contact of the molecules to be tested with the said ligand; followed thereafter by the identification of the molecules to be tested which have bound themselves to the said ligand.

Description

The present invention relates to a multi-well plate, the method of preparation thereof, and use thereof for cell culture or for screening of therapeutic molecules. In many fields of biology/pharmacy it is very evident that in vitro test conditions need to correspond more closely to physiological conditions. In particular, failure to appropriately adapt to these conditions is considered largely responsible for the failure of many molecules identified in vitro to successfully be transferred to the clinical phases, due to erroneous analysis of targets and efficacy of these molecules as well as underestimation of the toxicity thereof. The cellular behaviours giving rise to these erroneous predictions are at least partially linked to the preparation of cultures in vitro under conditions that are too far removed from actual physiological conditions. In this context, there is a need to develop multi-well plates that make it possible to perform tests under conditions that are as close as possible to physiological conditions while also retaining the advantages of such plates being used in tests, that is to say, the ease of use in implementation, reproducibility, high availability, ethics. Products are currently being developed that offer biomimetic environments that meet these specifications: 3D environments, geometric constraints to force physiological cell geometry, microfluidic perfusion chambers to mix cell secretions and mimic co-culture, biomimetic surface chemistry for 2D culture, support with elastic base/bottom to mimic the physiological mechanical properties. One plate production method for producing a multi-well plate for which the bottom of the wells are coated with hydrogel consists in preparing a hydrogel film on a flexible layer, then cutting the hydrogel film/flexible layer assembly in the form of pellets and inserting and assembling each pellet by means of an adhesive to the bottom of each well, as illustrated in Zustiak, S. et al. Biotechnology and Bioengineering, 2014, 111, 396-403 and in FIG. 1. The multi-well plate complies with the standard “Recommended Microplate Specifications” of the Society for Biomolecular Screening (SBS) and each well bottom may have a stiffness independent of that of other well bottoms. However, the preparation method requires a great deal of handling and manipulation. Thus the risk of contamination is very high, as is the risk of cutting residues causing degrading of the surface condition of the hydrogels. Finally, the presence of the adhesive+flexible layer+hydrogel film stack greatly deteriorates the images obtained by inverted microscopy. Alternatively, it is possible to prepare the hydrogel film directly at the bottom of the wells of a multi-well plate, as described in U.S. Pat. No. 8,871,499. The multi-well plate complies with the standard “Recommended Microplate Specifications” of the Society for Biomolecular Screening (SBS). Despite this however, it is likely that the gel thickness will differ from one well to another. The patent also reports that the edges of the gel in each well are softer. In addition, the monomer residues are very difficult to remove due to the narrowness of the wells, especially beyond 48 wells. However, these monomers are toxic to the cells. A product that neutralises free radicals is added, but comes with the risk of partially retaining the toxicity linked to the mutagenic, carcinogenic and reprotoxic properties of bis-acrylamide. Moreover, even if it is possible to vary the stiffness of the hydrogel between the wells by varying the monomer composition of the solution used, the preparation of soft hydrogels is difficult in practice due to the rapidness in reaching the limit at which the crosslinking is no longer covalent but entangled (“entanglement”), and the gel then exhibits strong spatial heterogeneities in stiffness. Finally, this method of preparation precludes creating controlled stiffness gradients within a well. Ahmed, N.; et al. Tissue engineering. Part C, Methods, 2016, 22, 543-551 describe the preparation of a multi-well plate comprising of the preparation by photopolymerisation through a photomask of a hydrogel in the form of pellets, followed by the assembly of these hydrogel pellets with a support consisting of wells drilled in a piece of plastic. The assembly is held in place by clamps, and annular seals located at the base of each well ensuring the sealing of each well. FIG. 2 illustrates such a plate. The resulting multi-well plate obtained is not compatible with robots and microscope incubators and does not comply with the standard “Recommended Microplate Specifications” of the Society for Biomolecular Screening (SBS), which greatly limits its use. In addition, it is necessary to position as many seals as there are wells on the plate, which makes the method very unsuitable for plates comprising a large number of wells. The patent application WO 2013/074972 describes a platform for biological assays including a substrate