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EP4536801A1 - Visualisation de collagène sur un dispositif microfluidique - Google Patents

Visualisation de collagène sur un dispositif microfluidique

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Publication number
EP4536801A1
EP4536801A1 EP23732527.9A EP23732527A EP4536801A1 EP 4536801 A1 EP4536801 A1 EP 4536801A1 EP 23732527 A EP23732527 A EP 23732527A EP 4536801 A1 EP4536801 A1 EP 4536801A1
Authority
EP
European Patent Office
Prior art keywords
cells
peg
polymer
cell
dextran
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23732527.9A
Other languages
German (de)
English (en)
Inventor
Ralph Müller
Xiao-hua QIN
Doris ZAUCHNER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eidgenoessische Technische Hochschule Zurich ETHZ
Original Assignee
Eidgenoessische Technische Hochschule Zurich ETHZ
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eidgenoessische Technische Hochschule Zurich ETHZ filed Critical Eidgenoessische Technische Hochschule Zurich ETHZ
Publication of EP4536801A1 publication Critical patent/EP4536801A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control

Definitions

  • the present application claims benefit of the priorities of EP22178671.8 filed 13 June 2022, and EP22183705.7 filed 7 July 2022, both of which are incorporated herein by reference.
  • Field The invention relates to a device, compositions and methods for generating and using biosystems that mimic osteogenesis in humans.
  • the invention provides compositions that can be used to generate macroporous hydrogels comprising mammalian cells, in microfluidic devices that allow the application of biophysical conditions that lead to differentiation of cells similar to the development of tissues in the human body.
  • Bone development — or osteogenesis is a complex process that involves changes in cellular behavior and extracellular matrix (ECM) organization induced by an intricate cellular signaling network and various physical factors.
  • ECM extracellular matrix
  • osteoid a matrix comprised mostly of collagen type I — is formed by a subset of osteoblasts. Mineralization of this matrix causes these cells to become embedded and differentiate into mature osteocytes that form a complex three-dimensional (3D) cellular network within the lacuno-canalicular network (LCN) system by reorganization of their cytoskeletal architecture.
  • LCN lacuno-canalicular network
  • FSS fluid shear stress
  • microfluidic chip was fabricated to study osteocyte-osteoclast interaction in 2D under physiological FSS.
  • microfluidic cell culture requires much fewer cells and reagents while enabling real-time analysis of transient cellular response to drug treatments by high-resolution live imaging. Given these merits, microfluidic organ-on-chip biosystems hold the potential to revolutionize the fields of disease modeling and in vitro drug discovery. Efforts to replicate the complex bone-like tissue environment within (micro-)fluidic systems have been actively sought. In a recent study, Nasello et al. (G. Nasello, P.
  • the microfluidic chamber comprises an inlet port and an outlet port, the inlet port being connectable to a cell culture medium influx, and the outlet port allow cell culture medium outflow to leave the chamber.
  • the medium may comprise a drug candidate molecule under evaluation.
  • This aspect of the invention may be regarded as the essential part of a method to assay or study such drug candidate molecules, or to evaluate their efficacy in addressing bone formation or other collagen-related disease.
  • the plurality of stem cells in a macroporous hydrogel is generated in a gelation step, by polymerization-induced phase separation process from an aqueous precursor solution comprising the stem cells.
  • the aqueous precursor solution comprises a. a first polymer susceptible to in situ crosslinking, and optionally, a crosslinking agent; and b.
  • the first polymer is miscible with the second polymer when the first polymer is not crosslinked.
  • the mixture comprised of the first polymer and the second polymer undergoes phase separation, separating said first and said second polymer into separate phases once the first polymer is crosslinked.
  • the first polymer In certain embodiments, the first polymer is susceptible to photo-crosslinking, and the precursor comprises a photoinitiator. In certain other embodiments, the first polymer is susceptible to thiol-Michael addition. Polymers susceptible to thiol-Michael addition have been described, inter alia, in Lutolf et al., Nature biotechnology 2003, 21, 513.
  • the vinylsulfone-modified PEG is a four-arm-PEG- vinylsulfone.
  • the inventors use a di-Cys peptide crosslinker when introducing specific MMP-sensitivity to the matrix to enable cell-matrix remodelling.
  • Non-MMP-sensitive peptides can be used as a non-degradable control.
  • PEG di-thiol may be used to make non-degradable matrices at significantly lower costs.
  • the vinylsulfone-modified poly(ethyleneglycol) (PEG) is present in the precursor solution at 1.5% to 3.0% (w/v).
  • the mammalian cells are selected from primary cells and cell culture cells.
  • the mammalian cells are stem cells.
  • the mammalian cells are human primary osteoblasts.
  • a related aspect of the invention provides a method for imaging or assaying cell development, cell differentiation and / or collagen secretion, comprising generating an in-vitro cell culture model of cell development or cell differentiation by a method according to any one of the preceding embodiments, and visualizing collagen secreted by cells.
  • the microfluidic chamber comprises an inlet port and an outlet port, the inlet port being connectable to a cell culture medium influx, and the outlet port allow cell culture medium outflow to leave the chamber.
  • the simplest form to generate a chamber for practicing the method of the invention is to situate the macroporous hydrogel between an inlet and an outlet.
  • a chamber with inlet and outlet is expected to provide the minimal functionality required for the generation of FSS, even though such simple setup might cause some technical difficulties when injecting the hydrogel precursor, since the medium channels are expected to be blocked relatively easily.
  • the flow through the gel in such a simple chip would vary a lot depending on the position along the channel since the pressure difference would be larger compared to a more complex geometry, wherein two fluid streams are provided on opposite sides of a chamber, the two streams creating a pressure gradient between them.
  • a more complex geometry wherein two fluid streams are provided on opposite sides of a chamber, the two streams creating a pressure gradient between them.
  • the polymer chains comprise a peptide molecule capable of promoting adherence of cells.
  • the peptide molecule capable of promoting adherence of cells is a peptide comprising a fibronectin-derived arginyl-glycyl-aspartic acid motif.
  • the peptide is or comprises SEQ ID NO 02 (GRCGRGDSPG) and SEQ ID NO 05 (CGRGDSP). This RGD peptide only has 1 C moiety, and is added to react with only 10%, at most, of vinylsulfone residues.
  • MIPs maximum intensity projections
  • e 3D view of actin-nuclei stained embedded cells as shown in d), scale bars: 200 ⁇ m.
  • Fig.6 shows a histological analysis of static hMSC culture within MMP-degradable and non-degradable PEG gels: 2.0% 4-PEG-VS, 0.5% HA, and cell density at 3.5 ⁇ 10 6 ml -1 .
  • Fig. 5c Quantification of mean cell area further evidenced the permissiveness of degradable gels.
  • the average cell area in the degradable gels was significantly larger compared to non-degradable gels.
  • MIPs maximum intensity protections
  • Fig. 5e shows the maximum intensity protections (MIPs) and 3D renderings of actin-nuclei stained cells, respectively.
  • the red color content significantly increased from day 8 to day 30 (p ⁇ 0.01). Alizarin red staining further indicated more pronounced matrix mineralization especially in close proximity to embedded cells within the degradable gels compared to non- degradable ones.
  • an increase in mineral deposition on day 30 implies the 3D osteogenic differentiation of hMSCs into a mature bone cell phenotype.
  • Osteocalcin a mature marker for osteoblasts, was predominantly expressed in MMP-degradable gels after cultivation for 30 days. In contrast, only limited expression of osteocalcin was observed in the non-degradable gels (Fig. 6c).
  • single hMSCs and hOBs sense the porous architecture to form an interconnected cellular network in 3D and then differentiate into an osteoid-like tissue.
  • macroporous PEG hydrogels are chemically defined.
  • the in situ PIPS process allows for the formation of interconnected pores in the presence of living cells, which is unachievable with other types of macroporous hydrogels formed by emulsification, porogen leaching and particle annealing.
  • the established tool could be used in the future to investigate the mechanisms of cell-matrix interactions as well as matrix defects in musculoskeletal disorders such as OI.
  • MMP-degradable acellular PEG gels (2.2% w/v 4-PEG- VS) were casted into microfluidic chips with attached luer connectors (AIM Biotech, LUC-1). After hydration for 24 h, flow imaging was performed on a wide field microscope (Olympus, IX83). Two different tracer solutions were prepared by diluting a 0.1% (w/v) stock solution of 70 kDa or 500 kDa FITC-dextran (both Sigma-Aldrich, FD70S-100MG and FD500S-100MG) 1:1000 in phenol red free DMEM.
  • Acellular PEG gels and collagen type I hydrogels were used.2 mg ml -1 collagen type I gel was prepared from an 8.91 mg ml -1 stock solution (rat-tail, Corning, 354249) as described by Shin et al. (Nature protocols 2012, 7, 1247) and casted on-chip. To determine the permeability, all gels were first hydrated in PBS for 24 h after crosslinking.
  • Ki67 Staining To stain cells embedded in PEG hydrogels for the cell proliferation marker Ki67, fixed samples from day 0 and day 2 of osteogenic culture were used. For immunohistochemistry, cells were first permeabilized for 10 min with 0.2% Triton X-100 (Sigma-Aldrich, 9002-93-1), then non-specific antibody binding was blocked with 1% BSA (Sigma, 9048-46-8) and 5% goat serum (Abcam, ab7481) for 1 h. The primary antibody (Invitrogen, MA5-14520) was diluted in PBS containing 1% BSA (1:250).
  • Actin-nuclei staining was performed to further investigate cellular and subcellular morphology and cellular network formation.
  • Gels were incubated in 1% BSA in PBS for 1.5 h at room temperature. Subsequently, cells were permeabilized in a solution of 0.2% Triton X-100 in 0.1% BSA in PBS for 10 min. Gels were washed 3 times with PBS.
  • the staining solution containing dilutions of 1:1000 Hoechst 33342 and 1:200 Phalloidin CruzFluor 647 Conjugate or Phalloidin-TRITC (Sigma-Aldrich, P1951) in 0.1% BSA was prepared.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Dispersion Chemistry (AREA)
  • Immunology (AREA)
  • Mechanical Engineering (AREA)
  • Cell Biology (AREA)
  • Computer Hardware Design (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé de génération d'un modèle de culture cellulaire in vitro de développement cellulaire ou de différenciation cellulaire, comprenant a) la fourniture d'une chambre microfluidique à travers laquelle un flux de milieu de culture cellulaire peut être conduit ; b) la fourniture d'une pluralité de cellules primaires incorporées dans un hydrogel macroporeux synthétique, l'hydrogel macroporeux comprenant des chaînes polymères réticulées par des molécules de liaison sensibles au clivage par une endopeptidase extracellulaire, c) l'application d'un flux de milieu de culture cellulaire à ladite pluralité de cellules souches à l'intérieur de la chambre microfluidique, soumettant ainsi lesdites cellules à une contrainte de cisaillement de fluide afin de déclencher une maturation fonctionnelle et une sécrétion de collagène. Un autre aspect de l'invention concerne un dispositif microfluidique, comprenant une chambre microfluidique qui comprend un hydrogel macroporeux dans lequel des cellules sont présentes. L'hydrogel macroporeux est composé de chaînes polymères réticulées par des molécules de liaison sensibles au clivage par une endopeptidase extracellulaire.
EP23732527.9A 2022-06-13 2023-06-13 Visualisation de collagène sur un dispositif microfluidique Pending EP4536801A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP22178671 2022-06-13
EP22183705 2022-07-07
PCT/EP2023/065802 WO2023242189A1 (fr) 2022-06-13 2023-06-13 Visualisation de collagène sur un dispositif microfluidique

Publications (1)

Publication Number Publication Date
EP4536801A1 true EP4536801A1 (fr) 2025-04-16

Family

ID=86896119

Family Applications (1)

Application Number Title Priority Date Filing Date
EP23732527.9A Pending EP4536801A1 (fr) 2022-06-13 2023-06-13 Visualisation de collagène sur un dispositif microfluidique

Country Status (2)

Country Link
EP (1) EP4536801A1 (fr)
WO (1) WO2023242189A1 (fr)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2934662C (fr) * 2013-12-20 2024-02-20 President And Fellows Of Harvard College Dispositifs microfluidiques a faible cisaillement et leurs procedes d'utilisation et de fabrication
US11229607B2 (en) * 2014-06-30 2022-01-25 President And Fellows Of Harvard College Hydrogel compositions comprising encapsulated cells and methods of use thereof
CN107108199B (zh) 2014-11-11 2020-01-10 微研生物科技有限公司 用于研究基于细胞的相互作用的微流体平台
EP3244929A1 (fr) * 2015-01-15 2017-11-22 Massachusetts Institute of Technology Hydrogel comprenant un macromère d'échafaudage réticulé avec un peptide et un motif de rèticulation
LT3347061T (lt) 2015-09-09 2021-09-10 ETH Zürich Įšvirkščiami makroporiniai hidrogeliai
US10988723B1 (en) * 2015-09-23 2021-04-27 National Technology & Engineering Solutions Of Sandia, Llc Modular assemblies and systems for cell cultures and methods thereof

Also Published As

Publication number Publication date
WO2023242189A1 (fr) 2023-12-21

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